The complete book - Cucurbit Breeding - North Carolina State ...

ORGANIZING COMMITTEE 

Gerald J. Holmes, Chair, North Carolina State University, Raleigh, North Carolina 

Jonathan R. Schultheis, North Carolina State University, Raleigh, North Carolina 

Todd C. Wehner, North Carolina State University, Raleigh, North Carolina 

SCIENTIFIC COMMITTEE & EDITORIAL BOARD 

Angela Davis, USDA-ARS, Lane, Oklahoma, USA 

Maria Luisa Gomez-Guillamon, Experiment Station ‘La Mayora’, Malaga, SPAIN 

Elzbieta Kozik, Research Institute of Vegetable Crops, Skierniewice, POLAND 

Ales Lebeda, Palacky University, Olomouc, CZECH REPUBLIC 

Donald Maynard, University of Florida, Bradenton, Florida, USA 

James McCreight, USDA-ARS, Salinas, California, USA 

Ray Martyn, Purdue University, West Lafayette, Indiana, USA 

Harry Paris, Newe Ya’ar Research Center, Ramat Yishay, ISRAEL 

Gordon Rogers, University of Sydney, AUSTRALIA 

Michel Pitrat, INRA, Montfavet cedex, FRANCE 

Susan Webb, University of Florida, Gainesville, Florida, USA 

Xingping Zhang, Syngenta Seeds, Woodland, California, USA 

SPONSORS 

Diamond Silver 

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Syngenta Chemtura Corporation 

Dow AgroSciences 

Hendrix and Dail, Inc. 

Hollar Seeds 

North Carolina Department of 

Agriculture and Consumer Services 

Valent BioSciences 

Willhite Seed Inc. 

Gold Bronze 

Abbott & Cobb, Inc. Addis Cates Company, Inc. 

DuPont Clifton Seed Company 

Harris Moran FMC Corporation 

Mt. Olive Pickle Company Nat’l Watermelon Promotion Board 

Nunhems North Carolina Watermelon Assoc. 

Sakata Seed USA North Carolina Pickle Producers 

Association 

ORO Agri 

Pickle Packers International, Inc. 

Rupp Seeds, Inc. 

UAP Carolinas 

Xenacom

PREFACE 

n behalf of the organizing committee of Cucurbitaceae 2006, I 

welcome you to Asheville, North Carolina for the 9 th O 

biennial 

gathering of cucurbitologists. This tradition began in 1989 

with Claude Thomas hosting the first conference in South Carolina, 

U.S.A. Poland hosted the meeting in 1992. Since then the conference 

has alternated between countries on both sides of the Atlantic in evennumbered 

years: Texas (1994), Spain (1996), California (1998), Israel 

(2000), Florida (2002), and the Czech Republic (2004). We are 

pleased to continue this rich tradition by hosting Cucurbitaceae 2006 

in the mountains of western North Carolina at the lovely Grove Park 

Inn, Resort and Spa in Asheville. 

Many people have contributed to the planning and execution of 

this conference. I am especially grateful to the organizing committee 

for assistance in planning all aspects of the conference and to the 

scientific committee for their prompt and careful editing of these 

proceedings. These committees are also responsible for selecting the 

three recipients of the Lifetime Achievement Award for outstanding 

contributions to the Cucurbitaceae. The Cucurbitaceae 2006 awardees 

are Harry Paris (breeding and genetics), Michel Pitrat (plant 

pathology) and Donald Maynard (crop production). Special thanks to 

Jane Dove Long of the Plant Pathology Department at NC State 

University for her tireless efforts and attention to detail in coordinating 

and executing the endless tasks, big and small, that make a conference 

successful. 

These proceedings contain 81 contributed papers from 236 

authors in 12 countries and 16 states within the U.S.A. and span the 

areas of research typical of any major horticultural crop. Special 

thanks go to Carolyn Currie for editing each manuscript, 

communicating with authors all over the world and especially for 

formatting the final document for printing. 

Lastly, I want to recognize the gracious support of our 

sponsors. Without their contributions, the cost of this conference 

would be much higher for every participant. Please take a moment to 

thank them for their generous support. 

On behalf of the Organizing Committee, we welcome you to 

Cucurbitaceae 2006 and look forward to a lively exchange of new 

ideas and discoveries in the Cucurbitaceae. 

GERALD J. HOLMES, CHAIR 

Cucurbitaceae 2006 Organizing Committee

Adkins, S. ............................. 309 

Akashi, Y. .................... 317, 372 

Alarcón, A. L. ...................... 146 

Álvarez, J. M. ....................... 146 

Ando, K. ............................... 347 

Andres, T. C. ................ 326, 333 

Aranda, M. ................... 180, 186 

Arús, P. .........146, 180, 186, 553 

Azulay, Y. .............................. 31 

Babadoost, M. ...................... 507 

Baker, C. A. ......................... 309 

Bar, E. .................................... 31 

Bascur, G. .............................. 65 

Becker, J. O. ......................... 395 

Becker, J. S. ......................... 395 

Bendahmane, A. ..................... 89 

Benyamini, Y. ........................ 31 

Besombes, D. ......................... 89 

Blanca, J. ...................... 180, 186 

Boualem, A. ........................... 89 

Bruton, B. ............................. 277 

Buckley, R. .......................... 539 

Burger, Y. .............................. 31 

Bustamente, R. ..................... 333 

Caboche, M. ........................... 89 

Caño, A. ............................... 180 

Cantliffe, D. J. ...................... 483 

Cavanagh, A. ........................ 232 

Chen, Q-J. ............................ 220 

Chou, T. T. ........................... 317 

Chung, S. M. ........................ 197 

Collins, J. K. 

...............545, 578, 585, 591, 597 

Colucci, S. J. ........................ 403 

Cortright, B. ......................... 427 

Cothran, A. ........................... 286 

Cowgill, W. .......................... 539 

Crosby, K. .........23, 70, 153, 165 

Cui, C. .................................. 133 

Dalmais, M. ............................ 89 

Dane, F. ............................ 48, 78 

Datnoff, L. E. ......................... 43 

Daunay, M.-C. ............. 235, 363 

Davis, A. .......125, 258, 412, 545 

Deleu, W. ............................. 180 

AUTHOR INDEX 

DeNicco, A. ........................... 301 

Dittmar, P. J. .......................... 241 

Dogimont, C. ............................ 89 

Dolan, K. ................................ 585 

Du, D. ..................................... 301 

Eduardo, I. ............................. 553 

Eubanks, M. D. .............. 146, 492 

Fergany, M. .............................. 89 

Fernández-Trujillo, J. P. . 146, 553 

Fish, W. ...................... 1, 277, 545 

Fujita, M. ................................. 14 

Fukino, N. ................................ 95 

Fukunaga, K. .......................... 372 

Gajdová, J. ................................. 9 

Gałecka, T. ................................515 

García-Mas, J. ................ 180, 186 

Garrido, D. ............................. 171 

Garza-Ortega, S. .................... 560 

Gasmanová, N. ......................... 51 

Gevens, A. J. .......................... 427 

Giovinazzo, N. ......................... 89 

Glala, A. ................................. 158 

Gómez, P. ............................... 171 

Gómez-Guillamón, M. L. 100, 108 

Gong, G. ......................... 133, 212 

González, D. .................. 180, 186 

González, M. .................. 180, 186 

González, V. .......................... 180 

González-Román, M. ............. 206 

Grajeda-García, L. F. ............. 560 

Grumet, R. ....................... 39, 387 

Guner, N. ............................... 468 

Guo, S. ................................... 212 

Haider, M. S. .......................... 527 

Hammar, S. .............................. 39 

Harp, T. L. ............................. 421 

Hassell, R. L. .......................... 591 

Hausbeck, M. K. .................... 427 

Hazzard, R. ............................ 232 

He, N. ..................................... 296 

Heckman, J. R. ....................... 539 

Henninger, M. R. ........... 116, 539 

Heredia, A. ............................. 108 

Hernandez, A. ........................ 125

Hofer, D. ...............................395 

Holmes, G. J. ........................403 

Holmstrom, K. E. ..................539 

Hopkins, D. L. ......................436 

Hossain, M. D. ........................14 

Huber, D. J. ...........................578 

Ibdah, M. ................................31 

Infante-Casella, M. L. ...116, 539 

Jamilena, M. .........................171 

Janick, J. .......341, 349, 358, 363 

Jester, B. ...............................591 

Jett, L. W. .............................249 

Jifon, J. ...................................23 

Jullian, E. ..............................358 

Kato, K. ........................317, 372 

Katzir, N. ........................31, 125 

Khaing, M. T. ...............317, 372 

King, S. .........................125, 258 

Kline, W. L. ..........................539 

Kołakowska, G. .......................515 

Korzeniewska, A. ....................515 

Kozik, E. U. .............................121 

Kuhn, P. J. ............................421 

Kunihisa, M. ...........................95 

Larkov, O. ...............................31 

Lebeda, A. ..........9, 51, 444, 453 

Leskovar, D. .....................23, 70 

Lester, Gene E. .......................70 

Levi, A. .........125, 380, 412, 468 

Lewinsohn, E. .........................31 

Li, H. .....................................133 

Li, Y. .....................................133 

Ling, K-S. .............................468 

Little, H. A. .............................39 

Liu, H-Y. ..............................139 

Liu, J. ......................................78 

López-Sesé, A. I. ..................100 

Loy, J. B. ..............................568 

Luo, F. ..................................272 

Maness, N. ....................578, 597 

Mao, A. .........................220, 225 

Martínez, J. A. ..............146, 553 

Marzec, L. ................................515 

Matsumoto, S. .........................95 

Maynard, D. N. .....................591 

McCreight, J. D. ...................139 

McGrath, M. T. ...................... 473 

Miller, M. ................................. 23 

Miranda, C. ............................ 553 

Mitchell, J. M. ........................... 483 

Monforte, A. ...........146, 180, 553 

Monks, D. W. ........................ 241 

Morton, H. V. ........................ 395 

Murphy, J. F. ......................... 492 

Napier, A. B. .......................... 153 

Navrátilová, B. ..................... 9, 51 

Niemirowicz-Szczytt, K. .......... 515 

Nishida, H. ......................317, 372 

Nitzsche, P. J. ........................ 539 

Nuez, F. ..........................180, 186 

Obando, J. .......................146, 553 

Ohara, T. ...........................95, 193 

Olson, S. ................................ 591 

Omran, S. ............................... 158 

Özdemir, Z. ............................ 498 

Paris, H. S. ......341, 349, 358, 363 

Park, S. O. .......................153, 165 

Pavon, C. ............................... 507 

Payán, M. C. .......................... 171 

Peñaranda, A. ......................... 171 

Perkins-Veazie, P. 

.................545, 578, 585, 591, 597 

Picó, B. ...........................180, 186 

Piskurewicz, U. ......................... 515 

Pitrat, M. ...........................89, 412 

Pompan-Lotan, M. ................... 31 

Portnoy, V. ........................31, 135 

Puigdomènech, P. ............180, 186 

Rajab, M. ................................. 89 

Ravid, U. .................................. 31 

Robbins, M. D. ...................... 197 

Roberts, W. .....................277, 597 

Robles, A. .............................. 180 

Roig, C. ...........................180, 186 

Rosales, R. ............................. 171 

Sakata, Y. ..........................95, 193 

Samulis, R. J. ......................... 539 

Sánchez-Estrada, A. ............... 560 

Sargent, S. A. ......................... 483 

Sarria, E. .........................100, 108 

Schaffer, A. A. ......................... 31 

Schultheis, J. R. ......241, 265, 591

Sedláková, B. ......................... 444 

Si, Y. ........................................ 48 

Siddiq, M. .............................. 585 

Skálová, D. ............................... 51 

Śmiech, M. ................................ 515 

Smith, M. ............................... 301 

Staub, J. E. ............................. 197 

Stephenson, A. G. .................. 301 

Stoffella, P. J. ......................... 483 

Sugiyama, K. .......................... 158 

Sugiyama, M. ......................... 193 

Sun, X. ................................... 272 

Sun, Z. .................................... 197 

Sztangret-Wiśniewska, J. ......... 515 

Tadmor, Y. ............... 31, 125, 545 

Tahir, M. ................................ 527 

Tanaka, K. ...................... 317, 372 

Taylor, M. .............................. 277 

Tetteh, A. ............................... 412 

Thies, J. A. ............................. 380 

Thimmapuram, J. ................... 125 

Thompson, C. M. ................... 146 

Tietjen, W. H. ........................ 539 

Tirpak, S. ............................... 539 

Tory, D. R. ............................... 42 

Trebitsh, T. ............................. 125 

Troadec, C. ............................... 89 

Troncoso-Rojas, R. ................ 560 

Turíni, T. A. ........................... 139 

Tzuri, G. ................................... 31 

Ugás, R. ................................. 333 

Urban, J. ................................. 453 

van der Knaap, E. ................... 146 

Wang, Y. .......................... 20, 225 

Wargo, J. ................................ 286 

Webb, S. E. ............................ 309 

Webber, III, C. ....................... 545 

Wechter, W. P. ....................... 125 

Wehner, T. C. . 121, 403, 412, 468 

Wenge, L. ............................... 296 

Wessel-Beaver, L. .................. 206 

Whipkey, A. ........................... 348 

Widrlechner, M. P. ................. 453 

Wien, H. C. .............................. 60 

Winsor, J. A. .......................... 301 

Wyenandt, C. A. .... 116, 534, 539 

Xu, Y. ............................. 212, 225 

Yan, Z. ................................... 296 

Yariv, Y. .................................. 31 

Yi, S. S. .................................. 317 

Yuste-Lisbona, F. J. ............... 100 

Zhang, F. ........................ 220, 225 

Zhang, H-Y. ... 133, 212, 220, 225 

Zhao, S. .................................... 96 

Zitter, T. A. ...................... 60, 498

TABLE OF CONTENTS 

Biotechnology and Physiology 

SPECTROPHOTOMETRIC QUANTITATION OF WATERMELON 

LYCOPENE EXTRACTED INTO AQUEOUS SODIUM DODECYL 

SULFATE 

Wayne W. Fish and Angela R. Davis ..........................................................................1 

PROTOPLAST FUSION IN GENUS CUCUMIS 

J. Gajdová, B. Navrátilová, and A. Lebeda .................................................................9 

EXPRESSION PROPERTIES OF THREE TAU-TYPE PUMPKIN 

GLUTATHIONE S-TRANSFERASES IN BACTERIA AND A SEARCH FOR 

THEIR INTRINSIC INHIBITORS 

M. D. Hossain and Masayuki Fujita ..........................................................................14 

PHYSIOLOGICAL CHARACTERISTICS OF GRAFTED MUSKMELON 

GROWN IN MONOSPORASCUS CANNONBALLUS-INFESTED SOIL IN 

SOUTH TEXAS 

John Jifon, Kevin Crosby, Marvin Miller, and Daniel Leskovar...............................23 

FUNCTIONAL GENOMICS OF GENES INVOLVED IN THE FORMATION 

OF MELON AROMA 

Nurit Katzir, Vitaly Portnoy, Yael Benyamini, Yoela Yariv, Galil Tzuri, Maya 

Pompan-Lotan, Olga Larkov, Einat Bar, Mwafaq Ibdah, Yaniv Azulay, Uzi Ravid, 

Yosef Burger, Arthur A. Schaffer, Yaakov Tadmor, and Efraim Lewinsohn ...........31 

STUDIES OF TRANSGENIC MELON EXPRESSING THE MUTANT 

ETHYLENE RECEPTOR, ETR1-1, INDICATE THAT ETHYLENE 

PERCEPTION BY STAMEN PRIMORDIA IS REQUIRED FOR CARPEL 

DEVELOPMENT IN MELON FLOWERS 

H. A. Little, S. Hammar, and R. Grumet ...................................................................39 

DROUGHT-INDUCED GENE EXPRESSION IN ROOTS OF CITRULLUS 

COLOCYNTHIS 

Ying Si and Fenny Dane ...........................................................................................48 

EMBRYO CULTURE AS A TOOL FOR INTERSPECIFIC 

HYBRIDIZATION OF CUCUMIS SATIVUS AND WILD CUCUMIS SPP. 

D. Skálová, B. Navrátilová, A. Lebeda, and N. Gasmanová .....................................51 

INIATIATING SUDDEN WILT DISORDER IN MUSKMELON WITH LOW- 

LIGHT STRESS 

H. C. Wien and T. A. Zitter .......................................................................................60

Breeding and Genetics 

DEVELOPMENT OF IMPROVED CULTIVARS OF ZUCCHINI SQUASH 

AND SWEETPOTATO SQUASH STARTING FROM CHILEAN 

LANDRACES 

Gabriel Bascur . .........................................................................................................65 

GENETIC VARIATION FOR BENEFICIAL PHYTOCHEMICAL LEVELS 

IN MELONS (CUCUMIS MELO L.) 

Kevin M. Crosby, Gene E. Lester, and Daniel I. Leskovar........................................70 

PHYLOGEOGRAPHY AND EVOLUTION OF WILD AND CULTIVATED 

WATERMELON 

Fenny Dane and Jiarong Liu ......................................................................................78 

FINE GENETICAL AND PHYSICAL MAPPING OF THE GENES A AND G 

CONTROLLING SEX DETERMINATION IN MELON 

M. Fergany, C. Troadec, M. Rajab, A. Boualem, M. Dalmais, M. Caboche, A. 

Bendahmane, D. Besombes, N. Giovinazzo, C. Dogimont, and M. Pitrat.................89 

QUANTITATIVE TRAIT LOCUS ANALYSIS OF POWDERY MILDEW 

RESISTANCE AGAINST TWO STRAINS OF PODOSPHAERA XANTHII IN 

THE MELON LINE ‘PMAR NO. 5’ 

N. Fukino, T. Ohara, Y. Sakata, M. Kunihisa, and S. Matsumoto.............................95 

LINKAGE ANALYSIS AMONG RESISTANCES TO POWDERY MILDEW 

AND VIRUS TRANSMISSION BY APHIS GOSSYPII GLOVER IN MELON 

LINE ‘TGR-1551’ 

M. L. Gómez-Guillamón, A. I. López-Sesé, E. Sarria, and F. J. Yuste-Lisbona 

.................................................................................................................................100 

EPICUTICULAR WAX MORPHOLOGY AND TRICHOME TYPES IN 

RELATION TO HOST PLANT SELECTION BY APHIS GOSSYPII IN 

MELONS 

M. L. Gómez-Guillamón, A. Heredia, and E. Sarria................................................108 

EVALUATION OF ZUCCHINI AND STRAIGHTNECK SUMMER SQUASH 

BREEDING LINES AND VARIETIES FOR POWDERY MILDEW AND 

DOWNY MILDEW TOLERANCE 

M. L. Infante-Casella, C. A. Wyenandt, and M. R. Henninger................................116 

INHERITANCE OF CHILLING RESISTANCE IN CUCUMBER SEEDLINGS 

Elzbieta U. Kozik and Todd C. Wehner ..................................................................121 

GENES EXPRESSED DURING DEVELOPMENT AND RIPENING OF 

WATERMELON FRUIT 

A. Levi, W. P. Wechter, A. Davis, A. Hernandez, J. Thimmapuram, T. Trebitsh, Y. 

Tadmor, N. Katzir, V. Portnoy, and S. King............................................................125

RESEARCH OF MOLECULAR MARKERS LINKED TO THE DWARF 

GENE IN SQUASH 

Haizhen Li, Haiying Zhang, Guoyi Gong, Yunlong Li, and Chongshi Cui.............133 

REACTION OF MELON PI 313970 TO CUCURBIT LEAF CRUMPLE VIRUS 

James D. McCreight, Hsing-Yeh Liu, and Thomas A. Turini .................................139 

THE MELON GENOMIC LIBRARY OF NEAR ISOGENIC LINES: A TOOL 

FOR DISSECTING COMPLEX TRAITS 

Antonio José Monforte, Iban Eduardo, Pere Arús, Juan Pablo Fernández-Trujillo, 

Javier Obando, Juan Antonio Martínez, Antonio Luís Alarcón, Esther van der Knaap, 

and Jose María Álvarez ...........................................................................................146 

MOLECULAR MARKERS ASSOCIATED WITH QUANTITATIVE 

CONTROL OF BETA-CAROTENE SYNTHESIS IN CUCUMIS MELO L. 

A. B. Napier, S. O. Park, and K. M. Crosby............................................................153 

A NEW EFFECTIVE TECHNIQUE FOR PRODUCING SEEDLESS 

WATERMELON FRUITS FROM SOME DIPLOID CULTIVARS 

S. Omran, K. Sugiyama, A. Glala............................................................................158 

QUANTITATIVE TRAIT LOCI FOR SUCROSE PERCENTAGE OF 

TOTAL SUGARS IN MELON CROSSES UNDER GREENHOUSE 

AND FIELD ENVIRONMENTS 

Soon O. Park and Kevin M. Crosby ........................................................................165 

ETHYLENE MEDIATES THE INDUCTION OF FRUITS WITH ATTACHED 

FLOWER IN ZUCCHINI SQUASH 

M. C. Payán, A. Peñaranda, R. Rosales, D. Garrido, P. Gómez, and M. Jamilena 

.................................................................................................................................171 

THE MELOGEN PROJECT: DEVELOPMENT OF GENOMIC TOOLS IN 

MELON 

Pere Puigdomènech, Ana Caño, Víctor González, Pere Arús, Wim Deleu, Jordi 

Garcia-Mas, Mireia González, Antonio Monforte, José Blanca, Fernando Nuez, 

Belén Picó, Cristina Roig, Miguel Aranda, Daniel González, and Antonio Robles 

................................................................................................................................180 

THE MELOGEN PROJECT: EST SEQUENCING, PROCESSING AND 

ANALYSIS 

Pere Puigdomènech, Pere Arús, Jordi Garcia-Mas, Mireia González, Fernando Nuez, 

José Blanca, Belén Picó, Cristina Roig, Miguel Aranda, and Daniel González 

.................................................................................................................................186 

DEVELOPMENT OF A POWDERY MILDEW-RESISTANT CUCUMBER 

Yoshiteru Sakata, Mitsuhiro Sugiyama and Takayoshi Ohara ...............................193 

HISTORY AND APPLICATION OF MOLECULAR MARKERS FOR 

CUCUMBER IMPROVEMENT 

J.E. Staub, M.D. Robbins, S.M. Chung, and Z. Sun ...............................................197

SILVERLEAF WHITEFLY AFFECTS LEAF MOTTLING IN CUCURBITA 

MOSCHATA DUCHESNE 

Linda Wessel-Beaver and Moisés González-Román ..............................................206 

QTL ANALYSIS OF SOLUBLE SOLIDS CONTENT IN WATERMELON 

UNDER DIFFERENT ENVIRONMENTS 

Yong Xu, Shaogui Guo, Haiying Zhang, and Guoyi Gong ....................................212 

MAPPING AND QTL ANALYSIS CONCERNING TOLERANCE TOWARD 

POOR LIGHT IN CUCUMBER 

(CUCUMIS SATIVUS L.) 

Yong-Jian Wang, Hai-Ying Zhang, Qing-Jun Chen, 

Feng Zhang, and Ai-Jun Mao .................................................................................220 

CONSTRUCTION OF A GENETIC MAP OF CUCUMBER USING RAPDS, 

SSRS, AFLPS AND MAPPING RESISTANCE GENES TO PAPAYA 

RINGSPOT, ZUCCHINI YELLOW MOSAIC, AND WATERMELON MOSAIC 

VIRUSES 

Haiying Zhang, Aijun Mao, Feng Zhang, Yong Xu and Yongjian Wang ...............225 

Crop Production 

PERIMETER TRAP CROP SYSTEMS USING BLUE HUBBARD SQUASH 

AS A CONTROL FOR STRIPED CUCUMBER BEETLE IN BUTTERNUT 

SQUASH 

Andrew Cavanagh and Ruth Hazzard ......................................................... 232 

CHARACTERIZATION OF COMMERCIALLY AVAILABLE 

WATERMELON POLLENIZERS 

Peter J. Dittmar, Jonathan R. Schultheis, and David W. Monks..............................241 

GALIA MUSKMELON PRODUCTION IN HIGH TUNNELS IN THE 

CENTRAL GREAT PLAINS USA 

Lewis W. Jett ...........................................................................................................249 

A COMPARISON OF NOVEL GRAFTING METHODS FOR 

WATERMELON IN HIGH-WIND AREAS 

Stephen R. King and Angela R. Davis.....................................................................258 

CUCURBITS AND THEIR IMPORTANCE IN NORTH CAROLINA 

Jonathan Schultheis..................................................................................................265 

APPROACHES TO MINIMIZE THE DEFECTS OF SEEDLESS 

WATERMELON 

Xiaowu Sun and Fuqing Luo ...................................................................................272 

COST BENEFIT ANALYSES OF USING GRAFTED WATERMELONS FOR 

DISEASE CONTROL AND THE FRESH-CUT MARKET 

Merritt Taylor, Benny Bruton, Wayne Fish, and Warren Roberts ...........................277

NITAMIN ® LIQUID: BACKGROUND AND USE ON CUCURBITACEAE 

FAMILY 

James Wargo and Anne Cothran .............................................................................286 

TRIPLOID SEEDLESS WATERMELON PRODUCTION IN CHINA 

Liu Wenge, Yan Zhihong, Zhao Shengjie, and He Nan .........................................296 

Entomology 

HERBIVORY BY CUCUMBER BEETLES AFFECTS POLLEN 

PRODUCTION AND POLLEN PERFORMANCE IN A WILD GOURD 

Andrew G. Stephenson, James A. Winsor, Daolin Du, Andrew DeNicco, and 

Matthew Smith ........................................................................................................301 

WHITEFLY TRANSMISSION OF A NEW VIRUS INFECTING CUCURBITS 

IN FLORIDA 

Susan E. Webb, Scott Adkins, and Carlye A. Baker ...............................................309 

Germplasm 

GENETIC DIVERSITY AND PHYLOGENETIC RELATIONSHIP AMONG 

MELON ACCESSIONS FROM AFRICA AND ASIA REVEALED BY RAPD 

ANALYSIS 

Y. Akashi, K. Tanaka, H. Nishida, K. Kato, M. T. Khaing, S. S. Yi, and 

T. T. Chou................................................................................................................317 

ORIGIN, MORPHOLOGICAL VARIATION, AND USES OF CUCURBITA 

FICIFOLIA, THE MOUNTAIN SQUASH 

Thomas C. Andres ...................................................................................................326 

LOCHE: A UNIQUE PRE-COLUMBIAN SQUASH LOCALLY GROWN IN 

NORTH COASTAL PERU 

Thomas C. Andres ...................................................................................................333 

OLD WORLD CUCURBITS IN PLANT ICONOGRAPHY OF THE 

RENAISSANCE 

Jules Janick and Harry S. Paris................................................................................341 

JONAH AND THE “GOURD” AT NINEVEH: CONSEQUENCES OF A 

CLASSIC MISTRANSLATION 

Jules Janick and Harry S. Paris ................................................................................349 

DEVELOPMENT OF AN IMAGE DATABASE OF CUCURBITACEAE 

Jules Janick, Anna Whipkey, Harry S. Paris, Marie-Christine Daunay, and E. Jullian 

.................................................................................................................................358 

FIRST IMAGES OF CUCURBITA IN EUROPE 

Harry S. Paris, Jules Janick, and Marie-Christine Daunay ......................................363

POLYPHYLETIC ORIGIN OF CULTIVATED MELON INFERRED FROM 

ANALYSIS OF ITS CHLOROPLAST GENOME 

K. Tanaka, K. Fukunaga, Y. Akashi, H. Nishida, K. Kato, and M. T. Khaing ........372 

RESISTANCE OF CITRULLUS LANATUS VAR. CITROIDES GERMPLASM 

TO ROOT-KNOT NEMATODES 

Judy A. Thies and Amnon Levi ..............................................................................380 

Phytopathology 

FACTORS INFLUENCING A CUCUMBER FRUIT SUSCEPTIBILITY TO 

INFECTION BY PHYTOPHTHORA CAPSICI 

Kaori Ando and Rebecca Grumet ............................................................................387 

EARLY PROTECTION AGAINST ROOT-KNOT NEMATODES THROUGH 

NEMATICIDAL SEED COATING PROVIDES SEASON-LONG BENEFITS 

FOR CUCUMBERS 

J. O. Becker, J. Smith Becker, H. V. Morton, and D. Hofer....................................395 

THE DOWNY MILDEW EPIDEMIC OF 2004 AND 2005 IN THE EASTERN 

UNITED STATES 

Susan J. Colucci, Todd C. Wehner, and Gerald J. Holmes......................................403 

WATERMELON RESISTANCE TO POWDERY MILDEW RACE 1 AND 

RACE 2 

Angela R. Davis, Antonia Tetteh, Todd Wehner, Amnon Levi, and Michel 

Pitrat.........................................................................................................................412 

DEVELOPMENT OF THE FUNGICIDE MANDIPROPAMID IN THE 

UNITED STATES FOR CONTROL OF DOWNY MILDEW OF CUCURBITS 

Tyler L. Harp, Donald R. Tory, and Paul J. Kuhn ...................................................421 

INTEGRATING CULTURAL AND CHEMICAL STRATEGIES TO 

CONTROL PHYTOPHTHORA CAPSICI AND LIMIT ITS SPREAD 

M. K. Hausbeck, A. J. Gevens, and B. Cortright .....................................................427 

TREATMENTS TO PREVENT SEED TRANSMISSION OF BACTERIAL 

FRUIT BLOTCH OF WATERMELON 

D. L. Hopkins and C. M. Thompson........................................................................436 

IDENTIFICATION AND SURVEY OF CUCURBIT POWDERY MILDEW 

RACES IN CZECH POPULATIONS 

A. Lebeda and B. Sedláková....................................................................................444 

INDIVIDUAL AND POPULATION ASPECTS OF INTERACTIONS 

BETWEEN CUCURBITS AND PSEUDOPERONOSPORA CUBENSIS: 

PATHOTYPES AND RACES 

A. Lebeda, M. P. Widrlechner, and J. Urban ..........................................................453

EVALUATING ZUCCHINI YELLOW MOSAIC VIRUS RESISTANCE IN 

WATERMELON 

Kai-Shu Ling and Amnon Levi, Nihat Guner and Todd C. Wehner ....................468 

OCCURRENCE OF FUNGICIDE RESISTANCE IN PODOSPHAERA 

XANTHII AND IMPACT ON CONTROLLING CUCURBIT POWDERY 

MILDEW IN NEW YORK 

Margaret Tuttle McGrath.........................................................................................473 

FRUIT YIELD, QUALITY PARAMETERS, AND POWDERY MILDEW 

(SPHAEROTHECA FULIGINEA) SUSCEPTIBILITY OF SPECIALTY 

MELON (CUCUMIS MELO L.) CULTIVARS GROWN IN A PASSIVE- 

VENTILATED GREENHOUSE 

J. M. Mitchell, D. J. Cantliffe, S. A. Sargent, L. E. Datnoff, and P. J. Stoffella......483 

INTEGRATION OF BIOLOGICAL CONTROL AND PLASTIC MULCHES 

TO MANAGE WATERMELON MOSAIC VIRUS IN SQUASH 

John F. Murphy and Micky D. Eubanks..................................................................492 

BACTERIAL LEAF SPOT (XANTHOMONAS CAMPESTRIS PV. 

CUCURBITAE) AS A FACTOR IN CUCURBIT PRODUCTION AND 

EVALUATION OF SEED TREATMENTS FOR CONTROL IN NATURALLY 

INFESTED SEEDS 

Zahide Özdemir and Thomas A. Zitter....................................................................498 

DETERMINING DENSITY OF PHYTOPHTHORA CAPSICI OOSPORES IN 

SOIL 

C. Pavon and M. Babadoost ....................................................................................507 

CHARACTERISTICS OF DOUBLE-HAPLOID CUCUMBER (CUCUMIS 

SATIVUS L.) LINES RESISTANT TO DOWNY MILDEW 

(PSEUDOPERONOSPORA CUBENSIS [BERK. ET CURT.] ROSTOVZEV) 

J. Sztangret-Wiśniewska, T. Gałecka, A. Korzeniewska, L. Marzec, G. Kołakowska, U. 

Piskurewicz, M. Śmiech, and K. Niemirowicz-Szczytt ................................................. 515 

NATURALLY OCCURRING STRAINS OF BIPARTITE BEGOMOVIRUSES 

AFFECT SOME MEMBERS OF CUCURBITACEAE FAMILY INSIDE AND 

OUTSIDE THE COTTON ZONE IN PAKISTAN 

M. Tahir and M. S. Haider ......................................................................................527 

EFFECT OF FUNGICIDE CHEMISTRY AND CULTIVAR ON THE 

DEVELOPMENT OF CUCURBIT POWDERY MILDEW ON PUMPKIN IN 

NEW JERSEY 

Christian A. Wyenandt ............................................................................................534

SURVEY FOR CUCURBIT VIRUSES IN COMMERCIAL FIELDS AND 

EVALUATION OF VIRUS-RESISTANT SUMMER SQUASH BREEDING 

LINES IN NEW JERSEY 

Christian A. Wyenandt, Michelle Infante-Casella, Melvin R. Henninger, Richard 

Buckley, Sabrina Tirpak, Kristian E. Holmstrom, Peter J. Nitzsche, William H. 

Tietjen, Raymond J. Samulis, Wesley L. Kline, Win Cowgill, and Joseph R. 

Heckman ..................................................................................................................539 

Postharvest 

A RAPID HEXANE-FREE METHOD FOR ANALYZING TOTAL 

CAROTENOID CONTENT IN CANARY YELLOW-FLESHED 

WATERMELON 

Angela R. Davis, Julie Collins, Wayne W. Fish, Charles Webber III, Penelope 

Perkins-Veazie, and Y. Tadmor...............................................................................545 

QUANTITATIVE TRAIT LOCI ASSOCIATED WITH SUSCEPTIBILITY 

TO POSTHARVEST PHYSIOLOGICAL DISORDERS AND DECAY OF 

MELON FRUIT 

J. P. Fernández-Trujillo, J. A. Martínez, J. Obando, C. Miranda, A. J. Monforte, I. 

Eduardo, and P. Arús ...............................................................................................553 

POSTHARVEST CHARACTERIZATION OF A CUSHAW SQUASH 

BREEDING LINE. 

L. Fernando Grajeda-García, Sergio Garza-Ortega, Alberto Sánchez-Estrada, and 

Rosalba Troncoso-Rojas ..........................................................................................560 

HARVEST PERIOD AND STORAGE AFFECT BIOMASS PARTITIONING 

AND ATTRIBUTES OF EATING QUALITY IN ACORN SQUASH 

(CUCURBITA PEPO) 

J. Brent Loy .............................................................................................................568 

RIPENING CHANGES IN MINIWATERMELON FRUIT 

Penelope Perkins-Veazie and Julie K. Collins Donald J. Huber, and Niels Maness 

.................................................................................................................................578 

CHANGES IN CAROTENOID CONTENT DURING PROCESSING OF 

WATERMELON FOR JUICE CONCENTRATES 

Penelope Perkins-Veazie, J. K. Collins, M. Siddiq, and K. Dolan...........................585 

VARIATION IN CAROTENOIDS AMONG MINIWATERMELONS 

PRODUCED IN FOUR LOCATIONS IN THE EASTERN U.S. 

Penelope Perkins-Veazie, Julie K. Collins, Richard L. Hassell, Donald N. Maynard, 

Jonathan Schultheis, Bill Jester, and Steve Olson....................................................591 

WATERMELON CAROTENOID CONTENT IN RESPONSE TO 

GERMPLASM, MATURITY, AND STORAGE 

Penelope Perkins-Veazie, Julie K. Collins, Angela Davis, Niels Maness, and Warren 

Roberts.....................................................................................................................597

SPECTROPHOTOMETRIC QUANTITATION OF 

WATERMELON LYCOPENE EXTRACTED 

INTO AQUEOUS SODIUM DODECYL 

SULFATE 

Wayne W. Fish and Angela R. Davis 

South Central Agricultural Research Laboratory, Agricultural 

Research Service, P.O. Box 159, Lane, OK 74555. 

ADDITIONAL INDEX WORDS. Citrullus lanatus 

ABSTRACT. The absorbance properties of aqueous sodium dodecyl sulfate 

(SDS) extracts of watermelon tissue were examined as part of an ongoing effort 

to develop simpler, more economical ways to quantify carotenoids in melon 

fruit. Levels of SDS >0.2% extracted and solubilized watermelon lycopenecontaining 

chromoplasts, and the absorbances at 565nm of the extracts obeyed 

Beer’s law. A method was developed for the detergent extraction/spectrophotometric 

determination of watermelon lycopene. Its application on 110 

watermelons representing 14 cultivars yielded results directly comparable to 

conventional lycopene methods (the regression coefficient for linear fit was 

~0.98). The methodology, its advantages, and its limitations are presented. 

L 

ycopene, a fat-soluble carotenoid, is a precursor of β-carotene 

(Sandmann, 1994) and has at least twice the antioxidant 

capacity of β-carotene (Di Mascio et al., 1989). A number of 

epidemiological studies suggest positive health benefits to be derived 

from the consumption of diets high in lycopene (Gerster, 1997), 

although a consensus for either its beneficial or detrimental role in the 

modulation of carcinogenesis remains to be established (reviewed in 

Bramley, 2000; Arab et al., 2001). Red-fleshed watermelon is a rich 

source of lycopene. Previous research has determined that watermelon 

cultivars vary in lycopene content depending on genotype and 

We wish to thank Rick Houser for his technical assistance and Julie Collins for 

performing the HPLC analyses. Mention of trade names or commercial products in 

this article is solely for the purpose of providing specific information and does not 

imply recommendation or endorsement by the U.S. Department of Agriculture. All 

programs and services of the U.S. Department of Agriculture are offered on a 

nondiscriminatory basis without regard to race, color, national origin, religion, sex, 

age, marital status, or handicap. The article cited was prepared by a USDA 

employee as part of his/her official duties. Copyright protection under U.S. 

copyright law is not available for such works. Accordingly, there is no copyright to 

transfer. The fact that the private publication in which the article appears is itself 

copyrighted does not affect the material of the U.S. Government, which can be freely 

reproduced by the public. 

Cucurbitaceae 2006 1

environmental conditions (Perkins-Veazie et al., 2006). To aid in 

breeding programs, plant breeders need a safe, economical, yet reliable 

method to determine lycopene content in individual watermelons. 

Presently, there are three principal experimental methodologies for 

lycopene quantitation. 

Conventional extraction methods coupled with spectrophotometry 

employ organic solvents for the extraction of lycopene from the tissue 

into the solvent. The extracted lycopene is then quantified 

spectrophotometrically by its absorbance in the visible region of the 

spectrum (Beerh and Siddappa, 1959; Adsule and Dan, 1979; Sadler et 

al, 1990; Fish et al., 2002). Though relatively simple and reliable, the 

extraction methodology requires the use of flammable, biologically 

hazardous organic solvents, and thus poses personnel-safety and 

environmental-waste issues. 

HPLC offers both separation/identification and quantitation of 

individual carotenoids (Craft, 2001). Although the research method of 

choice, HPLC requires significant technical expertise, expensive 

instrumentation, columns, and column standards, and the use of 

hazardous solvents. HPLC is too slow and too expensive for the 

routine screening of fruit from a breeding program. 

A recent methodology, Xenon flash spectrophotometry (Davis, et 

al., 2003), allows the direct spectrophotometric determination of 

lycopene in a puree of watermelon flesh. Therefore, it is rapid, simple, 

requires little sample preparation, and uses no hazardous chemicals. 

The two disadvantages of the method are the high cost of the 

instrumentation and the fact that for quantitation, the instrumental 

output initially must be calibrated with lycopene values determined by 

one of the previously mentioned methodologies. 

A recent investigation showed that aqueous solutions of the 

anionic detergent sodium dodecyl sulfate (SDS) extracted and 

solubilized lycopene-containing chromoplasts from watermelon fruit 

tissue. The findings from this study provided sufficient fundamental 

information to allow development of a detergent 

extraction/spectrophotometric method for lycopene quantitation (W. 

Fish, unpublished data). That methodology, its advantages, and its 

limitations are described below. 

Materials and Methods 

PLANT MATERIAL AND SAMPLING. Watermelons used for the 

study were from the 2003 harvest and were obtained from commercial 

producers in California, Texas, and Oklahoma. In addition, three 

watermelon cultivars were grown on research plots at Lane, OK. Two 

2 Cucurbitaceae 2006

to 10 watermelons were used from each of 14 cultivars for a total of 

110 individual watermelons. Seedless, hybrid seeded, and openpollinated 

seeded types of watermelon cultivars were employed. Prior 

to analysis, watermelon tissue was handled in one of two ways. In one 

method, two 100-g tissue samples were removed from the center of the 

watermelon heart and stored at –80 o C until assayed. When assayed, 

samples of frozen watermelon tissue were partially thawed and then 

ground to a homogeneous puree with an electric tissue grinder 

(Brinkman Polytron Homogenizer, Westbury, NY). In the second 

method, 40-g samples were removed from the heart of a fresh 

watermelon and immediately ground to a homogeneous puree with an 

electric tissue grinder. Tissue purees were kept on ice and out of light 

after preparation and until assayed. Purees were stirred on a magnetic 

stirring plate during replicate sampling. Sampling and weighing of 

replicates were performed in reduced room light. 

LYCOPENE ASSAYS BY CONVENTIONAL METHODS. The method of 

Fish et al. (2002) was employed for the conventional solvent 

extraction/analysis procedure. In a few instances, the HPLC elution 

profiles of watermelon carotenoids and their relative amounts 

extracted into SDS were compared to those obtained by direct 

extraction of the tissue with hexane. The method of Craft (2001) was 

employed for HPLC analyses, and was carried out in the laboratory of 

Penny Perkins-Veazie. 

LYCOPENE ASSAY BY SDS EXTRACTION. Approximately 2g of 

watermelon-tissue puree, prepared as described above, were removed 

from the total puree during rapid stirring. This material was weighed 

to the nearest 0.01g into a tared, 15-ml screw-capped plastic centrifuge 

tube (Becton Dickinson Labware, Franklin Lakes, NJ). At the same 

time, 0.5-g samples of puree were removed for lycopene assay by 

conventional organic solvent extraction/analysis. To the ~2-g weighed 

sample of puree were added 6ml of 0.4% SDS in H2O, and the total 

weight of the puree plus added solvent was recorded. The inclusion of 

0.02% sodium azide in the SDS solution to prevent microbial growth 

in solutions that stood at room temperature for long periods had no 

effect on the solubilizing effect of the SDS. The suspension was 

thoroughly mixed either by shaking or on a vortex mixer. After 

standing for 1 hr to allow denaturation of some of the cellular 

components and the solubilization of the lycopene-containing 

chromoplasts, the homogenate was centrifuged at room temperature 

for 15 min in the 15-ml plastic conical centrifuge tube at 1,500 x g 

(3,000rpm) in a Sorvall TC6 bench-top centrifuge (Kendro Laboratory 

Products, Asheville, NC). The chromoplasts were solubilized into the 

aqueous supernatant, and the colorless tissue residue was pelleted at 


the bottom of the tube. For watermelons that were noticeably overripe 

(i.e., orange coloration in the locule and tissue pulled away from the 

seeds), part of the lycopene in SDS was pelleted when centrifuged at 

1,500 x g. The result was a red pellicle on top of the extracted tissue 

residue at the bottom of the centrifuge tube. The amount of insoluble 

lycopene visually appeared to be roughly proportional to the degree of 

overripeness. Consequently, the lycopene contents of significantly 

overripe watermelons could not be as accurately quantified with the 

SDS extraction system (see Results). 

For spectrophotometric determination, ~5ml of each supernatant 

from the centrifugation step were pipetted into a cylindrical glass 

cuvette of 1.0cm path length (Milton Roy, Rochester, NY). The 

absorbance of each sample was measured at 565nm and 700nm on a 

Spectronic 21 spectrophotometer (Milton Roy, Rochester, NY) versus 

a blank composed of the SDS solvent diluted with water (6ml solvent 

+ 2ml H2O). The lycopene content of the watermelon was estimated 

from the absorbance readings by one of two relations: 

Not Corrected for Light Scattering: 

[Lycopene] 

(mg/kg tissue) 

( A565) 

1 { wt reagent + wt sample} 

= 

× × 

0.033 ml/ μg 

⋅cm 

1.0 

cm (wt sample) 

{ wt reagent + wt sample} 

= (30.3) × ( A 565) 

× 

(wt sample) 

Corrected for Light Scattering: 

[Lycopene] 

(mg/kg tissue) 

= 52.9 × 

Eqn. 1 

3 

A565 

-{A 

700 (700/565) } 1 { wt reagent + wt sample} 

= 

× × 

0.0189 ml/ μg 

⋅cm 

1.0 cm (wt sample) 

( A 

565 

− 

( 1. 

902)( 

A 

700 

{ wt reagent + wt sample} 

) ) × 

(wt sample) 

Eqn. 2 

The absorptivities, 0.0189ml/μg·cm with and 0.033ml/μg·cm 

without light-scattering correction, and the correction term for light 

scattering, (A565 – {A700(700/565) 3 }), were taken from earlier 

determinations (W. Fish, unpublished data). 

STATISTICAL ANALYSES. Statistical analyses were performed with 

the aid of Statistica software, version 6 (StatSoft, Tulsa, OK). 


Results and Discussion 

We initially tested to see if the SDS system could fulfill two 

requirements. First, that all of the lycopene present in watermelontissue 

puree could be extracted by the medium. Second, that the 

absorbance of the lycopene extracted into the aqueous SDS was a 

linear function of the amount of lycopene in the assay system, i.e., did 

it obey Beer’s law under this assay protocol? To test the latter 

requirement, we combined, in different ratios, purees from two 

watermelons, one of lycopene content (by hexane extraction) of 

21.6mg/kg and the other of lycopene content of 96.1mg/kg, and 

assayed each by SDS extraction/absorbance. The lycopene contents of 

the combination samples were also verified by hexane 

extraction/absorbance. When the absorbances of the SDS extracts at 

565nm, either corrected or not corrected for light scattering, were 

plotted versus the lycopene contents of the various combined purees, a 

linear relation was obtained (data not shown). 

The first requirement was tested indirectly by comparing the total 

amount of lycopene extracted into SDS versus the total amount of 

lycopene estimated to be in the tissue by direct hexane 

extraction/absorbance. Aliquots of lycopene-containing SDS extracts 

were themselves extracted with hexane, and the lycopene in the total 

SDS extract calculated from these results. The average recovery by 

SDS extraction of ripe and slightly underripe watermelons compared 

to that extracted by hexane was 100.3% + 1.3% (n = 6). Practical 

validation of the aqueous SDS extraction method was carried out by 

comparing the lycopene contents determined by this method with 

those determined by a conventional hexane extraction method for 110 

individual watermelons. When lycopene values determined by the 

SDS extraction method were plotted versus their corresponding values 

determined by a conventional hexane extraction assay, a linear relation 

was obtained (Figure 1). This same relationship was also observed for 

a group of over 180 watermelon samples examined in earlier studies 

by more sophisticated instrumentation while evaluating the state of 

lycopene in aqueous SDS (W. Fish, unpublished data). This one-toone 

linear relation is consistent with the hypothesis that results from 

the SDS extraction and the hexane extraction methods are directly 

comparable, and thus it supports the validity of the SDS extraction 

method. 

For the 90 individual non-overripe watermelons for which 

lycopene was quantified, the SDS extraction/absorption methodology 

yielded value differences that averaged –0.09% + 6.8% (n = 90) 

without the light-scattering correction and –1.50% + 10.0% (n = 90) 


with the light-scattering correction, as compared to those by hexane 

extraction/absorption. The differences from the earlier data referred to 

above were 0.1% + 5.2% (n = 181) without and –1.1% + 5.1% (n = 

181) with the light-scattering correction. All watermelon cultivars 

demonstrated normal scatter of data about the mean percent difference. 

This suggests that no individual cultivar exhibited aberrant behavior in 

the assay. Also, the average amount of lycopene determined for each 

cultivar was similar to that previously reported (Holden et al., 1999; 

Fish et al., 2002; Perkins-Veazie et al., 2006). The precision of the 

SDS extraction assay procedure appears to be comparable to that of 

the conventional hexane extraction assay. For 90 watermelon samples 

assayed by SDS extraction, the average standard error per triplicate 

was 1.12% + 1.07 % for determinations that employed light-scattering 

corrections and 1.00% + 1.26 % for the same samples not corrected for 

light scattering. As mentioned under Materials and Methods, part of 

the lycopene from noticeably overripe watermelons frequently 

sedimented when the extract was centrifuged. This resulted in an 

underestimation of the lycopene content of overripe melons. The 

absorbance responses of overripe samples in the SDS assay are 

demonstrated by the filled triangles in Figure 1A and B. The average 

difference between the SDS method and the conventional hexane 

extraction method for these overripe melons was –7.4% + 9.8% (n = 

20) with the light- scattering correction employed and –9.4% + 6.5% 

(n = 20) when the light- scattering correction was not employed. 

HPLC analysis of lycopene solubilized from watermelon tissue 

with aqueous 0.3% SDS exhibited the same levels of all-trans 

lycopene and its cis-isomers as those of direct hexane extracts of the 

tissue. Furthermore, higher levels of the more water-soluble 

carotenoids, such as lutein, were detected by HPLC analysis of SDS 

extracts of watermelon than were seen in direct organic solvent 

extracts of the tissue (data not shown). 

Previous results demonstrate aqueous solutions of SDS to be a 

marginal solvent for certain carotenoids (Takagi et al., 1982, 1996). 

Furthermore, our studies found that SDS was unable to extract 

lycopene from some types of plant fruit cell matrices, such as 

processed tomato products (W. Fish, unpublished data). In spite of 

such limitations, for selected systems in which a safe and inexpensive 

method to quantify lycopene is desired, the SDS extraction/absorption 

methodology offers an acceptable alternative to existing 

methodologies. This appears to be especially true for watermelon. 

One can reasonably anticipate that lycopene estimates for watermelon 

using the SDS system will be within +10% of estimates by organic 

solvent extraction/absorption. 


Because SDS is not toxic at the levels employed (it’s an ingredient 

in toothpaste), laboratory-personnel-safety concerns and hazardouswaste-disposal 

issues are eliminated. Furthermore, all of the 

equipment needed for the analysis, i.e., blender, tabletop centrifuge, 

and student lab-type spectrophotometer, can be purchased for under 

$3,500 at today’s prices. This methodology may thus afford the plant 

breeder or other investigator a means to quantify lycopene in 

watermelon or other fruits. 

A B 

Fig. 1. Lycopene levels determined for 110 individual watermelons by the SDS 

extraction/absorbance assay compared with the corresponding levels 

determined by conventional hexane extraction/absorbance. Open circles 

represent ripe or underripe melons and filled triangles represent overripe 

melons. A. The absorbance values of lycopene in SDS were adjusted for light 

scattering (see text). The equation of the linear least squares regression fit to 

the data is y = 0.569 + 0.976x. The regression coefficient for the linear fit of the 

data is r 2 = 0.969 (P < 0.05). B. The absorbance values of lycopene in SDS were 

not adjusted for light scattering. The equation of the linear least squares 

regression fit to the data is y = 3.163 + 0.929x. The regression coefficient for the 

linear fit of the data is r 2 = 0.981(P < 0.05). 

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analytical chemistry. John Wiley & Sons, New York. 

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biological carotenoid singlet oxygen quencher. Arch. Biochem. Biophys. 

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lycopene that utilizes reduced volumes of organic solvents. J. Food Comp. Anal. 

15:309–317. 

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PROTOPLAST FUSION IN GENUS CUCUMIS 

J. Gajdová, B. Navrátilová, and A. Lebeda 

Palacký University in Olomouc, Faculty of Science, Department of 

Botany, Olomouc, Czech Republic 

ADDITIONAL INDEX WORDS. Somatic hybridization, Cucumis sativus, cucumber, 

Cucumis melo, melon, polyethyleneglycol 

ABSTRACT. Protoplasts of Cucumis melo L. (MR-1, cv. Charentais D 132, cv. 

Charentais), Cucumis metuliferus ‘Meyer ex Naudin’, Cucumis sativus L. (line 

SM 6514, cv. Borciagovskij), and C. zeyheri ‘Sonder’ were isolated and then 

fused by PEG (polyethyleneglycol). Leaves were used along with growing 

apices, calli, and hypocotyls as sources of protoplasts. Various plant species and 

plant organs were combined for fusion. The most viable combinations were 

achieved using Cucumis sativus line SM 6514 (leaf) combined with either C. 

melo cv. Charentais (callus, leaf, growing apex), C. melo cv. Charentais D 132 

(callus), or C. metuliferus (leaf). Other successful combinations included: C. 

melo cv. Charentais (callus) with C. metuliferus (leaf), and C. sativus cv. 

Borciagovskij (leaf) with C. zeyheri (callus). These combinations successfully 

regenerated calli. Hypocotyl explants were the least viable for protoplast fusion. 

P 

rotoplast fusion offers a potential plant-breeding technique 

where traditional hybridization is not possible. Somatic 

hybridization in the genus Cucumis also may serve as a tool for 

introducing pathogen resistance against Pseudoperonospora cubensis 

to Cucumis melo and Cucumis sativus (Fellner et al., 1996). Previous 

work with somatic hybridization resulted in no regeneration in most 

observed cases (Gajdová et al., 2004). The regeneration of hybrid 

plants was achieved only in fusions of Cucumis melo with Cucurbita 

moschata × C. maxima (Yamaguchi and Shiga, 1993), and of C. melo 

with C. myriocarpus (Bordas et al., 1998). Yamaguchi and Shiga 

(1993) found that intergeneric hybrids did not retain Cucurbita 

features. Bordas et al. (1998) found the resulting plants were unable to 

produce roots. 

Our previous studies (Gajdová et al., 2006) have investigated and 

optimized the factors involved in regeneration. The aim of our recent 

work was to establish a reliable method of somatic hybridization in 

the genus Cucumis. All possible combinations of genotypes were 

tested and various combinations of plant organs were also investigated. 

Only genotypes that previously indicated good regenerative capabilites 

This research was supported by the following grants: (1) MSM 6198959215 

(Ministry of Education, Youth and Sports, Czech Republic); and (2) QD 1357 

(NAZV, Ministry of Agriculture, Czech Republic. 


(calli; in C. zeyheri microcalli obtained) (Gajdová et al., 2006) were 

selected for this study. 


Seeds of Cucumis melo (line MR-1 [CZ 09-H40-0600]), cv. 

Charentais D 132 (CZ 09-H40-1114), cv. Charentais (CZ 09-H40- 

1116), Cucumis metuliferus (CZ 09-H41-0587), Cucumis sativus (line 

SM 6514 [CZ 09-H39-0768]), C. sativus (cv. Borciagovskij [09-H39- 

0056]), and C. zeyheri (CZ 09-H41-0595, CZ 09-H41-0196) 

originating from different germplasm collections and recently 

maintained by the Research Institute of Crop Production (Dept. of 

Gene Bank, Olomouc, Czech Republic) were surface-sterilized with 

8% chloramin B and germinated on a 50% MS (Murashige and Skoog, 

1962) medium in the dark at 25°C. After 7 days, hypocotyls were 

detached and seedlings planted on MS medium in plastic boxes. 

Hypocotyls were used either for protoplast isolation or for callus 

derivation. Leaves and growing apices of 2–8-week-old plants served 

as sources of protoplasts. Calli were derived from leaves of in vitro 

plants, except in the case of C. melo (MR-1 and cv. Charentais), where 

they were derived from hypocotyls. Explants were placed on MSC 

medium (MS with 30g/l sucrose, 2.5mg/l NAA, 1mg/l BAP, and 0.8% 

agar) to induce callus growth and the calli subcultured every 2 weeks. 

Various combinations of plant species and plant organs were 

investigated for protoplast fusions. Protoplasts were isolated using a 

novel protocol (Gajdová et al., 2006). After isolation and viability 

assessment (by FDA staining), protoplasts were fused in Petri dishes 

(diameter 40mm) by 33% (v/v) PEG 6000 (polyethyleneglycol) 

solution (Christey et al., 1991) according to the following protocol: 

protoplasts in M+C solution (Christey et al., 1991) mixed in a test 

tube; 4 small drops (ca 100µl) of protoplasts were left in a Petri dish 

for 20 min of sedimentation; 20 min of PEG treatment - 50µl added to 

each protoplast drop; PEG pipetted off; Stop solution (Christey et al., 

1991) (200µl per 1 drop) treatment added for 20 min, then pipetted off. 

After the fusion liquid LCM1 medium (Debeaujon and Branchard, 

1992) was added into dishes (ca. 1.5ml per dish) and protoplasts were 

cultivated at 25°C in the dark. After 14 days samples were checked, 

liquid LCM 2 medium (Debeaujon and Branchard, 1992) was added, 

and dishes transferred under 16-hr light. Growing microcalli were 

transferred onto solid F medium (Pelletier et al., 1983) after 2–3 weeks 

and subcultured after 3 weeks. 



The investigation revealed that the best materials for protoplast 

fusion were Cucumis sativus line SM 6514 (leaf) combined either with 

C. melo cv. Charentais (callus, leaf, growing apex) or C. melo cv. 

Charentais D 132 (callus), and/or with C. metuliferus (leaf). Other 

successful combinations were: C. sativus line SM 6514 (growing apex) 

× C. metuliferus (growing apex); C. melo cv. Charentais (callus) × C. 

metuliferus (leaf); C. sativus cv. Borciagovskij (leaf) × C. zeyheri 0595 

(callus). It is unclear how these were combined. In these cases growing 

calli were regenerated after fusion but did not undergo organogenesis or 

somatic embryogenesis. The induction of shoots on the callus and their 

development into whole plants has yet to be achieved. These difficulties 

were also noted by some authors (Yamaguchi and Shiga, 1993). 

Table 1. Protoplast fusion in Cucumis melo cv. Charentais. * 

Cucumis melo cv. Charentais 

Callus Hypocotyl Leaf Growing apex 

C. metuliferus callus microcallus microcallus 

leaf 

C. sativus leaf callus callus callus 

Growing apex cell division microcallus 

Hypocotyl no 

regeneration 

C. melo MR-1 microcallus microcallus 

leaf 

Growing apex microcallus 

Hypocotyl cell division no 

regeneration 

Callus hyp. no 

regeneration 

* The highest level of regeneration is shown. 

Regeneration of calli in fusion of C. metuliferus with C. melo or with 

C. sativus has not been reported. Fusion of C. zeyheri has not been 

previously reported. In general, hypocotyls were found to be the least 

viable explant for successful protoplast fusion. Even in combination 

with leaves or growing apices they did not show regenerative capacity. 

They also indicated low regeneration ability when cultivated in pure 

culture (Gajdová et al., 2006). In C. melo cv. Charentais (Table 1) all 

other explants were equally suitable for fusion experiments. In C. melo 

MR-1 (Tables 1 and 2) growing apex or leaf is better than callus; 

however, calli grew well in vitro but plants did not. 


Table 2. Protoplast fusion in Cucumis melo MR-1.* 

Cucumis melo MR-1 

C. 

metuliferus 

leaf microcallus 

C. sativus 

Leaf 

Callus hyp. Hypocotyl Leaf 

Growing 

apex callus 

Hypocotyl first division 


no 

regeneration 

Growing 

apex 

In C. sativus (Tables 3 and 4) the leaf is the best choice for 

protoplast fusion (calli were not investigated as they were not viable 

[Gajdová et al., 2006]). To date cotyledons have been the most 

common source of protoplasts (Fellner and Lebeda, 1998). Cotyledons 

were not used in this study due to cotyledon protoplast regeneration 

problems reported. Previous studies suggested that this is due to their 

tetraploidy (Kubaláková et al., 1996). 

Electrofusion has been the most commonly used method of 

somatic hybridization. Polyethylene glycol (PEG) or high pH/Ca 2+ has 

previously been used to induce fusion (Gajdová et al., 2004). PEGinduced 

fusions yielded improved results over previously reported 

electrofusion (Greplová et al., 2005). Future research will be focused 

on enhancing organogenesis or somatic embryogenesis on regenerated 

calli. 

Table 3. Protoplast fusion in Cucumis sativus line 6514.* 

Cucumis sativus 

Leaf Growing apex Hypocotyl 

C. melo 

Charentais D 132 

callus 

C. metuliferus 

callus 

leaf callus microcallus microcallus 

Growing apex microcallus callus 



Table 4. Protoplast fusion in Cucumis sativus cv. Borciagovskij.* 

C. sativus 

Borciagovskij leaf 

C. metuliferus 

leaf 

C. zeyheri 595 

callus callus microcallus 

C. zeyheri 196 

callus microcallus 



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cytoplasmic male-sterile-nigra broccoli obtained by protoplast fusion between 

cytoplasmic male-sterile Brassica oleracea atrazine-resistant Brassica 

campestris. Theor. Appl. Genet. 83:201–208. 

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caulogenesis from cotyledons and leaf protoplast-derived colonies of melon 

(Cucumis melo L.). Plant Cell Rep. 12:37–40. 

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tissues of Cucumis sativus L. and C. melo L. seedlings. Biol. Plant. 41:11–24. 

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cucurbit genetics and breeding research. Proc. Cucurbitaceae 2004, 8th Eucarpia 

Meeting on Cucurbit Genetics and Breeding, Olomouc, Czech Republic. 

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source of protoplasts and media composition on Cucumis and Cucurbita 

protoplast isolation and regeneration. Acta Hort. (In press.) 

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EXPRESSION PROPERTIES OF THREE TAU- 

TYPE PUMPKIN GLUTATHIONE S- 

TRANSFERASES IN BACTERIA AND A 

SEARCH FOR THEIR INTRINSIC INHIBITORS 

M. D. Hossain and Masayuki Fujita 

Department of Plant Sciences, Faculty of Agriculture, Kagawa 

University, Miki-cho, Kita-gun, Kagawa 761-0795, Japan 

ADDITIONAL INDEX WORDS. Cucurbita maxima, E. coli, IPTG, substrate, 

ligand 

ABSTRACT. Expression levels of three tau-type pumpkin (Cucurbita maxima) 

glutathione S-transferases (GSTs) in bacteria and the presence of substances in 

pumpkin callus (PC) and leaf (PL) that inhibit activities of the GSTs were 

investigated. Bacterial expression systems based on E. coli transformed with 

CmGSTU1, CmGSTU2, or CmGSTU3 cDNA genes in pBluescripts [SK(-)] were 

used as enzyme sources. All GSTs were found to be expressed in E. coli XLI- 

Blue cells cultivated in the culture media with or without isopropyl-β-Dthiogalactopyranoside 

(IPTG). The chemical enhanced the expression of 

CmGSTU1 and CmGSTU2 but was found to suppress the CmGSTU3 expression 

level in bacterial cells. Inhibition studies indicated that alcoholic extracts of PC 

had strong inhibitory effects on CmGSTU1 and CmGSTU3 and a weak effect on 

CmGSTU2 activity toward 1-chloro-2,4-dinitrobenzene (CDNB). The PL extract 

showed a small inhibitory effect on CmGSTU3 but negligible or no effects on 

CmGSTU1 and CmGSTU2. Through diethylaminoethyl cellulose (anionexchange) 

column chromatography of PC and PL extracts, the highest 

inhibitions were obtained in 0.2M and 0.3M NH4HCO3-eluted fractions, 

respectively. HPLC analyses demonstrated that the fractions contain several 

inhibitors possessing hydrophobic or hydrophilic characteristics. These results 

suggest that some potent inhibitors charged negatively are present as substrates 

or physiological ligands of pumpkin GSTs in pumpkin callus and leaves. 

G 

lutathione S-transferases (GSTs, EC 2.5.1.18) are a family of 

multifunctional enzymes that catalyze the conjugation of 

reduced glutathione (GSH) with a wide range of hydrophobic, 

electrophilic, and usually cytotoxic substrates (Wilce and Parker, 

1994). In plants, GSTs are key enzymes in the detoxification of 

herbicides (Marrs, 1996). Some plant GSTs are involved in the 

metabolism of endogenous toxins and protect plants from oxidative 

damage. Furthermore, some plant GSTs act as binding proteins or 

ligandins. They are capable of binding and transporting plant 

hormones and a variety of secondary metabolites (Edwards et al., 

2000). There is some evidence that binding to ligands often inhibits 

GST activity (Lyon et al., 2003). Little is known, however, about the 


functions of GSTs with endogenous compounds, despite the fact that 

plants synthesize numerous toxic secondary metabolites that are 

potential GST substrates. 

From pumpkin (Cucurbita maxima Duchesne) callus cultured in a 

medium containing 2,4-D, three forms of tau-type pumpkin GSTs, 

Puga (CmGSTU1), Pugb (CmGSTU2), and Pugc (CmGSTU3), have 

been purified (Fujita et al., 1994, 1998) and their cDNAs have been 

cloned (Fujita and Hossain, 2003b). It has also been reported that 

CmGSTU1 is expressed at high levels in fully expanded mature organs 

and that CmGSTU2 is expressed preferentially in leaves and petioles, 

whereas expression of CmGSTU3 in pumpkin organs is under 

detectable levels (Fujita and Hossain, 2003b). Since GSTs play a role 

in detoxification of endogenous toxins, organs of pumpkin plants as 

well as the callus might contain substances that interact with GSTs. 

Little progress has been made previously in identification of inhibitors 

of pumpkin GSTs (Fujita and Hossain, 2003a). Recently, we found 

that tissues of pumpkin seedlings contain some substances that inhibit 

activities of pumpkin GSTs (Hossain et al., 2006). In this study, we 

investigated the effects of pumpkin callus and leaf extracts on 

activities of three tau-type pumpkin GSTs. The study would be helpful 

in predicting the physiological roles of pumpkin GSTs. 


PLANT MATERIALS. Callus was induced from sarcocarp tissues of 

pumpkin fruit on Murashige and Skoog’s solid medium containing 

4.5µM 2,4-D and 0.5µM kinetin at 25˚C in the dark (Fujita et al., 

1994). Fully expanded pumpkin leaves were harvested from mature 

plants at Kagawa University Farm. 

EXTRACTION. Extract was prepared from 25g of pumpkin callus 

(PC) using methanol : chloroform : water (12:5:3, v/v/v) and 70% 

ethanol solution following a method described in our previous study 

(Hossain et al., 2006). To prepare extract from the same quantity of 

pumpkin leaves (PL), 1.5-fold volume of each solvent was used. 

Finally, dried substances were dissolved in 10ml distilled water. 

COLUMN CHROMATOGRAPHY. To separate constituents into 

different fractions, alcoholic extracts of PC and PL were applied 

separately to a diethylaminoethyl cellulose (DE-52; Whatman, UK) 

column (5.0ml). After washing the column with a sufficient amount of 

distilled water, 1.9ml PC extract were applied to the column and eluted 

with different concentrations of NH4HCO3 (0, 0.05, 0.1, 0.2, 0.3, 0.4, 

0.5, 0.7, 0.9, 1.1, and 2.0M) and finally with 100% methanol, using a 

constant volume of 20ml for each fraction. The solvents of the 


fractions were thereafter removed by evaporation, and dried 

substances were dissolved in 0.5ml distilled water. To fractionate PL 

extract, a 1.0-ml sample was applied to the column and the same 

procedure was performed. Finally, dried substances were dissolved in 

1.5ml distilled water. Inhibitory activities of all fractions of PC and PL 

extracts were examined for CmGSTU3 toward 1-chloro-2,4dinitrobenzene 

(CDNB) as a substrate. 

PREPARATION OF ENZYME. E. coli cells containing CmGSTU1, 

CmGSTU2, and CmGSTU3 cDNAs in pBluescripts [SK 

(-)] were cultivated separately for about 16 hours at 37˚C in Luria- 

Bertani liquid medium with ampicillin (50µg/ml). To investigate the 

expression properties of GSTs, 0, 1, and 10mM IPTG were added to 

the culture medium. After incubation, the cells were harvested by 

centrifugation (2400×g for 10 min) and homogenized in 25mM Tris- 

HCl buffer (pH 8.0) containing 1mM EDTA, 1% (w/v) ascorbate, and 

10% (v/v) glycerol with a mortar and pestle. A small amount of sea 

sand was added to make grinding easier. Cellular debris was 

precipitated by centrifugation (10000×g at 4˚C for 10 min) and the 

supernatant was used as enzyme solution. 

ENZYME ASSAY, PROTEIN QUANTITATION, AND INHIBITION 

STUDY. GST activity was determined spectrophotometrically by the 

method of Booth et al. (1961) with some modifications. The reaction 

mixture contained 100mM potassium phosphate buffer (pH 6.5), 

1.5mM reduced glutathione, 1mM CDNB, and enzyme solution in a 

final volume of 0.7ml. To perform inhibition studies, various 

quantities of extracts were added to the reaction mixture. The enzyme 

reaction was initiated by the addition of CDNB, and A340 was 

monitored at 25˚C for 1 min. The protein concentration of each 

enzyme solution was determined by the method of Bradford (1976) 

using BSA as protein standard. Enzyme solutions of CmGSTU1 and 

CmGSTU2 extracted from E. coli cells with 1mM IPTG and 

CmGSTU3 with no IPTG in the culture media were used for inhibition 

studies. 

HPLC ANALYSIS AND ACTIVITY PROFILING. Fractions with the 

highest inhibitory potencies obtained from DE-52 column 

chromatography were analyzed on a LC-6AD Liquid Chromatograph 

(Shimadzu, Japan) fitted with a UV-VIS detector (SPD-6AV) and C- 

R6A Chromatopac. Separation was performed using a Shim Pack 

CLC-ODS column (4.6mm i.d. × 250mm, Shimadzu, Japan). The flow 

rate was 0.6ml min-1 and detection was carried out at 220nm. The 

column was eluted with a linear gradient of 0–80% methanol for 40 

min and with 100% methanol for an additional 20 min. 


Fifty µl of 0.2M NH4HCO3-eluted fraction of PC extract 

(corresponding to 475mg fresh callus) were injected onto the column. 

The effluent from the column was fractionated according to important 

peak areas or after some intervals (when no peak appeared). The 

solvent was removed by evaporation and dried substances were 

redissolved in 250µl distilled water. For PL extract, 20µl of 0.3M 

NH4HCO3-eluted fraction (corresponding to 30mg fresh materials) 

were injected onto the column and the same procedure was carried out 

for fractionation. In both cases, inhibitory activity of each of the 

HPLC-eluted fractions was assayed for CmGSTU3, using samples of 

50 and 100µl. 


In a previous investigation (Fujita and Hossain, 2003b), cDNAs of 

pumpkin GSTs, namely, CmGSTU1, CmGSTU2, and CmGSTU3, 

were subcloned in the multicloning site of the expression vector 

pBluescript [SK(-)], and the open reading frame of each clone was 

found to be in-frame with the α-complementation particle of the βgalactosidase 

gene. Each of the recombinant expression vectors was 

subsequently transformed into E. coli XLI-Blue cells and found to be 

expressed as a fusion protein in the presence of IPTG. In this study, we 

examined the effect of IPTG on expression levels of these GSTs as 

fusion proteins in bacterial cells. We cultivated bacterial cells 

containing cDNAs of the GSTs treated with 0, 1, and 10mM IPTG and 

then extracted proteins from the cells. Interestingly, all of the GSTs 

were found to be expressed without the addition of IPTG in the cells, 

and CmGSTU3 and CmGSTU2 showed the highest and the lowest 

activity, 6084 nmolmin- 1 mg- 1 protein and 60 nmolmin- 1 mg- 1 protein, 

respectively (Table 1). IPTG was found to significantly enhance the 

expression levels of CmGSTU1 and CmGSTU2 and, in contrast, to 

suppress the expression of CmGSTU3 in bacterial cells. In all cases, 

IPTG reduced the quantities of total proteins (data not shown). 

IPTG activates the expression of the α-complementation particle of 

the β-galactosidase gene in pBluescript [SK(-)], thereby increasing the 

production of the GST fusion protein. This is the main reason for the 

high expression levels of CmGSTU1 and CmGSTU2 in bacterial cells 

treated with IPTG. In the case of the suppression of CmGSTU3 

expression, IPTG might cause overexpression of the fusion protein, 

and the excessive quantity of the protein could be toxic to the bacterial 

cells, resulting in damage to the cells. In the control (no IPTG), 

bacterial growth appeared to be normal. 

To search for inhibitors of pumpkin GSTs, we examined the 


Table 1. Effect of IPTG on bacterial expression of pumpkin 

glutathione S-transferases. GSTs activities were measured with CDNB 

as a substrate. 

Specific activity (nmolmin- 1 mg- 1 protein) 

GSTs No IPTG 1mM IPTG 10mM IPTG 

CmGSTU1 944 3958 5315 

CmGSTU2 60 117 163 

CmGSTU3 6084 3473 2232 

effects of alcoholic extracts of PC and PL on the activities of the 

above-mentioned GSTs toward the model substrate CDNB. Our results 

showed that the PC extracts had strong inhibitory effects on 

CmGSTU1 and CmGSTU3 (Figure 1). In the assay system, 50% 

inhibition of CmGSTU1 and CmGSTU3 was achieved with extracts 

corresponding to 226 and 173mg of fresh callus, respectively. The 

extract showed only a small inhibitory effect on CmGSTU2. The 

specific activities of different GSTs can vary markedly, possibly 

causing differences in inhibition among them. In our previous study 

(Hossain et al., 2006), we found that inhibitory substances were 

Fig. 1. Inhibition of pumpkin GST activities toward CDNB by pumpkin callus 

(PC) and pumpkin leaves (PL) extracts. Results were obtained from two 

independent experiments; bars indicate standard error. Each µl of both extracts 

corresponds to the substances present in 2.5mg of fresh materials. 


present in pumpkin seedlings and that they had various inhibitory 

effects on the same GSTs as those used in the present study. We also 

obtained evidence that the variation of inhibitory effects on GSTs was 

due to the specific characteristics of the enzymes. 

The alcoholic extract of PL showed a marginal inhibitory effect on 

CmGSTU3 and negligible effects on the other two GSTs. Moreover, a 

larger volume of the extract in the assay system appeared to increase 

the activity of all three GSTs, suggesting that pumpkin leaves might 

contain inhibitors as well as activators of pumpkin GSTs. 

To obtain further information, we fractionated PC and PL extracts 

using a DE-52 column (as described in Materials and Methods). Potent 

inhibitory substances of PC extracts seemed to be eluted with 0.2M 

NH4HCO3 followed by 0.3M and 0.1M NH4HCO3 (Figure 2). In the 

assay system, 0.2M and 0.3M NH4HCO3-eluted fractions caused 50% 

inhibition of CmGSTU3, with samples corresponding to 264 and 

361mg fresh callus, respectively. In the case of PL extract, the 0.3M 

NH4HCO3-eluted fraction was found to contain the major inhibitory 

substances. However, the inhibitory effect of this fraction at a larger 

volume was beyond the detectable level due to the much thicker 

concentration of the sample.The 100% methanol-eluted fractions of 

PC and PL extracts were found to contain both water-soluble and 

water-insoluble substances. 

Fig. 2. Inhibition of CmGSTU3 activity toward CDNB by the fractions of 

pumpkin callus (A) and pumpkin leaves (B) extracts obtained from DE-52 

column chromatography. Fractionation was done by eluted columns with 

different concentrations of NH4HCO3 solutions and finally with 100% 

methanol. 


Although the water-soluble portion had almost no effect on 

CmGSTU3 activity (Figure 2), the insoluble counterpart showed an 

apparent high increase of GST activity (data not shown). HPLC 

analyses revealed that the water-insoluble substances present in the 

100% methanol-eluted fraction of PL extract neither increased the 

production of dinitrophenyl-glutathione conjugate (DNP-GS) nor 

produced any other conjugates (data not shown), suggesting that the 

increase in the absorbance does not depend on GST activity. 

In order to evaluate the inhibitory properties of the 0.3M 

NH4HCO3-eluted fraction of the PL extracts, we examined the 

production of DNP-GS by HPLC after incubating samples (0, 20, and 

60µl) with GSH, CDNB, and enzyme solution. We detected a decrease 

in DNP-GS production (peak area decreased to 66% with 60µl) along 

with an increase in unconjugated GSH concentration (peak area 

increased to 159% with 60µl) (Figure 3), but formation of other 

conjugates was not observed, suggesting that the fraction contains true 

inhibitors of the enzyme. 

Fig. 3. Effect of 0.3M NH4HCO3-eluted fraction of PL extract on the production 

of DNP-GS by CmGSTU3. Reactions were performed by adding 0, 20, and 60µl 

of inhibitor sample to 1.5mM GSH, 1mM CDNB, 100mM potassium phosphate 

buffer (pH 6.5), and 5µl enzyme solution to a final volume of 0.7ml. After 10 

min of reaction, 20µl of each product was injected to an HPLC column. The 

amounts of DNP-GS and unconjugated GSH among three reaction mixtures 

were compared by the peak areas (as shown by Chromatopac) detected at 

220nm. 


To gain some insight into the characteristics of the inhibitors, we 

analyzed 0.2M and 0.3M NH4HCO3-eluted fractions of PC and PL 

extracts, respectively, by HPLC. The results indicated that each sample 

contains at least two inhibitors that have small to almost no absorption 

at 220nm (Figure 4). From retention time, it was revealed that both 

hydrophilic and hydrophobic inhibitors are present in the sample. It 

was also reported earlier that inhibitors of GSTs that bind to the 

hydrophobic site are typically hydrophobic (Koehler et al., 1997), 

while mimic-substrate ligands are either hydrophobic or amphipathic 

(Ketley et al., 1975). The present study, as well as our previous 

investigation (Hossain et al., 2006), also interprets the presence of 

some inhibitory substances with hydrophobic as well as hydrophilic 

characteristics. 

We conclude that cDNAs of pumpkin GSTs in pBluescript are 

expressed in E. coli cells without IPTG. Furthermore, IPTG activates 

the expression of CmGSTU1 and CmGSTU2 but suppresses the 

expression of CmGSTU3. Pumpkin callus and leaves contain some 

hydrophilic or hydrophobic inhibitory substances that might be 

physiological substrates or true inhibitors of pumpkin GSTs. In a 

future study, we will determine the structural properties of the 

inhibitors and investigate their mechanisms of inhibition. 

Fig. 4. HPLC-based activity profiling with 0.2M and 0.3M NH4HCO3-eluted 

fractions of PC and PL extracts, respectively, for CmGSTU3 activity. The 

HPLC chromatograms show the absorbance of compounds detected at 220nm; 

the bar graphs above each of the chromatograms show the inhibitory activity of 

corresponding HPLC fractions. 



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Biochem. Biophys. Acta. 1205:1–18. 


PHYSIOLOGICAL CHARACTERISTICS OF 

GRAFTED MUSKMELON GROWN IN 

MONOSPORASCUS CANNONBALLUS- 

INFESTED SOIL IN SOUTH TEXAS 

John Jifon, Kevin Crosby, and Marvin Miller 

Vegetable and Fruit Improvement Center, Texas A&M University, 

College Station, TX 77845; Texas Agricultural Research and 

Extension Center, Texas A&M University, Weslaco, TX 78596; 

Department of Horticultural Sciences, Texas A&M University, College 

Station, TX 77843 

Daniel Leskovar 



Extension Center, Texas A&M University, Uvalde, TX 7; Department 

of Horticultural Sciences, Texas A&M University, College Station, TX 

77843 

ADDITIONAL INDEX WORDS. Vine decline, water potential, cantaloupe, 

rootstock, root vigor, hybrid squash, physiology 

ABSTRACT. Two commercial muskmelon varieties (‘Caravelle’ and ‘Primo’) 

were grafted on Cucurbita and Cucumis rootstocks and grown in fields with a 

history of vine decline disease caused by Monosporascus cannonballus to 

determine whether physiological responses of the scions are consistent with 

predictions of a high capacity for water uptake by the rootstocks. Three 

Cucurbita (hybrid squashes: HS 380, HS 286, and HS 1330) and six Cucumis (PI 

20488, PI 1207, MR1, PI 212210, PI 124104, and hybrid melon 3105) rootstocks 

were used. Vines of plants grafted on Cucurbita rootstocks were generally 

longer than those of nongrafted plants, but this effect was most significant only 

during the vegetative developmental stages. Leaf-water potentials (Ψleaf) of 

plants grafted on Cucurbita rootstocks were consistently higher than those of 

nongrafted plants, especially during the critical fruit- development and 

maturation phases. Melon plants grafted on PI 1207, PI 12404, and PI 20488 

also maintained relatively high Ψleaf. Grafted plants with high Ψleaf also had 

high leaf stomatal conductance and transpiration rates, indicating ample water 

supply from the root systems. Maintenance of high Ψleaf by grafted plants 

indicates that such plants could better tolerate root infection and damage by M. 

cannonballus without late-season collapse. 

V 

ine decline of melons caused by the soil-borne fungus 

Monosporascus cannonballus Pollack & Uecker has become a 

major production problem worldwide (Bruton, 1998; Martyn 

and Miller, 1996). Root infection and damage occur at all stages of 

development, but increased water demand during fruit development 


and maturation can lead to vine collapse due to loss of water-uptake 

capacity. Conditions that promote water loss such as high 

temperatures, high winds, and drought can exacerbate late-season vine 

collapse caused by root infection and damage (Bruton, 1998). The 

majority of commercial melon varieties lack resistance to M. 

cannonballus. Resistance to M. cannonballus among germplasm 

accessions of Cucumis melo L. has been linked to vigorous rootsystem 

development (Crosby et al., 2002; Crosby and Wolff, 1998). A 

vigorous root system presumably compensates for the reduced 

absorptive capacity, thus allowing infected plants to satisfy the 

increased water/nutrient demands during fruit maturation. Crosby et 

al. (2002) and Crosby et al. (2000) have reported differences in root 

morphology among susceptible and tolerant Cucumis cultivars and 

concluded that tolerance to root infection by M. cannonballus is 

closely linked to the integrity of the root structure. Grafting 

commercial melon and watermelon varieties on disease-resistant 

Cucurbita rootstocks is a common practice in the Mediterranean 

region and Southeast Asia to manage soil-borne diseases (Lee and 

Oda, 2003; Lee, 1994), including vine decline of melons caused by M. 

cannonballus (Cohen et al., 2005). Squash rootstocks have been the 

primary source of rootstocks for grafting melons; however, reports of 

poor fruit quality have reinforced the need to find compatible Cucumis 

rootstocks. The objective of this study was to characterize the 

physiological responses of two commercial melon varieties grafted on 

Cucurbita and Cucumis rootstocks and grown in fields naturally 

infested with M. cannonballus. The Cucumis accessions used were 

selected based on their high root-vigor ratings (Crosby and Wolff, 

1998; Crosby et al., 2002). 


Seeds of two western shipper-type muskmelon [Cucumis melo L. 

(Reticulatus group)] varieties (‘Primo’ and ‘Caravelle’) (scions) and 

nine rootstocks (including Cucumis and Cucurbita species; Table 1) 

were sown in 72-cell (0.04L) seedling trays filled with a commercial 

rooting medium (Sunshine #5 Plug Mix, Sun Gro Horticulture Inc, 

Bellevue, WA) and germinated in a greenhouse maintained at 28/20 

°C (day/night temperature regimes). Due to differences in 

This research was funded in part by a TDA-BARD Grant #TIE04-04, a USDA 

Grant: 2001-34402-10543, “Designing Foods for Health” and by the South Texas 

Melon Committee. We also thank technicians Yolanda Luna and Alfredo Rodríguez, 

Texas Agricultural Research and Extension Center-Weslaco, for their assistance. 


development rates, the squash rootstocks were sown 7 days later than 

the scions. At the two-leaf stage, scions were grafted on rootstocks 

using the cleft grafting technique (Lee and Oda, 2003). Grafted 

seedlings were healed in a mist chamber (28°C, >90%RH) for 7 days 

and hardened outdoors for 2 days before transplanting into field plots 

with a history of root rot/vine decline disease caused by M. 

cannonballus in Weslaco, Texas. 

Fifteen plants of each graft combination (including nongrafted 

scion varieties) were transplanted during fall 2005 in a randomized 

complete block design. Field-culture procedures were identical to the 

common commercial practices—namely, raised beds covered with 

plastic mulch, and subsurface drip irrigation. Fourteen days after 

transplanting (DAT), plots were evaluated for plant survival. At 30 

DAT, vine lengths of each graft combination were recorded. Leafwater 

potential (Ψleaf), stomatal conductance, and transpiration were 

measured before fruit set (~30 DAT) and during the fruit maturation 

period (~69 DAT). Leaf gas-exchange parameters were measured on 

at least four plants of each graft combination using a portable 

photosynthesis system (CIRAS-2, PP-Systems, Amesbury, MA). 

Leaf-water potentials (Ψleaf) of six to eight leaves from each graft 

combination were measured between 11:00–14:00h (local time) with a 

Scholander-type pressure bomb (Plant Moisture Systems, Santa 

Barbara, CA). Disease incidence (% wilted plants) was evaluated 

visually at the onset of fruit maturity. A plant was considered wilted if 

more than 50% of the canopy leaves had collapsed with wilt 

symptoms. Mature fruits were harvested at full-slip over an 8-day 

period and average fruit weight, size class, total soluble solids contents 

(TSS, an indicator of fruit quality), and yield were recorded. 


Survival rates were generally high among melon plants grafted on 

Cucurbita rootstocks (>85%) (Table 1). Survival rates varied among 

plants grafted on Cucumis rootstocks (33–86%) with PI 20488, PI 

124104, and PI 1207 having high rates and PI 3105 and MR1 

consistently having the lowest rates, regardless of scion variety. 

Nongrafted scion seedlings also generally had high (>80%) survival 

rates. 

Main-stem vine lengths also differed between Cucumis and 

Cucurbita rootstocks, with seedlings on Cucurbita rootstocks 

consistently having the longest vines compared to nongrafted plants. 

There were no differences in vine lengths among melons grafted on 

Cucurbita rootstocks, regardless of scion variety. Differences in vine 


Table 1. Transplant survival and vine length (four weeks after 

transplanting) of commercial muskmelon (‘Caravelle’ and ‘Primo’) 

grafted on Cucumis and Cucurbita rootstocks and grown in a field 

naturally infested with Monosporascus cannonballus. 

Survival Vine length 

Rootstock Genus % 

(m) 

Nongrafted 

‘Caravelle’ 

80.0 a 0.39 cd 

PI 20488 Cucumis 86.6 ab 0.43 bcd 

MR1 Cucumis 40.0 d 0.37 cd 

PI 1207 Cucumis 73.3 abc 0.45 abc 

PI 212210 Cucumis 53.3 cd 0.42 bcd 

PI 124104 Cucumis 60.0 bcd 0.46 abc 

PI 3105 a 

Cucumis 33.3 d 0.35 d 

HS 380 a Cucurbita 86.6 ab 0.52 a 

HS 286 a Cucurbita 86.6 ab 0.49 ab 

HS 1330 b Cucurbita 93.3 a 0.49 ab 

Nongrafted 

‘Primo’ 

86.6 ab 0.37 de 

PI 20488 Cucumis 73.3 abc 0.43 bcde 

MR1 Cucumis 46.6 cd 0.36 de 

PI 1207 Cucumis 73.3 abc 0.48 ab 

PI 212210 Cucumis 60.0 bcd 0.38 cde 

PI 124104 Cucumis 80.0 ab 0.43 bcd 

PI 3105 Cucumis 40.0 d 0.33 e 

HS 380 Cucurbita 93.3 a 0.47 abc 

HS 286 Cucurbita 86.7 ab 0.54 a 

HS 1330 Cucurbita 93.3 a 0.55 a 

a b 

Sakata Seeds; Abbot & Cobb Inc. 

Values for each scion variety (‘Caravelle’ or ‘Primo’) within a column followed by 

the same letter are not significantly different at the 0.05 probability level (Duncan’s 

multiple range test). 

lengths among plants grafted on Cucumis rootstocks were similar to 

those for plant survival. Vines of ‘Caravelle’ grafted on Cucumis 

rootstocks were slightly longer than those of nongrafted plants, but this 

trend was not significant. ‘Primo’ grafted on PI 1207 had the longest 

vines compared to nongrafted ‘Primo’ plants and to ‘Primo’ grafted on 

other Cucumis rootstocks. 

Midday leaf-water potentials (Ψleaf) measured prior to fruit set (30 

DAT) were significantly higher than those measured during fruit 

maturation (69 DAT) (Table 2). For both scion varieties, plants 


grafted on Cucurbita rootstocks had higher Ψleaf compared to 

nongrafted plants. Leaf-water potentials of ‘Caravelle’ plants grafted 

on Cucumis rootstocks were also generally high compared to 

nongrafted plants, but this trend was significant only for PI 1207 and 

PI 124014 during early and late development. For ‘Primo’, the trend 

was significant for plants grafted on PI 20488, PI 1207, and PI 

124014. 

Table 2. Leaf-water potential (Ψleaf), stomatal conductance (gs), and 

transpiration rates (E) of commercial muskmelon varieties (‘Caravelle’ 

and ‘Primo’) grafted onto different rootstocks and grown in a field 

naturally infested with Monosporascus cannonballus. Measurements 

were taken before fruit set (30 days after transplanting, DAT) and 

during fruit maturation (69 DAT). 

Ψleaf Ψleaf gs gs E E 

30 DAT 69 DAT 30 DAT 69 DAT 30 DAT 69 DAT 

Rootstock MPa mmolm -2 s -1 


Nongrafted -1.5 c -2.1 c 650 abc 274 de 4.0 d 1.7 c 

PI 20488 -1.2 bc -1.7 bc 584 bc 320 bcd 4.3 cd 1.9 bc 

MR1 -1.4 c -1.8 c 535 c 240 e 4.5 cd 1.9 bc 

PI 1207 -0.9 ab -1.3 a 663 abc 375 abc 5.1 abcd 2.2 abc 

PI 212210 -1.0 ab -1.4 abc 622 abc 313 cd 4.7 bcd 2.1 bc 

PI 124104 -0.9 ab -1.3 a 667 abc 334 bcd 5.2 abcd 2.2 abc 

PI 3105 -1.5 c -1.9 c 564 bc 217 e 4.1 d 1.8 c 

HS 380 -0.7 a -1.0 a 702 ab 392 ab 5.5 abc 2.4 ab 

HS 286 -0.8 a -1.2 a 764 a 420 a 6.1 a 2.7 a 

HS 1330 -0.8 a -1.2 a 693 ab 415 a 6.0 ab 2.6 a 

‘Primo’ 

Nongrafted -1.6 c -2.2 c 569 abc 321 bcd 4.5 ab 2.0 ab 

PI 20488 -1.0 ab -1.3 ab 666 abc 377 abc 5.2 ab 2.3 ab 

MR1 -1.5 c -2.1 c 546 bc 298 cd 4.9 ab 2.2 ab 

PI 1207 -1.1 ab -1.5 ab 754 a 355 abc 5.4 ab 2.8 a 

PI 212210 -1.2 b -1.6 b 604 abc 305 cd 4.5 ab 1.9 ab 

PI 124104 -1.2 b -1.6 b 643 abc 366 abc 4.9 ab 2.1 ab 

PI 3105 -1.6 c -2.0 c 525 c 241 d 3.7 b 1.6 b 

HS 380 -0.8 a -1.1 a 677 abc 410 a 5.9 a 2.6 ab 

HS 286 -0.9 a -1.2 ab 695 abc 429 a 6.4 a 2.8 a 

HS 1330 -1.0 ab -1.3 ab 723 ab 402 ab 5.8 a 2.2 ab 


the same letter are not significantly different at the 0.05 probability level (Duncan’s 



Leaf stomatal conductance (gs) and transpiration (E) values measured 

during the fruit maturation period were more than 50% 

lower than those measured during the vegetative growth phases. Both 

parameters were generally higher in the plants grafted on Cucurbita 

rootstocks than on Cucumis rootstocks but the trends were not 

consistent. 

Table 3. Average fruit weight, total soluble solids concentration 

(TSS), and vine decline disease incidence of commercial muskmelon 

varieties (‘Caravelle’ and ‘Primo’) grafted onto different rootstocks 

and grown in a field naturally infested with Monosporascus 

cannonballus. 

Fruit TSS Wilt 

Rootstock wt./kg % Incidence % 


Nongrafted 1.7 bc 7.9 bc 6.7 b 

PI 20488 2.2 ab 9.1 ab 13.3 ab 

MR1 1.9 abc 8.9 bc 20.0 a 

PI 1207 2.3 a 8.7 bc 13.3 ab 

PI 212210 1.6 c 7.7 c 13.3 ab 

PI 124104 2.4 a 8.4 bc 6.7 b 

PI 3105 2.0 abc 8.3 bc 20.0 a 

HS 380 2.4 a 9.2 ab 6.7 b 

HS 286 2.2 ab 10.1 a 6.7 b 

HS 1330 2.5 a 8.5 bc 

‘Primo’ 

6.7 b 

Nongrafted 1.5 d 9.1 a 13.3 ab 

PI 20488 2.5 ab 9.5 a 13.3 ab 

MR1 1.8 cd 9.3 a 20.0 a 

PI 1207 1.7 cd 7.8 bc 13.3 ab 

PI 212210 2.1 bc 9.2 a 20.0 b 

PI 124104 2.1 bc 9.1 a 6.7 b 

PI 3105 1.4 d 7.4 c 26.7 a 

HS 380 2.7 a 8.7 ab 6.7 b 

HS 286 2.5 ab 8.6 ab 6.7 b 

HS 1330 2.8 a 9.7 a 6.7 b 


the same letter are not significant different at the 0.05 probability level (Duncan’s 



The average fruit weight of nongrafted plants was significantly 

lower than that of plants grafted on Cucurbita rootstocks for both 

scion varieties (Table 3). For plants grafted on Cucumis rootstocks, 

the fruit-size differences were not consistent between the two scions 

varieties. The observed smaller average fruit weights for nongrafted 

plants was also associated with slightly higher fruit counts compared 

to grafted plants (data not shown). Fruit soluble solids concentrations 

varied among grafted and nongrafted plants for both rootstock types 

and scion varieties but there were no consistent trends in this 

parameter. 

Vine decline disease development was not severe due, perhaps, to 

generally cooler weather during the fruit maturation period. MR1 and 

PI 3105 had the highest incidence of plants with wilt symptoms among 

grafted plants. There were no statistically significant differences in 

total soluble solids (TSS) among the different graft combinations and 

between grafted and nongrafted plants. 

Vine decline disease of melons caused by M. cannonballus is 

believed to result from reduction in the root absorptive capacity 

following infection and root damage by the fungus (Bruton et al., 

1998). A vigorous root system presumably compensates for the root 

damage, thus allowing infected plants to meet the increased 

water/nutrient demands during fruit maturation (Dias et al., 2002). 

The leaf-water potential responses to grafting observed in this study 

are consistent with expectations of the effects of a vigorous root 

system on leaf-water status. Plants grafted on Cucurbita rootstocks 

had the highest Ψleaf, especially during fruit maturation, when plant 

water demands are high. Plants grafted on the Cucumis rootstocks PI 

1207, 124104, and 20488 also had higher Ψleaf values compared to 

nongrafted plants, and consistent with their vigorous root system 

classification (Crosby et al., 2002). Even though transpirational water 

loss of grafted plants was high, the symptoms of vine decline disease 

(wilting) were generally low. Maintenance of high Ψleaf by plants 

grafted on rootstocks with vigorous root systems indicates that such 

plants could better tolerate infection and root damage without collapse 

compared to nongrafted plants. 


Bruton, B. D. 1998. Soilborne diseases in Cucurbitaceae: pathogen virulence and 

host resistance, p. 143–166. In: J. McCreight (ed.). Cucurbitaceae 1998. ASHS, 

Alexandria, VA. 

Bruton, B. D., V. M. Russo, J. Garcia-Jimenez, and M. E. Miller. 1998. 

Carbohydrate partitioning, cultural practices, and vine decline diseases of 

Cucurbits, p. 189–200. In: J. McCreight (ed.). Cucurbitaceae 1998. ASHS, 

Alexandria, VA. 


Cohen, R., Y. Burger, C. Horev, A. Porat, and M. Edelstein. 2005. Performance of 

Galia-type melons grafted on to Cucurbita rootstock in Monosporascus 

cannonballus-infested and non-infested soil. Ann. Appl. Biol. 146:381–387. 

Crosby, K. and D. Wolff. 1998. Effects of Monosporascus cannonballus on root 

traits on susceptible and tolerant melon (Cucumis melo L.), p. 253–256. In: J. 

McCreight (ed.). Cucurbitaceae 1998. ASHS, Alexandria, VA. 

Crosby, K., D. Wolff, and M. Miller. 2000. Comparisons of root morphology in 

susceptible and tolerant melon cultivars before and after infection by 

Monosporascus cannonballus. HortSci. 35:681–683. 

Crosby, K., M. Miller, and D. Wolff. 2002. Screening plant introductions of Cucumis 

melo L. for resistance to Monosporascus cannonballus, p. 188–191 In: D. N. 

Maynard (ed.). Cucurbitaceae 2002. ASHS, Alexandria, VA. 

Dias, R. C. S., B. Pico, J. Herraiz, A. Espinos, and F. Nuez. 2002. Modifying root 

structure of cultivated muskmelon to improve vine decline resistance. HortSci. 

37:1092–1097. 

Lee, J. M. and M. Oda. 2003. Grafting of herbaceous vegetable and ornamental 

crops. Hort. Rev. 28:61–124. 

Lee, J. M. 1994. Cultivation of grafted vegetables. I. current status, grafting methods, 

and benefits. HortSci. 29:235–239. 

Martyn, R. D. and M. E. Miller. 1996. Monosporascus root rot and vine decline: an 

emerging disease of melons worldwide. Plant Dis. 80:716–725. 


FUNCTIONAL GENOMICS OF GENES 

INVOLVED IN THE FORMATION OF MELON 

AROMA 

Nurit Katzir, Vitaly Portnoy, Yael Benyamini, Yoela Yariv, 

Galil Tzuri, Maya Pompan-Lotan, Olga Larkov, Einat Bar, 

Mwafaq Ibdah, Yaniv Azulay, Uzi Ravid, Yosef Burger, Arthur A. 

Schaffer, Yaakov Tadmor, and Efraim Lewinsohn 

Department of Vegetable Crops, Agricultural Research Organization, 

Newe Ya’ar Research Center, Ramat Yishay, Israel 

ADDITIONAL INDEX WORDS. Cucumis melo, fruit quality, functional genomics 

ABSTRACT. A multidisciplinary approach aimed at the identification and 

characterization of key genes that direct the biosynthesis of aroma compounds 

in melon (Cucumis melo) is described. The aromas of fruits are normally due to 

complex mixtures of volatile compounds. Volatile esters, mainly acetate 

derivatives, are prominent in aromatic melon varieties, along with lower 

amounts of sesquiterpenes, norisoprenes, short-chain alcohols, and aldehydes. 

Nonaromatic melon varieties often have much lower levels of total volatiles, and 

especially lack the volatile esters. EST libraries of melon fruits were developed, 

representing the great variability of C. melo. Altogether, 4,800 ESTs from these 

libraries were sequenced and a melon EST database was developed 

(http://melon.bti.cornell.edu/). The database has been mined to identify 

candidate genes affecting fruit ripening and fruit-quality traits, among them 

genes involved in aroma formation from three gene families: (1) alcohol acetyl 

transferases, (2) sesquiterpene synthases, and (3) carotenoid cleavage 

dioxygenases. Full-length clones of candidate genes were isolated, and their 

expression patterns in various melon cultivars were studied. Biochemical 

characterizations of heterologously expressed gene products, coupled with the 

systematic analysis of aroma volatiles and corresponding biosynthetic enzymes, 

were used to identify the roles of these genes in aroma formation during 

ripening. 

F 

ruit quality is determined by numerous traits that affect taste, 

aroma, texture, pigmentation, nutritional value, and duration of 

shelf life (for review see Giovannoni, 2004). Recently, genomic 

tools, such as EST libraries and DNA microarrays, have been applied 

to various fruit species and have provided vast information on the 

patterns of gene expression throughout fruit development and 

This work was partially supported by the A.R.O Center for the Improvement of 

Cucurbit Fruit Quality, by an Israel Ministry of Science Grant, and by Research 

Grant No. IS-3333-02C from B.A.R.D, the United States-Israel Binational 

Agricultural Research and Development Fund. 


maturation (Aharoni et al., 2000; Alba et al., 2004; Fei et al., 2004). 

Melon (Cucumis melo L.) is a highly polymorphic species that 

comprises a broad array of wild and cultivated genotypes that differ in 

fruit traits such as ripening physiology (climacteric and 

nonclimacteric), sugar and acid content, and secondary metabolites 

associated with taste and aroma. In aromatic melon varieties the 

prominent compounds responsible for aroma formation are volatile 

esters, mainly acetate derivatives, and lower amounts of 

sesquiterpenes, norisoprenes, short-chain alcohols, and aldehydes 

(Shalit et al., 2001). The contribution of each component to the overall 

unique aromas is often difficult to assess due to the differences in odor 

intensity and thresholds of the different components. Thus, different 

components impart different notes and contribute in different ways to 

the overall aroma of melons. Nonaromatic varieties often have much 

lower levels of total volatiles, and especially lack volatile esters (Shalit 

et al., 2001; Burger et al., 2005; El-Sharkawy et al., 2005; Benyamini 

et al., unpublished). 

The aim of our project was to identify candidate genes that affect 

the aroma of melon, as part of a broader program aimed at identifying 

key genes that control melon quality and marketability. This project 

has helped to establish a platform for future marker-assisted selection 

and genetic modifications to generate superior varieties. 


PLANT MATERIAL. The melon cultivars investigated in this study 

included: (1) ‘Dulce’, ‘Noy Yizre’el’, ‘Arava’, and ‘En Dor’ of C. 

melo subsp. melo Group Reticulatus, (2) ‘Tam Dew’, ‘Rochet’, ‘Noy 

‘Amid’, and ‘Piel De Sapo’ of subsp. melo Group Inodorus, (3) 

‘Védrantais’ of subsp. melo Group Cantalupensis, (4) PI 414723 of 

subsp. agrestis Group Momordica, (5) ‘Dudaim’ of subsp. melo Group 

Dudaim, and (6) ‘Faqqous’ of subsp. melo Group Flexuosus. 

EST LIBRARIES. EST libraries were constructed using suppression 

subtractive hybridization (SSH, Diatchenko et al., 1996), PCR- 

SelectTM kit (Clontech Laboratories, Palo Alto, CA), or ZAP cDNA 

Synthesis Kit and ZAP Express® cDNA Gigapack ® III Gold Cloning 

Kit (Stratagene, La Jolla, CA) according to manufacturer’s 

recommendations. RNA was extracted using a modification of La 

Claire II and Herrin (1997). 

CLONE SELECTION AND CHARACTERIZATION. Expression of 

selected clones in different tissue types and developmental stages was 

further analyzed on Northern blots or Real-time PCR. Northern 

blotting (25μg of total RNA per lane, confirmed by methylene blue 


staining) and hybridization were performed as described by Sambrook 

and Russell (2001). Real-time PCR was performed as described by 

Ibdah et al. (2006). 

IDENTIFICATION OF VOLATILES: SAMPLE PREPARATION. Melon 

fruits were harvested at immature and mature stages. Immediately after 

harvest three fruits from each cultivar were picked randomly and cut 

into large pieces. Samples were prepared for analysis as described in 

Fallik et al. (2001). 

IDENTIFICATION OF VOLATILES: HEADSPACE-SOLID PHASE 

MICROEXTRACTION (HS-SPME). The volatiles were sampled by 

automated headspace-solid phase microextraction using an 

autosampler of CTC Pal system. The headspace phase was adsorbed 

for 30 min at ambient temperature and desorbed for 10 min into a gaschromatograph-mass 

spectrometer (GC-MS) injection port. The 

volatiles were analyzed according to Ibdah et al. (2006). 

ENZYMATIC ASSAYS OF ALCOHOL ACETYL TRANSFERASE, 

SESQUITERPENE SYNTHASE, AND CAROTENOID CLEAVAGE 

DIOXYGENASE ACTIVITY. Fruit extractions and enzymatic activity 

assays were performed as described by Shalit et al. (2001), Guterman 

et al. (2002), and Ibdah et al. (2006) respectively. 


Our project aimed at the discovery and application of important 

genes controlling the metabolism of fruit-quality components in 

Cucumis melo. EST libraries of melon fruits were developed, 

representing the great genetic variability of the species C. melo. These 

include subtracted and nonsubtracted, nonnormalized libraries from 

various genotypes. Altogether, 4,800 ESTs of these libraries were 

sequenced and a melon EST database was developed 

(http://melon.bti.cornell.edu/). 

The EST database has been mined to identify candidate genes 

affecting fruit ripening and fruit-quality traits, especially genes 

associated with aroma formation. To better understand the 

biochemistry and the molecular regulation of aroma biosynthesis, 

functional analyses of putative genes involved in aroma biosynthesis 

were studied. First we determined the main compounds in the 

headspace of the fruits. We then established biochemical assays to 

measure the enzymes putatively involved in the biosynthesis of key 

aroma compounds. Heterologous expression of the isolated genes in E. 

coli combined with enzymatic activity of their products enabled us to 

draw conclusions concerning their role in aroma biosynthesis. Some of 

our results are summarized below. 


ALCOHOL ACETYL TRANSFERASES. The major volatiles present in 

the aromatic melon varieties are volatile acetates (El-Sharkawy et al., 

2005; Benyamini et al., unpublished). Total volatile acetates present in 

mature melon varieties are shown in Figure 1A. These compounds are 

absent in unripe fruits and in the nonaromatic cultivars such as 

‘Rochet’, whether ripe or unripe. In accordance, cell-free extracts from 

aromatic melons contained alcohol acetyl transferase (AAT) activity, 

enabling acetylation of several alcohol substrates, while nonaromatic 

melons did not contain significant AAT activity with any of the 

substrates tested (Figure 1B). 

Therefore, the EST database was searched for putative AAT genes, 

leading to the identification of several members of this family. Two of 

them, namely CmAAT1 and CmAAT2, have been previously identified 

in melons (Yahyaoui et al., 2002; El-Sharkawy et al., 2005). Northern 

A 

B 

C 

µg acetates/gr f.w 

Pkat/gr f.w 

12000 

9000 

6000 

3000 

0 

20 

18 

16 

14 

12 

10 

8 

6 

4 

2 

0 

y m y m y m y m y m y m y m y m y m 

1 2 3 4 5 6 7 8 9 

Fig. 1. (A) Volatile acetate content in aromatic and nonaromatic melon 

cultivars. (B) AAT activity in crude extract of melon cultivars. (C) 

Northern blot of melon genotypes using probe of CmAAT1, y, young fruit; 

m, mature fruit. ‘Arava’ (1), ‘Noy Yizre’el’ (2), ‘Védrantais’ (3), ‘Dulce’ (4), 

and ‘Dudaim’ (5) have a typical melon aroma while ‘Tam Dew’ (6) and 

‘Rochet’ (8) are nonaromatic. PI 414723 (9) and ‘Faqqous’ (7) have distinct 

strong aromas. 


lots demonstrated expression of AAT in mature aromatic but not in 

nonaromatic genotypes (Figure 1C). This expression pattern fits well 

the composition of volatiles and AAT activity found in these melons: 

the majority of the volatiles of nonaromatic and unripe melons were 

short- and medium-chain alcohols, while the majority of the volatiles 

of mature aromatic melons were acetates, mostly aliphatic. 

CAROTENOID CLEAVAGE DIOXYGENASES. Comparative analysis of 

carotenoids and volatiles revealed an interesting association between 

carotenoid content and aroma of melon fruit flesh. The major volatile 

norisoprenoid present in orange-fleshed melons is β-ionone, a 

compound derived from the oxidative cleavage of β-carotene. 

β-ionone was not detected in any of the green- or white-fleshed 

melons. A search for a gene putatively responsible for the 

cleavage of β-carotene into β-ionone was carried out in our EST 

database, yielding a sequence (CmCCD1) highly similar (84%) to 

other carotenoid cleavage dioxygenase genes. Expression patterns 

of the gene were compared in the orange-fleshed ‘Dulce’, the green- 

fleshed ‘Tam Dew’ and ‘Noy Yizre’el, and the white-green-fleshed 

‘Rochet’ (Figure 2) in order to observe a possible relationship between 

mesocarp color and volatile production (Ibdah et al., 2006). CmCCD1 

gene expression is upregulated upon fruit development mainly in the 

orange melon ‘Dulce’ but also in the green and white-green melon 

varieties, despite the lack of norisoprenoid volatiles in the latter. Thus, 

the accumulation of β-ionone in melon fruit is probably limited by the 

availability of carotenoid substrate. 

Relative expression 

0.1 

0.08 

0.06 

0.04 

0.02 

0 

FR-1 FR-2 FR-3 FR-4 

Stage of development 

Tam Dew 

Dulce 

Noy Yizre'el 

Rochet 

Fig. 2. Expression patterns of CmCCD1 in melon fruit. Real-time PCR analysis 

of RNA extracted from four genotypes. Fruits at the following stages of develop- 

ment were sampled: FR-1, very young green fruits; FR-2, young green fruits; 

FR-3, full-size mature green fruits; FR-4, ripe fruits. Values are means ± SEM 

of 3 different evaluations carried out in duplicates with one set of cDNA. 


The clone was functionally active and overexpressed in E. coli 

strains previously engineered to produce β-carotene. The incorporated 

CmCCD1 gene product cleaved β-carotene, generating β-ionone. 

Interestingly, the gene product is able to cleave other carotenoids that 

normally do not accumulate in melons, such as lycopene, phytoene, 

and δ-carotene, into their respective breakdown products such as 

pseudoionone, geranylacetone, and α-ionone as determined by HPLC 

(carotenoids) and GC-MS (volatiles) (Ibdah et al., 2006). β-ionone is a 

very potent odorant and can be detected in concentrations as low as 

0.07ppm. Thus β-ionone accumulates to significant amounts in orange 

melons, and might have a profound effect on the aroma of the fruit. 

White-fleshed melons that lack β-carotene also lack β-ionone. 

Therefore, color has an effect on aroma. 

SESQUITERPENE SYNTHASES. In the case of sesquiterpenes, the 

volatile compounds, the appropriate enzymatic activity, and relevant 

gene expression were found to be cultivar-specific. Two novel 

sesquiterpene synthase genes have been isolated from two melon 

cultivars: CM-SS1 (‘Dulce’) and CM-SS2 (‘Noy Yizre’el’). The two 

genes were functionally expressed in E. coli and shown to encode for 

distinct proteins that convert farnesyl diphosphate into either farnesene 

(CM-SS1) or cadinene (CM-SS2). The details of the sesquiterpene 

study will be described in Benyamini et al. (unpublished). 

Conclusion 

We have undertaken an investigation aimed at rationalizing the 

biochemical and molecular events that lead to the formation of aroma 

volatiles in melon fruits. Cell-free enzymatic assays have enabled us to 

measure the potential of each melon variety to produce certain volatile 

compounds from their respective precursors. The expression level of 

the relevant genes as well as their enzymatic activity increases upon 

maturation, concomitantly with the appearance of their respective 

volatile compounds in fruit. 

By combining EST database mining, expression analyses, 

enzymatic assays, and volatile determinations, we have been able to 

establish a platform by which many genes typical of fruit maturation 

were isolated. Many of these genes play major roles in various 

metabolic pathways and physiological processes related to aroma 

volatile formation. The final confirmation of the biochemical role was 

assessed by heterologous functional expression. This interdisciplinary 

study has provided a good infrastructure to address questions related to 

aroma formation and other ripening processes taking place in melons. 



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STUDIES OF TRANSGENIC MELON 

EXPRESSING THE MUTANT ETHYLENE 

RECEPTOR, ETR1-1, INDICATE THAT 

ETHYLENE PERCEPTION BY STAMEN 

PRIMORDIA IS REQUIRED FOR CARPEL 

DEVELOPMENT IN MELON FLOWERS 

H. A. Little, S. Hammar, and R. Grumet 

Department of Horticulture, Michigan State University, 

East Lansing, MI 48824 

ADDITIONAL INDEX WORDS. Cucumis melo, sex expression, monoecious, 

monoecy 

ABSTRACT. Cucurbit species are characterized by a range of diverse sex 

phenotypes that are, at least in part, regulated by the plant hormone ethylene, 

which promotes increased femaleness, i.e., carpel development, on typically 

monoecious (male and female flowers) or andromonoecious (male and bisexual 

flowers) cultigens. We sought to determine the role of ethylene perception in 

carpel development by expression of the dominant negative Arabidopsis mutant 

ethylene receptor, etr1-1, under control of floral-targeted promoters in 

transgenic melon (Cucumis melo L.). Our previous studies with constitutively 

expressed etr1-1 demonstrated that etr1-1 blocked ethylene perception and 

prevented formation of carpel-bearing flowers when introduced into the 

andromonoecious melon cultivar Hale’s Best Jumbo, indicating the necessity of 

ethylene perception for carpel development. In these studies, we hypothesized 

that ethylene perception by the developing carpel primordium would be 

necessary for production of carpel-bearing flowers. In contrast to our 

prediction, expression of etr1-1 driven by the carpel-directed promoter, CRC, 

increased production of carpel-bearing buds, while etr1-1 driven by the stamen- 

and petal-specific promoter, AP3, abolished production of carpel bearing buds. 

These results suggest that ethylene perception by the stamen primordia is 

required for development of carpel primordia. 

C 

ucurbit species produce varying combinations of male, female, 

and bisexual flowers (Perl-Treves, 1999; Roy and Saran, 1990). 

Sex determination of individual flowers is hormonally 

regulated, with ethylene playing the predominant role (Rudich, 1990; 

Perl-Treves, 1999). Application of ethylene-releasing compounds 

We thank Drs. Eric Schaller, John Bowman, and Vivian Irish for providing the etr1- 

1 gene and CRC and AP3 promoters, respectively. This project was in part supported 

by research grants IS-3139-99 and US-3735-05C from BARD, the United States- 

Israel Binational Agricultural Research and Development Fund, and by a USDA- 

NNF fellowship to HAL. 


increases female or bisexual flower production, while treatment with 

inhibitors of ethylene biosynthesis or action increases maleness. 

In early stages of cucumber flower development, floral buds 

contain primordia of all four whorls (sepals, petals, stamens, and 

carpels) typical of a bisexual flower (Goffinet, 1990). In male flowers, 

arrest of carpel primordia occurs prior to differentiation of the ovary 

(Bai et al., 2004). In female flowers, initial differentiation of the 

filament and anther is followed by DNA degradation in the primordial 

anther (Hao et al., 2003). Response to ethylene-releasing or ethyleneinhibiting 

compounds indicates that the stage just preceding, or 

immediately following, differentiation of stamen primordia is critical 

for sex determination (Yamasaki et al., 2003; Bai et al., 2004; Hao et 

al., 2003). 

Our objective is to elucidate the role of ethylene perception on sex 

expression in melon. Melon plants are typically andromonoecious, 

with an initial phase of male flowers followed by a combination of 

bisexual and male flowers. We previously showed that melon plants 

transformed to constitutively express the dominant negative ethyleneperception 

mutant, etr1-1 from Arabidopsis, exhibited a range of 

phenotypes associated with reduced ethylene sensitivity, verifying that 

etr1-1 is able to confer ethylene insensitivity in the heterologous 

melon system (Papadopoulou, 2002). 

As predicted, the formation of carpel-bearing buds was essentially 

abolished in 35S::etr1-1 melons, demonstrating that ethylene 

perception is necessary to promote bisexual flower development in 

melon. 

In this work, we sought to identify the critical locations for 

ethylene perception within the developing flower bud. The etr1-1 

gene was introduced under the control of the Arabidopsis floral-organ 

specific Crab’s Claw (CRC) and Apetela3 (AP3) promoters. The CRC 

promoter drives expression in carpel and nectary primordia in 

Arabidopsis at the outset of gynoecium development (Bowman and 

Smyth, 1999; Lee et al., 2005). AP3 is expressed in cells destined to 

become petals and stamens (Irish and Yamamoto, 1995). It was 

predicted that blocking ethylene perception in the carpels would 

prevent carpel development, resulting in male flowers, but blocking 

ethylene perception in the stamens would not alter sex determination. 

In contrast to our prediction, CRC::etr1-1 plants showed increased 

female sex determination, while AP3::etr1-1 plants were nearly 

exclusively male. These results provide insight into the molecular 

basis for sex determination in melon, and indicate that ethylene 

perception by the stamen plays a critical role in regulating carpel 

development in the developing melon flower. 



PLASMID CONSTRUCTION, PLANT TRANSFORMATION, AND 

VERIFICATION OF TRANSFORMATION. Construction of the CRC::etr1- 

1 and AP3::etr1-1 plasmids is described in Little (2005). 

Agrobacterium tumefaciens-mediated transformation of 

andromonoecious melon (cv. Hale’s Best Jumbo, Hollar Seed, Rocky 

Ford, CO) was performed based on the methods of Fang and Grumet 

(1990) and Tabei et al. (1998). Regenerated plantlets (T0) were 

evaluated for the presence of the neomycin phosphotransferase protein 

by NPTII ELISA (Agia ® , Elkhart, IN) and the etr1-1 gene by 

polymerase chain reaction (PCR) using both etr1-1 and promoterspecific 

primers. PCR- and ELISA-positive T0 plants were transferred 

to the greenhouse and self-pollinated to produce T1 and T2 progeny. 

RNA was isolated from leaves, young male and female buds (3–5mm), 

and older male buds (10–12mm) of greenhouse-grown plants using 

Concert Plant RNA Reagent (Invitrogen, Carlsbad, CA). Gene 

expression was verified by northern analysis using standard procedures 

(Sambrook and Russell, 2001) and DIG-labeled probe (Roche 

Diagnostics, Germany). 

SEX EXPRESSION. The first 30 nodes of the main stem of 

transgenic T1 or T2 plants and nontransgenic controls were evaluated 

for the time of appearance of the first carpel-bearing flower bud; the 

total number of nodes with carpel-bearing or male buds; the number of 

carpel-bearing buds that reached anthesis; and, for aborted carpelbearing 

buds, the size reached prior to senescing. Each transgenic line 

was tested in at least three experiments using a randomized complete 

block design. 

Results 

GENE INTEGRATION AND EXPRESSION. Presence of the etr1-1 

gene and appropriate promoter was verified in T1 progeny of 

AP3::etr1-1 or CRC::etr1-1 plants by PCR analysis (Little, 2005). 

The observed segregation ratios were consistent with a single insertion 

site for all lines except AP3-3, which likely has two insertions (Little, 

2005). Northern hybridizations demonstrated that in AP3::etr1-1 

lines, etr-1-1 expression was absent in leaves (Figure 1A) and present 

in male buds (Figure 1B), as expected based on the petal and stamen 

specificity of AP3 in Arabidopsis (Jack et al., 1994). CRC::etr1-1 

lines showed hybridization in leaves, indicating leaky expression from 

the promoter in the heterologous melon system. etr1-1 mRNA was 

detected in both young male and carpel-bearing buds of CRC::etr1-1 

plants; higher levels were observed in the carpel-bearing buds, 


consistent with expected carpel expression. Detection of etr1-1 

message in male CRC::etr1-1 buds could be the result of expression in 

the nectaries (Bowman and Smyth, 1999; Lee et al., 2005) or 

leakiness, as is suggested by expression in the petals. 

SEX EXPRESSION OF TRANSGENIC ETR1-1 MELONS. 

CRC::ETR1-1. The typical progression of sex expression along the main 

stem of andromonoecious melon is several vegetative nodes, followed 

Fig. 1. Northern hybridization of etrl-1 gene expression. (A) Total RNA isolated 

from leaf tissue of nontransgenic (WT), 35s::etr1-1 (35S), CRC::etr1-1 (CRC11 

and CRC15), and AP3::etr1-1 (AP3-3 and AP3-8) plants. (B) Total RNA isolated 

from young male buds of WT, AP3-3, AP3-8, CRC5, and CRC15 plants and 

young female buds of WT, CRC5 and CRC15 plants. (C) Total RNA isolated 

from mature male buds of WT, CRC5, CRC11, and AP3-8 plants. Mature male 

buds were separated into petals and lower bud, which included sepals, stamens, 

nectary, and floral cup. The bottom band in each pair shows rRNA. 

by a phase of staminate buds, then a mixture of staminate and bisexual 

buds. ‘Hale’s Best Jumbo’ wild-type plants produced the first bisexual 

flowers at approximately Node 20 (Figure 2A). Examination of the 

CRC::etr1-1 plants did not show the predicted failure to produce 

carpel-bearing buds. Instead, carpel-bearing buds occurred 


significantly earlier (by 6–0 nodes) on the main stem of CRC::etr1-1 

plants than on the nontransgenic controls. CRC::etr1-1 plants also 

showed a significant increase in number of carpel-bearing buds on the 

first 30 nodes (Figure 2B). Thus, despite leaky expression from the 

CRC promoter, the phenotype of the CRC::etr1-1 plants was distinct 

from the 35S::etr1-1 plants, which failed to produce carpel-bearing 

buds (Papadopoulou et al., 2005; Little, 2005). 

Fig. 2. Sex expression of CRC::etr1-1 melon plants. (A) Node of first carpelbearing 

bud on main stem. (B) Total number of nodes with carpel-bearing buds 

on the main stem for ‘Hale’s Best Jumbo’ (WT), azygous nontransgenic plants 

(AZY), and CRC5, CRC11, and CRC15 lines. The experiment was repeated 

three times in a randomized complete block design. Data are means ± standard 

error of 16 plants/genotype. 

The effect of failure to perceive ethylene was, however, evident in 

the maturation of the carpel-bearing flowers. Typically, the majority 

of the buds bearing carpels initiated on the main stem of melon plants 

abort prior to reaching anthesis (Papadopoulou et al., 2005). 

Measurement of the size of the immature carpel-bearing bud at the 


time of abortion showed that carpel-bearing buds on CRC::etr1-1 

plants were four times more likely than buds on control plants to reach 

senescence at 4mm or less; a similar decrease was observed in the 

proportion of buds on CRC::etr1-1 plants that mature beyond 4mm or 

reach anthesis relative to controls (Figure 3). 

Fig. 3. Size reached by carpel-bearing buds before senescing for ‘Hale’s Best 

Jumbo’ (WT), azygous (AZY), and CRC::etr1-1 plants (lines CRC5, CRC11, 

and CRC15). The experiment was repeated three times in a randomized 

complete block design. Data are means ± standard error of 16 plants/genotype. 

AP3::ETR1-1. Inhibition of ethylene perception in the stamens was not 

predicted to affect sex determination in melon; however, all AP3-3 

plants and the majority of AP3-8 plants (61%) failed to produce any 

carpel-bearing buds within the first 30 nodes (Figure 4A). The AP3-8 

plants that did produce buds bearing carpels on the main stem reverted 

to male buds after producing only 1–2 buds containing a carpel. 

Examination of both main and lateral stems showed that less than 5% 

of the nodes on AP3::etr1-1 plants produced carpel-bearing buds vs. 

more than 50% of nodes evaluated in nontransgenic plants (WT and 

AZY) (Figure 4B). As was observed in previous experiments, 

CRC::etr1-1 plants that were included for comparison had earlier and 

increased formation of buds containing carpels. 


Discussion 

Expression of the etr1-1 gene under the direction of the 

Arabidopsis floral-targeted promoters CRC and AP3 conferred unique 

flowering phenotypes. CRC::etr1-1 plants exhibited enhanced 

femaleness as indicated by earlier and increased formation of buds 

containing carpels. This is in contrast to the expected inhibition of 

carpel-bearing buds, due to inhibited ethylene perception by the carpel, 

and the observed production of only male flowers on plants 

constitutively expressing etr1-1 (35S::etr1-1). The difference in the 

sex expression of 35S::etr1-1 and CRC::etr1-1 plants, despite leaky 

expression from the CRC promoter, may result from differences in 

timing and/or location of etr1-1 expression at key stages in sex 

determination, such that the CRC-driven etr1-1 expression does not 

prevent sex determination leading to initial carpel development. 

Conversely, and in contradiction to the predicted result that blocked 

ethylene perception in the stamen would not affect the production of 

bisexual buds, AP3::etr1-1 plants produced almost exclusively male 

flowers, suggesting that ethylene perception by stamen primordia is 

necessary for development of carpels. Collectively, these results with 

the CRC::etr1-1 and AP3::etr1-1 plants suggest that control of early 

carpel development resides within the stamens. 

Fig. 4. Carpel-bearing bud formation on AP3::etr1-1 transgenic plants. (A) 

Node position of first carpel-bearing bud on main stem. (B) Percentage of nodes 

bearing buds containing a carpel for ‘Hale’s Best Jumbo’ (WT), azygous (AZY), 

AP3::etr1-1 (lines 3 and 8), and CRC::etr1-1 (CRC15) plants. The experiment 

was repeated three times using a randomized complete block design with a total 

of 7–21 plants/genotype. 


Presumably, under normal growth conditions, 

andromoneocious melon plants would produce a phase of male flowers 

until sufficient levels of ethylene are perceived by the stamen 

primordia to promote bisexual buds. Morphological analyses of male 

and female bud development suggest that divergence from a bisexual 

to unisexual flower begins after the initiation of carpel primordia at 

Stage 5 (Bai et al., 2004), and the stage of floral development at which 

sex determination can be modulated by ethylene treatment just 

precedes, or immediately follows, differentiation of stamen primordia 

(Yamasaki et al., 2003). 

Perhaps the decision to promote or inhibit carpel development 

occurs at a stage in development when the stamen primordia, but not 

the less-developed carpel primordia, are able to perceive ethylene. 

Analysis of CRC::etr1-1 melon plants showed that although 

carpel-bearing buds were initiated, virtually all aborted very early, 

most before reaching 4mm in length. This suggests that ethylene plays 

a role in sustained maturation of the carpel. These results support our 

earlier observations made in transgenic melon constitutively 

expressing ACS (Papadopoulou et al., 2005). The 35S::ACS melon 

plants exhibiting increased ethylene production had earlier and 

increased production of carpel-bearing buds, and a marked increase in 

the proportion of carpel-bearing buds reaching anthesis. Thus, it 

appears that ethylene has separable functions for sex determination 

and pistillate bud-maturation in melon. 

In summary, the CRC::etr1-1 melon plants showed increased 

femaleness as demonstrated by earlier and increased carpel-bearing 

bud production while AP3::etr1-1 melon plants showed increased 

maleness as demonstrated by virtual elimination of bisexual buds. 

These results suggest that the critical site for ethylene perception for 

promotion of early carpel development does not reside within the 

carpel, and that perception by the stamens is required to prevent carpel 

arrest. Further study is necessary to determine the precise timing and 

localization of etr1-1 expression at key stages of sex determination. 


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DROUGHT-INDUCED GENE EXPRESSION IN 

ROOTS OF CITRULLUS COLOCYNTHIS 

Ying Si and Fenny Dane 

Department of Horticulture 

Auburn University, Auburn, AL 36849 

ADDITIONAL INDEX WORDS. Drought stress, cDNA-AFLP, real-time RT-PCR, 

bitter apple 

ABSTRACT. Citrullus colocynthis (L.) Schrad, closely related to watermelon, is a 

member of the Cucurbitaceae family. This plant, commonly known as the bitter 

apple or bitter gourd, is a tender perennial vine with a rich history as an 

important medicinal plant and as a source of valuable oil. It is a very drought 

tolerant species with deep root system, widely distributed in the Sahara- 

Arabian deserts in Africa and the Mediterranean region. A 20% polyethylene 

glycol 8000 (PEG) solution was used to induce drought stress in C. colocynthis 

seedlings. cDNA-AFLP analysis was used to study gene expression in roots. 

Genes with similarity to known function genes involved in transport facilitation, 

metabolism and energy, stress- and defense-related proteins, cellular 

organization, signal transduction and expression regulation were induced. The 

results suggest that C. colocynthis undergoes a complex adaptive process in 

response to drought stress. 

D 

rought is the major abiotic stress that has adverse effects on 

the growth of plants and productivity of crops. A combination 

of biochemical and physiological changes at the cellular and 

molecular levels, such as an increase in the plant stress hormone 

abscisic acid (ABA) and accumulation of various osmolytes and 

proteins coupled with an efficient antioxidant system, is known to 

result in stress tolerance. Plant cells have evolved to perceive different 

signals from their surroundings, to integrate them and respond by 

modulating the appropriate gene expression. The products of these 

genes are thought to function not only in stress tolerance but also in 

the regulation of gene expression and signal transduction (Bartels and 

Sunkar, 2005; Zhu, 2001). 

Although research has produced an enormous amount of 

information, we are far from understanding the complete mechanism 

of the stress reaction. It is proposed that our understanding of plant 

stress tolerance can be refined through gene isolation, characterization 

of individual genes and the assessment of their contribution to stress 

tolerance. 


Citrullus colocynthis (L.) Schrad, closely related to watermelon, is 

a member of the Cucurbitaceae family. This plant, commonly known 

as bitter apple or bitter gourd, is a tender perennial vine with a rich 

history as an important medicinal plant and as a source of valuable oil. 

It is a drought-tolerant species with deep root system, widely 

distributed in the Sahara-Arabian region in Africa and the 

Mediterranean region. 

In an effort to elucidate the molecular mechanisms of drought 

tolerance in C. colocynthis, we set out to examine genes induced 

during drought stress in seedling roots. 


Citrullus colocynthis seeds were sown in Metromix. Seedlings at 

5- to 6-leaf stage were cultured in 20% PEG 8000 solution for drought 

induction. Leaf and root samples were collected at 0, 4, 8, 12, 24 and 

48 h and stored at –80 o C immediately. RNA was extracted from 

control and 8 h treatment samples according to Spectrum Plant Total 

RNA Kit protocol (Ambion, Austin, TX), and purified by DNase I 

treatment. The concentration of RNA was measured using an 

Eppendorf Biophotometer (Brinkmann Instruments, NY), and quality 

was checked using formaldehyde/agarose gel electrophoresis. 

cDNA was synthesized using RETROscript (Ambion, TX) 

according to the manufacturer's instructions, and digested using the 

MseI/EcoRI enzyme combination. AFLP analysis was conducted 

according to the protocol of AFLP kit from Li-COR (Li-COR 

Biosciences, NE). Preamplified cDNA was diluted 40-fold, before 

amplification with selective primers. After selective PCR with E+A 

and M+C primers, selective PCR products were run on 6% 

polyacrylamide sequencing gel at 80 W for 5 h. Fragments were 

visualized by silver staining according to the Silver Sequence TM DNA 

Sequencing System Technical Manual (Promega, WI). 

Transcript-derived fragments (TDFs) were extracted from the gel 

and used as a matrix for reamplification by PCR. TDFs were ligated 

directly into the pGEM-T Easy Vector (Promega), and then 

transformed into competent Escherichia coli (Promega). Plasmids 

were isolated using Plasmid Mini Kit (BIO-RAD). Fragments were 

sequenced with the ABI 3100 DNA sequencer (AU Genomics Lab). 

Analysis of nucleotide sequence of fragments was carried out using the 

National Center for Biotechnology Information BLASTx search tool. 

Real-time quantitative RT-PCR was carried out using an AB 7500 

RealTime PCR System (AU Genomics Lab) and 7500 System 

software version 1.2.3 (Applied Biosystems, Foster City, CA) with β– 


actin gene used as reference in parallel with the target gene allowing 

gene expression normalization and providing quantification. PCR 

efficiencies of target and reference genes were determined by 

generating standard curves. 


cDNA-AFLP using 32 different primer combinations resulted in 42 

putative differentially expressed DNA fragments, which were cloned, 

sequenced, and analyzed. Genes with similarity to known function 

genes involved in transport facilitation, metabolism and energy, stress- 

and defense-related proteins, cellular organization, signal transduction, 

and expression regulation were detected. Thus far, 5 up-regulated 

fragments were confirmed to be differentially expressed in treated 

plants using quantitative relative real-time PCR. These genes show 

high similarities at the amino acid level to heat shock protein (Hsp70), 

putative senescence-associated protein, grpE-like protein, 

synaptobrevin-related protein, and putative pathogenesis-related 

protein. The drought-stress-induced genes are known to be regulated 

not only by drought signals, but also by osmotic stress and defense 

signals in other plants (Bartels and Sunkar, 2005; Zhu, 2001). More 

genes will be identified and confirmed in further studies and detailed 

characterization will be forthcoming. It is clear that C. colocynthis 

undergoes a complex adaptive process in response to drought. The 

recognition of specific genes and in-depth analysis of their function in 

the process of stress tolerance will enable their use in improving the 

stress-tolerance ability of other Citrullus species. 


Bartels D. and R. Sunkar. 2005. Drought and salt tolerance in plants. Crit. Rev. Plant 

Sci. 24:23–58. 

Zhu, J. K. 2001. Cell signaling under salt, water and cold stress. Curr. Op. Plant Biol. 

4:401–406. 


EMBRYO CULTURE AS A TOOL FOR 

INTERSPECIFIC HYBRIDIZATION OF 

CUCUMIS SATIVUS AND WILD CUCUMIS 

SPP. 

D. Skálová, B. Navrátilová, A. Lebeda, and N. Gasmanová 


Botany, Šlechtitelů 11, 783 71 Olomouc-Holice, Czech Republic 

ADDITIONAL INDEX WORDS. Cucumber, muskmelon, cross-pollination, embryo 

rescue, colchicine application, ploidy level, disease resistance 

ABSTRACT. One of the possible methods for introducing resistance to disease 

and pests from wild Cucumis species to cucumber genotypes is interspecific 

hybridization. Methods of embryo-rescue and/or ovule culture are of 

considerable interest for overcoming crossability barriers. In the present study, 

selected genotypes of Cucumis species (C. sativus L., C. melo L., C. anguria L., C. 

zeyheri Sonder, and C. metuliferus E. Meyer ex Naudin) were used for 

hybridization experiments. Cross-pollination between C. sativus (as a maternal 

parent) and wild Cucumis species (as paternal parent) to obtain fertile hybrid 

plants was unsuccessful but some callus formation (hybridization of C. sativus × 

C. melo) and morphological changes on the isolated embryos and seeds 

(hybridization of C. sativus × C. metuliferus) were observed. The most suitable 

medium for the culture of immature embryos and seeds was the GA medium 

(with the addition of gibberelic acid). The best results were obtained from 

hybridization between mixoploids (2n/4n) and tetraploids (4n) of C. sativus 

(produced by polyploidization based on colchicine application) with wild 

Cucumis spp. Callus formation on the isolated embryos was followed by 

rhizogenesis, leaf formation, and regeneration of plants (most frequently on the 

GA medium). 

T 

he genus Cucumis contains two economically important 

species, cucumber (Cucumis sativus L., 2n = 14; from the Asian 

group) and muskmelon (Cucumis melo L., 2n = 24; from the 

African group). These two species are different in their origins and 

basic chromosome numbers (Jeffrey, 2001). Cucumber is among the 

top 10 vegetables in world production (FAO, 2004; Lebeda et al., 

2006); however, it is a crop with a narrow genetic basis and is 

susceptible to many diseases and pests (Chen et al., 2004). Several 

valuable resistances have been found in wild African species of 

The authors thank Dr. H.S. Paris and Dr. M.P. Widrlechner for valuable comments 

on this manuscript. This research was supported by the following grants: (1) MSM 

6198959215 (Ministry of Education, Youth and Sports, Czech Republic); and (2) QD 

1357 (NAZV, Ministry of Agriculture, Czech Republic. 


Cucumis that are not known naturally in cucumber (Lebeda et al., 

2006). 

Because of the value of these resistances for cucumber breeding, a 

number of attempts have been made to cross them with cucumber. 

However, because of crossing barriers (Ondřej et al., 2001), most of 

the crosses have been rather unsuccessful (Lebeda et al., 2006; 

Skálová et al., 2004). The primary reason for these crossing barriers is 

thought to be related to the differing chromosome numbers of the 

parents. Yet, interspecific hybridization can be one of the most 

efficient ways to transfer useful characters from wild relatives to the 

cultivated species, and embryo-rescue culture can facilitate the process 

(Skálová et al., 2004). Embryos at the globular stage were observed in 

interspecific hybridization between C. sativus and C. melo 

(Niemirowicz-Szczytt and Wyszogrodska, 1976), and between C. 

sativus and C. metuliferus (Franken et al., 1988). Some successful 

interspecific hybridizations between cucumber and wild Cucumis spp. 

have been made via such unconvential techniques as embryo-rescue 

culture, e.g., with C. hystrix Chakr. (Chen et al., 2003) and with C. 

melo L. (Lebeda et al., 1996, 1999). Interspecific hybridization 

between C. sativus and wild Cucumis spp. is usually unsuccessful 

because of the different chromosome numbers of the parents. There 

are some possibilities for overcoming this problem; one of them is 

embryo-rescue culture (Skálová et al., 2004). Embryos at the globular 

stage were observed in interspecific hybridization between C. sativus 

and C. melo (Niemirowicz-Szczytt and Wyszogrodzka, 1976), and 

between C. sativus and C. metuliferus (Franken et al., 1988). 

Some attempts to improve the success of interspecific 

hybridization in Cucurbitaceae have been made by using 

polyploidization or haploidization of the parental lines to obtain 

similar chromosome numbers (Greplová et al., 2003; Yetisir and Sari, 

2003). Among the techniques used for chromosome manipulation, 

colchicine polyploidization is the most widely employed. Other 

doubling agents include podophyllin, oryzalin, pronamide, etc. (Rao 

and Suprasanna, 1996). Several methods for polyploidization by 

colchicine pretreatment have been used: wetted cotton-wool on the 

growth apex (Rao and Suprasanna, 1996); submersion of rootlets 

(Mathias and Röbbelen, 1991); submersion of the main stem (Yetisir 

and Sari, 2003); seed treatment (Kalloo, 1988); and incubation of 

embryos on MS-medium containing colchicine (Chen and Staub, 

1997). 

The main aim of this research was to contribute to the work of 

obtaining interspecific hybrids of Cucumis sativus with different wild 

Cucumis spp. via the embryo-rescue-culture technique, and to transfer 


esistance to some cucumber diseases (e.g., downy mildew). Crosses 

of C. sativus with wild Cucumis spp. without special pretreatment and 

using colchicine treatment of cucumber mother plants have been 

compared. 


PLANT MATERIAL. Selected genotypes of Cucumis species were 

used for hybridization experiments (Table 1). The plant material 

originated from the vegetable germplasm collection of the Research 

Institute of Crop Production (Prague), Department of Gene Bank, 

Workplace Olomouc, Czech Republic (, part 

databases, EVIGEZ) and the Plant Introduction Station, Iowa State 

University, Ames, IA. The plants were cultivated in a glasshouse and 

in insect-proof isolation cages. Various wild Cucumis species were 

used (Table 1) as a source of pollen, and C. sativus was used as the 

maternal component. The fruits were harvested on the 14th day after 

pollination. 

Table 1. Plant material. 

Cucumis spp. Abbreviation Accession number 

C. sativus (SM-6514/line) CS CZ 09H3900768 

C. anguria var. 

longaculeatus CA CZ 09H4100569 

C. metuliferus CME CZ 09H4100185 

C. zeyheri CZ CZ 09H4100196 

C. melo CM2 PI 124112 

C. melo (line MR1) CM1 CZ 09H4000600 

EMBRYO CULTURE. Fruits were surface-sterilized with 70% 

ethanol and seeds or embryos were excised. Embryos or seeds were 

cultivated on various media (Table 2) in test tubes for six weeks in at 

25 o C. After germination they were transferred to a culture room 

(22±2 o C and 16h-day/8h-night photoperiod). 

POLYPLOIDIZATION METHODS. Two C. sativus sowings were 

cultivated without colchicine pretreatment. Four other sowings were 

influenced by various types of polyploidization with colchicine 

(wetted cotton-wool on the growth apex with 0.5 % and 5% colchicine 

solution; submersion of rootlets for 24 h with 0.5 % colchicine 

solution). The ploidy of obtained plants was determined by flow 

cytometry. 


Table 2. Composition of culture media. 

Culture 

medium 

Basic 

medium Other components 

OK 

MS 20mg/l ascorbic acid, 0.01mg/l IBA, 

0.01mg/l BAP, 20mg/l sucrose, 8g/l agar 

(control medium) 

ON MS 1g/l caseinhydrolysate, 0.01mg/l IBA, 

0.01mg/l BAP, 20g/l sucrose, 6g/l agar 

CW MS 5% coconut water, 200mg/l α- glutamin, 

0.01mg/l IBA, 0.01mg/l BAP, 60g/l 

sucrose, 6g/l agar 

GA MS 0.3mg/l giberellic acid, 0.01mg/l IBA, 

0.01mg/l BAP, 20g/l sucrose, 8g/l agar 

MSNAA MS 2.5mg/l NAA, 1mg/l BAP, 30g sucrose, 

8g/l agar 

Abbreviations: MS = Murashige and Skoog (1962); OK, ON, CW, GA, MSNAA = 

abbreviations for MS-media supplemented with different type of additions 

supporting embryogenesis; IBA = indole butyric acid; BAP = benzylaminopurin; 

NAA = naphthalene acetic acid. 

ISOLATION AND STAINING OF NUCLEI. As an internal reference 

standard Cucumis sativus (CS) was used. Twenty mg of plant tissue 

were chopped together with the standard into small pieces in a Petri 

dish containing 0.5 ml OTTO I (Otto, 1990) (0.1M citric acid, 0.5% 

Tween 20). Then the suspension of isolated nuclei was filtered through 

nylon (40µm) and 50µ RNAse and 50µl PI (Propidium iodide) stock 

solution and 1ml OTTO II (Otto, 1990) (0.4M Na2HPO4) were added. 

The suspension was incubated for 5 min at room temperature. After 

incubation, each sample was run on a flow cytometer. 

ESTIMATION OF PLOIDY LEVEL. Relative fluorescence of the 

nuclei was measured using a PAS flow cytometer (Partec GmbH, 

Münster, Germany) equipped with a laser. The gain was adjusted so 

that the fluorescence peak of the standard (diploid) was placed on 

channel 100 of the 512-channel scale. Ploidy level was determined by 

comparing a position of peak corresponding to G1 nuclei of the sample 

with that of a diploid plant used as a standard. At least 5,000 nuclei 

were analyzed in each sample. 


The regeneration of embryos isolated from fruits obtained by 

interspecific hybridization of Cucumis spp. was efficient only in some 


cases. In total, the regeneration of seven embryos from interspecific 

crosses of C. sativus × C. melo was observed (Table 3). Five embryos 

developed small roots or a shoot meristem, however callus formation 

predominated. Two embryos developed into flowering plants and their 

hybrid origin was confirmed using isozyme analyses and flow 

cytometry, as in the previous experiments where development of 

hybrid embryo of C. sativus × C. melo was confirmed (Lebeda et al., 

1996, 1999). 

Some positive results were obtained in pollination of female 

flowers of C. sativus with pollen grains of C. melo and C. metuliferus 

(Table 3). Callus formation on some embryos from the fruits obtained 

after pollination of C. sativus with both Cucumis species was 

observed. Growth of isolated embryos (from the crosses of C. sativus 

× C. melo) without any subsequent regeneration was observed as well. 

However, the growth of embryos stopped very early. Regeneration of 

immature seeds from fruits of crosses of C. sativus with C. melo and 

C. metuliferus and some morphological changes on the seed coats 

and/or stems occurred. The most suitable media for the 

regeneration of isolated embryos and seeds were GA and CW (media 

with gibberelic acid and coconut water added) (Tables 2 and 3). 

Nevertheless, no direct or indirect regeneration of plants was observed 

from this type of hybridization. It can be concluded that interspecific 

hybridization between C. sativus and wild Cucumis spp. is rather 

difficult without using some special strategies of embryo-rescue 

culture. This conclusion is also supported in a literature survey focused 

on embryo-rescue culture of Cucumis spp. (Skálová et al., 2004). 

One possible strategy for overcoming crossing barriers is to use 

irradiated pollen for cross-pollination (Custers and Bergervoet, 1984). 

Custers et al. (1981) used AVG (aminoethoxyvinylglycine) with a 

positive effect on the success of reciprocal crosses between C. 

africanus and C. metuliferus ‘Megurk’ (2n = 19), and fertile F1 hybrid 

plants were derived from pollination of C. sativus with mixed pollen of 

C. sativus and C. melo (Van der Knaap et al., 1978). Embryo rescue 

after in vitro pollination has also been used to obtain hybrid calli from 

Cucumis spp. (Ondřej et al., 2002). Polyploidization of plant genera 

with lower chromosome numbers can be useful for overcoming the 

prezygotic barriers (Greplová et al., 2003). 

In this experiment we used different colchicine treatments of C. 

sativus (2n = 14) as a maternal parent. The main purpose of this 

treatment was to obtain plants with chromosome numbers closer to 

those of wild Cucumis spp. as sources of pollen. The polyploid 

character was confirmed by flow cytometry. Tetraploids (4n = 28) and 

mixoploids (2n/4n = 14/28) were obtained after these pretreatments. 


Table 3. Results of interspecific hybridization of Cucumis sativus with 

wild Cucumis spp. 

Partn. 

Type for 

of CT IH 1 

No. No. No. No./ Suc- 

No. obiso- isolated type cesspollitainedlatedemregenfulnation 

fruits seeds bryoser. media 2 

CM1 27 13 260 260 8 (C, OK, 

MC) CW, 

GA 

None 

CM2 

CA 

18 

11 

8 

2 

120 

80 

160 

0 

0 

0 

- 

- 

CZ 16 7 280 0 0 - 

CME 8 6 240 0 4 (C, CW, 

MC) GA 

Total 80 36 980 420 12 - 

CM1 9 6 219 20 1 (RE) GA 

CM2 7 6 240 16 7 (5 RE OK,O 

Wetted 

plants!; N, CW, 

cotton- 

2C) GA, 

wool, 

MSN 

0.5% col. CA 4 2 60 0 0 - 

CZ 3 2 70 0 0 - 

CME 2 2 45 25 1 (C) MSN 

Total 25 18 634 61 9 - 

CM1 28 16 505 68 6 (3RE, ON, 

Wetted 

cottonwool, 

5% 

col. 

CM2 

CA 

CZ 

9 

3 

12 

5 

1 

11 

185 

45 

302 

20 

0 

15 

3C) 

0 

0 

0 

CW, 

GA 

- 

- 

- 

CME 5 3 135 0 1 (MC) OK 

Total 57 36 1172 103 7 - 

CM1 12 7 180 80 1 (C) GA 

CM2 4 2 60 20 0 - 

Submer- CA 5 1 15 0 0 - 

sion of CZ 6 6 225 20 0 - 

rootlets, CME 2 2 90 0 4 (2C, 2 OK, 

0.5 % 

MC) ON, 

col. 

CW, 

GA, 

MSN 

Total 29 18 570 120 5 - 

Abbreviations: CT = colchicine treatment,; IH = interspecific hybridization; RE = 

regeneration and germination of embryos; C = callus formation; MC = 

morphological changes. 

1 2 

= particular genotypes of Cucumis spp. (Table 1). = different types of media 

(Table 2). 


Regeneration of embryos and seeds, obtained by hybridization of 

wild Cucumis spp. (Table 1) with cucumber maternal plants (after 

colchicine treatment), was relatively successful. In addition to calli on 

both immature above-mentioned explants and morphological changes 

on the seed explants, organogenesis was also recorded on calli 

(formation of roots or shoots on callus mass) and direct formation of 

whole plants (from embryos derived by interspecific hybridization C. 

sativus × C. melo, CM2). These embryos passed direct embryogenesis 

and germinated on all types of used media. The hybrid/nonhybrid 

origin of these plants will be confirmed by flow cytometry. 

Caseinhydrolysate (ON medium), coconut water (CW medium) 

and gibberelic acid (GA medium) promoted embryo germination, 

which was followed by callus formation (regeneration on the GA 

medium was the most frequent). Seed development was best on the 

ON and GA media, respectively (Table 3). 

The highest total number of successful regenerations occurred by 

pollination of C. sativus without colchicine treatment; however, no 

regenerated plants were obtained (Table 3). Only one type of 

colchicine treatment of C. sativus offered direct regeneration of 

embryos into plants (application of wetted cotton-wool on growth 

apex, 0.5% colchicine solution; crossing partner C. melo (CM2) (Table 

3). In this experiment, the most suitable partner for interspecific 

hybridization with C. sativus was C. melo (line MR1; CM1) (Table 1), 

because of the highest regeneration frequency (Table 3). Gibberelic 

acid (GA medium) appeared as the most efficient additive to 

cultivation media for embryos and seeds. 

It can be concluded that colchicine pretreatment of C. sativus is a 

possible way to improve the success and efficiency of interspecific 

hybridization with wild Cucumis species. The frequency of embryo 

and seed regeneration (organogenesis on the callus mass, direct 

regeneration to plants) was higher after colchicine treatment. From 

these experiments it is evident that the most suitable wild Cucumis 

partners for interspecific hybridization with C. sativus were C. melo 

(line MR1) and C. metuliferus (for both types of hybridization, i.e., 

with and without colchicine treatment in the maternal genotype of C. 

sativus with wild Cucumis spp. as the paternal genotype). The most 

efficient medium, for isolated and potentially interspecific embryos, 

seems to be the GA medium (added gibberelic acid). 


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(Cucumis melo). Cucurbit Gen. Coop. Rep. 1:8. 

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L.) dihaploidization. Sci. Hort. 98:277–283. 


INITIATING SUDDEN WILT DISORDER IN 

MUSKMELON WITH LOW-LIGHT STRESS 

H. C. Wien and T. A. Zitter 

Cornell University, Ithaca, NY 15853 

ADDITIONAL INDEX WORDS. Cantaloupe, vine collapse, Cucumber mosaic virus, 

Cucumis melo 

ABSTRACT. Sudden wilt or collapse of melon plants at a point close to harvest of 

the first mature fruit is a common disorder in the Northeastern U.S. Cucumber 

mosaic virus, Papaya ringspot virus, Fusarium oxysporum f. sp. melonis, and 

powdery mildew, among others, have been implicated as causal organisms, 

acting singly or in combination. The disorder is often triggered by unfavorable 

weather conditions, such as a period of cold rainy weather. To determine if the 

disorder could be initiated by conditions of low light that would occur during 

rainy weather, shading experiments were conducted in fields of ‘Athena’ melons 

in 2004 and 2005. In the first year, shading frames that reduced the incident 

radiation by 40 and 80% were placed over the crop for a week, starting either 7 

days before the first fruit harvest, or a week later. None of the treatments 

caused plant collapse nor affected yield in 2004. In 2005, the same two levels of 

shade were imposed on the field plots starting a week before first ripe fruit 

harvest for a duration of two weeks. This time, plant collapse was noticeable by 

one week after the start of shade treatments, and was proportional to the degree 

of shade. If confirmed by our 2006 experiment, the results indicate that plant 

collapse of Cucumis melo can result not only from direct attack from plant 

pathogens, but also from a carbohydrate stress. We speculate that under the 

latter conditions, the plants preferentially translocate assimilates to the ripening 

fruits rather than the root system, leading to an impairment of root function 

and plant collapse. 

T 

he sudden collapse of muskmelon plants close to harvest has 

concerned growers and frustrated researchers in the 

Northeastern U.S. for many years, and has defied simple 

answers as to causes and possible cures. Zitter (1995) summarized the 

state of knowledge at a previous Cucurbitaceae meeting, and listed a 

number of pathogens that have been associated with the disorder. 

Early reports of the disorder implicated Fusarium wilt (Fusarium 

oxysporum f. sp. melonis) (Crozier, 1963), but resistance to this 

disease in most modern cultivars has reduced the likelihood of 

involvement of this pathogen. Evidence that Cucumber mosaic virus 

(CMV) could be a causal agent for the collapse of melon plants has 

been more compelling. MacNab (1971) induced a vine decline of 

flowering melon plants by inoculating with CMV, and demonstrated 

the progress of wilting and necrosis of the leaves from the point of 

inoculation. Treatments did not lead to overall collapse of these 


plants, however. In other experiments with fruiting plants, MacNab 

(1971) could not induce collapse by late inoculation with CMV. His 

observations of sudden wilt in growers’ fields implicated a period of 

cold, rainy weather as a triggering factor. Unfortunately, his attempts 

to cause plant death by cooling the roots to 10 C produced only a 

temporary wilting, duplicating the results of similar experiments by 

Raleigh (1941). These cooling experiments were conducted with 

flowering plants without fruit load, so the question of whether cooling 

of roots of fruiting plants would lead to collapse has not been 

addressed. 

The presence of fruit on the plant can play a significant role in the 

health and vigor of plant root systems. Roots of greenhouse-grown 

tomatoes declined when fruits were actively growing (Hurd et al., 

1979), and similar observations have been made with cucumber (Van 

der Post, 1968). Under low-light stress, fruiting greenhouse cucumber 

plants allocated a smaller proportion of assimilates to roots, perhaps 

further impairing the capacity of the roots for water uptake (Marcelis, 

1994). If environmental stress factors such as low light or cold soils 

can contribute to the loss of function of melon roots on fruiting plants, 

it may be possible to stress a melon plant sufficiently with heavy shade 

to cause it to collapse even without the added stress of pathogens. The 

current experiments were conducted to test this premise. 


Plants for the field shading experiments were started in seedling 

trays in a greenhouse. Seeds of Cucumis melo cv. ‘Athena’ were sown 

in 72-cell trays in a commercial peat-vermiculite artificial-soil mix. 

Four weeks later, the seedlings were transplanted to the field, into a 

gravelly loam soil (mixed mesic, Glossoboric hapludalf), covered with 

IRT green polyethylene mulch 122cm wide, in rows 183cm wide. 

Plants were spaced 61cm apart in the row. Supplemental irrigation 

was provided by a single trickle irrigation line under the mulch, and in 

the very dry 2005 growing season, by additional sprinkler irrigation. 

At specific stages of plant growth in relation to fruiting, plots were 

shaded by wooden shading frames measuring 244cm wide, 488cm 

long, and 100cm tall, covered with black polyester shade fabric. Three 

degrees of shade were utilized: none, 40%, and 80%. Shade duration 

was one week in 2004, two weeks in 2005 and 2006. In 2004, shading 

treatments were begun either one week before or at first ripe fruit 

harvest; in 2005, shade was initiated at first fruit harvest; in 2006, 

shading treatments were applied when first fruits reached 3cm 

diameter, and at first fruit harvest. Fruits were harvested at full slip, 


weighed, and soluble solids measured using a refractometer. The 

degree of vine collapse was noted by counting the number of wilted 

plants in the harvest area at weekly intervals. The experimental design 

of the experiments with more than one shading time was a split plot 

with four replications, and shading time as main plots. Statistical 

analysis of the yield and soluble solids content was by analysis of 

variance using the Statistix software program. 

Results 

Climatic conditions in the 2004 fruiting and harvest season were 

cool and cloudy. Sudden wilt or late vine collapse did not occur in the 

experiment in 2004, regardless of the timing and degree of shade 

imposed. There was also no effect of the one-week shading treatments 

on marketable yield, which averaged 32MT/ha, or on soluble solids 

(10.83%). 

In 2005, the weather was dry and sunny most of the season, with 

higher temperatures than in 2004. From Aug. 1 to the end of fruit 

harvest, temperatures at the experimental farm averaged 19˚C in 2004 

and 21˚C in 2005. Rainfall totaled 220mm and 21mm in 2004 and 

2005 during the same period, respectively. 

First signs of plant collapse were noticed at first fruit harvest 

(August 10), 5 days after shade treatments were begun, and became 

progressively more marked (Table 1). The greater the shade level 

imposed, the more plants collapsed. From Aug. 22 to the final fruit 

harvest on Aug. 29, more of the plants subjected to 40% shade had lost 

turgor, and there appeared to be a slight recovery of some of the 

heavily shaded plants. This interaction of sampling date and shade 

level was significant at the 5% level. 

Table 1. Number of plants of ‘Athena’ melon collapsed (out of 8 

plants per plot) at specific dates in the harvest season of 2005, in 

relation to shading treatments applied. Treatment effect significant at 

the 0.1% level; interaction of sample date and treatment significant at 

the 5% level. 

Sample date 

Treatment Aug. 22 Aug. 29 

Control 0 1.2 

40% shade 2 4.5 

80% shade 6.2 5.2 


In spite of the plant collapse, marketable and total yields of fruit 

were not significantly affected by shading treatments. Yields averaged 

28MT/ha in 2005. Fruit soluble solids also did not vary significantly 

among treatments, and averaged 11.6%. 

Discussion 

The lack of response to shading in 2004 could be due to the shorter 

duration of the shade treatment in that year (one week compared to 

two weeks in 2005). This may not have depleted assimilates enough 

to affect root growth of the fruiting plants. 

If the 2005 results are more representative of the effects of a severe 

carbohydrate deficiency on fruiting melon plants, it would indicate that 

even apparently healthy melon plants can be subject to sudden wilt. 

‘Athena’ melon is resistant to the common races of Fusarium wilt 

(Races 0, 1, 2), and in our plantings did not show obvious symptoms 

of cucumber mosaic or powdery mildew (‘Athena’ has moderate 

powdery mildew resistance). The shade-induced collapse without 

obvious signs of disease would support the theory that melon plants 

near the time of fruit harvest are transferring such a high percentage of 

their assimilates to the fruits that support of the root system may be 

sacrificed, especially if the plants are under an assimilate stress. 

While normally one would not expect to encounter two weeks of 

severely low light conditions close to the time of melon harvest, under 

field conditions one could expect a combination of environmental 

stress and some plant diseases. Cucumber mosaic virus was shown by 

Bauerle (1971) to reduce root extension and water uptake. Powdery 

mildew in cucumber (Podosphaera xanthii syn. Sphaerotheca 

fuliginea) reduces photosynthetic rate of the leaves, and thus would 

contribute to an assimilate stress (Yurina et al., 1993). 

Given the apparent susceptibility of melon to collapse close to 

harvest, what practical measures can be taken to decrease its 

susceptibility? The development of multiple disease-resistant cultivars 

would be one useful solution. In addition, breeding lines that have 

more vigorous root systems should also help. This approach has been 

shown to be useful, when it was noted that grafting melon onto 

Cucurbita helped to lessen the incidence of sudden wilt disease caused 

by Monosporascus in Israel (Cohen et al, 2000). It remains to be 

determined if the establishment of a stronger sink in the root system of 

melon plants would reduce the sugar content of the fruits. 



Bauerle, W. L. 1971. Effect of the sudden wilt disease on the physiology of the 

muskmelon (Cucumis melo L. var. reticulates). PhD thesis, Cornell University, 

Ithaca, N.Y. 

Cohen, R., S. Pivonia, Y. Burger, M. Edelstein, A. Gamliel, and J. Katan. 2000. 

Various approaches toward controlling sudden wilt of melons in Israel. Acta 

Hort. 510:143–147. 

Crozier, J. A., Jr. 1963. Some factors affecting the development of a sudden wilt 

disease of muskmelon in New York State. PhD thesis, Cornell University, 

Ithaca, N.Y. 

Hurd, R. G., A. P. Gay, and A. C. Mountifield. 1979. The effect of partial flower 

removal on the relation between root, shoot and fruit growth in the 

indeterminate tomato. Ann. Appl. Biol. 93:77–89. 

MacNab, A. A. 1971. Symptomatology of decline, early-collapse, and late-collapse 

of muskmelons (Cucumis melo L. var. reticulates) in New York State and the 

etiology of decline. PhD thesis, Cornell University, Ithaca, N.Y. 

Marcelis, L. F. M. 1994. Effect of fruit growth, temperature and irradiance on 

biomass allocation to the vegetative parts of cucumber. Neth. J. Agric. Sci. 

42:115–123. 

Raleigh, G. J. 1941. The effect of culture solution temperature on water intake and 

wilting of the muskmelon. Proc. Amer. Soc. Hort. Sci. 38:487–488. 

Van der Post, C. J. 1968. Simultaneous observations on root and top growth. Acta 

Hort. 7:138–143. 

Yurina, T. P., V. A. Karavaev, and M. K. Solntsey. 1993. Characteristics of 

metabolism in two cucumber cultivars with different resistance to powdery 

mildew. Russian Plant Physiol. 40:197–202. 

Zitter, T. A. 1995. Sudden wilt of melons from a Northeast US perspective, p. 44– 

47. In: G. Lester and J. Dunlap (eds.). Cucurbitaceae ’94. 


DEVELOPMENT OF IMPROVED CULTIVARS 

OF ZUCCHINI SQUASH AND SWEETPOTATO 

SQUASH STARTING FROM CHILEAN 

LANDRACES 

Gabriel Bascur 

INIA C.R.I. La Platina, Santiago, Chile 

ADDITIONAL INDEX WORDS. Cucurbita pepo, Cucurbita maxima, inbred lines, 

fruit traits, new cultivar 

ABSTRACT. The CRI La Platina of INIA is developing zucchini squash 

(Cucurbita pepo L.) and sweetpotato squash (Cucurbita maxima Duch.) crops to 

provide cultivars with good horticultural characteristics in a market where 

there are few commercial cultivars available. Starting from selections made 

from commercial cultivars, inbred lines were evaluated in different seasons and 

locations. In zucchini squash, the cultivar Curital INIA was released. It is openpollinated, 

with a total yield 18.9% higher than ‘Black Chilean’. Its fruit are 

cylindrical, shiny, and dark green with clear green stripes. In sweetpotato 

squash, 300 inbred lines were produced and evaluated at Curacaví and La 

Platina locations. There is great variability among the inbreds both in fruit 

shape and size. Inbreds have been selected for adaptation, yield, and quality. 

Cultivars will be released for use by the growers. 

I 

n Chile, zucchini squash (Cucurbita pepo L.) and sweetpotato 

squash (Cucurbita maxima Duch.) have been used traditionally in 

the preparation of local dishes. Those dishes require that certain 

characteristics be present for success in the marketplace. As a result, 

certain cultivars that have maintained the preferences of local 

consumers have been grown for a long time. Those landraces have 

characteristics that are not present in the new cultivars available in the 

world market (Escaff, 2001). 

Since there are no suitable cultivars being developed for this 

market, INIA La Platina Regional Research Centre began a geneticimprovement 

program to produce cultivars with the characteristics and 

quality required by the Chilean consumer. The leading cultivar of 

zucchini squash is ‘Black Chilean’. It has dark green fruit with light 

green longitudinal stripes. This landrace has low yield and high 

variability for plant and fruit characteristics (Giaconi and Escaff, 

1997). 

In sweetpotato squash, a great diversity of fruit types exist that are 

marketed in the country. The landraces differ in fruit color, shape, size, 

and appearance. They also differ in thickness and color of the pulp, 

which determine the quality of the mature fruit. The Chilean market 


equires large fruit (over 15kg), clear grey to brown, with thick, dense, 

very dark orange pulp. One of our objectives is to develop a cultivar 

with these fruit characteristics but of smaller size. In this way, we hope 

to encourage the sale of this crop without having to cut the fruit for 

retail marketing. 

Zucchini squash was grown on approximately 6,000ha, and 

sweetpotato squash on 4,500 to 5,000ha annually during the period 

1995 to 2000 (ODEPA, 2006). The cultivation of these vegetables 

prevails at the small-farmer level of the center north zone of Chile. 


New zucchini squash cultivars were developed at CRI La Platina 

starting with the ‘Black Chilean’ landrace. Single-plant selections 

were made for fruit type. Crosses were made to combine traits from 

different plants, and inbreds developed through self-pollination as 

described by Whitaker and Robinson (1986). From this material, the 

best lines selected in preliminary evaluations were evaluated in trials 

established at La Serena (29°53'S; 71°19'W) IV Region; Curacaví 

(33°22S; 71°07'W); and CRI La Platina (33°34'S; 70°38'W) 

Metropolitan Region (RM). Trials were run in the 2002–2003 and 

2003–2004 seasons to evaluate phenotypic stability, horticultural 

characteristics, and yield based on number and weight of commercial 

fruit (10 to 15cm of long, immature fruit) per area. A randomized 

complete block design was used with four replications, where the 

control treatment was ‘Black Chilean’. The fruit were evaluated for 

shape, color, and shininess. 


ZUCCHINI SQUASH. Several inbred lines were selected for good 

yield, plant, and fruit characteristics, and one line (LPZI 2002-47) was 

released as ‘Curital INIA’ (Bascur, 2005). ‘Curital INIA’ has an erect 

growth habit with vigorous stems, medium-sized lobed leaves of 

variegated dark green and marked areas of grizzly white, with serrated 

borders, and the presence of trichomes. Sex expression is monoecious 

and flowers are an intense yellow. The fruit at the immature stage, as 

sold for the commercial product, are of cylindrical elongated shape, 10 

to 12cm long, of intermediate thickness, with an approximate weight 

of 200 to 250g, and no girdle. The fruit are shiny, dark green, with 

light reticulation, and light green stripes. 

‘Curital INIA’ had similar fruit yield to ‘Black Chilean’ at the La 

Platina and Curacaví locations (Table 1). In the 2003–2004 season, 

‘Curital INIA’ had significantly higher yield than ‘Black Chilean’ at 


the three locations. Averaged over all environments (year and 

location), ‘Curital INIA’ outyielded ‘Black Chilean’, with an 

advantage of 24,268 fruit/ha or 18.9% higher (Figure1). 

Upon comparing the standard deviation, a parameter that allows 

the estimation of the stability of the mean, it appears that ‘Curital 

INIA’ has a larger value than ‘Black Chilean’, which indicates that its 

production is more influenced by changes in environmental conditions 

such as year and location (Figure1). ‘Curital INIA’ has improved fruit 

shape and brightness relative to ‘Black Chilean’ (Table 2). 

SWEETPOTATO SQUASH. The breeding work carried out on 

sweetpotato squash resulted in the development of 300 inbred lines, 

which have a high uniformity of characteristics. However, there is 

great variability for fruit characteristics among lines, resulting in good 

opportunities for selection of outstanding lines for use by growers 

(Bascur, 2005). Preliminary data indicated that three lines excelled in 

number of fruit/ha and weight of fruit/ha compared to the two controls 

(Table 3). Line 37-1 had the largest fruit weight. 

Table 1. Yield (total number of fruit/ha) of ‘Curital INIA’ and the 

control (‘Black Chilean’) evaluated in different seasons and locations. 

La Platina Curacaví La Serena Curacaví La Platina 

Cultivar 

2002–2003 2002– 

2003 

2003– 

2004 

2003– 

2004 

2003– 

2004 

Curital INIA 

102,000 a* 

143,340 a 

177,333 a 

221,333 a 

120,667 a 

Black Chilean 104,000 a 142,000 a 148,667 b 164,000 b 84,667 b 

*Means followed by the same letter in column are not significantly different based 

on LSD test (P

Table 2. Fruit characteristics of Curital INIA and the control. 

Stripe 

Cultivar Width Shape Girdle Color color Brightness 

Curital 

INIA 

Black 

Chilean 

Medium 

Thick 

Cylindrical, 

elongated 

Conical, 

short 

No 

Yes 

Dark 

green 

Dark 

green 

Light 

green 

Light 

green 

Shiny 

Medium 

In the evaluations carried out in the 2003–2004 season, lines 

evaluated in the Curacaví location had higher fruit production, in 

number as well as in weight (Table 4). Also, the fruit produced was of 

larger size, in comparison with La Platina. At Curacaví, line 8-1 was 

better than the two controls. At La Platina, line 33-2 had the highest 

production. These results demonstrate that lines having better 

characteristics and productive potential than the ecotypes used by 

farmers have been generated. 

High fruit quality is defined by the presence of a thick pulp of dark 

orange. According to the evaluations, both characteristics are present 

in 28.8% of the total of the evaluated inbred lines. This result indicates 

that approximately one third of the improved germplasm combines 

two relevant aspects for obtaining a very good commercial fruit. 

We will continue testing the inbred lines to identify those of high 

productivity and fruit quality. In the future, we will determine 

combining ability and produce hybrid cultivars of sweetpotato squash 

for use by growers. 

Table 3. Number and weight of fruit per hectare, and average weight 

of fruit of sweetpotato squash inbred lines and controls in Curacavi, 

2002–2003 season. 

Inbred 

line 

Fruit 

number/ha -1 

Fruit weight 

(kg./ha -1 ) 

Average fruit 

weight (kg) 

79-1 2,250 abc 31,925 a 14.19 ab 

20-2 2,000 bcd 24,150 bcde 12.08 abcd 

20-1 2,250 abc 20,775 def 9.23 abcdef 

37-1 1,250 efg 20,000 defg 16.00 a 

33-1 1,500 def 17,850 efgh 11.90 abcd 

Farmer 1,188 fg 14,405 ghi 13.34 ab 

San Alfonso 1,000 fgh 10,125 ijk 10.55 abcde 

113-1 750 gh 3,325 k 4.43 ef 

*Means followed by the same letter in column are not significantly different based 

on LSD test (P

Table 4. Number and weight of fruit per hectare, and average weight 

of fruit of sweetpotato squash inbred lines and the controls in different 

locations, season 2003–2004. 

Curacavi La Platina 2003–2004 

Fruit 

weight 

Average 

fruit 

weight 

Number 

fruit/ha - 

1 

Fruit 

weight 

Average 

fruit 

weight 

(kg) 

Inbred Number 

line fruit/ha -1 

(kg./ha -1 ) (kg) 

(kg./ha -1 ) 

8-1 3,000 31,600 10.53 2,000 17,500 8.75 

11-2 1,750 17,250 9.86 2,000 20,600 10.30 

14-2 1,250 15,300 12.24 1,000 5,325 5.33 

20-2 2,250 20,925 9.30 2,250 14,750 6.56 

33-2 2,000 22,800 11.40 3,500 27,750 7.93 

34-1 2,000 16,300 8.15 2,250 17,075 7.59 

37-1 1,250 15,875 12.70 1,000 8,375 8.38 

Farmer 1,750 9,500 5.43 2,500 22,700 9.08 

San 

Alfonso 

1,000 

8,250 8.25 

1,000 9,100 9.10 


Bascur, B. G. 2005. Curital INIA: Nueva variedad de zapallito italiano tipo negro 

chileno. Hortic. Bras. 23(2):414(Abstr.). 

Bascur, B. G. 2005. Desarrollo de líneas puras en zapallo de guarda para la 

generación de variedades comerciales. Resúmenes 56º Congreso Agronómico de 

Chile:5(Abstr.). 

Escaff, G. M. 2001. Hortalizas, p. 717–757. In: SOQUIMICH (ed.). Agenda del 

salitre. Cousiño Asociados, Santiago, Chile. 

Giaconi, M. V. and M. G. Escaff. 1997. Cultivo de hortalizas. Editorial Universitaria, 

Santiago, Chile. 

ODEPA. 2006. Estadísticas agropecuarias. . 

Whitaker, W. T. and R. W. Robinson. 1986. Squash breeding, p. 209–242. In: J. M. 

Basset (ed.). Breeding vegetable crops. AVI Publishing Co., Westport, CT. 


GENETIC VARIATION FOR BENEFICIAL 

PHYTOCHEMICAL LEVELS IN MELONS 

(CUCUMIS MELO L.) 

Kevin M. Crosby 



Extension Center, Texas A&M University, Weslaco, TX 78596; 

Department of Horticultural Sciences, Texas A&M University, 

College Station, TX 77843 

Gene E. Lester 

U.S. Department of Agriculture, Agricultural Research Service, Kika 

de la Garza Subtropical Agricultural Research Center, 

Weslaco, TX 78596 

Daniel I. Leskovar 



Extension Center, Texas A&M University, Uvalde, TX 7; Department 

of Horticultural Sciences, Texas A&M University, 


ADDITIONAL INDEX WORDS. Vitamin C, beta-carotene, cultivars, western 

shipper, honeydew 

ABSTRACT. There is a paucity of published data on genetic variation for humanhealth-related 

phytochemicals in melon. Improvement of melon cultivars 

through traditional breeding depends on genetic variation for traits of interest. 

This research investigated the effects of cultivar and environment on the levels 

of vitamin C and beta-carotene in a diverse selection of melon cultivars and 

lines. Extensive variation was found for both compounds, with three- to fourfold 

differences between high and low entries. Several open-pollinated 

cultivars, including ‘TAM Uvalde’ and ‘TAM Perlita 45’, contained high levels 

of both these compounds, as did some older hybrid cultivars such as ‘Mission’. 

Many popular commercial hybrids such as ‘Cruiser’ and ‘Primo’ had lower 

levels of these phytochemicals, but larger fruit. Fruit grown at Uvalde were 

consistently higher in vitamin C than fruit grown at Weslaco. The highest levels 

of vitamin C recorded in this experiment (32mg·100g -1 ) were lower than the 

levels found in the USDA nutrition database, while the highest beta-carotene 

levels (62µg·g -1 ) were higher than USDA figures. There is good potential for 

genetic improvement of these phytochemical levels through conventional 

breeding and selection. 

This research was funded in part by USDA Grant: 2001-34402-10543, “Designing 

Foods for Health.” We acknowledge financial support from the South Texas Melon 

Committee. We also thank technicians Alfredo Rodríguez and Hyun J. Kang, Texas 

Agricultural Research and Extension Center-Weslaco, for their assistance. 


T 

he melon-breeding program at the Texas Agricultural 

Experiment Station in Weslaco focuses on improving fruit 

quality to meet demands of consumers and producers. The 

importance of melons in the human diet, coupled with their naturally 

high levels of vitamins, makes them ideal delivery systems for 

beneficial phytochemicals. Melons can provide vitamin C, betacarotene, 

potassium, calcium, sugars, and folate. Muskmelons are an 

excellent source of both vitamin C and beta-carotene (provitamin A). 

Among the top 10 most consumed fruits in the U.S., muskmelons are 

the only ones that provide the recommended daily allowance of both 

these vitamins (Lester, 1997). The stability of beta-carotene 

concentration over time is very high, which makes selection for this 

compound reliable (Lester and Bruton, 1986). In addition, there is a 

good correlation between levels of beta-carotene and desirable orange 

flesh color (Lester and Turley, 1990). This is both a selection and 

marketing advantage. Genotypic and environmental effects on betacarotene 

levels have been observed in field-grown fruit under 

commercial production practices. Lester and Eischen (1996) found 

that some melon varieties had higher levels of this compound than 

others. In addition, some varieties produced consistent beta-carotene 

levels across environments while others did not. Vitamin C is very 

susceptible to degradation over time or by exposure to high 

temperatures. Therefore, fresh melon fruit consumption will deliver 

the highest concentrations of this important compound. The species 

Cucumis melo L. is divided into six horticultural groups based on 

diverse fruit and vine characteristics (Robinson and Decker-Walters, 

1997). The great genetic diversity within this species is valuable for 

screening to identify genotypes high in these beneficial compounds. 


A diverse selection of melon lines and cultivars was planted in 

replicated field plots at Weslaco and Uvalde Texas during the spring 

of 2001. Seeds were sown on beds covered with black plastic mulch. 

Subsurface drip tape was utilized to irrigate and supply fertilizers 

throughout the growing season. Several applications of pesticides 

were applied to prevent mildew, gummy stem blight, and insect 

infestations. All fruit were harvested at the full-slip stage and 

processed within 24 hours. An effort was made to select fruit of 

similar size across entries when possible. Roughly 100-g flesh 


Table 1. Fresh fruit Vitamin C and beta-carotene contents of 15 melon 

cultivars and lines grown in field plots at Weslaco, Texas. 

Vitamin C 

Cultivar/line (mg·100g -1 Beta-carotene 

) (µg·g -1 ) Fruit type 

TAM Perlita 45 21.7 a z 62.2 a z 

Western shipper 

- size 15 y 


TAM Uvalde 15.2 b 57.5 ab 

- size 18 


Explorer 12.4 bc 45.0 cde - size 15 


Cruiser 12.1 bc 36.3 e 

- size 12 


Mission 11.9 bc 40.3 de 

- size 15 

Honeydew – 

Green Ice 11.8 bc 4.70 f 

size 15 

TAM Mayan 11.2 bcd 11.5 f Casaba - size 12 


Gold Mark 10.8 bcd 41.4 cde - size 12 


TXC 2015 10.0 bcd 49.8 bc 

- size 12 


HMX 9583 9.4 cd 46.0 cd 

- size 12 


Valley Gold 9.3 cd 48.5 bcd - size 12 


Mainpak 9.2 cd 43.5 cde - size 12 


Pronto 8.0 cd 43.0 cde - size 12 

Honeydew – 

TAM Dew Impr. 7.2 cd 4.70 f 

size 12 


Primo 7.0 cd 56.7 ab 

- size 12 

z 

Mean separations by LSD, P ≤ 0.05. Values followed by the same letter are not 

significantly different. 

y 

Sizes based on number of fruit that fit into a standard melon packing box. 

samples were taken from three replications per entry. These samples 

were stored in a –80 ºC freezer prior to chemical analysis. 

Vitamin C (total ascorbic acid) was extracted from 10g of frozen 

tissue by homogenizing in ice-cold meta-phosphoric acid (5% w/v) for 

5s in a polytron homogenizer (Brinkman Instruments, Westbury, NY). 

The homogenate was then centrifuged (7000gn) for 15min at 4 ºC. 

Detection of free and dehydro- ascorbic acid was at 525nm, and 


concentrations were calculated based on a standard curve (Hodges et 

al., 2001). Beta-carotene was extracted from lyophilized tissue using 

heptane under low-light conditions as described by Lester et al. (2005). 

HPLC was used to separate beta-carotene in a C18 column, with 

detection at 454nm. Total soluble solids were measured with a 

handheld refractometer (ATAGO USA, Inc., Bellevue, WA) at room 

temperature. Total ascorbic acid and beta-carotene concentration 

values were subjected to ANOVA and mean separations by LSD, 

utilizing STATGRAPHICS plus 4.1 software (Statpoint, Inc., 

Herndon, VA). 

Several families were developed with ‘TAM Uvalde’, ‘Mission’, 

‘Cruiser’, and ‘TAM Dew Improved’ as parents. The F2 progeny were 

screened to determine the potential for selection of larger fruit with 

genetically high levels of vitamin C and beta-carotene. Preliminary 

results demonstrated potential for transgressive segregation, with some 

progeny having higher concentrations of the phytochemicals and larger 

fruit than the parents (Crosby, unpublished data). Molecular markers 

linked to QTL affecting vitamin C have been developed in another F2 

family (Sinclair et al., 2006). Ideally, these RAPD markers, and others 

under development for beta-carotene QTL, will be useful to expedite 

selection of new melon cultivars with genetically enhanced levels of 

these two important phytochemicals. 


There is very little published information on genetic variation for 

fruit phytochemical traits in melon (Cucumis melo L). USDA 

statistics suggest that cantaloupe and honeydew melons are potentially 

good sources of vitamin C and beta-carotene, but do not address 

potential variation in these compounds across cultivars or horticultural 

groups (Adams and Richardson, 1981). Extensive genetic variation 

exists for many fruit-quality traits within the species Cucumis melo L. 

In addition, new cultivars and horticultural types are becoming 

available to U.S. consumers. It does not seem realistic to assume that 

all cultivars and types provide the same levels of beneficial 

phytochemicals. Both genetic and environmental components of 

variation for beta-carotene and vitamin C in melons were reported by 

Lester and Eischen (1996) and Lester and Crosby (2002). However, 

these studies included only a few cultivars of western-shipper 

cantaloupe or honeydew types. The present investigation included a 

larger number of commercial cultivars, germplasm accessions, and 

diverse horticultural types. 


Table 2. Fresh fruit vitamin C and total soluble solids of 39 melon 

cultivars and lines grown in field plots at Uvalde, Texas. 

Vitamin C 

Cultivar/line (mg·100g -1 Total soluble 

) solids (ºbrix) Fruit type 

TAM Uvalde 32.0 a z Western 

12.2 shipper 

Western 

Magnum 45 29.9 ab 8.5 shipper 

Western 

Mission 25.5 bc 12.3 shipper 

Western 

A-chapparal 22.5 cd 9.2 shipper 

Western 

TXC 2015 22.3 cd 10.4 shipper 

Western 

Durango 21.5 cde 9.0 shipper 

Western 

Tesoro 21.2 cdef 6.0 shipper 

Western 

Caravelle 21.0 cdef 11.0 shipper 

Western 

TXC 1053 20.6 cdefg 11.0 shipper 

Guadeloupe 20.3 cdefgh 10.5 Charentais 

TXC 807 19.7 defghi 10.2 Charentais 

Western 

TXC 1075 19.0 defghij 8.0 shipper 

Charlynne 18.8 defghijk 10.1 Ananas 

Western 

Sol Real 18.7 defghijk 10.0 shipper 

Ames 20488 18.2 defghijk 6.0 Momordica 

Western 

TXC 2040 17.5 defghijkl 8.5 shipper 

Western 

Primo 17.5 defghijkl 10.0 shipper 

Western 

TP 45 16.5 efghijklm 10.0 shipper 

Western 

TXC 1409 16.0 efghijklm 12.5 shipper 

Eastern 

Athena 15.7 fghijklm 8.0 shipper 

Western 

Impac 15.6 fghijklm 8.0 shipper 

TXC 1129 15.2 ghijklmn 13.0 Honeydew 

Western 

Cruiser 15.2 ghijklmn 8.3 shipper 

Trooper 14.8 hijklmno 7.7 Western 


(Table 2, 

continued) 

Vitamin C 

(mg·100g -1 ) 

Total soluble 

solids (ºbrix) 

Cultivar/line 

Fruit type 

shipper 

Western 

TXC 1143 14.6 ijklmno 10.5 shipper 

PI 124104 14.4 ijklmno 6.1 Honeydew 

Western 

RML 6483 14.0 ijklmnop 10.0 shipper 

Deltex 

Crème de 

13.4 jklmnop 9.0 Ananas 

Menthe 13.2 klmnop 13.3 Honeydew 

PI 127546 13.1 klmnopq 5.0 Momordica 

PI 165449 12.5 lmnopq 7.5 Casaba 

Western 

Copa de Oro 12.1 lmnopq 7.5 shipper 

Morning Ice 11.3 mnopq 13.0 Honeydew 

TAM Dew Impr. 11.3 mnopq 14.4 Honeydew 

TAM Mayan 10.9 mnopq 12.8 Casaba 

Mega Brew 9.6 nopq 14.0 Honeydew 

Santa Fe 9.2 opq 11.7 Honeydew 

Doublon 8.6 pq 11.0 Charentais 

Western 

TXC 1405 7.5 q 7.0 shipper 

z 

Mean separations by LSD, P≤0.05. Means followed by the same letters are not 

significantly different. 

Vitamin C concentrations at Weslaco demonstrated a threefold 

difference between highest and lowest entries (Table 1). This 

represents substantial genetic variation, considering that environmental 

parameters and growing conditions were presumably quite similar 

within the small field plots. The difference in vitamin C concentrations 

between the highest and lowest entries at Uvalde was even greater at 

32 vs. 7.5mg·100g -1 (Table 2). The mean vitamin C concentrations 

were higher at Uvalde than at Weslaco for the best cultivars, such as 

‘TAM Uvalde’ and ‘Mission’, and for those on the low side, such as 

‘TAM Dew Improved.’ In general, the trend for higher levels of 

vitamin C at Uvalde than Weslaco was evident. However, in neither 

location did the best entries produce as much vitamin C as was 

reported in western-shipper cantaloupe melons by the National 

Agricultural Library nutritional database Website (nal.usda.gov). This 


suggests that despite extensive genetic variation among cantaloupe 

types for vitamin C content, they may be quite susceptible to genotype 

x environment interaction for synthesis of this key phytochemical. 

This was found to be true with honeydews grown at different Texas 

locations (Lester and Crosby, 2002).Beta-carotene concentrations were 

assessed only from the Weslaco fruit. In contrast to the vitamin C 

data, the beta-carotene concentrations for the orange-fleshed entries 

were generally greater than the values reported by the USDA 

(nal.usda.gov.). Statistically significant differences were evident 

among orange-fleshed entries, with 1.7-fold greater levels in ‘TAM 

Perlita 45’ than in ‘Cruiser’. As expected, white- and green-fleshed 

honeydew cultivars had much lower levels of beta-carotene than the 

western-shipper types. The intermediate level in the casaba cultivar 

‘TAM Mayan’ is likely due to its pedigree, which includes an orangefleshed 

ancestor. Some light-orange coloration is evident in the 

placental region of this cultivar. At 62.2µg·g -1 , ‘TAM Uvalde’ 

contained roughly 1030 retinol equivalents or 100% of the RDA for 

provitamin A. This is better than the value of 676 retinol equivalents 

(3382 IU) for cantaloupe suggested by the USDA Website. This 

cultivar also had high total soluble solids and vitamin C, suggesting 

that selection for multiple fruit-quality and antioxidant traits 

simultaneously should be feasible. 


Adams, C. F. and M. Richardson. 1981. Nutritive value of foods. USDA Home and 

Garden Bul. 72. Government Printing Office, Washington D.C. 

Hodges, D. W., C. F. Forney, and W. V. Wismer. 2001. Antioxidant responses in 

harvested leaves of two cultivars of spinach differing in senescence rates. J. 

Amer. Soc. Hort. Sci. 126:611–617. 

Lester, G. E. 1997. Melon (Cucumis melo L.) fruit nutritional quality and health 

functionality. HortTech. 7:222–227. 

Lester, G. E. and B. D. Bruton. 1986. Relationship of netted muskmelon fruit water 

loss to postharvest storage life. J. Amer. Soc. Hort. Sci. 111:727–731. 

Lester, G. E. and F. Eischen. 1996. Beta-carotene content of postharvest orangefleshed 

muskmelon fruit: effect of cultivar, growing location and fruit size. Plant 

Foods for Hum. Nutr. 49:191–197. 

Lester, G. E., J. L. Jifon, and G. Rogers. 2005. Supplemental foliar potassium 

applications during muskmelon fruit development can improve fruit quality, 

ascorbic acid and beta-carotene contents. J. Amer. Soc. Hort. Sci. 130(4):649– 

653. 

Lester, G. E. and K. M. Crosby. 2002. Ascorbic acid, folic acid, and potassium 

content in postharvest green-flesh honeydew muskmelon fruit: influence of 

cultivar, fruit size, soil type and year. J. Amer. Soc. Hort. Sci. 127:843–847. 

Lester, G. E. and R. M. Turley. 1990. Chemical, physical and sensory comparisons 

of netted muskmelon fruit cultivars and breeding lines at harvest. J. Rio Grande 

Valley Hort. Soc. 43:71–77. 


Robinson. R. W. and D. S. Decker-Walters. 1997. Cucurbits. CAB International, 

New York. 

Sinclair, J. W., S. O. Park, G. E. Lester, K. S. Yoo, and K. M. Crosby. 2006. 

Identification and confirmation of RAPD markers and andromonoecious 

associated with quantitative 

trait loci for sugars in melon. J. Amer. Soc. Hort. Sci. 131(3):360–371 


PHYLOGEOGRAPHY AND EVOLUTION OF 

WILD AND CULTIVATED WATERMELON 

Fenny Dane and Jiarong Liu 

Department of Horticulture, Auburn University, Auburn, AL 36849 

ADDITIONAL INDEX WORDS. Citrullus lanatus var. lanatus, C. lanatus var. 

citroides, domestication, nucleotide polymorphism, cpDNA, nuclear sequence 

polymorphism 

ABSTRACT. Cultivated (Citrullus lanatus var. lanatus) and wild (C. lanatus var. 

citroides) watermelon accessions from different geographical areas were used to 

study the origin and domestication history of the cultivated watermelon. 

Chloroplast and nuclear DNA sequence analyses were conducted to infer 

evolutionary relationships and biogeographic patterns. Variability within C. 

lanatus was observed at regions of high AT content only. Distinct chlorotype 

lineages were identified separating the cultivated watermelon from several 

citroides lineages, indicating ancient splits between the cultivated and wild 

watermelon. While polymorphism at cpDNA regions within var. lanatus was 

lacking, sequence variation at glyceraldehyde 3-phosphate dehydrogenase and 

glutamine synthetase revealed variability, which was used to study colonization 

events. The egusi-type watermelon from Nigeria can be distinguished by a 

unique amino acid deletion at the transit peptide region of G3pdh. Variability 

among var. citroides accessions suggests a center of diversity in southern Africa. 

W 

ith the advent of agriculture began the process of plant 

domestication, which led from wild populations to plants 

adapted to cultivation and human use (Gepts, 2003). 

Human selection affected a wide array of morphological and 

physiological traits that distinguish cultivated crops from their wild 

ancestors. Research has revealed that a relatively small number of 

qualitative and quantitative trait loci with major effects control 

domestication-related traits in crops (Gepts, 2003). Understanding the 

process of domestication of species and colonization is fundamental to 

the study of evolutionary diversification. Studying the geographical 

distribution of plant lineages helps synthesize history and genetic 

exchanges and provides insights into the different factors that shape 

the genetic diversityof a species and the origins of crops (Avise, 2000; 

Gepts, 2003). 

Plant molecular evolution has been dominated by studies of the 

chloroplast (cp) genome because cpDNA is an abundant compenent of 

the total cellular DNA, and maternally inherited in most angiosperms. 

Chloroplast DNA is evolutionarily conservative in terms of genome 

The authors thank Rasima Bakhtiyarova for technical assitance. 


size, structure, gene content, and linear order of genes among plant 

lineages. Consequently, any change in structure or arrangement of the 

cp genome can have significant phylogenetic implications (Soltis et 

al., 1998). Both coding and noncoding sequences are used either 

through direct sequencing or PCR-restriction fragment length 

polymorphism (RFLP) analysis (Petit et al., 2005). The plant 

mitochondrial (mt) genome has with few exceptions not yielded useful 

amounts of population-level variation. Because sequences of cp- and 

mtDNA evolve more slowly than those of the nuclear genome in 

plants, nuclear gene markers are needed especially for phylogenetic 

reconstruction at low taxonomic levels. Noncoding regions of lowcopy 

nuclear genes have not been extensively explored in plants, 

although this genome can potentially provide multiple unlinked allele 

genealogies at the intraspecific level. Recent studies have 

demonstrated that rapidly evolving introns of low-copy nuclear genes 

can provide information to resolve intraspecific relationships (Olsen, 

2002; Zhang and Hewitt, 2003). 

Wild and cultivated watermelon (Citrullus lanatus [Thunb.] 

Matsum & Nakai) are native to Africa and can be divided into the 

botanical varieties lanatus and citroides (Jeffrey, 1990; Maynard, 

2001). The variety lanatus includes the cultivated watermelon, known 

for its large sweet red or yellow fruit, and the egusi-type, cultivated in 

West Africa for its oil- and protein-rich edible seeds. The variety 

citroides (Bailey) Mansf. includes the citron or preserving melon, 

which produces small spherical fruit with hard, inedible bitter flesh, 

and green or tan seeds. 

The primary gene center for watermelon is not known. One theory 

proposes that watermelon is derived from the perennial C. colocynthis, 

endemic to Africa; another that watermelon was domesticated from a 

putatively wild form of C. lanatus var. citroides (Maynard, 2001). 

The presence of 5000-year-old seeds of C. lanatus in Libya implies 

that domestication might have occurred in Northern Africa. The oldest 

published records of Citrullus remains come from the tomb of 

Tutankhamun in Egypt (ca. 1330 BC), and it was known in Sudan as 

early as 1500 BC (Hepper, 1990). Records of cultivated watermelon 

are known around the Mediterranean from the early first millennium 

BC. By the Roman period, records are widespread, ranging from 

Libya to Europe, but only a few are known from southern Africa. 

Watermelons have been cultivated in the Nile Valley since before 

2000 BC, and many landraces varying in fruit size, shape, flesh color, 

rind, and seed color were developed. From Africa, watermelons were 

introduced to India about 800 AD and to China about 1100 AD. 

Cultivation spread to Southeast Asia in the 15 th century, while the 


Moors introduced watermelon to Europe during their conquest of 

Spain. Watermelon was transported and introduced to the Americas in 

post-Columbian times by the early European colonists, and became 

readily accepted and disseminated by Native Americans, especially in 

the Mississippi Valley and the southwestern U.S. (Zohary and Hopf, 

2000). But despite the economic importance of watermelon, 

domestication events and phylogeographic relationships have received 

only limited scientific attention. 

Initial studies using PCR-RFLP analysis in Citrullus species were 

conducted using different chloroplast regions (Dane, 2002). 

Sequencing analysis of informative regions revealed four haplotypes 

within C. lanatus, one associated with the cultivated watermelon (var. 

lanatus), and three with var. citroides (Dane et al., 2004). The 

chlorotypes within C. lanatus suggest ancient splits between the 

cultivated and wild watermelon types (Liu, 2005). This study was 

conducted to provide a comprehensive review of DNA-sequence 

diversity and divergence within this wild/domesticated complex and to 

make inferences about the domestication history of the species. 

Variable nuclear regions were compared to information gained from 

cpDNA sequence analysis. 


Seeds from Citrullus accessions and cultivars were obtained 

through the Plant Introduction (PI) Station at Griffin, Georgia, the 

National Plant Genetic Resources Centre (NPGC) at Windhoek, 

Namibia, or from the Volcani Center in Israel (Table 1). Accessions 

were chosen based on an extensive survey of the Citrullus collection 

using PCR-RFLP and cpDNA sequencing analysis (Dane et al, 2004; 

Liu, 2005). 

DNA was extracted from seedlings or cotyledon tissues using the 

Qiagen plant DNA easy extraction kit (Qiagen, Valencia, CA). 

Chloroplast genome sections were amplified using universal primers 

(Liu, 2005). Nuclear DNA sequences were amplified with primers 

designed from published EST information. Primers were designed in 

exons to amplify fragments of around 600–800bp around intronic 

sequences (Table 2). PCR cycling conditions for the GS and Dip 

sequences were 95˚C for 2 min, followed by 10 cycles of 1 min at 

92˚C, 20 s at 73˚C, 20 s at 72˚C, followed by 25 cycles of 20 s at 92˚C, 

20 s at 55˚C, 20 s at 72˚C, and finally 5 min at 72˚C. PCR cycling 

conditions for the G3pdh sequences were 94˚C for 4 min, followed by 

35 cycles of 1 min at 94˚C, 1 min at 52.5˚C, 2 min at 72˚C, and finally 

10 min at 72˚C. 


Amplified fragments were purified using the Qiaquick PCR 

purification kit (Qiagen, Valencia, CA) and sequence analysis was 

performed using an ABI 3100 sequencer. Nucleotide sequences were 

submitted to GenBank and accession numbers are available from the 

senior author. Sequences were aligned using Vector NTI ® software 

(InforMax, Frederick, MD), followed by manual adjustments. 

Single base indels were crosschecked to the original chromatograms 

for accuracy. Indels were scored as single binary (0 vs. 1 characters 

appended to the main matrix. All character states were specified as 

unordered and equally weighted. To test for congruence of the 

cpDNA and different nuclear regions, partition homogeneity tests with 

heuristic searches were conducted in PAUP* with 100 replicates and 

tree-bisection-reconnection (TBR) branch swapping (Swofford, 2002). 

Table 1. Investigated Citrullus lanatus plant introductions (PI) and 

cultivars with their geographic origin. 

Taxon PI number Origin 

C. lanatus var. 

lanatus 

PI 179881 India 

PI 494529 Nigeria 

PI 295845 S. África 

AU Producer USA 

C. lanatus var. 

citroides 

PI 189225 Zaire 

PI 270563 S. Africa 

PI 271769 Transvaal 

PI 296343 Cape Province 

PI 485583 Botswana 

PI 288316 India 

PI 532667 Swaziland 

Nam1569 Namibia 

Nam1884 Namibia 

TCN1126 USA 


Since cpDNA sequence analysis did not uncover much variability 

within C. lanatus (Dane et al., 2004; Liu, 2005, Figure 1), wild and 

cultivated watermelon accessions were surveyed for sequence 

diversity at three nuclear regions. Characteristics of the studied 

nuclear regions are listed in Table 3. Glyceraldehyde 3-phosphate 


dehydrogenase (G3pdh) catalyzes the reduction of 1,3 

diphosphoglycerate to glyceraldehyde-3-phosphate in the cytosol or 

chloroplast. The N-terminal transit peptide is interrupted by three 

introns, two of which are conserved across gymnosperms and 

angiosperms. The sequenced G3pdh region in C. lanatus spans one 

intron (2), flanked by AG and GT motifs typical of nuclear introns, 

and a short section of the transit peptide region. Sequence divergence 

is high mainly at the intronic region, although one informative 3-bp 

deletion (glu) was detected at the transit peptide exon region in some 

var. lanatus accessions. 

Glutamine synthetase (GS) functions as the major assimilatory 

enzyme for ammonia produced from N fixation, and nitrate or 

ammonia nutrition. It also reassimilates ammonia released as a result 

of photorespiration and the breakdown of proteins and nitrogen 

transport compounds. GS is distributed in different subcellular 

locations (chloroplast and cytoplasm) and in different tissues and 

organs. The enzyme is the product of multiple genes with complex 

promoters that ensure the expression of the genes in an organ- and 

tissue-specific manner and in response to a number of environmental 

variables affecting the nutritional status of the cell (Miflin and Habash, 

2002). The GS primer pairs cover two intron regions of 574 and 

113bp, each flanked by GT-AG sequences. Variability between var. 

lanatus accessions was detected at one intron region, but very low 

between citroides accessions. 

Drought-induced polypeptide-1 or Dip-1 belongs to the 

ArgE/DapE/Acy1/Cpg2/YcsS protein family, and is strongly induced 

in wild watermelon leaves by drought/high-light-stress conditions 

(Kawasaki et al., 2000), but the catalytic property of Dip-1 remains to 

Table 2. Primer pairs used to sequence Citrullus lanatus nuclear 

regions. 

GenBank 

accession 

number Forward primer Reverse primer 

Nuclear region 

Glyceraldehyde- 

3-phosphate 

dehydrogenase 

Glutamine 

synthetase 

AI563173 CAGGCTAATGG/ 

AAAGGGTTT 

AI563060 ATGTCTCTGC/ 

TCTC 

Dip-1 AB036420 TGGGTTCTG/ 

TTCCATCCATT 

TTGTATCCTC/ 

CGCTCCTTCC 

TACCTGTCTT/ 

CTCC 

TTGTGAATCT/ 

GCTGTGTCAA 


e determined. The enzyme is known to be encoded by a single gene 

and resides within the plastid (Slocum, 2005). The Dip primer pair 

covers several introns (130, 111, and 529-566bp) and sequence 

divergence is high at both intronic and exonic regions (Table 3). One 

substitution at an exon results in an amino acid substitution from 

serine (at position 274) in var. citroides accessions (and C. 

colocynthis) to glycine in var. lanatus. 

The C. lanatus accessions are characterized by 26 substitution 

polymorphisms and six indels at the nuclear regions. These 

polymorphisms define a total of at least eight different haplotypes, 

while only four haplotypes were detected using cpDNA sequence 

analysis (Liu, 2005). Intravarietal differences were detected at the 

transit peptide region of G3pdh and intron region of GS and can 

subsequently be used to assess colonization patterns. Most of the 

accessions showed one nuclear haplotype, but heterozygosity was 

detected in PI 532667 and 596656 at the variable sites of the GS 

region. Since the PI accessions originate as seed collections and are 

maintained and increased under limited isolation, hybridization and 

recombination might have occurred and led to heterozygosity at 

nuclear regions. Alternatively, the C. lanatus GS shows high sequence 

homology with cytolosic GS1 forms of several species, which are 

known to be members of a small multigene family and haplotypes 

might not be orthologous. Sequence variability at GS was very low 

and not very informative. 

The 3-bp deletion at the G3pdh transit peptide region was found to 

be characteristic of egusi-type watermelon. Sequence analysis of 

several egusi-type watermelon originating in Nigeria showed this (glu) 

deletion in PIs 186490, 189318, 560012, 560023, 5559997, 494527, 

and 494529. The deletion was also detected in cultivars ‘Au- 

Producer’ and ‘Lucky’, but not in watermelons from any other 

geographic area (Syria, Afghanistan, India, or southern Africa). It can 

thus be hypothesized that the watermelon cultivars were domesticated 

from Nigeria. More studies will be forthcoming to test this hypothesis. 

The citroides accessions can be characterized by unique 

substitutions and indels at the nuclear regions, although a low number 

of substitutions were common to all citroides accessions. One major 

37 bp deletion at the Dip-1 intron is the result of replication slippage 

since a 13 bp section of this indel is homologous to contiguous 

flanking regions. This deletion was not detected in var. citroides PI 

296343, 189225, 288316, and NAM 1569. 

Partition homogeneity tests using Paup showed incongruence of 

the cpDNA and nuclear sequence data (P=0.01). The genomic regions 

may have been shaped by different evolutionary processes. Some 


Table 3. Characterization of sections of nuclear regions within C. 

lanatus. 

Glyceral- 

Nuclear Glutamine dehyde-3P 

regions synthetase dehydrogenase Dip-1 

aligned 620 (575bp 672 (600bp 650-687(428length(bp) 

intron) intron) 465 bp intron) 

intron AT 

content 

exon AT 

65.0% 64.6% 73.8% 

content 52.4% 51.4% 57.5% 

var. lanatus PI 1 2 Ts, 1 Tv, 2 ID 2 Ts, 2 Tv, 2 ID 6 Ts, 3 Tv, 1 ID 

var. citroides PI 1 Tv 3 Ts, 1 Tv 4 Ts, 1 Tv, 1 ID 

Nucleotide 

diversity (%)* 0.696 1.121 2.796 

1 PI = parsimony informative sites; Ts = transition; Tv = transversion; ID = indel 

*Nucleotide diversity refers to Π x 100, calculated at noncoding regions. 

forces such as population bottlenecks have a genome-wide effect, 

others like recombination and selection may have only a regional 

influence. Dating of indels and substitutions is difficult for nuclear 

data, but the presence of the large deletion at the dip intron adds 

phylogenetic weight to this nuclear marker. Nucleotide diversity was 

clearly not random along the nuclear genome (Table 3). This 

unevenness is associated with differences in recombination rate, gene 

density in the genomic region, transmission pattern, and selection 

strength (Zhang and Hewitt, 2003). The lowest number of nuclear 

polymorphic sites was observed in PI 288316 from India, possibly due 

to isolation and founder effect, the highest number (9) in accessions 

from Namibia and South Africa, probably the center of origin of 

watermelon. Minimum spanning trees (Figure 2) using substitutions at 

two nuclear regions divide the citroides accessions into two main 

groups. The small group of accessions from S. Africa (PI 189225, 

296343, and 271769) might be considered as ancestral, since they lack 

the large informative indel at dip. Information gained from the 

cpDNA analysis shows the ancestral chlorotype in PI 596656 and 

532667. Traditional phylogenetic analysis methods are not the most 

appropriate for analyzing intraspecific polymorphic data (Zhang and 

Hewitt, 2003) since rare polymorphisms such as singletons are treated 

as noninformative from a parsimony perspective. Results from cpDNA 


Fig. 1. Minimum spanning haplotype tree representing the relationships 

between Citrullus lanatus accessions inferred from sequence information at the 

trnS-trnG, atpA-trnR, trnE-trnT regions of the chloroplast genome (Liu, 2005). 

Large open circles represent observed haplotypes. Lines between haplotypes 

indicate mutational steps. Small circles, squares, and diamond indicate type of 

nucleotide substitution. The size of deletions is given in bp. The order of 

mutations on the branches is arbitrary. 

and nuclear DNA analyses support the divergence of var. lanatus and 

var. citroides from a common ancestor and little molecular exchange 

between them (Figures 1 and 2). 

The amount of genetic variation in cultivated crop plants is 

shaped in part by the way genetic material is passed from one 

cultivated generation to the next. In cucurbits, seeds from a limited 

number of wild plants were used to form cultivated populations. The 

resulting founder effect, coupled with ongoing selection of seeds from 


Fig. 2. Minimum spanning haplotype tree representing the relationships 

between Citrullus lanatus accessions inferred from sequence information of the 

glyceraldehyde- 3P-dehydrogenase and Dip nuclear genes. Observed haplotypes 

are in large open circles. Lines between haplotypes indicate mutational steps, 

small circles and squares indicate type of nucleotide substitution. The size of 

deletions is given in bp. The order of mutations on the branches is arbitrary. 

the initial pool of cultivated genotypes, produced a genetic bottleneck 

in the cultivated populations. Intense artificial selection during 

domestication results in a progressive narrowing of the genetic base of 

the cultivated populations. In recent times, the genetic base of 

cultivated populations most likely has been reduced further as modern 

crop-improvement programs have selectively bred only a small subset 

of the original cultivated gene pool (Gepts, 2003). This is clearly the 

case in watermelon, which is known for its narrow genetic base (Levi 

et al., 2004, 2005), indicating that bottlenecks have reduced the 

amount of genetic variability in the cultivated crop. The G3pdh and 

Dip primers used are applicable for intraspecific studies since they are 

evolutionarily conserved across different taxonomic groups and their 

target sequences are single or low-copy. At the nuclear regions 

substitutions in var. lanatus accessions were higher than in var. 

citroides accessions, indicative of the effects of selection and 

domestication. One major evolutionarily recent deletion at a nuclear 


intron points to citroides accessions from southern Africa as ancestral, 

supporting migration patterns from southern Africa to the U.S. and 

India. 


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FINE GENETICAL AND PHYSICAL MAPPING 

OF THE GENES A AND G CONTROLLING SEX 

DETERMINATION IN MELON 

M. Fergany, C. Troadec, M. Rajab, A. Boualem, M. Dalmais, M. 

Caboche, and A. Bendahmane 

INRA, URGV, Evry, France 

D. Besombes, N. Giovinazzo, C. Dogimont, and M. Pitrat 

INRA, GAFL, Avignon-Montfavet, France 

ADDITIONAL INDEX WORDS. Cucumis melo, flower biology, monoecious, 

gynoecious, hermaphrodite, andromonoecious, genetic map 

ABSTRACT. Sex expression in melon (Cucumis melo L.) is controlled by 2 major 

genes: andromonoecious controls the presence (allele a) or the absence (allele a + 

or A) of stamens in the female flowers and gynoecious controls the absence 

(allele g) or the presence (allele g + or G) of male flowers on a plant. A program 

of positional cloning has been developed for these 2 genes. AFLPs linked to the 2 

loci were selected by the Bulk Segregant Analysis method. The identified AFLPs 

were transformed into PCR specific markers and mapped on more than 5000 

plants segregating for a or g loci. In this analysis, the markers M23 and M58 

were mapped at 0.23 cM and 3 cM from the a and g loci, respectively. A melon 

BAC library of more than 120,000 clones was built and screened to isolate BAC 

clones carrying the targeted loci. 

M 

ost of the higher plants (angiosperms) have hermaphrodite 

flowers but some are monoecious or dioecious, for instance 

in the Betulaceae, Salicineae, Fagaceae, Juglandaceae, and 

Cucurbitaceae families. Understanding the genetic control and the 

biological processes leading to sex expression is of great interest for 

plant breeding and for producing hybrid seeds. 

Most species in the genus Cucumis are monoecious, with the 

exception of C. heptadactylus, C. kalahariensis, C. hirsutus, and C. 

baladensis, which are dioecious (Kirkbride, 1993). In cucumber (C. 

sativus L.), almost all traditional cultivars are monoecious. In the 

second part of the twentieth century, breeders have developed 

gynoecious cultivars. The genetic control of sex expression is based on 

two main genes (Rosa, 1928; Galun, 1961; Rowe, 1969): the gene 

Female (symbol F) controls the high frequency of pistillated flowers 

on a plant and the gene monoecious / andromonoecious (symbol m) 

controls the presence of stamens in pistillate flowers. Interaction 

between F and m results in four phenotypes: hermaphrodite (Fm), 

gynoecious (FM), monoecious (fM), and andromonoecious (fm). 

Another major gene (androecious, symbol a) controls the presence of 


mainly staminated flowers (phenotype androecious) if the recessive 

allele f is present. The gene F has been identified as an ACC synthase 

(Mibus and Tatlioglu, 2004). 

Wild melons (C. melo) are monoecious but about 60–70 % of 

cultivated melon accessions are andromonoecious. Monoecism is 

found mainly in the flexuosus, acidulus, chate, and mormordica 

groups. As in cucumber, andromonoecism is controlled by one gene 

(Rosa, 1928), which was first named monoecious (symbol M) and now 

andromonoecious (symbol a). Another phenotype, hermaphroditism, 

has been found in very few accessions. In 1937, Poole and Grimball 

identified in Charleston (SC) 5 hermaphroditic accessions from China, 

among 48 monoecious and 339 andromonecious ones. They studied 

the genetic control in crosses of monoecious x hermaphrodite and 

andromonoecious x hermaphrodite and they concluded to the action of 

a gene named gynoecious (symbol g), independent from a (Poole and 

Grimball, 1939). Interaction between the two loci is as follows: AG 

monoecious, Ag gynoecious, aG andromonoecious, and ag 

hermaphrodite. Gynoecious lines including WI 998 have been 

developed from this material (Rowe, 1969; Peterson et al., 1983). At 

the same time, Kubicki in Poland was also studying the genetic control 

of sex expression. He used an “uncultured hermaphroditic form” 

received from the All-Union Institute of Plant Industry in the Soviet 

Union. He also concluded at two loci with major effects and modifiers 

(Kubicki, 1969). Genetic controls in melon and in cucumber are very 

similar: alleles m and F in cucumber corresponding respectively to the 

alleles a and g in melon. It should however be noted that F in 

cucumber is dominant, whereas g in melon is recessive. 

We have developed a program of positional cloning for both genes 

a and g in melon. We will present here the first results of the fine 

genetical and physical mapping of these genes. 

Material and Methods 

PLANT MATERIALS. Five melon accessions were used: PI 124112 

is a monoecious line (genotype AAGG) from India; ‘Védrantais’ is an 

andromonoecious cultivar (genotype aaGG) in the Charentais type 

released by Vilmorin seed company; PI 161375 is an 

andromonoecious cultivar from Korea; ‘Gynadou’ is a gynoecious line 

(4 th backcross followed by selfing) in the Charentais type derived at 

INRA in Avignon-Montfavet from a white mealy flesh line received 

from B. Kubicki in 1967; ‘WI 998’ is a gynoecious line (genotype 

AAgg) released by the Michigan State University (Peterson et al., 


1983) and derived from an hermaphrodite accession from China 

studied by Poole and Grimball. 

MAPPING POPULATIONS. Population of recombinant inbred lines 

(RILs) ‘Védrantais’ x PI 124112 was already available (Perchepied et 

al., 2005). Backcross populations BCa = (‘Védrantais’ x PI 124112) x 

‘Védrantais’ and BCg = Gynadou x (Gynadou x PI 124112) have been 

developed. 

MOLECULAR MARKERS. AFLP analysis was carried out (Vos et 

al., 1995) using PstI and MseI primer combinations. Genomic DNA 

was extracted using standard procedures. Analysis was performed on 

bulked DNA samples of monoecious and andromonoecious plants for 

the a locus and monoecious and gynoecious plants for the g locus. To 

identify plants carrying recombination events linked to a and g loci, 

DNA samples were extracted from more than 5000 segregating plants 

and analyzed using the a- and g-flanking markers, respectively. Map 

distances are given in centimorgans, and represent the percentage of 

recombinant plants in the total number of plants analyzed. 

GENOMIC LIBRARY. A melon BAC library was prepared from 

nuclei extracted from line PI 124112, based on the method described 

in Peterson et al. (2000). The library consists of 120,000 BAC clones. 

To assess the insert size of the BAC clones, plasmid DNA was isolated 

from more than 100 randomly chosen clones, digested with NotI and 

analyzed by pulsed-field gel electrophoresis, as described previously 

(Kanyuka et al., 1999). The screening of the BAC library was 

performed as described by Kanyuka et al. (1999). 


ALLELISM TESTS. All the plants in F1 and F2 progenies between 

‘Gynadou’ and ‘WI 998’ were gynoecious. Previously, a 3 gynoecious 

vs. 1 hermaphrodite segregation has been observed in crosses between 

a “Polish hermaphrodite” and gynoecious material derived from a 

Chinese line studied by Poole and Grimball (Rowe, 1969). Thus it is 

highly probable that the same locus g is involved in plants studied by 

Poole and Grimball (1939) and by Kubicki (1969). 

MAPPING OF THE LOCUS a. The locus a has previously been 

mapped on linkage group II (Périn et al., 2002) and is linked to the 

locus Zym for Zucchini Yellow Mosaic-virus resistance. One thousand 

twenty-four combinations of primers were used on 2 bulks of 

monoecious and 2 bulks of andromonoecious plants derived from the 

BCa population including 15 plants per bulk. The promising markers 

were mapped on the RIL population ‘Védrantais’ x PI 124112 and on 

96 plants of the BCa population. M19 and M47 are 2 markers flanking 


0 

3 

3 

M17 

M47 

A M23 

M19 M64 

0 

9 

4 

5 

M17 

M47 

A M23 

M19 M64 

0 

3 

2 

Fig. 1. Genetic map around the locus a on LG II on 1 = 120 RILs Védrantais x 

PI 124112; 2 = 96 plants of the backcross population BCa = (Védrantais x 

PI 124112) x Védrantais; 3 = 5000 plants of BCa. Marker M23 cosegregates 

with the locus a on the first two populations and is at 0.23 cM from a on 

population 3; 4 = Construction of a BAC contig overlapping the a locus. 

Fig. 2. Genetic and physical map of the g locus: 1 = Fine genetic map around the 

g locus on 5000 plants of the BCg = Gynadou x (Gynadou x PI 124112) progeny; 

2 = Construction of a BAC contig overlapping the g locus. 

M47 

A 

M23 

M64 

1 2 3 4 


the locus a and M23 cosegregates with a (Figure 1). These 3 AFLP 

markers have been transformed in PCR specific markers and used to 

screen 5000 plants of the BCa population. Marker M23 could be 

separated from the locus a with 11 recombinant plants corresponding 

to 0.23 cM. The closest marker from a previously described was a 

SCAR derived from an AFLP at 5.5cM (Noguera et al., 2005). 

MAPPING OF THE LOCUS g. We identified AFLP markers tightly 

linked to the g locus by screening 1024 AFLP primer combinations on 

DNAs derived from bulked monoecious or gynoecious segregant 

plants from the BCg population. The obtained AFLPs were mapped 

relative to the g locus in a population of 5000 segregants to develop a 

high-resolution genetic map. In this analysis the g locus was delimited 

to a genetic interval of 5cM (Figure 2). 

PHYSICAL MAPPING OF THE a AND g LOCI. To isolate DNA clones 

carrying the a or g loci, a BAC library was constructed from nuclear 

DNA derived from a line homozygous for the a and g alleles. The 

library consists of 120,000 clones and represents the haploid melon 

genome at least 23 times over. Using a systematic PCR-based 

procedure, the library was screened with markers linked to a or g loci 

(Figs. 1 and 2). Positive BAC clones were isolated and aligned in 2 

single overlapping contigs. 

Conclusions 

As many recombinants have been identified in the vicinity of a and 

g the next step will include the anchoring of the BAC contigs to the 

genetic map. The outcome of this experiment will be the genetic 

delimitation of the a and g loci to single BAC clones. The identified 

BAC clones will be sequenced and annotated. The predicted genes will 

then be mapped relative to the identified recombination events. 

Complementation studies will be carried out in melon and 

Arabidopsis. 


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Genet. 98:679f–689. 

Kirkbride, J. H. (ed.). 1993. Biosystematic monograph of the genus Cucumis 

(cucurbitaceae). Parkway, Boone, NC. 


Kubicki, B. 1969. Sex determination in muskmelon (Cucumis melo L.). Genet 

Polonica. 10:145f–165. 

Mibus, H. and T. Tatlioglu. 2004. Molecular characterization and isolation of the F/f 

gene for femaleness in cucumber (Cucumis sativus L.). Theor. Appl. Genet. 

109:1669f–1676. 

Noguera, F. J., J. Capel, J.I . Alvarez, and R. Lozano. 2005. Development and 

mapping of a codominant SCAR marker linked to the andromonoecious gene of 

melon. Theor. Appl. Genet. 110:714f–720. 

Perchepied, L., M. Bardin, C. Dogimont, and M. Pitrat. 2005. Relationship between 

loci conferring downy mildew and powdery mildew resistance in melon 

assessed by QTL mapping. Phytopathology. 95:556f–565. 

Périn, C., L. S. Hagen, V. de Conto, N. Katzir, Y. Danin-Poleg, V. Portnoy, S. 

Baudracco-Arnas, J. Chadoeuf, C. Dogimont, and M. Pitrat. 2002. A reference 

map of Cucumis melo based on two recombinant inbred line populations. Theor. 

Appl. Genet. 104:1017f–1034. 

Peterson, C. E., K. W. Owens, and P. R. Rowe. 1983. Wisconsin 998 muskmelon 

germplasm. HortSci. 18:116. 

Peterson, D. G., J. P. Tomkins, D. A. Frisch, R. A. Wing, and A. H. Paterson. 2000. 

Construction of plant bacterial artificial chromosome (BAC) libraries: an 

illustrated guide. J. Agricult. Genom. 5:1f–3. 

Poole, C. F. and P. C. Grimball. 1939. Inheritance of new sex forms in Cucumis melo 

L. J. Hered. 30:21f–25. 

Rosa, J. T. 1928. The inheritance of flower types in Cucumis and Citrullus. 

Hilgardia. 3:233f–250. 

Rowe, P. R. 1969. The genetics of sex expression and fruit shape, staminate flower 

induction, and F1 hybrid feasibility of a gynoecious muskmelon. PhD Diss., 

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Vos, P., R. Hogers, M. Bleeker, M. Reijans, T. Lee, M. Hornes, A. Frijters, J. Pot, J. 

Peleman, M. Kuiper, and M. Zabeau. 1995. AFLP: a new technique for DNA 

fingerprinting. Nucleic Acid Res. 23:4407f–4414. 


QUANTITATIVE TRAIT LOCUS ANALYSIS OF 

POWDERY MILDEW RESISTANCE AGAINST 

TWO STRAINS OF PODOSPHAERA XANTHII 

IN THE MELON LINE ‘PMAR NO. 5’ 

N. Fukino, T. Ohara, Y. Sakata, M. Kunihisa, and S. Matsumoto 

National Institute of Vegetable and Tea Science, Tsu, Mie 514-2392, 

Japan 

ADDITIONAL INDEX WORDS. Cucumis melo, genetic mapping, recombinant 

inbred lines 

ABSTRACT. Due to the appearance of new races of Podosphaera xanthii (syn. 

Sphaerotheca fuliginea), leading melon varieties in Japan that had previously 

been considered resistant are now frequently infected by powdery mildew. The 

melon breeding line ‘PMAR No. 5’ (thought to be ‘AR5’) is resistant to these new 

races. To elucidate the relationship between resistance genes and strains, we 

constructed a genetic-linkage map of melon with a recombinant inbred line 

(RIL) population derived from a cross between ‘PMAR No. 5’ and the 

susceptible cultivar ‘Harukei 3’. Inoculations on leaf disks of this population 

were accomplished using two strains of P. xanthii (pxA and pxB). Quantitative 

trait locus (QTL) analysis allowed the detection of two QTLs on Linkage Groups 

II and XII at a significance threshold of 3.0. One QTL is responsible for 

resistance to both pxA and pxB at the cotyledon and the first-leaf stage, and the 

other QTL is responsible for pxA at both stages and pxB at the first-leaf stage. 

Therefore, it is more likely that the effects of the two QTLs differ depending on 

the strain and the stage. ‘PMAR No. 5’ will be useful as a source of resistance to 

powdery mildew in melon breeding in Japan. 

P 

owdery mildew caused by Podosphaera xanthii (Castagne) U. 

Braun & S. Takam and Erysiphe cichoracearum DC limits the 

production of melons (Cucumis melo L.) worldwide (Sitterly, 

1978), with E. cichoracearum being less important than P. xanthii as the 

causal agent of powdery mildew. Although many melon cultivars that 

are resistant to powdery mildew have been produced in Japan, they are 

now being severely infected with powdery mildew, and the appearance 

of new races of P. xanthii has been reported (Hosoya et al., 1999; 

Orihara et al., 2001). Accordingly, melon cultivars with resistance to the 

new races are needed. 

The melon breeding line ‘PMAR No. 5’ (Yoshida & Kohyama, 

1986) was introduced from the University of California in 1981 and is 

thought to be ‘AR5’. It shows strong resistance to powdery mildew and 

has been used for breeding. Fukino et al. (2004a) reported that two 


genes control the resistance of ‘PMAR No. 5’ to Race 1, but no reports 

have discussed DNA markers or provided a quantitative trait locus 

(QTL) analysis of powdery mildew resistance in ‘PMAR No. 5’. 

Because resistance to powdery mildew is greatly susceptible to 

environmental factors, marker-assisted selection of this trait is in great 

demand. Here we report the construction of a genetic-linkage map with 

a recombinant inbred line (RIL) population derived from a cross 

between ‘PMAR No. 5’ and ‘Harukei 3’ (type ‘Earl’s Favorite,’ which is 

susceptible to powdery mildew), and QTL analysis of powdery mildew 

resistance using two strains differing in their degree of virulence. 


PLANT MATERIALS. The population used in this study consisted of 

93 RILs derived from single seed descent of a cross between ‘PMAR 

No. 5’ and ‘Harukei 3’. RILs, their parents, and F1 plants were used for 

the test. Five seeds per line were sown in 34 × 48cm flats and were 

grown in a phytotron (28°C [day] and 20°C [night] with a 12-h 

photoperiod). 

POWDERY MILDEW RESISTANCE TEST. Two strains (tentatively 

denoted pxA and pxB) of P. xanthii were used for the test. The responses 

of these strains to the differential genotypes of melon are shown in 

Table 1. 

Leaf disks were cut from the cotyledons and first leaves and placed 

face up on medium with 8g/L agar in petri dishes. Three disks were used, 

and two independent trials were performed for each test. 

Table 1. Response of two strains of Podosphaera xanthii (pxA, pxB) to 

differential genotypes of melon. Genotypes were evaluated at the 

cotyledon (C) and first-leaf (FL) stages. 

pxA pxB 

Genotype C FL C FL 

Harukei 3 (‘Earl’s Favorite’) S S S S 

PMR45 M R R R 

WMR29 R R R R 

Edito47 M R R R 

PI414723 S S M R 

PMR5 R R R R 

MR1 M M R R 

R = resistant; M = moderately resistant; S = susceptible. 


Inoculation of powdery mildew was performed by spraying a 

conidial suspension (Morishita et al., 2003). The petri dishes were then 

covered and incubated at 25°C. After 10 to 14 days, a disease index (DI) 

indicating the degree of sporulation was scored on a scale of 0 (no 

sporulation) to 5 (entire disk covered with heavy sporulation) using a 

visual rating system. 

MOLECULAR MARKERS AND DATA ANALYSIS. Genomic DNA was 

extracted from leaf tissue as described by Fukino et al. (2002). Random 

amplification of polymorphic DNA (RAPD) and simple sequence 

repeat (SSR) analyses were performed as described by Nunome et al. 

(2001) and Fukino et al. (2004b), respectively. Cleavage amplified 

polymorphic sequence (CAPS) analysis followed the procedure of 

Kunihisa et al. (2003). Linkage and QTL analyses were performed 

using MAPMAKER/EXP 3.0 (Lander et al., 1987 and 

MAPMAKER/QTL 1.1 (Lincoln et al., 1993), respectively.) 

II XII 

pxA(C), pxA(FL), 

pxB(FL) 

pxA(C), pxA(FL), 

pxB(C), pxB(FL) 

Fig. 1. Map locations of QTLs involved in resistance against two strains of 

Podosphaera xanthii. 


In each test, ‘PMAR No. 5’ showed no symptoms, whereas ‘Harukei 

3’ was severely infected. F1 plants were moderately resistant to pxA and 

resistant to pxB. Average DI values of each RI line showed a continuous 

segregation from susceptible to resistant, suggesting the polygenic 

nature of this trait. 


The responses of 47 Japanese commercial F1 cultivars to both 

strains were tested (data not shown). Most cultivars were susceptible to 

pxA at both the cotyledon and first-leaf stages. The response to pxB was 

very different depending on the stage; at the cotyledon stage, 46 

cultivars were susceptible, whereas at the first-leaf stage, only 11 

cultivars were susceptible. This result indicates that the genetic system 

controlling resistance to the pxB strain differs depending on the growth 

stage. 

The constructed map spans 1,024cM consisting of 183 markers, 

which include 169 SSRs, 10 CAPs, and 13 RAPDs segregating into 18 

linkage groups. The localization of the QTLs is represented in Figure 1. 

Two QTLs were detected on Linkage Groups II and XII at a significance 

threshold of 3.0. Resistance to the pxA strain is conditioned by two 

QTLs at both the cotyledon and first-leaf stage, and the effects of two 

QTLs were similar and explained about 30% of the phenotypic variation. 

Resistance to pxB is controlled by one major QTL on Linkage Group 

XII at the cotyledon stage, and by two QTLs at the first-leaf stage. The 

effect of QTLs on Linkage Group XII was much larger than on LG II, 

explaining 46.7% (cotyledon) and 48.7% (first leaf) of the observed 

phenotypic variation. This suggests that the effects of the two QTLs 

differ depending on the strain and the stage. Further studies will be 

necessary to clarify the relationship between powdery mildew strains 

and resistance genes. Most Japanese cultivars were susceptible to pxA 

at both stages and pxB at the cotyledon stage. As ‘PMAR No. 5’ is 

resistant to both strains at both stages, introduction of the resistance 

genes of ‘PMAR No. 5’ into those cultivars will enhance the resistance 

of Japanese cultivars. 


Fukino, N., M. Kunihisa, and S. Matsumoto. 2004a. Characterization of recombinant 

inbred lines derived from crosses in melon (Cucumis melo L.), ‘PMAR No. 5’ × 

‘Harukei No.3.’ Breed. Sci. 54:141–145. 

Fukino, N., M. Kuzuya, M. Kunihisa, and S. Matsumoto. 2004b. Characterization of 

simple sequence repeats (SSRs) and development of SSR markers in melon 

(Cucumis melo). Proc. Cucurbitaceae 2004. 503–506. 

Fukino, N., M. Taneishi, T. Saito, T. Nishijima, and M. Hirai. 2002. Construction of a 

linkage map and genetic analysis for resistance to cotton aphid and powdery 

mildew in melon. Acta Hort. 588:283–286. 

Hosoya, K., N. Narisawa, M. Pitrat, and H. Ezura. 1999. Race identification in 

powdery mildew (Sphaerotheca fuliginea) on melon (Cucumis melo) in Jpn. Plant 

Breed. 118:259–262. 

Kunihisa, M., N. Fukino, and S. Matsumoto. 2003. Development of cleavage 

amplified polymorphic sequence (CAPS) markers for identification of strawberry 

cultivars. Euphytica. 134:209–215. 


Lander, E. S., P. Green, J. Abrahamson, A. Barlow, M. J. Daly, S. E. Lincoln, and L. 

Newburg. 1987. MAPMAKER: an interactive computer package for constructing 

primary genetic linkage maps of experimental and natural populations. Genomics. 

1:174–181. 

Lincoln, S., M. Daly, and E. S. Lander. 1993. Mapping genes controlling quantitative 

traits with MAPMAKER/QTL 1.1. Whitehead Institute technical report. 

Whitehead Institute for Biomedical Research, Cambridge, MA. 

Morishita, M., K. Sugiyama, T. Saito, and Y. Sakata. 2003. Powdery mildew resistance 

in cucumber. Jpn. Agric. Res. Quart. 37(1):7–14. 

Nunome, T., K. Ishiguro, T. Yoshida, and M. Hirai. 2001. Mapping of fruit shape and 

color development traits in eggplant (Solanum melongena L.) based on RAPD 

and AFLP markers. Breed. Sci. 51:19–26. 

Orihara, N., H. Uekusa, K. Kusano, K. Abiko, and M. Morishita 2001. Race 

differentiation of melon powdery mildew fungus (Sphaerotheca fuliginea) from 

Kanagawa Prefecture, and the relationship between races and resistance of 

commercial varieties. Ann. Rep. Kanto-Tosan Plant Protect. Soc. 48:45–48. 

Sitterly, W. R. 1978. Powdery mildew of cucurbits, p. 359–379. In: D. M. Spencer 

(ed.). The powdery mildews. Academic Press, New York. 

Yoshida,T. and T. Kohyama 1986. Mechanisms, genetics and selection methods of 

aphid resistance in melons, Cucumis melo. Bull. Veg. & Orn. Crops Res. Station 

Series C (Kurume). 9:1–12. 


LINKAGE ANALYSIS AMONG RESISTANCES 

TO POWDERY MILDEW AND VIRUS 

TRANSMISSION BY APHIS GOSSYPII 

GLOVER IN MELON LINE ‘TGR-1551’ 

M. L. Gómez-Guillamón, A. I. López-Sesé, E. Sarria, 

and F. J. Yuste-Lisbona 

Estación Experimental ‘La Mayora,’ CSIC, 29750 Algarrobo, Málaga, 

Spain 

ADDITIONAL INDEX WORDS. Genetic distance, Cucumis melo, Podosphaera 

xanthii 

ABSTRACT. The melon accession ‘TGR-1551’ shows resistance to Races 1, 2, and 

5 of powdery mildew and to virus transmission by A. gossypii. Linkage 

relationships of these four resistances were studied in a segregation F2 

population obtained from the cross between ‘TGR-1551’ and ‘Bola de Oro’. A 

very tight linkage (a distance of 2 to 4cM) among the powdery mildewresistance 

genes has been found. The segregation ratios (R:S) observed allow 

establishing the hypothesis that powdery mildew resistance is controlled by a 

double dominant and recessive interaction, where there could be three 

dominant genes tightly linked that provide specific resistance to Races 1, 2, and 

5 of P. xanthii, and one recessive gene that confers horizontal resistance to the 

three races. The estimated distance among powdery-mildew-resistance and 

virus-transmission-resistance genes was approximately 17cM. 

P 

owdery mildew is an important disease of cucurbits worldwide, 

with the reduction of quality and crop yield the most striking 

aspects of loss to the disease (Sitterly, 1978). Two fungal 

species, Podosphaera xanthii (DC.) VP. Gelyuta and Golovinomyces 

cichoracearum, (Castagne) U. Braun & N. Shishkoff have been 

reported as the common agents of powdery mildew in melon, although 

P. xanthii is the species usually found in regions with a temperate 

climate (Bertrand and Pitrat, 1989; Kenigsbuch and Cohen, 1992; 

Vakalounakis et al., 1994). Several physiological races of P. xanthii 

have been described according to the reactions of differential melon 

lines. To date, many races of this species have been differentiated 

(McCreight, 2006). The predominating race of powdery mildew 

changes depending on the melon cultivar, the cultivation season, and 

The authors thank the great collaboration of R. Tobar in all the experiments. This 

work has been financed by the CICYT Research Project: AGL2005-03850-C02-01. 


the geographical area studied (Hosoya et al., 2000), with Races 1, 2, 

and 5 being the most extensive in southern European regions (Bardin 

et al., 1997; Bertrand, 1991; Del Pino et al., 2002; Olalla, 2001). 

Viral diseases are the main cause of serious yield losses in 

cucurbits crops worldwide. Among them, the cucumovirus Cucumber 

mosaic virus (CMV) and the potyviruses Watermelon mosaic virus 

(WMV), Zucchini yellow mosaic virus (ZYMV), and Papaya ringspot 

virus-W (PRSV-W) are some of the most widespread in melon crops 

(Lovisolo, 1980; Luis-Arteaga et al., 1998; Nameth et al., 1986). These 

viruses are naturally transmitted by aphids in a nonpersistent manner 

(Pirone and Harris, 1970), with the cotton-melon aphid, Aphis gossypii 

Glover, being one of the most important vectors (Lecoq et al., 1979). 

Fungicide applications are the main means of powdery mildew 

control, but in spite of these measures, powdery mildew continues to 

impose serious limitations on cucurbit production throughout the 

world (Zitter et al., 1996). Control of nonpersistently transmitted 

viruses by means of insecticide spraying against their vectors or other 

crop-management practices is ineffective (Loebenstein and Raccah, 

1980). One of the most successful procedures for controlling plant 

diseases is the development of resistant cultivars. Genetic resistance 

to diseases is one of the main components of integrated plant 

protection systems (Heitefuss, 1989). 

Melon resistance to powdery mildew has been studied for a long 

time and several sources of resistance have been described for Races 1 

and 2 (Jagger et al., 1938; Bohn and Whitaker, 1964; Epinat et al., 

1993; Kenigsbuch and Cohen, 1992; Perchepied et al., 2005). 

Nevertheless, the inheritance of resistance remains somewhat 

confusing, due mainly to environmental conditions affecting trait 

expression (Cohen et al., 1996, 2002). There are not many virusresistance 

sources in melons, so indirect resistance to viruses could be 

incorporated by using genes conferring resistance to virus 

transmission. The Korean melon line PI 161375 was reported to show 

resistance to virus transmission by A. gossypii conferred by the Vat 

gene (Lecoq et al., 1979). 

The melon accession ‘TGR-1551’ from Zimbabwe shows 

resistance to Races 1, 2, and 5 of powdery mildew (Gómez-Guillamón 

et al., 1995, 1998). In addition, ‘TGR-1551’ presents resistance to 

virus transmission by A. gossypii, and this resistance seems to be 

controlled by a single dominant gene different from Vat (Soria et al., 

2003). Linkage between resistance genes has been reported in 

previous works. Thus, Pitrat (1991) described that resistance to virus 

transmission mediated by Vat appears to be tightly linked to resistance 

to powdery mildew provided by Pm-W (0cM). Bardin et al. (1999), 


Anagnostou et al. (2000), and Klinger et al. (2001) support the 

hypothesis of the existence of resistance-gene clusters in melon. 

Montoro et al. (2004) established that resistances to powdery 

mildew Race 5 and to virus transmission by aphid are linked in ‘TGR- 

1551’. In the current study, resistance to Races 1, 2, and 5 of P. 

xanthii and to virus transmission by A. gossypii have been evaluated in 

the F2 population obtained from the cross between ‘TGR-1551’ and 

the susceptible cultivar ‘Bola de Oro’. Linkage among the genes 

involved in the resistances to the different races of powdery mildew 

has been estimated, as well as the linkage to the virus-transmissionresistance 

gene. 


Ten plants of the resistant parental ‘TGR-1551’, the susceptible 

parental ‘Bola de Oro’, and their F1, and 295 plants of the F2 were 

evaluated. Scions from each plant were taken before inoculation in 

order to inoculate the same genotypes with both powdery mildew and 

virus transmission by aphid. 

Artificial inoculation of powdery mildew was done by depositing a 

small amount of conidia on the second true leaf (Ferriere and Molot, 

1988). All plants were inoculated on the same leaf with Races 1 

(isolate 27), 2 (isolate P-15.0), and 5 (isolate C8) of P. xanthii. The 

plants were maintained in a controlled growth chamber at 32ºC and 

22ºC with a 16:8h (light:dark) photoperiod. Plants were scored 12 

days postinoculation and classified as either resistant (R; no apparent 

fungal development) or susceptible (S; profuse sporulation). 

Virus inoculation by A. gossypii was carried out according to Soria 

et al. (2003) using CMV-M730 (‘Song’ pathotype). Ten days after 

inoculation, plants showing no symptoms were reinoculated. Presence 

or absence of virus symptoms was recorded 20 days after first 

inoculation, and plants with no or doubtful symptoms were tested by 

enzyme-linked immunosorbent assay (ELISA) test using CMV 

polyclonal antiserum (Loewe Biochemica GmbH). All plants showing 

clear symptoms of CMV infection, as well as plants testing positive in 

ELISA, were considered susceptible. The experiment was carried out 

in a greenhouse with temperatures that oscillated between 32ºC and 

20ºC under a 16:8h (light:dark) photoperiod. A. gossypii used for the 

experiments were taken from a nonviruliferous aphid colony 

maintained on plants of the susceptible accession ‘C-278’ growing at 

25–15ºC under a 16:8h (light:dark) photoperiod. 

Chi-square tests were performed to check segregation for each 

character in the F2 population. For linkage analysis, pairwise χ 2 tests 


were conducted and recombination frequency was estimated according 

to Fisher (1921). The Kosambi function (Kosambi, 1944) was used to 

convert recombination units into genetic distances. 


In order to verify that the simultaneous inoculation of the three 

races of powdery mildew on the same leaf could modify the plant 

response, previous work using differential melon genotypes was 

carried out and no interaction among resistances was detected 

(unpublished data). 

All plants of the cultivar ‘Bola de Oro’ showed clear symptoms of 

Races 1, 2, and 5 of powdery mildew and clear susceptibility to CMV- 

M730 infection. Plants of the accession ‘TGR-1551’ showed complete 

resistance to the three races of powdery mildew and to CMV-M730 

infection. The F1 plants showed a similar resistant response to the 

pathogens evaluated, indicating that these resistances are dominant. In 

the F2, segregation for these characters was observed (Table 1). 

Table 1. Segregation of resistance to Races 1, 2, and 5 of P. xanthii 

and to CMV-M730 transmission by A. gossypii in the F2 obtained 

from the cross between ‘TGR-1551’ and ‘Bola de Oro’. 

χ 2 

Observed 

Pathogen segregation Segregation 

ratio Value Probability 

P. xanthii Race 1 243/52* 13:3 0.244 0.621 

P. xanthii Race 2 242/53 13:3 0.119 0.730 

P. xanthii Race 5 247/48 13:3 1.189 0.275 

CMV-M730 190/77 3:1 2.098 0.147 

* Resistant plants/susceptible plants. 

The segregation ratios (R:S) observed after inoculation with the 

different races of powdery mildew were identical (13:3) (Table 1). 

Most of the F2 plants were clearly resistant or susceptible to all races, 

and only nine plants were recombinants. The presence of all the 

possible types of recombinants in the F2 population indicates the 

existence of at least one dominant resistance gene for each one of the 

races of powdery mildew. Although this hypothesis should be 


confirmed in F3 and backcross populations, it seems that powdery 

mildew resistance is controlled by a double dominant and recessive 

interaction, where there could be three dominant genes tightly linked 

that provide specific resistance to Races 1, 2, and 5 of P. xanthii, and 

one recessive gene that confers horizontal resistance to the three races. 

Segregation of resistance to powdery mildew Race 1 observed by 

Montoro (2005) in this material could adjust to our hypothesis; 

however, Montoro et al. (2004) described that resistance to Race 5 in 

‘TGR-1551’ is controlled by one dominant gene. In the current work, 

inoculations with the three races of powdery mildew have been done 

simultaneously, while Montoro (2005) inoculated each F2 plant with a 

different race. We found an excess of resistance for all the races, in 

comparison to the segregation observed by Montoro (2005); therefore, 

the possibility that simultaneous inoculation could have triggered a 

defensive response in the inoculation zone (Gozzo, 2003) in these F2 

plants cannot be discarded. The segregation ratio observed for virustransmission 

resistance (3:1) agrees with the observations of Soria et 

al. (2003), suggesting a dominant monogenic control (Table 1). 

In order to estimate the recombination fraction between resistance 

genes to different races of powdery mildew, we considered the 

probabilities of phenotypes according to our hypothesis of digenic 

inheritance with a common recessive gene. A very tight linkage (a 

distance of 2 to 4cM) among the powdery mildew-resistance genes 

present in ‘TGR-1551’ has been found (Table 2). Linkage between 

powdery mildew-resistance genes has been reported in previous 

works; genetic analysis done by Bardin et al. (1999) revealed that PI 

124112 possessed four genes for resistance to powdery mildew and 

that all these genes are linked in a cluster spanning 22cM. The 

estimated distance between powdery mildew-resistance and virustransmission-resistance 

genes was approximately 17cM (Table 2). In 

‘TGR-1551’, Montoro et al. (2004) established that the distance 

between the Race 5 powdery mildew-resistance gene and the virustransmission-resistance 

gene was 28,3cM with F2 populations, and 

15,9cM with backcrosses. Our results seem to agree with those 

observed by Montoro et al. (2004), and support the hypothesis of the 

existence of resistance-gene clusters in melon (Anagnostou et al., 

2000; Bardin et al., 1999; Klinger et al., 2001). 

The evaluation of more F2 plants could implement the number of 

representatives of each phenotype, including recombinants, for a more 

precise estimation of genetic distances. Segregation of resistance to P. 

xanthii and virus transmission by A. gossypii in the F3 populations and 

backcrosses obtained from the cross between ‘TGR-1551’ and ‘Bola 

de Oro’ is under study. These results and analysis of the RILs will 


Table 2. Linkage analyses between resistance genes to Races 1, 2, and 

5 of P. xanthii and between powdery mildew and CMV-M730 

transmission by A. gossypii in the F2 obtained from the cross between 

‘TGR-1551’ and ‘Bola de Oro’. 

Genetic distance 

Observed segregation Ratio (cM) 

R1R2 * Race 1/Race 

2 240 

R1S2 

3 

S1R2 

2 

S1S2 

50 

43:9:9:3 2.38 

Race 2/Race 

5 

R2R5 

241 

R2S5 

1 

S2R5 

6 

S2S5 

47 

43:9:9:3 4.27 

Race 1/Race 

5 

R1R5 

241 

R1S5 

2 

S1R5 

6 

S1S5 

46 

43:9:9:3 3.94 

P. xanthii / 

CMV-M730 

R R 

180 

R S 

34 

S R 

7 

S S 

43 

9.3:3.1 17.27 

* 

Ri: resistant to race i; Si: susceptible to race i. 

allow us to clarify powdery mildew genetics and estimate more precisely 

the distances among the genes involved. 

Selection of plant material resistant to several important pathogens 

is hard and time-consuming labor. The tight linkage among the 

different powdery mildew-resistance genes could allow the 

simultaneous selection of resistance to all three races with high 

probability. Moreover, the linkage between these genes and the virustransmission-resistance 

gene could likely allow selection of this gene. 


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EPICUTICULAR WAX MORPHOLOGY AND 

TRICHOME TYPES IN RELATION TO HOST 

PLANT SELECTION BY APHIS GOSSYPII IN 

MELONS 

M. L. Gómez-Guillamón 1 , A. Heredia 2 , 

and E. Sarria 1 

1 Experimental Station ‘La Mayora’ CSIC 29750-Algarrobo, 

Málaga, Spain 

2 Department of Molecular Biology and Biochemistry, 

Málaga University, Teatinos Campus, 29071-Málaga, Spain 

ADDITIONAL INDEX WORDS. Antixenosis, cotton-aphid, Cucumis melo, glandular 

trichomes, resistance, susceptibility 

ABSTRACT. Morphology of epicuticular wax layers and trichomes types and 

density in leaves of aphid-resistant cultivars of Cucumis melo were compared 

with the susceptible ‘Bola de Oro’. Epicuticular wax layer characteristics are 

similar in both resistant and susceptible cultivars. Three types of trichomes 

have been identified in Cucumis melo leaves. A relationship between the density 

of glandular trichomes Type I and the existence of antixenosis mechanisms to 

Aphis gossypii has been established. 

T 

he cotton-melon aphid, Aphis gossypii Glover, causes serious 

direct and indirect damage to many crops and is considered a 

serious pest of melon in Mediterranean countries. Growing 

resistant or tolerant cultivars is the most effective, environmentally 

compatible control strategy. Two main genetic resistance sources have 

been found in melons: the lines PI-161375, carrying the Vat gene, 

which confers resistance to A. gossypii colonization and virus 

transmission (Pitrat and Lecoq, 1980), and PI-414723, carrier of the 

Agr gene, which confers A. gossypii resistance (Kishaba et al., 1971). 

More recently, Garzo et al. (2002) described resistance to this aphid in 

the cultivar ‘TGR-1551’. Electrical Penetration Graphs (EPGs) of A. 

gossypii behavior in the C. melo cultivar ‘TGR-1551’ have shown the 

existence of phloematic specific factors disturbing the aphids feeding 

on resistant plants (Garzo et al., 2002). These authors also found 

antixenosis mechanisms in ‘TGR-1551’. 

Interaction between plants and aphids is very complex (Miles, 

The authors thank the valuable collaboration of R. Tobar and R. Camero in all the 

experiments. This work has been financed by the CICYT Research Project: 

AGL2005-03850-C02-01. 


1998). The selection or avoidance of potential host plants by aphids is 

guided by a complex combination of physical and chemical stimuli. 

The choice of host plants could be affected by the presence of 

chemicals, acting as repellents or deterrents. Also, the presence and 

density of trichomes in the leaves may become a physical barrier for 

aphid feeding and reproduction (Renwick, 1983). 

In other vegetable species, the role of the leaf epicuticular lipids 

composition in relation to aphid infestation and resistance has been 

studied (Powell et al., 1999; Ni and Quisenberry, 1997; Shepherd et. 

al., 1999; Bahlmann et al., 2003; Duetting et al., 2003), but no 

information exists for melons. Morphology, types, and density of leaf 

trichomes have also been related to the choice of host plants by aphids 

in other species (Levin, 1973; Bahlmann et al., 2003; Simmons et al., 

2003; Wagner et al., 2004) and a few authors have studied trichomes 

types in the Cucurbitaceae family (Kolb and Müller, 2004). 

Morphology and topography of epicuticular wax layers 

determination together with a description of the trichomes observed in 

leaves of ‘TGR-1551’ and the susceptible ‘Bola de Oro’ have been 

examined. Moreover, the relation between the density of glandular 

trichomes for three different resistant cultivars (‘TGR-1551’, PI- 

414723, and PI-161375), and the existence of antixenosis mechanisms 

to A. gossypii has been investigated. 


A. gossypii were reared on plants of the susceptible Spanish melon 

cultivar ‘ANC-57’, at 25ºC (day) and 20ºC (night) with a 16:8-hour 

(L:D) photoperiod. The melon cultivars used in the antixenosis 

experiments were the aphid-susceptible ‘Bola de Oro’ and the resistant 

cultivars ‘TGR-1551’, PI-414723, and PI-161375. In these cultivars, 

leaf trichome evaluations were also done. For the epicuticular wax 

observations only the cultivars ‘Bola de Oro’ and ‘TGR-1551’ were 

used. Plants were cultivated in a glasshouse in individual pots at 25ºC 

(day) and 18ºC (night) with a 14:10-hour (L:D) photoperiod. In the 

antixenosis experiment, 10 adult aphids (7–8 days old) were released 

on five plants from each melon line, at the five-full-expanded-leaf 

stage, following the methodology described by Martin and Fereres 

(2003). 

For the evaluation of epicuticular wax layer and trichomes, two 

leaf disks (5,94mm diameter) per plant were taken at three different 

levels in the plant, corresponding to young, medium-aged, and old 

leaves. Samples were fixed in glutaraldehyde (4% v/v, in phosphate 

buffer 0,2 M, pH 7) at 4ºC. They were thoroughly rinsed in fresh 


phosphate buffer and then dehydrated through acetone solutions series 

(25, 50, 75, and 100% v/v), 15 min each. Samples were mounted on 

scanning electron microscope tubes and coated with a thick layer of 

gold. A JEOL JSM-840 scanning electron microscope (SEM) operated 

at 15kV was used. 

Pubescence of the leaves was evaluated on fresh leaf disks using a 

stereoscopic magnifying glass. Using SEM, different types of 

trichomes were observed. To identify and count trichomes under a 

light microscope, staining of the samples was required. Leaf disks 

were decolorated using 100% alcohol and heating to 80ºC for three 

minutes. Samples were stained using an aqueous solution containing 

0,05% tolouidine blue O (O’Brien et al., 1964). 

Trichome numbers were analyzed using two-way ANOVA 

(p=0,05) and aphid data were analyzed using one-way ANOVA (p = 

0,05) to test for differences between antixenosis in different cultivars. 

Trichome density and antixenosis data were further analyzed by 

pairwise comparison using Student-Newman-Keuls test (p = 0,05). 


In the antixenosis experiments, the number of adults feeding on the 

inoculated leaves of the ‘Bola de Oro’ cultivar 72 hours after the 

aphids were released was always higher than the number observed in 

plants of ‘TGR-1551’, PI-414723, and PI-161375, which also 

confirmed the existence of antixenosis in the resistant cultivars (Table 

1). Similar results were also obtained by Garzo et al. (2002) in their 

experiments under free-choice and no-choice conditions using the 

same cultivars. 

Host selection by aphids is not a random process; these insects 

employ a variety of sensory and behavioral mechanisms to locate and 

recognize their host plants. Epicuticular waxes, trichomes exudates, 

substrate texture, topology, and color may influence aphid behavior 

before stylet insertion (Powell et al., 2006). The results obtained 

Table 1. Number of aphids (mean ± SE) on each inoculated leaf 72 h 

after 10 adults were released in the antixenosis experiment. 

Cultivar Average number of aphids 

‘Bola de Oro’ 7,42 b* ± 1,62 

‘TGR-1551’ 1,53 a ± 1,23 

PI-414723 1,80 a ± 0,79 

PI-161375 3,60 c ± 2,07 

*Means in columns followed by a common letter do not differ at the 5% level by 

SNK test. 


could suggest the existence of one or more of those stimuli. 

Observations using SEM suggested that the epicuticular wax layer 

is very thin. In young leaves no wax images were observed. As the 

leaves increase in age, some very small crystalline wax deposits were 

visible, but these appeared in leaves of both the susceptible cultivar 

and the resistant ‘TGR-1551’. The observed morphology was also 

similar for both cultivars, which indicated a similar chemical 

composition (Barthlott et al., 1998). These results suggest that neither 

the presence of a wax layer in leaves nor its composition seem to have 

a relation with the antixenotic effect on A. gossypii observed in ‘TGR- 

1551’. 

Three types of trichomes have been observed in the leaves (Figure 

1). The most frequent trichomes were identified as columnar trichomes 

(Figure 1a), similar in both the resistant and susceptible cultivars. 

Density of trichomes has been described in other species as a factor 

that impedes aphid feeding and reproduction (Donald, 1973; 

Bahlmann et al., 2003). The pubescence observed in the adaxial leaf 

surface of ‘Bola de Oro’ is lower than that in ‘TGR-1551’ at the three 

examined plant levels; however, the susceptible cultivar has the 

highest pubescence in the abaxial surface (differences were significant 

at the 5% level) (Table 2). Regarding these results, the pubescence 

does not seem to be an important factor in aphid resistance. 

Table 2. Number of columnar trichomes/cm² (mean ± SE) in abaxial 

and adaxial leaf surfaces of different melon cultivars. 

Cultivar Leaf surface Young leaf Medium leaf Old leaf 

Adaxial 377,1 a*± 70,4 225,5 a ± 40,6 106,8 a ± 14,5 

Bola de Oro 

Abaxial 2030,2 b ± 315,1 1346,4 b ± 295,6 782,3 b ± 128,6 

Adaxial 756,6 c ± 156,2 310,8 c ± 63,6 201,7 c ± 43,8 

TGR-1551 

Abaxial 1310,5 d ± 278,9 694,2 d ± 174,8 557,2 d ± 119,5 


SNK test. 

During SEM examination, two additional types of trichomes 

smaller than columnar trichomes were noticed in the leaf samples 

(Figure 1). These trichomes were located mostly on leaf veins on the 

abaxial and adaxial leaf surfaces in both cultivars. Regarding their 

morphology, one of them corresponded to Type I as described by Kolb 

and Müller (2004) in leaves of Cucurbita pepo ssp. pepo var. styriaca 

and observed on blooms of Cucurbita by Uphof and Hummel (1962). 


This type consisted of a basal cell, a uniseriate stalk, and a four-celled 

head region (Figure 1b). 

Type I trichomes have been described as glandular trichomes, with 

a secretory function. Using specific stains, Kolb and Müller (2004) 

observed that the head area of this trichome stored lipids, terpenoids, 

and other flavonoids. Terpenoids have also been identified in similar 

trichomes on leaves of Calceolaria adscendens (Sacchetti et al., 1999) 

and Teucrium scorodonia (Antunes and Sevinate-Pinto, 1991). These 

metabolites play a significant role in interactions between the plant and 

herbivorous or pathogenous organisms, respectively (Levin, 1973). 

The third type of trichome was identified as Type III as described 

by Kolb and Müller (2004) in oil pumpkin. Type III consisted of five 

cells in line, and a basal cell, and was observed very rarely on the 

abaxial surface of the leaves, in both the resistant and the susceptible 

cultivar (Figure 1c). Ascensao et al. (1999) have also found this type 

of glandular trichome in Plectranthus ornatus. 

Fig. 1. Different types of trichomes observed in melon leaves: (a) Columnar 

trichome, (b) trichome Type I, (c) trichome Type III. 

White scale-bar: 100µm; black scale-bar: 10µm 

In order to establish a correlation between the number of Type I 

trichomes in the leaves and antixenotic effects in the plants, density of 

these trichomes in the first three young leaves was evaluated. Results 

showed that Type I trichomes were also found in leaves of PI-414723 

and PI-161375 in numbers similar to those observed on plants of 

‘TGR-1551’. Density of Type I trichomes was similar in both leaf 

sides and they were in any case located on leaf veins. Number of 

trichomes/cm² was higher in younger leaves, as expected. But the most 

interesting observation was that their density was always higher in the 


esistant cultivars than in the susceptible ‘Bola de Oro’, and these 

differences were clearly significant (Table 3). 

When trichomes were stained with diphenylboric acid-2aminoethyl 

ester (DPBA) (results not shown), the presence of 

flavonoids inside them was observed. Flavonoids have been described 

as chemicals disturbing the behavior of aphids, forcing them to avoid 

plants in which they detect their presence (Levin, 1973; Dreyer and 

Jones, 1981; Mitchell et al., 1993; Francis et al., 2004). The higher 

number of Type I trichomes found in resistant cultivars could be 

related to higher flavonoid content in the leaf environment, and that 

could force the aphid to leave the plant in the antixenosis experiment. 

The density of these trichomes in resistant cultivars could also explain 

the results obtained by Garzo et al. (2002) in their free-choice 

experiment. Their experiments showed that aphids preferred the leaf 

disks of the susceptible cultivar for feeding. 

Table 3. Number of trichomes type I/cm² (mean ± SE) in the abaxial 

and adaxial leaf surfaces of different melon cultivars. 

Cultivar Surface leaf 1 st leaf 2 nd leaf 3 rd leaf 

‘Bola de Adaxial 44,0 a* ± 6,3 17,9 a ± 5,2 21,7 ab ± 3,5 

Oro’ Abaxial 17,6 a ± 3,7 14,3 a ± 7,2 1 3,6 a ± 6,7 

PI-161375 

PI-414723 

Adaxial 344,9 b ± 63,0 127,1 bc ± 11,9 41,4 b ± 7,5 

Abaxial 230,8 b ± 29,7 97,0 c ± 11,5 33,8 b ± 2,7 

Adaxial 311,7 b ± 130,3 116,9 b ± 44,2 55,2 b ± 17,4 

Abaxial 251,7 b ± 89,2 120,6 bc ± 43,4 61,1 b ± 12,0 

Adaxial 313,5 b ± 103,3 226,8 d ± 10,1 122,4 c ± 32,2 

‘TGR-1551’ 

Abaxial 223,7 b ± 111,7 171,9 b ± 37,8 110,9 c ± 20,1 


SNK test. 

With these observations, the density of Type I trichomes could be 

related to antixenosis against A. gossypii in ‘TGR-1551’, PI-414723, 

and PI-161375. Results suggest that the number of Type I 

trichomes/cm² could be considered as a useful morphological marker 

for antixenosis selection in melon breeding. 



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EVALUATION OF ZUCCHINI AND 

STRAIGHTNECK SUMMER SQUASH 

BREEDING LINES AND VARIETIES FOR 

POWDERY MILDEW AND DOWNY MILDEW 

TOLERANCE 

M. L. Infante-Casella 

Rutgers Cooperative Research and Extension of Gloucester County, 

Clayton, NJ 08312 

C. A. Wyenandt 

RCRE, Rutgers Agricultural Research and Extension Center, 

Bridgeton, NJ 08302 

M. R. Henninger 

RCRE, Foran Hall, New Brunswick, NJ 08901 

ADDITIONAL INDEX WORDS. Cucurbits, Cucurbita pepo, disease tolerance 

ABSTRACT. Field research was conducted with 22 green zucchini and yellow 

straightneck summer squash (Cucurbita pepo) hybrids to evaluate tolerance to 

powdery mildew (Podosphaera xanthii) and downy mildew (Pseudoperonospora 

cubensis) infection under field conditions in 2005. Powdery mildew (PM) and 

downy mildew (DM) are important diseases of cucurbit crops in the mid- 

Atlantic region of the United States annually during summer months. In New 

Jersey, PM is first observed in mid-July and DM is usually first observed in 

mid-August. However, during the 2004 and 2005 production seasons, DM was 

identified approximately two months earlier than expected. In 2005, AUDPC 

values for PM development were lowest in the straightnecks ‘Sunray’, ‘General 

Patton’, and ‘Patriot II’ and in the zucchinis ‘Wildcat’, ‘Judgment III’ 

(formerly ‘EX04629728’, released December 2005), ‘Justice III’, and ‘Payroll’. 

AUDPC values for DM development were lowest in the straightnecks ‘Lioness’, 

‘Conqueror III’, and ‘Cougar’ and in the zucchinis ‘Judgment III’, ‘SSX6593’, 

‘Lynx’, and ‘Leopard’. 

Z 

ucchini and straightneck yellow squash (Cucurbita pepo L.) are 

important crops for many vegetable farmers in New Jersey. 

Over 1000 hectares of green zucchini and 360 hectares of 

yellow straightneck summer squash are grown annually in the state, 

with a farm-gate value of 12.8 million dollars, making New Jersey the 

fifth highest in the nation in production value (National Agricultural 

Statistics Service, 2000). Powdery mildew (Podosphaera xanthii) 

(PM) and downy mildew (Pseudoperonospora cubensis) (DM) are two 

of the most important foliar diseases of summer squash in New Jersey 

(Infante-Casella et al., 2003). Without the use of fungicides, losses to 

PM and DM can be extremely high in the mid-Atlantic region. 


Unfortunately, in recent years, PM resistance to certain fungicide 

chemistries has been detected in the U.S. (McGrath and Thomas, 

1996). Additionally, some fungicide chemistries used to control DM 

are also at risk for resistance development. This study was done to 

evaluate zucchini and straightneck summer squash hybrids for 

tolerance to PM and DM. 


A trial was conducted in the field during the summer and fall of 

2005 at the Rutgers Agricultural Research and Extension Center 

(RAREC) in Bridgeton, New Jersey, on a Sassafras sandy loam soil. 

All squash were seeded on 14 July by hand with 75cm spacing into 

black plastic mulch on high raised beds. Rows were spaced 150cm 

apart. In total, there were 22 hybrids with six plants per block, with 

four replications. Drip irrigation was used for supplying water and 

fertilizer. Prefar 4E (bensulide) at a rate of 11.2L/ha was applied on 

the soil surface just before laying plastic for preemergent weed 

control. For control of aphids and cucumber beetles, Admire 

(imidacloprid) was applied in the seed hole at a rate of 1.7L/ha after 

planting using a backpack sprayer. On August 26, Gavel (zoxamide) 

at 2.4kg/ha tank-mixed with Phostrol (phosphite salts) at 5.6L/ha was 

applied to slow downy mildew infection. Green zucchini hybrids 

planted included: ‘Tigress’, ‘Leopard’, ‘Lynx’, ‘Wildcat’, ‘SSX6560’, 

‘SSX6593’, ‘SSX6609’, ‘SSX6713’, ‘Payroll’, ‘Revenue’, ‘Senator’, 

‘Independence II’, ‘Justice III’, and ‘Judgment III’ (formerly 

EX04629728). Yellow straightneck hybrids planted were ‘General 

Patton’, ‘Sunray’, ‘XPHT1832 III’, ‘Patriot II’, ‘Liberator III’, 

‘Conqueror III’, ‘Cougar’, and ‘Lioness’. Disease evaluations began as 

soon as the plants showed visual signs of PM (1 Sep.) and DM (18 

Aug.) infection. Thereafter, weekly evaluations were done to assess 

PM and DM development for each of the 22 hybrids. Leaves and 

stems of plants were visually rated on a scale of 0.0 (no sign of 

infection) to 10.0 (100% infection) at 0.5 increments for presence of 

PM and DM infection. Arcsine-transformed AUPDC values were 

calculated for PM and DM development during the late-season 

evaluation period for observing differences in disease development. 

Zucchini and straightneck squash data were analyzed separately using 

SAS (ANOVA, P=0.05). 


There were significant differences in AUDPC values for PM and 

DM development among both straightneck (Table 1) and zucchini 


(Table 2) squash cultivars. Of the straightnecks, AUDPC values for 

PM development were lowest in ‘Sunray’, ‘General Patton’, and 

‘Patriot II’, respectively. AUDPC values for DM were lowest in 

‘Cougar’, ‘Lioness’, and ‘Conqueror III’. No straightneck squash had 

acceptable levels of both PM and DM tolerance. Zucchini squash that 

had the lowest AUDPC values for PM development included 

‘Wildcat’, ‘Judgment III’, ‘Justice III’, ‘Payroll’, and ‘SSX6560’. 

Zucchini squash with the lowest AUDPC values for DM development 

included ‘Judgment III’, ‘SSX6593’, Lynx’, and ‘Leopard’, 

respectively. Of all the squash evaluated, ‘Judgment III’ was the only 

one that had a relatively low AUDPC value for both PM and DM. 

Table 1. AUDPC values z for powdery mildew (PM) and downy 

mildew (DM) on straightneck summer squash cultivars grown at the 

Rutgers Agricultural Research and Extension Center (RAREC), 

Bridgeton, New Jersey, in 2005. 

Variety Seed company PM DM 

Cougar Harris Moran 1164.3 1531.8 

Lioness Harris Moran 1085.2 1432.9 

Conqueror III Seminis 1078.1 1531.8 

XPHT 1832 III Seminis 1038.84 1861.0 

Liberator II Seminis 1017.2 1697.9 

Patriot II Seminis 897.2 1674.7 

General Patton Seminis 684.5 1838.4 

Sunray Seminis 595.6 1805.5 

LSD 154.5 210.6 

z AUDPC values arcsine transformed. 

The identification of PM- and DM-tolerant and -resistant squash 

varieties is important to the future of summer squash production in 

New Jersey and other production areas in the mid-Atlantic region. 

With the high cost of fungicides and the threat of fungicide-resistance 

development, summer squash growers are looking for cultivars that 

have tolerance to both PM and DM. Vegetable producers can reduce 

the need for fungicides that are at high risk for resistance development 

by selecting resistant varieties for production (McGrath, 2005). 

Breeders identify disease tolerance or resistance by continually testing 

new lines. Much of the breeding is aimed at developing PM-tolerant 

or -resistant squash lines, with less effort in finding DM tolerance or 


esistance (T. Pagels, personal communication). While there are 

fungicides available to control PM, it is still a problem for many 

growers (Elkner and Young, 2004). The same can be said for control 

fungicides available to control PM, it is still a problem for many 

growers (Elkner and Young, 2004). The same can be said for control 

of DM on susceptible cucurbits, due to the difficulty of properly 

timing application and the potential for fungicide-resistance 

development. DM resistance exists in some cucumber cultivars. 

However, some of these cultivars appear to be losing resistance. The 

cucumber ‘Speedway’, thought to be DM resistant, was found to be 

infected with DM in southern New Jersey in 2005. There is a need to 

evaluate all cucurbit varieties for DM tolerance or resistance, even 

though DM resistance may not be the main target of many cucurbitbreeding 

programs. 

Table 2. AUDPC values z for powdery mildew (PM) and downy 

mildew (DM) on zucchini squash cultivars grown at the Rutgers 

Agricultural Research and Extension Center (RAREC), Bridgeton, 

New Jersey in 2005. 

Variety Seed company PM DM 

Revenue Siegers 1355.1 1835.5 

Leopard Harris Moran 1181.7 1635.2 

Lynx Harris Moran 1145.6 1628.6 

Independence II Seminis 1140.2 1670.5 

SSX6593 Siegers 1076.9 1622.3 

Senator Seminis 1065.1 1722.7 

Tigress Harris Moran 1046.4 1732.4 

SSX6713 Siegers 1023.3 1863.7 

SSX6560 Siegers 992.3 1825.4 

Payroll Siegers 815.4 1698.0 

Justice III Seminis 757.6 1752.1 

Judgment III Seminis 702.1 1522.9 

Wildcat Harris Moran 657.1 1677.7 

LSD (P=0.05) 192.4 161.0 

z AUDPC values arcsine transformed. 

PM and DM have continued to be a challenge when producing 

summer squash in New Jersey. Identifying PM and DM diseasetolerant 

or -resistant cultivars is necessary to improve the economics 

of producing summer squash in New Jersey and the mid-Atlantic 

region of the U.S. 



Elkner T. E. and L. Young. 2004. Mildew tolerant pumpkin variety evaluations in 

Pennsylvania, p. 35–38. In: Proc. 35 th Annual Mid-Atlantic Vegetable Workers 

Conference. 

Infante-Casella, M. L., G. M. Ghidiu, and B. A. Majek. 2003. Crop profile for 

summer and winter squash in New Jersey. 

. 

McGrath, M. T. 2005. Managing fungicide resistance is key to controlling powdery 

mildew, p. 63–66. 2005. Proc. New Jersey Annual Vegetable Meeting. Rutgers 

Cooperative Research and Extension. 

McGrath, M. T. and C. E. Thomas. 1996. Powdery mildew, p. 28–30. In: T. A. Zitter, 

D. L. Hopkins, and E. E. Thomas (eds.). Compendium of cucurbit diseases. APS 

Press, St. Paul, MN. 

National Agricultural Statistics Service. 2000. 2000 National rankings, squash, fresh 

market and processing. . 


INHERITANCE OF CHILLING RESISTANCE IN 

CUCUMBER SEEDLINGS 

Elzbieta U. Kozik 

Department of Genetics, Breeding and Biotechnology, Research 

Institute of Vegetable Crops, 96-100 Skierniewice, Poland 

Todd C. Wehner 

Department of Horticultural Science, North Carolina State University, 

Raleigh, NC 27695-7609 

ADDITIONAL INDEX WORDS. Cold injury, genetics, vegetable breeding 

ABSTRACT. The inheritance of the resistance in cucumber inbred NC-76 

(originating from PI 246930) was studied. The results clearly confirm the 

presence of a high level of resistance to chilling injury in NC-76. The 

segregation data of F2 and backcross progenies of the resistant NC-76 crossed 

with the susceptible ‘Chipper’ and Gy 14 demonstrated that a high level of 

resistance from NC-76 was conferred by a single, incompletely dominant gene 

Ch. 

C 

ucumber (Cucumis sativus L.) is susceptible to chilling (low 

temperatures above freezing). The first symptoms of chilling 

injury to the leaves of cucumber plants are rapid leaf wilting 

and development of sunken, necrotic patches within a few hours of 

chilling exposure. When plants return to warm temperatures, the leaf 

margins and necrotic patches dry out, giving the leaf a mottled and 

brittle appearance. Chilling injury may occur in the spring in many 

geographical regions where the cucumber crop is planted before the 

risk of frost has ended. In order to determine whether chillingresistant 

cultivars can be developed, a method for testing genetic 

variation in chilling sensitivity was developed, and germplasm having 

partial chilling resistance was identified, including AR75-79 

(sometimes called ‘Little John’) and ‘Chipper’ (Smeets and Wehner, 

1997). Using this method, Chung et al. (2003) investigated 

inheritance of chilling injury in genetic progenies of resistant 

‘Chipper’ and AR75-79 in crosses with susceptible Gy 14. Their data 

suggested that chilling resistance was maternally inherited. In the 

meantime, a high level of resistance was identified in cucumber 

accession PI 246930, and the genetic basis of resistance in this 

accession is presented here. 


The plant material used in this study consisted of reciprocal F1, F2, 

and backcross progenies from three groups of crosses between 


esistant inbred NC-76 (P1) (selected from PI 246930) and ‘Chipper’ 

(P2) and Gy 14 (P3) (susceptible). All crosses were made by handpollination 

in a greenhouse. 

Chilling-resistance tests were conducted under controlledenvironment 

conditions in the growth chambers of the Southeastern 

Plant Environment Laboratory at North Carolina State University. 

Seeds were sown in peat pots (57mm 2 , 100-ml volume) filled with a 

standard substrate of gravel and peat in a 1:1 ratio and placed in trays. 

One seed was sown in each pot. 

After their germination, the plants were placed in growth chambers 

set at 26/22°C (day/night) temperatures under long days, consisting of 

12 hours of combined fluorescent and incandescent light (from 8 AM 

to 8 PM). Light intensities (PPFD) were 650 and 44mmol . m -2. s -1 , 

respectively. Plants were watered with the standard phytotron nutrient 

solution (Thomas et al., 2005). The experiment was divided into 10 

sets for ease of handling. Each set contained 3 plants of each parent 

(P1, P2, P3), 6 F1, 6 BC1P1, 6 BC1P2/BC1P3, and 30 plants of the F2 

generation. Data from all sets were pooled for the analysis. 

When the plants reached the first-true-leaf stage, they were moved 

from the main growth chamber to the chilling chamber for treatment at 

4°C under a light intensity of 500mmol . m -2. s -1 PPFD during 7 hr. 

After the chilling treatment, they were returned to the main growth 

chamber and placed under the same light and temperature regime as 

before. Plants were evaluated 14 days after chilling, rating the damage 

on the first true leaf. The scale used was 0 to 9: 0 = no damage; 1–2 = 

trace of damage; 3–4 = slight damage; 5–6 = moderate damage; 7–8 = 

advanced damage; 9 = plant dead. Data were collected as means over 

all true leaves (not cotyledons) for each plant within each generation. 

Plants from Classes 0, 1, and 2 were considered highly resistant (R), 

those from Classes 3, 4, 5, and 6 were considered moderately resistant 

(M), and those from Classes 7, 8, and 9 were considered susceptible 

(S). Data from these three categories of F2, BC1P2, and BC1P3 

populations were tested for goodness-of-fit to theoretical ratios using 

the chi-square tests. 


Analysis of the data showed that plants of NC-76 exhibited high 

but nonabsolute resistance level (89% highly resistant and only 11% 

moderately resistant plants) in response to low temperatures. In 

contrast, plants of either the ‘Chipper’ or Gy 14 line showed high 

susceptibility (100% Class 9) (Table 1). 


The F1, reciprocal F1, and BC1 to the resistant parent NC-76 had 

highly resistant and moderately resistant plants, with some excess of 

highly resistant plants, which is in accordance with a dominantly 

Table 1. Segregation ratios for chilling-resistant leaves in cucumber 

seedlings of populations derived from crosses between NC-76 and ‘Chipper’, 

and NC-76 and Gy 14. 

No. of plants 

Parent/ 

segregating Segregation ratio 

Cross R M S Total Expected Obtained Chi 2 df Prob. 

P1 NC-76 47 6 0 53 100% R+M 100% R+M 

P2 Chipper 0 1 56 57 100% S 100% S 

P3 Gy14 0 0 27 27 00% S 100% S 

F1 

(P1xP2/P3) 53 30 1 84 00% R+M 100% R+M 

F1R 

(P2/P3xP1) 42 41 1 

BC1P1 

84 100% R+M 100% R+M 

(F1xP1) 49 34 0 83 100% R+M 100% R+M 

BC1P2/P3 

(F1xP2/P3) 1 45 49 95 

F2 

(F1xself) 125 247 113 485 

50%R+M : 

50%S 

1R+M : 1S 

25%R:50% 

M:25%S 

1R : 2M : 1S 

48%R+M : 

52%S 

1R+M : 1S 0.100 1 

26%R:51% 

M:23%S 

0.752 

1R : 2M : 1S0.76 2 0.684 

inherited trait. The pooled F2 population had 125 highly resistant 

plants, 247 moderately resistant plants, and 113 susceptible plants. 

These data fit a ratio of 1 highly resistant : 2 moderately resistant : 1 

susceptible with a probability of 0.684 (pooled chi square = 0.76). 

The pooled BC1 to resistant NC-76 parent was considered resistant 

(100% highly resistant and moderately resistant, with an excess of 

highly resistant plants).In the pooled BC1 to both susceptible parents 

‘Chipper’ and Gy 14, there were 1 highly resistant, 45 moderately 

resistant, and 49 susceptible plants. These data fit a ratio of 1 

moderately resistant : 1 susceptible with a probability of 0.752 (chi 

square = 0.100). 

The segregation pattern in the F2 (1R : 2M : 1S), BC1P1 (100% 

resistant plants), and BC1P2/P3 (1M : 1S) populations demonstrated 

that a high level of resistance was conferred by a single, incompletely 

dominant gene from NC-76. These results are different from the ones 

obtained by Chung et al. (2003), who suggested that chilling resistance 

was maternally inherited. It seems that there is a different genetic 

system for chilling resistance in NC-76 and ‘Chipper’. Other 

differences in the two studies include chilling-test methods and plant 


stage. In our studies, seedlings at the first fully expanded true leaf 

were subjected to a chilling treatment of 7 h at 4°C, while Chung et al. 

(2003) treated seedlings whose first true leaf was apparent but not 

fully expanded for 5.5 h at 4°C. Thus, the tests they ran were less 

severe than ours, and ‘Chipper’ showed slight resistance under those 

conditions. 

The symbol Ch is proposed to designate the incompletely 

dominant gene from C. sativus NC-76 for resistance to chilling 

conditions in accordance with gene nomenclature rules (Wehner, 

1993). The results are promising, since resistance conferred by the 

incomplete resistance gene Ch was effective under extreme chilling 

conditions. The gene should be useful in breeding programs for the 

development of new cultivars adapted to planting in the field in early 

spring. 


Chung, S. M., J. E. Staub, and G. Fazio. 2003. Inheritance of chilling injury: A 

maternally inherited trait in cucumber. J. Amer. Soc. Hort. Sci. 128(4):526–530. 

Smeets, L. and T. C. Wehner. 1997. Environmental effects on genetic variation of 

chilling resistance in cucumber. Euphytica. 97:217–225. 

Thomas J. F., R. J. Downs, and C. H. Saravitz. 2005. Phytotron procedural manual 

for controlled environment research at the southeastern plant environment 

laboratory. NCARS Tech. Bull. 244 (revised), 44 p. 

Wehner, T. C. 1993. Gene list update for cucumber. Cucurbit Gen. Coop. Rpt. 

16:92–97. 


GENES EXPRESSED DURING DEVELOPMENT 

AND RIPENING OF WATERMELON FRUIT 

A. Levi and W. P. Wechter 

USDA, ARS, U.S. Vegetable Laboratory, 2700 Savannah Highway, 

Charleston, SC 29414 

A. Davis 

USDA, ARS, P.O. Box 159, Lane, Oklahoma 74555 

A. Hernandez and J. Thimmapuram 

University of Illinois at Urbana-Champaign, Roy J. Carver 

Biotechnology Center, W.M. Keck Center for Comparative and 

Functional Genomics, 

1201 W. Gregory Drive, Urbana, IL 61801 

T. Trebitsh 

Department of Life Sciences, Ben-Gurion Univ. of the Negev, 

Beer-Sheva, 84105, Israel 

Y. Tadmor, N. Katzir, and V. Portnoy 

Agricultural Research Organization, P.O. Box 1021, 

Ramat Yishay 30095, Israel 

S. King 

Department of Horticulture, Texas A&M University, 

College Station, Texas 77845 

ADDITIONAL INDEX WORDS. cDNA, gene expression, ESTs, Citrullus lanatus 

ABSTRACT. A cDNA library was constructed using watermelon fruit (flesh) 

mRNA from 3 distinct developmental time-points and was normalized and then 

subtracted by hybridization with leaf cDNA. Random cDNA clones of the 

watermelon flesh subtraction library were sequenced from the 5’ end in order 

to identify potentially informative genes associated with fruit setting, 

development, and ripening. One-thousand and forty-six 5’-end sequences 

(expressed sequence tags; ESTs) were assembled into 832 nonredundant 

sequences, designated as “EST-unigenes.” Of these 832 EST-unigenes, 254 

(~30%) have no significant homology to sequences of other plant species. 

Additionally, 168 EST-unigenes (~20%) correspond to genes with unknown 

function, whereas 410 EST-unigenes (~50%) correspond to genes with known 

function in other plant species. Microarray analysis indicated that a large 

number of the ESTs (about 15%) are differentially expressed during the 

development of watermelon fruit. This study provides new genetic information 

for watermelon as well as an expanded pool of genes associated with fruit 

development in watermelon. These genes will be useful targets in future 

functional genomic studies dealing with watermelon fruit development. 


W 

atermelon fruits are diverse in shape and size, in rind and 

flesh color, and in flesh texture, aroma, flavor, and nutrient 

composition. Ripening watermelon fruits undergo changes 

in pigment accumulation, flavor and aromatic volatiles, conversion of 

starch to sugars, and in increased susceptibility to postharvest 

pathogens (Karakurt and Huber 2004). Significant knowledge has 

accumulated in a number of plant species with respect to genes 

associated with fruit development, including genes associated with 

cell-wall metabolism, ethylene biosynthesis, signal transduction, and 

hormones affecting fruit setting, growth, and ripening (Giovannoni 

2001). However, there is little information on genes controlling these 

processes in cucurbit fruits including watermelon. Identifying, 

mapping, and characterizing these genes will be useful to research and 

breeding efforts in this crop. 

In this study we report the development of 832 expressed 

sequenced tags (ESTs) for watermelon fruit, their classification based 

on their putative function in other plant species, and their differential 

expression during fruit development and ripening. 


PLANT MATERIAL AND RNA ISOLATION. Fruits at early 

development stage (white flesh; 12 days postpollination), at maturing 

stage (light pink flesh; 24 days postpollination), and ripe fruits (red 

flesh; 36 days postpollination) of the watermelon heirloom cultivar 

‘Illiniwake Red’ were used for RNA isolation. RNA was isolated from 

freeze-dried tissue of watermelon according to the procedure described 

by Callahan et al. (1989). 

CDNA SYNTHESIS, SIZE SELECTION, AND CLONING. cDNA 

synthesis and library construction was performed using Stratagene kits 

(Stratagene, Inc., La Jolla, CA), as described by Soares and Bonaldo 

(1998). The primary cDNA library was normalized as described by 

Bonaldo et al. (1996). The library was first normalized in order to 

enrich it with the genes that have low expression levels in the fruit 

tissue. Then the normalized library was subtracted using leaf cDNAs 

in order to enrich the library with genes that are differentially 

expressed in the fruit tissue. 

SEQUENCING OF CDNA CLONES AND ASSEMBLING OF ESTS. The 

cDNA clones were sequenced from the 5’ ends using standard T7 

primer and ABI BigDye terminator chemistry on ABI 3700 capillary 

systems (Applied Biosystems, Foster City, CA). The sequenced cDNA 

clones were designated as expressed sequenced tags (ESTs). The EST 

clusters were assembled into contigs (contiguous sequence) by 


multiple-sequence alignment, which generates a consensus sequence 

for each cluster with criteria of 95% identity over 30 nt overlap. The 

ESTs remaining in a cluster after the formation of contigs are 

designated as cluster singlets. The set of nonredundant sequences for 

the library includes the contigs, cluster-singlets, and singlets and was 

designated as “EST-unigenes.” These sequences were used to query 

the GenBank database for homologs using the Basic Local Alignment 

Search Tool (BLAST) (Altschul et al. 1990), using e-value of 0.01, to 

ensure a high level of confidence that each sequence represents a 

nonredundant gene transcript. 

MICROARRAY ANALYSIS. Each of the 832 ESTs was represented 

on the microarray chip by 15 oligos (24 bp) x 3 replications. 

Microarray design, chip production, hybridizations, staining, and data 

extractions were performed by NimbleGen, Inc. (Madison, WI). 


Of the 832 watermelon EST-unigenes analyzed, 254 (~30%) had 

no detectable homologs (E 0.1) to any other plant genomes or protein 

sequences reported so far in GenBank (Figure 1). Some of these ESTunigenes 

may represent untranslated (UTR) 3’regions (Mignone et al. 

2005). However, further studies are needed to determine if they are 

typical to watermelon and other cucurbit species. The majority of the 

watermelon fruit EST-unigenes reported in this study could be 

grouped into abundantly expressed gene families. However, a 

considerable number of the EST-unigenes could not be classified 

(Table 1; Figure 1). Extensive genome sequencing is still needed for 

cucurbit species to identify the genes that may be distinct to this 

family. 

A large number of the ESTs identified in this study are 

homologous to genes previously reported to be important in fruit 

growth and ripening in other plant species. The 1,046 random cDNA 

clones sequenced produced 832 EST-unigenes. Of these 832 ESTunigenes, 

747 were single ESTs (singletons; nonassembled sequencing 

reads), and 85 were contigs generated by computer-based assembly of 

sequence fragments from several clones (contigs). Of the 832 ESTunigenes, 

578 have significant homology to amino acid sequences 

from the GenBank nonredundant protein database. A large number of 

these homologous sequences have previously been ascribed to 

Arabidopsis proteins, and were successfully annotated using gene 

ontology (GO) analysis. The length of the ESTs ranges from 338 to 

699 bases, whereas contigs range from 555 to 2823 bases. The 

individual sequences of 1046 ESTs have been submitted to NCBI 


(Accession numbers: DV736965–DV738010, to be released on 

October 1, 2006). 

A functional class was assigned to each EST-unigene based on the 

degree of similarity (E-value) to the closest counterpart sequence 

found in other plant species. Of the 578 EST-unigenes that had 

significant homology to the nucleotide database (nt), 168 are 

homologous to genes with unknown nucleotide database (nt), 

Cell wall and 

division 

5% 

Membrane transport 

8% 

Amino acid 

synthesis 

7% 

Primary metabolism 

9% 

DNA/RNA 

Transcription 

8% 

No significant 

homology to genes 

in other plant 

species 

30% 

Signal transduction 

8% 

Defense/stress 

response 

4% 

Secondary 

metabolitesm 

1% 

Unknown function 

20% 

Fig. 1. Distribution of watermelon flesh ESTs according to their function. 

168 are homologous to genes with unknown function, while 410 are 

homologous to genes with known function. These 410 EST-unigenes, 

based on GO annotation, were assigned to one of the following 

functional classes: (1) primary metabolism (74 EST-unigenes); (2) 

amino acid synthesis and processing (57 EST-unigenes); (3) 

membrane and transport (66 EST-unigenes); (4) cell division, cell wall 

and metabolism, cytoskeleton, and cellular organization (41 ESTunigenes); 

(5) DNA/RNA transcription and gene expression (63 ESTunigenes); 

(6) cellular communication/signal transduction (70 ESTunigenes); 

(7) defense- and stress-related proteins (31 EST-unigenes); 

and (8) secondary metabolism (8 EST-unigenes) (Figure 1). 


Table 1. Expression levels of expressed sequence tags (ESTs) during 

watermelon fruit development, including early-stage (12 days 

postpollination [PP]), maturing (24 days PP), and ripened fruit (36 

days PP). The levels shown are relative to the expression level in the 

leaf, which is determined as 1.0. 

EST Early Maturing Ripe 

AL01006B2C02.f1 22.09 26.07 16.16 

AL01005A1F10.f1 22.07 13.24 11.62 

AL-Contig4 18.98 21.89 13.91 

AL-Contig47 17.45 32.16 9.39 

AL-Contig60 17.34 5.27 2.78 

AL010001000B12 15.15 10.18 3.93 

AL01005B1D07.f1 14.93 27.32 22.52 

AL-Contig9 10.24 14.15 11.72 

AL01005B2A05.f1 10.16 10.28 7.81 

AL01006A2D05.f1 10.12 8.63 7.82 

AL01006A1C09.f1 9.94 8.83 7.99 

AL01006B1H12.f1 9.61 5.84 13.00 

AL-Contig71 9.37 10.36 3.97 

AL01003X1E03.f1 9.27 16.11 11.37 

AL01005A2B07.f1 9.03 16.71 16.33 

AL010002000G10 9.00 7.17 5.03 

AL01003X1E07.f1 7.60 9.15 8.81 

AL01003X1A05.f1 7.58 2.25 0.84 

AL01005B1E08.f1 7.49 12.53 4.36 

AL-Contig41 7.30 12.03 7.93 

AL-Contig66 7.04 13.59 9.87 

AL01005A2H08.f1 7.03 7.28 7.61 

AL01006B2E04.f1 5.85 4.81 0.73 

AL01005B2F12.f1 5.66 4.89 3.81 

AL01006A2G09.f1 4.92 22.20 5.32 

AL-Contig83 4.88 1.10 0.81 

AL01005B2E08.f1 4.80 3.33 2.46 

AL010002000E03 4.70 1.67 1.48 

AL-Contig78 4.70 8.01 7.95 

AL010001000D09 4.60 2.36 0.92 

AL-Contig64 4.41 2.22 5.22 

AL01006B2F11.f1 4.34 6.23 4.74 

AL01005A2F02.f1 3.64 11.18 8.66 

AL-Contig27 3.56 16.38 3.80 

AL01005B2D01.f1 3.47 5.55 6.52 

AL-Contig56 3.45 4.04 4.07 

AL-Contig79 3.42 7.21 6.39 


(Table 1, continued) 

EST Early Maturing Ripe 

AL-Contig46 3.22 5.42 3.98 

AL01006A1H06.f1 2.80 7.01 5.01 

AL01006A2H11.f1 2.80 3.05 2.35 

AL010001000C07 2.80 2.90 2.03 

AL010002000A05 2.80 3.17 3.42 

AL010001000H02 2.74 2.70 1.80 

AL-Contig42 2.15 25.60 18.29 

AL010002000B05 2.09 2.50 2.47 

AL010002000B08 2.07 1.91 2.09 

AL01006B1A07.f1 2.06 3.58 1.55 

AL01006A1F01.f1 2.04 3.98 8.38 

AL01006B1G08.f1 1.90 2.14 2.27 

AL010001000E12 1.89 1.84 1.86 

AL-Contig15 1.81 2.30 2.03 

AL01005A2A11.f1 1.80 2.48 2.13 

AL010002000A07 1.70 1.45 1.55 

AL01004X1H02.f1 1.60 4.06 3.97 

AL-Contig73 1.56 2.06 1.94 

AL01005B2G04.f1 1.55 1.09 1.09 

AL010001000F05 1.48 3.75 2.17 

AL01005A1D10.f1 1.46 1.58 1.66 

AL01005B2G02.f1 1.43 1.70 1.67 

AL01005A2D06.f1 1.42 2.14 1.70 

AL010001000G11 1.34 2.58 2.80 

AL01006B2G09.f1 1.22 1.29 1.23 

AL01003X1C10.f1 1.22 1.79 1.49 

AL01005B2F05.f1 1.00 1.80 1.36 

AL01006B1G06.f1 1.00 1.51 1.36 

AL01005A2E07.f1 0.99 1.27 1.18 

AL01006A1C10.f1 0.99 1.64 1.56 

AL01004X1B12.f1 0.96 1.09 0.87 

AL01004X1A07.f1 0.96 1.52 1.25 

AL010002000H11 0.94 1.64 1.24 

AL01006B1B11.f1 0.93 1.48 1.50 

AL01005A1G08.f1 0.83 1.16 1.03 

AL-Contig32 0.83 3.79 1.82 

AL01003X1E11.f1 0.73 0.82 0.80 

AL01005B1G08.f1 0.60 0.84 0.62 


RNA from each fruit stage including early development stage 

(white flesh; 12 days postpollination), maturing stage (light pink flesh; 

24 days postpollination), ripe fruit (red flesh; 36 days postpollination), 

and young leaf was used for hybridization with the microarray chip 

containing the 832 EST oligos (15 oligos of 24 bases each; 3 

replications for each oligo). One-hundred and thirty-six ESTs showed 

high differential expression (expression level of 3–22 folds compared 

with the leaf RNA) during watermelon fruit development (as shown 

for a sample of ESTs in Table 1). All other ESTs (696) showed low 

expression (expression levels ranging from 0.11 to 2.9 folds compared 

with that in the leaf) (as shown in Table 1). These results 

indicate that normalizing the cDNA library, and subtracting the fruit 

fleshcDNA library with a leaf cDNA, was effective in obtaining 

cDNA clones that are differentially expressed, and at the same time 

obtaining cDNA clones that have low expression (in a low-copy 

number). The results indicate that about 10–15% of ESTs (watermelon 

genes) are differentially expressed during watermelon fruit 

development and ripening. Watermelon may have a large number of 

ESTs with no significant sequence homology to gene sequences in 

other plant species reported an NCBI. The genes discovered in this 

study will be useful in future functional genomic studies dealing with 

watermelon fruit development and ripening. 


Alba, R., Z. Fei, P. Payton, Y. Liu, S. L. Moore, P. Debbie, J. Cohn, M. D'Ascenzo, 

J. S. Gordon, J. K. Rose, G. Martin, S. D. Tanksley, M. Bouzayen, M. M. Jahn, 

and J. Giovannoni. 2004. ESTs, cDNA microarrays, and gene expression 

profiling: tools for dissecting plant physiology and development. Plant J. 

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local alignment search tool. J. Mol. Biol. 215:403–410. 

Bonaldo, M. F., G. Lennon, and M. B. Soares. 1996. Normalization and subtraction: 

two approaches to facilitate gene discovery. Genome Res. 6:791–806. 

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RNAs from developing peach fruit. HortSci. 24:356–358. 

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Physiol. Plant Mol. Biol. 52:725–749. 

Karakurt, Y. and D. J. Huber. 2004. Ethylene-induced gene expression, enzyme 

activities, and water soaking in immature and ripe watermelon (Citrullus 

lanatus) fruit. J. Plant Physiol. 161:381–388. 

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Harbor, NY. 2:49–157. 


RESEARCH OF MOLECULAR MARKERS 

LINKED TO THE DWARF GENE IN SQUASH 

Haizhen Li, Haiying Zhang, and Guoyi Gong 

National Engineering Research Center for Vegetables (NERCV), 

Banjing, Haidian, Beijing 2443#, Beijing, 100089, P. R. China; 

Yunlong Li and Chongshi Cui 

Department of Horticulture, Northeast Agricultural University, 59 

Mucai Street, Gongbing Road, Xiangfang, Haerbing, Heilongjiang 

province, 150030, P.R. China 

ADDITIONAL INDEX WORDS. Near isogenic line, dwarf gene D, RAPD, SCAR 

ABSTRACT. In this study, the first discovered dwarf mutant (Ai 10) of Cucurbita 

moschata Duchesne was used as a donor parent and the normal C. maxima 

Duchesne (cv. MengRi) was used as a recurrent parent. Nine pure lines of the 

near isogenic lines (NILs) S1 through S9 were obtained through six backcrosses 

and two self-crosses. 

W 

ith the development of the world economy, rapid increase of 

global population, and decrease in arable land, an increase in 

food production per unit of land is especially important. 

Short-straw and half short-stalked crops with short and thickset stems 

that do not easily fall over can improve crop yields significantly (Liu, 

2005). With the development of intensive-cultivation agriculture, there 

is an urgent need for more dwarfing stock varieties and dwarf cultivars. 

Recently, many good dwarf and half-dwarf varieties have been 

developed, and some dwarf genes of Rht short-straw wheat and rice and 

a half short-straw sd-1 gene and Arabidopsis GA3 have been cloned 

(Peng et al., 1999; Sasaki et al., 2002; Helliwell et al., 1998). Because 

the mechanisms of short-straw and half-dwarf straw genes are not 

understood completely, their applications in crop improvement are 

limited. A search for new sources of dwarf genes revealed the dwarf 

China pumpkin (Cucurbita moschata Duchesne). This rare resource, 

found in Shanxi Province in 1982, is the first dwarf mutant to be 

discovered in the world. Up to now, Chinese and foreign botanists have 

described the Chinese pumpkin (Cucurbita moschata D.) as having long 

This research was supported by the Natural Science Foundation of Beijing (5042008 ) 

and by the Special Foundation Support of Beijing’s Excellence person. 


vines. The discovery of a dwarf mutant has enriched the germplasm 

resources of pumpkin greatly. It may become a standard for breeding 

dwarf pumpkin. It has been proven that the short-vine and long-vine 

characteristics of Chinese pumpkin (C. moschata D.) are controlled by a 

pair of corresponding hereditary characters. Vinelessness (dwarfism) is 

a dominant gene (indicated with D), while long-vining is a recessive 

gene (indicated with d) (Zhou and Li, 1991). Mapping and cloning this 

gene may be helpful in understanding its mechanism, enriching plant 

genetic resources, and in developing dwarfs through genetic 

engineering, leading to improved varieties. 

Cloning and separation of genes often depends on the molecular 

genetic marker tightly linked to the gene. In this study, our objective 

was to find a marker that is tightly linked to the gene for dwarfism using 

near isogenic lines (NILs ) and RAPD technology (Fang et al., 2002). 


PLANT MATERIALS. Plant materials were provided from the 

pumpkin-breeding laboratory of the National Engineering Research 

Center for Vegetables (NERCV) in China. The plants used included 

‘MengRi’ (C. maxima Duchesne), the dwarf mutant Ai 10 (C. moschata 

Duchesne), nine pure NILs designated S1 through S9 (obtained through 

six backcrosses and two self-crosses) and 306 F2 individuals derived 

from the single-cross progeny of the NIL S2×‘MengRi’ and five 

long-vine C. maxima. 

METHODS. Genomic DNA was extracted from young expanding 

leaves following the CTAB protocol (Murray and Thompson, 1980). 

DNA concentration and quality were estimated by agarose gel 

electrophoresis. Final concentrations were adjusted to 30ng/ul. PCR 

reactions for RAPD markers were performed using standard procedures 

in the DNA PTC-100 (Dongsheng Chuangxin Biotechnology Co., Ltd). 

Products were analyzed on 1.5% agarose gels using a Kodak EDAS-120 

for imaging analysis and recording the polymorphic fragment. 

Data were analyzed Using JoinMap3.0 software (Van Ooijen and 

Vorrips, 2001) to calculate the genetic distance between the RAPD 

marker and the dwarf gene D. The data were denominated according to 

the rules of the JoinMap3.0 software. RAPD reamplification was 

performed using the marker linked to dwarf gene D. The product was 

detected on a 1% agarose gel. Bands were excised and purified by the 

Takara Agarose Gel DNA Purification Kit. PCR fragments were 

subcloned using the Promega PGEM-T easy Vector system. The DNA 

of the recombinant plasmid was verified by PCR after enzyme 

digestion. 


Subcloned fragments were sequenced in ShangHai ShengGong 

Biological Engineering Services Limited and AoKe Biological 

Engineering Services Limited. 

PCR amplification was performed using SCAR primers on parents, 

nine pure lines of the NILs and 306 F2 generation plants, and visualized 

by agarose gel electrophoresis. The standard molecular weight marker 

is GeneRuler TM 100bp DNA Ladder Plus (MBI packing). 

Results 

ANALYSIS OF GENETIC DWARF GENE D. The experiments used a 

total of 306 separated F2 generation plants. Our field survey determined 

that 223 of the plants were dwarf and 83 were long-vine plants. We 

calculated X 2 =0.736< X 2 (0.05,1) =3.841, which is consistent with a 3:1 

separated ratio. This data suggests that the dwarf character is controlled 

by a single dominant gene, supporting the conclusions of Zhou et al. 

(1991). 

RAPD PRIMER SCREENING. Through screening 640 randomly 

primed amplifications, a total of 13 RAPD primers generated specific 

repeatable bands that appeared to associate with the drawf gene D 

phenotype. Selecting seven dwarf and seven long-vine plants randomly 

from the F2 progenies, we performed initial link analysis. A RAPD 

fragment amplified using primer S1225 (band 548bp, and named 

S1225-548) was present in the dwarf but not in the long-vine plants 

(Figure 1). The primer S1225 was used to carry out RAPD 

amplification and certification of 306 F2 plant progenies. Of these, 

seven plants had a dwarf phenotype inconsistent with the predicted 

RAPD band (Table 1). These seven plants allowed us to map the 

distance between the RAPD marker and the dwarf gene D to 2.29 cM. 

The appearance of seven plants inconsistent with the predicted RAPD 

band may be due to an exchange between markers with dwarf gene D, 

which lost the linkage relationship. We further verified primer S1225s 

linkage to the dwarf gene D by screening the eight original NILs (Figure 

2) and the six C. maxima (Figure 3). 

LINKAGE ANALYSIS OF RAPD MARKER AND DWARF GENE D. The 

F2 generation was analyzed with JoinMap3.0 software. The results 

suggest that the dwarf gene D has a genetic distance of 2.29cM to 

marker S1225-548. A SCAR marker (SCAR3-398) was developed from 

the sequencing of the S1225-548 RAPD marker using these primers: 

upstream primer sequence 5’-CTTGTCCATTCCTCCTCC-3’, and 

downstream primer sequence 5’-TCCACTCCCTCTTTTTCA-3’. The 

amplification product fit to the RAPD expansion profile (Figure 4). 


Fig. 1. Patterns of ‘MengRi’ Ai10 S2 and some F2 individuals with RAPD primer 

S1225 (arrow indicates marker S1225-548). M = Marker; 1= ‘MengRi’; 2 = Ai10; 

3 = S2; 4–10 = normal plants in the F2 population; 11–17 = dwarf plants in the F2 

population. 

Fig. 2. Patterns of ‘MengRi’, Ai10, S2, and the other eight pure lines of NILs with 

RAPD primer S1225 (arrow indicates the marker S1225-548). M = Marker; 1= 

‘MengRi’; 2 = Ai10; 3 = S2; 4–11 = the other eight pure lines of the NILs (S1, 

S3–S9). 


Fig. 3. Patterns of ‘MengRi’, Ai10, S2 and the cultivars of C. maxima with RAPD 

primer S1225 (arrow indicates the marker S1225-548). M = Marker; 1= 

‘MengRi’; 2 = Ai10; 3 = S2; 4–8 = the other five cultivars of C. maxima. 

Fig. 4. SCAR patterns of ‘MengRi’, Ai10, and some F2 individuals with primer 

SCAR3 (arrow indicates the marker SCAR3-398). M = Marker; 1= ‘MengRi’; 

2 = Ai10; 3–10 = normal plants in the F2 population; 11–14 = dwarf plants in the 

F2 population. 


Table 1: Cosegregation between S1225-548 and phenotype in the F2 

population from MengRi×Ai10 progenies. 

S1225-54 

Genetic 

Phenotype No. plant Presence Absence distance 

Long 83 4 

79 

Dwarf 223 220 3 

2.29cM 

Discussion 

Dwarf gene D is a dominant gene, though growing conditions can 

affect the growth habit of seedlings, sometimes making it difficult to 

accurately pheontype the plants for up to 35 days. 

Our RAPD and SCAR markers can be a useful tool to verify the D 

phenotype at the seedling stage. Bulked segregation analysis and NIL 

analysis allowed the linking of our RAPD marker to 2.29cM from the 

objective gene. Through theoretic calculation, the proportion of 

transmigration parents should be 98.438%. Currently we are screening 

more backcrosses to find a closer RAPD marker. 


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REACTION OF MELON PI 313970 TO 

CUCURBIT LEAF CRUMPLE VIRUS 

James D. McCreight and Hsing-Yeh Liu 

U.S. Agricultural Research Station, U.S. Department of Agriculture, 

Agricultural Research Service, 1636 E. Alisal St., 

Salinas, California 93905 

Thomas A. Turini 

University of California Cooperative Extension, Imperial County, 

1050 E. Holton Rd., Holtville, CA 92250-9615 

ADDITIONAL INDEX WORDS. Cantaloupe, Cucumis melo, muskmelon, disease, 

Cucurbit leaf curl virus, inheritance 

ABSTRACT. Cucurbit leaf crumple virus (CuLCrV) is a recently described 

sweetpotato whitefly (Bemisia tabaci [Gennadius]) biotype-B-transmitted 

begomovirus (Geminiviridae family) of cucurbits in the desert southwest U.S.A. 

‘Top Mark’, a standard, orange-flesh, western U.S. shipping-type melon 

(Cucumis melo L.) was susceptible and expressed severe symptoms in two 

naturally infected field tests in Imperial Valley, CA, in 2003 and 2004. PI 

313970, a kakri-type melon, exhibited resistance to CuLCrV in these tests; it 

was asymptomatic in 2003, but 20 of 30 plants evaluated in 2004 expressed 

symptoms. The difference in the reaction of PI 313970 could be due to seasonal 

differences in symptom expression, disease pressure, recovery from initial 

infection, and additional experience in assessing CuLCrV symptoms. Diseasesymptom 

data from a cross of ‘Top Mark’ and PI 313970 (F1, F2, and 

backcrosses to each parent) indicated recessive genetic control of the resistance 

reaction of PI 313970 to CuLCrV. 

S 

weetpotato whitefly, Bemisia tabaci (Gennadius), adversely 

affects yield and quality of a wide range of vegetable and 

agronomic crops worldwide directly through feeding damage or 

indirectly as a virus vector (Henneberry et al., 1998). Three new B. 

tabaci biotype-B-transmitted begomoviruses of cucurbits were 

observed in commercial cucurbit fields in the southwest U.S.A., 

northern Mexico, and Guatemala beginning in 1998. Cucurbit leaf 

crumple virus (CuLCrV) was observed in Imperial Valley, California, 

first on watermelon (Citrullus lanatus [Thunb.] Matsum. & Nakai) in 

1998 (Guzman et al., 2000; Hernandez et al., 2000), and then on melon 

We thank Patti Fashing for assistance in the field and greenhouse tests, and Jeff Wasson and 

John Sears for assistance in the greenhouse and laboratory tests. Mention of a trade name, 

proprietary product, or specific equipment does not constitute a guarantee or warranty by the 

USDA and does not imply its approval to the exclusion of other products that may be suitable. 


(Cucumis melo L.) in 1999 (Guzman et al., 2000). Cucurbit leaf curl 

virus (CuLCV) occurred on pumpkin (Cucurbita spp.), honeydew, and 

muskmelon in Arizona, Texas, and Coahuilla, Mexico, in 1998 

(Brown et al., 2000). Melon chlorotic leaf curl virus (MCLCV) 

occurred on melon in Guatemala in 2000 (Brown et al., 2001). 

CuLCrV and CuLCV are identical based on percent nucleotide and 

amino acid similarities for open reading frames, and are distinct from 

Squash leaf curl virus (SLCV) on Cucurbita spp. (Flock and Mayhew, 

1981) and MCLCV (Brown et al., 2002). CuLCrV symptoms are 

variable and include stunted and chlorotic tips and leaves (Figure 1A, 

B), crumpling (Figure 1B, C), and interveinal yellowing of leaves 

(Figure1D). 

C D 

Fig. 1. Cucurbit leaf crumple virus (CuLCrV) in a naturally infected field test: 

(A) susceptible plant showing stunted and chlorotic growing points; (B) close-up 

of chlorotic terminal bud and crumpled leaves; (C) crumpled leaves; and (D) 

interveinal yellowing. 


Although the foliar symptoms have been widespread and severe 

during the fall cantaloupe season, from August to November, the 

presence of CuLCrV has not been associated with obvious decreases in 

cantaloupe yield or quality in Imperial Valley (T. A. Turini, 

unpublished). Severe and uniform infection of melons was observed in 

the fall of 2003 at the University of California, Desert Research and 

Education Center, Holtville (DREC), and the University of Arizona, 

Yuma Agricultural Research Center, Yuma. Symptoms were similar to 

SLCV, CuLCrV, and CuLCV, and were preceded by unusually high 

numbers of B. tabaci biotype B at the two research stations, 

documented at the Yuma site (J. C. Palumbo, unpublished data). 

Population density trends between Yuma and Imperial sites are similar 

(Yee et al., 1997). 

We report here data on the inheritance of the resistant reaction of 

PI 313970 to CuLCrV in two naturally infected field tests. PI 313970 

is a kakri-type melon from India that possesses resistance to B. tabaci 

biotype B (Boissot et al., 2000), Lettuce infectious yellows virus 

(LIYV) (McCreight, 2000), and powdery mildew incited by 

Podosphaera xanthii (syn. Sphaerotheca fuliginea) (McCreight, 2003). 


VIRUS CONFIRMATION AND IDENTIFICATION. Symptomatic melon 

leaf samples were taken from ‘Top Mark’ in both years. Total nucleic 

acids were isolated from 100mg of infected melon leaf tissue 

according to the method described by Li et al. (1998). The polymerase 

chain reaction (PCR) parameters used to amplify the CuLCrV DNA 

fragment were as follows: an initial denaturing step at 94°C for 5 min 

followed by 94°C, 40 sec for denaturing; 60°C, 40 sec for annealing; 

and 72°C, 1 min for extension for 35 cycles, with a final extension step 

of 72°C for 10 min. PCR-amplified DNA fragments were analyzed by 

electrophoresis in 1% agarose gels in 1X TAE buffer (40mM Trisacetate 

and 2mM EDTA, pH 8.0), stained with ethidium bromide and 

visualized with UV light. The primer pairs FA-908 (5’- 

ACCCCGTGTATGCGACATTG-3’), RA1-1419 (5’-CGACGAATA 

GACTTGGACTGCG-3’), and RA2-1601 (5’- 

AGGAATCCCATCAATCGTGC-3’) were designed from the 

sequence of CuLCrV DNA-A component (GenBank accession no. 

AF224760) to amplify AC2 and AC3 open reading frames. The 

specific primer pair for CuLCrV, FB-1324 (5’- 

TTCTTCTGGTAAAATATGGC-3’) and RB-2370 (5’- 

CCGACGAGATATGTCAACG-3’), was designed from the sequence 

of CuLCrV DNA-B component (GenBank accession no. AF224761). 


The amplified products from AC2 and AC3 open reading frames were 

purified using QIAquick Gel Extraction Kit (QIAGEN Inc. Valencia, 

CA) and sequenced by a commercial company (MCLAB, South San 

Francisco, CA). Sequences were analyzed by the program MacVector 

7.0 (Accelrys Inc., San Diego, CA). 

INHERITANCE STUDY. Two naturally infected field tests were 

conducted in the fall melon seasons of 2003 and 2004. The studies were 

planted at DREC on 11 Sept. 2003 and 15 Sept. 2004, and irrigated, as 

needed, using subsurface drip. Each plot was 366cm long and consisted of 

three hills spaced 91cm apart with 91-cm buffers on each end; beds were 

203cm wide. Two seeds were planted per hill and thinned to one seedling 

at the first-true-leaf stage of growth. Each of the seven replications 

included one plot of each parent, one plot each of four reciprocal F1 

progenies (one TM x PI and three PI x TM), five plots of one F2 progeny, 

one plot of the backcross to ‘Top Mark’ (BCTM), and two plots of the 

backcross to PI 313970 (BCPI). Plants were inoculated by naturally 

occurring B. tabaci biotype B. Disease symptoms were recorded on 20 

Nov. 2003 and 7 Nov. 2004, and noted as present or absent. 

All F1, F2, and backcross progenies used in the field and greenhouse 

tests were produced using standard hand-pollination techniques for melon 

(Robinson and Decker-Walters, 1997). Data were analyzed using chisquare. 

Yates correction was used for the analysis of the 2003 data from 

the F2 and BCPI families where the number of expected segregants in one 

class was less than 20 (Yates, 1931). 


VIRUS CONFIRMATION AND IDENTIFICATION. The CuLCrV-specific 

primer pair from BC1 region was amplified by PCR. A product size of 

about 1000bp was expected (Figure 2, lane 3). No products were found in 

similar preparations from healthy plants (Figure 2, lane 2). The expected 

product sizes from primer pairs used to amplify AC2 and AC3 open 

reading frames were obtained (Figure 2, lanes 4 and 5). The sequence 

analysis of AC2 and AC3 revealed a high degree of sequence identity with 

CuLCrV (100% and 99.0% for nucleotides and 100% and 97.7% for 

amino acids, respectively). The PCR and sequence analyses confirmed 

that the virus present in the inheritance studies was CuLCrV. 

INHERITANCE OF RESISTANCE IN PI 313970. ‘Top Mark’ was 

highly susceptible in both years (Table 1) and exhibited pronounced 

CuLCrV symptoms (Figure 1). PI 313970 was asymptomatic in 2003, 

but 20 of the 31 plants in the test were symptomatic in 2004 (Table 1). 

PI 313970 was, therefore, not immune to CuLCrV. The difference in 

the observed reactions of PI 313970 in the two years could be due to 


one or more factors, including seasonal differences in symptom 

expression, disease pressure, recovery from initial infection, and 

additional experience in assessing CuLCrV symptoms. 

In 2003, whitefly and disease pressures were very high. CuLCrV 

generally occurred in isolated fields and to a very limited extent during 

May and June (Turini, unpublished). The planting in 2003 was subject 

to very high whitefly pressure and it is unlikely that PI 313970 escaped 

infection due to low or nonuniform insect pressure. Disease recovery 

may be a factor. Cucurbita spp. were observed to recover from SLCV, 

Table 1. Reactions of ‘Top Mark’, PI 313970, and their F1, F2, and 

backcross families to natural infection by Cucurbit leaf crumple virus 

in two field tests, Holtville, CA, Fall 2003 and Fall 2004 z . 

Entry 

Asympto- 

matic 

Sympto 

matic - Expected χ 2 P 

2003 

Top 

Mark 

(TM) 0 15 all S 

PI 

313970 

(PI) 18 0 all A 

F1 TM x PI 1 17 all S 

F1 PI x TM 2 52 all S 

F2 15 64 1 A:3 S 1.09 0.30 

BCTM 0 15 all S 

BC1PI 15 20 1 A:1 S 0.46 0.49 

2004 

Top 

Mark 

(TM) 3 17 all S 

PI 

313970 

(PI) 11 20 all A 

F1 TM x PI 6 24 all S 

F1 PI x TM 4 28 all S 

F2 38 129 1 A:3 S 0.08 0.78 

BCTM 2 19 all S 

BC1PI 23 34 1 A: 1 S 2.12 0.15 

z Yates correction applied to the 2003 analysis of F2 and BCPI segregation data. 


a related geminivirus, in field and greenhouse tests (McCreight and 

Kishaba, 1991). The 2003 test was evaluated 70 days after planting 

whereas the 2004 test was evaluated 53 days after planting. The 

additional 17 days in 2003 may have been sufficient for PI 313970 to 

recover and appear asymptomatic. 

Regardless of the discrepancy in the reactions of PI 313970 to 

CuLCrV between years, the reciprocal F1 progenies were susceptible, 

and the F2 segregated in acceptable fits to a 3 susceptible:1 resistant 

(asymptomatic) ratio in both years (Table 1), clear indication of 

recessive control of the resistance reaction in PI 313970. The 

backcrosses provided additional evidence for a recessive gene. The 

backcross to ‘Top Mark’ was susceptible with only two escapes in 

2004 (Table 1). The backcross to PI 313970 segregated in an 

acceptable fit to a 1 susceptible:1 resistant (asymptomatic) in both 

years (Table 1). 

Fig. 2. A 1.0% agarose gel showing the products of polymerase chain reaction 

(PCR) for detection of Cucurbit leaf crumple virus (CuLCrV) in melon leaf 

tissue with CuLCrV-specific primer pairs. Lane 1, BRL 1kb ladder; Lane 2, 

from healthy melon leaf tissue; Lane 3 to Lane 5 from CuLCrV-infected melon 

leaf tissue. The primer pair for Lanes 2 and 3 was FB-1324 and RB-2370; Lane 

4 was primers FA-908 and RA1-1419; Lane 5 was primers FA-908 and RA2- 

1601. 


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resistance in Cucumis melo accession 90625. Proc. 7 th EUCARPIA meeting on 

Cucurbit Genetics and Breeding. Acta Hort. 510:297–304. 

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cucurbit-infecting Begomovirus species capable of forming reassortants with 


elated viruses in the Squash leaf curl virus cluster. Phytopathology. 92:734– 

742. 

Brown, J. K., A. M. Idris, D. Rogan, M. H. Hussein, and M. Palmieri. 2001. Melon 

chlorotic leaf curl virus, a new begomovirus associated with Bemisia tabaci 

infestation in Guatemala. Plant Dis. 85:1027. 

Brown, J. K., A. M. Idris, M. W. Olsen, M. E. Miller, T. Isakeit, and J. Anciso. 2000. 

Cucurbit leaf curl virus, a new whitefly-transmitted geminivirus in Arizona, 

Texas, and Mexico. Plant Dis. 84:809. 

Flock, R. A. and D. E. Mayhew. 1981. Squash leaf curl, a new disease of cucurbits 

in California. Plant Dis. 65:75–76. 

Guzman, P., M. R. Sudarshana, Y. S. Seo, M. R. Rojas, E. Natwick, T. Turini, K. 

Mayberry, and R. L. Gilbertson. 2000. A new bipartite geminivirus 

(Begomovirus) causing leaf curl and crumpling in cucurbits in the Imperial 

Valley of California. Plant Dis. 84:488. 

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Aleyrodidae) in the United States history, pest status, and management. Recent 

Res. Dev. Ent. 2:151–161. 

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Generation and characterization of infectious clones of Cucurbit leaf crumple 

virus, a new bipartite geminivirus from the Imperial Valley of California. 

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techniques for detection of tomato infectious chlorosis virus. Plant Dis. 82:84– 

88. 

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melon. HortSci. 35:1118–1120. 

McCreight, J. D. 2003. Genes for resistance to powdery mildew races 1 and 2 U.S. in 

melon PI 313970. HortSci. 38:591–594. 

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Yates, F. 1931. Contingency tables involving small numbers and the χ2 test. Suppl. J. 

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249. 


THE MELON GENOMIC LIBRARY OF NEAR ISOGENIC 

LINES: A TOOL FOR DISSECTING COMPLEX TRAITS 

Antonio José Monforte, Iban Eduardo, and Pere Arús 

Laboratori de Genètica Molecular Vegetal CSIC-IRTA, Cabrils, Spain 

Juan Pablo Fernández-Trujillo, Javier Obando, 

Juan Antonio Martínez, and Antonio Luís Alarcón 

Technical University of Cartagena (ETSIA) and Institute of Plant 

Biotechnology Cartagena, Spain 

Esther van der Knaap 

The Ohio State University/OARDC, Wooster, OH 

Jose María Álvarez 

Centro de Investigación y Tecnología Agroalimentaria de Aragón, 

Zaragoza, Spain 

ADDITIONAL INDEX WORDS. Cucumis melo, QTL, fruit quality, introgression 

line, marker- assisted selection 

ABSTRACT. A doubled haploid line (DHL) population of melon derived from a 

cross between the Korean accession PI 161375 (SC) and the Spanish Inodorus 

cultivar ‘Piel de Sapo’ (PS) was used to develop a collection of near isogenic 

lines (NILs). Selected DHLs were backcrossed to PS and further backcrossing 

and selfing was performed, monitoring introgressions from SC using molecular 

markers. A final collection of 57 NILs was obtained, containing a unique 

independent introgression from SC in the PS genetic background. The 

introgressions within the collection cover at least 85% of the SC genome, with 

an average introgression size of 41cM, corresponding to 3.4% of the SC genome. 

The melon NIL genomic library was used to dissect the genetic control of melon 

fruit shape. A total of 15 quantitative trait loci (QTLs) were detected, 8 of them 

producing round melons and 7 producing elongated melons. These results 

demonstrate that the melon NIL genomic library is a powerful tool for the study 

of QTLs involved in important complex traits, introduction of new genetic 

variability in modern cultivars from exotic sources, or the development of 

precompetitive breeding lines in melon-breeding projects. 

M 

elon (Cucumis melo) is the most diversified species of the 

genus Cucumis, and this variability is reflected at the 

morphological, physiological, biochemical (Kirkbride, 

1993; Liu et al., 2004), and molecular (Stepansky et al., 1999; Mliki et 

al., 2001; Akashi et al., 2002; Monforte et al., 2003) levels. A high 

proportion of the genetic variability can be found in African, Indian, 

and Oriental germplasm, which are considered exotic relative to 

European and North American cultivars. Exotic germplasm has been 

used to search for resistance genes, but its potential as a source of new 

major gene and quantitative trait locus (QTL) alleles with favorable 


effects on fruit quality has not been thoroughly investigated. 

Furthermore, the genetic control of fruit morphological variation is 

largely unknown. 

QTLs involved in fruit-quality traits have been detected in three 

different crosses involving European cultivars and exotic Asian 

accessions (Périn et al., 2002; Monforte et al., 2004). However, a more 

thorough analysis is needed to assess the suitability of incorporating 

new alleles from exotic germplasm into elite melon cultivars. 

Werhahn and Allard (1965) demonstrated that individual QTL 

effects can be efficiently estimated using backcross inbred lines, each 

having a low proportion of the donor genome. Eshed and Zamir (1994, 

1995) further developed the idea, constructing an introgression line 

(IL) population consisting of a set of lines where each contained a 

single homozygous chromosome segment from a donor parent, in the 

genetic background of an elite cultivar. A NIL population can be 

defined as a genomic library where inserts are chromosome fragments 

of the donor-parent genome and the vector is the recurrent-parent 

genome. QTL analysis is very efficient using NIL collections (Eshed 

and Zamir, 1995). Fine mapping and cloning of genes underlying the 

QTL is more expeditious when introgression lines are used (Frary et 

al., 2000). 

In the current report, we present the construction of a NIL 

collection in melon. A Spanish ‘Piel de Sapo’ cultivar (PS), belonging 

to the horticultural group Inodorus, was chosen as the recipient 

genotype. PS fruits are very sweet, oval, white-fleshed, and 

nonclimacteric (Monforte et al., 2004). The Korean cultivar 

‘Songwhan Charmi’ accession PI 161375 (SC), included in the 

horticultural group Conomon, was chosen as the donor genotype 

The authors thank A. Montejo, A. Ortigosa, E. Moreno, F. García, E. Truque, M. C. 

García-Abellán, M. Cánovas (CIFACITA S. L.), A. B. Pérez, and N. Welty for 

technical assistance. This work was funded in part by grants AGL2003-09175-C02- 

01 and AGL2003-09175-C02-02 from the Spanish Ministry of Education and 

Science and Fondo Europeo de Desarrollo Regional (FEDER, European Union), and 

the Fundación Séneca de la Región de Murcia (00620/PI/04). AJM was supported in 

part by a contract from Instituto Nacional de Investigación y Tecnología Agraria y 

Alimentaria (INIA) and by a fellowship from the Departament d’Universitats, 

Recerca i Societat de la Informació (Generalitat de Catalunya, Spain). IE and JO 

were supported by fellowships from the Spanish Ministry of Education and Science 

and the Spanish Ministry of Foreign Affairs, respectively. Field space and personnel 

at the WO location were provided by the Ohio Research and Development Center of 

Ohio State University. Thanks are due to Semillas Fitó S. A. for providing the seeds 

of the parental PS line. 


ased on phenotypic and molecular data. The genetic distance between 

PS and SC is one of the highest described between two cultivars within 

melon germplasm (Garcia-Mas et al., 2000; Monforte et al., 2003). SC 

presents some resistance to some diseases and pests, and its fruits are 

pear-shaped, green-fleshed, with low sugar content (Monforte et al., 

2004). The NIL collection was used to study the genetic control of 

melon fruit shape. 


The development of the melon NIL genomic library is detailed in 

Eduardo et al. (2005). Briefly, 30 selected doubled haploid lines 

derived from the cross between a ‘Piel de Sapo’ (PS) genotype and the 

accession PI 161375 (SC) were backcrossed to PS. The selected 30 

DHLxPS were considered as our initial BC1 (first generation of 

backcrossing) population. All 30 BC1 genotypes were backcrossed to 

PS. Next, 12 BC1 progenies were chosen to found the BC2 generation 

(192 plants in total). BC2 plants were backcrossed to PS and genotyped 

with 65 molecular markers, covering the whole genome (see below). 

BC3 families with candidate introgressions and a lower proportion of 

donor genome were selected according to BC2 genotypes. Seedlings 

from each BC3 family were genotyped in order to select the target 

regions and discard other exotic introgressions. Several rounds of 

marker-assisted selection (MAS), backcrossing with PS, and selfpollination 

were performed to obtain a set of NILs with a single 

introgression from the SC donor genome. 

DNA was extracted as described by Klimyuk et al. (1993) and 

Garcia-Mas et al. (2000) from cotyledons and young leaves. The 

molecular markers included 62 simple sequence repeats (SSRs), one 

cleavage amplified polymorphic sequence (CAPS), and two sequence 

characterized amplified regions (SCARs) selected from the map of 

Gonzalo et al. (2005). SSRs were amplified as in Gonzalo et al. (2005) 

and visualized using a LI-COR IR 2 sequencer (Li-Cor Inc., Lincoln, 

NE). Bands were scored visually and the molecular weight of each 

band was estimated by comparing its migration on electrophoresis 

with the IRD-labelled STR molecular size marker (Li-Cor Inc., 

Lincoln, NE). The SCAR markers were genotyped as described by 

Morales et al. (2004). 

A selected set of 27 NILs was evaluated in four locations in the 

summer of 2004: Cabrils, Spain (CA), Zaragoza, Spain (ZA), 

Cartagena, Spain (CT), and Wooster, Ohio, USA (WO). In CA, 10 

plants of each NIL and 100 controls (PS) were grown in a greenhouse 

in peat bags, drip-irrigated. In ZA, four plots of 3 plants for each NIL 


and 14 plots of PS were randomized in the field. In CT and WO, 10 

plants of each NIL and 50 plants of PS were transferred to the field in 

a completely randomized design. 

Fruit-shape index (FS) was calculated as the ratio between 

maximum fruit length and maximum fruit diameter. NIL mean values 

were compared with the control genotype PS using the Dunnet contrast 

(Dunnet, 1955) with Type-I error α ≤ 0.05 for each location. 


The melon NIL genomic library includes 57 NILs, each of them 

containing a single homozygotic introgression from SC, with an 

average introgression size of 41.0cM (approximately 3.4% of the SC 

genome), ranging from 6.8 to 100.8cM (0.6% to 8.9% of the donor 

genome) in the PS genetic background (Eduardo et al., 2005). Each 

linkage group was represented by an average of 4.75 NILs with 

overlapping introgressions. The average genetic resolution to map new 

molecular markers or QTLs is 18.9cM, although the resolution can be 

increased easily by generating new recombinants from selected NILs 

allowing fine mapping or map-based cloning of QTLs (Fridman et al., 

2000). 

Three NILs were already used to verify the QTLs involved in 

several fruit traits: fruit shape (fs1.1 and fs9.1), external color (ecol9.1), 

and flesh color (gf1.1) (Monforte et al., 2004). The effects of these 

QTLs were verified using NILs with introgressions in the candidate 

regions (Eduardo et al., 2004). To assess further the suitability of the 

NIL library to dissect complex traits, the genetic control of fruit shape 

was studied with a selected set of NILs evaluated in four locations. 

The results of this experiment are summarized in 

Table 1. FS of the recurrent parent was moderately elongate. The 

overall mean of the NIL library was similar to PS; however there was 

important variation among NILs, ranging from completely round to 

extremely elongate, demonstrating the high genetic variability within 

this population for this trait (Figure 1). 

Sixteen NILs were significantly different in at least one location. 

Eight of them had fruits that were between 11 and 52% more 

elongated than the control, and the other 8 produced 10–28% rounder 

fruits. Three showed significant effects in all locations, and 5 showed 

significant effects in only one location. The minimum number of 

QTLs estimated as affecting this trait was 15. Périn et al. (2002) 

detected seven QTLs for FS, as did Monforte et al. (2004). All these 

QTLs have been detected with the NIL genomic library in at least one 

location. Furthermore, the number of detected QTLs was significantly 


Table 1. Fruit-shape index means and standard deviations for recurrent 

genotypes ‘Piel de Sapo’ (PS), the overall NIL library mean, and the 

range among NILs among four locations. 

Cabrils Cartagena Wooster Zaragoza 

PS 1.26 ± 0.08 1.39 ± 0.10 1.42 ± 0.08 1.48 ± 0.06 

NIL library 1.30 ± 0.16 1.40 ± 0.16 1.45 ± 0.21 1.45 ± 0.18 

Range 0.98–1.72 1.00–1.74 1.11–2.03 1.09–1.86 

higher using the NIL genomic library, demonstrating the high 

efficiency of this novel population to dissect complex traits in melon. 

The results reported in the present article are only a first example 

of the possible applications of the melon NIL genomic library. This 

resource could also be useful for studying traits useful to consumers, 

such as nutrition, pharmaceutical-compound content, aroma, flavor, 

texture, external aspect, shelf life, and sensitivity to internal disorders, 

as well as for detecting new allelic variability that could also be a 

resource for modern breeding programs. For example, designing 

cultivars with appropriate fruit shape for automatic fruit processing is 

critical for reducing mechanical damage, and fruit shape is also an 

important factor in consumer preferences. In the current work, some 

highly heritable QTLs modifying FS have been identified that would 

allow the design of melons adapted for different markets by 

introducing these QTLs in modern cultivars, using marker-assisted 

selection. 

The allelic variability detected in this NIL population is obviously 

only a small portion of the melon genetic variability. New crosses 

between distantly related cultivars or accessions are necessary to 

understand globally the genetic variability in melon germplasm. 

Further analysis of this NIL population, including fine mapping, 

candidate gene analysis, transcriptomics, metabolomics, and mapbased 

cloning, will provide a body of data comparable to other model 

species. 

In summary, we present a study of the genetics of fruit shape in 

melon, using a newly available melon NIL genomic library. The 

results presented here can provide a starting point for further 

characterization of the genetic factors involved in melon fruit quality. 

Future studies with this and other similar populations will allow 


efficient, systematic, and straightforward manipulation of the allelic 

variability present in melon germplasm. 

Fig. 1. Images of typical fruits from the parent genotypes PS and PI 161375 and 

from several NILs showing the variability in fruit shape within the NIL genomic 

library. 


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melon. Theor. Appl. Genet. 101:860–864. 

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P. Arus, and A. J. Monforte. (2005). Simple-sequence repeat markers used in 

merging linkage maps of melon (Cucumis melo L.). Theor. Appl. Genet. 

110:802−811. 

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(Cucurbitaceae). Parkway, Boone, NC. 

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treatment for rapid preparation of plant material for reliable PCR analysis. Plant 

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of fruit. Euphytica. 135:305–313. 

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(Cucumis melo L.): an evaluation of African germplasm. Genet. Res. Crop. Ev. 

48:587–597. 

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based on microsatellite variation. Plant Breed. 122:153–157. 

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Arus. 2004. Identification of quantitative trait loci involved in fruit quality traits 

in melon (Cucumis melo L.). Theor. Appl. Genet. 108:750–758. 

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wheat. Genetics. 51:109–119. 


MOLECULAR MARKERS ASSOCIATED 

WITH QUANTITATIVE CONTROL OF 

BETA-CAROTENE SYNTHESIS IN 

CUCUMIS MELO L. 

A. B. Napier, S. O. Park, and K. M. Crosby 

Vegetable and Fruit Improvement Center, Department of Horticultural 

Sciences, Texas A&M University, College Station, Texas, 77843 

Texas Agricultural Experiment Station, Weslaco, Texas, 78596 

ADDITIONAL INDEX WORDS. Inheritance, segregation, progeny, polymorphism 

ABSTRACT. Flesh color in muskmelon (Cucumis melo L.) is considered to be 

controlled by two genes, gf (green flesh) and wf (white flesh). The epistatic 

interactions between the genes produce orange flesh color. The intensity of 

orange flesh color, caused by the levels of beta-carotene, appears to be 

quantitatively inherited. Our study of two F2 populations, formed from the 

initial cross of ‘Sunrise’ (white fleshed) with ‘TAM Uvalde’ (orange fleshed), 

resulted in a continuous distribution of beta-carotene concentrations from high 

to low, which suggests quantitative inheritance for this trait. A chi-square test 

for our segregating F2 populations corresponded with expected segregation 

ratios. From an F2 population, two DNA bulks were composed of either eight 

high or eight low beta-carotene F2 individuals. The bulks were screened for 

detecting polymorphic molecular markers using 128 primer combinations with 

the polymerase chain reaction-amplified fragment length polymorphism 

technique (PCR-AFLP); this technique did not provide useful polymorphisms. 

Additional bulks were created using six high and six low beta-carotene F2 

individuals. These bulks were screened using random amplified polymorphic 

DNA (RAPD) primers. Of 510 primers screened, 47 produced marker 

polymorphisms between parents and bulks. 

M 

uskmelons play an important role in the American diet. 

Ranked as one of the top 10 most-consumed fruits by the 

USDA, muskmelons have the highest amount of betacarotene 

among these top 10 fruits. Beta-carotene, also called provitamin 

A, is an essential nutrient required for eye health, and may 

have the potential as an antioxidant to reduce the risks associated with 

cancer, heart disease, and other illnesses (Lester, 1997). 

This material is based upon work supported by the Cooperative State Research, 

Education, and Extension Service, U.S. Department of Agriculture “Designing Foods 

for Health” under Agreement No. 2004-34402-14768 or 2005-34402-16401. We 

would also like to acknowledge Dr. Monica Menz for her assistance with AFLP 

procedures, techniques, and supplies. 


Breeding melon varieties with increased levels of beta-carotene 

will benefit consumer health. Many phytonutrients are most 

bioavailable when consumed in their fresh form, rather than as vitamin 

supplements. The higher level of beta-carotene found in some melons 

has a genotypic component, which may be used to breed melons high 

in beta-carotene. Molecular markers and marker-assisted selection 

(MAS) can be used to increase the efficacy of the breeding process, 

while lowering breeding costs. Developed markers can also be used to 

easily screen existing lines for high beta-carotene content. 


An F2 population was created by self-pollinating F1 progeny of 

‘Sunrise’, the white-fleshed female parent, crossed with ‘TAM 

Uvalde’, a high beta-carotene variety. A field population consisting of 

117 F2 individuals and a greenhouse population containing 90 F2 

individuals were grown. The resulting fruit samples were subjected to 

hexane extraction followed by spectrophotometric analysis (Fish et al., 

2002) and ranked according to beta-carotene content. Genomic DNA 

was extracted from young leaf tissue by the method of Skroch and 

Nienhuis (1995). Two eight-plant DNA bulks composed of either high 

or low beta-carotene F2 individuals were screened for polymorphic 

molecular markers (Michelmore et al., 1991). Primers based on 

EcoRI+3 and MseI+3 were used to create 128 combinations, which 

were screened using the amplified fragment length polymorphism 

(AFLP) technique described by Vos et al. (1995). 

Additional bulks were created consisting of six high and six low 

beta-carotene F2 individuals. Random amplified polymorphic DNA 

(RAPD) primers were also used to screen between bulks, using the 

method described by Skroch and Nienhuis (1995). A total of 510 

RAPD primers were screened. 


The levels of beta-carotene found in the parents were as expected. 

‘Sunrise’ had consistently low levels while the beta-carotene quantities 

found in ‘TAM Uvalde’ averaged about 26µg/g. Beta-carotene levels 

within the F2 populations were quite variable, and in some cases much 

higher than ‘TAM Uvalde’ (Table 1). This indicates that the quantity 

of beta-carotene produced in each progeny fruit is quantitatively 

controlled. 

The segregation ratios of our F2 populations further confirm the 

results presented by Clayberg (1992) and Monforte et al. (2004) (Table 

2). If orange flesh is always dominant to white and green flesh color, 


Table 1. Beta-carotene content in fruit of field and greenhouse F2 

populations. 

Beta- 

Generation/ 

Average beta- Standard carotene 

description 

carotene, μg/g deviation range, μg/g 

P1, Sunrise 1.41 0.41 

P2, TAMU 25.91 8.20 

F2, (P1 × P2) greenhouse 12.81 9.32 1.13-40.17 

F2, (P1 × P2) field 15.32 9.36 1.41-50.25 

Table 2. Expected and actual melon-flesh color segregation ratios 

Generation/ 

Description Total Orange White Green 

P1, Sunrise 9 0 9 0 

P2, TAMU 6 6 0 0 

F2, (P1 × P2) 

green-house 

79 58 13 8 

F2, (P1 × P2) 

field 

117 92 12 13 

Ratios χ2 

Expected Actual Accept Ho Actual ά 

F2 g-house 

o:w:g 12:3:1 12:3:1 5.99 2.15 0.05 

o:no z F2 field 

3:1 3:1 3.84 0.11 0.05 

o:w:g 12:3:1 7:1:1 5.99 9.14 0.05 

o:no 

z 

no = not orange. 

3:1 3:1 3.84 0.82 0.05 

then a simple segregation of 3:1 (orange: white+green) is expected. 

The segregation ratios for both F2 populations match this. The 

epistatic interaction between the genes wf and gf create the three 

observed flesh colors in a ratio of 12:3:1. The segregation ratio of the 

greenhouse F2 population corresponds to this ratio. The chi-square test 

indicates that the field-grown F2 population did not have the expected 

segregation ratio of 12:3:1. The variation found between the field and 

greenhouse populations could be caused by uncontrollable 

environmental factors or, as mentioned by Clayberg (1992), the 

inherent difficulties associated with differentiating white and green 

flesh color. The ideal ratios from the greenhouse plants are also 


indicative of the previously assumed parental genotypes. To achieve 

the 12:3:1 ratio, the genotypes of ‘TAM Uvalde’ and ‘Sunrise’ would 

be wf+wf+/gf-gf- and wf-wf-/gf+gf+, respectively. 

After performing the initial AFLP primer screenings, no useful 

polymorphic data could be obtained. Of the 510 random primers 

screened, 47 primers have generated marker polymorphisms; an 

example of RAPD marker OAC09.900 is shown in Figure 1. Of those 

markers, 19 were derived from ‘Sunrise’ and 28 were derived from 

‘TAM Uvalde.’ Further screening of these primers is needed to 

determine their significance throughout our F2 populations, and their 

potential utility for marker-assisted selection within other breeding 

lines. The 47 RAPD primers generating clear polymorphic bands will 

be used to screen the entire greenhouse F2 population. RAPD markers 

demonstrating linkage to the high beta-carotene phenotype within the 

greenhouse population, will be screened within the field population to 

determine the extent of environmental affects on relevant genes or 

QTL. 

1 2 3 4 5 

- 1500 bp 

- 900 bp 

- 600 bp 

Fig. 1. RAPD marker OAC09.900 expressing polymorphism between two DNA 

bulks from high and low beta-carotene F2 plants. 1 = Sunrise (low parent); 2 = 

TAM Uvalde (high parent); 3 = DNA bulk from low beta-carotene F2 plants; 4 = 

DNA bulk from high beta-carotene F2 plants; and 5 = molecular size marker. 

Secondary evaluations of the primer combinations used in the 

RAPD screenings are needed before firm conclusions can be drawn 


egarding genetic linkage relationships. Our current findings comply 

with already well-known flesh-color genes, but we are still pursuing 

the quantitative genetic aspects of beta-carotene content. 


Clayberg, C. D. 1992. Interaction and linkage tests of flesh color genes in Cucumis 

melo L. Cucurbit Gen. Coop. 15:53–54. 

Fish, W. W., P. Perkins-Veazie, and J. K. Collins. 2002. A quantitative 

assay for lycopene that utilizes reduced volumes of organic solvents. 

J. Food Comp. Anal. 15:309–317. 

Lester, G. E. 1997. Melon (Cucumis melo L.) fruit nutritional quality and health 

functionality. HortTech. 7:222–227. 

Michelmore, R. W., L. Paran, and R. V. Kesseli. 1991. Identification of markers 

linked to disease resistance genes by bulked segregant analysis: a rapid method 

to detect markers in specific genomic regions using segregating populations. 

Proc. Natl. Acad. Sci. USA. 88:9828–9832. 

Monforte, A. J., M. Oliver, M. J. Gonzalo, J. M Alvarez, R. Dolcet-Sanjuan, and P. 

Arus. 2004. Identification of quantitative trait loci involved in fruit quality traits 

in melon (Cucumis melo L.). Theor. Appl. Genet.108:750–758. 

Skroch, P. W. and J. Nienhuis. 1995. Qualitative and quantitative characterization 

of RAPD variation among snap bean genotypes. Theor. Appl. Genet. 

91:1078–1085. 

Vos, P., R. Hogers, M. Bleeker, M. Reijans, T. van de Lee, M. Hornes, A. Frijters, 

J. Pot, J. Peleman, M. Kuiper, and M. Zabeau. 1995. AFLP: a new technique 

for DNA fingerprinting. Nuc. Acids Res. 23:4407–4414. 


A NEW EFFECTIVE TECHNIQUE FOR 

PRODUCING SEEDLESS WATERMELON 

FRUITS FROM SOME DIPLOID CULTIVARS 

S. Omran 

Horticulture Research Institute Breeding and Genetics of Vegetable 

Dept. Agriculture Research Center, Egypt 

K. Sugiyama 

National Agricultural Research Center for Hokkaido Region 

Hitsujigaoka, Toyohira-ku, Sapporo, 062-8555, Japan 

A. Glala 

Horticulture Technology Department 

National Research Center, Cairo, Egypt 

ADDITIONAL INDEX WORDS. Tetraploid, pollen grains, pollenizer 

ABSTRACT. In this paper, we present a new technique for the production of 

diploid seedless watermelon (Citrullus lanatus) from some diploid watermelon 

cultivars by using tetraploid plants as the pollen source. Diploid seedless 

watermelon fruits were obtained from ‘Crimson Sweet’ and ‘Aswan’ when their 

female flowers were pollinated with pollen grains from tetraploid watermelon 

(Lines SS100 and SS200). However, this technique was not effective with those 

cultivars producing large seeds, such as ‘Giza1’ and ‘Targi’. Large, empty seed 

coats were obtained in their fruits when their female flowers were pollinated 

with pollen grains from tetraploid watermelon (Lines SS100 and SS200). Almost 

no difference was obtained between seeded and seedless fruits of diploid 

watermelon, with regard to appearance, fruit maturity, fruit weight, fruit 

shape, flesh color, and rind thickness. The exception was that seedless fruits 

recorded a slightly higher percentage of total soluble solids. No hollow heart or 

misshapen fruits were found when the new technique was used. This new 

technique has potential utility for commercial production of inexpensive 

seedless watermelon fruits. 

W 

hile seedless watermelon fruits have become very popular 

in the developed countries because they are easier to eat 

(Sugiyama and Morishita, 2000), seedless watermelon fruits 

are still very expensive for most watermelon consumers around the 

world due to high production costs. Moreover, the breeding of 

commercial triploid cultivars is more time consuming, challenging, 

and expensive than the breeding of diploid cultivars (Kihara 1951). 

Hence the diversity of triploid watermelon genetics is not as large as 

that of the diploid. To overcome this problem, many have investigated 

the potential of producing seedless watermelon fruits from diploid 


plants directly. Seedless watermelon fruits can be produced by using 

plant growth regulators such as NAA (1-naphthalene acetic acid) 

(Buttrose and Sedgley, 1979) and CPPU (N-[2-chloropyridyl]-Nphenylurea) 

(Hayata et al., 1995; Kano, 2000). However, using plant 

growth regulators has occasionally resulted in deformed fruits, because 

the female flowers (ovaries) were injured by directly spraying or 

rubbing growth regulators on them. This method also poses potential 

food-safety problems, and therefore has not been used. Producing 

seedless watermelon fruits on diploid plants by using pollen irradiation 

with soft-X-ray has been recently developed (Sugiyama and Morishita, 

2000; Watanabe et al 2002). This method makes it possible to produce 

seedless fruits from any diploid cultivars with no loss in fruit quality. 

One of the shortcomings of the technique lies in the occurrence of 

empty seed coats in the fruit (Watanabe, et al., 2002). The number and 

size of empty seed coats varies among cultivars (Sugiyama and 

Marishata, 2000). Also the application of pollen irradiation with soft- 

X-ray can be implemented only in specialized laboratories because of 

safety and health considerations. 

The current study aims to develop a simple technique for 

producing seedless fruits from diploid cultivars for commercial 

production in the fields that can easily and safely be applied by small 

producers and that will eliminate the potential danger of using either 

growth regulators or soft X-ray-irradiation. 


Seedlings of four diploid watermelons cultivars (‘Giza1’, ‘Crimson 

Sweet’, ‘Targi’, and ‘Aswan’ F1) and two tetraploid lines (SS100 and 

SS200) were transplanted in the field at a spacing of 1m in-row and 

2m between rows on 1 March in 2004 and 2005. Five plants of each 

genotype were transplanted in every treatment. Three replications in 

complete randomized block design were conducted. Other agriculture 

practices were carried out based on commercial recommendations for 

the growing area. At flowering stage, each male flower of the 

tetraploid plants and each female flower of the diploid plants was 

covered with a paper bag the day before anthesis. All bagged male 

flowers of the tetraploid plants were collected in the morning on the 

day of anthesis and used for hand-pollinating the bagged diploid 

female flowers for 40 days. In addition to the bagged flowers that were 

pollinated, some female flowers were artificially self-pollinated on all 

diploid genotypes as a comparison (control) treatment. All handpollinated 

female flowers were quickly re-covered with a bag for 3–5 

days to prevent contamination with insect-borne pollen (Sugiyama and 


Morishita 2000). Mature diploid fruits of ‘Giza 1’ were harvested 45 

days postpollination; ‘Crimson Sweet’ and ‘Aswan’ 40 days; and 

‘Targi’ 35 days. 

Fruit weight, shape, rind thickness, sugar content (total soluble 

solids; TSS), and hollow heart were recorded for all treatments on the 

day of harvesting. All recorded data were analyzed using the Statistical 

Analysis System (SAS Version10.2; Cary, NC). 


The response of the four diploid genotypes to the tetraploid 

pollenizer is shown with respect to the number of fully developed 

(hard) and empty (seed coats only) seeds per fruit. Note the hard seed 

produced with the diploid pollenizers and no seed produced with the 

tetraploid pollenizers in ‘Crimson Sweet’ and ‘Aswan’. Empty seed 

coats and some hard seed, however, were produced when the tetraploid 

was used as a pollenizer for ‘Giza1’ and ‘Targi’. The effect of the 

tetraploid pollenizer on the appearance and inner quality of fruits is 

shown in Figure 1. The fruits had similar rind and flesh 

characteristics, except for the presence of hard seeds in three of four 

cases. 

The response of maturity period and various quality characteristics 

of watermelon fruits to diploid and tetraploid pollenizers is provided in 

Table 2. There were no observable differences in terms of fruit set 

between tetraploid and diploid pollenizers. 

All genotype entries were different at the 1% level of significance 

in terms of hard-seed and empty-seed-coat counts per fruit when a 

diploid was used (Table 1). ‘Crimson Sweet’ fruit had the highest 

number of hard seed and empty seed coats per fruit, while fewer were 

produced by (in descending order) ‘Aswan’, ‘Targi’, and ‘Giza1’. No 

hard seeds were found in any of the cultivars when pollen was 

obtained from the tetraploid. More empty seed coats per fruit were 

detected with ‘Giza1’ and ‘Targi’ when the tetraploid was used as the 

pollenizer. The fruits of ‘Giza1’ had the highest number of empty seed 

coats, followed by ‘Targi’, ‘Aswan’, and ‘Crimson Sweet’ fruits, 

respectively. When the tetraploid pollenizer was used, all empty seeds 

were white and very soft (i.e., edible) for all cultivars except ‘Targi’, 

which contained some colored and hard seeds. 

The seeds in ‘Giza1’ and ‘Targi’ were derived from fruit set 

though self-pollination. The low seed counts per fruit of these 

cultivars may be due to the large size of their seeds compared to 

‘Crimson Sweet’ and ‘Aswan’ seeds. The same rationale may explain 

the high number of empty seed coats in ‘Giza1’ and ‘Targi’ versus 


‘Crimson Sweet’ and ‘Aswan’ cultivars when tetraploid-pollinated 

(Figure 2). 

A similar response was obtained by Henderson (1977). He 

reported that the triploid reciprocal cross [(2x) female x (4 x) male] in 

watermelon has not been successful at producing hard seeds. The 

results we obtained also agree with those reported by Sugiyama and 

Morishita (1998a, b) when they used soft X-rays to produce diploid 

seedless watermelon fruits. 

While tetraploid pollen germinated on the stigma, the pollen tube 

didn’t elongate to reach the embryo sac, because of the wide pollentube 

size, compared with the diploid pollen tube. Hormones and 

auxins produced during the tetraploid pollen germination led to 

improved fruit set without any hard seeds (seedless fruit). 

1. A B 2. A B 

3. A B 4. A B 

Fig. 1. Exterior and interior quality of diploid watermelon in response to diploid 

and tetraploid pollenizers. 1 = ‘Aswan’; 2 = ‘Crimson Sweet’; 3 = ‘Giza 1’; 4= 

‘Targi’. 

A = diploid (normal fruit); B = seedless fruit by tetraploid pollenizers. 


350 

300 

250 

200 

150 

100 

50 

0 

D.Normal 

seeds 

D.Empty 

seeds 

T.Normal 

seeds 

T.Empty 

seeds 

Fig. 2. Number of hard and empty seed coats across cultivars in response to 

whether the pollenizer was diploid or tetraploid. D = Diploid watermelon 

(control); T = diploid watermelon set by tetraploid pollen. From left to right: 

‘Aswan’ F1, ‘Crimson Sweet’, ‘Giza 1’, and ‘Targi’. 

Generally, the use of a tetraploid pollenizer is an effective method 

for producing diploid seedless watermelon fruits. However, some 

diploid genotypes respond better than others. 

Time to fruit maturity was not affected by pollenizers; however, it 

varied among genotypes. ‘Targi’ had the shortest period to maturity 

(35 days from pollination), followed by ‘Aswan’, ‘Giza1’, and 

‘Crimson Sweet’, respectively (Table 1). ‘Aswan’ produced the 

heaviest fruits, followed by ‘Crimson Sweet’, ‘Giza1’, and ‘Targi’, 

respectively (Table 2). 

Our results agree with those reported by Hayata et al., (1995). 

They produced seedless watermelon fruits using artificial hormones, 

resulting in fruits that were deformed and small compared with fruit 

that were set with pollen. Fruits produced for each cultivar from the 

different pollenizer types were round and free from hollow heart. 

There were significant differences in TSS among all fruit cultivars. 

The tetraploid pollenizer increased significantly in both ‘Giza1’ and 

‘Crimson Sweet’. There was no significant effect of tetraploid 

pollenizer on rind thickness. 

Conclusion 

The use of tetraploid pollenizers may have potential for 

commercial production of high-quality diploid seedless watermelon 

fruits since this was successfully demonstrated with ‘Aswan’, 

‘Crimson Sweet’, and ‘Giza1’. In general, the application of this new 

technique not only reduces total costs of seedless watermelon fruits, 

but also increases net income compared to triploid watermelon 


(traditional method). The new technique seems to be a promising 

solution for many economic problems at different levels. 

Table 1. Hard-seed and empty-seed-coat counts per fruit and maturity 

period of diploid watermelon fruits in response to diploid and 

tetraploid pollenizers. 

Second season 2005 First season 2004 

Anthesis 

to 

Empty 

seed 

Anthesis 

to 

Empty 

seed 

Hard 

Hard 

harvest coats seeds harvest coats seeds Pollenizer Cultivar 

40.0b 21.0d 291.0b 40.0b 21.0d 293.0b Diploid Aswan 

40.0b 10.9e 0.0e 40.0b 10.8f 0.00e Tetraploid Aswan 

46.0a 27.0c 306.7a 46.0a 27.0c 310.0a Diploid Crimson 

Sweet 

46.0a 5.0g 0.0e 46.0a 5.0g 0.0e Tetraploid Crimson 

Sweet 

45.0a 15.0ef 181.0d 45.0a 15.0ef 180.3d Diploid Giza 1 

44.7a 120.2a 0.0e 45.0a 120.2a 0.00e Tetraploid Giza 1 

35.0c 17.0ed 255.0c 35.0c 17.0ed 253.0c Diploid Targi 

36.0c 85.4b 0.0e 36.0c 85.4b 0.00e Tetraploid Targi 

** ** ** ** ** ** Significance level 

NS= nonsignificant; * = significant at 5 %; **= significant at 1 %. 

Table 2. Response of maturity period and various quality parameters 

of diploid watermelon fruits to diploid and tetraploid pollenizers. 

TSS 

% 

Second season 2005 First season 2004 

Rind 

thickness 

cm 

Fruit 

weight 

kg 

TSS 

% 

Rind 

thickness 

cm 

Fruit 

weight 

kg Pollenizer Cultivar 

11.7cd 1.2a 7.4a 11.5 d 1.2a 7.5a Diploid Aswan 

12.6b 1.2a 7.4a 12.3 b 1.2a 7.4a Tetraploid Aswan 

11.3de 1.3a 7.0b 11.5de 1.3a 7.0b Diploid Crimson 

Sweet 

12.2c 1.3a 6.9b 12.2bc 1.3a 6.9b Tetraploid Crimson 

Sweet 

12.0d 1.3a 6.1c 12.0c 1.3a 6.1c Diploid Giza 1 

13.2a 1.3a 5.8c 13.0a 1.3a 5.8c Tetraploid Giza 1 

11.5d 0.8b 4.5d 11.5d 0.8b 4.5d Diploid Targi 

12.4bc 0.8b 4.8d 12.1c 0.8b 4.4d Tetraploid Targi 

* * ** * * ** Significance level 

NS= nonsignificant; * = significant at 5 %; **= significant at 1 %. 



Buttrose, M. and M. Sedgley. 1979. Anatomy of watermelon embryo sacs following 

pollination, non-pollination or parthenocarpic induction of fruit development. 

Ann. Bot. 43:141–146. 

Hayata,Y., Y. Niimi, and N. Iwasaki. 1995. Synthetic Cytokinin-1-(2-chloro-4pyridyl)-3-phenylurea 

(CPPU) - promotes fruit set and induces parthenocarpy in 

watermelon. J. Amer. Soc. Hort .Sci. 120:997–1000. 

Henderson, W. R. 1977. Effect of cultivar, polyploid and reciprocal hybridization on 

characters important in breeding triploid seedless watermelon hybrids. J. Amer. 

Soc. Hort. Sci. 102(3):293–297. 

Kano, Y. 2000. Effect of CPPU treatment on fruit and rind development of 

watermelons (Citrullus lanatus Matsum. et Nakai ). J. Hort. Sci. & Biotech. 

(6):651–654. 

Kihara, H. 1951. Triploid watermelon. Proc. Amer. Soc. Hort. Sci. 58:217–230. 

Sugiyama, K. and M. Morishita, 1998a. Induction of seedless watermelon by 

pseudogamy, p. 297–299. In: J. D. McCreight (ed.). Cucurbitaceae ’98, 

evaluation and enhancement of cucurbit germplasm. ASHA Press, Alexandria, 

VA. 

Sugiyama, K. and M. Morishita. 1998b. Fruit set and seed characteristics of diploid 

seedless watermelon (Citrullus lanatus) cultivars produced by soft-x-irradiation 

pollen. J. Jpn. Soc. Hort. Sci. 69(6):684–689. 

Sugiyama, K. and M. Morishita. 2000. Production of seedless watermelon using 

soft-x-irradiation pollen. Sci. Hort. 84:255–264. 

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QUANTITATIVE TRAIT LOCI FOR SUCROSE 

PERCENTAGE OF TOTAL SUGARS IN MELON 

CROSSES UNDER GREENHOUSE AND FIELD 

ENVIRONMENTS 

Soon O. Park and Kevin M. Crosby 

Vegetable & Fruit Improvement Center, Texas A&M University, 


Texas Agricultural Research and Extension Center, Texas A&M 

University, Weslaco, TX 78596 

ADDITIONAL INDEX WORDS. Cucumis melo, glucose percentage of total 

sugars 

ABSTRACT. Our objectives were to identify RAPD markers associated with 

QTL for sucrose percentage of total sugars (SPTS) using bulked segregant 

analysis (BSA) in an F2 population derived from the melon (Cucumis melo L.) 

cross of ‘TAM Dulce’ (high SPTS) × TGR1551 (low SPTS) in a greenhouse 

experiment, and confirm the association of RAPD markers with QTL for the 

sweetness trait in an F2 population from the cross of ‘Deltex’ (high SPTS) × 

TGR1551 in a field experiment. Continuous distribution for SPTS was observed 

in the genetic populations indicating quantitative inheritance for the trait. A 

significant positive correlation was found between SPTS and sucrose or soluble 

solids. Seven RAPD markers, 3 amplified from ‘TAM Dulce’ and 4 amplified 

from TGR1551, were detected to be significantly associated with QTL for SPTS 

in the greenhouse population. Three unlinked markers were significant in the 

full model, and accounted for 31% of the total phenotypic variation for SPTS. 

Four RAPD markers were confirmed in the field population to be consistently 

associated with QTL for SPTS. These RAPD markers associated with QTL for 

the sweetness trait identified in the greenhouse and confirmed in the field could 

be useful in melon breeding for improving the mature fruit sweetness. 

D 

ue to consumer preference for sweet fruit, sugar content is a 

highly important quality trait of different melon classes. The 

improvement of sugar content is one of the most significant 

goals in breeding programs of most melon types worldwide. Sucrose, 

glucose, and fructose are major factors determining mature melon fruit 

sweetness. The ratio of individual sugar compositions is also an 

important sweetness trait. 

This research was funded in part by USDA Grant 2001-34402-10543, “Designing 

Foods for Health.” We acknowledge financial support from the South Texas Melon 

Committee. We also thank technicians Alfredo Rodríguez and Hyun J. Kang, Texas 

Agricultural Research and Extension Center-Weslaco, for their assistance. 


Bulked segregant analysis (Michelmore et al., 1991) is an efficient 

method of rapidly identifying molecular markers linked to a specific 

gene using DNA bulks from F2 plants. Molecular markers linked to 

genes for sweetness traits may improve the breeder’s ability to recover 

sugar genotypes and aid in the development of sugar cultivars. 

However, markers associated with QTL affecting SPTS present in 

‘TAM Dulce’, a western-shipper muskmelon type, have not been 

reported. Our objective was to identify RAPD markers associated with 

QTL for SPTS by means of BSA in an F2 population derived from the 

melon cross of ‘TAM Dulce’ (high SPTS) × TGR1551 (low SPTS) in 

a greenhouse experiment. Park et al. (1999) emphasized the 

importance of confirming the marker-QTL associations in different 

populations and environments before using molecular markers for 

marker-assisted selection in breeding programs. Thus, our additional 

goal was to confirm the associations of RAPD markers with QTL for 

the sweetness trait in an F2 population from the cross of ‘Deltex’ (high 

SPTS) × TGR1551 in a field experiment. 


PLANT MATERIAL. For identification of QTL, 105 F2 plants 

derived from the cross of ‘TAM Dulce’ × TGR1551 were planted in a 

greenhouse at the Texas Agricultural Research and Extension Center- 

Weslaco, Texas A&M University, on 15 Oct. 2002. ‘TAM Dulce’ is a 

western-shipper muskmelon type with high fruit quality, while 

TGR1551, originally obtained from Zimbabwe, is a wild type with low 

fruit quality. For confirmation of QTL, 64 F2 plants from the cross of 

‘Deltex’ × TGR1551 were planted on black plastic mulch with drip 

irrigation on sandy clay loam soil at Weslaco, Texas on 10 March 

2005. The high-sugar ‘Deltex’ parent is a commercial ananas cultivar 

(Nunhems, Parma, ID). Data for SPTS were obtained from the two 

parental pairs as well as the 105 and 64 F2 plants. 

BULKED SEGREGANT ANALYSIS USING RAPD. Fully expanded 

leaves of the 105 and 64 F2 plants along with their parental pairs were 

collected at 21 days after planting. Total genomic DNA was extracted 

from the leaf tissue using the method of Skroch and Nienhuis (1995). 

A total of 500 random 10-mer primers (Operon Technologies, 

Alameda, CA) were used. Polymerase chain reactions (PCR) were 

performed on 96-well plates in an MJ Research thermalcycler (model 

PTC-0100; MJ Research, Waltham, MA). Protocols for PCR and the 

composition of the final volume of reactants were the same as those 

described by Skroch and Nienhuis (1995). Two low and high bulks 

were prepared from equal volumes of standardized DNA (10ng. L-1) 


from 8 selected F2 plants of the ‘TAM Dulce’ × TGR1551 cross with 

the highest and lowest SPTS values, respectively. The 500 primers 

were used to simultaneously screen between the low and high DNA 

bulks, and between the parents ‘TAM Dulce’ and TGR1551. Primers 

that generated marker polymorphisms between the low and high DNA 

bulks were tested in the F2 population from the cross between ‘TAM 

Dulce’ and TGR1551 for identifying QTL. Six primers were tested in 

the F2 population of the ‘Deltex’ × TGR1551 cross for confirming 

QTL. 

DETECTION OF QTL. To detect segregation distortion of markers, 

F2 population marker data were tested for goodness-of-fit to a 3:1 ratio 

using the chi-square test. Due to the dominant nature of RAPD 

markers, the linkage analyses of three markers obtained from ‘TAM 

Dulce’ and four markers obtained from TGR1551 were separately 

performed on the data for 105 F2 plants of the ‘TAM Dulce’ and 

TGR1551 cross using MAPMAKER version 3.0 (Lander et al., 1987). 

We also executed separately the linkage analyses of three markers 

from ‘Deltex’ and three markers from TGR1551 on the data for 64 F2 

plants of the ‘Deltex’ × TGR1551 cross. Simple linear regression 

(SLR) for each pairwise combination of quantitative traits and marker 

loci was used to analyze the greenhouse and field data for detection 

and confirmation of QTL for SPTS. Significant differences in trait 

associations were based on F-tests (P

Table 1. Pearson correlations of sucrose percentage of total sugars 

(SPTS) with other fruit-sweetness traits in two F2 populations derived 

from melon crosses of ‘TAM Dulce’ × TGR1551 (TT) and ‘Deltex’ × 

TGR1551 (DT) in greenhouse and field experiments. 

Sweetness traits 

Trait Cross TSS z Sucrose Glucose Fructose GPTS x FPTS 

SPTS TT 0.33** 0.89** -0.03 NS 0.18 NS -0.85** -0.58** 

DT 0.53** 0.98** -0.77** -0.59** -0.95** -0.92** 

z 

TSS = total soluble solids. 

x 

GPTS = glucose percentage of total sugars; FPTS = fructose percentage of total 

sugars. 

**Nonsignificant or significant at P < 0.01. 

IDENTIFICATION OF QTL. A total of 500 primers were used for 

the RAPD analysis of two different bulks for SPTS along with their 

parents ‘TAM Dulce’ and TGR1551. Seven RAPD markers were 

polymorphic between the two DNA bulks. Three displayed an 

amplified DNA fragment in the high bulk that was absent in the low 

bulk. Four showed an amplified DNA fragment in the low bulk that 

was absent in the high bulk. These seven marker fragments segregated 

in the F2 population of the ‘TAM Dulce’ × TGR1551 cross. A 

goodness-of-fit to a 3:1 ratio for band presence to band absence for 

each of the seven markers was observed in 105 F2 plants. These 

markers were unlinked based on linkage analysis, suggesting that they 

are located on different chromosomes. 

Seven significant associations of RAPD marker loci with QTL 

influencing SPTS were identified in this greenhouse population based 

on SLR (Table 2). The ‘TAM Dulce’ parent contributed high SPTS 

alleles for these seven markers. Markers OAU13.1350, OAT03.250, 

and OAW06.1250 from ‘TAM Dulce’ explained 4% to 13% of the 

variation for this trait. Only one of the three unlinked markers 

accounting for 13% of the variation was noted using the SMR analysis. 

Markers OAW06.600, OAA09.350, OAU05.600, and OAQ13.750 

from TGR1551 explained 6% to 17% of the variation for this trait. 

Three of the four unlinked markers were significant in the full model, 

and accounted for 31% of the total variation for the trait. Six and two 

of the seven RAPD markers associated with QTL for SPTS were also 

found to be significantly associated with QTL for GPTS and FPTS, 

respectively. However, andromonoecious (a) on Linkage Group 4 of 

the classical melon map regulating stamen absence or stamen presence 

in female flowers was not associated with SPTS in the greenhouse 

population. 


Table 2. Simple linear regression (SLR) and stepwise multiple 

regression (SMR) analyses of marker and data for detection of QTL 

for sucrose percentage of total sugars (SPTS) in an F2 population 

derived from the melon cross of ‘TAM Dulce’ (high SPTS) × 

TGR1551 (low SPTS) in the greenhouse experiment. 

Marker Source P 

OAU13.1350 

SLR Average value SMR 

R 2 

(%) Presence Absence P 

R 2 

(%) 

TAM 

Dulce 0.000 13 12.6 z 5.5 y 0.000 13 

OAT03.250 TAM 

Dulce 0.027 5 12.4 8.9 

OAW06.1250 TAM 

Dulce 0.04 4 12.4 9.1 

Cumulative R 2 = 13 

OAW06.600 TGR1551 0.000 17 9.8 16.5 0.000 17 

OAA09.350 TGR1551 0.000 13 10.1 17.1 0.000 9 

OAU05.600 TGR1551 0.007 7 10.5 14.4 0.008 5 

OAQ13.750 TGR1551 0.013 6 10.4 15.4 


z An average value of F2 plants with band presence for the marker. 

y An average value of F2 plants with band absence for the marker. 

Table 3. Confirmation of RAPD markers associated with QTL for 

sucrose percentage of total sugars (SPTS) in an F2 population derived 

from the melon cross of ‘Deltex’ (high SPTS) × TGR1551 (low SPTS) 

in the field experiment. 

Simple linear Stepwise multiple 

regression 

regression 

R 

Marker Source P 

2 

R 

(%) P 

2 

(%) 

OAW06.1250 Deltex 0.000 10 0.000 10 

OAU13.1350 Deltex 0.047 4 0.047 4 


OAA09.350 TGR1551 0.000 12 0.000 12 

OAU05.600 TGR1551 0.041 4 0.045 4 


Four RAPD markers associated with the sweetness trait in the 

greenhouse experiment were confirmed in the F2 population of the 

‘Deltex’ × TGR1551 cross in the field experiment to be consistently 

associated with QTL for the trait on the basis of SLR (Table 3). 

Marker OAA09.350 from TGR1551 associated with QTL for the trait 

in the greenhouse population was also found to be significantly 

associated with QTL for the trait in the field population, and accounted 


for 12% of the phenotypic variation for the trait. A significant 

association of OAW06.1250 from ‘Deltex’ with QTL for the 

sweetness trait was consistently expressed in our populations derived 

from two different crosses under greenhouse and field environments. 

However, markers OAT03.250 and OAQ13.750, slightly associated 

with the sugar trait identified in the greenhouse F2 population, were 

not confirmed in this field F2 population. The RAPD markers linked to 

the SPTS QTL identified and confirmed in two populations and 

environments here should be more reliable for marker-assisted 

selection than those evaluated in a single population and environment. 


Edwards, M. D., C. W. Stuber, and J. F. Wendell. 1987. Molecular marker-facilitated 

investigations of quantitative trait loci in maize. I. numbers, genomic 

distribution, and types of gene action. Genetics. 116:113–125. 

Lander, E. S., P. Green, J. Abrahamson, A. Barlow, M. J. Daly, S. E. Lincoln, and L. 

Newburg. 1987. MAPMAKER: an interactive computer package for 

constructing primary genetic linkage maps with experimental and natural 

populations. Genomics. 1:174–181. 

Michelmore, R. W., I. Paran, and R. V. Kesseli. 1991. Identification of markers 

linked to disease resistance genes by bulked segregant analysis: a rapid method 

to detect markers in specific genomic regions using segregating populations. 


Park, S. O., D. P. Coyne, N. Mutlu, G. Jung, and J. R. Steadman. 1999. Confirmation 

of molecular markers and flower color associated with QTL for resistance to 

common bacterial blight in common beans. J. Amer. Soc. Hort. Sci. 124:519– 

526. 

Paterson, A. H., S. Damon, J. D. Hewitt, D. Zamir, H. D. Rabinowitch, S. E. 

Lincoln, E. S. Lander, and S. D. Tanksley. 1991. Mendelian factors underlying 

quantitative traits in tomato: comparison across species, generations, and 

environments. Genetics. 127:181–197. 

Skroch, P. W. and J. Nienhuis. 1995. Qualitative and quantitative characterization of 

RAPD variation among snap bean genotypes. Theor. Appl. Genet. 91:1078– 

1085. 


ETHYLENE MEDIATES THE INDUCTION OF 

FRUITS WITH ATTACHED FLOWER IN 

ZUCCHINI SQUASH 

M. C. Payán 1 , A.Peñaranda 1 , R. Rosales 2 , D. Garrido 2 , P. Gómez 1 , 

and M. Jamilena 3 

1 Departamento de Biotecnología, IFAPA de Almería. Autovía del 

Mediterráneo, La Mojonera, Almería, Spain 

2 Departamento de Fisiología Vegetal, Facultad de Ciencias, 

Universidad de Granada, 18071 Granada, Spain 

3 Departamento de Biología Aplicada, Escuela Politécnica Superior, 

Universidad de Almería, 04120 Almería, Spain 

ADDITIONAL INDEX WORDS. Cucurbita pepo, flower maturation, flower 

abscission, sex expression, high temperature 

ABSTRACT. We previously reported that high temperature during the springsummer 

growing season is the main factor increasing the incidence of zucchini 

fruits with attached flower, a trait that reduces the shelf life and commercial 

quality of the fruits produced in greenhouses. In this work, the increase in the 

number of fruits with attached flowers is caused by reduced ethylene 

production in female flower buds. Treatments with ethylene inhibitors such as 

aminoethoxyvinylglycine (AVG) and silver thiosulphate (STS) promote the 

production of fruits with an attached flower in a number of cultivars. In 

addition, the flowers that remain attached to the harvested zucchini fruits are 

found to be transformed into bisexual, with a lower internal ethylene level than 

female ones. Alterations of the development of attached flowers, which exhibit 

different degrees of stamen development and are arrested in their maturation, 

indicate that ethylene is required for both the maintenance of stamen arrest and 

for completing the maturation of female flowers. Moreover, the incidence of 

this character is genotype-dependent, and the internal level of ethylene is 

reduced in cultivars that produce the highest proportion of fruits with attached 

flowers. This opens the possibility of direct or indirect contraselection of this 

trait in current breeding programs of zucchini squash. 

T 

he production of zucchini squash in greenhouses requires 

cultivars in which the abscission of the flower occurs in the 

earlier stages of fruit development and always before 

harvesting. The delay in abscission of zucchini flowers is now a major 

problem for greenhouse cultivation of zucchini squash in SE Spain. 

This work was supported by grants C03-180 and AGL2005-06677-CO2, awarded by 

the Consejería de Agricultura y Pesca de la Junta de Andalucía (Andalucía, Spain), 

and the Ministerio de Educación y Ciencia (Spain), respectively. 


When the enormous flower of zucchini remains attached to the fruit 

after harvesting, it must be manually removed by growers, inducing 

wounding and making the fruit more susceptible to infection and 

rotting during storage and transport, thus diminishing its shelf life and 

its commercial quality and value. 

Previous results from two trials carried out under different 

environmental conditions in greenhouses in SE Spain indicate that 

high greenhouse temperature during the spring-summer growing 

season is the major environmental factor that induces the production of 

zucchini fruits with attached flowers (Gómez et al., 2004). 

Nevertheless, the incidence of this trait is genotype-dependent, and 

while some cultivars produce more than 80% of fruits with attached 

flowers under high temperatures, others scarcely reach 5% of such 

fruits (Gómez et al., 2004). Given that attached flowers are arrested in 

their maturation, not reaching anthesis but remaining green and closed 

even in harvested fruits, and are also transformed into bisexual 

flowers, showing different degrees of stamen development, we 

assumed that ethylene was involved in this flower-development 

alteration. 

Ethylene plays a key role in the sex expression of cucurbits. 

Treatments with ethylene or ethylene-releasing agents increase the 

production of female flowers, while treatments with inhibitors of 

ethylene biosynthesis or perception induce the production of a higher 

number of male flowers (Robinson et al., 1969; Rudich et al., 1969; 

Owens et al., 1980). Accordingly, the levels of endogenous ethylene in 

gynoecious cultivars of cucumber are two- to threefold higher than in 

monoecious and andromonoecious genotypes (Trebitsh et al., 1987; 

Yamasaki et al., 2001). In fact, the additional copy of the 1aminocyclopropane-1-carboxylate 

(ACC) synthase (ACS) gene in 

cucumber, one of the key enzymes of the ethylene biosynthesis 

pathway, cosegregates with the F gene, conferring gynoecy in this 

species (Trebitsh et al., 1997; Mibus and Tatlioglu, 2004). 

It has recently been reported that ethylene is involved in the 

development and maturation of flowers in Arabidopsis (Hall and 

Bleecker, 2003) and melon (Papadopoulou et al., 2005; Little et al., 

2005). Thus, flower organs of the double mutants for the ethylene 

receptors ers1 etr1 are arrested in development, remaining as 

immature closed flowers in which petals and stamens have not been 

elongated and anthers have not been dehisced (Hall and Bleecker, 

2003). Moreover, andromonoecious melon lines that overexpress an 

ACS transgene not only have altered sex-expression patterns but also 

an increased number of bisexual buds that reach anthesis, indicating 

that ethylene is necessary to maintain the development of ovary- 


earing flowers in species of the Cucurbitaceae family (Papadopoulou 

et al., 2005). 

In this paper we study the implication of ethylene in the induction 

of zucchini fruits with attached flowers using two different 

approaches. On the one hand, we analyze the effects of ethylene 

inhibitors on flower development and abscission in different zucchini 

squash grown under winter conditions. On the other hand, we compare 

endogenous ethylene production in different cultivars and different 

environments. Data demonstrate that the delay in flower abscission 

occurs concomitantly with the inhibition of female flower-bud 

maturation, a process accompanied by conversion of the female bud 

into a bisexual one and reduction in the levels of ethylene production. 


The four hybrid cultivars used, ‘Cora’, ‘Cavili’, ‘Consul’, and 

‘Xsara’, were selected because they differ in the production of fruits 

with attached flowers when grown under high temperatures. For 

treatments with ethylene inhibitors, seeds were germinated in 

rockwool cubes (Grodan), and when three to four leaves had 

developed, they were transferred to 1-m rockwool slabs at a density of 

two plants/slab. Plants were grown in a greenhouse in La Mojonera 

(Almería, Spain) in winter 2005, following standard local practices for 

both plant nutrition and pest management. 

Treatments with aminoethoxyvinylglycine (AVG) and silver 

thiosulphate (STS) were carried out when the fifth leaf was 2cm long. 

The apical meristems of these plants were treated with 25μl of a 

solution containing 1mM AVG and 0.1% Tween 20, or 0.5 M STS 

and 0.1% Tween 20. Control plants were treated with a solution 

containing only the same concentration of Tween 20. 

The release of ethylene was measured in mature flowers at anthesis 

in spring-summer 2005, and winter-early spring 2006. In bisexual 

flowers with arrested maturation, ethylene production was measured in 

closed flower buds, in which fruit length and width were similar to 

those of mature female flowers. The ethylene produced by the flowers 

after incubation at room temperature for 24h in the dark was measured 

in a Perkin-Elmer 8600 gas chromatograph, fitted with a flame 

ionization detector and 2m×3.2mm column packed with 80/100 mesh 

porapak R. The instrument was calibrated with standard ethylene gas. 

At least four replicates were made for each measurement. 



EFFECTS OF ETHYLENE INHIBITORS. The cultivars ‘Cora’, 

‘Consul’, ‘Cavili’, and ‘Xsara’ were used to analyze the effect of 

ethylene-inhibitor treatments on the incidence of fruits with attached 

flowers. These cultivars were selected because they represent cultivars 

producing low (‘Cora’ and ‘Consul’) and high (‘Cavili’ and ‘Xsara’) 

proportions of fruits with attached flowers when grown under springsummer 

conditions. The study was carried out under winter conditions 

since, under such conditions, all the cultivars except ‘Cavili’ produce a 

very low proportion of fruits with attached flowers. 

Number of flowers 

Female flowers Male flowers Bisexual flowers 

12 

Cora Cavili 

12 

10 

8 

6 

4 

2 

0 

1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 

12 

10 

8 

6 

4 

2 

0 

1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 

10 

8 

6 

4 

2 

Node number in the main stem 

0 

1 3 5 7 9 111315 1719 21 23 25 2729 

12 

10 

8 

6 

4 

2 

0 

1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 

Control 

AVG 

Fig. 1. Effects of AVG on sexual expression in two cultivars of zucchini squash. 

Treatments increase the production of bisexual and male flowers in the nodes 

after the treatment (Node 5). Bisexual flowers remain attached to the fruits. 

The inhibition of ethylene biosynthesis or perceptions by means of 

AVG or STS, respectively, induced the production of fruits with 

attached flowers in all the studied cultivars. AVG altered the sexual 

expression of the plants, inducing a partial or complete conversion of 

female flowers into bisexual ones and thus increasing the maleness of 

the plants (Figure 1). The effect of the treatment was similar in all four 

cultivars. In the nodes immediately above the treatment, AVG induced 

the development of bisexual flowers, while in the following nodes the 

treatment increased the production of male flowers (Figure 1). The 

bisexual flowers, showing partial or complete development of stamens, 


were those that remained attached to the harvested fruits (Figure 2). 

These bisexual flowers were all arrested in flower maturation, 

remaining as closed floral buds even in fruits of commercial length 

and width (Figure 2). 

The effect of the inhibition of ethylene perception by STS was 

very similar, inducing the production of male and bisexual flowers in 

all cultivars (Figure 3). Nevertheless, STS treatments induced a higher 

proportion of bisexual flowers than AVG (Figure 3). Moreover, 

besides the partial or complete development of stamens and the arrest 

of flower maturation, these bisexual flowers were also altered in the 

development of pedicel, sepals, and carpels. The pedicel and sepals of 

these bisexual flowers resembled those of male flowers, being larger 

than those of female ones (Figure 2). Moreover, in addition to the 

normal inferior ovary, which may be more or less developed, some of 

these flowers developed superior ovary tissue (Figure 2). 

The flower phenotypes of bisexual flowers induced by AVG and 

STS treatments were very similar to those found by Gomez et al. 

(2004) in different cultivars of zucchini grown under high 

temperatures. Given that these bisexual flowers appeared in the nodes 

just above the treatment, it is likely that they result from a reduction of 

ethylene in already determined female floral buds. In more immature 

floral buds the inhibition of ethylene biosynthesis or perception would 

produce a complete reversion of female into male flowers, such as was 

observed in higher nodes of AVG- and STS-treated plants. 

ETHYLENE PRODUCTION IN MALE AND FEMALE FLOWERS. The 

production of ethylene was measured in female and male flowers of 

‘Cavili’ and ‘Cora’ cultivars of zucchini grown under winter or springsummer 

conditions. These cultivars were selected because they 

produce the highest and lowest proportion of fruits with attached 

flowers, respectively, when grown at high temperatures. The 

production of ethylene in all flower sex phenotypes of ‘Cavili’ was 

significantly lower than in those of ‘Cora’ under both environmental 

conditions, which may explain the higher susceptibility of ‘Cavili’ 

plants to spring-summer environmental conditions (Gómez et al., 

2004). 

Male flowers of both cultivars produced less ethylene than female 

ones (Figure 4), which is not surprising given that ethylene promotes 

femaleness in this species. The production of ethylene in bisexual 

flowers that appeared attached to the harvested fruits under very high 

temperatures during spring-summer conditions was intermediate 

between that of perfect female or male flowers. Moreover, in 

comparison with winter conditions, spring-summer conditions promote 

a reduction in internal ethylene in female flowers (Figure 4). 


Fig. 2. Effects of AVG (A, B, and C) and STS (D, E, and F) treatments on flower 

development in zucchini squash. Both treatments induced the production of 

fruits with attached flowers. These flowers were arrested in their maturation, 

remaining green and closed even in harvested fruits (A and D). Fruits with 

attached flower were also bisexual, exhibiting different degrees of stamen 

development (B, C, E, and F). The pedicel and sepals of STS-treated plants 

resembled those of male flowers (D). STS also alters carpel development, 

inducing the growth of superior ovary tissue (E and F). 

The effects of ethylene inhibitors on flower development and the 

measurements of ethylene production in the bisexual flowers that 

remain attached to the fruits have indicated that the induction of fruits 

with attached flowers in zucchini is caused by a reduction in the 

internal level of ethylene in female flower buds. Differences in 

susceptibility to summer environmental conditions and in the 

incidence of fruits with attached flowers among genotypes of zucchini 

squash (Gómez et al., 2004) appear to be correlated with a decrease in 

the base level of ethylene. Indeed, we have observed that ethylene 


production in all the studied flowers of ‘Cavili’, a cultivar with one of 

the highest percentages of fruits with attached flowers in the spring- 

summer growing season, was significantly lower than in flowers of 

‘Cora’, a cultivar among those with the lowest production of fruits 

with attached flowers. Therefore, genotypes that have a reduced level 

of ethylene production could be more susceptible to high temperatures, 

not being able to reach the threshold level necessary for normal 

development of female flowers. 

Number of flowers 

10 

8 

6 

4 

2 

0 

1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 

10 

8 

6 

4 

2 

Female flowers Male flowers Bisexual flowers Bisexual flowers with 

Cora 10 

male characteristics 

Cavili 

0 

1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 

Fig. 3. Effects of STS on sexual expression in two cultivars of zucchini squash. 

Treatments induce the production of bisexual flowers with male secondary 

characteristics immediately after the treatment (Node 5). 

Given that the production of ethylene in the flowers that remain 

attached to the harvested fruits was lower than that of female flowers, 

the developmental arrest of these flowers before maturation supports 

the idea that ethylene is required to maintain the development of 

ovary-bearing flowers, and for the transition from immature to mature 

flowers in C. pepo. This conclusion agrees with other studies on 

Arabidopsis and melon indicating that ethylene is involved in flower 

maturation (Hall and Bleecker, 2003; Papadopoulou et al., 2005). 

The conversion of female into bisexual flowers observed in AVG- 

or STS-treated zucchini squash and the lower level of internal ethylene 

in these transformed flowers also support the conclusion that a 

threshold ethylene level is required to arrest stamen growth during 

female-flower development in C. pepo. The specificity of ethylene 

8 

6 

4 

2 

0 

1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 

10 

8 

6 

4 

2 

Node number in the main stem 

0 

1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 

Control 

STS 


function on stamen arrest in female flowers of C. pepo may depend on 

the internal levels of this hormone in specific flower sex types, as we 

have observed in zucchini flowers, but also on a differential sensitivity 

of male and female flowers to ethylene as proposed by Yamasaki et al. 

(2001) in cucumber. 

nl ethilene/g fw*d 

20 

15 

10 

5 

0 

Females Bisexuals Males 

Cora Summer 13,6928 11,8959 10,3283 

Cavili Summer 3,686 5,127666667 

Cora Winter 16,2097 8,6609 

Cavili Winter 6,6658 9,8858 

Fig. 4. Ethylene production of flowers in anthesis of the zucchini 

cultivars ‘Cora’ and ‘Cavili’ in winter or spring-summer 

conditions. Data represent the mean of at least 6 measurements 

from 3 replicates. Vertical bars represent standard deviation of 

the means. Bisexual flowers appeared only in the springsummer 

culture. Under these conditions, some of the female 

flowers of ‘Cora’ and nearly all the female flowers of ‘Cavili’ 

were transformed into bisexual ones. 


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THE MELOGEN PROJECT: DEVELOPMENT 

OF GENOMIC TOOLS IN MELON 

Pere Puigdomènech, Ana Caño, and Víctor González 

Laboratori de Genètica Molecular Vegetal CSIC-IRTA, Departament 

de Genètica Molecular, Barcelona, Spain 

Pere Arús, Wim Deleu, Jordi Garcia-Mas, 

Mireia González, and Antonio Monforte 

Laboratori de Genètica Molecular Vegetal CSIC-IRTA, Departament 

de Genètica Vegetal, Cabrils, Spain 

José Blanca, Fernando Nuez, Belén Picó, and Cristina Roig 

COMAV-UPV, Departamento de Biotecnología, 

Valencia, Spain 

Miguel Aranda and Daniel González 

CEBAS-CSIC, Departamento de Biología del Estrés y Patología 

Vegetal, Murcia, Spain 

Antonio Robles 

UPV, Departamento de Informática de Sistemas y Computadores, 

Valencia, Spain 

ADDITIONAL INDEX WORDS. Cucumis melo, ESTs, TILLING, SNP, oligo chip 

ABSTRACT. The Spanish Initiative in Genomics and Proteomics started The 

MELOGEN Project: Development of Genomic Tools in Melon (C. melo L.) in 

2004 for the analysis of resistance to pathogens and fruit-quality traits. This is a 

collaborative effort involving five different research groups. After two years, 

several of the project objectives have been achieved including (1) EST 

sequencing; (2) development of a Webpage and a database that integrates the 

EST and genetic map information; (3) expanding the melon genetic map 

resolution with EST-derived SNP markers; (4) construction of a melon physical 

map by anchoring BAC clones with genetic markers; (5) development of BAC 

contigs in regions containing clusters of disease-resistance genes; (6) 

development of an EMS-mutagenized melon population for TILLING; and (7) 

construction of an oligo-based chip containing the EST unigene set. 

M 

elon (Cucumis melo L.) is a diploid species (2n=24) that 

belongs to the family Cucurbitaceae and is a vegetable crop 

widely distributed in temperate, subtropical, and tropical 

climates. It is, after the tomato, the most widely grown vegetable crop 

in Spain in both area and production. Spain is the fifth largest melon 

producer worldwide and the leader in Europe. An important aspect of 

the melon industry in Spain is the production and commercialization of 

melon hybrid seed. Melon seeds command the highest prices among 

all vegetable crop seeds. Melon breeding is focused mainly on the 


incorporation of disease resistance and fruit-quality traits into 

commercial cultivars. 

The melon genome is relatively small. Some studies have 

estimated its genome size to be 450–500Mb (Arumuganathan and 

Earle, 1991), similar to that of rice (419Mb), and three times the size 

of the model plant species Arabidopsis thaliana (125Mb). The melon 

genetic map has an estimated size of 1197cM (Oliver et al., 2001), 

which represents approximately 375kb per cM of physical map vs. 

genetic map. These data make it an attractive species to study from a 

genomics point of view. 

Our genetic map was obtained from an F2 population of the cross 

PI 161375 (Korean accession) x ‘Piel de sapo’ T111 cultivar (Gonzalo 

et al., 2005). The genetic map is based mainly on Restriction Fragment 

Length Polymorphism (RFLP) and Simple Sequence Repeat (SSR) 

markers. Recently, several methods for the detection of single 

nucleotide polymorphisms (SNPs) have been developed. SNPs are 

good markers for detecting polymorphism and they can be developed 

from genes with known function, which gives them added value. SNP 

markers will likely be the reference marker type in the near future and 

we have started to use them in melon (Morales et al., 2004). The 

primary objective of this project was to generate a high-resolution 

genetic map of melon based on SNPs that will complement an earlier 

map. This new map will be important for the achievement of the rest 

of objectives of the project. 

One of the main objectives in melon breeding in Spain is 

incorporating resistance to a number of plant diseases, both fungal 

(e.g., powdery mildew, soil-borne fungal diseases, and melon collapse) 

and viral (e.g., Cucumber mosaic virus [CMV], Watermelon mosaic 

virus [WMV-2], Zucchini yellow mosaic virus [ZYMV], and Cucurbit 

aphid-borne yellows virus [CABYV] in the field and Melon necrotic 

spot virus [MNSV], Cucumber vein yellow virus [CVYV], and 

Cucumber yellow stunting disorder virus [CYSDV] in glasshouse 

production). It is important to develop melon varieties resistant to 

these pathogens (Nuez et al., 1999). There are several known 

monogenic disease-resistance genes in melon. A few have been 

characterized by positional cloning (Joobeur et al., 2004; Nieto et al., 

unpublished). Thus, it is important to have BAC libraries, such as the 

one obtained from a melon DHL containing 23,000 clones with an 

average size of 141kb (van Leeuwen et al., 2003). The cloning and 

analysis of the genomic distribution of some disease-resistance genes 

in several plant species has revealed the existence of clusters of these 

genes in some genomic regions (Leister et al., 1996). We have 

detected at least three regions in melon that contain several known 


disease-resistance genes and homologous sequences (Garcia-Mas et 

al., 2001). These regions will be further studied during this project 

after building BAC contigs spanning 1-Mb regions, which will lead to 

a better understanding of the organization of the melon genome (van 

Leeuwen et al., 2003, 2005). 

On the other hand, the availability of large collections of ESTs in 

model species has facilitated the correct assembling of whole genomes 

after sequencing (Arabidopsis Genome Initiative, 2000). EST 

collections may allow the construction of microarrays with thousands 

of ESTs representing an organism, a very powerful tool for highthroughput 

genetic-expression studies (Seki et al., 2001). During this 

project we anticipate sequencing 30000 ESTs and developing a melon 

ESTs oligo chip. The analysis of the expression data obtained with the 

chip will be complex, so we will also establish a bioinformatics 

platform. 

The analysis of gene function requires the existence of mutant 

alleles for the genes analyzed. Plant breeding also needs genetic 

variability, which is not always available naturally. This can be 

important in the cucurbit family, a group of species relatively isolated 

among the cultivated crops. For this reason the TILLING approach has 

been developed (McCallum et al., 2000). Using this methodology we 

can search for allelic variants for a candidate gene. In this project we 

will also establish a melon mutant population and a TILLING 

platform. 


The melon genotypes used for contructing the cDNA libraries were 

the Korean accession PI 161375, two ‘Piel de sapo’ cultivars, the C35 

cantaloupe line, and the C. melo ssp. agrestis accession pat81. RNAs 

were extracted using the Tri-reagent kit (Sigma). cDNA libraries were 

normalized and constructed in the pBSK cloning vector. The EST 

sequences were produced in Macrogen (Seoul, Korea). EST annotation 

and analysis was performed and stored in . 

For SNP discovery, primers were designed from melon ESTs, and 

the amplified products in PI 161375 and T111 were sequenced and 

aligned. SNPs were genotyped using CAPS or the SNaPshot kit. SNPs 

were mapped in the PI 161375 x T111 genetic map using bin-mapping 

(Howad et al., 2005). 

cDNA clones corresponding to melon RFLP markers in Oliver et 

al. (2001) were sequenced. Specific primers were designed and used to 

screen the melon BAC library (van Leeuwen et al., 2003). 


Melon seed from a ‘Piel de sapo’ breeding line from Semillas Fitó 

was treated with ethyl-methane sulfonate (EMS) using standard 

protocols. The M1 plants were selfed and M2 seed was stored. Ten M2 

individuals from each M2 family were grown and DNA was extracted 

(QIAGEN) and pooled for each family. 


MELON ESTS. We have constructed eight normalized cDNA 

libraries from five different melon genotypes (see Materials and 

Methods section). Two cDNA libraries corresponded to melon fruit 

tissue 15 and 46 days after pollination (DAP). Four cDNA libraries 

were obtained from healthy roots and roots inoculated with 

Monosporascus cannonballus in two different melon genotypes. Two 

cDNA libraries were prepared from healthy leaves and leaves infected 

with CMV. After sequencing 3,500–4,000 EST clones from each 

library (5’ sequencing), 30,675 high-quality ESTs were obtained. 

WEBPAGE AND EST DATABASE. Once clustered and annotated, the 

ESTs were classified in 6,023 contigs and 10,615 singletons, yielding 

16,637 unigenes. A Webpage that contains all of the EST information 

has been constructed (www.melogen.upv.es). The database is 

searchable in a user-friendly way to provide sequence information for 

the members of MELOGEN. For example, the system provides tools 

to easily identify ESTs containing SSR motifs or “in silico” SNPs. 

SNP DISCOVERY AND DETECTION. ESTs were selected either 

randomly or because they may be candidate genes for interesting 

agronomic traits. Two systems were used to discover SNPs between 

the melon genotypes PI 161375 and ‘Piel de sapo’: (a) specific primer 

design from each EST and resequencing the amplicons from PI 

161375 and ‘Piel de sapo’, and (b) in silico SNP discovery using the 

MELOGEN database. Because many EST contigs contain ESTs from 

different melon genotypes, it is possible to identify putative SNPs that 

can be validated later. For SNP detection and mapping we used 14 

DHL individuals from the PI 161375 x ‘Piel de sapo’ mapping 

population, which allows mapping the SNP in a known interval of the 

genetic map with lower resolution: selective mapping or “binmapping” 

(Howad et al., 2005). SNPs have been detected using either 

CAPS markers or, when there was no restriction enzyme differentially 

recognizing the SNP sequence, the SNaPshot kit. At this time we have 

discovered and mapped approximately 100 new SNPs, which are 

evenly distributed in the 12 melon linkage groups. 

PHYSICAL MAP. We have anchored to the genetic map 100 BAC 

clones using RFLP probes. For each cDNA producing an RFLP, 


specific primers were designed. These primers were used to PCR 

amplify the melon BAC library (van Leeuwen et al., 2003), which is 

arranged in superpools and pools of DNA, making it easier to identify 

a positive BAC clone for each RFLP. These BAC clones are the 

original skeleton of the melon physical map. 

MELON MUTANT POPULATION. A ‘Piel de sapo’ breeding line was 

chemically mutated with EMS at different concentrations to determine 

the optimum EMS concentration for an efficient mutation of the melon 

genome. From 17,000 M0 mutagenized seed, 8,000 M1 plants were 

grown and selfed in two different locations. We have obtained M2 seed 

from 3,000 M1 plants. This is the basis of our melon TILLING 

population. At the beginning of 2006, 10 to 20 seeds have been grown 

for 1,000 M2 families. DNA has been extracted and pooled for each 

M2 family. Experiments to identify mutants for several genes of 

interest using the heteroduplex digestion method based on the Cel1 

enzyme have been started. 

FUTURE PROSPECTS. The EST unigene set is ready for the design 

of the first melon oligo chip. For this purpose we will use Nimblegen 

technology. Expression experiments will be performed using the 

melon oligo chip with RNA samples from melon fruits in different 

stages of development, and leaf and root tissues infected with viruses 

and fungi, respectively. We will continue the discovery and mapping 

of SNPs in order to reach 300 new markers in the melon genetic map. 

At the same time, the SNP markers will be used to identify new BAC 

clones anchored to the genetic map. If the mutation rate in the EMStreated 

population is acceptable, we will continue extracting DNA 

from the M2 families until we reach an initial collection of 3,000. 


Arabidopsis Genome Initiative. 2000. Analysis of the genome sequence of the 

flowering plant Arabidopsis thaliana. Nature. 408:796–815. 

Arumuganathan, K. and E. D. Earle. 1991. Nuclear DNA content of some important 

plant species. Plant Mol. Biol. Rep. 9:208–218. 

Garcia-Mas, J., H. van Leeuwen, A. Monfort, M. C. de Vicente, P. Puigdomènech, 

and P. Arús. 2001. Cloning and mapping of resistance gene homologues in 

melon. Plant Sci. 161:165–172. 

Gonzalo, M. J., M. Oliver, J. Garcia-Mas, A. Monfort, R. Dolcet-Sanjuan, N. Katzir, 

P. Arús, A. J. Monforte. 2005. Simple-sequence repeat markers used in merging 

linkage maps of melon (Cucumis melo L.). Theor. Appl. Genet. 110:802–811. 

Howad, W., T. Yamamoto, E. Dirlewanger, R. Testolin, P. Cossont, G. Cipriani, A. 

J. Monforte, L. Georgi, A. G. Abbot, and P. Arús. 2005. Mapping with a few 

plants: using selective mapping for microsatellite saturation of the Prunus 

reference map. Genetics. 171:305–1309. 


Joobeur, T., J. J. King, S. J. Nolin, C. E. Thomas, and R. A. Dean. 2004. The 

fusarium wilt resistance locus Fom-2 of melon contains a single resistance gene 

with complex features. Plant J. 39:283–297. 

Leister, D., A. Ballvora, F. Salamini, and C. Gebhardt. 1996. A PCR-based approach 

for isolating pathogen resistance genes from potato with potential for wide 

application in plants. Nature Gen. 14:421–429. 

McCallum, C. M., L. Comai, E. A. Greene, and S. Henikoff. 2000. Targeted 

screening for induced mutations. Nat. Biotech. 18:455–457. 

Morales, M., E. Roig, A. J. Monforte, P. Arús, and J. Garcia-Mas. 2004. Single 

nucleotide polymorphisms detected in ESTs of melon (Cucumis melo L.). 

Genome. 47:352–360. 

Nuez, F., B. Picó, A. Iglesias, J. Esteva, and M. Juarez. 1999. Genetics of melon 

yellows virus resistance derived from Cucumis melo spp. agrestis. Eur. J. Plant 

Pathol. 105:453–464. 

Oliver, M., J. Garcia-Mas, M. Cardús, N. Pueyo, A. I. López-Sesé, M. Arroyo, H. 

Gómez-Paniagua, P. Arús, and M. C. de Vicente. 2001. Construction of a 

reference linkage map for melon. Genome. 44:836–845. 

Seki, M., M. Narusaka, H. Abe, M. Kasuga, K. Yamaguchi-Shinozaki, P. Carninci, 

Y. Hayashizaki, and K. Shinozaki. 2001. Monitoring the expression pattern of 

1300 Arabidopsis genes under drought and cold stresses by using a full-length 

cDNA microarray. Plant Cell. 13:61–72. 

van Leeuwen, H., A. Monfort, H. B. Zhang, and P. Puigdomènech. 2003. 

Identification and characterisation of a melon genomic region containing a 

resistance gene cluster from a constructed BAC library. microlinearity between 

Cucumis melo and Arabidopsis thaliana. Plant. Mol. Biol. 51:703–718. 

van Leeuwen, H., J. Garcia-Mas, M. Coca, P. Puigdomènech, and A. Monfort. 2005. 

Analysis of the melon genome in regions encompassing TIR-NBS-LRR 

resistance genes. Mol. Gen. Genom. 273:240–251. 


THE MELOGEN PROJECT: EST 

SEQUENCING, PROCESSING AND ANALYSIS 

Pere Puigdomènech 

Laboratori de Genètica Molecular Vegetal CSIC-IRTA, 

Departament de Genètica Molecular, Barcelona, Spain 

Pere Arús, Jordi García-Mas, and Mireia González 

Laboratori de Genètica Molecular Vegetal CSIC-IRTA, 

Departament de Genètica Vegetal, Cabrils, Spain 

Fernando Nuez, José Blanca, Belén Picó, and Cristina Roig 

COMAV-UPV, Departamento de Biotecnología, Valencia, Spain 

Miguel Aranda and Daniel González 

CEBAS-CSIC, Departamento de Biología del Estrés y Patología 

Vegetal, Murcia, Spain 

ADDITIONAL INDEX WORDS. Cucumis melo, expressed sequence tabs, cDNA 

libraries, bioinformatics, qPCR, gene expression 

ABSTRACT. Thirty thousand melon (Cucumis melo) ESTs have been sequenced 

from eight normalized cDNA libraries corresponding to different tissues in 

different physiological conditions: healthy leaf and root, Monosporascus 

cannonballus-infected root, cotyledons inoculated with Cucumber mosaic virus 

(CMV), and fruits of 15 and 46 days after pollination. A database and a Web 

page have been created (www.melogen.upv.es), with statistical information 

about libraries, 16,600 unigenes, 350 putative SNPs, and 1,000 putative 

microsatellites. In addition, we have made gene-expression analysis by Real- 

Time-qPCR for 20 unigenes of the database, including genes known to respond 

to pathogen infection such as a 70-KDa heat shock protein (Hsp70), a WRKY 

transcription factor, the eukaryotic translation initiation factors 4E and (iso)4E 

described as susceptibility factors for some plant viruses, and several other 

genes involved in fruit development. 

S 

pain is fifth in the world in the production of melons (Cucumis 

melo L.) and is the largest producer in Europe. In Spain, melon 

is the second most widely grown vegetable crop after tomato 

(F.A.O., 2006). The agricultural importance of melon and the 

possibilities that plant genomics offers (e.g., Rensink and Buell, 2005) 

have led to the establishment of a Spanish consortium to develop 

genomic tools in melon, focusing on resistance to pathogens and on 

fruit-quality traits (Melogen). The research that we present in this 

paper has been performed within the framework of this consortium. 

Partial sequencing of cDNA inserts of expressed sequence tags 

(ESTs) has been used as an effective method for gene discovery, 

molecular-marker generation, and transcript pattern characterization. It 


is an efficient approach for identifying sets of plant genes expressed 

during different developmental stages and/or responding to 

environmental stimuli. Despite the importance of the Cucurbitaceae, 

relatively little EST information is currently available. For this reason, 

we have undertaken a survey of the melon transcriptome by analyzing 

ESTs from eight normalized cDNA libraries corresponding to different 

tissues in different physiological conditions: healthy leaf and root, 

Monosporascus cannonballus-infected root, cotyledons inoculated 

with Cucumber mosaic virus (CMV), and fruits of 15 and 46 days after 

pollination. Here we present the sequencing of 30,000 melon ESTs 

from these libraries and also a gene-expression analysis by Real-TimeqPCR 

for 20 unigenes of the database. 


PLANT MATERIAL. The genotypes used for the construction of the 

libraries were the ‘Piel de Sapo’-type cv. T111 (Semillas Fitó, SL, 

Barcelona, Spain), the cantaloupe accession C35 (germplasm 

collection of “La Mayora” Experimental Station, Málaga, Spain), and 

C. melo ssp. agrestis accession pat81 (germplasm collection of 

COMAV, Valencia, Spain). Inoculations with the pathogens were 

carried out using standard procedures (Table 1). This plant material 

was used as the RNA source for the construction of libraries and for 

gene-expression analysis by RT-qPCR. 

Table 1. Description of the cDNA libraries. 

Name Accession Tissue Physiol. cond Kind 

A agrestis pat_81 root healthy normalized 

AI agrestis pat_81 root M cannonballus normalized 

PS Piel de Sapo root healthy normalized 

PSI Piel de Sapo root M cannonballus normalized 

HS c35 leaf healthy normalized 

CI c35 cotyledon CMV infected normalized 

15d t111 Piel_de_Sapo 15 days fruit healthy normalized 

46d t111 Piel_de_Sapo 46 days fruit healthy normalized 

CDNA LIBRARIES. Total RNA was prepared using TriReagent 

(Sigma Chemical Co., St. Louis, MO). Poly(A) RNA from total RNA was 

purified with a cellulose-oligo(dT)-based method [MicroPoly(A) 

Purist, (Ambion, Austin, TX)]. Integrity and quality of both total and 


poly(A) RNA were tested by gel electrophoresis. cDNA libraries were 

constructed with the SMART cDNA Library Construction kit 

(Clontech, Mountain View, CA), with a modified primer to include an 

Sfi I enzyme restriction site. The normalization step was carried out 

with TRIMMER kit (Evrogen, Moscow, Russia). The cDNA 

fractionation step was made with SizeSep 400 Spun Columns 

(Amersham, Buckinghamshire, England). cDNA was digested with Sfi 

I generating Sfi IA-Sfi IB cohesive ends for directional cloning into a 

modified version of BlueScript SK plasmid vector (Stratagene, La 

Jolla, CA). The product of ligation was transformed into E. coli 

electrocompetent cells DH10B (Invitrogen, Carlsbad, CA) by 

electroporation. Title of libraries was evaluated plating an aliquot on 

LB agar plates with ampicillin at 100μg/ml. Only libraries of 

10 5 cfu/ml or more were accepted. In order to check the average insert 

size, 24 plasmid DNA minipreps from randomly picked colonies were 

analyzed by Eco RI-Hind III restriction analyses. 

SEQUENCING OF ESTS. Sequencing was carried out from the 5énd 

of the insert without library amplification using the universal M13 

reverse primer. An external custom service was contracted for this task 

(Macrogen Inc., Seoul, Korea). Approximately 6,000 clones were 

sequenced from the “inoculated cotyledon” (CI) library, and 3,500 

clones from each of the other libraries (Table 2). 

Table 2. cDNA libraries statistical information. 

Raw 

sequen 

High- 

quality 

Redundancy 

Libspecificuni- 

Libraryces 

ESTs 

SingletonsContigsUnigenes 

(%) genes 

15d 3936 3582 1064 1875 2939 18 1312 45 

46d 3840 3493 1070 1787 2857 18 1291 45 

A 3936 3666 1344 1844 3188 13 1424 45 

Ai 3647 3255 974 1644 2618 20 1494 57 

Hs 3648 3012 989 1564 2553 15 1120 44 

Ci 6605 5664 2177 2509 4686 17 2618 56 

Ps 3840 3377 1245 1702 2947 13 1408 48 

Psi 3840 3555 1339 1768 3107 13 2000 64 

Total 34417 30675 10614 6023 16637 46 12844 77 

Nov- 

elty 

(%) 

GENE-EXPRESSION ANALYSIS BY RT-QPCR. Real-time 

quantitative PCR was performed with an AB 7500 System (Applied 

Biosystems, Foster City, CA) to quantify mRNA of some transcripts of 

interest. Twenty ESTs representing these transcripts were chosen from 

the database and used to generate gene-specific primers with Primer 


Express Software (Applied Biosystems, Foster City, CA). The 

chemistry used for PCR product detection was the Power SYBR green 

dye (Applied Biosystems, Foster City, CA) and ROX as passive 

reference. Cyclophilin served as endogenous control (sequence 

extracted from the database) and ΔΔCt was the method of calculation 

used to perform relative quantification. Melting curves analyses at the 

end of the process and No Template Controls (NTC) were carried out 

to ensure product-specific amplification and no primer-dimer 

quantification. A control reaction as for reverse transcription but 

without the enzyme was performed to evaluate genomic DNA 

contamination. 


Almost 35,000 rough sequences were generated. After processing 

to eliminate vector sequences, short PCR products, and poor-quality 

sequences, 30,675 high-quality ESTs were identified. These were used 

for clustering, resulting in 16,637 unigenes (10,614 singletons and 

6,023 contigs) (Table 2). This high proportion of singletons could be 

due to the normalization step. Traditionally, normalization protocols 

are based on reassociation of denatured double-stranded DNA, so that 

most of the abundant DNA molecules will form double-stranded (ds) 

molecules, whereas the single-stranded (ss) fraction results equalized 

to a considerable extent. The normalization protocol that we have used 

here consists of a novel method based on a DNase treatment that, in 

contrast with other methods, does not require physical separation of 

the two fractions for selecting the ss-molecules, increasing yield and 

probably efficiency. As shown by electrophoresis in agarose gels, 

bands corresponding to the more abundant transcripts are efficiently 

eliminated in normalized samples (Figure 1, arrow in lane 4). 

Once processed and annotated, an EST database and a Web page 

were created to facilitate access to information (www.melogen. 

upv.es). In addition to the unigenes identified, clustering and 

redundant sequences have served as a resource for identification of 

single nucleotide polymorphisms (SNPs) by comparing EST 

sequences pertaining to contigs from different melon genotypes. More 

than 1,000 putative short sequence repeats (SSRs) and 356 putative 

SNPs have been generated and have the potential to be used as 

molecular markers. 

Gene-expression analyses were made by RT-qPCR to quantify 

transcript accumulation for some genes of interest (Table 3). 


1 2 3 4 5 

Fig. 1. Construction of the healthy leaf library (HS). Lanes 1, 2, 3: cDNA after 

normalization. Lane 4: cDNA prior to normalization. Lane 5: molecular weight 

marker. 

Sequences were extracted from the database searching by keyword or 

BLAST result with an orthologue sequence from another species (e.g., 

Arabidopsis thaliana). Transcripts were quantified altogether in the 

tissues and conditions used for generation of the libraries. Cyclophilin 

was used for normalization. Its expression pattern showed very little 

variation when tissues in different physiological conditions were 

compared (e.g., healthy vs. inoculated roots), though variation 

increased when different tissues were compared (e.g., root vs. leaf) 

(data not shown). Therefore, it seemed reasonable to compare 

expression profiles for different physiological conditions but for a 

given tissue. For example, Figure 2 shows the effect of CMV 

inoculation on cotyledon gene expression. As already described for 

other plant-virus combinations, we identified an increase in the 

expression of the 70-KDa and 101-KDa heat shock proteins 

Table 3. Genes analyzed by RT-qPCR. 

Gene Description Gene Description 

4A Eukar.init.fact. 4A Hsp101 Heat shock prot. 

4E Eukar.init.fact. 4E Hsp70 Heat shock prot. 

4E novel Eukar.init.fact. 4E nov. Lsm Sm-like protein 

Aqp Aquaporine Ly e-lycopene cyclase 

Aux Auxine responsive MADS MADS box protein 

Chi Chitinase Tom1 Tobamovirus multip. 1 

Eth Ethylene recept ETR2 Tom2A Tobamovirus multip. 2A 

Ga2ox gibberellin 2-oxidase Tom3 Tobamovirus multip. 3 

Glu UDP-glucose epimerase WRKY WRKY DNA-binding 

Hyp hypersensitive response Xyl Xyloglucan endotransgl. 


(HSP70 and HSP101) associated with CMV infection (Aranda et al., 

1996). Notably, the transcription factor WRKY also responded with an 

increased transcript accumulation (Park et al., 2006). We were 

interested in analyzing the response of genes described as factors 

required for viral infection, such as eukaryotic translation initiation 

factors (Robaglia and Caranta, 2006), Tom genes, and Lsm (Kushner 

et al., 2003). Except for Tom2A, no significant modification on their 

patterns of transcript accumulation could be detected after CMV 

infection (Figure 2). 

10 

9 

8 

7 

6 

5 

4 

3 

2 

1 

0 

4A 

4e 

4enovel 

cy 

hsp101B 

hsp70 

Fig. 2. Pattern of transcript accumulation for 10 selected genes in healthy (CS) 

and CMV-infected melon cotyledons (CI). RNA accumulation is expressed 

relative to that of healthy cotyledons. 

FUTURE PROSPECTS. The unigene set will be used to construct an 

oligo-based microarray to perform transcriptomic analysis in melon. 

An international project has been initiated to further increase the 

collection of melon ESTs and to create a common database to continue 

developing genomic tools in this species. 


Aranda, M. et al. 1996. Induction of HSP70 and polyubiquitin expression associated 

with plant virus replication. Proc. Natl. Acad. Sci. U.S.A. 93:15289–15293. 

F.A.O. 2006. Food And Agriculture Organization Of The United Nations. 15 May, 2006, 

. 

Kushner, D. et al. 2003. Systematic, genome-wide identification of host genes 

affecting replication of a positive-strand RNA virus. PNAS. 100:15764–15769. 

Cucurbitaceae 2006 191 

lsm 

tom1 

CS 

CI 

tom2A 

tom3 

WRKY

Park, C. et al. 2006. A hot pepper gene encoding WRKY transcription factor is 

induced during hypersensitive response to Tobacco mosaic virus and 

Xanthomonas campestris. Planta. 223:168–179. 

Rensink, W. and R. Buell. 2005. Microarray expression profiling resources for 

plant–genomics. Trends Plant Sci. 10(12):603–608. 

Robaglia, C. and C. Caranta. 2006. Translation initation factors: a weak link in plant 

RNA virus infection. Trends Plant Sci. 11:40–45. 


DEVELOPMENT OF A POWDERY MILDEW- 

RESISTANT CUCUMBER 

Yoshiteru Sakata, Mitsuhiro Sugiyama, and Takayoshi Ohara 

National Institute of Vegetable and Tea Science 

Kusawa, Ano, Tsu, Mie. 514-2392, Japan 

ADDITIONAL INDEX WORDS. Cucumis sativus, Podosphaera xanthii, temperatureindependent 

resistance 

ABSTRACT. We developed a cucumber (Cucumis sativus L.; accession CP-02) 

with resistance to powdery mildew (Podosphaera xanthii [Castaggne] U. Braun 

& N. Shishkoff) as potential breeding material. This cucumber is the progeny 

from crosses among CS-PMR1, ‘Sharp 1’ (Saitama Gensyu Ikuseikai), and 

‘Rira’ (Enza Zaden). CP-02 shows a high level of resistance to powdery mildew, 

not only at higher temperatures (above 25°C), but also at relatively cool 

temperatures (20°C). This resistance to powdery mildew is thought to be 

controlled by one major recessive gene and one incompletely dominant gene, 

which enhance the resistance. 

P 

owdery mildew (Podosphaera xanthii [Castaggne] U. Braun & 

N. Shishkoff) limits the production of cucumber (Cucumis 

sativus L.) worldwide. In Japan, the damage caused by 

powdery mildew is severe, as most Japanese commercial greenhouse 

cultivars are susceptible to the disease during the winter-to-spring 

period (Morishita et al., 2002). In addition, tolerance to powdery 

mildew declined after the adoption of bloomless rootstocks for the 

production of bloomless fruits (Hazama et al., 1993; Sakata et al., 

2006). 

Generally, fungicides are used to control powdery mildew in 

cucumber production, but as daily harvests are common in Japanese 

cultivation it is often difficult to time the spraying of fungicides 

properly. The mistiming of fungicide application can lead to a decline 

in yield, and the labor and cost of fungicide application is an 

undesirable burden for growers. In addition, the demand for food 

produced with fewer agrochemicals has been increasing. 

For production with reduced agrochemicals, the use of diseaseresistant 

cultivars is desirable. Although some European greenhousetype, 

Asian open-field, and Beit Alpha-type cucumber cultivars show 

resistance to powdery mildew at higher temperatures (above 25°C), no 

known cultivars are resistant under relatively cool conditions (20°C). 

Morishita et al. (2002) identified an accession, CS-PMR1 (Figure 1), 

that showed a temperature-independent resistance to powdery mildew. 

This accession was derived from PI 197088, a wild-type cucumber of 


Indian origin, and resistance was observed at both higher and lower 

temperatures. Morishita et al. (2003) have started breeding powdery 

mildew-resistant cucumber by using CS-PMR1, but it may require a 

long time to breed practical cultivars, as the resistance of CS-PMR1 is 

controlled by one recessive gene and one incompletely dominant gene. 

Therefore, we intend to release an accession, CP-02, with temperatureindependent 

resistance to powdery mildew, as breeding material. 

Fig. 1. Fruit of CS-PMR1, a resistant source material for breeding 

powdery mildew-resistant cucumber. The fruit was harvested 8 days 

after anthesis. The white bar indicates 5cm. 

Origin 

The source of the resistance to powdery mildew was CS-PMR1. 

Progenies from crosses among CS-PMR1, ‘Sharp 1’ (Saitama Gensyu 

Ikuseikai), and ‘Rira’ (Enza Zaden) were evaluated for powdery 

mildew resistance at 20°C, and one fixed accession, CP-02, was 

selected (Figure 2). 

1996 1997 1998 1999 2005 

PI 197088 CS-PMR1 

F1 F2 F3 Sharp 1 F1 F2 F8 F9 Rira CP-02 

Fig. 2. Pedigree of the powdery mildew-resistant cucumber accession CP-02 


Table 1. Powdery mildew resistance of CP02 and control cultivars at 

20°C and 26°C. 

Disease index z 

Temperature 

20°C 

Cultivar 0 1 2 3 4 5 6 7 8 9 Avg. 

CP02 12 0.00 

Sharp 1 

Suisei- 

1 7 3 8.18 

fushinari 

Kurume 

3 2 8.40 

65 

Poinsette 

2 1 5 5 2 6.27 

76 

Asomidori 

3 5 1 6.78 

5 

Natsu- 

3 6 3 2 1 4.47 

fushinari 

Freedom 

2 3 1 1 1 4.50 

House 2 3 1 2 1 5.14 

CS-PMR1 1 11 0.92 

26°C CP02 12 0.00 

Sharp 1 

Suisei- 

1 11 5.92 

fushinari 

Kurume 

1 1 6 1 5.78 

65 

Poinsette 

12 6.00 

76 

Asomidori 

12 6.00 

5 

Natsu- 

2 8 2 2.00 

fushinari 

Freedom 

4 6 2 1.83 

House 2 1 4 4 1.33 

CS-PMR1 12 0.00 

z 

Disease index: 0 = no symptoms; 9 = completely covered with mildew. 

Inoculation of powdery mildew performed as in Morishita et al., 2002. 

Description 

The characteristics of CP-02 were evaluated in a greenhouse from 

spring to summer 2005. A single stem was trained vertically and 

pinched at 180cm, and all lateral branches were pinched at the first 

node. The Japanese standard cultivar ‘Tokiwa’ was used as a control. 

The plant height, length of internodes, and leaf size of CP-02 were 

similar to those of ‘Tokiwa’ (data not shown), but petiole length was 

relatively longer. The plant was monoecious. The fruit harvest began 


approximately 60 days after seeding, as is characteristic of an earlyharvest 

type. Eighty percent of the lateral branches bore fruit, which 

was cylindrical and similar to those of a Beit Alpha cucumber (Figure 

3). Fruit was small, weighing approximately 60g. No warts or spines 

were found on the surface. The fruit skin was dark green and tough, 

the flesh relatively soft. Parthenocarpy was evident. 

Fig. 3. Fruit of CP-02. The fruit was harvested 8 days after anthesis. The white 

bar indicates 5cm. 

Under growth-chamber conditions, using artificial inoculation of 

Podosphaera xanthii derived from naturally infected melon leaves, the 

powdery mildew resistance of CP-02 was found to be temperatureindependent, 

with resistant reactions observed at both 20°C and 26°C 

(Table 1). We have tested with various isolates and populations; to 

date, no breakdown of resistance has been observed. The resistance to 

powdery mildew is thought to be controlled by one major recessive 

gene and one incompletely dominant gene, enhancing the resistance 

(data not shown). 


Hazama, W., S. Morita, and T. Kato. 1993. Resistance to Corynespora target leaf 

spot in cucumber grafted on a bloomless rootstock. Soc. Phytopath. Soc. Jpn. 

59:243–248. (In Japanese with English summary.) 

Morishita, M., K. Sugiyama, T. Saito, and Y. Sakata. 2002. An improved evaluation 

method for screening and selection of powdery mildew resistant cultivars and 

lines of cucumber (Cucumis sativus L.). J. Jpn. Soc. Hort. Sci. 71:94–100. (In 

Japanese with English summary.) 

Morishita, M., K. Sugiyama, T. Saito, and Y. Sakata. 2003. Powdery mildew 

resistance in cucumber. JARQ. 37:7–14. 

Sakata, Y., M. Sugiyama, T. Ohara, and M. Morishita. 2006. Influence of rootstocks 

on the resistance of grafted cucumber (Cucumis sativus L.) scions to powdery 

mildew (Podosphaera xanthii U. Braun & N. Shishkoff). J. Jpn. Soc. Hort. Sci. 

75:135–140. 


HISTORY AND APPLICATION OF 

MOLECULAR MARKERS FOR CUCUMBER 

IMPROVEMENT 

J. E. Staub, M. D. Robbins, S. M. Chung, and Z. Sun 

USDA/ARS, Vegetable Crops Unit, Department of Horticulture, 

University of Wisconsin-Madison, WI 53706 

ADDITIONAL INDEX WORDS. Marker development, map construction, QTL 

analysis, marker-assisted selection 

ABSTRACT. The history of marker development, map construction, and the 

utility of marker-assisted selection (MAS) is presented. Experimental results 

indicate that the identification of marker-trait associations will continue to be 

extremely expensive and time consuming, but will likely pay large dividends for 

use in MAS. MAS for quantitative trait improvement will require a populationspecific 

evaluation of genotypic background, and an understanding of 

physiology (source-sink relationships), epistasis, and heritabilities. 

C 

ucumber has a narrow genetic base (3–8%) as defined by DNA 

assessments of genetic diversity (Dijkhuizen et al., 1996; 

Horejsi and Staub, 1999; Meglic et al., 1996). Nevertheless, 

cucumber is amenable to MAS because of its rapid life cycle (3–4 

months), its low chromosome number, and relatively small genome (~ 

750cM map length). The development of genetic marker systems 

(Horejsi et al., 1999; Fazio et al., 2002) and unique genetic stocks 

(Staub et al., 1996 & 2002a) have allowed for the slow but consistent 

development of genetic maps for use in marker-assisted selection 

(MAS) (Serquen et al. 1997; Fazio et al., 2003a). 

The pyramiding of simply inherited genes (e.g., disease resistance) 

during germplasm enhancement is common, and has proven useful in 

the improvement of many crop species. Less well reported and 

understood are genetic approaches for the incorporation of 

quantitatively inherited traits. In cucumber, quantitative trait loci 

(QTL) conditioning yield components have been identified and 

mapped for potential use in MAS (Serquen et al. 1997; Fazio et al., 

2003a). The use of MAS in cucumber improvement has been effective 

for increasing gain from selection for single (Fazio et al., 2003b) as 

well as multiple quantitatively inherited traits (Fan et al., 2006; 

Robbins, 2006). Because marker development and the implementation 

of MAS in cucumber has spanned nearly 2.5 decades, reports are often 

not synchronous and, therefore, a historical understanding of this 

process is not self-evident. This paper seeks to provide a historical 


understanding of marker development, mapping, and MAS in 

cucumber. 


The results presented herein are a synthesis of reports on marker 

development (Horesji et al., 1999; Staub et al., 2002b; Robbins, 2006), 

map construction (Serquen et al., 1997; Bradeen et al., 2001; Fazio et 

al., 2003a; Sun et al., 2006), and their use in MAS (Robbins and Staub, 

2004; Robbins, 2006; Fazio et al., 2003b; Fan et al., 2006). While the 

description of marker development focuses on the difficulties 

encountered in marker identification and characterization, Linkage 

Group (LG) 1 is used to validate its importance in MAS. Previously 

unreported data are presented, and are used to synthesize concepts to 

define the use of marker systems for cucumber improvement. 


The development and use of molecular markers for cucumber 

improvement in our laboratory is depicted in Figure 1. The process for 

the application of genetic markers in MAS has followed three major 

recurring cycles, regardless of marker type. Potentially useful markers 

are identified and then developed into efficient and effective systems. 

These markers are then placed on a genetic map and associated with 

QTL through progeny analysis for their subsequent use in MAS. 

1980 

Isozymes, 

RFLP 

1990 

RAPD, 

AFLP, 

SSR 

2000 

SNP 

Resource 

allocation 

New Technologies New Technologies New Technologies 

Methods 

development 

Marker 

Development 

Methods 

evaluation 

New Technologies 

Methods 

assessment 

Map 

construction 

Mapping & 

QTL analysis 

QTL 

analysis 

Field 

evaluation 

Analysis of 

gain 

Markerassisted 

selection 

Experimental 

design 

Population 

development 

New Technologies New Technologies 

Fig. 1. Schematic of marker development and application in cucumber 

breeding. 


MARKER DEVELOPMENT. Marker development has occurred in 

several marker systems [isozymes, restriction fragment length 

polymorphisms (RFLP), random amplified polymorphic DNA 

(RAPD), sequenced characterized amplified region (SCAR), amplified 

fragment length polymorphisms (AFLP), simple sequence repeats 

(SSR), and single nucleotide polymorphisms (SNP)], where changes 

have been driven by steady advances in technology. In each case, the 

cost of development has been critical, since funding for markerdevelopment 

projects has not been abundant in U.S. public plantbreeding 

programs. Much-needed funding to support our laboratory 

was received from international seed companies between 1984 and 

1999 for the development of isozyme, RFLP, RAPD, and SSR 

technologies, with the goal of developing a moderately saturated 

genetic map (~150 to 200 markers to provide 90–95% coverage at 10– 

15-cM intervals). 

Between 1984 and 1995, isozyme and RFLP markers were 

developed and placed on unsaturated genetic maps (Knerr and Staub, 

1992; Meglic and Staub, 1996; Kennard et al., 1994). The use of these 

codominant markers was assessed, but their development was 

minimized because of their high utilization costs and the paucity of 

markers detected when compared to newly developed RAPD 

technologies (Figure 1). Dominant RAPD, and subsequently AFLP 

markers, were attractive because of their comparatively low 

technological costs and simple methodologies. In the case of RAPD 

technology, putative polymorphism declaration (i.e., number of bands) 

were relatively high (10–15 bands per primer), but reproducibility was 

low, as was their fit to 3:1 genetic ratios for many putative marker loci 

(recovery rate ≈ 50 markers per 1,000 evaluated) (Staub et al., 1996). 

This level of recovery is not unusual in other marker systems. Sun et 

al. (2006) found relatively few polymorphisms in RAPD, SCAR, and 

SSR markers between elite mapping parents in a narrow cross (Table 

1). 

Dominant markers (RAPD and AFLP) were initially useful in the 

development of more advanced maps (Serquen et al., 1997; Bradeeen 

et al.,2001), but are not preferred in breeding programs. The mapped 

RAPD loci were, nevertheless, strategically important (Serquen et al., 

1997), and were subjected to conversion to SCAR markers by silver 

staining-mediated sequencing (Horejsi et al., 1999). Although 62 

(83%) of the 75 RAPDs were successfully cloned, only 48 (64%) 

RAPD markers were successfully converted to SCAR markers and 11 

(15%) of these reproduced the polymorphism observed in the original 

RAPD PCR product. The appearance of automated sequencing 

technologies led to the development of codominant SSR and SNP 


Table 1. Marker recovery from comparative genotype screening 

between two elite parents (2A and Gy8) used in mapping 

parthenocarpy (Sun et al. 2006). 

Total 

Marker type examined 

No. 

polymorphic 1 

No. 

mapped 2 

RAPD 1077 77 17 

SCAR 3 77 2 1 

Melon SSR 4 89 1 0 

Cucumber 

SSR5 135 4 3 

1 At least one major band polymorphism between parents. 

2 Fitted to either 1:2:1 (codominant) or 3:1 (dominant) ratio at α = 0.01. 

3 SCAR according to Horejsi et al., 1999. 

4 Melon SSR sequences according to Danin-Poleg et al., 2000. 

5 Cucumber SSR sequences according to Fazio et al., 2002. 

technologies (Fazio et al., 2002, 2003a) and a reassessment of the 

conversion of RAPD to SCAR markers (Robbins, 2006; Figure 1). 

Two methods (sequencing and BAC library hybridization) were 

employed to convert RAPD to SCAR, and SCAR to SNP markers for 

increased efficiency (multiplexing) and effectiveness (stable and 

codominant) (Robbins, 2006). A total of 39 new markers (SCAR and 

SNP) were developed, seven of which have proven effective when 

multiplexed in MAS (Figure 2; Robbins, 2006). 

MAPPING AND QTL ANALYSIS. The cucumber maps of Kennard et 

al. (1994; wide- and narrow-based), Serquen et al. (1997), and Park et 

al. (2000) spanned 766 (narrow-based) and 480 (wide-based), 600, and 

1904 

1584 

1375 

947 

831 

564 

AW14SCAR (H) 

L19-2-SCAR (H) 

AW14SCAR (G) 

L18-SNP-H19 (H) 

Fig. 2. Example of a multiplexing reaction in a population. The far left lane is a 

molecular weight marker (in base pairs). The four bands (top to bottom) are: 

the H19 allele of AW14SCAR (a codominant marker); L19-2-SCAR (H19 allele 

is present, GY7 allele is absent); the GY7 allele of AW14SCAR; and L18-SNP- 

H19 (H19 allele is present, GY7 allele is absent). 


816cM, respectively. The map constructed by Serquen et al. (1997) 

defined 9 linkage groups with an average distance between RAPD 

markers of 8.4cM. Information from the Serquen et al. (1997) map 

was recently merged with other maps (Fanourakis and Simon, 1987; 

Horejsi et al. 2000; Kennard et al., 1994; Knerr and Staub, 1992; 

Meglic and Staub, 1996) to synthesize a consensus map spanning 10 

linkage groups with 255 markers, including morphological traits, 

disease resistance loci, isozymes, RFLPs, RAPDs, and AFLPs 

(Bradeen et al., 2001). The mean marker interval in this consensus 

map was 2.1cM with a total length of 538cM. Fazio et al. (2003a) 

then constructed a map containing 14 SSR, 24 SCAR, 27 AFLP, 62 

RAPD, 1 SNP, and 3 morphological markers (131 total markers) 

spanning 7 linkage groups (the theoretical number) using recombinant 

inbred lines (RIL). This map spanned 706cM with a mean marker 

interval of 5.6cM. More recently, Sun et al. (2006) constructed a map 

to identify the map location of parthenocarpy using an F2:3 design 

employing lines 2A (parthenocarpic) and Gy8 (nonparthenocarpic) as 

mapping parents. Linkage maps having 7 linkage groups (LG) were 

constructed, consisting of 47 (2A-coupling phase), 48 (Gy8-coupling 

phase), and 54 (coupling plus repulsion phase) DNA markers, 

spanning 388.6cM, 412.2cM, and 496.6cM, with an average marker 

interval of 8.1cM, 8.8cM, and 9.2cM, respectively. The general 

marker order is conserved (collinearity) in the maps of Fazio et al. 

(2003a), Sun et al. (2006), and Bradeen et al. (2001) (Table 2), 

indicating the potential utility of the consensus map of Bradeen et al. 

(2001). Three genomic regions conditioning parthenocarpic QTLs in 

Sun et al. (2006) were also mapped to the same genomic regions as 

QTLs detected for fruit yield at first-harvest as reported by Fazio et al. 

(2003a). 

MARKER-ASSISTED SELECTION. Two experiments have been 

reported using marker-trait associations identified by Serquen et al. 

(1997) and Fazio et al., (2003a) for MAS of yield components. Five 

QTL for a single trait, multiple lateral branching (MLB), were linked 

with SNP, SSR (2), and RAPD (2) markers on different linkage 

groups. These associations were used during backcrossing to increase 

MLB. The means of MLB after phenotypic (BC2PHE = 3.0, BC3PHE 

= 3.3) and marker-aided selection (BC2MAS = 3.1, BC3MAS = 3.1) 

were not significantly different. Thus, markers linked to MLB are 

effective tools in marker-assisted cucumber improvement. 

Multiple-trait MAS in cucumber was examined during phenotypic 

recurrent mass selection (population development) followed by MAS 

(SSR, RAPD, SCAR, and SNP) backcrossing (line extraction) (Fan et 

al., 2006). Similar gain from selection was detected as a result of 


Table 2. Common genetic markers in Linkage Group 1 across four 

linkage maps in cucumber (Cucumis sativus L. var. sativus and var. 

hardwickii [R.]) Alef. 

Sun et al., 2006 

(2A × Gy8) F2 

var. sativus × 

var. sativus 

Linkage group 1 

Updated Fazio et al. 

2003a 

(Gy7 × H19) 

RIL var. 

sativus × 

var. sativus 

Bradeen et al. 2001 Bradeen et al. 2001 

Narrow- 

based 

consensus 

F 2/BC var. 

sativus × var. 

sativus 

Broad- 

based 

consensus 

F 2/BC 

var. sativus 

× var. hardwickii 

F (LG1,0.0) F (LGA,0.0) 

CSWCT25-350 CSWCT25-350 

(LG1,6.5) 

(LG1,9.4) 

J5-SCAR 

(LG1,11.2) 

J5_1 (LGA,5.6) 

de (LG1,28.8) de (LGA,15.6) 

E14M62-214 E14/M62-F-214-P2 E14/M62-F-214-P2 

(LG1,37.2) 

(LGA,52.2) 

(LGA,77.9) 

E14M62-112 E14/M62-F-112-P1 

(LG1,43.0) 

(LGA,41.2) 

E12M62-230 E12M62-230 

(LG1,56.2) 

(LG1,49.0) 

E18M48-188 E18M48-188 

(LG1-2A,64.6) (LG1,54.7 

I1B-SCAR 

(LG1,57.5) 

I1_1 (LGA,54.4) 

OP-AJ6 (LG1,59.5) AJ6 (LGA,52.2) 

E12M48-107 E12/M48-F-107-P2 

(LG1,62.2) 

(LGA,53.0) 

BC523-SCAR 

(LG1,64.1) 

BC523 (LGA,52.2) 

OP-AD12-1 AD12 (LGA,49.2) 

(LG1,68.4) 

OP-W7-2 

(LG1,76.2) 

W7_2 (LGA,71.8) 

E14M62-224 

(LG1,76.9) 

ll (LG1,82.0) 

E14/M62-F-224-P2 

(LGA,48.2) 

ll (LGA,68.5) 

E18M48-303 (LG1- 

2A,68.5) 

E18M48-303 

(LG1,84.0) 

BC551 (LGA,69.1) BC551 (LGA,92.1) 

BC592-SCAR 

(LG1,100.1) 

BC592 (LGA,81.8) 

OP-AH14 

(LG1,112.7) 

AH14 (LGA,96.2) 

* Underline = single sequence repeat; italic = amplified fragment length 

polymorphism; bold = random amplified polymorphific DNA or sequence 

characterized region; and bold & underlined = restriction fragment length 

polymorphism. Parentheses indicate linkage group and position. 


phenotypic and MAS selection for MLB (~0.3 branches/cycle), and 

fruit length-to-diameter ratio (L:D; ~0.1 unit increase/cycle) with 

concomitant changes in frequency at linked marker loci. MAS 

operated to fix favorable alleles that were not exploited by PHE, thus 

MAS could be applied for altering plant architecture in cucumber to 

improve its yield potential. 

The value of MAS in population development of cucumber was 

examined by Robbins (2006) using the same traits and markers 

employed by Fan et al. (2006). Four inbred lines, complementary for 

the target traits, were intermated and the resulting populations 

underwent three cycles of MAS and PHE. Selections by PHE were 

visually made for all four traits at the whole-plant level and MAS was 

based on individuals possessing the highest number of desired marker 

genotypes from 20 marker loci. Both MAS and PHE provided 

improvements in all traits under selection in at least one population, 

except for earliness by MAS. Overall, PHE was most effective for 

gynoecious sex expression, earliness, and L:D, while MAS was most 

effective for MLB. The most effective selection method, however, 

varied by trait and between populations (Figure 3). 

No. of lateral branches 

3.50 

3.25 

3.00 

2.75 

2.50 

2.25 

2.00 

Population 1 

Multiple lateral branching 

C0 C1 C2 C3 

Cycle of selection 

No. of lateral branches 

3.50 

3.25 

3.00 

2.75 

2.50 

2.25 

2.00 

Population 3 

Multiple lateral branching 

C0 C1 C2 C3 

Cycle of selection 

MAS 

PHE 

RAN 

Linear (MAS) 

Linear (PHE) 

Linear (RAN) 

Fig. 3. Response to three cycles of selection (C0–C3) by marker (MAS), 

phenotype (PHE), and no selection (RAN) for the number of lateral branches in 

two populations. 

MAS in cucumber can be effective, but requires high resource 

inputs to develop markers, define valuable marker-trait associations, 

and conduct appropriate experiments to determine marker efficacy. 

Although initial marker development efforts were largely ineffective, 

sequencing technologies and the availability of BAC libraries (Nam et 

al., 2006), and expressed sequence tags (ESTs) will allow for the 


development of codominant SSR- and SNP-based markers that will be 

extremely useful in MAS in cucumber. 


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PhD Thesis, University of Wiscosin-Madison. 

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quantitatively inherited yield components in cucumber, p. 401–410. Proc. 8th 

Eucarpia Conference, Cucurbitaceae 2004: progress in cucurbit genetics and 

breeding research. Olomouc, Czech Republic. 

Serquen, F. C., J. Bacher, and J. E. Staub. 1997. Mapping and QTL analysis of a 

narrow cross in cucumber (Cucumis sativus L.) using random amplified 

polymorphic DNA markers. Molec. Breeding 3:257–268. 

Staub, J. E., J. Bacher, and K. Poetter. 1996. Factors affecting the application of 

random amplified polymorphic DNAs in cucumber (Cucumis sativus L.). 

HortSci. 31:262–266. 

Staub, J. E., L. K. Crubaugh, and G. Fazio. 2002a. Cucumber recombinant inbred 

lines. Cucurbit Gen. Coop. Rpt. 25:1–2. 

Staub, J. E., M. D. Robbins, and A. I. López-Sesé. 2002b. Molecular methodologies 

for improved genetic diversity assessment in cucumber and melon. Proc. 26th 

IRC, Horticulture: art and science for life-advances in vegetable Breeding. Acta 

Hort. 642:41–47. 

Staub, J. E., V. Meglic, L. K. Crubaugh, and L. D. Knerr. 1996. Cucumber 

germplasm: isozyme genetic stocks W6743, W6744, W6745. HortSci. 

31:1243–1245. 

Sun Z., R. L. Lower, S.M. Chung, and J. E. Staub. 2006. Identification and 

comparative analysis of quantitative trait loci (QTL) associated with 

parthenocarpy in processing cucumber. Plant Breed. (In press.) 


SILVERLEAF WHITEFLY AFFECTS LEAF 

MOTTLING IN CUCURBITA MOSCHATA 

DUCHESNE 

Linda Wessel-Beaver and Moisés González-Román 

University of Puerto Rico, Mayagüez, Puerto Rico 

ADDITIONAL INDEX WORDS. Bemisia argentifolii , recessive epistatis, 

complementary gene action 

ABSTRACT. In Cucurbita moschata, leaf mottling (silver patches in the leaf-vein 

axils) is reported to be controlled by a single gene M (mottle-leaf) that is 

dominant over m (green-leaf). In the presence of the silverleaf whitefly (Bemisia 

argentifolii Bellows and Perring), some green-leaf lines express a yellow-mottle 

(yellow patches in the leaf-vein axils), while other lines remain green-leaf, 

suggesting a more complex inheritance of leaf mottling. We studied the 

relationship between whiteflies and leaf mottling by observing segregations in 

populations derived from crosses of mottle-leaf, yellow-mottle, and green-leaf 

phenotypes. In the presence of whiteflies, an F2 population of mottle-leaf × 

green-leaf segregated 9 mottle-leaf: 3 yellow-mottle: 4 green-leaf. F2 populations 

of mottle-leaf × yellow-mottle segregated 3 mottle-leaf: 1 yellow-mottle. F2 

populations of yellow-mottle × green-leaf segregated 3 yellow-mottle: 1 greenleaf. 

These segregations suggest that mottling in the presence of the silverleaf 

whitefly is controlled by two genes, M (previously reported) and proposed M-2 

(mottle leaf-2), where allele m-2 changes M/M to green-leaf (recessive epistatis 

of m-2 to M and m). In the absence of whiteflies, locus M-2 and M exhibit a 

complementary gene action where both dominant alleles are needed to produce 

the mottle-leaf phenotype. 

S 

ilver-grey patches in leaf-vein axils are seen in many different 

Cucurbita including wild species. In C. moschata, the presence 

of silver patches is reported to be controlled by a single gene M 

(mottle-leaf) dominant over m (green-leaf) (Coyne, 1970). Coyne 

(1970) studied this trait in two crosses using four parents: ‘Crookneck 

Butternut’ × ‘Golden Cushaw’ and ‘New Hampshire Butternut’ × 

‘Hercules.’ All four parents are temperate types of C. moschata and 

likely represent a narrow genetic background. Three of the parents are 

butternut types. The first author, in her breeding program, noticed that 

some genotypes classified as green-leaf (m/m) would sometimes 

The authors thank Mr. Obed Roman Hernandez for his technical assistance. This 

research was supported in part by the USDA/CSREES Special Grant Program in 

Tropical/Subtropical Agriculture Research, USDA Hatch funds, and by the Puerto 

Rico Agricultural Experiment Station. 


express an unusual type of yellow leaf mottling, not described by 

Coyne (1970). This yellow-mottle phenotype occurred only in the 

presence of the silverleaf whitefly. Other m/m genotypes would remain 

green-leaf in the presence of the insect. The objective of this study was 

to determine the relationship between the presence of whiteflies and 

the expression of leaf mottling by observing segregations in 

populations derived from crossing parents with various combinations 

of leaf-mottling phenotypes (mottle-leaf, yellow-mottle, and greenleaf). 


Crosses were made among 12 cultigens that differed in leafmottling 

phenotype (Table 1). With the exception of ‘Waltham’ and 

BN111, most parents were tropical, subtropical, or mixed (tropical × 

temperate) in origin. The F2 and backcross (BC) populations were 

evaluated at 5 weeks after planting for leaf mottling under conditions 

of high natural populations of whiteflies in the field in Isabela, Puerto 

Rico, in either June 2001 or May 2005, or in Juana Díaz, Puerto Rico, 

in May 1999. Whitefly populations are typically very high during the 

late spring/early summer in Puerto Rico. The parents and F1s were 

phenotyped in those plantings and several others over the past 5 years, 

both in the presence and absence of whiteflies. Leaf mottling was 

classified from 0 to 3 (0 = green-leaf; 1 = weakly mottle-leaf; 2 = 

intermediate mottle-leaf; 3 = strong mottle-leaf) after Coyne (1970). 

This classification was used only when leaf-axil patches were silvergrey. 

Plants with yellow-mottle (light yellow patches in the leaf-vein 

axils) were simply classified as Y. Observed versus expected 

segregations were compared using chi-square tests. Classes 1 to 3 for 

silver-grey mottle-leaf were combined, leaving three mottle-leaf 

classes: silver-grey mottle (SM), yellow-mottle (YM), and green-leaf 

(G). The G phenotype included plants that had silver veins but no grey 

patches in the leaf-vein axils. 

Results 

Four parents (‘Waltham’, BN11, 9706, and Bol-370), classified as 

green-leaf (m/m) in the absence of whiteflies, express a yellow-mottle 

phenotype when planted in fields naturally infested with whiteflies 

(Table 1). Two other genotypes (Col-5 and PI 162889) remain greenleaf 

(defined as having no leaf-vein-axil patches) even in the presence 

of whiteflies. Col-5 had a slight silver color that followed, but did not 

concentrate in, leaf-vein axils (no patches of silver). The latter 

phenotype was very distinct from that of the six mottle-leaf genotypes 


Table 1. Leaf mottling phenotypes expressed by Cucurbita moschata 

parents in the presence and absence of the silverleaf whitefly Bemesia 

argentifolii 

Parent 

Type of 

germplasm 

Phenotype/ 

whiteflies 

absent 

Phenotype/ 

whiteflies 

present 

Coyne 

(1970) 

genotype 

Soler Tropical 

Tropical × 

SM SM M/M 

TP411 Temperate 

Tropical × 

SM SM M/M 

TP312 Temperate SM SM M/M 

Col-11 

Nigerian 

Tropical SM SM M/M 

Local Tropical SM SM M/M 

CG-1 Tropical SM SM M/M 

Waltham Temperate G YM m/m 

BN111 Temperate 

Tropical × 

G YM m/m 

9706 Temperate G YM m/m 

Bol-370 Tropical G YM m/m 

Col-5 Tropical G G m/m 

New 

proposed 

genotype 

M-2/M- 

2M/M 

M-2/M-2 

M/M 

M-2/M-2 

M/M 

M-2/M-2 

M/M 

M-2/M-2 

M/M 

M-2/M-2 

M/M 

M-2/M-2 

m/m 

M-2/M-2 

m/m 

M-2/M-2 

m/m 

M-2/M-2 

m/m 

m-2/m-2 

M/M 

PI162889 Subtropical G G m/m 

m-2/m-2 

m/m 

SM = silver-grey mottle [“mottle-leaf” of Coyne (1970)]; YM = yellow mottle-leaf; 

G = no patches in leaf-vein axils [“green-leaf” of Coyne (1970)]. 

that displayed large silver-grey patches in the leaf-vein axils. The Col- 

5 phenotype is clearly a variation of green-leaf, and not of the silvergrey 

mottle-leaf. The genotypes classified as M/M according to Coyne 

(1970) expressed the same degree of silver mottling whether whiteflies 

were present or not (data not presented). 

Segregating F2 populations produced ratios of 3:1 or 9:3:4 (Table 

2). Crosses of silver-grey mottle (SM) × yellow-mottle (YM) 

segregated 3 silver-grey mottle: 1 yellow-mottle; crosses of silver-grey 

mottle (SM) × green-leaf (G) segregated 9 silver-grey mottle: 3 

yellow-mottle: 4 green-leaf or 3 silver-grey segregated 3: yellowmottle: 

1 green-leaf. Backcross populations segregated either 1 silver- 


grey mottle: 1 yellow-mottle or 1: silver-grey mottle: 1 green-leaf, 

depending on the cross. 

Table 2. Segregation for leaf mottling in F2 and BC populations of Cucurbita 

moschata in the presence of silverleaf whiteflies (Bemesia argentifolii). 

Observed frequency of: 

F2 or BC 

population 

Description of 

parental 

phenotypes 

Mottleleaf 

(SM) 1 

Yellow 

mottleleaf 

(YM) 

Greenleaf 

(G) 1 

Exp. 2 

Nigerian 

Local (NL) × 

Chi-square 

(probability) 

Col-11 

Waltham × 

SM × SM 49 0 0 all M - 

TP411 

BN111 × TP 

YM × SM 80 22 0 3:1 0.64 (0.42) 

312 

YM × SM 60 17 0 3:1 0.35 (0.55) 

9706 × Soler 

TP312 × 

YM × SM 24 12 0 3:1 1.33 (0.25) 

Waltham 

PI162889 × 

YM × SM 84 0 0 all M - 

Soler 

G × SM 34 16 14 9:3:4 1.69 (0.43) 

NL × Col-5 

CGold-1 × 

SM × G 39 0 8 3:1 1.60 (0.21) 

Col-5 

Bol-370 × 

SM × G 35 0 16 3:1 1.10 (0.29) 

PI162889 YM × G 1 38 10 3:1 0.44 (0.50) 3 

(9706 × (YM × SM) × 

Soler) × 9706 

(CG-1 × Col- 

YM 27 15 0 1:1 2.57 (0.11) 

5) × Col-5 

(NL × Col-5) 

(SM × G) × G 25 0 25 1:1 0.00 (1.00) 

× Col-5 (SM × G) × G 22 0 28 1:1 0.72 (0.40) 

(CG-1 × Col- (SM × G) × 

5) × CG-1 SM 50 0 0 all M - 

1 

Mottle-leaf refers to silver-grey patches and green-leaf refers to the lack of silvergrey 

patches in the leaf-vein axils, as described by Coyne (1970). Terminology is 

also that of Coyne (1970). 

2 

Expected segregation. 

3 

Because the chi-square test is inappropriate for models with expected frequencies of 

zero, single observations that did not fit into the 3:1 model were not included in the 

calculation of the statistic. 


Discussion 

Three phenotypic classes suggest that leaf mottling is controlled 

either by a single locus with incomplete dominance or additivity 

(producing an “intermediate” phenotype), or by two loci with epistasis. 

The ratio observed for the silver-grey mottle × green-leaf cross, 9:3:4 

rather than 1:2:1, indicates that the latter explanation is more likely. 

We propose that mottling in the presence of the silverleaf whitefly is 

controlled by two genes, a new gene M-2 (mottle-leaf-2) and the 

previously reported M. When silverleaf whiteflies are present, m-2/m- 

2 causes M/M to produce a green-leaf phenotype and causes 4/16 of 

the F2 progeny in a SM × G cross to be green-leaf. Fehr (1991) calls 

this type of between-locus interaction “recessive epistatis.” In the 

absence of whiteflies, locus M-2 and M exhibit a complementary gene 

action, where both dominant alleles are needed to produce the mottleleaf 

phenotype. Under this scheme, in the presence of whiteflies, the 

SM phenotype corresponds to M-2/M-2 M/M and the YM phenotype 

corresponds to M-2/M-2 m/m. The G phenotype corresponds to two 

possible genotypes: m-2/m-2 M/M or m-2/m-2 m/m. The segregations 

of crosses involving the two parents with the G phenotype (Col-5 and 

PI162889) indicate that Col-5 is M/M while PI162889 is m/m (Tables 

1 and 2). 

The genotypes Coyne (1970) used in his study were probably 

homozygous for M-2/M-2. This would explain why Coyne (1970) 

reported leaf mottling to be controlled by a single locus. What Coyne 

(1970) described as M/M and m/m were likely the genotypes M-2/M-2 

M/M and M-2/M-2 m/m, repectively. In the experience of the first 

author (evaluating hundreds of PIs and other germplasm of C. 

moschata), the m-2/m-2 genotype is very rare. We have observed it 

only in tropical or subtropical germplasm from South America. Col-5 

and PI 162889 are landraces from Colombia and Paraguay, 

respectively. These materials are of special interest because they often 

appear to be resistant to the whitefly-induced silverleaf disorder 

(Wessel-Beaver, 1998). 


Coyne, D. P. 1970. Inheritance of mottle-leaf in Cucurbita mochata Poir. HortSci. 

5(4):226–227. 

Fehr, W. R. 1991. Principals of cultivar development, vol. I. Iowa State University, 

Ames, Iowa. 

Wessel-Beaver, L. 1998. Sources of whitefly-induced silvering resistance in 

Cucurbita. In: J. D. McCreight (ed.). Cucurbitaceae ’98: evaluation and 

enhancement of cucurbit germplasm. ASHS, Alexandria, VA. 


Wessel-Beaver, L. 2000. Inheritance of silverleaf resistance in Cucurbita moschata. 

In: N. Katzir and H. S. Paris (eds.). Proc. Cucurbitaceae 2000, Acta Hort. 

510:289–295. 


QTL ANALYSIS OF SOLUBLE SOLIDS 

CONTENT IN WATERMELON UNDER 

DIFFERENT ENVIRONMENTS 

Yong Xu, Shaogui Guo, Haiying Zhang, and Guoyi Gong 


Banjing, Haidian, Beijing 2443#, Beijing, 100089, P. R. China 

ADDITIONAL INDEX WORDS. C. lanatus, quantitative trait locus, genotype × 

environment interaction 

ABSTRACT. Soluble solid content (SSC) is an important factor affecting the 

quality of watermelon. It is necessary to confirm the genetic stability of the 

quantitative trait loci (QTLs) under different environments. An F2S8 RIL 

population was derived from the cross of PI 296341-FR × ‘97103’ and a highdensity 

molecular genetic-linkage map covering 1383.8cM with an average 

distance 6.8cM was constructed. QTL analysis of watermelon SSC was 

conducted under three environments. In total, 18 QTLs were detected, located 

on the Linkage Groups 1, 2, 3, 5, 14, 15, and 19. Six QTLs were detected under 

two environments. Only 2 QTLs, qSSC-1a and qSSC-1b (explaining the 19% 

and 17.6% average phenotypic variation, respectively), were comparatively 

stable and can be detected under the three environments observed. These two 

QTLs, which may be the main loci controlling the gene expression of SSC, were 

mapped between the markers N02_800a and Z03_250a. The results are 

advantageous for using MAS for SSC in watermelon. 

S 

oluble solids content (SSC) is an important factor affecting the 

quality of watermelon and many breeders attach importance to 

this trait. But SSC is a quantitative trait locus (QTL) and 

controlled by several genes. The stable expression of QTL under 

different environments is an important factor in the use of QTL as the 

target gene in marker-assisted selection (MAS). Twenty-nine QTLs 

related to SSC were detected in the F2 and F3 populations of tomato 

under three different environments, but only 4 QTLs had outstanding 

expression under three environments (Paterson et al., 1991). QTLs are 

sensitive to environment and are expressed with different stability. 

That is to say, the QTLs with high heritability are easily detected 

under several different environments (Huang et al., 1997). The SSC 

genes of watermelon have been mapped using impermanent 

populations, and several QTLs have been detected recently (Fan et al., 

This research was supported by the National Natural Science Foundation of China 

(30471186, 30570997) and the Natural Science Foundation of Beijing (5062008, 

5050001). 


2000; Hashizume et al., 2003). The SSC genes of watermelon have 

been mapped using the F2 population derived from a crossing between 

a cultivated inbred line ‘97103’ and an African wild form PI 296341- 

FR, and 4 related QTLs have been mapped (Fan et al., 2000). A QTL 

for the SSC of watermelon had been mapped using the BC1 population 

derived from a crossing between a cultivated inbred line (H-7; C. 

lanatus) and an African wild form (SA-1; C. lanatus) (Hashizume et 

al., 2003). QTL analysis of watermelon SSC needs to be conducted 

under different environments to confirm their inheritance stability. 

However, the report about the analysis of the SSC expression in 

watermelon under different environments using permanent population 

has not been found up to now. 

In the present study, a high-density genetic map has been 

constructed based on the permanent population derived from a 

crossing between a cultivated inbred line ‘97103’ and an African wild 

form PI 296341-FR. The SSC of watermelon under three different 

environments was analyzed and their respective QTLs were compared 

to find the QTLs with high heritability and stability. 


PLANT MATERIALS. The high-SSC cultivated inbred line ‘97103’ 

(Citrullus lanatus [Thunb., Mansfeld] var. lanatus) was crossed with 

the low-SSC African wild form PI 296341-FR (Citrullus lanatus 

[Thunb., Mansfeld] var. citroides). One F1 plant was self-pollinated to 

produce 120 F2 progeny, which were then self-pollinated by the singleseed 

descent method to obtain 117 F2S8 recombinant inbred lines 

(RILs). The RILs were planted in a randomized block design under 

three open-air environments: (1) Daxing, Beijng in May, 2002 (E 

116°13′-116°43′ N 39°26′-39°51′; altitude 54.7m; warmtemperature/semihumid 

climate region; annual mean temperature 13°C; 

annual duration of sunshine 2000–2800 hours; average annual 

precipitation 507.7mm; frost-free period 189 days); (2) Changji, 

Xinjiang in May, 2003 (E 86°24’-87°37’, N 43°06’-45°20’; altitude 

918.7m; midtemperate continental arid climate region; annual mean 

temperature 6.8°C; annual duration of sunshine 2500–3550 hours; 

average annual precipitation 190mm; frost-free period 160 days); and 

(3) Changji, Xinjiang in May, 2004. Thirty plants of every line were 

planted in three rows with 10 individuals in each row according to 

standard conventional methods. The central flesh juices of the mature 

fruits were analyzed for SSC using a hand refractometer (ATAGO 

ATC-1E, Atago Co., Ltd., Tokyo, Japan). The RILs were used in map 

construction and QTL analysis. 


MAP CONSTRUCTION AND QTL ANALYSIS. Two molecular linkage 

maps have been constructed in our laboratory (Zhang et al., 2004; Yi 

et al., 2004). In the first, a total of 87 RAPD markers, 13 ISSR markers, 

and 4 SCAR markers were arranged into 15 linkage groups. A total of 

1027.5cM in the population was covered with an average interval 

length of 11.7cM (Zhang et al., 2004). In the second map, 150 AFLP 

markers were arranged into 17 linkage groups. A total of 1240.2cM in 

the population was covered with an average interval length of 8.3cM 

(Yi et al., 2004). Combining with the 24 polymorphic SSR markers 

and a morphological marker screened in our laboratory, a new 

molecular genetic-linkage map of watermelon was constructed using 

JoinMap3.0 software. In this map, 79 AFLP markers, 86 RAPD 

markers, 24 SSR markers, 10 ISSR markers, 3 SCAR markers, and a 

morphological marker were arranged into 19 linkage groups. A total of 

1383.8cM in the population was covered with an average interval 

length of 6.8cM. The distribution of SSC data of watermelon in 

parents and the RILs was analyzed using SYSTAT 10 software and 

overall genome QTL screening was conducted using MapQTL 5 

software. The significance of each QTL interval was tested by a 

likelihood-ratio statistic (LOD) score of 3.0. The variation explanation 

and the additive effect of the QTLs were calculated simultaneously. 

These QTLs were denominated following the universal rules 

(McCouch et al., 1997). 

Results 

DISTRIBUTION OF SSC IN TWO PARENTS AND RIL POPULATION. 

The SSC of ‘97103’ is higher than SSC of PI 296341-FR under the 

three environments, and the differences between both values in the 

three environments were 7.75ºBrix, 7.90ºBrix, and 7.80ºBrix, 

respectively. No significant differences were detected between the 

values of SSC under the three environments. The SSC of the RIL 

population distributed broadly and there was a significant difference 

between the two end values. A continuous distribution near to normal 

for SSC was observed in the RIL population (Table 1). The 

correlations between the SSC values observed in the RILs population 

under the three different environments were 0.621, 0.651 and 0.773 

(Table 2). 

There were significant and positive correlations at the level α = 

0.01 between the SSC values under the three environments. The result 

demonstrated that the SSC of the parents and the RILs are comparative 

stable and fit to be used in this study and the SSC is a typical 

quantitative trait. However the different correlation coefficients 


Table 1. Distribution of SSC of the two parents and the RIL population 

in ºBrix 

97103 

Parents RIL population 

PI 

296341- 

FR Mean Range 

Mean 

±SD 

Kurtosis 

Skewness 

Beijing, 

2002 10.50 2.75 3.39 1.40~7.50 3.39±1.26 0.37 0.89 

Xinjiang, 

2003 10.70 2.80 3.95 2.00~8.40 3.95±1.40 0.42 1.20 

Xinjiang, 

2004 10.30 2.50 5.20 2.92~10.40 

5.20±1.48 0.89 0.95 

indicated that there was GE interaction between the SSC of 

watermelon and the environments. 

QTL ANALYSIS. Interval mapping was performed analyzing the 

SSC data (Figure 1). A total of 18 QTLs were detected, 14 in Beijing, 

2002, 6 in Xinjiang, 2003, and 8 in Xinjiang, 2004; these QTLs were 

located on the Linkage Groups 1, 2, 3, 5, 14, 15, and 19. Two QTLs 

explained 19% and 17.6% of the phenotypic variation detected under 

the three environments. Six QTLs were detected under two of the 

observed environments. 

Two QTLs detected under the three environments, qSSC-1a and 

qSSC-1b, were mapped in Linkage Group 1, and their peaks were 

mapped in the same location of markers N02_800a and Z03_250a, 

respectively. The average additive effects of qSSC-1a and qSSC-1b 

were -0.6039ºBrix and -0.5764ºBrix, respectively. The corresponding 

positive alleles of the QTLs came from ‘97103’. The average variation 

explanation value of qSSC-1a is 19% and its allele, coming from 

‘97103’, could increase the SSC in 0.6039ºBrix. Its additive effect and 

the variation explanation were the highest among the detected QTLs. 

Two QTLs, qSSC-2a and qSSC-2b, were mapped in Linkage 

Group 2; their peaks were mapped in the same location of markers 

H08_1500 and Q05_675a, respectively. The average additive effect of 

qSSC-2a and qSSC-2b were -0.5759ºBrix and -0.5323ºBrix, and their 

average variation explanations were 12.9% and 13.7%. The 

corresponding positive alleles of the QTLs came from ‘97103’. The 

QTL qSSC-2a could be detected under two environments (Beijing, 

2002 and Xinjiang, 2004). The QTL qSSC-2b could be detected under 

two environments (Xinjiang, 2003 and Xinjiang, 2004). 

Five QTLs were mapped in Linkage Group 3. Two QTLs, qSSC-3a 

and qSSC-3b, could be detected under two environments (Beijing, 


Table 2. Correlation coefficients of watermelon SSC characters under three 

different environments. 

SSC 

Beijing, Xinjiang, Xinjiang, 

Location 

2002 

2003 

2004 

Beijing, 2002 - 

Xinjiang, 2003 0.621 ** c 

Xinjiang, 2004 0.651 ** 0.773 ** ** Significant at α = 0.01 

- 

2002 and Xinjiang, 2004). The peak of qSSC-3a was mapped in the 

same location of the marker R05_750. The QTL qSSC-3b was mapped 

near the marker Z12_1800 with a distance of 2cM. Their average 

additive effects were -0.5491ºBrix and -0.5835ºBrix, and their 

variation explanations were 16.7% and 18.1%. The corresponding 

positive alleles of the QTLs came from ‘97103’. The other three QTLs 

could be detected in Beijng, 2002, with obvious GE interaction. 

The QTL qSSC-5 was detected in the Linkage Group 5 with a 

distance of 2.1cM away from marker P17_1000. This QTL could be 

detected under two environments (Beijing, 2002 and Xinjiang, 2004). 

Its average additive effects were -0.5751ºBrix and the average variation 

explanation is 16.3%. The corresponding positive alleles of the QTLs 

came from ‘97103’. 

Three QTLs were mapped in Linkage Group 14 under two 

environments (Xinjiang, 2003 and Xinjiang, 2004) and the peak of 

qSSC-14a was mapped in the same location of marker Z01_2100. The 

corresponding positive alleles of the QTLs came from ‘97103’ and 

could increase the SSC by an average of 0.5689ºBrix; their average 

variation explanation value is 15.6%. The other two QTLs could be 

detected only in Xinjiang, 2003. 

Two QTLs for SSC were mapped in Linkage Group 15 in Beijing, 

2002, with obvious GE interaction. The corresponding positive alleles 

of the QTLs coming from ‘97103’ could increase the SSC in 0.4864 to 

0.4956ºBrix, and their variation explanations were 14.9%–15.6%. 

Three QTLs mapped in Linkage Group 19 could be detected only 

in Beijing, 2002. The positive alleles of the QTLs came from ‘97103’ 

and could increase the SSC in 0.4781 to 0.5499ºBrix; their variation 

explanations were 14.4%–19%. 

EFFECT OF DIFFERENT ENVIRONMENTS ON THE QTLS FOR SSC. 

Among the 18 QTLs detected, only 2, qSSC-1a and qSSC-1b, were 

detected under all three environments. The directions of these genetic 

effects were identical, whereas the additive effects and the variation 


explanation were different because of the influence of the 

environments. Six of the QTLs were detected under two environments 

and 10 QTLs under only one environment. The results demonstrated 

that these QTLs are easily affected by environmental conditions. It is 

necessary to do the experiments in different years and places to 

identify the stable major gene. 

Causal GE interactions can usually reduce general response to 

MAS across environments, and the reduction in the cumulative 

response is a function of the proportion of GE interactions involved in 

the improved trait. The QTL analysis of the agronomic traits in rice 

indicated that the results of the same traits were different when 

observed in different environments (Zhuang et al., 1997). Some 

researchers considered that the results of QTL mapping depend on 

many factors, such as the planting environment, the size of the 

population, the number of the markers in the genetic map, and the 

general heritability (Tanksley, 1993). Thus, new breeding strategies 

based on QTL evaluation among different environments will be 

necessary to realize the potential of MAS. 

In this study, the QTL for the SSC of watermelon was analyzed 

under three different environments. The significant climate distinction 

of the two locations, Beijing and Xinjiang, benefits the identification 

of the stable major genes. Comparing the QTL detected under three 

different environments, only 2 QTLs were comparatively stable and 

could be detected in all the environments. The two most stable QTLs, 

qSSC-1a and qSSC-1b, may be the major QTLs for SSC in 

watermelon. It is noted that these QTLs were mapped in the same 

range between markers N02_800a and Z03_250a. The result is 

advantageous for the use of MAS for SSC in watermelon. The QTL 

qSSC-1a was mapped near the flanking marker IX_334. The band of 

the marker IX_334 was present in the parent ‘97103’ and the distance 

between the marker and qSSC-1a is only 3cM. Considering that the 

positive allele of qSSC-1a came from ‘97103’ could increase the SSC 

in 0.6039ºBrix, the marker IX_334 can be used in the MAS of the 

corresponding positive allele of qSSC-1a came from ‘97103’. 

Due to the differences in the populations used, molecular markers, 

genetic maps, and planting environments, no identical QTLs were 

found between this study and other reports (Fan et al., 2000; 

Hashizume et al., 2003). Nevertheless, in the F2 population derived 

from the same parents used in this study, two QTLs were detected in 

which alleles from ‘97103’ could increase SSC (Fan et al., 2000). 

Follow-up studies will be performed to analyze the relation 

between the two QTLs detected in this F2 population and those found 

in the RILs using the flanking marker of the QTLs. The stability and 


eliability of qSSC-1a and qSSC-1b will be further validated using 

different analysis models and different combinations. 

N02_800a 

T13_850a 

IX_347 IX_334 

Z03_250a 

LG1 

0 

10 

AG_TT320 

AG_TT288 

AG_AT127 

CC_AA341 

AC_TT143 

AC_TC100 

X01_900a 

A08_1100a 

AG_AC350 

CC_AA110 

CC_AA244 

AC_TC122 

AG_TC157 

C06_1300a 

Z10_1900 

I835_210 I835_209 

Q05_500 

SCP01_570 X20_700 

K14_1500 

J11_1200 

LG4 

A05_800 

AI15_850a 

AG_TA260 

I835_565 

qSSC-1a 

0 

10 

20 

30 

40 

50 

60 

70 

80 

90 

100 

110 

120 

130 

140 

150 

160 

170 

180 

190 

LG13 

qSSC-1b 

A10_1031a 

P03_600a 

E08_600a 

AC_AA79 

B13_1400a 

AH03_1031a 

AL11_650a 

C04_1300a 

IXX_192 

H08_1500 I856_300 

I04_1200 

AC_TT141 

AH12_2000 

S09_650 

AG_AT104 

CA_AC195 

AG_AC120 

H17_550a 

AG_AC402 

Q05_675a 

AC_AA302 

N02_325a 

AC_TT117 AC_TT116 

P17_1000 

M18_620 

H02_690a 

AC_TC139 

I842_1500a 

B12_830a 

AG_TC107 

O13_350a 

IX_583 

AG_TT117 

AG_TA156 

0 Z01_2100 

10 X04_450 

20 

30 

AG_TC128 

P17_2400a 

LG8 

LG14 

LG2 

LG5 

0 

10 

20 

30 

40 

0 

10 

20 

30 

40 

0 

10 

20 

30 

40 

50 

60 

70 

80 

90 

100 

110 

120 

130 

140 

150 

160 

170 

180 

190 

200 

210 

220 

0 

10 

20 

30 

qSSC-14a 

qSSC-5 

qSSC-2a qSSC-2b 

R05_750 

Z12_1800 

B12_1100a 

P17_940a 

AC_TT166 

AH13_1200a 

F08_2500a 

AC_AA347 

AG_AC124 

T17_1200a 

T17_800 

I842_525 

AC_AA207 

N02_1600 

CC_AA114 

AC_TT126 

A12_600 

H07_950 

XXI_283 

XXI_267 

E08_950a 

AG_AT122 

AC_AA613 

AC_TC92 

Z03_1200a 

AG_TA157 

AG_TC79 

CA_AC230 

CC_AA247 

AG_TA212 

AC_TT99 

AH03_1600a 

I856_625a 

CA_AC354 

CA_AC347 

C09_900a 

CC_AA163 

CC_AA162 

qSSC-14b 

qSSC-14c 

LG9 

LG6 

0 

10 

20 

30 

40 

50 

60 

70 

80 

90 

100 

110 

120 

LG3 

0 

10 

20 

30 

Fig. 1. The genetic-linkage map of watermelon and the distribution of QTLs for 

SSC of watermelon. 


Fan, M., Y. Xu, H. Y. Zhang, H. Z. Ren, G. B. Kang, Y. J. Wang, and H. Chen. 2000. 

Identification of quantitative trait loci associated with fruit traits in watermelon 

[Citrullus lanatus (Thanb) Mansf] and analysis of their genetic effects. Yichuan 

Xuebao (Acta Genetica Sinica). 27(10): 902–910. 

Hashizume, T., I. Shimamoto, and M. Hirai. 2003, Construction of a linkage map and 

QTL analysis of horticultural traits for watermelon [Citrullus lanatus (Thunb.) 

0 

10 

20 

30 

40 

50 

60 

70 

80 

90 

100 

110 

120 

130 

140 

150 

160 

170 

180 

190 

qSSC-3a 

qSSC-3b qSSC-3c 

qSSC-3d 

AG_TC195 AG_TC194 

AG_TC206 AG_TC205 

F09_800 

AG_AC112 

IXX_298 IXX_296 

Z16_500a 

AL03_800a 

M05_1200a 

Subi 

LG15 

LG10 

0 

10 

20 

30 

0 

10 

20 

qSSC-15a 

AG_AT80 

K12_400a 

Z10_500a 

K12_900a 

CA_AC91 

CC_AA157 

II_147 

II_155 

AG_TA105 

AG_TT219 

qSSC-3e 

LG7 

0 

AG_AT207 AG_AT206 

A10_500 

qSSC-15b 

LG18 

H08_550 

M18_850a 

XII_205 

0 

AG_TA154 0 

10 

AC_AA121 10 

20 

30 

40 

V_240 

V_228 

S15_1000a 

VI_678 

20 

30 

40 

50 

60 

70 

VI_950 VI_1030 

H07_540a 

AG_TT223 

AC_TC80 

50 

60 

70 

80 

CC_AA332 80 

90 

A07_1100 

O05_480 Q17_1500 

A18_1600 

AG_TT79 

90 

100 

110 

V_405 V_395 120 

N04_600 130 

I835_430 140 

V_257 

IX_689 IX_678 

AC_TT106 

AC_TT208 

150 

160 

170 

AC_AT280 180 

O16_550a 

SCP219_1600 

AC_AT257 

AC_AT258 

190 

200 

210 

I835_298 220 

O16_475 

Z12_850 V03_800 

B20_1200 

230 

240 

X04_1100 250 

Z04_1600 

Z04_900a G10_850a 

N04_850 N04_800a 

I835_328 

260 

270 

280 

N02_1031a Q05_1500 290 

Q05_1600a 

X01_650a 

SCP01_700a 

300 

310 

AC_AA375 320 

AC_TC84 

AG_AT557 

AC_TC314 

AG_AT89 

330 

340 

350 

O03_480a 360 

Q05_1200a 370 

M07_400a M07_590 

AC_TT327 380 

LG11 

LG12 

LG16 

0 

0 

10 

I_268 

I_224 I_276 

AG_TT280 

AG_TT98 

AG_TA93 

AG_TT278 

CA_AC143 

218 Cucurbitaceae 2006 

LG17 

LG19 

0 

0 

10 

20 

30 

40 

qSSC-19a 

qSSC-19b 

qSSC-19c

Matsum & Nakai] using RAPD, RFLP, and ISSR markers, Theor. Appl. Genet. 

106:779–785. 

Huang, N., E. R. Angeles, J. Domingo, G. Magpantay, S. Singh, G. Zhang, N. 

Kumaravadivel, J. Bennett, and G. S. Khush. 1997. Pyramiding of bacterial 

blight resistance genes in rice: marker-assisted selection using RFLP and PCR. 

Theor. Appl. Genet. 95:313–320. 

McCouch, S. R., Y. G. Cho, M. Yano, E. Paul, M. Blinstrub, H. Morishima, and T. 

Kinosita. 1997. Report on QTL nomenclature. Rice Genet. Newslett. 14:11–13. 

Paterson, A. H., S. Damon, J. D. Hewitt, D. Zamir, H. D. Rabinowitch, S. E. Lincoln, 

E. S. Lander, and S. D. Tanksley. 1991. Mendelian factors underlying 


environments. Genet. 127:181–197. 

Tanksley, S. D. 1993. Mapping polygenes. Ann. Rev. Genet. (27):205–333. 

Yi, K., Y. Xu, X. Y. Lu, L. T. Xiao, X. L. Xu, G. Y. Gong, and H. Y. Zhang. 2004. 

Construction of AFLP molecular genetic map for RIL population of watermelon. 

Yuanyi Xuebao (Acta Horticulturae Sinica). 31(1): 53–55. 

Zhang, R. B., Y. Xu, K. Yi, H. Y. Zhang, L. G. Liu, G. Y. Gong, and A. Levi. 2004. A 

genetic linkage map for watermelon derived from recombinant inbred lines. J. 

Amer. Soc. Hort. Sci. 129(2):237–243. 

Zhuang, J. Y., H. X. Lin, J. Lu, H. R. Qian, S. Hittalmani, N. Huang, and K. L. 

Zheng. 1997. Analysis of QTL × environment interaction for yield components 

and plant height in rice. Theor. Appl. Genet. 95:799–808. 


MAPPING AND QTL ANALYSIS CONCERNING 

TOLERANCE TOWARD 

POOR LIGHT IN CUCUMBER (CUCUMIS 

SATIVUS L.) 

Yong-Jian Wang, Hai-Ying Zhang, Qing-Jun Chen, 

Feng Zhang, and Ai-Jun Mao 


Banjing, Haidian, Beijing 2443#, Beijing, 100089, P. R. China 

ADDITIONAL INDEX WORDS. AFLP, RAPD, RIL, SSR, genetic map 

ABSTRACT. An AFLP and RAPD genetic map with 235 markers and an RIL 

(recombinant inbred lines) population from the cross of two cultivated lines were 

employed in mapping and analysis of quantitative trait loci (QTL) concerning 

tolerance toward poor light in cucumber using the interval mapping method. 

Leaf area in seedlings under low light intensity was used as phenotypic value to 

detect the QTL concerning tolerance. Five QTL were mapped in four linkage 

groups. Four loci, including La-1, La-2, La-3, and La-4, showed a negative 

additive effect, while La-5 showed a positive additive effect. Of the five QTL, 

phenotypic variation explained by La-5 was the largest, La-2 was the second. 

C 

ucumber is an important greenhouse vegetable. The main goal 

of breeding for greenhouse cucumber cultivars is to increase 

tolerance to poor light and low temperature. The impact of 

poor light on cucumber growth and yield is usually stronger than that 

of low temperature (Chen et al, 2003). Several indexes, such as 

photosynthetic rate, light compensation point, and photosynthetic yield, 

under chilling and poor light have been reported (Aoki et al., 1988; Xu 

et al., 1997, Wang et al., 2001). The increase in leaf area in seedlings 

under low light intensity was a reliable phenotypic value to evaluate 

tolerance to poor light, according to our study. 

Tolerance to poor light was believed to be conditioned by many 

genes. Wang et al. (1998) indicated that light compensation at 15ºC 

could reflect tolerance to poor light at low temperature; the inheritance 

agreed with an additive-dominant epistatic model. It was impossible, 

using classical genetic methods, to know how many genes concerning 



5050001). 


tolerance are involved and where they are located. 

Recently, molecular markers and related technologies have allowed 

plant geneticists and breeders to devise strategies for extensive 

mapping of plant genomes (Staub et al., 1996). In commercial 

cucumber, the application of molecular-marker technologies has 

increased the understanding of its genome (Kennard et al., 1994; 

Kennard and Havey, 1995; Meglic and Staub, 1996). Serquen et al. 

(1997) conducted a molecular mapping with QTLs for length of the 

stem and number of fruits in cucumber based on a F3 population. Up to 

now little information has been reported on mapping QTLs of traits 

concerning tolerance to poor light. 

The objective of this study was to map genes concerning tolerance 

to poor light in cucumber using the RIL population obtained from the 

cross of the lines ‘European 8’ and ‘Qiu Peng’ and based on a linkage 

map with 235 markers (AFLP, RAPD, and SSR) that had already been 

constructed by our research group. 


MATERIALS. The inbred line ‘European 8’, a European 

greenhouse-cucumber cultivar with tolerance to poor light, was 

crossed with the inbred line ‘QiuPeng’, an open-field Chinese 

cucumber cultivar sensitive to poor light. Single F1 plants were 

randomly selected and self-pollinated to generate F2 families. A single 

F2 family was randomly chosen and individual plants were 

self-pollinated for four more generations to generate an F2-derived F6 

RIL population. Those RILs were used for all DNA isolations and 

evaluated for tolerance to poor light. 

EVALUATION OF TOLERANCE TO POOR LIGHT. Cucumber seeds 

were germinated for 24 hr at 28ºC and sown in plastic pots of 8cm 

diameter with a steamed commercial peat medium. About 8 days after 

sowing, 24 uniform seedlings at the cotyledon fully expanded stage 

were treated under low illumination (80µmol·m-2·s-1, 8 h, 25ºC/18ºC) 

and 3 replications were conducted. Seedling leaf area was measured 12 

days after treatment. 

LINKAGE ANALYSIS. Using SYSTAT 7.0 software, a graphic of the 

frequency distribution in the RIL population for leaf area was drawn. 

The data of the mean of leaf area were used for QTL analysis. The 

whole genome was scanned and the QTLs for tolerance were detected 

by using Windows QTL Cartograph vi20 at the threshold LOD score 

2.0. 



PHENOTYPIC VARIATION OF THE MATERIAL. There were significant 

differences between both parental lines and significant segregation 

among RILs (Table 1). The average increase in leaf area of RIL was 

45,187cm 2 and the standard deviation was 11,348cm 2. . The graphic of 

the frequency distribution (Kurtosis/SEK = 1.33; Skewness/SES = 0.78) 

fits normal distribution (Figure 1). 

Variance analysis indicated that there were no significant 

differences among replications for the increase of leaf area, while the 

differences among RILs were significant and the broad heritability was 

77.5%. The high heritability of leaf-area increase under low 

illumination suggested that the above index could be used as an 

acceptable criterion to evaluate tolerance to poor light in cucumber as 

well as for QTL analysis. 

40 

30 

20 

10 

0 

0.0 

10 20 30 40 50 60 70 80 

Fig. 1. The frequency distribution in cucumber RIL population for 

low-light-tolerance trait. 

Abscissa = phenotypic value; ordinate = number of lines on the left side and 

the percentage on the right. 

QTL ANALYSIS OF TOLERANCE TO POOR LIGHT IN CUCUMBER. 

QTL analysis was conducted by using Windows QTL Cartograph. The 

scanning of gene tolerance to poor light in cucumber in the whole 

genome (Figure 2) indicated that there were five QTLs at the threshold 

LOD score 2.0 concerning tolerance to poor light: La-1, La-2, and 

La-3 on Linkage Group 1, La-4 on Group 7, and La-5 on Group 9. 


0.3 

0.2 

0.1

Table 1. Parent values and distribution in cucumber RIL population for 

low-light- tolerance trait. 

Parent values Distribution in RIL population 

Agronomic 

traits 

Leaf-area 

No. 8 

EuropeanQiupeng 

Mean SD Range 

Kurtosis/ 

SEK 

Skewness/ 

SES 

increase 

(cm 2 ) 61.00±3.6 22.00±2.9 45.19 11.348 55.94 1.33 0.785 

The result of the scanning showed that four putative QTLs were 

negative (Table 2). La-5 was a positive locus contributing in 5.54 

additive effects to tolerance. 

The expression of quantitative traits is the result of the reaction 

between genotype and environment. It is important to limit the 

influence of environment to achieve accurate QTL mapping. The 

results derived from RILs analysis could be more accurate than those 

using F2 populations, since replications could be conducted to reduce 

environment error (Paterson et al.,1991). In classical quantitative 

genetics, a theoretical model assumed that genes controlling quantitative 

traits had the same direction and similar effects. However, many studies 

have indicated that the genes are diverse in effect and direction. This 

study showed that only one locus expressed a positive effect and that the 

others expressed negative effects. Yu et al. (2003) also found that the 

effect direction of QTLs controlling some morphological traits in 

Chinese cabbage were opposite. 

Fig. 2. LR plots and additive-effect plots on cucumber genome for low-light 

tolerance. 


Table 2. QTLs controlling low-light tolerance and its effect in cucumber. 

Agronomic 

traits QTL 

Leaf area La-1 

La-2 

La-3 

La-4 

La-5 

Linkage 

group 

1 

1 

1 

7 

9 

QTL 

position 

6.0 

10.0 

50.2 

36.0 

54.0 

LR 

value 

9.8 

10.8 

12.4 

13.5 

11.0 

Variance 

explained 

(%) 

10.6 

11.9 

7.3 

8.3 

20.2 

Additive 

effect 

-4.58 

-5.17 

-4.02 

-4.34 

5.47 


Aoki, S., M. Oda, and M. Naga 1988. Chilling and heat sensitivities in cucumber 

seedlings measured by chlorophyll fluorescence. Bull. Natl. Res. Inst. Veg. 

Ornam. Plants Tea. A(2):81–92. 

Chen, Q. J., F. M. Zhang, Y. J. Wang, and K. Kenji. 2003. Studies of physiological 

characteristics of the reaction of cucumber to low temperature and poor light. 

Sci. Agr. Sinica. 36(1):23-26. 

Kennard, W. C. and M. J. Havey. 1995. Quantitative trait analysis of fruit quality in 

cucumber: QTL detection, confirmation, and comparison with mating-design 

variation. Theor. Appl. Genet. 91:53-61. 

Kennard, W. C., K. Poetter, A. Dijkhuizen, V. Meglic, J. Staub, and M. Havey. 1994. 

Linkages among AFLP, RAPD, isozyme, disease resistance, and morphological 

markers in narrow and wide crosses of cucumber. Theor. Appl. Genet. 91:53–61. 

Meglic, V. and J. E. Staub. 1996. Inheritance and linkage relationships of isozyme 

and morphological loci in cucumber (Cucumis sativus L.). Theor. Appl. Genet. 

92(7):865–872. 

Paterson, A. H., S. Damon, J. D. Hewitt, D. Zamir, H. D. Rabinowitch, S. E. Lincoln, 

E. S. Lander, and S. D. Tanksley. 1991. Mendelian factors underlying 


environments. Genome. 127:181–197. 

Serquen, F. C., J. Bacher, and J. E. Staub. 1997. Mapping and QTL analysis of a 

narrow cross in cucumber (Cucumis sativus L.) using random amplified 

polymorphic DNA marker. Molec. Breeding. 3:257–268. 

Staub, J. E., F. C. Serquen, and M. Gupta. 1996. Genetic markers, map construction, 

and their application in plant breeding. Hort Sci. 31:729–741. 

Wang, Y. J., H. Y. Zhang, Y. W. Jiang. 2001. Effects of low temperature and low light 

intensity stress on photosynthesis in seedlings of different cucumber varieties. 

Acta Hort. Sinica. 28(3):230–234. (In Chinese.) 

Wang, Y. J., Y. W. Jiang, G. S. Wu, F. Zhang, and L. R. Zhang. 1998. Relationships 

between photosynthetic light compensation point and tolerance to low 

temperature and low irradiance in cucumber. Acta Hort. Sinica. 25(2):199–200. 

(In Chinese.) 

Xu, C.-H., Z.-Q. Chen, K.-B. Wang, F.-H. Zhao, S.-Q. Lin, C.-Q. Tang, and T.-Y. 

Kuang. 1997. Effects of chilling injury on the thylakoid membrane of cucumber 

chloroplasts. Acta Bot. Sinica. 39(12):1143–1146. 

Yu, S.-C., Y. J. Wang, and X. Y. Zheng. 2003. Mapping and QTL analysis controlling 

some morphological traits in Chinese cabbage (Brassica campestris L. ssp. 

pekinensis). Acta Gen. Sinica. 30(12):1153–1160 (In Chinese.) 


CONSTRUCTION OF A GENETIC MAP OF 

CUCUMBER USING RAPDS, SSRS, AFLPS 

AND MAPPING RESISTANCE GENES TO 

PAPAYA RINGSPOT, ZUCCHINI YELLOW 

MOSAIC, AND WATERMELON MOSAIC 

VIRUSES 

Haiying Zhang, Aijun Mao, Feng Zhang, Yong Xu, 

and Yongjian Wang 


Beijing, China 

ADDITIONAL INDEX WORDS. Cucumis sativus, genetic distance, linkage map. 

ABSTRACT. The watermelon strains of Papaya ringspot virus (PRSV-W), 

Zucchini yellow mosaic virus (ZYMV), and Watermelon mosaic virus (WMV) are 

potyviruses that cause significant losses in cucumber. F6 recombinant inbred 

lines (RILs) were generated from a cross between Cucumis sativus L. line 

‘European 8’ and a line from ‘QiuPeng’, which is resistant to the three viruses. 

A 244-point genetic map of cucumber was generated, delineating nine linkage 

groups and spanning 759cM, with an average distance of 3.1cM. Resistance to 

PRSV-W, ZYMV, and WMV were mapped to Linkage Group 2. The order of 

the three virus resistances was PRSV-W, ZYMV, and WMV. The distance 

between PRSV-W- resistant and ZYMV-resistant genes was 8cM, and the 

distance between ZYMV- and WMV- resistant genes was 4cM. The results 

indicate that this genetic map will be useful for cucumber genetics and breeding 

for disease resistance. 

T 

he watermelon strains of Papaya ringspot virus (PRSV-W), 

Zucchini yellow mosaic virus (ZYMV), and Watermelon 

mosaic virus (WMV) are potyviruses that cause significant 

losses in cucumber (Cucumis sativus L.) throughout China and 

the world (Provvidenti et al., 1984; Wang et al., 1997). The genetics of 

potyvirus resistance in cucumber have been well studied. Provvidenti 

(1987) reported that resistance to ZYMV from TMG1 was inherited as 

a recessive allele at the zym single locus. ZYMV resistance from the 

cultivar ‘Dina’ was also inherited as a recessive allele at the same 

locus (Abul-Hayja and Al-Shahwan, 1991). Wang et al. (1984) 

reported that resistance to PRSV-W (originally named Watermelon 



5050001). 


mosaic virus 1) in the ‘Surinam’ cultivar was inherited as a recessive 

allele at the prsv-1 locus. Wai and Grumet (1995a) found that the prsv- 

2 locus in the line TMG1 inhibited PRSV-W symptoms. Wai et al. 

(1997) reported that resistance to PRSV-W from ‘Surinam’ (prsv-1) 

and TMG1 (Prsv-2) were allelic. Cohen et al. (1971) reported that 

resistance to WMV was conditioned by a dominant allele at a single 

locus from cultivar ‘Kyoto 3 Feet’. Several studies demonstrated that 

numerous potyviruses such as PRSV-W, WMV, Moroccan 

watermelon mosaic virus, Zucchini yellow fleck virus, and ZYMV that 

are resistant in TMG1 are inherited at the same locus (Gilbert- 

Albertini et al., 1995; Wai and Grumet 1995b; Kabelka et al., 1997; 

Wai et al., 1997). 

Conventional plant breeding had a significant impact on improving 

cucumber breeding for resistance to potyviruses, but the timeconsuming 

process of making crosses, backcrosses, and the selection 

of desired resistance progeny makes it difficult to react adequately to 

the evolution of new virulent pathogens. Thus, marker-assisted 

selection (MAS) may be easier for the breeding of multiple-virusresistant 

cucumbers. Since the 1980s, molecular markers are being 

used widely as a principal tool for the breeding of many crops. In 

particular a great work has been realized in the construction of the 

molecular map of cucumber and in finding molecular markers linked 

to disease-resistance genes. Park et al. (2000) reported that resistances 

to PRSV-W and ZYMV were tightly linked (2.2cM) and mapped to 

the end of one linkage group. The AFLP marker E15/M47-F-197 

cosegregated with ZYMV resistance and should be a useful indirect 

selection tool for potyvirus resistance in cucumber. These mapping 

results support the hypothesis that potyvirus resistance is controlled by 

different alleles at clustered loci in cucumber (Kabelka and Grumet 

1997; Wai et al., 1997). The mapping of other resistance genes to 

potyviruses such as WMV should be one of the major topics for 

further study. 

Chinese cucumber and European greenhouse cucumber are two 

important ecological types cultivated widely. Many Chinese cucumber 

cultivars possess genetic resistance to four or more viruses, but the 

genetics of potyvirus resistance of these cultivars have not been well 

studied. Most of the European greenhouse-cucumber cultivars are not 

resistant to potyviruses. In our laboratory, we have generated 

recombinant inbred lines (RILs) from the cross between ‘European 8’ 


(an inbred line of European greenhouse-cucumber cultivars susceptible 

to ZYMV, PRSV-W, and WMV) and ‘QiuPeng’ (an inbred line of 

Chinese cucumber cultivars resistant to these viruses). The two main 

goals of our research were: (a) to construct the first cucumber map in 

China, a narrow-based genetic map using random amplified 

polymorphic DNAs (RAPDs), amplified fragment length 

polymorphism (AFLP), and simple sequence repeat (SSR), and (b) to 

map resistance genes to ZYMV, PRSV–W, and WMV. 


CROSSES. ‘European 8’, an inbred European greenhouse-cucumber 

cultivar susceptible to ZYMV, PRSV-W, and WMV, was crossed with 

‘QiuPeng’, an inbred open-field cucumber cultivar of China with 

resistances to ZYMV, PRSV-W, and WMV. A total of 140 RIL6 

families were used for all DNA isolations and virus evaluations. 

VIRUS MAINTENANCE AND EVALUATION. ZYMV-CH, PRSV, and 

WMV were kindly provided by Dr. R. Provvidenti (Cornell 

University, Geneva, NY). The viruses were maintained by transferring 

to zucchini squash (Cucurbita pepo L.) regularly. For virus evaluation, 

cucumber seeds were germinated for 24 hr at 28 ο C. Fourteen days after 

sowing, cotyledons and the first expanding leaves were dusted with 

carborundum and inoculated with each of the viruses. Fourteen to 20 

days after inoculation, the plants were scored as: 0 = no symptoms; 1 = 

slight symptoms limited to lower leaves; 3 = slight mosaic on upper 

leaves; 5 = clear mosaic on lower leaves and moderate mosaic on 

upper leaves; 7 = severe mosaic on most of leaves; 9 = severe mosaic 

on all leaves, new leaves aberrant. Ten plants from each F6 family 

were evaluated in each of the three replications. Disease index (DI) 

was calculated by using the formula DI = (∑score x number of 

plants/9x total number of plants) x 100. The graphic of the frequency 

distribution in the RIL population for DI of ZYMV, WMV, and 

PRSV-W was drawn by using Systat 7.0 software, 

DNA ISOLATION. An improved CTAB procedure (Murray and 

Thompson, 1980) was used . 

AFLP ANALYSIS. The AFLP procedure was accomplished 

according to Vos et al. (1995). 

RAPD ANALYSIS. The RAPD procedure was accomplished 

according to Park et al. (2000). 


SSR ANALYSIS. The sequence of primers was obtained from Dr. J. 

E. Staub, University of Wisconsin. PCR amplification was conducted 

according to Danin-Poleg et al. (2000). 

LINKAGE-MAP CONSTRUCTION. Linkage-map construction was 

performed using Join Map3.0 for Kosambi mapping function. 


INHERITANCE OF RESISTANCE TO ZYMV, WMV AND PRSV-W 

IN CUCUMBER. The frequency distribution in the RIL population for 

DI of three viruses showed two peaks (Figure 1). These results 

demonstrated that resistances from ‘QiuPeng’ to ZYMV, WMV, and 

PRSV-W were all inherited as a recessive allele at a single locus. It is 

obvious that the resistance from ‘QiuPeng’ to WMV differed from the 

resistance from ‘Kyoto 3 Feet’, which was dominantly inherited. 

MAP CONSTRUCTION. The 244 markers, including AFLPs, 

RAPDs, and SSRs, formed nine linkage groups at LOD 3.0, and the 

nine linkage groups spanned 759cM, with an average distance of 

3.1cM (Figure 2). Since there are seven pairs of chromosomes in 

cucumber, we delineated nine linkage groups, and this map is not fully 

saturated. 

For the map construction, even distribution of markers with small 

intervals between was expected. Instead, however, marker clusters 

formed in several crops, such as tomato and maize. Our study showed 

that some markers, especially AFLP markers, were distributed 

unequally. Large intervals (>20cM) between adjacent markers were 

also found in some groups. Populations from various parents or more 

types of molecular markers, including RFLPs and SSRs, would be 

helpful for constructing a saturated map. 

MAPPING RESISTANCE TO POTYVIRUSES ZYMV PRSV-W AND 

WMV. In addition to resistances to ZYMV and PRSV-W, resistance 

to WMV was mapped on the genetic map. This was the first mapping 

of the gene for resistance to WMV, and we found that resistances to 

ZYMV, PRSV-W, and WMV all were closely linked in Linkage 

Group 1 (Figure 2). The orders of three virus resistances were PRSV- 

W, ZYMV, and WMV. The distance between PRSV-W and ZYMV 

resistances was 8cM, and the distance between ZYMV and WMV 

resistance genes was 4.0cM. Park et al. (2000) reported that the 

distance between the two resistances to ZYMV and PRSV-W was 

2.2cM. In our study, the distance was 8cM. Although there is some 

difference in genetic distance, both studies show that resistance to 

ZYMV is linked closely to resistance to PRSV-W. A series of studies 


demonstrated that numerous potyvirus (PRSV-W, WMV, ZYMV) 

resistances in TGMT are inherited at the same locus; however, we 

scored about 10 families as recombinant for resistance to PRSV-W and 

ZYMV, to PRSV-W and WMV, and to WMV and ZYMV among 140 

families. Our results indicate that three important virus resistances in 

cucumber are conditioned by a major chromosome region, which 

further supports the hypothesis that potyvirus resistance is controlled 

by different alleles at clustered loci in cucumber (Kabelka and 

Grumet 1997; Kabelka et al, 1997; Wai and Grumet, 1995b). 

Count 

40 

30 

20 

10 

ZYMV 

0 

0 10 20 30 40 

DI 

50 60 70 

0.0 

80 

Count 

40 

30 

20 

10 

0.3 

0.2 

0.1 

Proportion per bar 

WMV 

Fig. 1. The frequency distribution in cucumber RIL populations for DI traits 

involving resistance to the watermelon strain of Papaya ringspot virus (PRSV- 

W), Watermelon mosaic virus (WMV), and Zucchini yellow mosaic virus 

(ZYMV). Abscissa = DI; ordinate = number of lines on the left side and the 

percentage on the right. 

Count 

PRSV-W 

0.3 

0.2 

0.1 

0 

0.0 

0 10 20 30 40 50 60 70 80 90 100 

DI 

0 

0 10 20 30 40 50 60 70 80 

0.0 

90 

DI 


30 

20 

10 

Proportion per bar 

0.2 

0.1 

Proportion per bar

Fig. 2. A linkage map for cucumber derived from an RIL population (‘QiuPeng’ 

× ‘European 8’). 



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gene effective against races of bacterial blight, maps to a cluster of previously 

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Melon. 1:45–47. 

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mosaic virus in cucumber line TMG1 and cosegregation with zucchini yellow 

mosaic virus resistace. Euphytica. 95:237–242. 

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plant DNA. Nucl. Acids Res. 8:4321–4325. 

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genetic map of cucumber composed of RAPDs, RFLPs, AFLPs, and loci 

conditioning resistance to papaya ringspot and zucchini yellow mosaic viruses. 

Genome. 43:1003–1010. 

Provvidenti, R., D. Gonsalves, and H. Humaydan. 1984. Occurrence of zucchini 

yellow mosaic virus in cucurbits from Connecticut, New York, Florida, and 

California. Plant Dis. 68:443–446. 

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Plant Path. 66:425–429. 

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Pot, J. Peleman, M. Kuiper, and M. Zabeau. 1995. AFLP: a new technique for 

DNA fingerprinting. Nuleic Acids Res. 23:4407–4414. 

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papaya ringspot virus in the cucumber line TMG1. HortSci. 30:338–340. 

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virus in the cucumber line TMG1: tissue-specific expression and relationship to 

zucchini yellow mosaic virus resistance. Theor. Appl. Genet. 91:699–706. 

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alleles in cucumber. J. Hered. 88:454–458. 

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PERIMETER TRAP CROP SYSTEMS USING 

BLUE HUBBARD SQUASH AS A CONTROL 

FOR STRIPED CUCUMBER BEETLE IN 

BUTTERNUT SQUASH 

Andrew Cavanagh and Ruth Hazzard 

Department of Plant, Insect, and Soil Science, University of 

Massachusetts, Amherst, MA 01003 

ADDITIONAL INDEX WORDS. Acalymma vittatum, Cucurbita maxima, Cucurbita 

moschata, IPM 

ABSTRACT. Striped cucumber beetle, Acalymma vittatum (Fabricius), is the 

primary insect pest of cucurbit crops in the Northeastern U.S. Adult beetles 

colonize squash crops from field borders, causing feeding damage at the 

seedling stage and transmitting the bacteria Erwinia tracheiphila. Conventional 

control methods rely on pesticide applications to the entire field. Acalymma 

vittatum demonstrates a marked preference for Blue Hubbard squash 

(Cucurbita maxima) over butternut squash (Cucurbita moschata). Perimeter 

trap cropping with a single or double row of Blue Hubbard squash concentrates 

beetles in the border where they can be controlled with timely perimeter 

treatments, limiting the need for pesticide in the main crop of butternut squash. 

We evaluated this system in commercial butternut fields in 2003 and 2004, 

comparing fields using perimeter trap cropping to conventionally managed 

fields. Equivalent control of striped cucumber beetles was achieved with >90% 

reduction in pesticide rates compared to conventional control methods. 

T 

he value of vegetable crops sold in the United States was $12.7 

billion in 2002 (USDA, 2002 Census of Agriculture). 

Northeastern states have a high proportion of their vegetablecrop 

industry invested in cucurbit crops, including squash, melons, 

cucumbers, and pumpkins; in Massachusetts, 40% of the vegetablecrop 

acreage is devoted to cucurbit crops (USDA, 2002). Yields of 

winter squash, one of the major cucurbit crops, exceed 20,000lb/acre, 

with a wholesale value estimated at $3400/acre, or greater than $5 

million for the state (Hollingsworth, 1998). Butternut squash is the 

primary winter squash crop, in part because of strong market demand 

and excellent storage capability. 

Striped cucumber beetles (Acalymma vittatum) constitute one of 

most serious pests of cucurbit crops in the world (Metcalf and Metcalf, 

1992). These beetles are ranked as the most important insect pest in 

cucurbit crops in the Northeast, and are the primary target of 

insecticide applications used by growers (Hoffmann et al., 1996; 

Stivers, 1999). The Northeast Vegetable IPM Commodity Working 


Group, with representatives from 12 northeastern states, ranked the 

cucumber beetle and bacterial wilt complex as a regionwide problem 

that causes significant reduction in yield and results in high pesticide 

use (IPM Priorities for Vegetables in the Northeast: 

http://northeastipm.org/work_vegepriority.cfm). Conventional pest 

management for many cucurbit crops requires two to eight 

applications of insecticides such as carbaryl (Brust and Foster, 1995) 

or other carbamates or synthetic pyrethroids (Howell et al., 2004). 

Recently, growers in the Northeast have adopted use of systemic 

insecticides (e.g., imidacloprid) in the furrow at planting, which 

protects cucurbit crops from early feeding damage (Hazzard, 2003). 

This has the advantage of less work for the grower; however, the 

insecticide cost is higher than with foliar applications, and widespread 

adoption of systemic insecticides may lead to resistance. 

Cucumber beetles can reduce yield in cucurbits through direct 

damage or by transmitting the causal agent of bacterial wilt. 

Cucumber beetles overwinter as adults, colonize cucurbit fields in 

early to mid-June, and may completely destroy newly germinated 

plants (Ferguson et al., 1983). Damage by adults to leaves and early 

flowers takes place over a four–five-week period in New England, 

followed by a period of inactivity aboveground after the overwintering 

generation of adults has laid eggs in the soil and died. Summer adults 

emerge in mid- to late summer and continue to feed, damaging leaves, 

flowers, and fruit, before leaving the fields for overwintering sites. 

Although yield losses to direct damage can be extensive, the most 

damaging effect of cucumber-beetle infestation may be through 

transmission of the bacteria Erwinia tracheiphila. Yield losses from 

bacterial wilt in winter squash and pumpkins have increased in the past 

decade (Hazzard et al., 2001; Zitter, 2001), increasing the necessity for 

developing control methods that prevent transmission of wilt during 

the most susceptible stages of plant development. To address these 

issues, in the spring of 2003 we began looking at the effectiveness of a 

perimeter-trap-cropping system to reduce the time, expense, and 

potential hazards that are inherent in conventional management 

strategies. 

Perimeter trap cropping, or PTC, is based on the fact that insects 

prefer one species or variety of plant over another, due to morphology, 

relative concentration of feeding stimulants, or other factors. The 

more attractive crop is planted around the outer edge of the main cash 

crop, surrounding it on all sides. Striped cucumber beetles overwinter 

in the woods edges surrounding fields, and move into a crop from the 

edges. This makes them a good candidate for this method of control. 

It is also likely that by reducing the field area sprayed, we provide a 


efuge for beetles that are still susceptible to chemical controls, thus 

delaying resistance. Blue Hubbard squash is highly attractive to 

striped cucumber beetles in comparison with butternut squash and has 

low susceptibility to bacterial wilt (McGrath and Shishkoff, 2000; 

Cavanagh and Hazzard, unpublished data). It has been used as an 

effective perimeter trap crop in summer squash with great success 

(Boucher and Durgy, 2002). It is similar to butternut squash in terms 

of its temperature and spacing requirements, and days to maturity. 

These factors make it an ideal perimeter trap crop for butternut squash. 


On-Farm Experiment 2003 

EXPERIMENTAL DESIGN. To assess the value of perimeter-trapcropping 

systems in commercial agriculture, we monitored 12 

commercial butternut fields, 6 using a PTC system and 6 using 

conventional chemical controls (foliar treatment with carbaryl). Every 

week from 3 June 2003 to 14 July 2003 (peak period for striped 

cucumber beetle activity), 25 plants were randomly selected and 

scouted along the field border, and another 25 were randomly selected 

from a row halfway between the border and the center of the field. 

Perimeter-trap-crop growers were advised to spray their borders at the 

first arrival of the cucumber beetle. Additional border sprays were 

applied at weekly intervals if the beetle pressure in the border 

exceeded an average of one beetle per plant. Growers in all fields 

were advised to spray their main crop if beetles reached a threshold 

number of one beetle per plant on average. 

RESPONSES MEASURED. Both the border and a randomly selected 

row halfway between the border and the center of the field were 

scouted weekly for number of live beetles, number of dead beetles, 

presence of cotyledon damage, and percent defoliation. The 

defoliation scale used was as follows: 0 = no damage, 1 = 1–20% 

damage, 2 = 21–40% damage, 3 = 41–60% damage, 4 = 61–80% 

damage, 5 = 81–100% damage. At the end of the season, growers 

rated their satisfaction with yield and management costs under this 

system and how it compared to expectations and past methods. 

FIELD MANAGEMENT. All the farmers participating in the 

experiment planted their fields as they normally would, except for the 

inclusion of a single- or double-row Hubbard border in the PTC fields. 

Double rows were used along woods edges where beetles were likely 

to have overwintered, or in fields close to previous cucurbit crops. No 

fields had been planted to cucurbits in the previous year. Cultivation 


and nutrient management were performed by the grower, according to 

the needs of the field and standard management practices. 

STATISTICAL ANALYSIS. All data in 2003 were subjected to 

ANOVA analysis using PROC GLM in SAS V.9.1. Beetle counts 

were subjected to log transformation to more closely meet 

expectations of normality. P values less than or equal to 0.05 were 

considered significant. 


EXPERIMENTAL DESIGN. The experimental design in 2004 was 

the same as in 2003, except that in 2004 we chose to also look at the 

effects of treating the border with a systemic insecticide 

(imidacloprid). This was done at the request of several growers who 

thought that a systemic would alleviate some of the difficulty in timing 

the initial spray correctly. The design was adapted so that there were 

now two pesticide treatments—systemic (imidacloprid) and foliar 

(carbaryl). The intention was to have three PTC foliar fields, three 

PTC systemic fields, three conventional foliar treated fields, and three 

conventional systemic treated fields. One of the PTC systemic fields 

failed to germinate due to poor field conditions, so we were left with 

only two fields in the systemic PTC category. During the 2004 trials 

we were also able, through increased contact with the growers during 

the critical early part of the season, to address many of the problems 

we experienced in 2003. 

RESPONSES MEASURED. Fields were scouted from 4 June 2004–16 

July 2004, which represents the period of peak beetle activity. The 

same scouting methods were used and the same responses measured as 

in 2003. 

FIELD MANAGEMENT. Field management was essentially the same 

as in 2003, with the exception of a furrow drench of imidacloprid 

applied at planting to the systemic conventional fields and the Hubbard 

borders of the systemic PTC fields. 

STATISTICAL ANALYSIS. Analysis for the 2004 data was 

conducted in the same manner as in 2003. 



The on-farm trials showed significant differences in total beetle 

numbers between both the systems overall (PTC vs. conventional; 

P=0.02) as well as location overall (border vs. main crop; P=0.02). 

This was complicated by the interaction between system and location 

being highly significant (df 1,10; P=0.007). When the means for each 


location within each system were separated, it was clear that the 

number of beetles in the PTC borders was much higher than in either 

of the main crops or in the borders of the conventional fields (Table 1; 

Figure 1). We chose to use total beetles (live + dead beetles) as the 

measure of beetle pressure because dead beetles accumulate wherever 

pesticides are used, while only live beetles are present in unsprayed 

fields; the sum indicates total population pressure in both treated and 

untreated fields. There were no significant differences in either 

defoliation or cotyledon-damage rankings. In three out of six plots 

with a Hubbard border, beetle populations in the main crops exceeded 

the action threshold. This occurred for several reasons. In one field 

the beetle pressure was high enough to overwhelm and destroy the 

single-row border. Plants were eaten down to the ground over a period 

of two days, before the border was sprayed. In two irregularly shaped 

fields the border was planted with wide gaps. 

Table 1. Average number of live plus dead beetles per plant, summed 

over six weeks from germination. 

Beetles per plant, 2003 

Border Main 

PTC 2.86a 0.39b 

Conventional 0.38b 0.38b 

Means with the same letter are not significantly different, P=0.05. 

Looking at the colonization of the field over time, we saw an 

apparent difference in the pattern of beetle dispersion. The main crop 

in the PTC systems had comparable levels of beetles to the other main 

crops, despite being in such close proximity to the highly infested 

Hubbard and despite the fact that half of these fields were never 

sprayed. All conventional fields were treated with insecticide at least 

once. 


The modifications we made to the system based on our experiences 

in 2003 led to greatly improved effectiveness. Beetle pressures were 

similar to 2003; however, the main crop of PTC fields did not exceed 

threshold and no PTC fields required a full-field spray. The total 

number of beetles was again higher in the Blue Hubbard borders, 

regardless of what chemical the border was treated with (P=0.037; df 


1,7) (Table 2). There were no differences in beetle densities in the 

main crops regardless of whether or not the field was surrounded 

Avg per Plant Beetle count 

9 

8 

7 

6 

5 

4 

3 

2 

1 

0 

Average Total (live+dead) beetles per plant 

wk1 wk2 wk3 wk4 wk5 wk6 

border - conv 

border - PTC 

main - conv 

main - PTC 

Fig. 1. Average adult beetles (sum of live plus dead beetles) per plant, by week, 

in the border and main crop of PTC and conventional fields, 2003. 

Table 2. Average number of live plus dead beetles per plant, summed 

over six weeks from germination. 

Beetles per plant, 2004 

Border Main 

PTC 3.13a 0.32b 

Conv 0.35b 0.30b 

Means with the same letter are not significantly different. 

Table 3. Average defoliation ratings, summed over six weeks from 

germination, 2004. 

Defoliation 

Pesticide Border Main 

Conventional carbaryl 0.44a 0.39a 

imidacloprid 0.52abc 0.33a 

PTC carbaryl 0.70c 0.49ab 

imidacloprid 0.65bc 0.63bc 

Means with the same letter are not significantly different. 

by a Hubbard border or what chemical the border was sprayed with. It 

is important to note that the beetle populations in the main crop of 

each field were equivalent despite the fact that the main crops of the 


PTC fields were not sprayed or treated with insecticide in any way, 

and the main crops of the other fields were either treated with 

imidacloprid at planting or sprayed with a foliar insecticide on 

multiple occasions through the growing season. There was a small but 

statistically significant increase in the defoliation rating (Table 3) for 

the main crop of the imidacloprid treated PTC field vs. the 

conventional main crops (P=0.015; df 1,7). When comparing only at 

the vulnerable seedling stages, the difference was not significant. 

In 2004 the main crops of the PTC fields never exceeded the spray 

threshold (Figure 2). It appeared that the pressure in the main crops of 

the PTC fields was actually lower than in any other field at the onset of 

beetle colonization (Weeks 1 and 2). This has important implications 

for the success of the system, because beetle feeding has a 

disproportionate impact on young plants. 

GROWER-SURVEY RESULTS. When evaluating an experimental 

system that is designed, ultimately, to be adopted by growers, it is 

vitally important to evaluate the participating growers’ opinions of the 

effectiveness of the system. This type of evidence, while anecdotal, 

often carries more weight in the target community than more objective 

forms of measurement. In 2004, we surveyed our Massachusetts 

growers, along with several growers in Connecticut who had been 

using the Blue Hubbard/ butternut PTC system, with the following 

results: 

• 100% of surveyed growers found the PTC system and training to 

be good or excellent overall. 

• Eight out of 10 said that using PTC saved them money. The other 

2 said it cost about the same. 

• Eight out of the 10 growers we surveyed were very satisfied or 

thrilled with the way PTC worked for them. The remaining 2 

growers were satisfied. 

• 100% said using PTC took less or the same amount of time as 

using conventional methods. 

• 100% said they used less pesticide. 

Coupled with the evidence from the on-farm experiments, the 

survey results indicate that perimeter trap cropping can provide 

adequate striped cucumber beetle control while greatly reducing 

pesticide use in commercial butternut squash fields. The results of this 

study show that PTC is an excellent candidate for adoption by growers 

wishing to reduce their pesticide costs and exposure. 


Average per Plant Beetle count 

8 

7 

6 

5 

4 

3 

2 

1 

0 

Average Total (live + dead) beetles per plant 

1 2 3 4 5 6 

ptc - border 

ptc - main 

conv - border 

conv - main 

Fig. 2. Average adult beetles (sum of live plus dead beetles) per plant, by week, 

in the border and main crop of PTC and conventional fields, 2004. 


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system fact sheet. University of Connecticut Extension, Storrs, CT 

Brust, G. E., and R. E. Foster. 1995. Semiochemical based toxic baits for control of 

striped cucumber beetle (Coleoptera: Chrysomelidae) in cantaloupe. J. Econ. 

Ent. 88:112–116. 

Ferguson, J. E., E. R. Metcalf, R. L. Metcalf, and A. M. Rhodes. 1983. Influence of 

cucurbitacin content in cotyledons of Cucurbitaceae cultivars upon feeding 

behavior of Diabroticina beetles (Coleoptera, Chrysomelidae). J. Econ. Ent. 

76:47–51. 

Hazzard, R. V. 2003. Imidacloprid for striped cucumber beetle control, p. 187–190. 

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Association, Kirkville, NY 

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management program annual report. University of Massachusetts Extension 

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with kairomone-baited traps for cucumber beetles and corn rootworms in 

cucurbits. Env. Ent. 25:1173–1181. 

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McGrath, M. T. and N. Shishkoff. 2000. Comparison of cucurbit crop types and 

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CHARACTERIZATION OF COMMERCIALLY 

AVAILABLE WATERMELON POLLENIZERS 

Peter J. Dittmar, Jonathan R. Schultheis, and David W. Monks 

North Carolina State University, Department of Horticultural Science, 

Raleigh, NC 27695-7609 

ADDITIONAL INDEX WORDS. Citrullus lanatus, triploid watermelon, seedless 

ABSTRACT. Pollen from triploid (seedless) watermelon (Citrullus lanatus 

[Thunb.] Matsum. & Nak.) is nonviable. Diploid (seeded) watermelons produce 

viable pollen for seedless-watermelon production. In 2004, markets continued 

to increase for triploid but decreased for diploid watermelons. Seed companies 

are commercializing diploid cultivars (pollenizers) designed as pollen sources 

for triploid-watermelon production. The objectives of this research were to 

characterize the vegetative, floral, and fruit growth and development of these 

pollenizers. Five pollenizers were evaluated: ‘Companion’, ‘Mickylee’, ‘Mini 

Pool’, ‘SP-1’, and ‘Jenny’. ‘Companion’ produced the smallest plants, reaching 

a maximum vine length of 183cm, 5 weeks after transplant (WAT). ‘Mickylee’, 

‘Mini Pool’, ‘SP-1’, and ‘Jenny’ reached maximum lengths of 294–335cm, 5 

WAT. ‘Companion’ had the shortest internode length, 3cm. ‘SP-1’ produced 

the most male flowers, 30–40 per plant per day 5 to 8 WAT. ‘Companion’, 

‘Mickylee’, ‘Mini Pool’, and ‘Jenny’ produced male-flower at similar rates 

early in the season; after 5 WAT, however, ‘Companion’ had the lowest level of 

all pollenizers. ‘SP-1’ produced the most female flowers and had the most fruit 

(8 per plant). ‘Companion’ produced the fewest female flowers and produced 

two fruit per vine. ‘Mickylee’ had the largest fruit, 5.9kg; ‘SP-1’ and ‘Jenny’ 

produced the smallest, 3.1kg. 

T 

hree quarters of total watermelon production in the United 

States is triploid (seedless) (USDA Economic Research 

Service, 2005). Triploid watermelons do not provide adequate 

amounts of viable pollen for fruit set to occur (Kihara, 1951; 

Robinson and Decker-Walters, 1997; Rubatzky and Yamaguchi, 

1997). Diploid or pollenizer plants are placed next to or near triploid 

watermelon plants to provide viable pollen for fruit set on the triploid 

plants (Kihara, 1951; Robinson and Decker-Walters, 1997; Rubatzky 

and Yamaguchi, 1997). 

The pollenizer plants are planted in separate rows or in the same 

row as the triploid watermelon plants (Maynard and Elmstrom, 1992; 

NeSmith and Duval, 2001). Spacing of triploid watermelon plants has 

an effect on fruit number and size. Closer in-row spacing (eight per 1– 

2ft) of seedless cultivars ‘Crimson Jewel’ and ‘Honeyheart’ lowered 

the number and individual fruit weight per vine (Motsenbocker and 

Arancibia, 2002). The rind pattern of the pollenizer and triploid fruit 


must be different so that they can be separated and correctly marketed 

(Robinson and Decker-Walters, 1997; Rubatzky and Yamaguchi, 

1997). 

Honeybees (Apis mellifera L.) are used to promote pollination and 

fruit set of watermelon. Diploid flowers require 6 to 8 bee visits for 

fruit set (Adlerz, 1966; Stanghellini et al., 1997). However, fruit set in 

triploid watermelons requires over 24 bee visits (Walters, 2005). The 

objectives of this research were to evaluate several commercially sold 

diploid cultivars used exclusively for pollination of triploid 

watermelons. To evaluate the potential of these cultivars for 

pollination purposes, we measured vegetative growth, flower 

production, and individual fruit characteristics. These measurements 

are important because the pollenizer plant must effectively compete 

with but not out-compete triploid-watermelon plant growth in the 

production field. Additionally, the amount of flower production is 

important with respect to supplying viable pollen to optimize triploid 

fruit set. The production of female flowers on the diploid pollenizer 

plant is important as substantial fruit set may inhibit pollen production 

while the fruit must be easily distinguished from triploid fruit. 


The study included five commercially available diploid 

pollenizers. ‘Companion’, a cultivar marketed by Seminis Seed 

Company, was released in 2003 and is sold in the United States. 

‘Jenny’ is a cultivar released in 2005 by Nunhems Seed and marketed 

worldwide. ‘Mickylee’ is an open-pollinated cultivar available from 

many seed distributors, and is often used as a pollenizer in Central 

America and Florida. ‘Mini Pool’ was released from Hazera Seed and 

is predominately sold and used in Israel. ‘Super Pollenizer 1’ (‘SP1’) 

was released by Syngenta in 2004 and is utilized in the United States 

and throughout the world. 

Cultivars were planted 20 April, 2004, into LE1803 transplant 

trays (Landmark Plastics Corp., Akron, OH). Plants were transplanted 

17 May, 2004 (27 days after seeding) at the Central Crops Research 

Station, Clayton, NC, on black polyethylene mulch. The soil type was 

a Norfolk loamy sand (fine-loamy, kaolinitic, thermic typic 

kandiudults). Each plot had four plants spaced 1.3m in-row and 3.1m 

between-row. The plots were arranged in a randomized complete 

block design and replicated five times. 

Vegetative and floral growth and fruit production were evaluated 

for each pollenizer. Vegetative growth was characterized by vine and 

internode length. Vine length was measured using a measuring tape, 


from the crown of the plant to the tip of the longest vine on a given 

plant. Internode length was measured between the third and fourth 

fully expanded leaf on the same vine. Vine and internode length were 

collected three, four, and five weeks after transplanting (WAT). Floral 

growth was characterized by the number of female and male flowers 

produced per plant per date. Male and female flowers were counted on 

a weekly basis beginning the 3 rd WAT and ending the 8 th WAT. The 

number of fruit set was counted beginning 3 WAT and concluding 8 

WAT. Final fruit weights were measured at time of harvest, 19 July, 

2004. Fruit were considered as set when petals had completely dried 

and fallen off a healthy ovary. All data were evaluated using GLM 

procedures and means were separated using Duncan’s Multiple Range 

Test. 


Plant size was measured by vine and internode length. The 

cultivar ‘Companion’ was the smallest of the pollenizers evaluated 

(Figure 1). The internodes of ‘Companion’ were the shortest, 3.9cm, 

while the other pollenizers had internodes 9.3 to 10.0cm long (Figure 

2). ‘Mickylee’ and ‘Mini Pool’ produced the longest vines (Figure 1). 

‘SP1’ had growth early in the season that was similar to that of 

‘Mickylee’ and ‘Mini Pool’. However, later in the growing season (5 

WAT), ‘SP1’ had a shorter vine (295cm) than ‘Mickylee’ and ‘Mini 

Pool’ (Figure 1). Although ‘SP1’ vines were not as long as those of 

the other pollenizers, we observed that ‘SP1’ had extensive side 

branching. The cultivars with the longest vines had the greatest 

internode length (Figure 2). 

Floral growth was characterized by male- and female-flower 

production (Figures 3 and 4). ‘SP1’ produced the most male flowers 

throughout the experiment, with as many as 40 male flowers per plant 

per day by 4 WAT, with male-flower production never falling below 

30 (Figure 3). All other cultivars produced fewer male flowers. 

‘Companion’ had the same number of male flowers as ‘Jenny’, 

‘Mickylee’, and ‘Mini Pool’ 4 and 5 WAT; however, its production 

lagged behind that of the other cultivars 6 WAT and thereafter. 

‘Companion’ had 13.5 male flowers per plant per day 5 WAT and 5 or 

fewer flowers for the remainder of the experiment. In comparison, 

‘Jenny’, ‘Mickylee’, and ‘Mini Pool’ produced between 8 and 12 

flowers per plant per day 5 WAT. 

Of all the pollenizers evaluated, ‘SP1’ produced the most female 

flowers, with 3 per plant per day (Figure 4). ‘Companion’ had the 

lowest number. ‘Jenny’, ‘Mickylee’, and ‘Mini Pool’ produced the 


Fig. 1. Vegetative growth of watermelon pollenizers. Length of the longest vine 

from three to five weeks after transplanting (WAT). Bars with a different letter 

are significantly different for pollenizers tested for a given week (P>.05). 

Fig. 2. Vegetative growth of watermelon pollenizers. Length of internode 

between the third and fourth internode from the tip, averaged over the season. 

Bars with a different letter are significantly different for pollenizers tested for a 

given week (P>.05). 

same number of female flowers as ‘SP1’ 3 and 4 WAT but produced 

fewer after 5 WAT. 

‘SP1’ set the most fruit and produced eight fruit per plant by the 

end of the season (Figure 5). The fruit, however, were the smallest, 

10kg (Figure 6). The other four cultivars set fruit at similar times. 

‘Jenny’, ‘Mickylee’, and ‘Mini Pool’ continued to set additional fruit 7 

and 8 WAT, while ‘Companion’ remained at 2 fruits per vine 


(Figure 5). ‘Mickylee’ had the largest fruit, 22.9kg (Figure 6). 

At time of harvest, fruit rind pattern was determined for each 

cultivar. ‘Companion’ fruit has a ‘Charleston Gray’ rind, a boxy, 

blocky oblong shape, and red flesh. ‘Jenny’ is round with distinct dark 

green stripes on a light green background and red flesh. ‘Mickylee’ 

Fig. 3. Floral growth of watermelon pollenizers. Number of male flowers per 

plant per day. Points with a different letter are significantly different (P>.05). 

z 

‘SP1’ is in Group a; ‘Jenny’, ‘Mickylee’, and ‘Mini Pool’ are in Group ab; 

‘Companion’ is in Group c. 

y 

‘Jenny’, ‘Mickylee’, ‘Mini Pool’, and ‘Companion’ are in Group b. 

Fig. 4. Number of female flowers per plant per day. Points with a different 

letter are significantly different (P>.05). 

z 

‘Jenny’ is in Group b; ‘Mickylee’ and ‘Mini Pool’ are in Group ab; 

‘Companion’ is in Group c. 

y 

All pollenizers are in Group a. 

x 

‘Jenny’, ‘Mickylee’, ‘Mini Pool’, and ‘Companion’ are in Group b. 


Fig. 5. Fruit set on watermelon pollenizer vines from three to eight WAT. 

‘Jenny’, ‘Mickylee’, and ‘Mini Pool’ had the same number on all dates except 5 

WAT, and are shown on the same line. Points on the lines with a different letter 

are significantly different (P>.05). 

y 

All pollenizers are in Group a. 

x 

‘Companion’, ‘Jenny’, and ‘Mini Pool’ are in Group ab; ‘Mickylee’ is in 

Group b. 

z 

‘SP1’ is in Group a; ‘Companion’, ‘Jenny’, ‘Mickylee’, and ‘Mini Pool’ are in 

Group b. 

Fig. 6. The weight of individual watermelon at time of harvest. 

and ‘Mini Pool’ have round to oval fruit, a ‘Charleston Gray’ rind 

pattern, and red flesh. ‘SP1’ is round, with indistinct medium green 

stripes on a light green background and white flesh. Its rind is brittle 

and can split when very little pressure is applied to the fruit. 

Vegetative characteristics of the pollenizers provide some clues as to 

how competitive these cultivars will be when grown among triploid 

cultivars. The dwarf growing habit of ‘Companion’ provides less 

competition with triploid plants. However, the dense canopy produced 

by its shorter internodes may deter bees from accessing the pollenizer 


flowers and pollinating the triploid flowers. ‘Jenny’, ‘Mickylee’, and 

‘Mini Pool’ had longer vines and a more open growth habit. With 

these cultivars, the male flowers could be easily accessed by bees. If 

interplanted with the triploid watermelons, the more vigorous vines 

might compete more with triploid plants for water and nutrients 

thereby reducing triploid fruit set and/or size. This practice would 

likely reduce seedless-watermelon yields. If competition is a problem, 

it could be minimized by planting the pollenizers in a dedicated hill or 

in separate rows from the triploid plants. ‘SP1’ had slightly shorter 

vines than ‘Jenny’, ‘Mickylee’, and ‘Mini Pool’; however, the 

additional side branching could cause competition with the triploidwatermelon 

plants. 

In this research, these pollenizers were given optimal space for 

light interception, nutrition, and water. They need to be tested and 

evaluated further by interplanting them with triploid plants. We are 

currently conducting such studies on several pollenizers to determine 

their effects of triploid-watermelon production. 

The production of sufficient male flowers with viable pollen is 

important for fruit set in triploid watermelons. ‘Companion’ produces 

a comparable number of male flowers to other diploid pollenizers early 

in the season; however, the number of male flowers is reduced later in 

the season. If this cultivar is used as the pollenizer, triploid plants may 

have an earlier fruit set and harvest. An extended harvest season may 

be questionable since male-flower production levels were substantially 

reduced after fruit set occurred on ‘Companion’ plants. Both ‘Jenny’ 

and ‘Mini Pool’ hybrids have the same number of male flowers as the 

open-pollinated cultivar ‘Mickylee’. The high number of ‘SP1’ male 

flowers produced throughout the season may result in steady 

production of triploid fruit throughout the extended growing season. 

The characterization of the fruit is important for harvesting. A 

large number of unharvestable pollenizer fruit can interfere with 

harvest operations in the field, with workers tripping over or stepping 

on the fruit. Additionally, pollenizer cultivars must have a distinct 

rind pattern if used in a triploid-production field so that they can easily 

be distinguished from seedless fruit. This is even more imperative 

should miniwatermelons be produced and be of similar size as the 

pollenizers. 


Adlerz, W. C. 1966. Honey bee visit number and watermelon pollination. J. Econ. 

Ent.. 59:28–30. 

Kihara, H. 1951. Triploid watermelons. Proc. Amer. Soc. Hort. Sci. 58:217–230. 

Maynard, D. N. and G. W. Elmstrom. 1992. Triploid watermelon production 


practices and varieties. Acta Hort. 318:169–173. 

Motsenbocker, C. E. and R. A. Arancibia. 2002. In-row spacing influences triploid 

watermelon yield and crop value. HortTech. 12:437–440. 

NeSmith, D. S. and J. R. Duval. 2001. Fruit set of triploid watermelons as a 

function of distance from a diploid pollinizer. HortSci. 36:60–61. 

Robinson, R. W. and D. S. Decker-Walters. 1997. Cucurbits. CAB, New York. 

Rubatzky, V. E. and M. Yamaguchi. 1997. World vegetables, 2 nd ed. Chapman & 

Hall, New York. 

Stanghellini, M. S., J. T. Ambrose, and J. R. Schultheis. 1997. The effects of honey 

bee and bumble bee pollination on fruit set and abortion of cucumber and 

watermelon. Amer. Bee J. 137:386–391. 

USDA Economic Research Service. 2005. Vegetables and melons situation and 

outlook yearbook/VGS-2005/July 21, 2005: x. 

Walters, S. A. 2005. Honey bee pollination requirements for triploid watermelon. 

HortSci. 40:1268–1270. 


GALIA MUSKMELON PRODUCTION IN HIGH 

TUNNELS IN THE CENTRAL GREAT PLAINS 

USA 

Lewis W. Jett 

Department of Horticulture, University of Missouri, 

Columbia, MO 65211 

ADDITIONAL INDEX WORDS. Thermal water tubes, row covers, transplants, 

direct seeding 

ABSTRACT. High tunnels are rapidly being adopted as a season-extension 

technology for horticulture producers in the Central Great Plains. A distinct 

advantage of high tunnels is their use in growing diverse crops that could not 

normally be grown in the field environment of the Central Great Plains. 

Specialty melons such as Galia muskmelons (Cucumis melo L. var. reticulatus) 

are a high-value, warm-season vegetable that has the potential for early-season 

high-tunnel production. Research was conducted in 2004–2005 at the 

University of Missouri-Columbia using ‘Galia 152’ muskmelon within passively 

vented, solar-heated high tunnels. Transplanting significantly accelerated early 

harvest of Galia muskmelons relative to direct seeding within a high tunnel. 

When transplanting was combined with the use of thermal water tubes and row 

covers, ambient air temperature and early-season yield was significantly 

increased. Early-season harvest of quality Galia muskmelons within high 

tunnels can be profitable for growers in the Central Great Plains. 

H 

igh tunnels are a low-cost season-extension technology used 

for producing a diversity of horticulture crops (Lamont et al., 

2003). Specifically, high tunnels are passively vented, solar 

greenhouses covered with one layer of greenhouse plastic. Crops are 

grown directly in the soil beneath the high tunnel, and the only 

external connection is the drip irrigation system. In addition to 

accelerating crop growth and maturity, high tunnels protect the crop 

from a capricious environment where extremes in temperature, wind, 

rainfall, pests, and light intensity can severely reduce marketable 

yields and quality. Using a high tunnel, crops can be harvested at peak 

horticulture maturity over a longer growing season because the crops 

are not weakened by insects, weeds, or diseases. 

The choice of which crops to grow within a high tunnel is based on 

market potential, economics, yield per plant, and improved quality 

(Waterer, 2003). In a survey of high-tunnel producers in the Central 

Great Plains, the dominant crops growers produced within high tunnels 

were tomatoes, peppers, salad greens (lettuce, spinach, etc.), and 

cucurbits, respectively (Jett and Carey 2006). Most high-tunnel 


producers in the Central Great Plains use high tunnels for 1–2 crops 

per year and desire early-season harvest of high-value warm-season 

vegetables. Each crop occupies the high tunnel for approximately four 

months or one-third of each calendar year. While early-season 

tomatoes may be the dominant crop grown within high tunnels in the 

Central Great Plains, growers are interested in diversifying their 

production with other warm-season crops that can be double-cropped 

or rotated with tomatoes and peppers. There is a premium price for 

early muskmelons in the Midwest, and many specialty melons are 

becoming increasingly popular with consumers (Simon et al., 1993). 

Galia muskmelons (Cucumis melo L. var. reticulatus) are greenfleshed 

melons with a yellow, netted rind, high soluble solids, and a 

robust aroma that are adapted to warm, dry growing environments 

(Karchi, 2000). Although not widely found in U.S. markets, they are 

very popular in Europe, and production has been evaluated in Florida 

within passively vented greenhouses (Cantliffe, et al., 2002). Rainfall 

during flowering and fruit formation significantly lowers quality of 

Galia melons, and they should be harvested at the vine-ripe stage for 

maximum quality (Cantliffe, et al., 2002). Hochmuth et al. (1998) 

noted less cracking of Galia muskmelons when grown in protected 

culture versus the field in Florida. High tunnels may provide an 

optimal environment for growing Galia muskmelons in the Central 

Great Plains. 

Muskmelons are susceptible to chilling injury at temperatures 

between 41–59°F (Mitchell and Madore, 1992). There are several 

production practices that are used to protect muskmelons from lowtemperature 

stress and to increase early yields. Transplants can 

significantly increase early, marketable yield of muskmelons relative 

to direct seeding (Wiedenfeld et al., 1990). Spunbonded and perforated 

polyethylene row covers used in combination with black plastic mulch 

significantly increased early and total marketable yield of muskmelons 

(Waterer, 1993; Hemphill and Mansour, 1986; Loy and Wells, 1982; 

Wiebe, 1973). Water is a very efficient collector of radiant energy. 

Water-filled polyethylene tubes have been shown to reduce 

greenhouse heating costs by accumulating energy during the day and 

releasing heat during the night time (Pavlou, 1990). The use of waterfilled 

poly tubes in conjunction with clear plastic mulch and 

nonperforated polyethylene row covers significantly increased early 

yield of field-grown muskmelon in Quebec, Canada (Jenni et al., 

1998). Many of these techniques have been evaluated in the field 

environment but have not been evaluated in concert with a high tunnel. 

The objective of this research project was to evaluate the production 


and economic potential of early-season Galia muskmelons within a 

high tunnel and to determine which combination of season-extension 

practices is the most effective in accelerating early harvest in the 

Central Great Plains. 


HIGH-TUNNEL STRUCTURES AND PLOT ESTABLISHMENT. Three 

freestanding, Quonset high-tunnel units (Stuppy Greenhouse 

Manufacturing, Kansas City, MO) were constructed in early March 

2002 at the University of Missouri Bradford Research and Extension 

Center near Columbia, MO (lat. 90’11” W; long. 38’37” N). Each 

high tunnel was covered with a single layer of greenhouse-grade, 6-mil 

(0.006-in. thick) polyethylene plastic (K-50, Klerk’s Plastic 

Manufactures, Inc., Richburg, SC). The dimensions of each high 

tunnel were 6m (wide) x 11m (long) x 3.7m (high), with each arch 

spaced 1.2m apart. The high tunnels were oriented in a north-south 

direction with 3m between each high tunnel. Temperature and 

humidity were managed by manually rolling up the sidewalls or 

removing two end-wall panels from each end of the high tunnel. 

Whenever air temperatures inside the high tunnel exceeded 35°C, the 

sidewalls were rolled up to lower temperature and humidity. 

The soil within each high tunnel was a Mexico silt loam with a pH 

of 6 that was tilled and formed into raised beds 15cm high x 61cm 

wide beginning in mid-March of 2004 and 2005. Each high tunnel 

accommodated 5 rows of raised beds on 1.2-m centers. Preplant 

fertilizer (13N-5.7P-11K) was top-dressed and incorporated into each 

raised bed at a rate of 336g/90 m 2 nitrogen (N), 1120g /90 m 2 

potassium (K2O), and 1120g/90m 2 phosphorus (P2O5) (Jett, 2006). 

Calcium nitrate (15.5N-0P-0K-19Ca) was fertigated at a rate of 

560g/90m 2 /week commencing 2 weeks after seeding or transplanting 

and continuing through harvest. 

Black embossed plastic mulch (0.9m width) was laid over each 

raised bed. Each row was irrigated with one 8-mil (0.008-in.) T-Tape 

(T-Systems International, San Diego, CA) plastic drip tube with 

drippers spaced on-center 31cm apart. Irrigation was scheduled using 

a tensiometer placed 31cm deep in the center of each bed (Jett, 2004). 

TRANSPLANTS VERSUS DIRECT SEEDING WITHIN A HIGH TUNNEL. 

‘Galia 152’ (Hazera Genetics, El Segundo, CA) was chosen for this 

experiment based on performance in cultivar trials (Shaw et al., 2004). 

In early February of 2004 and 2005, ‘Galia 152’seeds were planted in 

the greenhouse in 72-cell polystyrene trays (Speedling, Sun City, FL) 

filled with a moistened soilless mix (Pro-Mix BX, Premier Brands, 


Yonkers, NY) and allowed to grow for 6 weeks before transplanting 

within each high tunnel. The plants were fertilized once per week with 

a 20N-8.7P-16.6K (Peter’s 20-20-20 water-soluble fertilizer) 

containing 200ppm N. The transplants were watered as needed. 

The minimum temperature for muskmelon seed germination is 

16°C (Maynard and Hochmuth, 1997). When the soil temperature at 

the 5-cm depth equaled or exceeded this temperature level, the plants 

or seeds were planted within the high tunnel. To evaluate direct 

seeding within a high tunnel, two seeds of ‘Galia 152’ were handseeded 

approximately 1.9cm deep and 61cm apart in 5-cm diameter 

planting holes on each raised bed. After emergence, the muskmelons 

were thinned to one vigorous seedling per hill. Six-week-old ‘Galia 

152’ melon transplants were planted at the same spacing. Both 

seeding and transplanting occurred on 30 March 2004 and 2005. 

Cultural practices consisted of standard recommendations for growing 

melons within a high tunnel (Jett, 2006). 

ROW COVERS AND THERMAL WATER BAGS. Immediately after 

seeding and transplanting, low tunnels were fashioned using wire 

hoops (#9 gauge) covered with two layers of lightweight, spunbonded 

polypropylene row cover (AG-19; Agribon Inc., Mooresville, NC). 

The row covers remained on the plants continuously until anthesis, 

defined as the time when the first perfect muskmelon flower was 

observed. After anthesis, the row covers were held in reserve in the 

event of a frost event. 

Water was evaluated as a solar collector that could increase the 

microclimate temperature around each muskmelon plant rather than as 

a technique to affect the air temperature within the entire high-tunnel 

structure. Two 3.8-L Ziploc storage bags (2-mil thickness) holding 

3.6kg of water per bag were filled with water and placed on either side 

of each planting hole immediately after planting or seeding. Initially a 

7.6-cm-diameter polyethylene water tube was used but was discarded 

after discovering the tube leaked water and failed to remain stationary 

on the raised beds. 

To facilitate harvest, the muskmelon vines were trellised using a 2m-high 

plastic mesh trellis (Johnnys’ Selected Seeds, Albion, ME) 

supported by tensile wire and metal posts. The vines were not pruned, 

and each lateral was trained or directed to grow on the trellis using 

plastic vine clips. 

EXPERIMENTAL DATA. Treatments consisted of combinations of 

two planting methods, transplanting (TRP) and direct seeding (DS), 

with or without spunbonded row covers (RC), and with or without 

thermal water bags (WB). Individual plots were one raised bed, 2.4m 


long, containing four muskmelon plants. Treatments were randomized 

within each of the three high tunnels. 

Replicated air temperature was recorded 46cm above the crop 

canopy using Hobo H8 data loggers (Onset Computers, Bourne, MA) 

in 2005. Temperature was measured under row covers, (with or 

without thermal water bags), within the high tunnel, as well as the 

ambient air temperature. Temperature was logged hourly and 

averaged to determine average daily temperature. 

Since muskmelons require physical transfer of pollen from 

staminate to perfect flowers to set fruit, honeybee (Apis mellifera L.) 

colonies were placed 300ft from the high tunnels to facilitate 

pollination. In 2005, small bumblebee (Bombus impatiens Cresson) 

colonies (Koppert Biological Systems, Ann Arbor, MI) containing 

approximately 150 bees per colony were placed within each high 

tunnel on 10 April to aid in pollination after it was determined 

honeybees were not entering the high tunnels in sufficient numbers to 

pollinate early, perfect flowers in 2004. 

Thirty days after flowering, most muskmelons hanging on the 

trellis were supported with mesh onion sacks cradled around the fruit 

and secured to the trellis. Fruits were harvested at full slip twice per 

week and individually weighed beginning 22 June 2004 and 15 June 

2005. Fruits weighing less than 0.9kg were categorized as 

unmarketable. Harvest continued until 31 July of each year. 

STATISTICAL ANALYSIS. The experiment was a randomized 

complete block design with three blocks (high tunnels). Data were 

analyzed using analysis of variance (ANOVA) and orthogonal 

contrasts (SAS Institute, Cary, NC). 


EFFECT ON AIR TEMPERATURE. Throughout most of the vegetative 

growth in 2005, high tunnels maintained an average temperature of 

15.8°C compared with an outside temperature of 11.7°C. These 

temperatures are consistent with research conducted at Penn State 

where the average temperature difference between a single-layer 

plastic high tunnel and the outside was 4.3°C (Lamont, et al., 2003). 

The addition of two layers of row covers increased the average 

microclimate temperature by 2.7°C (18.5°C), while row covers 

combined with thermal water bags increased the microclimate 

temperature by 3.3°C (19.1°C). Photosynthesis and anthesis of Galia 

muskmelons is reduced when temperatures decrease below 18.3°C (D. 

Cantliffe, personal communication). 


High tunnels alone generally don’t provide significant frost 

protection but may protect against chilling temperatures. Minimum 

temperatures within a high tunnel eventually equilibrate with the 

outside temperature, as was observed in Kansas during March (Kadir, 

et al., 2006). Ambient temperatures did not decrease below 0°C in 

2004. However, on 24 April 2005 a frost event occurred, and the air 

temperature within the high tunnel was < 0°C for 5h and < 5°C for 

11h. Muskmelons with only a row cover had microclimate 

temperatures

Table 1. Marketable yield of Galia muskmelons grown within a high 

tunnel combined with inputs to accelerate harvest. 

Early yield Total marketable yield 

fruits/plant kg/plant fruits/plant kg/plant 

Treatment z 

2004 2005 2004 2005 2004 2005 2004 2005 

DS 0.0 0.8 0.0 1.6 2.0 1.9 2.0 3.8 

DS+RC 0.2 0.6 0.4 1.1 1.5 0.8 2.9 1.4 

DS+WB 0.5 0.8 1.1 1.5 2.9 1.3 5.8 2.4 

DS+WB+RC 0.4 0.5 0.9 0.8 1.0 0.7 2.1 1.0 

TRP 0.7 1.0 1.2 1.8 1.8 2.3 3.6 3.8 

TRP+ RC 0.2 0.8 0.3 1.7 1.9 2.0 3.2 3.6 

TRP+WB 0.3 1.0 0.4 1.7 2.7 1.9 4.4 3.2 

TRP+WB+RC 

Treatments y 

0.5 2.7 1.0 4.4 3.8 3.2 6.9 5.3 

Orthogonal 

contrasts 

NS ** NS ** ** ** ** ** 

TRP vs. DS 

TRP+WB+RC 

NS ** NS ** ** ** ** ** 

vs. TRP NS ** NS ** NS NS NS NS 

WB vs. NWB NS NS NS NS NS NS ** NS 

RC vs. NRC NS NS NS NS NS NS NS NS 

z 

TRP = Transplanted; DS = Direct Seeded; RC = Row cover; WB = (Thermal) water 

bag. 

y 

Significant at P ≤ 0.05. 

marketable yield. Plants that had only thermal water bags showed no 

signs of chilling injury relative to the controls (data not shown). 

EFFECT ON TOTAL MARKETABLE YIELD. Fruit quality was 

excellent, with most melons averaging 14° brix regardless of 

treatment. Due to the longer harvest period, transplanted melons had a 

significantly higher total-season yield in both years of this study. 

Direct-seeded Galia muskmelons averaged 1.2–1.9 melons per plant 

while transplanted muskmelons averaged 2.4–2.6 melons per plant. 

Despite earlier flowering and harvest of transplanted melons, average 

fruit mass did not differ with planting method. In both years, fruit 

mass averaged 1.7kg across all treatments. 

In a commercial high tunnel, which is typically 225m 2 , 

approximately 300 muskmelon plants can be planted. If each plant 

yielded three marketable fruits, 900 Galia muskmelons can be 

harvested per high tunnel. In 2004, inadequate pollination delayed 

early fruit set on transplanted Galia melons with row cover and 

thermal water bags. Transplanted melons produced only 20–25% of 

their total yield before early July in 2004, but in 2005, more than 50% 

of the total yield was early. Given adequate density of pollinators in 


2005, 86% of the total marketable yield of transplanted melons with 

row cover and thermal water bags was harvested before 4 July. 

Galia muskmelons are a potentially profitable warm-season crop 

for high-tunnel production in the Central Great Plains. Galia 

muskmelons can be rotated or double- cropped with popular hightunnel 

vegetables such as tomatoes, peppers, and lettuces. Production 

practices that increase microclimate temperature and early growth, 

such as using transplants, row covers, and thermal water bags, will 

increase both early and total marketable yield. The increased yields 

will offset the increase in production costs. Wholesale prices may not 

be high enough for a significant profit to occur. Most high-tunnel 

producers in the Central Great Plains market their produce via local 

farmers’ markets where a price premium for vine-ripe early 

muskmelons may be attained. In addition, high tunnels enable Galia 

melons to be grown using fewer pesticides, which may further increase 

the price premium. 


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A COMPARISON OF NOVEL GRAFTING 

METHODS FOR WATERMELON IN HIGH- 

WIND AREAS 

Stephen R. King 

Vegetable & Fruit Improvement Center, Department of Horticultural 

Sciences, Texas A & M University, College Station, TX 77843-2133 

Angela R. Davis 

USDA, ARS, South Central Agriculture Research Laboratory, P.O. 

Box 159, Lane, OK 74555 

ADDITIONAL INDEX WORDS. Citrullus lanatus 

ABSTRACT. Grafted watermelon transplants are not commonly used by growers 

in the U.S., despite the advantages associated with their use. The two major 

disadvantages of using grafted watermelons are the high cost associated with 

performing the graft, and the potential for increased transplant losses, 

particularly under high-wind conditions. We found that modifying the grafting 

techniques to provide additional support to the graft union resulted in 

nonsignificant increases in transplant survivors one week after transplanting 

compared to a nonsupported grafting technique. The effort required for the 

modifications was slightly increased for the tube-supported method, and slightly 

decreased for the toothpick-supported method. More study is needed to identify 

grafting modifications that will reduce costs and increase transplant 

survivability under high-wind conditions. 

G 

rafting of watermelon to various rootstocks is a common 

practice in many parts of the world but, despite the reported 

advantages, such as resistance to soil-borne diseases and 

increased water-use efficiency, the practice is not common in the U.S. 

The use of rootstocks has been shown to enhance the vigor of the scion 

through avoidance of soil pathogens, tolerance of low soil 

temperatures and/or salinity, and increased scavenging of soil nutrients 

(Ruiz et al., 1997). The type of rootstock has been shown to affect 

watermelon plant growth and yields (Yetisir and Sari, 2003). Yetisir 

and Sari (2004) demonstrated that the survival rate of grafted plants 

was inversely correlated with the difference in diameters of scion and 

rootstock; although they could not show that survival rate was affected 

by the number of vascular bundles, the number of vascular bundles 

positively affected the growth rate of the grafted watermelon plants. 

Edelstein et al. (2004) showed that stem diameter and number of 

vascular bundles of the rootstock did not correlate with scion-plant 

fresh weight for C. melo scions and 22 Cucurbita sp. rootstocks. 


Increases in soil-borne pathogens accompanied by the loss of 

effective pesticides such as methyl bromide may necessitate the use of 

alternative forms of disease control, such as the use of disease-resistant 

rootstocks. Other advantages, such as increased water-use efficiency 

and tolerance to salt and/or low temperatures, that may accompany the 

use of grafted watermelon would most likely speed acceptance of 

grafting in the U.S. One disadvantage that has been observed by 

growers in the U.S. is a higher mortality of grafted watermelon plants 

when transplants are subjected to high winds shortly after 

transplanting. It is suspected that the graft union is more susceptible to 

breakage under high-wind conditions. Despite the potential benefits of 

using grafted watermelon, little research has been done in the U.S. on 

local cultivars utilizing local cultural practices. The purpose of this 

research was to investigate the potential of various grafting procedures 

for establishing local diploid and triploid cultivars in the field using 

different rootstocks under established growing conditions that include 

high wind shortly after transplanting. 


PLANT MATERIAL. Scions included ‘Royal Sweet’ (diploid 

watermelon), ‘Sugar Time,’ ‘Super Seedless 7167’, and ‘Tri-X 313’ 

(triploid watermelons). Rootstocks used included ‘Strongtosa’ (an 

interspecific hybrid between Cucurbita maxima and C. moschata), 

‘Destiny III’ (C. pepo), ‘Red Kuri’ (C. maxima), ‘Long Island Cheese’ 

(C. moschata), ‘Musque de Provence’ (C. moschata), ‘Rouge vif 

D’etampes’ (C. maxima), and ‘Birdhouse’ (Lagenaria siceraria). 

Seeds were sown into flats with individual cell sizes of 4.4cm x 3.8cm 

x 6cm deep (1006 X-Line insert, BWI, Shulenberg, TX) filled with 

Pro-Mix PGX germination medium (Premier Horticulture, Dorval, 

We would like to thank Anthony Dillard, Amy Helms, and Ashley Gammon for 

providing valuable technical support. Special thanks go to Tom Williams, and Glen 

Price for supplied seed. Partial funding was provided by USDA-CSREES grant 

2004-34402-14768. Mention of trade names or commercial products in this article is 

solely for the purpose of providing specific information and does not imply 

recommendation or endorsement by the U.S. Department of Agriculture. All 



age, marital status, or handicap. The article cited was prepared by a USDA employee 

as part of his/her official duties. Copyright protection under U.S. copyright law is not 

available for such works. Accordingly, there is no copyright to transfer. The fact that 

the private publication in which the article appears is itself copyrighted does not 

affect the material of the U.S. Government, which can be freely reproduced by the 

public. 


QC, Canada) or into 3.8cm square x 6.3cm deep Speedling flats 

(#F128A, Speedling, Inc., Sun City, FL) containing Redi-earth growth 

medium (SunGro, Vancouver, BC, Canada). The watermelon cultivars 

were sown approximately 7days prior to sowing the rootstock, except 

for ‘Destiny III’, which was sown on the same day as the watermelon. 

All watermelon and‘Destiny III’ squash plants were approximately 4w 

old at time of grafting, and the remaining rootstocks were 

approximately 3weeks old. 

Fig. 1. Tube-grafting method as described in text. Tube is placed over rootstock 

and scion is inserted into tubing (A, B). A binder clip holds the tube in place for 

ca. 72 hours (C). The tube is left in place at transplanting. 

TUBE-MODIFIED SLANT-CUT METHOD AT COLLEGE STATION, 

TX. Scions were grafted using modifications of the slant-cut method. 

The slant-cut method incorporated the use of 0.64-cm vinyl tubing cut 

into approximately 2-cm lengths and slit lengthwise, and then inserting 

the rootstock and scion into the tubing, which was held in place with 

¾”-wide binder clips (Figure 1). ‘Super Seedless 7167’ and ‘Tri-X 

313’ were grafted onto ‘Destiny III’, ‘Red Kuri’, ‘Long Island 

Cheese’, ‘Musque de Provence’, ‘Rouge vif D’etampes’, and 

‘Birdhouse’ using the vinyl-tubing method. Grafted plants were placed 

in flats covered with clear plastic domes for approximately 48h in the 

greenhouse, when the domes were removed and plants were left in the 

greenhouse. The binder clips were removed after approximately 72h, 

but the vinyl tubing was left in place, with the slit allowing for stem 

expansion. Temperatures ranged from a high of 28 o C to a low of 20 o C. 

Survivability counts were taken prior to transplanting in the field, and 

at 3 days and 7 days after transplanting. Seedlings were fieldtransplanted 

into raised beds covered with black plastic mulch and drip 

irrigation 2 weeks after grafting in a randomized complete block 

design with two replications in a serpentine arrangement. Beds were 

200cm apart and plant spacing was 90cm. The field soil was Robco 

loamy fine sand. There were no windbreaks in the field. 


TONGUE-AND-GROOVE METHOD AT LANE, OK. Scions were 

grafted using the tongue-and-groove method with or without the aid of 

a wooden pin. The tongue-and-groove method involved cutting a slit 

in the rootstock and sharpening the scion to fit the groove made in the 

rootstock. Clamps were unavailable, so the two were held together 

either with Parafilm or with a toothpick inserted into both the scion 

and rootstock to align the two and then Parafilm (Figure 2). 

Fig. 2. The toothpick-modified tongue-and-groove procedure. A groove is made 

in the rootstock and a tongue is made in the scion to fit the groove. For the 

modification, a toothpick is inserted into the rootstock and the scion inserted 

into the toothpick (A) and pressed down to make contact with the rootstock. 

The nonmodified method uses Parafilm to hold the graft in place. 

‘Royal Sweet’ and ‘Sugar Time’ were grafted onto the 

‘Strongtosa’ rootstock. Grafted plants were placed in flats and were 

placed in a warm environment for 48h. Temperatures ranged from 

23°C to 28°C, but the environment was not sufficiently humid and we 

had a high loss of plants early. Seedlings were planted in the field 

3weeks later in a randomized complete block design with four 

replications in a serpentine arrangement. The field had Bernow fineloamy, 

siliceous, thermic, glossic palendalf soil. There were no 

windbreaks in the field. Survivability counts were taken prior to 

transplanting in the field, and at 3 days and 7 days after transplanting. 

STATISTICS. Means were calculated for grafting frequency by 

dividing the number of graft attempts by successful grafts (prior to 

transplanting) for each variety and method, and 7-day-survival 

frequencies were calculated by dividing the number of transplants 

surviving after 10 days by the number of transplants for each variety. 

Data were analyzed using SAS statistical software (Cary, NC). 


Results 

TUBE-MODIFIED SLANT-CUT METHOD. It was hoped that the tube 

method would speed the grafting procedure by not using clips; 

however, it was found that for most grafts, a clip was needed to 

support the scion on the rootstock, which resulted in an increase in 

time compared to using the clips alone. Survivability counts showed 

that grafting efficiency prior to transplant ranged from a low of 25% to 

a high of 42% (Table 1). Stand counts 7 days after transplanting 

showed a survival range from 78 to 84%, which was significantly 

reduced from the 7-day transplant survival of the nongrafted control 

plants. The primary cause of this reduction appeared to be breakage 

and/or tissue tearing at the graft union. Wind conditions in College 

Table 1. Comparison of grafting frequency and 7-day transplant 

survival frequency for cultivars and grafting methods. 

Grafting 

7-daysurvival 

Location Variety 

Tri-X 

Rootstock Method N Frequency frequency 

Texas 313 

Tri-X 

Multiple Tube 36 0.42 0.78 

Texas 313 None Nongrafted 12 NA 1.00 

Texas S.S.7167 Multiple Tube 36 0.25 0.84 

Texas S.S.7167 None 

Royal 

Nongraftedf 12 NA 1.00 

Oklahoma Sweet 

Royal 

Strongtosa Parafilm 75 0.35 0.83 


Royal 

Strongtosa Toothpick 0 0.83 


Sugar 

None Nongrafted 33 NA 1.00 

Oklahoma Time 

Sugar 

Strongtosa Parafilm 75 0.40 0.70 

Oklahoma Time 

Sugar 

Strongtosa Toothpick 75 0.39 0.73 

Oklahoma Time None Nongrafted 33 NA 0.98 

*Grafting frequency = successful grafts/attempted grafts. There were no significant 

differences at the P0.5 level of probability. 

**Frequency of plants surviving 7 days after transplanting. 

Means followed by the same letter are not significantly different at the P0.5 level of 

probablilty. 


Station following transplanting ranged from a daily high of 13mph to 

32mph, with maximum gusts of 37mph (Table 2). 

TONGUE-AND-GROOVE METHOD. The toothpick modification of 

the tongue-and-groove method did speed grafting compared to the 

nonmodified method because the toothpick provided sufficient 

support. Survivability of the tongue-and-groove and toothpickmodified 

tongue-and-groove method were similar, ranging from 33 to 

40% success (Table 1), which indicates that the toothpick modification 

did not hinder the healing process. The two methods were also similar 

for transplant survival after 7 days, with a range from 70 to 83%, 

which was significantly reduced when compared to the 7-day survival 

of nongrafted plants. The primary cause of transplant loss appeared to 

be tissue breakage at the graft union. Wind conditions in Lane 

following transplanting ranged from a daily high of 9 to 25mph with 

maximum gusts of 32 mph (Table 2). 

COMPARISON OF THE METHODS. While the different methods were 

used on different rootstocks and varieties at different locations, 

statistical analysis showed no significant interactions among variety, 

rootstock, or location (data not shown). A comparison of the methods 

showed no significant differences (Table 3). All methods did have a 

lower 7-day transplant survival than the nongrafted control, but the 

range was still fairly high at 77 to 81%. The primary cause of 

transplant loss at both locations appeared to be tissue breakage at the 

graft union. Wind conditions were similar at both locations, with gusts 

in the 30+mph range. 

Table 2. Wind-speed weather data for the two locations for 7 days 

following transplanting. 

College Station, TX Lane, OK 

Days after Average Gust Average Gust 

transplant (mph) (mph) (mph) (mph) 

0 10 33 5 18 

1 10 26 8 27 

2 8 17 5 32 

3 14 35 9 22 

4 11 37 8 19 

5 6 16 9 20 

6 11 24 8 21 

7 11 23 7 24 


Table 3. Grafting frequency and 7-day-survival frequency of the 

transplant methods. 

Grafting 7-day-survival 

Grafting method* frequency** frequency*** 

Control NA 0.99 

Tube 0.40 0.81 

Toothpick 0.36 0.79 

Parafilm 037 0.77 

*Grafting methods described in text. **Successful grafts/attempted grafts. All 

frequencies n.s. at the P0.5 level of probability. 

***Frequency of plants surviving 7 days after transplanting. 

Means followed by the same letter are not significantly different at the P0.5 level of 

probability. 

Discussion 

This study showed that the grafting modifications we used result in 

survival frequencies similar to those of conventional grafting methods. 

The goals of any grafting modifications should be to reduce input costs 

and increase survivability in the field. The stabilized grafting methods 

did not show a marked increase in survivability, but the toothpick 

method did decrease the amount of time required to make the grafts, 

which will result in lower costs. The tube method did not reduce 

grafting time because of the need to clip the plants, but further 

investigation may reduce the need for the clip, which may reduce 

grafting time. Further study is needed to dertermine whether the added 

graft-union support afforded by the tube-modified and/or toothpickmodified 

grafting methods will result in increased survival of grafted 

watermelon plants under high-wind conditions. 


Edelstein, M., Y. Burger, C. Horev, A. Porat, A. Meir, and R. Cohen. 2004. 

Assessing the effect of genetic and anatomic variation of Cucurbita rootstocks 

on vigour, survival and yield of grafted melons. J. Hort. Sci. Biotech. 79:370– 

374. 

Lee, J. M. and M. Oda. 2003. Grafting of herbaceous vegetables and ornamental 

plants. Hort. Rev. 28:61–124. 

Ruiz, J. M., A. Belakbir, I. Lopez-Cantarero, and L. Romero. 1997. Leafmacronutrient 

content and yield in grafted melon plants. a model to evaluate the 

influence of rootstock genotype. Sci. Hort. 71:227–234. 

Yetisir, H. and N. Sari. 2003. Effect of different rootstock on plant growth, yield and 

quality of watermelon. Austral. J. Expt. Agr. 43:1269–1274. 

Yetisir, H. and N. Sari. 2004. Effect of hypocotyl morphology on survival rate and 

growth of watermelon seedlings grafted on rootstocks with different emergence 

performance at various temperatures. Turkish J. Agric. & For. 28:231–237. 


CUCURBITS AND THEIR IMPORTANCE IN 

NORTH CAROLINA 

Jonathan R. Schultheis 

North Carolina State University, Department of Horticultural Science, 

Raleigh, NC 27695-7609 

ADDITIONAL INDEX WORDS. Acreage, cucumber, Cucumis sativus, melon, 

Cucumis melo, pumpkin, squash, Cucurbita pepo, Cucurbita maxima, Cucurbita 

moschata, watermelon, Citrullus lanatus, dollar value 

ABSTRACT. North Carolina agriculture is very diversified, accounts for 22% of 

the state’s income, and employs 20% of its work force. Net farm income 

accounts for $2 billion, while annual vegetable farm-gate value is approximately 

$300 million, with cucurbits accounting for approximately $60 million. In the 

United States, North Carolina is the second largest state producer of pickling 

cucumber, fourth in fresh market cucumber, sixth in muskmelon, and seventh 

in watermelon. It is a leading state when it comes to the production, marketing, 

and research of traditionally grown cucurbits as well as in the development and 

marketing of new or specialty cucurbit crops. 

W 

orldwide, cucurbits have always been of importance to 

mankind. They have been used in a variety of ways; 

utensils, sponges, filters, musical instruments, and 

especially for food and fiber (Robinson and Decker-Walters, 1997). 

Many of the cucurbits contain needed dietary components that are rich 

in vitamins A and C. Squashes (Cucurbita sp.) and melons (Cucumis 

melo) are especially rich in vitamin A and/or C (Rubatzky and 

Yamaguchi, 1997). Other cucurbits, such as certain watermelon 

cultivars, have recently been documented to be a rich source of 

lycopene (Perkins-Veazie et al., 2001), an antioxidant that purportedly 

reduces cancer (Sies and Stahl, 1998). The diversity of cucurbits is 

large and various regions in the world have certain crop types that are 

more commonly grown and consumed (Robinson and Decker-Walters, 

1997). In the United States (US), some of the most commonly grown 

and/or consumed cucurbits are fresh market and processing cucumbers 

(Cucumis sativus), various kinds of winter squash (Cucurbita sp.) (i.e, 

acorn, butternut, Hubbard, kabocha) and summer squash (Cucurbita 

pepo) (yellow and zucchini), melons [primarily muskmelon (Cucumis 

melo var. reticulata) and honeydew (Cucumis melo var. inoderous)], 

pumpkin (Cucurbita pepo), and watermelon (Citrullus lanatus). Other 

minor cucurbit crops are grown and sold in the US. New cucurbit 

crops continue to be discovered, grown, and markets developed. A 

review of cucurbit production worldwide and in the US was given 


during Cucurbitaceae 2002 in Naples, Florida (Taylor and Brant, 

2002). The purpose of this manuscript is to give some perspective of 

the importance of the role that cucurbits have in North Carolina (NC) 

horticulture with respect to the state’s agricultural economy and 

compared with US production. 

Discussion 

North Carolina has a diversified agriculture industry. The industry 

accounts for 22 percent of the state's income, and employs over 20 

percent of the work force (http://www.ncagr.com/stats/general/ 

general.htm). This includes food production, fiber, and forestry, which 

contribute $62.6 billion annually to the state's economy. 

Approximately 56,000 farmers grow over 80 different commodities. 

North Carolina produces more tobacco and sweetpotatoes than any 

other US state and ranks second in the production of hogs, turkeys, 

Christmas trees, trout, and pickling cucumbers. The state ranks sixth 

nationally with respect to farm profits. Net farm income for the state 

is over $2 billion, while net income per farm in the state is over 

$36,000. 

In NC, the vegetable industry makes up an important part of the 

agricultural industry, with farm-gate values totaling about $308 

million in 2003 (North Carolina Department of Agriculture and 

Consumer Sciences, 2004). Key vegetable crops produced within the 

state of North Carolina in which reported values are more than $5 

million are: bell pepper (Capsicum annuum), cabbage (Brassica 

oleracea var. capitata), cucumbers, potato (Solanum tuberosum), snap 

beans (Phaseolus vulgaris), squash (Cucurbita pepo), sweet corn (Zea 

mays var. rugosa), sweetpotato (Ipomoea batatas), tomato 

(Lycopersicon esculentum L.), and watermelon. 

There are approximately 2,550 commercial farmers who grow 

vegetables in North Carolina (U.S. Department of Agriclture, 2002). 

Individual farms range from one hectare up to a thousand hectares, and 

produce is marketed locally, nationally, and internationally. In the last 

10 years, decreasing revenues due to lower profit margins on federally 

subsidized crops such as corn, soybeans, cotton, and tobacco have 

stimulated NC growers to look for more profitable alternative 

agricultural enterprises. In some cases, this includes vegetables. 

North Carolina is the largest US producer of sweetpotatoes, 

growing about 17,000 hectares valued at nearly $85.3 million in 2003 

and accounting for 37% of the national production (North Carolina 

Department of Agriculture and Consumer Services, 2004, 2005). 


The second highest value vegetable crop to NC is cucumber at 

$30.7 million (North Carolina Department of Agriculture and 

Consumer Services, 2004; Table 1). Thus, cucumber is the most 

important cucurbit crop with respect to farm-gate value and acreage in 

NC. North Carolina is the second largest US producer of pickling 

cucumbers, growing about 6883 hectares valued at $19.4 million in 

2004 (North Carolina Department of Agriculture and Consumer 

Services, 2004). Two large pickling cucumber-processing companies, 

Mt. Olive Pickle Company (Mt. Olive, NC) and Bay Valley Foods 

(Faison, NC), are located less than 15km apart in eastern NC. Labor 

has until recent years generally been quite adequate for hand-harvest 

operations. The availability and accessibility of migrant labor, 

primarily from Mexico, has been reduced and may not be available 

pending legislation at the national level that may further restrict or 

eliminate the accessibility of Mexican workers into the US. It is 

interesting to note that Michigan, number one producing state of 

pickling cucumbers, hand-harvested all their production acreage in 

1964 (Motes, 1977). By 1972, over 90% of its pickling-cucumber 

acreage was once-over machine-harvested. A similar scenario may 

occur in NC as less than five years ago, there were few acres that were 

once-over machine-harvested. Currently, in 2006, about one-third of 

NC's pickling-cucumber acreage is once-over machine-harvested. It is 

possible that most pickling-cucumber acreage will be machineharvested 

by 2010 in NC. All of NC’s pickling-cucumber production 

is in the eastern part of the state due to its large, flat expanses of land 

that are typically composed of a sandy loam soil and conducive for 

pickling-cucumber production (Schultheis et al., 1998). There is a 

state growers association, the NC Pickle Producers Association, which 

promotes and supports various research and marketing efforts related 

to pickling cucumbers. 

In NC, “slicer” cucumbers were planted on an additional 2834 

hectares for fresh market, placing it fourth nationally in area planted, 

with the value of production totaling $11.3 million in 2003 (North 

Carolina Department of Agriculture and Consumer Sciences, 2004; 

Table 1). Fresh market cucumbers are produced both on bare ground 

and on plasticulture (black polyethylene mulch and drip irrigation) 

(Schultheis et al., 1998). All fresh market cucumbers are handharvested. 

Important production areas are located in both eastern and 

western North Carolina. In 2002, there were nearly 700 commercial 

growers who produced either fresh market or processing cucumbers in 

North Carolina (U.S. Department of Agriculture, 2002). North 

Carolina State University has the only national public breeding 

program for both slicing and pickling cucumbers. 


Following cucumbers, the cucurbit that is most widely produced in 

NC is watermelon (North Carolina Department of Agriculture and 

Consumer Services, 2004). The land devoted to watermelon 

production in NC from 1994 to 2004 has ranged from 3238 to 4615 

hectares (Arney et al., 2006). North Carolina is the seventh leading 

state with respect to watermelon production and value compared with 

other US producing states (Table 1). North Carolina primarily 

produces red-flesh, seedless watermelon since market demand has 

shifted away from seeded, red-flesh cultivars (U.S. Department of 

Agriculture, Economic Research Service, 2005). NC also produces 

and sells specialty watermelon melon types: yellow-flesh, orangeflesh, 

and miniwatermelons. Marketing and production of 

watermelons is supported by a growers' association (the North 

Carolina Watermelon Association), North Carolina State University, 

and the North Carolina Department of Agriculture and Consumer 

Services. The 2004 watermelon crop was valued at $6.3 million 

(Arney et al., 2006). 

In 2004, summer squash was reportedly grown on over 1,500 

hectares in NC and valued at $8.4 million (North Carolina Department 

of Agriculture and Consumer Services, 2004; Table 1). This reference 

report accounts for only summer squash, which includes the yellow 

straightneck and crookneck types and zucchini. There are many 

winter squashes that are grown in NC for which no published statistics 

are kept. These winter types include butternut, acorn, spaghetti, 

cushaw, and kabocha. The summer squash types are marketed 

nationally, mainly along the east coast, while most of the winter 

squash types are sold in more localized markets. 

Pumpkins are grown across the state of NC. In 2002, 

approximately 280 farms produced pumpkins valued at $2.7 million 

(Table 1) on a total of 540 hectares (U.S. Department of Agriculture, 

2002). Pumpkins are grown primarily for decorative/display purposes. 

The fruit are used to adorn the home and festivals and are one 

indicator that the autumn season has arrived. In addition, the primary 

use of pumpkins by US citizens is as jack o’lanterns. Jack o’lanterns 

are used for display or are carved at Halloween. The primary specie 

used for jack o’lanterns is Cucurbita pepo. C. maxima and C. 

moschata are also used, but to a lesser extent. 

The primary melon produced in NC is muskmelon. Muskmelon 

(Cucumis melo var. reticulatus), commonly referred to in the US as 

cantaloupe, was produced by over 600 NC growers on approximately 

1100 hectares and valued at approximately $15 million in 2002 (U.S. 

Department of Agriculture, 2002; Table 1). The primary cultivar 

grown in NC and throughout the southeastern US is ‘Athena’. Most 


Table 1. Production of cucurbits in North Carolina in comparison with 

United States production. Z 

Crop 

genera & 

species 

North Carolina United States 

Rank 

in 

value 

among 

US 

states 

Hectares 

planted 

Farmgate 

value 

(in 

millions 

$US) 

Hectares 

planted 

Farmgate 

value 

(in 

millions 

$US) 

Cucumbers Cucumis sativus 

Fresh 4 2834 11.3 47,085 157.1 

Processing 2 6883 19.4 22,741 212.7 

Muskmelon Cucumis melo var. reticulatus 

6 1133 15.0 38,420 315.6 

Squash Cucurbita sp. 

Fresh & 

processing 6 1579 8.4 21,296 222.7 

Pumpkin Cucurbita sp. 

Fresh & 

processing 

Y 

537 2.7 18,502 91.7 

Watermelon Citrullus lanatus 

7 3441 6.3 65,182 343.1 

z 

Data were obtained for cucumbers and squash for 2004 from U.S. Department of 

Agriculture, Economic Research Service, 2005; for muskmelon for 2002 from U.S. 

Department of Agriculture, 2002; watermelon for 2004 from Arney et al., 2006. 

Y 

Data not available. 

melons in NC are produced in the eastern or south central part of the 

state, where the soils have a sandy or sandy loam texture. 

Most US melon, especially honeydew, production is located in the 

more arid regions of the country in Arizona and California (U.S. 

Department of Agriculture, 2005). The irregular and often heavy 

rainfalls during the production season in NC and the southeastern US 

often cause the honeydew fruit to split (Elmstrom and Maynard, 1992). 

In recent years, honeydew production area has increased in NC. The 

development of new hybrids that have better disease and splitting 

resistance has contributed to this increase (Jester, 2004). 

To a much lesser extent, NC also produces other melon types— 

crenshaw, casaba, galia, juan canary, and piel de sapo. Most of these 

melons have limited markets, which need to be developed for more 

sales to occur. The oriental, crisp-flesh melon, cultivar Sprite, is an 

exception. The ‘Sprite’ melon was researched and marketed through 


the Specialty Crops Program at NC State University (Schultheis et al., 

2001; Jester and Schultheis, 2004). ‘Sprite’ has many positive 

attributes including crisp flesh, unique flavor, high soluble solids (up 

to 18), and small size (0.6 to 0.8kg)—about one-third to one-fourth the 

size of most marketed muskmelon fruit (2 to 3kg) or honeydew fruit 

(2.5 to 3.5 kg). Production practices for ‘Sprite’, such as spacing 

(Jester and Taylor, 2005) and time of harvest, were determined by 

researchers at North Carolina State University. On-farm testing was 

conducted on small acreages with several growers in collaboration 

with extension personnel, and marketing was conducted by the North 

Carolina Department of Agriculture and Consumer Services in 

collaboration with NC State University faculty and NC growers 

(Schultheis et al., 2001). Through these collective efforts, a new 

product was developed with its own price look-up number (PLU#) and 

specifications. Other program melons are currently under 

development in the Specialty Crops Program. More production of 

specialty and honeydew melons in NC and along the east coast is 

likely as increasing transportation costs continue to drive up the price 

of melons from western states. 

Summary 

North Carolina is a leading producer of cucurbits in the US. 

Cucurbits contribute nearly $60 million to the state’s agricultural 

industry. North Carolina is the second leading state producer of 

pickling cucumber in the US, fourth in muskmelon production, and 

seventh in watermelon production. In addition to growing the 

commonly grown cucurbit crops, NC is a leader in the development of 

new and improved cucurbit crops for production and market. 


Arney, M., S. R. Fore, and R. Brancucci. 2006. Watermelon reference book. 

National Watermelon Promotion Board. 80pp. 

Elmstrom, G. W. and D. N. Maynard. 1992. Exotic melons for commercial 

production in humid regions. Second Int. Symp. Specialty & Exotic Vegetable 

Crops. 318:117–124. 

Jester, B. and J. Schultheis. 2004. The commercialization of an oriental crisp flesh 

‘Sprite’ through the NC specialty crops program. HortSci. 39(3)660(Abstr.). 

Jester, B. 2004. Honeydew melon: a new niche for melon growers in the Southeast. 

In: G. Holmes (ed.). North Carolina vegetable growers association 2004 

yearbook. 6:35(Abstr.). 

Jester, W. R. and B. Taylor. 2005. The effect of in-row transplant spacing on sprite 

melon yield and sizes. HortSci. 40(3):887(Abstr.). 

Motes, J. E. 1977. Picking cucumbers production harvesting. Coop. Ext. Serv. 

Mich. St. Univ. Printing. Ext. Bull. E-837. 8pp. 


North Carolina Department of Agriculture and Consumer Sciences. 2004. 

Agricultural Statistics Division. 130pp. . 

North Carolina Department of Agriculture and Consumer Sciences. 2005. 

Marketing North Carolina sweetpotatoes including Louisiana, 2003–2004 crop. 

North Carolina Dept. Agric. & Consumer Services. 28pp. 

Perkins-Veazie, P., J. K. Collins, S. D. Pair, and W. Roberts. 2001. Lycopene 

content differs among red-fleshed watermelon cultivars. J. Sci. Food Agric. 

81:1–5. 

Robinson, R. W. and D. S. Decker-Walters. 1997. Cucurbits. CAB Intl., New 

York. 

Rubatzky, V. E. and M. Yamaguchi. 1997. World vegetables. 2 nd ed. Chapman & 

Hall, New York. 

Schultheis, J. R., C. W. Averre, M. D. Boyette, E. A. Estes, G. J. Holmes, D. W. 

Monks, and K. A. Sorensen. 1998. Commercial production of pickling and 

slicing cucumbers in North Carolina. NC Coop. Ext. Serv. AG-552. 

Schultheis, J. R., W. R. Jester, and N. J. Augostini. 2001. Screening melons for 

adaptability in North Carolina, p. 439–444. In: J. Janick and A. Whipkey (eds.). 

Trends in new crops and new uses. Proc. 5th Nat. Symp. new crops & new uses 

strength in diversity. ASHS Press, Alexandria, VA. 

Sies, H. and W. Stahl. 1998. Lycopene: antioxidant and biological effects and its 

bioavailability in the human. Proc. Exp. Biol. Med. 218:121–124. 

Taylor, M. J. and J. Brant. 2002. Trends in world cucurbit production 1991 to 2001, 

p. 373–379. In: Cucurbitaceae 2002. Amer. Soc. Hort. Sci. 

North Carolina Department of Agriculture and Consumer Sciences Web site. 

(accessed June 2006). 

U.S. Department of Agriculture, Economic Research Service. 2005. Vegetables and 

melons situation and outlook yearbook/VGS-2005. 

U.S. Department of Agriculture. 2002. 2002 Census of Agriculture. National 

Agriculture Statistics Service. 


U.S. Department of Agriculture. 2005. Vegetables 2004 summary. National 

Agriculture Statistics Service. 71 pp. 

 



APPROACHES TO MINIMIZE THE DEFECTS 

OF SEEDLESS WATERMELON 

Xiaowu Sun and Fuqing Luo 

Melons Institute of Hunan Province, Shaoyang, Hunan 422001, 

P. R. China 

ADDITIONAL INDEX WORDS. Polyploid, triploid hybrid, Citrullus lanatus 

ABSTRACT. Triploid seedless watermelon (Citrullus lanatus) is a polyploid F1 

hybrid. Triploid seedless watermelon offers both the benefits of polyploid and 

the heterosis of F1 hybrids. However, it has the defects of low hybrid seed yield, 

low germination, weak and slow seedling development, misshapen fruit, 

variable rind thickness, hard seed coats, and hollow heart. The authors have 

learned, from over 25 years of breeding and seedless-watermelon production, 

that these defects can be reduced through breeding and cultural practices. 

These include using F1 hybrids of similar tetraploid lines as the female parent, 

selecting proper hybrid combinations, improving seed-production protocol, and 

utilizing proper seed treatment and application of fertilizer. With all these 

approaches we have improved seed yield over 80%, seed germination and 

usable plants to over 95%, and fruit yield over 30%, as well as improving the 

stability of marketable yield. 

T 

riploid seedless watermelon is a polyploid hybrid created 

through chromosome engineering. Triploid seedless 

watermelon can offer the benefits of polyploid and the heterosis 

of a properly selected hybrid (An and Sun, 1991; Sun, 1998; Gong 

1993; Yu, 1975). The key benefits of triploid seedless watermelon 

include high quality derived from seedlessness and high sugar content, 

high yields, vigorous plants, tolerance to diseases and humidity, better 

shipping ability, and economic return. However, triploid seedless 

watermelon also has its special defects (Sun, 1998). The main defects 

include low hybrid seed yield, slow development of seedlings, hard 

seed coats in the flesh, hollow heart, thick rind, misshapen fruit, late 

maturity, and the requirement for excellent cultural practices. This 

article outlines the approaches to minimize the defects of seedless 

watermelon, which we learned from our over 25 years of breeding and 

production of triploid seedless watermelon. 

Key Approaches To Minimize The Defects Of 

Seedless Watermelon 

DEVELOPMENT OF DESIRABLE TETRAPLOIDS. Improvement of 

chromosome-doubling techniques has allowed development of many 


tetraploid watermelon varieties (An, 1991; Sun, 1998; Gong, 1993). 

However, availability of desirable tetraploids is limited. We have 

learned from our own and other breeders’ research that the fruit set and 

quality of tetraploids are critical for the fruit set and quality of triploid 

hybrids. Better tetraploids can be selected from the progenies that are 

derived from the crosses between tetraploids. The seed yield of 

tetraploids Beijing No.1, Zhengguo 401, and Shaohuan 4201, 

developed from the crosses of tetraploids, is almost double that of 

Tetra 1, the tetraploid derived directly from chromosome doubling. 

This can be an effective approach to improving the fruit set of yellowfleshed 

and small-fruited tetraploids (Deng and Sun, 2003). Selection 

of tetraploid progenies of chromosome-doubled F1 hybrids may take 

more generations to fix a desirable genotype; however, these kinds of 

populations provide more genetic variation from which to select. 

UTILIZATION OF TETRAPLOID HYBRIDS. Our breeding practices 

have shown that hybrids of similar tetraploid lines can be desirable in 

female parents for overcoming the defects of low seed yield, low 

germination, and low seedling vigor of a triploid hybrid. The seed 

yield of the hybrid between Tetra 452 and 404 is about 50% higher 

than its parents. Improvement of triploid hybrid seed yield over 80% 

was achieved by using tetraploid hybrids of similar lines as the female 

parent (Sun, 1998). 

SELECTION OF DESIRABLE TRIPLOID HYBRID COMBINATIONS. 

Triploid hybrids are sterile (An and Sun, 1991; Gong, 1993; Yu, 1975). 

The proper combination of tetraploid and diploid is critical for the 

performance of the triploid hybrid. Significant hybrid seed-yield 

variation was observed among hybrids of the same tetraploid female 

parent and different diploid male parents (Sun et al., 1998). When 

‘Sugar Baby’, ‘Crimson Sweet’, and ‘Du No. 3’ were used as male 

parents for the same tetraploid female parent, Tetra 404, the average 

number of hybrid seed per fruit was 76, 84, and 63, respectively. We 

also observed significant effects of hybrid combinations on triploid 

seed germination, seedling vigor, hollow heart, rind thickness, size of 

empty seed coats, fruit shape, flesh quality, and fruit yield. 

IMPROVEMENT OF SEED-PRODUCTION TECHNIQUES. Seedproduction 

techniques are not only important for genetic purity, but 

also critical for seed yield, germination, and seed vigor. Fermentation 

of harvested seed in the flesh significantly affects the germination of 

triploid seed. Extended fermentation will kill most of the triploid 

embryos (Sun et al., 1998). Larger seeds in the same seedlot with welldeveloped 

embryos germinate much better than small seeds with lessdeveloped 

embryos. Fruit set and seed yields are improved when the 

tetraploid female parent plants are grafted onto Lagenaria gourd. 


Increased applications of phosphorous fertilizer significantly enhance 

embryo development, and therefore increase seed weight and 

germination. Humidity and temperature during pollination are 

important for triploid seed yield. We usually get better seed yield from 

the spring crop than from the fall crop in Hunan. We get more than a 

20% seed yield increase by properly managing plant density. 

DEVELOPMENT OF SEEDLESS MINIWATERMELON VARIETY. 

Regular seedless watermelons are later than seeded varieties, and are 

not suitable for early production in the spring and late production in 

the fall. We have been breeding seedless watermelon with small fruit 

size since 1995 (Deng and Sun, 2003). We have released over 10 

triploid hybrids with small fruit size, e.g., Snow Peak TM XiaoYuHong 

Seedless. Two to three crops of seedless watermelon can be produced 

a year in subtropical areas with small-fruited seedless watermelon 

varieties. This significantly extends supplies of seedless fruit from the 

concentrated months of July and August to the months of May through 

November (Sun et al., 1997). 

IMPROVEMENT OF SEED-PRODUCTION PROTOCOL. Triploid hybrid 

seeds are produced by using a tetraploid as the female parent and a 

diploid as the male parent in a ratio of 10 female to 1 male (Sun, 1998). 

The female and male parents are usually grown in different sections of 

the seed-production field. Manual emasculation, for some tetraploids 

and/or at some production periods, and controlled hand-pollination are 

the common practices for hybrid triploid-seed production. 

Uncontrolled pollination is also used to reduce the cost of triploid-seed 

production. The tetraploid female and diploid male parents are mixedplanted 

in the same row in the ratio of 2:1 or 4:1. Male flowers of the 

male parent are collected in the early morning, 6 to 8 AM, to pollinate 

the female flowers of the female parent. Seeds are harvested only from 

the fruit of female parent. This kind of production can be done only in 

isolated fields. The tetraploid seeds are removed from the triploid 

seedlot based on seed morphology by experienced workers. The 

tetraploid plants or fruits can also be identified later in commercial 

seedless-watermelon production fields based on seedling morphology 

or fruit characteristics (Sun, 1998). 

We investigated the possibility of producing triploid hybrid seed 

using a diploid as the female parent and a tetraploid as the male parent. 

However, the pollinated fruit produced only a normal-sized hard seed 

coat, with no visible embryo. Zhang also found a similar result; 

however, about 10% of the seeds studied produced triploid plants in 

vitro (X. P. Zhang, personal communication). More investigation is 

needed to understand this phenomenon. It would be very helpful if 


triploid hybrid seed could be produced by using a diploid line as the 

female parent. 

IMPROVEMENT OF CULTURAL PRACTICES OF SEEDLESS- 

WATERMELON PRODUCTION. Cultural practices are very important for 

the success of seedless-watermelon production (Li et al., 1994; Sun, 

1990, 1992, 1998; Sun et al., 1997). We recommend these tips to 

improve germination of triploid hybrid seed: (1) Carefully control the 

temperature and moisture during seed germination. The temperature 

should be set at 32–35˚C during germination. High moisture levels 

will damage the triploid seeds with less-developed embryos. (2) 

Mechanically cut open the hard seed coat of the triploid seed before 

germination. The seed coat should be removed once the seedling has 

emerged from soil. (3) Carefully disinfect the triploid seed using 

fungicides or disinfectant before germination. More than 90% 

germination and 85% usable seedlings have been achieved by using 

these three techniques. 

Cultural practices have significant impact on yield, fruit quality, 

hollow heart, and hard seed coat of seedless watermelon (Li et el., 

1994; Sun, 1990, 1992, 1998; Sun et al., 1997). Stable nutrient and 

water supplies are critical for fruit yield, fruit shape, and fruit quality. 

Drought stress during fruit development reduces fruit size and 

increases rind thickness. Over-fertilization and -irrigation cause 

vegetative overgrowth, delayed fruit set, small fruit size, misshapen 

fruit, hard flesh, and hollow heart. Early-stage mismanagement of 

fertilizer and water are difficult to correct at the late stage. Overuse of 

nitrogen increases rind thickness and reduces sugar content. The hard 

seed-coat defect is low at moderate phosphate levels; higher levels of 

phosphate will increase it. Potassium improves the sweetness of 

seedless watermelon. Fruit quality is also affected by production 

season, plant population, and grafting. 

MICROPROPAGATION OF SEEDLESS WATERMELON USING TISSUE 

CULTURE. The combination of tissue-culture micropropagation and 

grafting can provide a solution to the problems of low seed yield, low 

germination, and low seedling vigor (Deng, 1987; Tang et el., 1994; 

Gao, 1983; Xu, 1979). The shoot tips of in vitro germinated seedlings 

were harvested and cultured on MS media containing 2–5mg/l BA for 

proliferation. Ploidy change and somaclonal variation were not 

observed during the subculture period of 10 months. As many as 

50,000–100,000 plantlets can be obtained from single shoot tip in 6 

months. However, the higher cost of tissue-culture plants and 

relatively low propagation rate prevents the commercial application of 

this technology. More research is needed in this area. 


Conclusion 

With the techniques described above we have been able to 

overcome the problems of low hybrid seed yield, low seed germination, 

and low seedling vigor of triploid seedless watermelon. Hybrid seed 

yields have increased from an average 2kg per 1000m 2 in the 1970s– 

1980s to an average 7kg per 1000m 2 today. Seed germination has been 

enhanced from 60% to 95%, and the usable-seedling rate is 

approaching 95%. We have developed several seedless varieties with 

small fruit size. Seedless plants are more vigorous and more tolerant to 

diseases and unfavorable conditions than seeded varieties, and their 

yields are 30% higher. Seedless watermelon also produces better 

quality fruit with a longer shelf life and better marketability. All these 

qualities make triploid seedless watermelon a strong-growing segment 

of the market. However, more tetraploids with desirable horticultural 

traits and good combining ability are needed. Tetraploid male sterile 

lines can be helpful for hybrid seed production. An improved seedproduction 

protocol is needed to ensure high seed quality and seed 

health. 


An, P. S. and X. W. Sun. 1991. Breeding and utilization of polyploid watermelon. 

Acta Huan Agric. Coll. 17(addition):126. 

Deng, D. C, X. W. Sun, P. Y. Zuo, Y. H Liu, and X. D. Xie. 2003. A new smallsized 

seedless watermelon “Xuefeng Xiaoyuhong”. Acta Hort. Sinica. 30(3):375. 

Deng, Z. P. 1987. Studies on asexual propagation of seedless watermelon and 

grafting. Northern Hort. (2):9–11. 

Gao, X. Y. 1983. Studies on asexual propagation of seedless watermelon. China 

Agric. Sci. (2):5–17. 

Gong, Z. J. 1993. Advances of polyploid watermelon breeding. China Watermelon & 

Melon. (3):22-23. 

Li, Z. S., C. H. Ling, and H. L. Gao. 1994. Cultural practices affect quality of 

seedless watermelon. China Watermelon & Melon. (1):16-18. 

Sun, X. W. 1990. Growing seedless watermelon for high quality. China Watermelon 

& Melon. (1):39–41. 

Sun, X. W. 1992. Cultural management of seedless watermelon. Hunan Agric. (4):14. 

Sun, X. W., F. Q. Luo, and X. P. Muo. 1997. Cultivation models for seedless 

watermelon production, p 54–59. Melon & Watermelon Working Group of 

CSHS (ed.). Advances of China watermelon and melon research. China 

Agriculture Press. Beijing. 

Sun, X. W. 1998. Studies on cultivation model of seedless watermelons, p. 388. Proc. 

25 th Int. Hort. Congress (IHC). 2–7 August, 1998, Brussels. 

Sun, X. W., F. Q. Luo, and S. Y. Tan. 1998. Solutions to the defects of seedless 

watermelon, p. 229–232. In H. Y. Luo and X. W. Sun (eds.). Proc. Hort. Sci. 

Hunan Sci. & Tech. Press. Hunan. 

Tang, S. H., Y. Liao, and Y. C. Xu. 1994. Screening of tissue culture medium and 

micropropagation of seedless watermelon. Acta Southwest Agric. Univ. Sinica. 

16(6):540–542. 

Xu, Z. H. 1979. Propagation of triploid seedless watermelon in vitro. Acta Plant 

Physiol. Sinica. 15(3):245–251. 

Yu, Z. X. 1975. Achievements of watermelon breeding in Taiwan. Agric. Sci. 

23.(3/4):123–131. 


COST BENEFIT ANALYSES OF USING 

GRAFTED WATERMELONS FOR DISEASE 

CONTROL AND THE FRESH-CUT MARKET 

Merritt Taylor 1 , Benny Bruton 2 , Wayne Fish 2, and Warren 

Roberts 1 

1 Oklahoma State University, Wes Watkins Agricultural Research and 

Extension Center, Lane, OK 

2 USDA/ARS, South Central Agricultural Research Laboratory, 

Lane, OK 

ADDITIONAL INDEX WORDS. Risk management, Fusarium wilt, soil-borne, 

pathogens, Cucurbita sp., Lagenaria sp. 

ABSTRACT. Soil-borne diseases such as Fusarium wilt continue to plague 

watermelon growers in intensive-production areas where land resources are 

scarce and rotation of various crops is limited. Risk-management alternatives 

available to the farmer have been reduced by the loss of soil-fumigation 

chemicals such as methyl bromide. Grafting of watermelon onto resistant 

rootstock onto certain scions has been found to provide effective resistance to 

Fusarium wilt but at an increased cost of production. There is potential for 

these grafted plants to have an increased demand by the cut-fruit industry, in 

the long run, due to the superior quality of the flesh. Fruit from certain 

scion/rootstocks may even bring a premium from the cut-fruit industry as they 

are recognized for their superior shelf life and firmness of flesh. The resistance 

of these plants to soil-borne diseases provides the farmer a viable riskmanagement 

strategy as an alternative to methyl bromide as a means of disease 

control. 

G 

rafting watermelon onto other Cucurbitaceous crops for soilborne 

disease and nematode control has been practiced for 

many years (Oda, 1999). Because of limited ability for crop 

rotation, the practice of grafting has provided a useful method for 

growing watermelons on land that would otherwise require fumigation 

or be abandoned. Grafting has been routinely utilized in Japan and 

Korea since the late 1920s for the control of Fusarium wilt, caused by 

Fusarium oxysporum f. sp. niveum (Lee, 1994). Grafting watermelons 

in the United States had not been attempted because of sufficient land 

for rotation, availability of methyl bromide, and increased costs of 

grafted transplants. However, we have experienced in recent years 

constraints of new land for rotation, loss of methyl bromide, and an 

increased incidence and severity of soil-borne diseases (Bruton, 1998). 


Evolution of Grafting Watermelons 

Historically, Cucurbita, Benincasa, Lagenaria spp., and some 

hybrid rootstocks have been utilized for grafting watermelon. 

However, these rootstocks are not resistant to all diseases. In Spain, 

Armengol et al. (2000) noted that Fusarium solani f. sp. cucurbitae 

Race 1 could be particularly damaging to the rootstock of grafted 

watermelon. Bottle gourd (Lagenaria siceraria) is often used as 

rootstock for watermelon and is susceptible to the seed-borne 

(Kuniyasu, 1981) fungus F. oxysporum f. sp. lagenariae (Matsuo and 

Yamamoto, 1967). Cucurbit yellow vine disease (CYVD), caused by 

Serratia marcescens, causes a vine decline of watermelon, squash, and 

pumpkin (Bruton et al., 2003). More recently, CYVD has been 

observed in watermelon grafted onto Lagenaria or Cucurbita sp. 

rootstocks (B. Bruton, 2006, unpublished data). 

More than 95% of the watermelon in Japan, Korea, and Taiwan is 

being grafted onto squash and gourd rootstocks (Lee et al., 1998). 

However, rootstock-scion selection has a profound effect on yield and 

fruit-quality attributes (Huh et al., 2003; Pulgar et al., 2000; Yetisir 

and Sari, 2003). When squash (Cucurbita sp.) was used as the 

rootstock, the yield and/or quality of the fruit was often inferior to fruit 

of plants grafted on bottle gourd (Lagenaria siceraria) or of 

nongrafted watermelon (Yetisir et al., 2003). 

Despite some advantages, the use of methyl bromide has been 

associated with major problems, including the depletion of the ozone 

layer (Montreal Protocol, 1987). Because of this, its use was scheduled 

to be phased out on a worldwide scale—by the end of 2005 in the E.U. 

and other developed countries, including the United States, and by 

2015 in the developing countries (Rowlands, 1993). Therefore, there is 

an urgent need to define and implement alternative solutions for 

managing soil-borne pathogens. Grafting is one such alternative 

practice that has potential for cucurbits. 

Benefits of Grafting Other Than Disease Control 

Ioannou et al. (2000) stated, “Although growing grafted 

watermelon represents a novel horticultural practice for Cyprus, the 

technique has been quickly and widely adopted by growers. Presently, 

over 80% of watermelons grown in the open field and under low 

tunnels are grafted on various rootstocks. Grafted plants are produced 

by fully equipped commercial nurseries. Research efforts have been 

directed towards the utilization of wilt-resistant rootstocks for offseason 

watermelon production in heated greenhouses. Although there 

are still many technical aspects that need further investigation, results 


so far are quite promising since this method enables watermelon 

production as early as March, when prices are very high.” They also 

stated, “The yield of grafted Crimson Sweet watermelon reached a 

record level of 150 tons/ha.” 

The use of grafted watermelon as a technique to prevent loss due to 

diseases is widespread throughout the world, but other positive aspects 

may be equally important to the producer/decision maker regarding the 

potential to harvest watermelons during a market window where high 

prices exist that were previously unreachable (Ioannou et al., 2000). 

In Greece, grafting is highly popular for watermelon and melons, 

especially in the southern areas, where early cropping under low 

tunnels is practiced (Traka-Mavrona et. al, 2000). 

Findings at Lane, Oklahoma 

Researchers at Lane, Oklahoma, completed two years of 

experiments that included tests of five watermelon (Citrullus lanatus) 

scions on four rootstocks of squash (Cucurbita sp.)or gourd 

(Lagenaria sp.) to test for resistance to Fusarium wilt in fields where 

heavy disease pressure would make production of nongrafted cultivars 

impractical. 

Treatments consisted of watermelon cultivars SF800, SS5244, 

SS7167, SS7177, and SS7187 from Abbott & Cobb Seed Co., grown 

as nongrafted plants or grafted onto rootstocks of RS1330, RS1332, 

RS1420, or RS1421. The grafting was done by Alamo Transplants Inc. 

of Alamo, Texas. Additional controls consisted of the nongrafted 

cultivars ‘Sangria’, ‘Royal Sweet’, ‘Jubilee’, and ‘Jamboree’. Two 

fields were planted each year with three replications of each treatment 

per field. Plants were grown with 1-m spacing between plants, with 

rows 3m apart. 

There were no grafted plants lost to Fusarium wilt. However, the 

other cultivars, especially ‘Jubilee’, did exhibit varying degrees of wilt 

incidence. Yields of grafted plants were generally equal to or greater 

than those of the nongrafted plants. Tests were run on firmness, 

lycopene content, sugar, storability, etc. Sugar content, measured as 

soluble solids, was affected minimally, if any, by grafting. Lycopene 

content of fruit from grafted plants was equal to, or marginally better 

than, fruit from nongrafted plants. Fruit firmness, as measured by a 

penetrometer, was significantly greater in the grafted fruit than in the 

nongrafted fruit. The firmest fruit occurred with SS7167 and SS7187 

scions grafted onto RS1420 rootstock, which had a value of about 2.0 

x 10 5 Pa. Fruit from nongrafted plants had values of about 1.0 x 10 5 Pa 


or less. Matching of scions with appropriate rootstocks was important, 

as interactions did occur. Certain combinations were significantly 

superior to other combinations. 

Fresh-Cut Market Potential 

According to the International Fresh-Cut Produce Association, 

fresh-cut fruits and vegetables make up one of the fastest-growing 

food categories in U.S. supermarkets. U.S. sales of fresh-cut produce 

sprang from $3.3 billion in 1994 to $11 billion in 2000, and are 

projected to reach $15 billion in 2005 (Peabody, 2005). On average, 

sales of fresh-cut products increased by 8% in 2003 compared to 2002. 

Of the three fresh-cut categories, fresh-cut fruits had the largest 

increase at 15%, followed by packaged-salad sales at 9%, and freshcut 

vegetables at 6% (Clement, 2003). 

The market for fresh-cut fruit continues to grow at a rapid rate. 

Sanitation and microbial contamination are of particular concern due 

to the high water/sugar content of watermelon fruit. Because of 

microbial concerns and product deterioration, the shelf life of fresh-cut 

watermelon is generally considered by the industry to be about five to 

seven days. However, product safety is the major concern. 

Several minimum quality criteria appear to be required for 

watermelon to be acceptable for sale as a fresh-cut product. The flesh 

must be firm, with a minimal juice loss, and have the “crunch” of a 

freshly cut piece of watermelon. It should have a soluble solids 

measurement of Brix greater than 10%. It should be seedless. It 

should have a positive visual perception of red and be high in 

lycopene. It should have the taste and aroma of a freshly cut 

watermelon. It should be safe, with minimal levels of fungal and 

bacterial contaminants. 

In our studies at Lane, Oklahoma, the shelf life and overall quality 

of fresh-cut watermelon from two cultivars grafted onto one of four 

rootstocks were compared with fresh-cut fruit from the nongrafted 

cultivars. Fresh-cut cubic pieces of about 4.5cm per side were prepared 

from ripe watermelons grown at the Lane Research Center and were 

stored at 5 o C in 35-oz. PETE containers. Quality attributes of firmness, 

soluble solids content, lycopene content, and bacterial counts of the 

pieces were measured after 0, 5, and 10 days of storage. Sugar content 

of the cut fruit was independent of rootstock and remained constant 

over the 10 days of storage. Lycopene content of the fruit decreased by 

5–10% during the storage period, regardless of treatment. Bacterial 

count on the fruit from all treatments remained low and variable 

during the 10 days at 5 o C. Firmness of cut pieces from fruit originating 


from the grafted plants was dependent upon the rootstock employed. 

Watermelons from grafted plants possessed firmer flesh than did those 

from the nongrafted plants. Overall, the firmness of fruit from all 

sources decreased 20–30% during the 10 days of cold storage. 

However, the firmness of fruit from some of the rootstocks after 10 

days of storage was equal to or significantly higher than that of the 

fruit from nongrafted plants when it was initially cut. Thus, these 

studies suggest that grafting to a proper rootstock will produce freshcut 

watermelon that is equal in sweetness and lycopene content to its 

nongrafted counterpart, but that will maintain greater crispness 

throughout its storage on the supermarket shelf. 

A separate study currently underway of fresh-cut watermelon 

being sold in several retail locations in Oklahoma and Texas provided 

the following preliminary general information: Brix ranged from 6.2 to 

13.2, with the average being 9.6. Firmness ranged from 0.4x10 5 Pa to 

3.1x10 5 Pa, with an average of 1.4x10 5 Pa. Lycopene ranged from 

22.6µg/g to 79.8µg/g, with an average of 44.3µg/g. The cost of this 

fresh-cut watermelon ranged from a low of $1.17/lb ($2.58/kg) to a 

high of $12.00/lb ($26.46/kg), and an average cost to the consumer of 

$3.13/lb ($6.90/kg). Assuming the average cost of whole watermelon 

to be $0.11/lb ($0.24/kg) and a 50% recovery percentage, the actual 

cost of the watermelon in the package would be approximately 

$0.22/lb ($0.485/kg) or 19% of the total cost of the package at the lowcost 

package, 1.8% of the high-cost package, and approximately 7% of 

the total cost of the average package. 

What does this mean to the grower? The discovery by the Lane, 

Oklahoma, scientists that some of the grafted watermelon 

scion/rootstock combinations provided superior firmness at the end of 

10 days shelf life compared to the firmness of the nongrafted 

watermelons at the beginning of the test has huge ramifications for the 

potential of these watermelons in the cut-fruit industry. Although 

industry officials state that maintaining quality and firmness during 

shelf life is one of the critical issues for maintaining or increasing 

market share, the question arises regarding the willingness of the 

industry to pay the farmer a premium price for a superior-quality 

product. The cost per acre of growing grafted watermelons is greater 

than that for nongrafted. 

We estimate that the cost to purchase a grafted seedling plant from 

a seedling supplier currently would be $0.75, which would include the 

cost of the seed and the grafting operation. Nongrafted seedless plants 

cost approximately $0.28 per plant. Assuming 1,500 plants per acre 

(3,706 per hectare), grafted seedless transplants would cost 

approximately $705.00 per acre ($1,743 per hectare) more than the 


nongrafted plants (Table 1). Since methyl bromide usage is not an 

option in the United States, comparing the costs between planting 

grafted plants versus fumigating with methyl bromide and planting 

conventional transplants is a moot point for US growers, but should be 

of interest to international growers. The following costs of production 

are estimated with the single variable being whether grafted or 

nongrafted seedless watermelons are planted. All other costs are held 

constant. 

Table 1. Comparative cost in $US of production per acre and per 

hectare. 

Nongrafted Grafted Nongrafted Grafted 

seedless/acre seedless/acre seedless/ha seedless/ha 

Preplant 

Growing 

250 250 618 618 

season 842 1,546 2,080 3,823 

Total 1,092 1,797 2,698 4,441 

Difference $705 per acre $1,743 per hectare 

Assuming variable yields and prices per acre (per hectare), the 

following sensitivity analyses were developed to evaluate the breakeven 

yield, or price required for the farmer to consider planting grafted 

seedless watermelons versus nongrafted seedless watermelons. 

Calculations (Table 2) indicate that a farmer producing 25,000 

pounds per acre (28,021 kg/ha) would be breaking even at $0.08/lb 

($0.1764/kg) with nongrafted transplants but would need $0.11/lb 

($0.2425/kg) to break even with the grafted transplants (Table 3). In 

the calculations of the fresh-cut products above, if the fresh-cut facility 

paid the farmer $0.08/lb ($0.1764/kg) for a 50% recovery on 

traditional seedless watermelon and sold the product at the average 

retail price of $3.13/lb ($6.90/kg), the actual watermelon in the 

package would cost $0.16/lb ($0.35/kg) or 5.11% of the average retail 

price. Alternatively, if the grafted watermelon, with superior 

crunchiness/firmness and shelf life, were purchased at a $0.02/lb 

($0.044/kg) premium ($0.10/lb or $0.22/kg), the 50% recovery 

watermelon would cost the processor $0.20/lb ($0.485/kg) or 6.38% of 

the average retail price. The question is thus raised whether the freshcut 

processor would be willing to assure the farmer of a premium price 

of $0.02–$0.03/lb ($0.044–$0.066/kg) higher than the spot market 

price for a contract to provide a superior product to their customers. 


Table 2. Expected net return per hectare for nongrafted seedless 

watermelon at different yields and prices. 

$2,698 production 

cost per hectare 

$US price per kg 

$0.0661 cost/kg 

harvest, pack, and 

ship 

Yield 

kg/ha $0.1323 $0.1543 $0.1764 $0.1984 $0.2205 $0.2425 

11,209 -1,956.68 -1,709.57 -1,462.47 -1,215.36 -968.25 -721.15 

16,813 -1,586.01 -1,215.36 -844.70 -474.04 -103.38 267.28 

22,417 -1,215.35 -721.14 -226.93 267.28 761.49 1,255.70 

28,021 -844.69 -226.93 390.84 1,008.60 1,626.36 2,244.13 

33,626 -474.03 267.29 1,008.60 1,749.92 2,491.24 3,232.55 

39,230 -103.37 761.50 1,626.37 2,491.24 3,356.11 4,220.98 

44,834 267.30 1,255.72 2,244.14 3,232.56 4,220.98 5,209.40 

50,438 637.96 1,749.93 2,861.91 3,973.88 5,085.85 6,197.83 

56,043 1,008.62 2,244.15 3,479.67 4,715.20 5,950.73 7,186.25 

Risk Management and Disease Control 

A more practical question facing a farmer is the potential of losing all 

or part of the crop after the entire costs of production have been 

expended. In most cases of an outbreak of Fusarium wilt, the plants 

begin to decline late in the production season after virtually all 

production costs have been spent. This is one of the conditions 

frequently faced by a farmer when symptoms of Fusarium wilt are 

observed. Returning to the sensitivity tables, let us now assume a 

current market price of $0.10/lb ($0.22/kg) and an average yield of 

30,000lbs per acre (33,626 kg/ha). Under the two scenarios, the farmer 

would make a profit of $303/acre ($748/ha) with the grafted plants 

(Table 3) and $1,008/acre ($2,491/ha) with the nongrafted plants 

(Table 2) when no Fusarium wilt occurred. Now assume conditions of 

heavy Fusarium wilt disease in the field such that 50% of the 

nongrafted production is lost before harvest. In the grafted-watermelon 

fields, where the plants are resistant to Fusarium wilt, the farmer could 

expect a yield of 30,000lbs per acre (33,626kg/ha) and obtain 

approximately $303 per acre ($748/ha) profit (Table 3). In the 


nongrafted field, the farmer may be lucky to harvest 15,000lbs per acre 

(16,812 kg/ha) and thus show a net loss of -$42.00 per acre 

(-$103.00/ha) (Table 2). As disease pressures increase over time, 

grafted disease-resistant plants, such as the ones tested at Lane, will 

become part of risk-management decisions for the farmer. 

Table 3. Expected net return per hectare for grafted seedless 

watermelon at different yields and prices. 

$4,440.00 production 

cost per hectare 

$0.0661 cost/kg harvest, 

pack, and ship 

$US price per kg 

Yield kg/ha $0.1323 $0.1543 $0.1764 $0.1984 $0.2205 $0.2425 $0.2646 

11,209 -3,698.68 -3,451.57 -3,204.47 -2,957.36 -2,710.25 -2,463.15 -2,216.04 

16,813 -3,328.01 -2,957.36 -2,586.70 -2,216.04 -1,845.38 -1,474.72 -1,104.07 

22,417 -2,957.35 -2,463.14 -1,968.93 -1,474.72 -980.51 -486.30 7.91 

28,021 -2,586.69 -1,968.93 -1,351.16 -733.40 -115.64 502.13 1,119.89 

33,626 -2,216.03 -1,474.71 -733.40 7.92 749.24 1,490.55 2,231.87 

39,230 -1,845.37 -980.50 -115.63 749.24 1,614.11 2,478.98 3,343.85 

44,834 -1,474.70 -486.28 502.14 1,490.56 2,478.98 3,467.40 4,455.82 

50,438 -1,104.04 7.93 1,119.91 2,231.88 3,343.85 4,455.83 5,567.80 

56,043 -733.38 502.15 1,737.67 2,973.20 4,208.73 5,444.25 6,679.78 


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host resistance, p. 143–166. In: J. D. McCreight (ed.). Cucurbitaceae ’98. ASHS 

Press, Alexandria, VA. 

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Brady, B. Bextine, and T. H. Popham. 2003. Serratia marcescens, a phloemcolonizing, 

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characteristics of watermelon grafted onto Citrullus rootstocks selected for 

disease resistance. J. Kor. Soc. Hort. Sci. 44:649–654. 

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ammonium fertilizers. EPPO Bull. 30:223–230. 


Kuniyasu, K. 1981. Seed transmission of Fusarium wilt of bottle gourd, Lagenaria 

siceraria, used as rootstock of watermelon. Jpn. Ag. Res. Quart. 14:157–162. 

Lee, J. M. 1994. Cultivation of grafted vegetables. I. current status, grafting 

methods and benefits. HortSci. 29:235–239. 

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Hort. Sci. 67:1098–1104. 

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63. 

Montreal Protocol on Substances that Deplete the Ozone Layer. 1987. International 

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Secretariat, UNEP, 2000. xv, 367p. 

Oda, M. 1999. Grafting of vegetables to improve greenhouse production. Ext. Bull. 

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nutrition in grafted watermelon plants: nitrogen metabolism. Biol. Plant. 

43:607–609. 

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(Cucurbita spp.) as rootstock for melon (Cucumis melo L.). Sci. Hort. 83:353– 

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and quality of watermelon. Aust. J. Exp. Agri. 43:1269–1274. 

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NITAMIN ® LIQUID: BACKGROUND AND USE 

ON CUCURBITACEAE FAMILY 

James Wargo and Anne Cothran 

Georgia-Pacific Resins, Inc., Decatur, Georgia 

ADDITIONAL INDEX WORDS. Citrullus lanatus, Cucumis sativus, cucumber, 

watermelon, ammonium nitrate, urea, fertilizer, slow-release nitrogen 

ABSTRACT. Nitamin ® is a slowly available nitrogen source developed by 

Georgia-Pacific that contains 60% slowly available nitrogen (AAPFCO, Rules 

and Regulations-Fertilizer, 3 (b), 2004). The patented urea polymer-based 

fertilizer is broken down by soil microbes and releases nitrogen over 60–75 

days. The release profile is dependent on soil texture, temperature, and 

moisture. Nitamin 30L demonstrates increased nitrogen use efficiency (NUE) 

compared to quick-release fertilizers by reducing nitrogen loss via ammonia 

volatilization and leaching. In 2005, Nitamin 30L liquid fertilizer demonstrated 

increased yields in three studies conducted on cucurbits on a range of soil types. 

Split applications were required with watermelon on sand in FL, but single 

applications prior to planting were sufficient in trials on watermelon in TX and 

pickling cucumbers in NC. The higher value created through increased yield 

was demonstrated to have positive economic results and suggests that slowly 

available fertilizers such as Nitamin are feasible for moderate to high value 

vegetable production. 

itamin ® N 

30L is a 30-0-0 liquid N fertilizer with slow-release 

properties. It is composed of 40% urea and 60% slowly 

available nitrogen in the form of polymeric chains of 

methylene urea and various triazone species (closed ring structures). 

The combination of readily available N and slow-release N provides a 

steady N release over a period of 60 to 75 days. Nitamin polymers are 

converted from organic nitrogen to plant-available forms through 

microbial degradation. This process is dependent on soil temperature, 

texture, water availability, and oxygen content—all of which can 

affect the N-release profile. The breakdown begins with the simplest 

molecules (urea), then methylene urea compounds, and eventually the 

triazone ring structures with strong carbon-to-nitrogen bonds. Once 

The universities listed as conducting studies on Nitamin ® do not 

endorse Georgia-Pacific, Nitamin, or the use of Nitamin. Costs given are 

not guaranteed prices but estimates on current raw material costs available. 

Nitamin ® is a registered trademark and STEADY-DELIVERY is a 

trademark of Georgia-Pacific Resins, Inc. All rights reserved. U.S. patent 

nos. 6,632,262; and 6,900,162. Additional domestic and international 

patents pending. 


the monomers are cleaved from the chain or ring structures 

they undergo normal N-transformation processes in the soil to 

convert to nitrate. 

The steady release of N can provide several agronomic 

and environmental advantages over quick-release N fertilizers. 

University studies with Nitamin have shown more consistent 

plant nitrogen status during the growing season, higher 

cumulative N uptake, and reduced ammonia volatilization 

(Figure 1) (Miguel Cabrera, unpublished data) and reduced 

nitrate leaching (Figure 2) (Elizabeth Guertal, unpublished 

data). All these factors combine to result in higher N use 

efficiency. Other potential benefits include the need for less 

frequent N applications during the growing season and 

reduced N application rates compared to the standard grower 

program. In many cases improved yield and fruit quality have 

been achieved by limiting fluctuations in soil N status and 

increasing N availability to the plants. 

Studies on watermelon and cucumbers in TX, FL, and NC showed 

that Nitamin could supplant the current N fertilization practice and 

improve fruit yield and quality. The results of those trials are 

discussed below. 


FLORIDA STUDY. A watermelon trial was conducted by Dr. George 

Hochmuth with the University of Florida at the IFAS North Florida 

Research Center in Live Oak, FL (George Hochmuth, unpublished 

data). The soil was Lakeland sand (96% sand, 0% silt, and 4% clay) 

with a pH of 6.0, and organic matter less than 1%. Beds were formed 

with black plastic mulch and fumigated with methyl bromide: 

chloropicrin at 449kg/ha. The test area was drip irrigated to maintain a 

soil moisture of -8cb to -12cb measured by a tensiometer at 15.2cm 

soil depth. The plots were irrigated for periods of 15 min twice/day 

from planting until 10 May, 30 min twice/day from 10 May to 26 May, 

and 38 min twice/day through harvest. Transplants (variety 

‘Mardigras’) were planted on March 10, 2005, in a single row with 

0.91m spacing. The experimental design was randomized complete 

block with four replicates in 2.4m x 10.7m plots. One block was 

eliminated from the results due to poor plant stand caused by poor 

water drainage. 

The treatments included ammonium nitrate and Nitamin 30L 

applied at different rates and times. Nitrogen rates were 117, 168, and 


Urea 

UAN 

Nitamin 30L 

Fig. 1. Ammonia volatilization results from UGA incubation. Study conducted 

by Dr. Miguel Cabrera, University of Georgia, 2005. Data was analyzed weekly 

for significant difference by Fisher LSD (0.05) by week 1) 3.2, 2) 0.3, 3) 0.2 NS 

4) 0.05 NS, 5) 0.02 NS. 

211kg N/ha for Nitamin and 168 and 211kg N/ha for ammonium 

nitrate. Ammonium nitrate was applied through weekly fertigation for 

12 weeks with 25% of the N applied to the bed preplant. All three 

Nitamin rates were applied to the bed prior to planting and 

incorporated before plastic mulch was laid. An additional Nitamin 

30L treatment was injected through the irrigation system in three 

applications spaced 2 weeks apart beginning 3 weeks after planting. 

The total rate for the injected treatment was 168kg N/ha with 25% of 

the N total applied to the bed before planting. 

TEXAS STUDY. A second watermelon study with Nitamin 30L 

liquid fertilizer was conducted by Russ Wallace at the Texas 

Agricultural Research & Extension Center (AREC) in Lubbock, 

Texas, in 2005 (Russ Wallace, unpublished data). The soil was an 

Amarillo clay loam (47% sand, 20% silt, and 33% clay) with 0.9% 

OM, pH 8.1, and a CEC of 16.5. Core samples were taken 

pretreatment from 15.2cm–20.3cm depth and showed 9kg NO3/ha 

were available in April 2005. The test plots were treated with Prefar 

4E herbicide and other standard practices were used for disease and 

pest control in addition to weeding by hand. Plot size was 5.1m x 

7.62m with four replicates in a randomized complete block design. 

The plots were transplanted with 3-week old watermelon seedlings 

(cv. Sugar Baby) on June 9 and drip irrigated as needed. Harvest 

occurred once, on August 9, 2005. Other variables measured were 

vigor, rated on July 10 and August 4, and vine length, rated on June 

28. 


Urea 

UAN 

Nitamin Liquid 

Fig. 2. Auburn University leaching results. Cumulative mineral 

nitrogen (mg) in leachate as affected by fertilizer. Study conducted by 

Dr. Elizabeth Guertal, Auburn University, 2005. 

The treatments included urea broadcast preplant and incorporated 

(PP) at rates of 89 and 135kg N/ha; Nitamin 30L applied all at planting 

(AP) or as split applications (S) at planting and just before vine run on 

June 27 at rates of 45, 89, and 135kg N/ha. Nitamin was applied to 

open trenches 10cm deep and 10cm to the side of each row with a 

pressurized backpack sprayer. 

NORTH CAROLINA STUDY. The third cucurbit study was a trial 

conducted by Dr. Jonathan Schultheis, North Carolina State 

University, on cucumber cultivar ‘Jackson’ at Kinston, NC (Jonathan 

R. Schultheis and Bradfred W. Thompson, unpublished data). The soil 

classification is Norfolk loamy sand (85% sand, 10% silt, and 8% 

clay) with 1.3% organic matter and pH 6.5. The plots were 25’ X 3.5’ 

having 10’ alleyways between plots. Each treatment consisted of five 

replicates in a randomized complete block design. Plots were thinned 

to a target plant stand of 50 plants per plot in a single row. The seeds 

were hand-sown on April 26, 2005, and thinned on May 20 and June 

15. Weed control was applied after planting, while insecticides and 

fungicides were applied as needed. Irrigation was supplemented as 

necessary to maintain 1 inch per week, and no excessive rain events 

occurred. Measurements included: vine length, petiole nitrate by 

Cardy meter, and graded yield. Vine length was measured on June 10. 

Twenty petiole samples from each plot were collected June 10 at the 

fruit bloom, June16 at first harvest, and June 29 after the fourth 

harvest. The cucumbers were harvested eight times (biweekly) from 

June 16 through July 8, 2005. The yield was graded into US No. 1, 

No. 2, No. 3, and No. 4, and marketable yield included No. 1, 2, and 3 

(all noncull fruit). Some of the plots were infected with powdery 

mildew, bacterial wilt, or poor plant stand, resulting in poor yields. 

These plots were excluded from the yield calculations. 


The treatments included ammonium nitrate and Nitamin 30L 

applied at rates of 45, 89, 135, and 180kg N/ha. An untreated control 

was also included. Recommended rates are 89 to 135kg/ha for 

pickling cucumbers in NC. Nitamin treatments were applied once just 

prior to planting on April 26, 2005. The ammonium nitrate treatments 

were applied in split applications in 45-kg increments except the 45-kg 

rate, which was applied 31 days after planting at the third-leaf-growth 

stage on May 26, 2005. The 89kg and 135kg rates were applied at the 

first-true-leaf stage (May 19) and again on May 31. Part of the 135 

rate was applied at planting. Applications of the 180-kg rate started at 

planting and continued (fourth application) until “vine out” growth 

stage on June 7, 2005. The ammonium nitrate treatment schedule was 

intended to mimic a typical grower application program. 

Results 

FLORIDA STUDY. Nitamin produced the highest yield of any 

treatment when 75% of the N was injected through the irrigation 

system biweekly during the first half of the growing season (Table 1). 

Yield increases with the injected Nitamin treatment over the two 

ammonium nitrate treatments ranged from 60 to 86cwt(kg)/ha. The 

injected Nitamin treatment also tended to have fewer cull fruit than 

ammonium nitrate and all at-planting Nitamin treatments. The 

Nitamin treatments that consisted of a one-time preplant application 

underneath the plastic produced the lowest yields regardless of N rate. 

These treatments produced higher yields in the first harvest, but had a 

much lower second-harvest yield, suggesting insufficient nitrogen 

availability late in the growing season. The results demonstrate that 

split N applications are necessary on sand with low organic matter and 

low cation-exchange capacity. However, it was evident that a 

Table 1. University of FL Watermelon Trial, Live Oak. 

Total-season yield 

(metric tons/ha) 

Treatment Marketable Culls 

Ammonium Nitrate injected 12 times – 168kg N/ha 45.3 3.4 


Nitamin 30L preplant – 117kg N/ha 37.7 1.2 



Nitamin 30L injected 3 times – 168kg N/ha 53.9 0.8 

LSD 0.05 9.8 NS 


educed application schedule can be employed since only 3 Nitamin 

injections were made compared to 12 weekly injections of ammonium 

nitrate. Furthermore, the last Nitamin injection was made 7 weeks 

after transplanting, ending 5 weeks earlier than the ammonium nitrate 

treatments. The results demonstrate that injecting Nitamin 30L 

through the drip-irrigation system can be more effective than spoonfeeding 

ammonium nitrate every week over the growing season. 

TEXAS STUDY. Nitamin liquid fertilizer increased total yield and 

marketable yield compared to urea (Figure 3). Yields were slightly 

higher when Nitamin was applied in split applications compared to a 

single application, but the difference was not statistically significant. 

The high clay content and cation-exchange capacity of the soil at the 

experimental site may have resulted in some ammonium fixation when 

applied all preplant. Yield differences between fertilizers were 

greatest at the highest N rates, and split applications of Nitamin at 89 

and 135kg N/ha increased average fruit size compared to the 135kg 

N/ha urea treatment. Vigor ratings and vine-length measurements in 

late June and early July showed that Nitamin plants got off to a faster 

start, which may have resulted in earlier fruit set and increased fruit 

size (Table 2). 

NORTH CAROLINA STUDY. All of the N treatments promoted 

increased fruit set and higher yield than the untreated (0 N) control 

treatment (Table 3). Ammonium nitrate did not demonstrate a yield 

response with respect to N rate, while the Nitamin treatments did 

produce a more typical yield response curve. 

With Nitamin, marketable yield increased up to 89kg N/ha, then 

leveled off at 135kg N/ha until decreasing at the highest (180kg N/ha) 

nitrogen- application rate. It is not unusual for yields to decrease when 

such a high rate of N is applied all in one application. No plant burning 

Table 1. University of FL Watermelon Trial, Live Oak. 

Total-season yield 

(metric tons/ha) 

Treatment Marketable Culls 






Nitamin 30L injected 3 times – 168kg N/ha 53.9 0.8 

LSD 0.05 9.8 NS 


was noted at the highest Nitamin rate even though yields were 

depressed. 

Numerically, Nitamin applied at 89kg N/ha was the highest 

yielding treatment, followed by Nitamin applied at 135kg N/ha, and 

then ammonium nitrate applied at 45kg N/ha. It is not clear why the 

lowest ammonium nitrate treatment was one of the highest yielding in 

metric tons / hectare 

30 

25 

20 

15 

10 

5 

0 

Urea preplant Nitamin liquid preplant 

Nitamin liquid split application 

Total Marketable 

Fig. 3. Watermelon yield at Texas AREC. LSD (0.05) Total 9.1, marketable 9.1. 

this study. While yields were not always statistically significant 

between N sources, the Nitamin rates of 89 and 135kg N/ha tended to 

produce increased vine growth, higher early-season yields, and higher 

total yield compared to ammonium nitrate. 

Nitrogen rate resulted in some differences in vine growth. The 

lowest N rate, the no-N treatment, and the 180kg/ha Nitamin treatment 

had the least vine growth (Table 4). The highest Nitamin rate 

(180kg/ha) appeared to inhibit vine growth while the ammonium 

nitrate at 180kg/ha did not. This appears to be a function of how the 

material was applied–Nitamin all in one application vs. ammonium 

nitrate split evenly in four applications. The nitrate samples for all N 

treatments except the 45- and 89-kg/ha Nitamin treatments were at or 

above sufficiency ranges (Figure 4). Although the petiole nitrate was 

not at sufficient levels for 89kg/ha Nitamin, it was numerically the 

highest-yielding treatment. 

ECONOMIC ANALYSIS. One factor that must be considered with 

any new or existing technology is the cost to implement it. 

Controlled-release technology has been in existence for several 

decades but its use has been primarily restricted to the turf, 


Table 2. Watermelon vine length and vigor Texas AREC. 

Length 

Average 

June 28 Vigor Vigor size 

Treatment 

(cm) July 10 Aug. 4 (kg) 

Urea 89kg N/ha 86.45 6.75 8.00 4.98 

Urea 135kg N/ha 76.23 7.25 9.25 4.20 

Nitamin 89kg N/ha (AP) 106.58 8.25 9.75 4.68 

Nitamin 135kg N/ha (AP) 108.98 9.25 9.5 5.02 

Nitamin 89kg N/ha (S) 90.43 7.75 9.25 5.10 

Nitamin 135kg N/ha (S) 96.45 8.75 9.00 5.11 

LSD (0.05) 25.12 1.92 1.24 0.86 

Vigor ratings: 1= dead; 3 = poor; 6 = fair; 7 = good; 10 = excellent. AP = at 

planting; S = split application. 

ornamental, and greenhouse industries because of cost. As 

environmental concerns with nitrogen contamination increase and the 

cost of controlled-release fertilizer decreases, this barrier is coming 

down. An evaluation of fertilizer costs, crop value, and increased 

grower profit comparing Nitamin to ammonium nitrate or urea is 

presented in Tables 5 and 6. As the data show, the increased 

production with Nitamin results in significantly higher returns. 

Discussion 

Nitamin 30L increased yields in three studies but differences were not 

always statistically significant at p=0.05. In two cases, applying 

Nitamin all at planting outperformed up to four split applications of 

quick-release fertilizer. It appears the 60–75 day Nitamin release rate 

matched the plants’ N-uptake pattern in those studies. For soils that 

ppm nitrate 

1600 

1400 

1200 

1000 

800 

600 

400 

200 

0 

45 51 64 

Days after planting 

Ammo. Nitrate 45 Kg/ha 

Ammo. Nitrate 89 Kg/ha 

Ammo. Nitrate 135 

Kg/ha 

Ammo. Nitrate 180 

Kg/ha 

Nitamin 45 Kg/ha 




No N fertilizer 

Fig. 4. Cucumber petiole nitrate. Statistical data by SAS. LSD (0.05) 

values: Day 45, 274; Day 51, 218; Day 64, 300. 


Table 3. Weight of cucumbers in metric tons per hectare by grade. 

Market- 

Treatment kg/ha No. 1 No. 2 No. 3 No. 4 able total 

No N fertilizer 2.1 3.8 4.6 1.9 10.5 

NH4NO3, 45 3.5 6.7 7.5 2.7 17.7 

NH4NO3, 89 2.8 6.4 6.2 2.0 15.4 

NH4NO3, 135 3.1 6.1 7.3 2.6 16.4 

NH4NO3, 180 2.7 5.6 7.6 2.1 15.9 

Nitamin, 45 2.6 5.0 6.1 2.4 13.6 

Nitamin, 89 3.1 6.1 9.9 3.3 19.1 

Nitamin, 135 3.1 6.1 9.1 2.7 18.3 

Nitamin, 180 2.5 4.5 5.4 1.7 12.3 

Average 2.8 5.6 7.1 2.4 15.5 

LSD (0.05) 0.9 1.9 3.0 1.2 4.2 

Table 4. Cucumber-vine measurements. 

Vine 

Vine 

length Treatment length 

(cm) (kg/ha) 

(cm) 

No N fertilizer 39 Nitamin, 45 39 

NH4NO3, 45 38 Nitamin, 89 48 

NH4NO3, 89 39 Nitamin, 135 46 

NH4NO3, 135 43 Nitamin, 180 36 

NH4NO3, 180 42 

LSD (0.05) 9 

are over 90% sand and < 1.0% organic matter, split applications of 

Nitamin should be used. Application rates of 135 to 168kg N/ha from 

Nitamin appeared to be adequate for watermelon, whereas rates as low 

as 89kg N/ha were sufficient for pickling cucumbers. In general, 

Nitamin application rates should be 75–100% of the local cooperative 

extension recommendation for each crop. Further research is needed 

to refine application rates and scheduling in each geographical area. 

The potential profit advantages of using affordable slow-release N 

products such as liquid Nitamin demonstrates their feasibility for use 

on cucurbits and other vegetables, especially considering such benefits 

as reduced N losses through leaching and ammonia volatilization. 

Growers may also find advantages in reducing the number of sidedress 

applications or lowering overall N rates with slow-release products. 


Table 5. Value proposition of Florida watermelons per hectare based 

on N source. 

Fertilizer 

Grower profit 

Fertilizer kg/ha cost Crop value increase 

Nitamin 30L 168 $370 $10420 $1479 

Ammo. nitrate 168 $128 $8941 0 

Ammo. nitrate 211 $138 $9452 $511 

Watermelon price of $2.002/metric ton, Nitamin 30L = $600/ton, Nitamin 42G = 

450/ton (ton is U.S. short, not metric), ammonium nitrate = $208/ton. 

Table 6. Value proposition of Texas watermelons per hectare based on 

N source. 

Fertilizer 

Grower profit 

Fertilizer kg/ha cost Crop value increase 

Nitamin 30L 89 $196 $6076 $39 

Nitamin 30L 135 $297 $7499 $1,462 

Urea 89 $63 $6037 0 

Urea 135 $95 $5787 0 

Watermelon price of $2.816/metric ton, Nitamin 30L = $600/ton, urea 

$224/ton (ton is U.S. short, not metric). 


Green Markets, May 8, 2006. 30(19):4, col. 1 & 2. 

Official Publication AAPFCO. 2004. 57:40. 

U.S. Department of Agriculture. 2006. Agricultural statistics for 2005. 

USDA, Washington, DC. . 


TRIPLOID SEEDLESS WATERMELON 

PRODUCTION IN CHINA 

Liu Wenge, Yan Zhihong, Zhao Shengjie, and He Nan 

Zhengzhou Fruit Research Institute, 

Chinese Academy of Agricultural Sciences, 

Zhengzhou, Henan 450009, P. R. China 

ADDITIONAL INDEX WORDS. Citrullus lanatus, watermelon, China, triploid, 

seedless, production 

ABSTRACT. More than 1,000,000ha of watermelon are grown annually in China. 

Seedless watermelon is an important part of watermelon production in China, 

accounting for 20% of total watermelon production acreage. Ninety percent of 

seedless watermelon production is south of the Yellow River. Chinese consumers 

like red- or deep pink-fleshed seedless watermelon with black or dark green skin 

color. More recently, yellow-fleshed seedless watermelons have been introduced 

and gradually accepted. Small-fruited seedless watermelons are also becoming 

popular. Some easy yet effective technologies aimed at helping China increase its 

production of seedless watermelon have been developed and implemented . 

W 

atermelon is a warm-season crop similar to cantaloupe, 

squash, cucumber, and pumpkin. In China, watermelons are 

grown on any well-drained soil throughout the country. 

Watermelons are cultivated year-round by utilizing low tunnels and 

greenhouses. Watermelons are transported throughout the country. 

More than 1,000,000 hectares of watermelon are produced in China 

annually, accounting for 58% of the world watermelon production area 

and 73% of world watermelon yield in 2003. Yields of 45 to 50 metric 

tons per ha are common (Liu, 2005), similar to those of the United 

States (Maynard, 2001). 

Seedless watermelon is an important part of watermelon production 

in China. Commercial varieties of seedless watermelon are triploid 

hybrids (Tan, 2002). There were 7,000ha of seedless watermelon in 

1990. Since 1995, seedless watermelon has become more popular, and 

the production acreage has increased to 200,000ha in 2005. Seedless 

production now accounts for 20% of total watermelon acreage, 

providing a tremendous boost to the watermelon industry (Liu, 2005). 

Major Seedless Watermelon Production Areas in 

China 

The major seedless watermelon cultivation areas, based on a 2005 

survey, are listed Table 1. More than 200,000ha of seedless watermelon 

were produced in 2005. Henan, Hunan, Hubei, Hainan, and Jiangxi 


provinces are the top suppliers of seedless watermelon. Anhui, Guangxi, 

Guangdong, Guizhou, Shaanxi, Shandong, Beijing, and Jiangsu are the 

second for harvested production areas. The provinces with fewer than 

1000ha of seedless watermelon include Hebei, Sichuan, Jilin, Shanghai, 

Zhejiang, Chongqing, Yunnan, Tianjin, Heilongjiang, Gansu, Ningxia, 

Shanxi, Xinjiang, and Inner Mongolia. Hainan is the primary supplier of 

seedless watermelon in December, January, and February. Seedless 

watermelon production has increased even though total watermelon 

area is decreasing (Liu, 2005). Eighty percent of seedless watermelon 

production is located south of the Yangtze River, with 90% south of the 

Yellow River. This is because of seedless watermelon’s higher tolerance 

to humidity and disease resistance than that of diploid seeded 

watermelon (Tan, 2002). Seedless watermelon production is growing 

rapidly in some new areas such as the provinces of Yunnan, Sichuan and 

Shaanxi, where there was little seedless watermelon cultivation in the 

past. 

Seedless Watermelon Varieties Used in China 

The key seedless watermelon varieties grown in different regions of 

China are listed in Table 2. Consumers traditionally like red- or 

deep-pink-fleshed seedless watermelon. Yellow-fleshed seedless 

watermelons have recently been introduced and gradually accepted by 

Chinese consumers. Small-fruited seedless watermelon is also 

becoming popular. The major seedless watermelon varieties used in 

China in 1990s were ‘Mimeiwuzi 1’, ‘Guangxi 2’, ‘Heimi 2’, ‘Xuefeng 

304’, ‘Dongting 1’, ‘Xuefenghuapi’, and ‘Xinxiu 2’. Since the 

registration of ‘Heimi No. 5’ in the national variety identification 

system in 2000, Chinese watermelon breeders have made significant 

efforts to develop new seedless varieties. In 2002 the following varieties 

were registered in the state variety identification system: 

‘Zhengkangwuzi No. 1’, ‘Zhengkangwuzi No. 2’, ‘Zhengkangwuzi No. 

3’, ‘Mihuangwuzi’, ‘Mihongwuzi’, ‘Xiaoyuhongwuzi’, ‘Fenglewuzi 

No. 1’, ‘Fenglewuzi No. 2’, ‘Fenglewuzi No. 3’, and ‘Jinmi 20’. Some 

varieties have been registered in the provincial variety identification 

system. Midseason and later-maturity varieties have also been released 

recently. The major objectives for triploid watermelon breeding include 

disease resistance, yield, fruit type, high soluble solid contents, free of 

hard seed coats, eating quality, and shipping ability. The new varieties 

have facilitated rapid growth of seedless watermelon production in 

China. 


Table 1. Seedless watermelon production in China. 

Acreage Yield Harvesting 

Province (ha) (kg/ha) period 

Henan 33000 45,000–75,000 June–Aug. 

Hunan 33000 37,500–52,500 June–July 

Hubei 32000 45,000–75,000 June–July 

Hainan 26600 37,500–75,000 Dec.–Feb. 

Jiangxi 21300 30,000–45,000 June–July 

Anhui 8000 45,000–75,000 June–July 

Guangxi 6600 30,000–52,500 April–May 

Guangdong 4000 30,000–52,500 April–May 

Guizhou 2600 30,000–52,500 July–Aug. 

Shaanxi 2600 60,000–75,000 May–June 

Shandong 2000 45,000–75,000 June–July 

Beijing 2000 60,000–75,000 July–Aug. 

Jiangsu 1000 45,000–75,000 June–July 

Impact of Research on Triploid Seedless 

Watermelon Production 

Low germination, low seedling vigor, and low seed yield of triploid 

hybrids are the three major obstacles for seedless watermelon 

production. Scientists have tried to find solutions to these problems 

(Liu, 2005). Key cultivation techniques have been developed for 

growing seedless watermelon in different climatic regions in China. 

These techniques include (1) Pregerminating triploid seed with high 

temperature (30–32°C). Triploid seeds are placed between moistened 

towels, placed on an electric blanket and/or under heating lights, until 

the radical has emerged from seed coat. (2) Using plastic mulch to 

improve survival rate of triploid seedlings in the field. (3) Producing 

triploid hybrid seed under dry climates. These changes have lead to 

acceptable seed germination rates (>85%), seedling survival rates 

(>80%), and triploid seed yields (>6kg/667m 2 ). These improvements 

have made commercial seedless watermelon production possible in 

different regions in China. (Tan, 2002). 

Transplants and hand-pollination are used in most production areas 

where labor is abundant. Bee-pollination is not common for seedless 

watermelon production in China. National and provincial (such as 

Anhui, Guangxi, and Jiangsu) standards for seedless watermelon 

cultivation have been established. Seedless watermelon is also suitable 


for plastic-tunnel and greenhouse production. A year-round supply of 

seedless watermelon is achieved through protected production in 

northern regions and open-field production in southern regions. 

Examples of protected seedless watermelon production include 

double-plastic-mulch cultivation in Beijing and greenhouse cultivation 

in Shannxi. Hainan island is the most important seedless watermelon 

production area for the winter season. Guangxi and Yunnan are the 

important production areas for the spring season. 

Table 2. Triploid seedless watermelon varieties grown in major 

production areas in China. 

Fruit type Varieties Characteristics Production areas 

Crimson 

Sweet 

Zhengkangwuzi No. 3, 

Fenglewuzi No. 2, 

Mihongwzui 

Oval-shaped Zhengkangwuzi No. 1, 

Cuibao No. 5, Xuefeng 

wuzi 

Sugar Baby Zhengkangwuzi No. 5, 

Dongting No. 1, Jinmi 

No. 20 

Hei Mi Heimi No. 5, 


Shubao, Zhengkang 

2008 

Xin Yihao Fenglewuzi No. 3, 

Quality No. 1, Guangxi 

No. 3 

Yellow 

flesh 

Yellow 

skin 


Xiangxigua No. 19, 

Mihuang wuzi, 

Huangbaoshi 

Round, dark narrow 

stripes on a light 

green background, 

red flesh 

Large, round, dark 

wide stripes on a light 


red flesh 

Large, round, no 

stripes, solid dark, 

red flesh 

Large, round, dark 

wide stripes on a dark 


red flesh 

Large, Round, Dark 

medium stripes on a 

medium green 

background, red flesh 

Large, round, yellow 

flesh 

Henan, Hubei, 

Jiangxi, 

Guangdong 

Henan, Hubei, 

Hunan, Guizhou, 

Xinjiang 

Henan, Hubei, 

Jiangxi, Hunan, 

Anhui, Guangxi, 

Shanxi 

Hunan,Hubei, 

Jiangxi,Anhui, 

Shaanxi,Beijing, 

Jiangsu, Hebei, 

Liaoning 

Hainan,Guangxi, 

Shandong 

Hubei, Hunan, 

Guangxi 

Jin Taiyang No. 1 Yellow rind, red flesh Sichuan, Gansu, 

Jiangxi, Henan 



Liu, W. G. 2005. Review and perspectives of scientific research and production 

cooperation of seedless watermelon in China. China watermelon and melon. 

2005(2):19–20 

Maynard, D. N. (ed.). 2001. Watermelon. characteristics, production, and marketing. 

ASHS Press, Alexandria, VA. 

Tan, S. Y. (ed). 2002. Seedless watermelon culture and breeding. Agriculture Press, 

Beijing, China. 


HERBIVORY BY CUCUMBER BEETLES 

AFFECTS POLLEN PRODUCTION AND 

POLLEN PERFORMANCE IN A WILD GOURD 

Andrew G. Stephenson, James A. Winsor, Daolin Du, 

Andrew DeNicco, and Matthew Smith 

Department of Biology, The Pennsylvania State University, 

University Park, PA 16802 

ADDITIONAL INDEX WORDS. Cucurbita pepo ssp. texana, diabroticite beetles, 

Erwinia tracheiphila, fitness, male function, pollen competition 

ABSTRACT. The impact of herbivory and other biotic stress on fitness through 

the male function is rarely studied in wild plants. To examine the effects of 

cucumber-beetle herbivory on the male function of wild gourds (Cucurbita pepo 

ssp. texana [Bailey] Andres), inbred and outbred plants from five families were 

grown in one-acre experimental plots for four field seasons. On these plants we 

nondestructively assessed foliar beetle damage and recorded staminate-flower 

production, pollen production per male flower, and in vitro pollen-tube growth. 

We also examined in vivo pollen performance in a pollen-competition 

experiment. Our results reveal that as beetle damage increases on plants, both 

pollen production and pollen performance decreases. Moreover, we found that 

as beetle damage increases, the probability that the plants will survive to 1 

September decreases, as a result of increased exposure to the causative agent of 

bacterial wilt disease, Erwinia tracheiphila. These findings suggest that foliar 

beetle damage is likely to decrease fitness through the male function by 

reducing both the amount of pollen that a plant donates to conspecifics and the 

probability that the pollen will sire a seed after deposition onto a stigma. 

M 

ost flowering plants are hermaphrodites and consequently 

achieve half of their fitness through the male function 

(Yampolsky and Yampolsky, 1922). In cultivated species 

for which the flowers, fruits, or seeds are the desired commodity, there 

have been few studies of the impact of soil nutrients, abiotic stress, 

herbivory, disease, etc., on the male function. Although there have 

been many ecological studies using noncultivated species that have 

examined the effects of various environmental stresses on growth and 

We thank T. Kinney, J. Thaller, N. Myers, and B. Leyshon for field and greenhouse 

assitance; R. Oberheim and his staff for use of The Pennsylvania State University 

Agriculture Experiment Station at Rock Springs, PA (Horticulture Farms); and A. 

Omeis for use of the Buckhout greenhouse. This research was supported by NSF 

grant DEB02-35217 to AGS and JAW and by a China Scholarship Council 

Fellowship to DD. 


eproduction through the female (fruit and seed) functions, only 

recently have ecologists begun to study the effects of environmental 

stress on pollen production per flower, pollen performance, or the 

number of seeds sired in the population (Delph et al., 1997; 

Stephenson et al., 2003). This neglect is most likely due to the 

difficulty of assessing pollen production, pollen performance, and 

patterns of gene flow in species for which biochemical, molecular, and 

genetic resources are unavailable. Moreover, for most wild species 

little is known about the herbivores and even less about the pathogens 

that are likely to be the causes of the biotic stress. 

Because of the comparative wealth of resources available in 

cultivated species (e.g., knowledge of the full suite of herbivores and 

pathogens and their vectors, and of the volatile compounds; 

biochemical and genetic resources; and, in some cases, commercially 

available immunological test kits for the pathogens, transgenes for 

disease resistance, microarrays, and other genomic, proteomic, and 

metabolomic tools), ecologists have recently begun to transfer some of 

this information and technology to wild relatives of the cultivated 

species in order to address fundamental ecological and evolutionary 

questions (Richman et al., 1995; DeMoraes et al., 1998; Halitschke et 

al., 2003). Over the last five years, we have been investigating the 

interrelationships among inbreeding, herbivory, and disease 

establishment and transmission in a wild gourd (free-living squash, 

Cucurbita pepo ssp. texana). These studies have revealed that there is 

genetic variation for resistance to cucumber beetles in families of 

seeds collected from the wild; that inbred plants suffer higher levels of 

herbivory by cucumber beetles and harbor larger aphid populations 

than outbred plants; that inbred plants are more likely to be infected 

with aphid-transmitted viral diseases than outbred plants; and that 

outbred plants produce more floral volatiles than inbred plants (Hayes 

et al., 2004; Stephenson et al., 2004; Ferrari, 2006). Here, we use data 

from four field seasons to examine the impact of herbivory and disease 

on the male function of this wild gourd. 

Cucurbita pepo ssp. texana (also known as C. pepo ssp. ovifera 

var. texana) is an annual monoecious vine with indeterminate growth 

and reproduction. It is native to Texas and adjacent areas and is 

thought to be either the wild progenitor of the cultivated squashes (C. 

pepo ssp. pepo, also known as C. pepo ssp. ovifera var. ovifera) or an 

early escape from cultivation (Decker, 1988). When the two 

subspecies are grown in the same vicinity, they frequently produce 

hybrid progeny, and the F1 progeny are highly viable and fertile 

(Quesada et al., 1993). 


After a period of vegetative growth (5–7 nodes), the wild gourd 

produces one large yellow flower (either staminate or pistillate) in the 

axil of each leaf. The flowers last for only one morning and are 

pollinated by bees, especially squash bees. The leaves and other organs 

of the wild gourd produce bitter compounds called cucurbitacins 

(oxygenated tetracyclic triterpenes) that deter most herbivores 

(Tallamy, 1985; Metcalf and Rhodes, 1990). Interestingly, 

cucurbitacins are phagostimulants to cucumber beetles from the 

Luperini subtribes Diabroticina and Aulacophorina (Chyrsomelidae: 

Galerucinae). It is generally thought that by consuming cucurbitacins, 

cucumber beetles embitter themselves and make themselves less 

attractive to potential predators (Tallamy and Krischik, 1989). 

Furthermore, cucurbitacins may play a role in the beetle mating system 

(Tallamy et al., 2002). These cucumber beetles cause a characteristic 

pattern of holes (typically 1–1.5cm in diameter) in the portions of the 

leaf serviced by the smallest veins. Aphids also feed on the leaves, leaf 

axils, and growing tips of the vine. 

Cucumber beetles and aphids are also known to transmit the most 

important growing-season diseases of Cucurbita. Consequently, the 

full impact of herbivory on wild gourds includes increased exposure to 

a variety of pathogens. Cucumber beetles are the primary vector of the 

bacterial wilt pathogen Erwinia tracheiphila (Yao et al., 1996). In 

Pennsylvania, the bacterium is transmitted via the feeding of the 

striped and spotted cucumber beetles (Acalymma vittata and 

Diabrotica undecimpunctata howardi) (Fleischer et al., 1999). 

Erwinia proliferates in the xylem and secretes a mucilaginous matrix 

that cuts off water supply, resulting in wilting and eventual death of 

the plant. Wilt symptoms typically develop 10–15 days following 

infection in mature plants and are nearly always fatal. Aphids are the 

sole vectors for four of the most common viral diseases of cucurbits, 

Cucumber mosaic virus, Papaya ringspot virus-Type W, Watermelon 

mosaic virus, and Zucchini yellow mosaic virus. 


An experimental population of C. pepo ssp. texana was initiated 

from seeds sampled randomly from a natural population in Texas. A 

random sample of five progeny was used to found five maternal lines, 

and the remaining lines were reserved as potential pollen donors. A 

multiyear crossing program was used to generate plants with a range 

of inbreeding coefficients to study inbreeding depression in a host of 

traits (Stephenson et al., 2001; Hayes et al., 2005). For the purposes of 

this study, we were concerned only with offspring from outcrossed 


matings (ƒ = 0) and first generation self-matings (ƒ = 0.5 ) in each 

year. In the summers of 2002–2005 we conducted field experiments to 

examine the effects of inbreeding and genetic variability on herbivory 

and patterns of disease spread. In each year, we germinated selfed and 

outcrossed progeny from the five maternal lines in a greenhouse and 

transplanted to 2–4 fields of 0.4-ha each at the Pennsylvania State 

Agricultural Experimental Station at Rock Springs, Pennsylvania, on 

18–22 May. Plants were arrayed in a grid of 12 plants by 15 plants in a 

systematic pattern to assure mixing of genotypes. 

To assess the male function of each plant in each year we (1) 

recorded male (staminate) flower production on each plant in each 

field during each year two days per week until 31 August (because the 

flowers last only a single morning, this provides an unbiased estimate 

of male-flower production). (2) In late July 2002, we collected the 

anthers from one flower on each plant, placed them into a glass vial, 

and placed the vial into a drying oven. Subsequently, we counted the 

pollen produced by the staminate flowers using an Elzone Particle 

Counter (Micromeritics Corp., Norcross, GA). (3) In early August of 

2002, 2004, and 2005, we again collected the anthers from one flower 

on each plant in one field, and then brushed the pollen onto Petri plates 

containing a modified Brewbaker and Kwack (1963) medium with 

12% sucrose and 1% agar. After 20 minutes, we added a few drops of 

80% ethanol to stop the germination and growth of pollen tubes (see 

Stephenson et al., 2001). We recorded the lengths of a sample of 30 

pollen tubes on each plate using an image analysis system. (4) In 2003, 

we assessed the in vivo performance of pollen by collecting pollen 

from outbred plants growing in the same fields that were hand-sprayed 

weekly with esfenvalerate (Asana XL, DuPont Corp., Wilmington, 

DE) (which decreased both herbivory and disease symptoms compared 

to unsprayed plants; see Stephenson et al., 2004) and from unsprayed 

outbred plants from each of the five families used in the study. Pollen 

from each of the 10 combinations (5 families x 2 spray/no spray 

treatments) was then placed onto stigmas of an inbred line of zucchini 

(‘Black Beauty’) along with an equal amount of zucchini pollen 

(Figure 1). Three replicate pollinations of each type were made on 

different plants and the resulting fruits were harvested at the end of the 

growing season. A sample of the progeny was germinated, grown, and 

scored for paternity based on the shape of the ovary and other singlegene 

traits (see Quesada et al., 1993, for pollination and scoring 

techniques). 

Three times during the growing season (mid-June, mid-July, and 

mid-August) each year, we nondestructively recorded the amount of 

beetle damage on the new growth along the main stem of each plant 


Fig. 1. Diagram of design of pollen-mixture experiment. Equal amounts of wildgourd 

pollen and zucchini pollen were placed onto zucchini stigmas. 

using a 0–5 scale in which 0 = most leaves with no beetle damage and 

no leaf with more than 5% of the leaf area removed, and 5 = all leaves 

damaged and at least one leaf with > 50% of the leaf area removed 

(Stephenson et al., 2004). Similarly, two times during the 2002 

growing season (late June and early August), we nondestructively 

recorded symptoms of viral infection on a 0–5 scale by inspecting the 

leaves on the new growth along the main stem. During the twiceweekly 

flower counts (above) in each field during each year, we also 

recorded the onset of bacterial wilt symptoms and the date in which 

the plant died (bacterial wilt disease is 100% fatal once visual 

symptoms appear). 

Separate analyses of variance were conducted for the three 

components of male reproductive output (staminate flowers/plant, 

pollen/flower, and in vitro pollen-tube growth) using a mixed-effects 

ANOVA (Proc GLM, SAS Institute Inc., 2002). Type III sums of 

squares were used to test the significance of family (random), f (the 

coefficient of inbreeding, 0 or 0.5), beetle damage (a covariate 

summed for the three dates that it was assessed), and the two-way 

interactions. For brevity, the results and discussion will focus only on 

the effects of beetle damage on the male function. 



The results of our mixed-effects model analysis of variance using 

only those plants that survived until 31 August revealed that beetle 

damage had no significant effect on staminate-flower production 

(F1,606 = 0.47; p > 0.10). However, when we analyzed the data using all 

plants that survived to 1 July (the approximate date of first flower 

production in each year), we found that beetle damage significantly 

decreased staminate-flower production (F1,1076 = 5.22; p < 0.003). 

These findings suggest that the plants that died prior to the end of the 

growing season had significantly higher levels of beetle damage than 

the plants that survived. In fact, a preliminary analysis that will be 

explored in more detail in another publication indicates that the plants 

that died in the month following each assessment of beetle damage had 

significantly higher levels of beetle damage than those that survived. 

In our fields, the greatest cause of mortality during the growing season 

was due to E. tracheiphila—a pathogen that is vectored by cucumber 

beetles. In the four years of this study, 8 to 35% of the plants died after 

exhibiting symptoms of wilt disease before 31 August (Ferrari, 2006). 

Our analyses of variance also reveal that beetle damage 

significantly decreases both pollen production per flower (F1,112 = 4.70; 

p < 0.04) and in vitro pollen-tube growth rate (F1,396 = 4.48; p < 0.04). 

These findings indicate that beetle damage decreases total pollen 

production per plant even though those plants that survive to August 

do not differ in total staminate-flower production. Consequently, 

damaged plants have fewer pollen grains to donate to conspecifics. 

Moreover, the in vitro pollen-tube-growth data suggest that as beetle 

damage increases on plants, the plants have fewer or lower quality 

resources for their developing pollen grains (Delph et al., 1997; 

Stephenson et al., 2003). The amount of stored nutrient and energy 

compounds, in turn, affects the growth of pollen tubes in vitro and the 

initial (autotrophic phase) growth of pollen tubes in vivo (Stephenson 

et al., 2003). A chi-square ANOVA (Proc CATMOD; SAS Institute, 

2002) reveals that pollen produced from sprayed plants (plants with 

significantly less cucumber beetle and aphid herbivory and 

significantly lower levels of the diseases transmitted by aphids and 

cucumber beetles; Stephenson et al., 2004) sired significantly more 

seeds (53%) in competition with zucchini pollen than pollen from 

nonsprayed plants (37%). 

Although the male function of cultivated species rarely merits 

attention, the ecological impacts of biotic stress on the male function 

and its evolutionary implications are very important to their 

noncultivated relatives. The findings from this study suggest that 


damage by cucumber beetles can impact fitness by decreasing pollen 

production, which in turn decreases the probability that a plant’s 

pollen will be deposited onto stigmas of conspecifics. Moreover, the in 

vitro pollen-tube-growth data and the in vivo pollen-competition data 

suggest that beetle damage decreases the probability that those pollen 

grains that are deposited onto a stigma will actually get their genes into 

the next generation. Because studies of the wild species in the 

Cucurbitaceae can draw upon the comparative wealth of information 

and resources that are available in the cultivated species, it is likely 

that the wild relatives will become important as model systems for 

addressing basic issues in evolutionary biology, disease ecology, and 

population genetics. 


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WHITEFLY TRANSMISSION OF A NEW 

VIRUS INFECTING CUCURBITS IN FLORIDA 

Susan E. Webb 

University of Florida, Entomology and Nematology Department, 

Gainesville, FL 32611-0620 

Scott Adkins 

United States Department of Agriculture-Agricultural 

Research Service, Fort Pierce, FL 

Carlye A. Baker 

Florida Department of Agriculture and Consumer Services, 

Division of Plant Industry, Gainesville, FL 

ADDITIONAL INDEX WORDS. Cucurbita pepo, Cucurbita spp. Citrullus lanatus, 

Bemisia tabaci, Potyviridae 

ABSTRACT. A virus isolated from squash collected in Hillsborough County, FL 

in 2003 was subsequently determined to be an ipomovirus. The virus was 

transmitted by the silverleaf whitefly, Bemisia tabaci B strain, in laboratory 

experiments. The virus was acquired by whiteflies after a 3-h access period on 

infected plants. After sequential inoculation access periods on healthy seedlings, 

whiteflies (15–20 per clip cage) transmitted the virus during the second and 

subsequent access periods, with the highest rate of transmission during the 

second access period (2–4h postacquisition). A limited host-range study 

suggested that the virus is restricted to cucurbits. All Cucurbita species tested 

showed vein yellowing and were stunted compared to healthy controls. The 

Cucumis species tested showed transient vein yellowing and apparent recovery. 

Watermelon (Citrullus lanatus) leaves yellowed, petioles collapsed, and plants 

became necrotic and died 7–10 days after inoculation. The relationship between 

this virus and the highly damaging disease known in Florida as mature 

watermelon vine decline is being explored. 

D 

uring a survey of cucurbit viruses in Florida from 2000 to 

2003 (Webb et al., 2003), leaf samples were collected from a 

field of yellow summer squash (Cucurbita pepo L.) in 

Hillsborough County. All samples were tested by DAS ELISA for the 

common cucurbit viruses in Florida (Watermelon mosaic virus 2, 

Zucchini yellow mosaic virus, Papaya ringspot virus watermelon 

strain [PRSV-W]) and for five other viruses that have been found 

occasionally (Cucumber mosaic virus, Watermelon leaf mottle virus, 

Tobacco streak virus, Squash mosaic virus, Tomato spotted wilt virus). 

Of the 40 samples tested from the field, 39 tested positive for PRSV- 

W and one was negative for all 8 viruses, although it had symptoms 

typical of a viral infection (Whidden and Webb, 2004). This sample 

was used to mechanically inoculate 'Prelude II' squash, and the 


esulting infection maintained in the greenhouse for further study by 

weekly transfer to fresh plants. 

Tissue samples were sent to Agdia (Elkhart, IN) to be tested for 14 

viruses, including 6 of the viruses for which we had already tested. 

Results, including those from a general potyvirus test, were negative. 

Agdia also performed group tests, using PCR, for carmoviruses, 

nepoviruses, and geminiviruses, with negative results. Our initial 

efforts to detect viral inclusions in epidermal strips (Christie and 

Edwardson, 1994) and to detect particles in leaf dip preparations by 

electron microscopy were not successful. However, we were later able 

to purify the virus, and preliminary sequence comparisons showed that 

it was an ipomovirus (unpublished data), a genus in the family 

Potyviridae whose members are transmitted by whiteflies (Bemisia 

tabaci). 

Experiments described in this paper confirm that the virus found in 

Florida is also transmitted by whiteflies and begin host-range 

comparisons with the other ipomovirus found in cucurbits in Europe 

and the Middle East, Cucumber vein yellowing virus (CVYV) (Al- 

Musa et al., 1985; Cuadrado et al., 2001; Harpaz and Cohen, 1965; 

Louro et al., 2004; Yilmaz et al., 1989). 


WHITEFLIES, PLANTS, AND VIRUS. Adult whiteflies (Bemisia 

argentifolii Bellows & Perring, also known as the B strain of Bemisia 

tabaci) of mixed ages (newly enclosed to 4d old) were obtained from a 

colony maintained on cotton, Gossypium hirsutum L. (‘DPL 90’) and 

collard, Brassica oleracea var. acephala (‘Georgia’) as described by 

Chen et al. (2004). 

The yellow crookneck squash cultivar ‘Prelude II’, Cucurbita pepo 

L. (Seminis Seeds, Oxnard, CA) was used for all experiments. Plants 

were grown in a 3:1 (vol/vol) mixture of Metro-Mix Ag Lite Mix (Sun 

Gro Horticulture, Bellevue, WA) and pasteurized sand (Quikrete C., 

Atlanta, GA). 

Virus was maintained in ‘Prelude II’ plants in the greenhouse by 

mechanical transmission, using 20mM sodium phosphate buffer (pH 

7.0) containing 0.1% (wt/vol) sodium sulfite and 1% (wt/vol) Celite. 

The culture used for transmission experiments was started from 

purified virus. 

TRANSMISSION EXPERIMENTS. An infected source plant (10d 

postinoculation) and a healthy plant of the same age were placed in 

separate cages (61cm x 61cm x 61cm) consisting of frames 

constructed of PVC pipe (1.3cm diameter) enclosed in organdy covers. 


Approximately 100 adult whiteflies were added to each cage for a 24-h 

acquisition period. At the end of that time, six 2-w-old squash plants 

were added to each cage. Plants were removed from the cages after 2d 

and moved to a greenhouse after the remaining adult whiteflies were 

destroyed. Plants were monitored for symptom development for 10d. 

A second experiment was done to compare the level of 

transmission and retention time after a much shorter acquisition access 

period with fewer whiteflies based on the results of Mansour and Al- 

Musa (1993) with a Jordanian isolate of CVYV, one of the three 

described ipomoviruses. Five source plants, 10d postinoculation with 

strong vein yellowing symptoms, were placed in one of the cages 

described above. Approximately 2000 whiteflies were released in the 

cage. A second cage, set up in a different room, contained mockinoculated 

healthy plants and approximately 500 whiteflies. After a 3h 

acquisition access period, whiteflies were aspirated into glass tubes 

using a hand-operated vacuum pump and transferred to cylindrical 

polystyrene clip cages that were screened on both top and bottom with 

a mesh that allowed feeding and oviposition through the mesh, but did 

not allow whiteflies to escape. Approximately 15–20 whiteflies were 

placed in each of 25 cages. Each cage was clipped to the youngest 

expanded leaf of a healthy 20-d-old squash plant, so that whiteflies had 

access to the underside of the leaf. After 2h, cages were removed and 

placed on a second set of plants. After 2h on the second set of 25 

plants, cages were moved to a third set of plants and left overnight 

(16h). The cages were moved to a fourth set of plants for 2h on the 

following morning. Control plants were exposed to whiteflies that had 

access to the mock-inoculated plants for 22h. Plants were moved to a 

greenhouse and treated with pymetrozine (Fulfill, Syngenta, 

Greensboro, NC). Presence or absence of symptoms was monitored for 

12d and samples of all plants were tested by RT-PCR (unpublished) 

for the presence of virus. 

HOST RANGE. Fifteen species in six families were mechanically 

inoculated with the virus. Twenty plants of each species were grown 

from seed in the greenhouse in the growing medium described earlier 

and monitored for typical symptoms for 2 to 3w after inoculation. 

Tissue from some plants of each species not showing symptoms was 

used to mechanically inoculate ‘Prelude II’, which was observed for 

2w for symptom development. 

Results 

WHITEFLY TRANSMISSION. In the initial whitefly transmission 

experiment, 3 of 6 plants developed typical vein clearing symptoms, 


eginning 8d after exposure to whiteflies. Infection was confirmed by 

RT-PCR (data not shown). 

In the second experiment, no plants from the first 2-h inoculation 

access period developed symptoms. Of the 25 plants from the second 

inoculation access period (2–4h postacquisition), 3 developed typical 

symptoms. One of 25 plants in the third access period (4–20h 

postacquisition) developed symptoms, and no plants in the last group 

(20–22h postacquisition) showed any symptoms. However, when 

plants were tested by RT-PCR, 1 additional plant without symptoms 

from the third time period was positive, as were 2 of the plants from 

the final time period. 

HOST RANGE. Only plants in the Cucurbitaceae developed 

symptoms (Table 1) and only Cucurbita species and Luffa acutangula 

developed strong, systemic vein clearing (Figure 1). Cucumber, 

Cucumis sativus, and Cucumis melo showed vein-clearing symptoms 

on only the first leaf to develop after inoculation. Virus could be 

occasionally recovered from this leaf by using it to mechanically 

inoculate ‘Prelude II’. When watermelon (Citrullus lanatus) was 

inoculated, the first symptoms consisted of yellowing of the youngest 

leaves and petioles, followed by collapse of the petioles, and finally 

necrosis and death of the whole plant (Figure 2). 

Table 1. Experimental host range of new ipomovirus. 

Family Species Common name Symptoms a 

Amaranthaceae Gomphrena 

globosa 

Globe amaranth - 

Chenopodiaceae Chenopodium 

- 

amaranticolor 

Cruciferae Brassica oleracea 

var acephala cv. 

Georgia 

Cucurbitaceae Citrullus lanatus 

cv. Sangria 

Citrullus lanatus 

cv. Crimson Sweet 

Cucumis melo cv. 

Athena 


cv. Dasher II 

Cucurbita maxima 

cv. Big Max 

Collard - 

Watermelon SWN 

Watermelon SWN 

Muskmelon TVY 

Cucumber - 

Pumpkin VY 


Table 1 cont’d 

Family Species Common name Symptoms a 

Cucurbita 

moschata cv. El 

Calabaza VY 

Dorado 

Cucurbita pepo 

cv. Prelude II 

Yellow 

crookneck 

squash 

Luffa acutangula Luffa VY 

Fabaceae Phaseolus 

vulgaris cv. Bush 

Blue Lake 

Green bean - 

Pisum sativum cv. 

Alaska Pea 

English pea - 

Solanaceae Nicotiana 

benthamiana 

- 

Lycopersicum 

esculentum cv. 

Sunny 

Tomato - 

Datura 

stramonium 

Jimson weed - 

a 

Symptoms on uninoculated leaves: = no infection, TVY = transient vein yellowing, 

VY = vein yellowing, SWN = systemic wilt and necrosis. 

VY 

Discussion 

The new ipomovirus isolated from squash is transmitted by 

silverleaf whitefly, a common pest of cucurbits in Florida. The 

efficiency of transmission was low (50%) in our first experiment, even 

when whiteflies (100 per 6 plants) were given free access to plants for 

relatively long periods of time. Mansour and Al-Musa (1993) found a 

similar level of transmission (56%) of CVYV when they used 15–20 

whiteflies and 24-h acquisition and inoculation access periods. To 

begin to determine the lower limits of acquisition and inoculation 

access times and the period of retention of the virus, we allowed 

whiteflies a 3-h acquisition access period followed by several serial 

inoculation access periods. That we obtained transmission under these 

conditions suggests that it does not take long for whiteflies to acquire 

the virus. The very low rates of transmission (a high of 12% at 2–4 h 

postacquisition) suggest that either the screened clip cages interfered 

with transmission, or that there were not enough actively feeding 

whiteflies to attain higher levels of transmission. However, it was 


Fig. 1. Symptoms on squash, ‘Prelude II’. 

Fig. 2. Symptoms on watermelon, ‘Crimson Sweet’. 


surprising to find plants that tested positive for virus by PCR in the 

group receiving whiteflies that were 20h postacquisition. Both 

Mansour and Al-Musa (1993) and Harpaz and Cohen (1965) found 

that whiteflies were unable to transmit CVYV after 6h. 

Harpaz and Cohen (1965) found that, for CVYV, a complete cycle 

of acquisition and inoculation could be completed within 60 min. This 

and the short time that the whiteflies remained viruliferous led them to 

suggest a semipersistent mode of transmission. We are now beginning 

a systematic exploration of the transmission parameters of the Florida 

ipomovirus in order to more fully compare it with CVYV and to 

develop management recommendations for growers. 

The host range of the virus so far appears to be limited to the 

Cucurbitaceae. Unlike CVYV, which causes yellowing, stunting, and 

sudden plant death in Cucumis melo (Louro et al., 2004), the Florida 

ipomovirus causes only mild and transitory vein yellowing in melon. 

In contrast, infection of watermelon results in death of the plant. A 

mature watermelon vine decline and fruit rot has resulted in serious 

crop losses in Florida in the past few years (Roberts et al., 2004). The 

virus described in this paper has been found in watermelon in a limited 

survey (unpublished data) and may be the causal agent of the disease. 


Al-Musa, A. M., S. J. Qusus, and A. N. Mansour. 1985. Cucumber vein yellowing 

virus on cucumber in Jordan. Plant Dis. 69:361. 

Chen, J., H. J. McAuslane, R. B. Carle, and S. E. Webb. 2004. Effects of Bemisia 

argentifolii (Homoptera: Aleyrodidae) infestation and squash silverleaf disorder 

on zucchini yield and quality. J. Econ. Entomol. 97:2083–2094. 

Christie, R. G. and J. R. Edwardson. 1994. Light and electron microscopy of plant 

virus inclusions. University of Florida, Institute of Food and Agricultural 

Sciences. Monograph 9, rev. 

Cuadrado, I. M., D. Janssen, L. Velasco, L. Ruiz, and E. Segundo. 2001. First report 

of cucumber vein yellowing virus in Spain. Plant Dis. 85:336. 

Harpaz, I. and S. Cohen. 1965. Semipersistent relationship between cucumber vein 

yellowing virus (CVYV) and its vector, the tobacco whitefly (Bemisia tabaci 

Gennadius). Phytopathol. Z. 54:240–248. 

Louro, D., A. Quinot, E. Neto, J. E. Fernandes, D. Marian, M. Vecchiati, P. Caciagli, 

and A. M. Vaira. 2004. Occurrence of cucumber vein yellowing virus in 

cucurbitaceous species in southern Portugal. Plant Pathol. 53:241. 

Mansour, A. and A. Al-Musa. 1993. Cucumber vein yellowing virus; host range and 

virus vector relationships. J. Phytopathol. 137:73–78. 

Roberts, P., R. M. Muchovej, P. Gilreath, G. McAvoy, C. A. Baker, and S. Adkins. 

2004. Mature vine decline and fruit rot of watermelon. Citrus and Vegetable 

Magazine. Dec. 12. 


Webb, S. E., E. Hiebert, and T. A. Kucharek. 2003. Identity and distribution of 

viruses infecting cucurbits in Florida. Phytopathology. 93:S89(Abstr.) 

Whidden, A. and S. Webb. 2004. Virus in yellow squash in Hillsborough County. 

Vegetarian 4(1). Horticultural Sciences Dept., Vegetable Crops Extension 

Newsletter, University of Florida, Gainesville. 

. 

Yilmaz, M. A., M. Ozaslan, and D. Ozaslan. 1989. Cucumber vein yellowing virus 

in Cucurbitaceae in Turkey. Plant Dis. 73:610. 


GENETIC DIVERSITY AND PHYLOGENETIC 

RELATIONSHIP AMONG MELON 

ACCESSIONS FROM AFRICA AND ASIA 

REVEALED BY RAPD ANALYSIS 

Y. Akashi, K. Tanaka, H. Nishida, and K. Kato 

Faculty of Agriculture, Okayama University, 1-1-1 Tsushima, Naka, 

Okayama, Japan 

M. T. Khaing, S. S. Yi, and T. T. Chou 

Vegetable and Fruit Research and Development Center (VFRDC), 

Yemon, Indaingpo, Hlecuts, Yangon, Myanmar 

ADDITIONAL INDEX WORDS. Cucumis melo, Group conomon, polyphyletic 

origin, small-seed type, transmission route, 

ABSTRACT. The genetic diversity of melon from the Middle East and Africa was 

studied by RAPD analysis, and compared with that of Asian landraces. 

Polymorphic index was higher in Africa, Middle East, and India, and decreased 

toward East Asia. A total of 291 samples were classified into seven clusters, 

among which Cluster II consisted of the conomon group and small-seed type of 

East India and Southeast Asia. Cluster III consisted mostly of the small-seed 

type of areas from Yunnan (China) to Pakistan, as well as the southern part of 

Africa. Cluster V consisted mostly of the large-seed type from Southeast Asia to 

the Middle East and North Africa. Cluster VII consisted of the small-seed type 

from the central and southern parts of Africa. From these results, it appeared 

that the small-seed type was transmitted from the southern part of Africa to 

India, and that the primitive form of Group Conomon originated in East India. 

Another type of melon introduced from North Africa to Europe and India via 

the Middle East was the large-seed type. Small- and large-seed melons appeared 

to be established allopatrically and then differentiated into several varieties. 

M 

elon (Cucumis melo L.) is an important horticultural crop 

cultivated in various areas of the world. Great morphological 

variation exists in melon for fruit characteristics such as size, 

shape, color, and taste of the fruit. Therefore, C. melo is considered to 

be the most diversified species in the genus Cucumis (Bates and 

Robinson, 1995). Melon cultivars commonly cultivated in Europe and 

America are classified as Group Cantalupensis or Group Inodorus, 

We would like to thank K. R. Reitsma, Iowa State University, for kindly supplying 

the seed. This study was partly supported by a Grant-in-Aid for International 

Scientific Research of Ministry of Education, Science, Culture and Sports, Japan 

(No. 15255011), titled “Genetic Assay and Study of Crop Germplasm In and Around 

China”. This is Contribution number 5 from the Sato Project of Research Institute for 

Humanity and Nature (RIHN), Japan. 


and are characterized by sweet flesh and seeds longer than 9.0mm 

(large-seed type). Other groups of melon, such as flexuosus, 

momordica, chito (dudaim), and Group Conomon, are cultivated in the 

Middle East, and are characterized by smooth skin and small seeds 

shorter than 9mm (small-seed type). 

The genetic relationship among different types of melon has been 

analyzed in various ways. Fujishita et al. (1993) classified melon 

accessions into three groups by the expression of bitterness in the 

young fruit placenta of intraspecific F1 hybrids. Cultivated and wild 

melons in East Asia were both classified as Type I, which was different 

from Type III (South and Southeast Asia) and Type II (the other areas 

including the western part of East Asia). Stepansky et al. (1999) 

detected the largest divergence between North American and European 

melon and Asian melon. Furthermore, Akashi et al. (2002) and 

McCreight et al. (2004) evaluated genetic variation in East and South 

Asian melon by the analysis of isozyme, and indicated that Indian 

melon was richer in genetic diversity compared with those in East 

Asia. Akashi et al. (2002) also suggested that Group Conomon could 

have originated from the small-seed type melon (

from Africa, using USDA-GRIN database, indicated that a dessert-type 

melon is cultivated in the northern part of Africa, while melons with 

small seeds and a sour taste are common in the southern part of Africa. 

Genetic differentiation among melon accessions from northern and 

central parts of Africa and those from the southern part of Africa was 

confirmed by RAPD analysis (Mliki et al., 2001). 

Therefore, in this study, RAPD polymorphism was surveyed for 

melon accessions from the Middle East and Africa, and genetic 

diversity and phylogenetic relationships among local melon 

populations were investigated by integrating RAPD data of Asian 

landraces (Tanaka et al., unpublished) and cytoplasmic genotype 

(Tanaka et al., 2006). The origin and transmission routes of cultivated 

melon were also considered. 


PLANT MATERIALS. Melon (Cucumis melo L.) accessions 

examined in the present study were 108 landraces collected from 

various areas of the world, consisting of 31 landraces from Africa, 20 

from the Middle East, 1 from Southeast Asia, 51 from South Asia, and 

5 from East Asia. In addition, 2 accessions of C. sativus. var. 

hardwickii were examined as wild relatives distributed in India. The 

details of plant materials are shown in Table 1. All landraces of 

unknown variety were classified into large-seed type (≥9.0 mm) and 

small-seed type (


The proportion of melon accessions having small seeds in the 

present study was 0% in the northern part of Africa and 15% in the 

Middle East (Table 1), as low as in Europe and America where the 

small-seed type is not cultivated. On the other hand, the proportion 

increased drastically in areas east of the Middle East, being 67.6% in 

South Asia, 73.3% in Southeast Asia, and 100% in East Asia. It was 

also different within Africa; nearly as high as in South and East Asia in 

the central (90.9%) and southern (73.3%) parts, and completely 

different from that in the northern part (0%). The same pattern of 

geographical distribution was observed by the analysis of seed-weight 

data of 7396 samples obtained from USDA-GRIN database. 

Twenty-seven polymorphic bands observed by Tanaka et al. 

(unpublished) were produced using 18 random primers. Polymorphic 

index (PI) within each population was calculated, and the highest PI 

was observed in the large-seed type from India (average = 0.300) and 

in accessions from the Middle East (0.300). On the other hand, PI was 

0.2 0.1 

0 

Africa-South-L 

Japan-S 

Korea-S 

China-S 

Maldives-S 

Yunnan-S 

Myanmar-S 

India-East-S 

India-Center-S a 

India-South-S 

India-West-S 

Southeast Asia-S 

Nepal and Bangladesh-S 

Myanmar-L 

India-East-L 

India-North-L 

India-Center-L b 

India-South-L 

India-North-S 

India-West-L 

Pakistan 

Middle East-S 

Africa-Center-S 

Africa-South-S 

Southeast Asia-L 

Middle East-L 

Africa-North-L 

Fig. 1. Genetic relationship between 27 populations of melon landraces, as 

revealed by cluster analysis with UPGMA method based on RAPD. L = largeseed 

type; S = small-seed type. 


I 

II 

III 

IV 

V

0.221 and 0.222 in large- and small-seed-type populations from Africa, 

respectively, as high as those in the small-seed type from India and 

accessions from Southeast Asia. However, PI calculated from the 

pooled data of all accessions from Africa was 0.399, and as high as 

those in the Middle East (0.323) and India (0.332). 

Genetic distance between 27 populations of local melon ranged 

from 0.013 to 0.729 and was 0.229 on average. Twenty-seven 

populations were classified into five clusters using cluster analysis 

based on GD (Fig. 1). Cluster I consisted of a large-seed-type 

population from the southern part of Africa. Cluster II consisted of 

three populations of Group Conomon from East Asia. Cluster III was a 

large cluster consisting of populations from India, Myanmar, and 

Southeast Asia, further divided into subclusters based on their seed 

size: IIIa, small-seeded, and IIIb, large-seeded. Clusters IV and V 

consisted of populations from the Middle East and Africa, and they 

were divided based on their seed size rather than by their cultivated 

area. As summarized above, melon populations from geographically 

related areas were grouped together and divided into subclusters based 

on their seed size. As to melon populations from Africa, they were 

divided into two clusters, and the small-seed type from central and 

southern parts of Africa was closely related with South and East Asian 

melons rather than with the large-seed type from the northern part of 

Africa. 

By principal coordinate analysis (PCO) based on similarity matrix 

among each group, up to 53.5% of the total variation was explained by 

the first two eigenvectors, which accounted for 37.4% and 16.1%, 

respectively. Melon populations were mostly separated into small- and 

large-seed types by the first principal axis (Fig. 2). In contrast, by the 

second principal axis, populations were separated by the area of origin; 

one group of Africa, Middle East, and East Asia and another group of 

India and Southeast Asia. Interestingly, the small-seed type of East 

Asia and South Asia appeared close to that of southern Africa on the 

PCO-plot, though these populations were clustered rather distantly by 

UPGMA methods. The close relationship can be seen from GD for the 

small-seed type from southern Africa and small-seed type from India 

and Southeast Asia, which ranged from 0.122 to 0.294 and was 0.166 

on average. 

Genetic relationship among the 291 samples was visualized by 

cluster analysis, and they were classified into seven clusters. The 

number of melon accessions classified into each cluster is summarized 

in Table 1. Cluster II consisted of Group Conomon from East Asia, and 

also included the small-seed type of East India and Southeast Asia. 


PCO 2 

0.6 

0.4 

0.2 

0.0 

-0.2 

× 

-0.4 

-0.6 -0.4 -0.2 0.0 0.2 0.4 

PCO 1 

Fig. 2. Principal coordinate plot of first two axes based on the genetic similarity 

matrix of 27 populations of melon landraces from Africa to Asia, based on 

RAPD. Geographical group of each population is indicated by the following 

symbols: = East Asia; = Southeast Asia, Myanmar, Nepal, and 

Bangladesh; × = Pakistan and the Middle East; = Africa; = India;. 

Black and open symbols indicate small- and large-seed type, respectively. 

Cluster III consisted mostly of the small-seed type (85.7%) of the areas 

from Yunnan in China to Pakistan, and also included small-seed type 

of southern Africa. Cluster V consisted mostly of the large-seed type 

(69.0%) of the areas from Southeast Asia to the Middle East; the largeseed 

type of North Africa was also included. Cluster VII consisted of 

the small-seed type from central and southern parts of Africa and C. 

sativus var. hardwickii. 

Tanaka et al. (2006) analyzed PS-ID sequence of the chloroplast 

genome for 221 accessions among 291 accessions examined in this 

study, and determined their cytoplasmic genotype. Most of the 

accessions of Clusters II and III proved to be of A-type, and their 

proportion was 93.2% and 90.4%, respectively, suggesting that smallseed-type 

accessions of both clusters belong to the same maternal 

lineage, which is different from that of the large-seed type. The results 

summarized in Table 1 indicate that small-seeded melons from 

southern Africa seemed to relate with those from India, as also 

suggested by Mliki et al. (2001). Small-seeded melons from East India 

seemed to relate with Group Conomon from China, Korea, and Japan, 

as also indicated by Yashiro et al. (2005) and Tanaka et al. 

(unpublished). Based on these results, we propose the following 

hypothesis about the transmission route of small and large seed types: 

The A-type with small seeds was transmitted from southern Africa to 


× 

×

Table 1. Number of melon accessions classified into seven 

clusters in each population. 

Country Area 

Seed 

size 

Cluster 

No. of 

accessions I II III IV V VI VII 

Africa 

Algeria North L 1 - - - - 1 - - 

Egypt North L 5 - - - - 5 - - 

Mali North L 1 - - - - 1 - - 

Morocco North L 2 - - - - 2 - - 

Cameroon Center S 2 - - - - - - 2 

Chad Center S 1 - - - - - - 1 

Ghana Center S 1 - - - - - - 1 

Senegal Center S 5 - - - - 1 - 4 

Sierra Leone Center L 1 - - - - 1 - - 

Sudan Center S 1 - - - - - - 1 

South Africa South L 2 - - 1 - 1 - - 

Zimbabwe South S 5 - - 3 - - - 2 

Zambia South L+S 4 - - 1 - - - 3 

Middle East 

Afghanistan L 5 - - - - 3 - 2 

Iran L+S 5 - - 1 - 4 - - 

Iraq L+S 2 1 - - - 1 - - 

Lebanon L 2 - - 1 - 1 - - 

Syria L 2 - - - - 2 - - 

Turkey L 4 - - - - 4 - - 

South Asia 

Nepal L 6 - - 5 - 1 - - 

Maldives S 4 - - 4 - - - - 

Bangladesh L+S 12 - - 10 - 2 - - 

Pakistan S 4 - - 2 - 2 - - 

India West L 10 - - 2 2 4 - 2 

North L 9 - - 2 - 5 2 - 

Center L 6 - - 2 1 3 - - 

South L 10 - - 1 - 8 - - 

East L 13 - 1 3 1 8 - - 

India West S 10 - - 7 1 2 - - 

North S 7 - - 2 2 3 - - 

Center S 10 - - 8 1 1 - - 

East S 24 - 2 18 2 2 - - 

Myanmar L 5 - - 2 - 3 - - 

S 36 - 1 26 1 8 - - 

Southeast Asia 

Indonesia L&S 4 - 1 1 - 2 - - 

Laos L 1 - - 1 - - - - 

Malaysia L&S 2 - - 1 - 1 - - 

Thailand S 6 - - 4 - 2 - - 

Vietnam S 2 - 1 1 - - - - 


Table 1, continued 

Country Area 

Seed 

size 

No. of 

accessions I II III IV V VI VII 

East Asia 

China S 24 - 23 1 - - - - 

China-Yunnan S 5 - - 5 - - - - 

Korea S 9 - 9 - - - - - 

Japan S 15 - 15 - - - - - 

C. sativus var. hardwickii 2 - - - - - - 2 

Total 291 1 48 119 11 73 2 21 

L = large-seed type; S = small-seed type. 

India by the sea route, and the prototype of Group Conomon originated 

from small-seeded melons under wet conditions in East India. 

African melon accessions were divided into three groups by RAPD, 

which classified their respective cytoplasm type as follows: A-type 

(100%) in Cluster III, T-type (82.3%) in Cluster V, and NA-type 

(78.6%) in Cluster VII. Tanaka et al. (2006) suggested that NA is the 

primitive type of cultivated melon, and that the large-seeded T-type and 

small-seeded A-type have been independently established in northern 

and southern Africa, respectively, during the long history of cultivation 

and transmission of melon. The result obtained by the analysis of the 

nuclear genome supports this hypothesis, and suggests that large- and 

small-seed types had a polyphyletic origin, and thereafter differentiated 

into different varieties. Therefore, for further analysis of origin, 

domestication, and differentiation of cultivated melon, more accessions 

of melon collected from various parts of Africa should be analyzed for 

nuclear and cytoplasm genomes. 


Akashi, Y., N. Fukuda, T. Wako, M. Masuda, and K. Kato. 2002. Genetic variation 

and phylogenetic relationships in East and South Asian melons, Cucumis melo 

L., based on the analysis of five isozymes. Euphytica .125:385–396. 

Apostol, B. L., W. C. Black IV, B. R. Miller, P. Reiter, and B. J. Beaty. 1993. 

Estimation of the number of full sibling families at an oviposition site using 

RAPD-PCR markers: applications to the mosquito Aedes aegypti. Theor. Appl. 

Genet. 86:991–1000. 

Bates, D. M. and R. W. Robinson. 1995. Cucumber, melons and water-melons, 

Cucumis and Citrullus (Cucurbitaceae), p. 89–111. In: J. Smartt and N. W. 

Simmonds (eds.). Evolution of crop plant. John Wiley and Sons, New York. 

Fujishita, N., H. Furukawa and S. Morii. 1993. Distribution of three genotypes for 

bitterness of F1 immature fruit in Cucumis melo. Jpn. J. Breed. 43(Suppl.2):206 

(in Japanese). 


Gower, J. C. 1966. Some distance properties of latent root and vector methods used 

in multivariate analysis. Biometrika. 53: 325–338. 

McCreight, J. D., J. E. Staub, A. López-Sesé, and S. Chung. 2004. Isozyme variation 

in Indian and Chinese melon (Cucumis melo L.) germplasm collections. J. Amer. 

Soc. Hort. Sci. 129:811–818. 

Mliki, A., J. E. Staub, Z. Sun, and A. Ghorbel. 2001. Genetic diversity in melon 

(Cucumis melo L.): an evaluation of African germplasm. Genet. Res. Crop Evol. 

48:587–597. 

Nakata, E., J. E. Staub, and A. I. López-Sesé. 2005. Genetic diversity in Japanese 

melon (Cucumis melo L.) as assessed by random amplified polymorphic DNA 

and simple sequence repeat markers. Genet. Res. Crop Evol. 52:405–419. 

Nei, M. 1972. Genetic distance between populations. Am. Nat. 106:283–292. 


New York. 

Silberstein, L., I. Kovalski, R. Huang, K. Anagnostou, M. Kyle, and R. Perl-Treves. 

1999. Molecular variation in melon (Cucumis melo L.) as revealed by RFLP and 

RAPD markers. Sci. Hort. 79:101–111. 



Plant Syst. Evol. 217:313–332. 

Tanaka, K., A. Nishitani, Y. Akashi, Y. Sakata, H. Nishida, H. Yoshino and K. Kato. 

2006. Molecular characterization of South and East Asian melon, Cucumis melo 

L., and the origin of two varieties makuwa and conomon revealed by RAPD 

analysis. (in preparation). 

Tanaka, K., Y. Akashi, H. Nishida, K. Fukunaga and K. Kato. 2006. Polyphyletic 

origin of cultivated melon inferred by the analysis of chloroplast genome. 

Cucurbitaceae 2006. 

Weir, B. S. 1996. Genetic data analysis II. Sinauer Associates, Sunderland, 

Massachusetts. 

Yashiro, K., H. Iwata, Y. Akashi, K. Tomita, M. Kuzuya, Y. Tsumura, and K. Kato. 

2005. Genetic relationship among East and South Asian melon (Cucumis melo 

L.) revealed by AFLP analysis. Breed. Sci. 55:197–206. 


ORIGIN, MORPHOLOGICAL VARIATION, AND 

USES OF CUCURBITA FICIFOLIA, THE 

MOUNTAIN SQUASH 

Thomas C. Andres 

The Cucurbit Network, Bronx, New York, 10471 

Additional Index Words. Fig leaf squash, black-seeded pumpkin, Malabar 

gourd, chilacayote, crop domestication 

Abstract. Cucurbita ficifolia Bouché is the most important domesticated species 

of squash in the highland regions of the Neotropics, including the seven Andean 

countries in South America. In addition to this extensive area of traditional 

cultivation, this species has spread within the last few centuries throughout the 

highland tropical regions of the world. This popularity appears to be due to ease 

of cultivation in cool regions under extreme conditions, both dry and moist. The 

fruits possess no more nutritional or flavor qualities than other cultivated 

Cucurbita species, but they do have a particularly long shelf life. Usage varies in 

different regions, but mostly it is eaten where it is grown under small, 

sustainable agricultural systems. Otherwise, the fruits are sold in local markets. 

More commercial uses occur in Mexico, Costa Rica, Colombia, Ecuador, and 

Argentina, where it is made into confections and jams as well as cooked in 

nonsweetened traditional dishes. The archaeological record shows that it was 

extensively used and traded in pre-Incan times in northern Peru. Morphological 

variation in the species, while comparatively small for a domesticated 

Cucurbita, is greatest in the region from Peru to Colombia, which indicates its 

potential domestication in this area. The wild ancestor is unknown. 

T 

he biosystematics of the cultigen Cucurbita ficifolia Bouché 

has been reviewed (Andres, 1990). Since then, no discoveries 

have been made of a possible wild ancestor of the species. A 

survey of its uses and morphological diversity throughout its range in 

Latin America is reported here, along with evidence from the 

archaeological record, to ascertain where the species may have been 

domesticated. Field investigations have been undertaken over the last 

20 years specifically in Mexico, Panama, Ecuador, Peru, and Bolivia. 

Herbarium specimens from numerous countries have been examined. 

The survey of C. ficifolia in Peru and Bolivia was made possible by the Amy 

Goldman Ancestors of Squashes grant through the New York Botanical Garden. Also 

thanks to Sumru Aricanli and Craig Morris at the American Museum of Natural 

History for their permission to examine the Junius Bird Huaca Prieta archaeological 

collection and to the curators of the New York Botanical Garden, particularly 

Michael Nee, for their assistance in making the field and herbarium work possible. 


Like all other species of Cucurbita, C. ficifolia originated in the 

Americas. It is known only under cultivation or as recent escapes. Its 

wild ancestor has been hypothesized to be from southern Mexico 

(Sauer, 1969) or the Andes (Nee, 1990). Present-day distribution 

ranges from northern Mexico to central Chile and northern Argentina, 

or from 30° north to 30° south latitude. 

Elsewhere in the world, it is most frequently grown in Eurasia, 

Africa, and Oceania in the following countries: Portugal, France, 

Germany, India, China, Japan, Korea, Ethiopia, Kenya, Tanzania, 

Angola, New Zealand, and the Philippines. It is planted elsewhere 

primarily as a curiosity or ornamental. 

In Latin America, the common names most frequently used are 

chilacayote (Mexico and Guatemala), chiverre (Costa Rica and 

Honduras), victoria (Colombia), zambo (Ecuador), chiclayo (Peru), 

lacayote (Bolivia), and cayote (Argentina). But perhaps the most 

descriptive name is calabaza serrana in Peru, which means “mountain 

squash.” The common name most applied in English is “fig leaf 

squash,” derived from the scientific specific epithet. But since this leaf 

shape is not unique among Cucurbita as the extreme high altitudes 

where it is cultivated, perhaps mountain squash makes a better name. 

Another frequently used English name for C. ficifolia is “Malabar 

gourd,” derived from the once-held mistaken belief that it originated in 

India. Portuguese trade routes in the 16 th and 17 th centuries extended 

between the Americas, where C. ficifolia was collected, and both India 

and Europe, where it was introduced. The Malabar region includes wet 

tropical mountains where C. ficifolia is still grown. 

There are no production statistics on C. ficifolia since it is typically 

grown on a small, home-garden scale as landraces, i.e., locally adapted 

and propagated populations by traditional farmers. Due to their 

aggressive growth habit, they are often planted on marginal 

agricultural land, such as steep embankments, not suitable for other 

crop plants. There is little need to attend to the plants during their long 

growing season of six to eight months. Therefore, C. ficifolia is 

commonly dismissed as an unimportant crop plant. But throughout the 

Andes, it is widely grown and popular in most communities lying 

between 1000 and nearly 4000m in altitude. It is more tolerant of 

cooler temperatures than the other domesticated species of Cucurbita, 

although all Cucurbita are frost-tender. 

The plants seem to do best where conditions are moist, particularly 

in cloud forests. In such habitat, the fruits, flowers, and vines reach 

their greatest size. But at the other extreme, they are also cultivated in 

arid regions where there may be only a brief wet season just enough 

for stand establishment. Fruit size varies from as short as 12cm under 


dry conditions to 60cm long and weighing 20kg in cloud forests; 

roadside vendors have reported specimens of up to 100cm long. It has 

not been ascertained whether this difference in fruit size is strictly 

environmentally induced or whether there are genetic differences 

between populations. All C. ficifolia fruits have a very hard or 

lignified rind. The flesh is fibrous and nearly white. These 

characteristics are typical for wild Cucurbita species and primitive 

landraces; yet C. ficifolia also has some of the largest pollen grains and 

flowers in the genus. One possibility is that there is no wild population 

that differs genetically from the cultivated plants. This seems unlikely 

since all other wild Cucurbita species are known to have fruits with 

bitter flesh, and the plants are never as big in leaf size, stem, flower, 

and fruit. 

Morphological diversity is small for a domesticated plant, 

especially compared to the other domesticated Cucurbita species. The 

fruit shape is nearly round to somewhat elongate like a watermelon. 

There are basically three fruit coloration patterns, (1) all white, (2) a 

distinct reticulated green/white pattern, and (3) dark green. Ten white 

longitudinal stripes spreading from the floral end are usually present in 

the latter two (Figure 1). The three fruit colorations occur not only 

throughout its range, but also generally in the same field. In Costa 

Rica, large fields are sometimes planted with only all-white fruits. The 

seeds are most commonly black or dark brown, a color not found in 

any other species of Cucurbita. But tan seeds, more typical of 

Cucurbita, also occur throughout its range. The genetics of these 

differences have not been studied nor has the plant undergone any 

modern breeding. 

In my survey, other fruit characteristics were found. In Peru and 

Bolivia, uniform light green fruits were seen, in addition to all white 

and all dark green fruits. A unique fruit shape was found in northern 

Peru. A few populations had elongated fruits that were bottle-shaped 

or somewhat pyriform with a length to diameter ratio of close to three 

(Figure 1). Cárdenas (1989) reports seeing “victoria” squashes with 

“necks like a bottle” in Colombian markets. No other reports in the 

literature record the occurrence of this fruit shape in C. ficifolia. It is 

not known how widespread this morphology is in northwest South 

America and whether it represents a primitive character or derives 

from an ancestral population. 

The archaeological record is sparse, except in Peru where abundant 

pedicels and seed fragments dating back to 5000–6000 B.C. are found 

along the central and northern coast. While C. ficifolia is not grown in 

this lowland region, trade between the Andean cultures and the coastal 

people were evidently widely practiced (Cohen, 1977). Still today, C. 


ficifolia is brought down from the highlands and sold in coastal 

markets, such as at Trujillo (Figure 2). Since the coastal region is arid, 

ancient plant remains are well preserved. Archaeological sites in the 

highly humid cloud forests are less likely to contain any plant 

macromaterial. There is no apparent change to a smaller seed size in 

the earlier archaeological strata, thus suggesting that the imported 

squash was always a domesticate. There are reports that the Aztecs 

used C. ficifolia during the Spanish conquest of Mexico (Sahagun, 

1956), but no definitive archaeological remains have been found north 

of South America. 

Fig. 1. Comparison between a typical nearly round-shaped fruit and a white 

elongated pyriform or bottle-shaped fruit from the Department of Cajamarca, 

Peru. 

The present-day uses of C. ficifolia are diverse and vary in 

different regions. The most popular is in the preparation of jams, 

puddings, and confections, or dulce, made by cooking the pulp of the 

mature fruit in sugar or honey, for example, dulce de cayote in 

Argentina (Figure 3). There are many variations on this recipe ranging 

from the addition of mineral lime to make solid candies to teasing 

apart the spaghetti squash (Cucurbita pepo)-like fibers by boiling to 

make a dessert called cabello de angel—angel’s hair—in Spanish. 

Other ingredients, including coconut, tamarind, and cream, are often 


added to these desserts. The sweetened products are used in 

empanadas and other baked products in South America. 

Other uses of the mature fruits are for making an alcoholic drink 

and for adding to stews or picadillos and soups. In regions of more 

recent introduction, such as parts of Africa, the long-lasting fruits are 

considered unfit for human consumption but are fed to livestock 

during the dry season. Elsewhere, most notably in the Chiriquí 

province of Panama, C. ficifolia is not used at all; however, the plants 

persist in weedy vegetable fields, suggesting that it was once used 

there. 

The large protein-rich seeds are popularly consumed in a few 

regions, including Ecuador and China, but ignored elsewhere. They are 

used along with honey in Chiapas, Mexico to make palanquetas, a 

dessert snack. 

Nearly as popular as the mature fruits are the tender young fruits 

(Figure 2), which are consumed like summer squash (C. pepo) 

throughout Latin America and the Philippines. These fruits are said to 

fetch a good price in Bogotá (National Research Council, 1989). Other 

sporadic uses are made of the flowers and as a green leafy vegetable. 

In Japan, Germany, and the Netherlands, cucumber is grafted onto 

rootstock of C. ficifolia for winter production in greenhouses. 

Fig. 2. Diversity of immature C. ficifolia fruits in the main market in Trujillo, 

Peru. 


The most commercial use occurs in southern Mexico, Costa Rica, 

Colombia, Ecuador, and northern Argentina. In Costa Rica, C. ficifolia 

is an Easter holiday staple, commemorated by fairs, such as Feria del 

Chiverre. Up to 20-hectare plantations are cultivated. 

While the fruits do not possess as many nutritional benefits as 

other cultivated Cucurbita species, or any notable flavor qualities, they 

do have an especially long shelf life of well over a year. During this 

postharvest period, the flesh becomes sweeter. This long storage 

period makes the fruits useful during times of need, such as during 

long dry seasons and ocean voyages, a property that may have 

facilitated their spread throughout pre-Incan and Aztec empires. 

This survey of C. ficifolia throughout Latin America broadens our 

understanding of the biology and uses of this species. Not only is C. 

ficifolia widely cultivated throughout the highlands of the Neotropics 

for a wide range of uses, but it is a more important crop than generally 

recognized. It should perhaps be renamed the mountain squash. 

Fig. 3. Cucurbita ficifolia jam (left) and candy with walnuts (right) from the 

Province of Jujuy, Argentina. 

Evidence from the archaeological record and the geographical 

distribution of the limited morphological variation supports the 

hypothesis that domestication first took place on the eastern slope of 

the northern Andes in northwestern South America. This region 

represents the latitudinal midpoint of the range of C. ficifolia in Latin 

America. Also, this region contains one of the most species-rich 


endemic floras of the world. Since this terrain is quite rugged with 

complex, often inaccessible microenvironments, it is quite possible 

that wild ancestral populations still occur there. 


Andres, T. C. 1990. Biosystematics, theories on the origin, and breeding potential of 

Cucurbita ficifolia. p. 102–119. In: D. M. Bates, R. W. Robinson, and C. Jeffrey 

(eds.). Biology and utilization of the Cucurbitaceae. Cornell University Press, 

Ithaca, New York. 

Cárdenas, M. 1989. Manual de las plantas economicas de Bolivia. 2 nd Edition. Los 

Amigos del Libro, La Paz, Bolivia. 

Cohen, M. N. 1977. Population pressure and the origins of agriculture: an 

archaeological example from the coast of Peru. In: C. A. Reed (ed.). Origins of 

agriculture. Mouton, The Hague. 

Nee, M. 1990. The domestication of Cucurbita (Cucurbitaceae). Econ. Bot. 44:56– 

68. 

National Research Council. 1989. Lost crops of the Incas: little-known plants of the 

Andes with promise for worldwide cultivation. National Academy Press, 

Washington, DC. 

Sahagun, F. B. 1956. Historia General de las Cosas de Nueva España. Tom. 1, Lib. 

1, Cap. 21:13. A. M. Garibay K. Editorial Porrua, Mexico City. 

Sauer, C. O. 1969. Agricultural origins and dispersals. M.I.T. Press, Cambridge, 

MA. 


LOCHE: A UNIQUE PRE-COLUMBIAN 

SQUASH LOCALLY GROWN IN NORTH 

COASTAL PERU 

Thomas C. Andres 

The Cucurbit Network, Bronx, New York 10471 

Roberto Ugás and Fiorela Bustamante 

Programa de Hortalizas, 

Universidad Nacional Agraria La Molina, Lima, Peru 

ADDITIONAL INDEX WORDS. Cucurbita moschata, zapallo, vegetative 

propagation, seedlessness 

ABSTRACT. The loche is a high-quality landrace of Cucurbita moschata 

Duchesne grown only on the north coast of Peru and practically unknown 

elsewhere. Moche pottery depicting the fruit shows that it has been used for 

thousands of years, yet it is not a primitive landrace. The fruits today, indicated 

as well by the archaeological record, have some morphological diversity. 

Typical loche fruits are small, straight, and warted along longitudinal ridges 

with nonlignified rinds. They contain few to no seeds, and the fruits vary 

respectively in having a slightly swollen end to being tapered at both ends. The 

orange flesh is very high in total soluble solids and is used only in small amounts 

for flavoring traditional regional dishes. In the local markets, loche is sold in 

cut-up pieces at premium prices. The entire fruit, rind included, is consumed. 

Propagation using shoot cuttings has been practiced for at least the past 100 

years. Fruits from such plants are considered more desirable than those from 

direct-seeded plants. This unique crop is at risk of being displaced by industrial 

agriculture of export crops, but at the same time plays a clear role in the revival 

of Peruvian gastronomy within the country and abroad. 

L 

ittle is known about the highly regarded landrace of squash 

from the north coast of Peru called loche or zapallo loche. The 

literature is sparse except for mention of loche in publications 

Renán Valega Rosas, Michael Nee, Victor Vásquez, and Teresa Rosales all helped 

with fieldwork, while Miguel Holle, Abundio Sagástegui, Eric Rodríguez, Karen E. 

Stothert, Dolores Piperno, and Craig Morris were important consultants. Rolando 

Santa Maria provided reproductive material and extremely useful loche horticultural 

information. Thanks also to Pilar Kuriyama and Cecilia Ono for their kind help in 

processing the fruits and seeds. Special thanks to the Amy Goldman “Ancestors of 

Squashes” grant through the New York Botanical Garden for making this study 

possible, and to the Vegetable Crops Research Program of UNALM and the project 

for the conservation of Andean plant genetic resources executed by UNALM and the 

Council of Francophone Universities (CIUF) of Belgium, for their support of 

fieldwork in Illimo and La Molina. 


about the distinct gastronomy of the region. Even there, it is usually 

assumed that the reader knows what a loche is, and therefore no 

description is given. Some recipes state that if loche is not available, 

other orange-fleshed squashes (in most South American countries 

called zapallo) may be substituted. Even more confusingly, the 

meaning of the word “loche” has become more generalized to apply to 

any desirable orange-fleshed squash, especially if the seller does not 

have any good loches. For instance, along the central coast of Peru, as 

early as 1957, a crookneck squash with a dark green, smooth rind was 

called loche (Diaz, 1957). Therefore, for clarification, adjectives are 

sometimes applied, such as “true loche” or “black loche” when 

referring to the traditional genuine loche. 

Loche is listed on Web sites and in literature as being either 

Cucurbita maxima Duchesne (Soukup, 1987; Custer, 2000) or 

Cucurbita moschata Duchesne, but there is a common understanding 

in Peru that all loches are C. moschata (Diaz, 1957; Ugás et al., 2000). 

Both species are cultivated in the region. And both have been found in 

local archaeological sites, although C. moschata, along with Cucurbita 

ficifolia Bouché, is much more abundant in earlier layers back to 5000 

B.P. The traditional cultivar of C. maxima, called ‘macre’, with fruits 

that can weigh up to about 80kg, is by far the most cultivated squash in 

Peru. 

Methods 

Starting in 2003, in both northern Peru and in Lima, we visited 

marketplaces where loches are sold. We interviewed farmers in loche 

fields and cooks in restaurants in northern Peru. We also visited 

national and regional archaeological museums that contain ceramic 

pieces identified as depicting the loche. Fieldwork has begun at the 

Vegetable Crops Research Program of Universidad Nacional Agraria 

La Molina (UNALM), Lima, Peru to observe and describe the growth 

habit and diversity of loche under uniform field conditions, both from 

seed and from shoot cuttings. 


The shape of the leaves and flowers, seed morphology, and 

indumentation of the loche show that it belongs to C. moschata. The 

leaves are unlobed or shallow-lobed. The male flower hypanthium is 

campanulate rather than conical as in C. maxima. The seeds are typical 

of light-colored C. moschata seeds, with a slightly coarse margin of a 

color a shade darker. The indument along the primary nerves on the 


abaxial surface of the leaf blade as well as on the petiole and on the 

floral bud is short to long pilose or villous and soft. 

The one character that is not typical of other C. moschata is the 

shape of the pedicel attachment to the fruit. This species typically has 

a rounded expanded pedicel where it attaches to the fruit, but in loche 

the shape of the pedicel varies from typical C. moschata shape to not 

expanded, but extending along its ridges down a couple of centimeters 

of the fruit. This may be due to the shape of the fruit base, which is 

acute rather than flattened or rounded like most cultivars. 

Loche plants are viny, but have smaller parts than most other C. 

moschata cultivars, including the leaves, flowers, and fruits. The fruits 

are up to 1.2kg, 20 to 30cm long and 10cm wide, oblong to oblate, and 

not crooknecked, but sometimes shaped like butternut squashes with a 

bulbous end. They start out light green but gradually turn to dark green 

with a glaucous waxy covering at maturity. A diagnostic character is 

10 loosely defined warty longitudinal ridges that are often a darker 

shade than the rest of the fruit and thus make the fruit subtly striped. 

The rind is nonlignified, which is unusual for warty Cucurbita fruits. 

The flesh is various shades of orange, but never as deeply orange 

as some other local landraces of C. moschata. But the total soluble 

solids or sugar content has been measured with a handheld 

refractometer to be 18°Brix, which is extremely high for any cucurbit. 

The flavor is excellent. 

Loche is unique to the species in having naturally occurring 

seedless fruits. Fruits that are tapered at both ends are seedless and 

solid, whereas those with a bulbous end generally contain a small seed 

cavity with up to 20 fully developed seeds and additional undeveloped 

seeds (Figure 1). The seedless fruits with their soft rinds are consumed 

in their entirety with no waste. However, the bulbous fruits are 

considered to be of higher quality. 

In general, seedlessness in edible fruits is becoming more 

widespread and more popular. This is the trend as well with cucurbits, 

such as watermelon, cucumber, and some summer squash. In winter 

squash, except for minor exceptions, there are no seedless commercial 

cultivars. Some butternut cultivars have small seed cavities containing 

a decreased number of seeds. Commercially produced interspecific 

hybrids between C. maxima and C. moschata may lack seeds. 

The cause of infertility in loche is not known. In the field, loche 

plants are monoecious and develop normal-looking male and female 

flowers with abundant pollen on the stamens. There are many more 

male flowers than female flowers per plant. An abundance of bee 

activity in the morning transfers pollen between male and female 

flowers so that stigmas are covered with pollen by midmorning. 


Fig. 1. Longitudinal section through a loche fruit showing the seed cavity at the 

apical end and the total number of seeds in the fruit, consisting in this case of 

four or five probably viable seeds (the darker colored seeds to the left) and four 

aborted seeds to the right. 

Pollination, therefore, does occur naturally of both selfs and sibs. Fruit 

set and productivity appear to be comparable to seed-bearing winter 

squash of the region. But the plants in loche fields are usually not 

uniform, with fruits varying somewhat in size, shape, color, and 

wartiness, thus indicating some genetic diversity. Therefore, a theory 

among growers that the loche is a sterile hybrid does not seem 

plausible, but needs to be tested further. It is not known whether the 

fruits are parthenocarpic or the ovules abort after fertilization. 

Another unique feature of loche is that it is commercially 

reproduced vegetatively. Shoot-tip cuttings of around 50–70cm long 

are made after the fruit is harvested. These are planted and watered, 

and roots grow from the underground nodes where the leaves have 

been removed. If the dry season is particularly severe, not only may a 


crop season be lost, but no further cuttings will be available. Saved 

seeds are then sowed. But the fruit quality of this first generation is 

considered inferior and generally not harvested. Cuttings are made 

from this generation to produce more-desirable fruits in subsequent 

generations. To avoid direct seeding, loche farmers cooperate by 

trading cuttings. Sometimes a few irrigated “mother plants” of a region 

are used solely as a backup source for cuttings. The difference between 

direct-seeded plants and those propagated asexually is being 

investigated at UNALM. 

The practice of vegetative propagation for improving quality of the 

loche has been in use at least since the late 19 th century, according to 

the notes of Enrique Brüning, a German engineer whose private 

archaeological collection is now housed in the Brüning Museum, 

Lambayeque (Schaedel, 1988). Brüning also reported that growers 

would burn a stone shaped like the fruit in the pile of soil where loche 

was planted as an amulet to encourage growth. 

The main pest problems in loche are pickleworm (Diaphania sp.) 

and whitefly (Bemisia sp). Insecticides are commonly applied. The 

most common diseases appear to be wilt (Fusarium sp., Phytophthora 

sp.) and fruit rot (Phythium sp). Watermelon mosaic virus (WMV) has 

been isolated from loche plants, and its symptoms are often observed 

in the field. Loche is apparently highly susceptible to root-knot 

nematode (Meloidogyne incognita). 

Because of the high value of the crop, loche fields are often kept 

hidden from roads. There is a problem with poachers stealing not only 

the fruits, but also stems for transplanting. The fields range in size 

from backyard gardens to a few hectares (Figure 2). Loche is 

cultivated almost exclusively in the lowland valleys of northern Peru 

in the departments of Lambayeque, Piura, and La Libertad. The 

climate is temperate to hot in the north, with no rain except during a 

possible short rainy season or when El Niño occurs. Surprisingly 

under such arid conditions, irrigation is normally not used. The fruits 

are sold in local markets and in Lima. Loche is neither 

grown in nor exported to neighboring countries. 

The most productive region lies around Chiclayo, the capital city 

of Lambayeque. In the past, this was a major trade center between the 

coast and the highlands and the Amazon beyond, as well as settlements 

to the north and south. The area is rich in important pre-Inca 

archaeological sites where Cucurbita remains have been found. 

Unfortunately, the loche, unlike other squashes, does not preserve 

well. Squash seeds, pedicels, and rind macrofragments are often found 

well preserved in the dry coastal archaeological sites. But the loche 

produces few seeds; the pedicels are weak and do not separate from 


Fig. 2. Rolando Santa María, expert grower from Illimo, Lambayeque, Peru, in 

his field of loche. 

the decaying fruit, and the rinds are nonlignified. Microremains of 

squash rind phytoliths are abundant in these sites, but there are no 

phytoliths in loche rind. Reevaluation by Andres and Nee 

(unpublished) since the study of Whitaker and Bird (1949) of the 

thousands of Cucurbita macroremains from Huaca Prieta, Chicama 

Valley, Peru, where some of the earliest such specimens have been 

found in South America (now housed at the American Museum of 

Natural History in New York), show that some of these pedicels 

resemble the distinct pedicels of loche. 

A better source of information in archaeological records comes 

from the Moche people, a pre-Inca culture that flourished in the coastal 

valleys of northern Peru approximately A.D. 50–800. They were highly 

skilled in ceramics and left a legacy in tombs of realistic life-sized fruit 

and vegetable sculptures, as well as depictions of all aspects of their 

daily life. Whilst farming in the region using desert irrigation began in 

pre-Moche times, the Moche ceramics show that they had a rich 

assortment of crop plants, including a few species of cucurbits 

composed of a diversity of landraces. The most common squash 

depicted is a warty crooknecked C. moschata. But there are some 

ceramic fruits that are identical to loche. Figure 3 shows one such fruit, 

photographed at the Universidad Nacional de Cajamarca-Jaén, found 


at Batan Grande, an important archaeological site located only 40km 

northeast of Chiclayo, where in the adjacent picture the fresh fruits in 

the main market were photographed. This is evidence that the loche 

has an ancient origin. The warty crooknecked squashes are also found 

in the region today, as well as other landraces depicted in the hundreds 

of surviving Moche ceramics. 

Fig. 3. Comparison between an archaeological ceramic loche fruit (left) and 

present-day seedless and seeded fruits (right). 

Brüning (Schaedel, 1988) considered the loche semiwild, and 

believed that it was crossed with cultivated squashes to produce a 

more appetizing fruit. There is no known wild Cucurbita in the region 

today except for C. ecuadorensis H. C. Cutler & Whitaker. 

Furthermore, except for its relatively small size, loche does not have 

any primitive characteristics of a wild cucurbit. On the contrary, the 

following characterististics indicate an advanced state of 

domestication: reduced to no seed cavity, fruit that does not readily 

“slip” or abscise from the vine, no lignification of the rind, and 

nonbitter flesh. It is widely believed that squash was domesticated 

initially for the edible seeds since all wild species of Cucurbita have 

large seed cavities full of numerous seeds, with a small amount of 

bitter flesh, and hard rinds. Although this may be true in North and 

Central America, south of Ecuador, Cucurbita seeds are rarely 

consumed and were therefore probably never an important source of 

food, although now they are commonly used in traditional medicine. 


Area under loche production appears to be decreasing, although the 

word itself, being misapplied to a broader range of cultivars, is in more 

frequent use. The agriculture of the region is shifting to industrialscale 

crop production mostly for export, primarily of rice, sugarcane, 

cotton, asparagus, mango, and Capsicum peppers. 

Like the loche itself, the highly prized cuisine of northern Peru 

deserves to be better known elsewhere. The dishes include a rich 

assortment of ingredients, including a number of endemic crop plants. 

The loche is used in small amounts to flavor desserts, soups, and 

stews, most notably the traditional regional dish made with kid goat 

called seco de cabrito a la norteña. In the local markets, loche is sold 

in cut-up pieces at premium prices; for example, in April 2006, prices 

in supermarkets in Lima were 16.50 soles per kg versus 2.5 soles per 

kg for macre (C. maxima). Loche is one of the landmarks of trendy 

“nouveau Andean cuisine” and, because of its distinctive taste, is an 

ingredient in new dishes oriented to international palates. For instance, 

in a recent gastronomic showroom in Vienna organized by the 

government of Peru, European chefs highlighted loche and 

‘escabeche’ chili pepper (Capsicum baccatum var. pendulum) as their 

most remarkable discoveries. 

A broader effort should be made to promote traditional dishes from 

northern Peru incorporating loche, increasing demand for it, so that 

more farmers will grow it. Northern Peru is expected to become a 

major tourist area in the near future because of major archaeological 

discoveries, new museums, and beach hotels, as well as an interesting 

endemic living culture. This will contribute to new demand for loche 

and other local crops. The loche deserves study from ethnobotanical, 

taxonomic, and horticultural standpoints. 


Custer, T. 2000. The art of Peruvian cuisine. Ediciones Ganesha, Lima. 

Diaz, A. 1957. Estudio de las variedades nacionales de zapallos. Tesis para Ingeniero 

Agrónomo, Escuela Nacional de Agricultura, Lima. 

Schaedel, R. P. 1988. La etnografía muchik en las fotografías de H. Brüning,1886– 

1925. Corporación Financiera de Desarrollo, Lima. 

Soukup, J. 1987. Vocabulario de los nombres vulgares de la flora peruana y catálogo 

de los géneros. Editorial Salesiana, Lima. 

Ugás, R., S. Siura, F. Delgado de la Flor, A. Casas, and J. Toledo. 2000. Hortalizas, 

datos básicos. Programa de Hortalizas, Universidad Nacional Agraria La 

Molina, Lima. 

Whitaker, T. W. and J. B. Bird. 1949. Identification and significance of the cucurbit 

materials from Huaca Prieta, Peru. Amer. Mus. Novit. 1426:1–15. 


OLD WORLD CUCURBITS IN PLANT 

ICONOGRAPHY OF THE RENAISSANCE 

Jules Janick 

Department of Horticulture & Landscape Architecture,Purdue 

University,625 Agriculture Mall Drive, West Lafayette, IN 47907-2010 

Harry S. Paris 

Agricultural Research Organization, Newe Ya’ar Research Center, 

P. O. Box 1021, Ramat Yishay 30-095, Israel 

ADDITIONAL INDEX WORDS. Citrullus lanatus, Cucumis melo, Cucumis sativus, 

Ecballium elaterium, Lagenaria siceraria, Momordica balsamina, plant 

iconography 

ABSTRACT. Old World cucurbits include Citrullus lanatus (watermelon), 

Cucumis melo (melon), Cucumis sativus (cucumber), Ecballium elaterium 

(squirting cucumber), Lagenaria siceraria (bottle gourd), and Momordica 

balsamina (balsam apple). Fruit images of each of these cucurbits appear in 

festoons painted on a ceiling of the Villa Farnesina in Rome between 1515 and 

1518. These cucurbits are also depicted in paintings and botanical herbals of the 

Renaissance. These images provide information on cucurbit history, dispersal, 

and genetic diversity. 

I 

n 1505, one of the richest men in Europe, the Sienese banker 

Agostino Chigi (1466–1520), began construction of an extravagant 

new home on the west bank of the Tiber River in Rome. Although 

not particularly well-educated, Chigi was attracted to the rediscovery 

of ancient Greek and Latin writings and science. The decorations in his 

villa, now known as the Villa Farnesina because it was sold to 

Cardinal Farnese by Chigi’s heirs, were a revocation of the classical 

world, the rooms filled with paintings, statues, and opulent 

furnishings. Attached was a repository or garden of rare plants, the 

viridarium. The ceiling of one room, decorated with scenes of the 

heavenly adventures of Venus, Cupid, and Psyche, was executed in 

fresco in the spaces between the arches by the workshop of Raphael 

Sanzio (1483–1520). Festoons or wreaths painted by Giovanni 

Martini da Udine (1487–1564) on the ribs of the arches contain 

thousands of images of individual fruits, vegetables, and flowers 

encompassing about 170 species (Caneva, 1992a,b; Janick and 

Caneva, 2005; Janick and Paris, 2006). The festoon images have been 

deconstructed by scanning the images and collating each species. Our 

objective is to describe and discuss the Old World cucurbits in the 

ceiling festoons of the Villa Farnesina and compare them with other 


Renaissance images of these plants appearing in art and botanical 

herbals. 

Watermelon, Citrullus lanatus (Thunb.) Matsum. & 

Nakai 

There are five images of watermelons in the festoons of the Villa 

Farnesina (Figure 1A). All of the depicted fruits are round, medium 

green with narrow darker stripes. Four appear to be “icebox” size, 

Fig. 1. Old World cucurbit images from the Loggia of Cupid and Psyche ceiling 

of the Villa Farnesina: (A) Citrullus lanatus. (B) Cucumis melo Cantalupensis 

Group. (C) Cucumis melo Reticulatis Group. (D) Cucumis melo Flexuosus 

Group. (E) Cucumis sativus. F. Ecballium elaterium. (G) Lagenaria siceraria 

(bottle-shaped). (H) Lagenaria siceraria (elongate). (I) Momordica balsamina. 


about 4kg, and one is smaller and covered by what appears to be white 

fungal growth. Overall, little genetic variability is depicted. 

The festoon images in the Villa Farnesina appear to be the first 

Renaissance paintings of watermelons. Depictions of watermelon 

fruits are common in 16 th - and 17 th -century European paintings and 

appear to be similar in size, shape, and rind coloration to those of the 

Farnesina festoons. These include a watermelon fruit in the frieze of 

the chamber of Dominio Fiorentino painted by Jacopo Zucchi circa 

1586 (Janick, 2004a), Caravaggio’s 1603 painting Fruits and 

Vegetables on a Ledge (Janick, 2004b), and a painting of Pensionante 

del Saracceni titled Still Life with Melons and Carafe of White Wine, 

painted between 1615 and 1620. Black-and-white images of 

watermelons are found in the printed botanical herbals of the 16 th 

century. These images do not show rind color or pattern but do include 

the foliage, allowing them to be easily identified as watermelons by 

the characteristic pinnatifid leaves. Such images can be found in the 

tomes of Fuchs (1542), Bock (1546), Dodoens (1554), Mattioli (1558), 

du Pinet (1561), L’Obel (1576), and many others. 

Melon, Cucumis melo L. 

Sixteen melons from three cultivar-groups (Pitrat et al., 2000) are 

depicted in the festoons of the Villa Farnesina, reflecting great genetic 

diversity. Represented are 11 cantaloupes (Cantalupensis Group), three 

muskmelons (Reticulatus Group), and two snake melons (Flexuosus 

Group) (Figure 1B, C, D). 

The 11 cantaloupes represent four different cultivars, all of which 

have distinct longitudinal furrows. One of them, represented by four 

fruits with dark green furrows alternating with tan lobes, resembles 

‘Cantalun’ (syn. ‘Charentais’), the leading market type of melon in 

France and some other countries. Three of the four fruits are split at 

the furrows, revealing their deep orange flesh (mesocarp). A second 

cultivar, represented by five smaller, round to slightly oblate fruits, 

lacks the dark green coloration in the furrows, resembling ‘De 

Bellegarde’ (Goldman, 2002). One of these five fruits has a split at the 

peduncle end that reveals orange flesh. A third cantaloupe cultivar, 

represented by one oval fruit beginning to rot at the split calyx end, is 

dark green splashed with yellow, revealing orange flesh, and 

resembles ‘Noir des Carmes’. The fourth cantaloupe cultivar, 

represented by two dark green, warted fruits, one large and one small, 

resembles the ‘Black Rock’ melon (Stuart, 1984; Paris and Janick, 

2005). 


The three muskmelons are most easily distinguished from the 

cantaloupes by their reticulate (netted) rind. Of the three muskmelons 

depicted in the festoons, two are oblong and the other quite oblate, 

indicating that two distinct cultivars or market types are represented. 

Although the two oblong melons differ in external color, one having 

an orange cast and the other yellow-green, this color difference might 

be attributable to different stages of fruit maturity, the orange one 

being fully ripe or overripe. The oblate melon has a section cut out, 

revealing orange flesh. 

The snake melons are represented by two images. One of them 

shows clearly that the fruit has a warty skin, an unusual characteristic 

for this cultivar-group. 

Warty cantaloupes appeared in an Italian painting from 1580, 

Fruttivendola (Fruit Seller) by Vincenzo Campi (Paris and Janick, 

2005). Cantaloupes appear, in black and white, in many of the early 

Renaissance herbals, including those of Fuchs (1542), Bock (1546), 

Dodoens (1554), Cordi (1561) depicting two different cultivars, 

L’Obel (1576), Camerarius (1586), and many others. Muskmelons 

seem to have been introduced into Europe later than the cantaloupes. 

An illustration of what appears to be a long oval muskmelon appears 

in Tabernaemontanus (1591) and in Gerard (1597) as “Spanish 

Melons.” Chabrey (1666) presented this illustration too, together with 

an illustration of another netted melon, named Melo Reticulatus (with 

the label and description misplaced on the page). An image of a warty 

flexuosus appeared in the work of Camerarius (1586); this image may 

have been derived from the estate of Conrad Gesner (1516–1565). 

Smooth flexuosus appeared in the herbals of L’Obel (1576), two 

images in Dalechamps (1587), Tabernaemontanus (1591), Gerard 

(1597), Dodoens (1616), and Chabrey (1666). 

Cucumber, Cucumis sativus L. 

Thirteen clusters of cucumber (Figure 1E) are included in the 

festoons, all resembling fruits of cultivars commonly grown today in 

the U.S.A. for pickling. Fruits are prominently warted, short, 

cylindrical but tapering to narrow at the stylar end, straight or slightly 

curved, and some have distinct furrows. Some are completely light 

green, apparently immature. Others are mature (ripe) and completely 

orange and yet others are turning from green to orange. Some of the 

immature fruits show whitening of the stylar end, which extends as 

striping in the furrows (where present) along part of the length of the 

fruit. Many of the fruits show remnants of the corolla at the distal end. 

The slight differences in fruit striping and furrowing suggest the fruits 


were derived from several plants, but this modest variation may have 

been typical of that found within an open-pollinated cultivar. 

Nearly all European Renaissance depictions of cucumbers are of 

the American pickling type, like those of the festoons and those 

sculpted in the ceramics of Luca della Robbia (1399–1482) and 

depicted in 15 th century paintings by Carlo Crivelli of the Madonna 

(Impelluso, 2004). They appear in a painting, Still Life with Flowers, 

Fruits and Vegetables, attributed to the Master of Hartford (thought to 

be the young Caravaggio) and in Caravaggio’s Lute Player (1586). A 

famous Still life by the Spanish artist Juan Sanches Cotán (ca. 1600) 

prominently features cucumbers. Similar sculpted cucumbers, dating 

to 1601, appear on the bronze doors of the Cathedral in the Piazza 

Miracoli in Pisa. Plants bearing similar fruits appear in the works of 

Fuchs (1542), Bock (1546), Dodoens (1554), Mattioli (1558), L’Obel 

(1576), Camerarius (1586), Tabernaemontanus (1591), Gerard (1597), 

and Chabrey (1666). However, one other type of cucumber, much less 

common, appears in Europe during the Renaissance. This is a 

pyriform cucumber, painted between 1505 and 1508 in the single-copy 

prayer book Grandes Heures d’Anne de Bretagne (Bilimoff, 2001). An 

illustration of a cucumber plant bearing pear-shaped fruits also 

appeared in the herbals of Tabernaemontanus (1591), Gerard (1597), 

and Chabrey (1666). Cucumer minor pyriformis (Chabrey, 1666) was 

probably an andromonoecious cultivar, a possible forerunner of 

‘Lemon’. Overall, little diversity is depicted for the cucumbers of 

Renaissance Europe, perhaps indicative of only one or two prior 

introductions. 

Squirting Cucumber, Ecballium elaterium A. Rich. 

One festoon image with five fruits is identifiable as squirting 

cucumber (Figure 1F). Used as a medicinal plant in Roman antiquity, 

this cucurbit was occasionally cultivated for this purpose in 

Renaissance Europe (Jeffrey, 2001). Frequently encountered growing 

wild in Mediterranean countries, it is unusual for a wild cucurbit 

because it has a bushy rather than a viney growth habit. The fruits of 

squirting cucumber are small, light yellow-green ovals, approximately 

2 or 3cm long, five-furrowed, and stiffly hairy. We have not found 

images of squirting cucumber in Renaissance paintings. Depictions of 

squirting cucumber appeared in the herbals of Fuchs (1542), L’Obel 

(1576), Dodoens (1616), Gerard (1597), and others. 


Bottle Gourd, Lagenaria siceraria (Molina) Standley 

Bottle gourds are the most abundant cucurbit images in the 

festoons (Figure 1 G, H), with at least 37 fruits depicted. Three 

distinct morphological variants of bottle gourd—large bottle-shaped, 

small bottle-shaped, and elongate—are depicted,. 

The large bottle-shaped type is represented by portraits of eight 

fruits. Some are light gray-green whilst others are tan, the latter 

probably reflecting more advanced fruit maturity. Three images are 

associated with white flowers, typical of the genus. There are 

differences in shape: three have a short squat neck, four have a 

pinched neck, and one has a thin tapering neck. One fruit shows 

splitting at the calyx end, while another, a mature, yellowed fruit, is 

covered with dark fungal spots and shows an oblong slit on the surface 

displaying yellow flesh. 

The small bottle-shaped type is represented by seven fruits. They 

are tan, tan-orange, or green-yellow, again probably reflecting 

differing fruit maturity. 

The elongate, cocuzzi type is represented by 23 fruits, most of 

which are slightly curved, but one is quite contorted. Unlike the bottleshaped 

types, which are used as vessels, utensils, and a variety of other 

purposes, fruits of the cocuzzi type are consumed when young and 

tender. There are differences in shape among the 23 fruits, suggesting 

that some of them might have been hybrids between the elongate and 

bottle-shaped types. At least 4 fruits are associated with white flowers. 

One of them, the well-known phallic representation of an elongated 

gourd penetrating a fig, is based on an imaginary cocuzza. Two others 

depict the gourds wrapped with strands of red currant berries (Ribes 

rubrum), a not-too-subtle erotic association of the serpentine gourd 

with the worship of Priapus (Morel, 1985). After completing the 

images in the Villa Farnesina, Raphael and Giovanni da Udine, at the 

request of Leo X, decorated a loggia of the Vatican, including many 

cocuzzi in the borders. This was repeated in the Villa Medici by 

Jocobo Zucchi and the Villa d’Este in Tivoli (Morel, 1985; Janick, 

2004a). 

Each of the three fruit forms of bottle gourds depicted in the 

festoons appeared in printed botanical herbals of the 16 th century, 

among them those of Fuchs (1542), Bock (1546), Dodoens (1554), 

Tabernaemontanus (1591), and Gerard (1597). La Zucca nella Natura 

Morta dal Cinquecento al Novecento (Ravelli, 2004) contains 

reproductions of a number of Renaissance artworks depicting longfruited 

Lagenaria siceraria. Caravaggio’s 1603 painting Fruits and 

Vegetables on a Ledge also includes a cocuzza (Janick, 2004b). An 


early color depiction of a bottle-shaped gourd, painted between 1505 

and 1508, appeared in the Grandes Heures d’Anne de Bretagne 

(Bilimoff, 2001). There are a number of depictions of L. siceraria, 

both bottle-shaped and elongate, from Europe, Asia Minor, and North 

Africa, that considerably antedate these. 

Balsam Apple, Momordica balsamina L. 

Four images of balsam apple (Figure 1I) appear in the festoons 

with a total of 12 fruits. The fruits are slightly warty, with a pointed 

end, and some of them are red and others are orange. Balsam apple 

has bitter fruit and is grown for vegetable or medicinal purposes in 

relatively dry tropical areas, but it is not well adapted to most parts of 

Europe. This species is depicted in the painting Stilleben mit 

Kűrbissen, 1657, at the Kunsthistorisches Museum in Vienna (T. C. 

Andres, personal communication). We have not found any other 

Renaissance paintings of balsam apple, but depictions of this plant 

appeared in the herbals of Fuchs (1542), Dodoens (1616), and Chabrey 

(1666), among others, and it was also described by Ray (1686). 

Conclusions 

A number of the images of cultivated cucurbits in the festoons of 

the Villa Farnesina are similar to fruits of existing market types, which 

would seem to have been maintained over the course of the past 500 

years. This is remarkable considering the tendency of cucurbit 

cultivars of the same species to cross with one another, thereby 

mutually diluting their distinguishing characteristics. Moreover, the 

same market types of cucurbit fruits depicted in the festoons can also 

be found in works of art and botanical herbals that appeared decades 

later. 

The intraspecific diversity exhibited in the festoons of the Villa 

Farnesina differs among the cucurbit species, with watermelon and 

cucumber showing little and bottle gourd and melon showing 

extensive variation. The festoons appear to contain the first European 

paintings of sweet melons, documenting a wide phenotypic range that 

includes cultivars of both the Cantalupensis and Reticulatus groups, 

within a century of their introduction from southwestern Asia. 


Bilimoff, M. 2001. Promenade dans des jardins disparus: les plantes au moyen age, 

d’après les Grandes Heures d’Anne de Bretagne. Editions Ouest-France, 

Rennes. 

Bock, H. 1546. Kreuter Buch. Rihel, Strasbourg. 


Camerarius, J. 1586. Kreutterbuch. Calceolario, Frankfurt-on-Main. 

Caneva, G. 1992a. La Loggia di Psiche. Att. Accadem. Nazl. Linc. 9(3):163–172. 

Caneva, G. 1992b. Il mondo di Cerera nella Loggia di Psiche. Fratelli Palombi, 

Rome. 

Chabrey, D. 1666. Stirpium sciagraphia et icones. Gamoneti & de la Pierre, 

Geneva. 

Cordi, V. 1561. Annotationes in Pedacii Dioscoridis Anazarbei de medica materia 

libros V. Rihel, Strasbourg. 

Dalechamps, J. 1587. Historia generalis plantarum. Roville, Lyons. 

Dodoens, R. 1554. De stirpium historia. Plantin, Antwerp. 

Dodoens, R. 1616. Stirpium historiae pemptades. Plantin, Antwerp. 

Fuchs, L. 1542. De historia stirpium. Isingrin, Basel. 

Gerard, J. 1597. The herball or generall historie of plants. Norton, London. 

Goldman, A. 2002. Melons for the passionate grower. Artisan, New York. 

Impelluso, L. 2004. Nature and its symbols. Getty Publications, Los Angeles. 

Janick, J. 2004a. Erotic use of Lagenaria in Renaissance art. Cucurbit Network 

News. 11(2):7. 

Janick, J. 2004b. Caravaggio’s fruits: mirror on Baroque horticulture. Chron. Hort. 

44(4):9–15. 

Janick, J. and G. Caneva. 2005. The first images of maize in Europe. Maydica. 

50:71–80. 

Janick, J. and H. S. Paris. 2006. The cucurbit images (1515–1518) of the Villa 

Farnesina, Rome. Ann. Bot. 97:165–176. 

Jeffrey, C. 2001. Cucurbitaceae. In: P. Hanelt et al., Mansfeld’s encyclopedia of 

agricultural and horticultural crops, p. 1510–1557. Springer, Berlin. 

L’Obel, M. de. 1576. Sev stirpium historia. Plantin, Antwerp. 

Mattioli, P. A. 1558. Apologia adversus Amathum Lusitanum, cum censura in 

eiusdem enarrationes. Valgrisi, Venice. 

Morel, P. 1985. Priape à la Renaissance. Les guirlandes de Giovanni da Udine à la 

Farnésine. Revue de L’Art. 69:13–28. 

Paris, H. S. and J. Janick. 2005. Early evidence for the culinary use of squash 

flowers in Italy. Chron. Hort. 45(2):20–21. 

Pinet, A. du. 1561. Historia plantarum. Coter, Lyons. 

Pitrat, M., P. Hanelt, and K. Hammer. 2000. Some comments on infraspecific 

classification of cultivars of melon. In: N. Katzir and H. S. Paris (eds.). Proc. 

Cucurbitaceae 2000. Acta Hort. 510:29–36. 

Ravelli, L. 2004. La zucca nella natura morta dal cinquecento al novecento. 

Sometti, Mantova, Italy. 

Ray, J. 1686. Historia plantarum, vol. 1. Clark, London. 

Stuart, D. C. 1984. The kitchen garden, a historical guide to traditional crops. 

Guernsey Press, Guernsey, UK. 

Tabernaemontanus, D. J. T. 1591. Kraeuter Buch. Basse, Frankfurt-on-Main. 


JONAH AND THE “GOURD” AT NINEVEH: 

CONSEQUENCES OF A CLASSIC 

MISTRANSLATION 

Jules Janick 

Department of Horticulture & Landscape Architecture, Purdue 

University, 625 Agriculture Mall Drive, 

West Lafayette, IN 47907-2010 




ADDITIONAL INDEX WORDS. Citrullus colocynthis, Lagenaria siceraria, Ricinus 

communis, plant iconography 

ABSTRACT. The fast-growing plant referred to in the biblical Book of Jonah is 

most often translated into English as “gourd.” However, this is a mistranslation 

that dates to the appended Septuagint, the Greek translation of the Hebrew 

Bible, in which the Hebrew word qiqayon (castor, Ricinus communis, 

Euphorbiaceae) was transformed into the somewhat similar-sounding Greek 

word kolokynthi (colocynth, Citrullus colocynthis). In translation of the Greek 

into Latin, kolokynthi became the similar-sounding cucurbita (gourd). This is 

reflected in early iconography, the plant most often depicted being a longfruited 

Lagenaria siceraria (bottle or calabash gourd), a fast-growing climber. 

C 

ucurbits are frequent subjects of art, literature, and myth. Since 

ancient times, people the world over have been fascinated by 

the fast growth of cucurbits, from a seed to a rampant vine 

bearing prominent, attractive fruits within two or three months. 

Metaphorically, the cucurbits are associated with warmth, sunshine, 

health, vitality, fertility, sexuality, and abundance, leading to mirth and 

laughter (Norrman and Haarberg, 1980). 

Cucurbit fruits have been valued by humans for thousands of 

years, for food and a multitude of other uses. The Cucurbitaceae are 

extremely polymorphic for fruit size, shape, and color and the fruits of 

some species can exhibit great similarity to those of others (Chester, 

1951). Often, the result has been different names for the same species 

and the same name for different species, resulting in the confusion that 

has afflicted cucurbit terminology since ancient times. This confusion 

has been enhanced by the translation of names to different languages 

and by mistranslations. One of the most striking cases of 

mistranslation occurred when the biblical Book of Jonah, which is read 

in its entirety by Jews as part of the afternoon prayer of the Day of 

Atonement, was translated into other languages. 


Jonah, more accurately Yona the son of Amittay, is one of the 

remarkable figures of the Hebrew Bible. He was ordered by God to get 

up and go to Nineveh (a destroyed Assyrian city near the present-day 

Mosul, Iraq), the great city, and call upon it because its wickedness 

has risen up to Me. But Jonah disobeyed the Divine command and 

tried to run away, boarding at the port of Jaffa (Yafo, Israel) a boat 

bound for the city of Tarshish (a Mediterranean port, perhaps in 

Spain). However, the craft was soon overtaken by a squall, for which 

Jonah admitted responsibility and persuaded the crew of the boat to 

throw him overboard. Swallowed by a providential fish in whose belly 

he remained for three days and three nights, Jonah composed a psalm 

of thanksgiving. He was then vomited out on the shoreline. The Divine 

command to preach to Nineveh was repeated. As a result of Jonah’s 

exhortations, the populace heeded the warning of destruction and 

atoned, and the city was spared. Jonah, who had withdrawn and 

watched the city from a booth, was displeased at the Divine mercy 

toward Nineveh. A fast-growing plant had provided him with muchneeded 

shade, but at the break of dawn one day a worm attacked the 

plant, causing it to wither. Jonah was rebuked for his distress at the 

loss of the plant, in view of his displeasure toward the Divine mercy 

that spared 120,000 Ninevans their lives (Jonah 4:6–11). 

The Hebrew name for the fast-growing plant that provided relief 

for Jonah is qiqayon. Derived from ancient Egyptian, this word 

signifies castor, Ricinus communis L. (Euphorbiaceae), castor oil being 

shemen qiq in Hebrew. However, in a number of biblical translations, 

including the King James Version of 1611, qiqayon is translated as 

“gourd”: And the LORD God prepared a gourd, and made it to come 

up over Jonah, that it might be a shadow over his head, to deliver him 

from his grief. So Jonah was exceeding glad of the gourd. But God 

prepared a worm when the morning rose the next day, and it smote the 

gourd that it withered (Jonah 4:6–7). 

A translation more faithful to the Hebrew is given in the Jerusalem 

Bible (Fisch, 1992): And the LORD God appointed a castor oil plant, 

and made it to come up over Yona, that it might be a shade over his 

head, to deliver him from his distress. And Yona was exceeding glad of 

the plant. But God appointed a worm when the dawn came up the next 

day, and it attacked the plant, so that it withered. 

The story of Jonah is one of the best-known biblical tales and is 

referred to both in the New Testament and in the Qur’an. The giant 

fish, referred to as a whale by Jesus (Matthew 10:39–40), has captured 

the imagination of children, like the marvelous but less well-known 

miraculous “gourd” that resonates in the story of Jack and the 

Beanstalk. It is not our purpose here to reflect on the theological 


meaning of Jonah or its historical accuracy. Our objective is twofold: 

first, to trace the story of how the fast-growing qiqayon eventually 

became translated as “gourd” and second, to identify the cucurbit 

depicted in ancient images of the story of Jonah. 

Translation from Hebrew to Greek: Septuagint 

The translation of the Hebrew Bible to Greek began in the 3 rd 

century BCE in Alexandria. Philadelphus II (Ptolemy, 285–247 BCE) 

requested of El’azar, the High Priest in Jerusalem, a translation from 

Hebrew into Greek for the library at Alexandria. Initially, only the 

Tora (Instruction), known in Greek as the Pentateuch or Five Books of 

Moses, was translated. This Greek translation became known as the 

Septuagint (Seventy) as it was supposedly conducted by a committee 

composed of six translators from each of the 12 tribes of Israel. Only 

later were the other books comprising the Jewish scriptures translated 

and appended to the original Septuagint, becoming an inseparable part 

of it. The Septuagint is still a part of the Bible of the Eastern Orthodox 

Christian churches. 

In the Septuagint translation of the Book of Jonah, the Hebrew 

qiqayon is translated as the somewhat similar-sounding word kolokynthi 

(also kolokynthan), colocynth, Citrullus colocynthis (L.) Schrader. The 

intensely bitter colocynth is referred to in the Bible as the Hebrew 

paqqu’ot sade. During a famine, its fruit was gathered from vines in the 

field and were then mistakenly included in a pottage prepared for the 

disciples of the prophet Elisha’: And they poured for the people to eat 

and it was when eating from the pottage that they yelled and said death 

is in the pot of the man of God and they could not eat (2 Kings 4:39–40). 

Elisha’ was able to ameliorate the taste of the pottage by adding flour. 

Citrullus colocynthis is a prostrate, relatively small vine with 

pinnatifid leaves. It is not adapted to climbing nor could it be thought 

of as a good provider of shade. Possibly, the ancient Greek usage 

could have been less specific and included a broader spectrum of 

cucurbits. The colocynth was used by the ancients medically as a 

purgative, and is specifically mentioned in three works of the first 

century CE: Materia Medica of Pedanius Dioscorides, Historia 

Naturalis (Book 20) of Pliny the Elder (Jones, 1951), and the Mishna, 

a series of six books of commentary on Jewish Law. In Book 2 of the 

Mishna an entirely different use of the paqqu’ot is revealed. Oil was 

pressed from the seeds, for illumination, but was deemed inappropriate 

for lighting the Sabbath (Mishna 2, Massekhet Shabbat). In Book 6 yet 

another usage is described. Young shoots of paqqu’ot plants were 

eaten after pickling in salt or vinegar (Mishna 6, Massekhet ‘Oqazin) 


(Feliks, 1957). An ancient illustration labeled with Greek Kolokynthis 

clearly depicting a plant of Citrullus colocynthis (Figure 1) is in the 

Codex Vindobonensis (Anciae Juliana), a 512 CE rendition of 

Dioscorides’ Materia Medica. 

Citrullus colocynthis was described as being used for medicinal 

purposes in botanical herbals of the Renaissance and was depicted in 

the herbals of Fuchs (1542), Bock (1546), Dodoens (1616), Chabrey 

(1666), and others. Subsequently, the usage of colocynth or coloquinte 

was expanded to encompass other bitter cucurbits, most often the 

small-fruited Cucurbita pepo L. gourds (Gerard 1597; 

Tabernaemontani 1664). Bauhin (1651) illustrated and described the 

true colocynth and listed and described briefly nine other gourds that 

were also denoted colocynth. Seven were described as having rough 

foliage, probably indicative of C. pepo, an eighth was pyriform, 

probably also C. pepo. The last was described as having soft foliage 

and white flowers, evidently a small, bitter gourd of Lagenaria 

siceraria (Molina) Standley. The loose usage of the word colocynth in 

the later Renaissance gives credence to the supposition that the word 

kolokynthi was occasionally used loosely for cucurbits in ancient 

times. 

Translation to Latin 

The translation of the Hebrew Bible into Latin was originally by 

way of the Septuagint. The Greek kolokynthi was translated with the 

similar-sounding cucurbita (gourd) (Norrman and Haarberg, 1980). 

Eusebius Hieronymus Sophronius, 340–420 CE (later Saint Jerome), 

who was fluent in Greek, Hebrew, and Latin, translated directly from 

Hebrew to Latin. His translation is known as the Vulgate. The plant in 

the Book of Jonah was to become a source of contention in early 

Christianity between Augustine of Hippo, 354–430 CE (later Saint 

Augustine), and Jerome. Augustine was concerned about differences 

that could arise about biblical mistranslations, and the precise name of 

the plant in the Book of Jonah became the basis of a heated exchange 

of letters between Augustine and Jerome starting in 394 and 

continuing through 406. Augustine stridently objected to Jerome 

translating the Bible into Latin without adding notes and was 

especially vexed about mistranslations of the fast-growing plant of the 

Book of Jonah that Jerome translated as hedera (ivy). The English 

translations of Augustine’s letter of 403 and Jerome’s response of 406 

are quoted below (http://www.bible-researcher.com/vulgate2.html): 


Augustine: A certain bishop, one of our brethren, having 

introduced in the church over which he presides the reading of 

your version, came upon a word in the book of the prophet 

Jonah, of which you have give a very different rendering from 

that which had been of old familiar to the senses and memory of 

all the worshippers, and had been chanted for so many 

generations in the church. Thereupon arose such a tumult in the 

congregation, especially amongst the Greeks, correcting what 

had been read, and denouncing the translation as false, that the 

bishop was compelled to ask the testimony of the Jewish 

residents (it was in the town of Oea). These, whether from 

ignorance or from spite, answered that the words in the Hebrew 

manuscripts were correctly rendered in the Greek version, and 

in the Latin one taken from it. What further need I say? The man 

was compelled to correct your version in that passage as if it 

have been falsely translated, as he desired not to be left without 

a congregation—a calamity which he narrowly escaped. From 

this case we also are led to think that you may be occasionally 

mistaken. You will also observe how great must have been the 

difficulty if this had occurred in those writings which cannot be 

explained by comparing the testimony of languages now in use. 

Jerome: You tell me that I have given a wrong translation of 

some word in Jonah, and that a worthy bishop narrowly escaped 

losing his charge through the clamorous tumult of his people, 

which was caused by the different rendering of this one word. At 

the same time, you withhold from me what the word was which I 

have mistranslated; thus taking away the possibility of my saying 

anything in my own vindication, lest my reply should be fatal to 

your objection. Perhaps it is the old dispute about the gourd 

which has been revived, after slumbering for many long years 

since the illustrious man, who in that day combined in his own 

person the ancestral honours of the Cornelii and of Asinius 

Pollio, brought against me the charge of giving in my translation 

the word “ivy” instead of “gourd.” I have already given a 

sufficient answer to this in my commentary on Jonah. At present, 

I deem it enough to say that in that passage, where the 

Septuagint has “gourd,” and Aquila and the others have 

rendered the word “ivy” (kissos), the Hebrew MS. has 

“ciceion,” which is in the Syriac tongue, as now spoken, 

“ciceia.” It is a kind of shrub having large leaves like a vine, 

and when planted it quickly springs up to the size of a small tree, 


standing upright by its own stem, without requiring any support 

of canes or poles, as both gourds and ivy do. If, therefore, in 

translating word for word, I had put the word “ciceia,” no one 

would know what it meant; if I had used the word “gourd,” I 

would have said what is not found in the Hebrew. I therefore put 

down “ivy,” that I might not differ from all other translators. 

But if your Jews said, either through malice or ignorance, as you 

yourself suggest, that the word is in the Hebrew text which is 

found in the Greek and Latin versions, it is evident that they 

were either unacquainted with Hebrew, or have been pleased to 

say what was not what was not true, in order to make sport of 

the gourd-planters. 

Clearly, then, as now, misuse and mistranslations of plant names, 

especially names of cucurbits, have led to misunderstandings and 

controversy. 

The Translation in the Qur’an 

In the Qur’an, written in the 7 th century, the plant at Nineveh is 

identified as yaqtin: there grew over Jonah a kind of yaqtin (Sura 

37:139–146). Usually, the Arabic yaqtin is identified with Lagenaria 

siceraria. Commentators of the Qur’an have offered a spectrum of 

opinions concerning the identity of the yaqtin, which can be 

summarized as indicating an herbaceous, summer-annual plant lacking 

support tissue, a climbing, quick-growing vine, having large foliage. 

The identification of the Hebrew qiqayon as Ricinus communis, Arabic 

kharua’, is not accepted in Islamic tradition (Amar, 1998), echoing 

Latin translations of the Septuagint rather than the Hebrew Bible or the 

Vulgate. 

English Translations 

The famous Authorized Version of King James I, 1611, has many 

mistranslations of biblical plants (Moldenke and Moldenke, 1952). 

The erroneous use of “gourd” for qiqayon in the Book of Jonah was 

copied in a number of subsequent English translations. The English 

translations based on the French Douay version of Roman Catholics 

use “ivy,” following Jerome’s Vulgate. A number of new translations 

based on modern scholarship equivocate and call it either “plant” 

(Revised Standard Version) or “vine” (New International Version). 


Images from Antiquity 

A gourd is used for the story of Jonah in Judeo-Christian 

iconography of the 3 rd to the 16 th centuries. One example is a sculpture 

from 3 rd -century Phrygia (Central Turkey) showing Jonah under a vine 

bearing a fruit that is clearly an elongated Lagenaria siceraria (Figure 

2). Two others are mosaics, one from Tunisia dating to the end of the 

3 rd or beginning of the 4 th century (Figure 3) and the other from Italy 

(4 th century) (Figure 4). Their similarity suggests that a standard 

representation of the incident was copied. The two mosaics show a 

nearly nude figure of the reclining Jonah under a trellis from which 

hang eight elongate fruits. Based on the swollen peduncular ends the 

fruits (Figure 4), these too appear to be L. siceraria. The young fruits 

of elongate L. siceraria are a food crop in some regions, such as Sicily, 

to the present day. Long-fruited L. siceraria are also depicted in 

Roman mosaics of the 2 nd to 3 rd century, indicating that they were a 

familiar esculent in the ancient Mediterranean region, and specific 

mention of the use of trellises for gourds is made by Columella and 

Pliny. A 12 th century image (Figure 5) captures the entire story of 

Jonah, including his being tossed overboard, being swallowed by the 

fish, and reclining under a cucurbit vine, but the image is too crude to 

make an identification of the species. Later images, from the 14 th and 

16 th centuries, depict Jonah’s gourd as the more familiar bottle-shaped 

L. siceraria. 

Conclusion 

The identification of the fast-growing plant in the Book of Jonah as 

a gourd is due to a mistranslation of the Hebrew word qiqayon (castor) 

to the Greek word kolokynthi and then to the Latin word cucurbita. 

The error is reflected in early iconography, the qiqayon being depicted 

as a long-fruited Lagenaria siceraria. The confusion over the plant 

shows how, then as now, misuse and mistranslation of plant names, 

especially names of cucurbits, have led to misunderstandings and 

controversy. The qiqayon of Jonah was a lush, fast-growing provider 

of shade. By rendering it an edible-fruited cucurbit, it became, in 

addition, a symbol of sustenance, well-being, and life itself. 


Fig. 1. Painting of Citrullus colocynthis from Codex Vindobonensis, 512 CE (Der 

Wiener Dioskurides, 1998). 

Fig. 2. Jonah at Nineveh in a paleo-Christian sculpture from Phrygia (Central 

Turkey), ca. 270–280 (Cleveland Museum of Art). 

Fig. 3. Jonah at Nineveh in a 3 rd –4 th -century mosaic at Tunis (Baggio et al., 

1995). 

Fig. 4. Jonah at Nineveh in a mosaic at Aquileia, Italy (Rossi, 1968). 

Fig. 5. The story of Jonah from a 12 th century manuscript. Sinai Peninsula, 

Monastery of Saint Catherine, Ms. 1186, fol. 110r. Photo Richard Cleave. 

(Encyclopedia Judaica, 1972). 



Amar, Z. 1998. Zihuy hazomeah hamiqra’i bir’i parshanut haQur’an [Scriptural 

plant identification mirrored in Qur’an commentary]. Bet Miqra. 43(1):67–77. 

Baggio, M., M. De Paoli, M. T. Lachin, M. Salvadori, and S. Toso. 1995. Mosaici 

Romani di Tunisias. CNRS Editions, Paris. 

Bauhin, J. 1651. Historiae plantarum universalis. Graffenried, Yverdon. 

Bock, H. 1546. Kreuter Buch. Rihel, Strasbourg. 

Chabrey, D. 1666. Stirpium sciagraphia et icones. Gamoneti & de la Pierre, Geneva. 

Chester, K. S. 1951. Selected writings of N. I. Vavilov. The origin, variation, 

immunity and breeding of cultivated plants. Chronica Botanica, Waltham, MA. 

Der Wiener Dioskurides. 1998. Codex medicus Graecus 1 der Österreichischen 

Nationalbibliothek. Graz. 

Dodoens, R. 1616. Stirpium historiae pemptades. Plantin, Antwerp. 

Encyclopedia Judaica. 1972. Jerusalem. 

Feliks, J. 1957. ‘Olam hazomeah hamiqra’i [Plant world of the Bible], 1 st ed. 

Massada, Tel Aviv. 

Fisch, H. 1992. The holy scriptures. The Jerusalem Bible. Koren, Jerusalem. 


Gerard, J. 1597. The herbal or generall historie of plants. Bollifant, London. 

Jones, W. H. S. 1951. Pliny natural history. Harvard Univ. Press, Cambridge, MA. 

Moldenke, H. N. and A. L. Moldenke. 1952. Plants of the Bible. Chronica Botanica, 

Waltham, MA. 

Norrmann, R. and J. Haarberg. 1980. Nature and language: a semiotic study of 

cucurbits in literature. Routledge & Kegan Paul, London. 

Rossi, F. 1968. Il mosaico: pittura a de pietra. Alflieri & Lacroix, Milan. 

Tabernaemontani, J. T. 1664. Kraeuter-Buch. Koenigs, Basel. 


DEVELOPMENT OF AN IMAGE DATABASE 

OF CUCURBITACEAE 

Jules Janick and Anna Whipkey 


University, 

625 Agriculture Mall Drive, West Lafayette, IN 47907-2010 


Agricultural Research Organization, Newe Ya'ar Research Center, 


Marie-Christine Daunay and E. Jullian 

INRA, Unité de Génétique & Amélioration des Fruits et Légumes, 

Domaine St Maurice, BP 94, 84143 Montfavet cedex, France 

ADDITIONAL INDEX WORDS. plant iconography, taxonomy, crop history, crop 

evolution 

ABSTRACT. The systematic collection of plant iconography would be an 

invaluable resource to researchers, providing significant information on 

taxonomy, crop history and evolution, lost traits, and genetic diversity. To this 

end a project, “Plant Image”, is being organized to assemble a searchable 

database of plant images, focusing on the Cucurbitaceae and Solanaceae. 

Searches have been made from various sources including art (mosaics, 

paintings, and sculpture), illustrated manuscripts, and hand illustrated and 

printed herbals and books. We are concentrating our search on antiquity (Old 

and New World), medieval, and Renaissance sources but we intend to include 

more recent images as well. Bibliographic information on primary and 

secondary sources will be associated with each image and, in the case of herbals, 

associated text material will eventually be included. We hope to receive images 

and information from all persons interested in the Cucurbitaceae and 

Solanaceae, so that the database will be a living, dynamic document. The 

working database is online: www.hort.purdue.edu/newcrop/iconography. 

P 

lant iconography is a valuable resource for such fields as 

taxonomy, genetics, crop domestication and crop evolution, and 

genetic diversity. It is one of the tools for assessing the 

historical presence of botanical taxa in a particular region (Eisendrath, 

1961). A large, maybe even enormous, amount of ancient illustrative 

material exists, but it is fragile, rare, and widely scattered among 

libraries and collections, public and private, and is often difficult to 

access. Hence, there is no easy way for one person to assemble all of 

the illustrations for any plant family. Moreover, copyrights to images 

are often involved, and policy for access to the images varies from one 

library or collection to another. The viewing and usage of the many 

images are restricted. 


On the other hand, the digitizing of information by some of the 

major world libraries has greatly facilitated the search for ancient 

illustrations and, for the first time, made their collection feasible. 

Collection of all of the ancient illustrations of such a large, diverse, 

and attractive family as the Cucurbitaceae would especially be too 

huge for any single individual to undertake and therefore a joint effort 

is necessary. Our goal is to develop a database assembling plant 

images based on botanical names, so that plant researchers can easily 

focus their iconographic research on taxa of interest. We call this 

project “Plant Image” and have initiated it with two plant families: 

Cucurbitaceae and Solanaceae. 

Description 

We are concentrating our searches on antiquity (Old and New 

World), and Medieval, Renaissance, and Baroque periods but we 

intend to include images from the 18 th century to the present. The 

images collected so far are from various sources, including art 

(mosaics, paintings, and sculpture), illustrated manuscripts, printed 

herbals, books, and private collections. In the case of herbals, we hope 

to eventually associate text material. We are aware that many of the 

images from printed herbals are merely copies of preexisting images 

but we will include as many as feasible; for works that went through 

many editions, we will concentrate on the primary sources. Our 

intention is to include the following bibliographic information 

associated with each image: 

Bibliographic Information for Images: 

Family: family name 

Species: modern binomial with authority 

Common name: common name or names, English first 

Original label: designation in original document 

Original document: where image was first published or created 

Time period: time when image was produced 

Associated text: yes/no (list separately) 

Author: author of text of original document 

Illustrator: creator of image 

Color: black and white, hand tinted, painted, printed in color 

Library/collection: source from where document was obtained 

Library reference: library identification of source document 

Secondary source: used if image was obtained from a later document 

Comments: additional information 

Donor: contributor of image 

Web link: original Website 


Operation 

The database was created using FileMaker Pro®, currently on the 

server of Purdue University. The database is maintained by Anna 

Whipkey of the Department of Horticulture and Landscape 

Architecture (awhipkey@purdue.edu). All images need to be defined 

with the bibliographic information described above. The donor of the 

image will be acknowledged and identified. We hope to have the 

highest resolution possible, but we are aware that in many cases the 

highest resolution images are available only by purchase from specific 

institutions. 

The project must be a living, dynamic collection and success will 

depend on images obtained from willing scientists, art historians, 

archivists, enthusiasts, and others. We invite anyone to contribute 

images and are willing to acknowledge and identify each donor. The 

database is intended to be freely open to all. We are attempting to 

solicit modest grants to facilitate this project. As an example of the 

database contents, representative cucurbit images with associated 

information are shown in Figures 1 to 5. The information we are 

compiling is freely available as part of the Purdue NewCROPWebsite: 

. 

Fig. 1. Typical page of database. 


Fig. 2. Ecballium elaterium 

(squirting cucumber) from 

Dioscorides, De Materia Medica, 

Codex Vindobonensis 512 CE. 

Source: Der Wiener Dioskurides, 

1998. 

Fig. 4. Cucumis melo, 

cantalupensis Group, painted by 

Giovanni da Udine from the Villa 

Farnesina (1515–1518). Source: 

Janick and Paris, 2006. 

Fig. 3. Lagenaria siceraria (bottle 

gourd). Source: Schoeffer, 1485. 

Fig. 5. Citrullus lanatus 

(watermelon), woodcut from De 

Historia Stirpium (Fuchs, 1542) and 

republished in Fuchs’ New 

Kreuterbuch, 1543, coloring from 

his personal copy (Meyer et al., 

1999) 



Der Wiener Dioskurides. 1998. Codex medicus Graecus 1 der Österreichishchen 

Nationalbibliothek. Graz. 

Eisendrath, E. R. 1961. Portraits of plants. a limited study of the “icones”. Ann. 

Missouri Bot. Gard. 48:291–327. 

Schoeffer, P. 1485. Gart der gesundheit, p. 83. Schoeffer, Mainz, Germany. 

Janick, J. and Paris, H. S. 2006. The cucurbit images (1515–1518) of the Villa 


Meyer, F. G., Trueblood, E. E., and Heller, J. L. 1999. The great herbal of Leonhart 

Fuchs, vol. 1, commentary, p. 522–523, 669–670, pl. 64. Stanford Univ. Press, 

Stanford, CA. 


FIRST IMAGES OF CUCURBITA IN EUROPE 




Jules Janick 


University, 625 Agriculture Mall Drive, 

West Lafayette, IN 47907-2010 

Marie-Christine Daunay 

I.N.R.A., Unité de Génétique & Amélioration des Fruits et Légumes, 

Domaine St. Maurice, BP 94, 84143, Montfavet cedex, France 

ADDITIONAL INDEX WORDS. Cucurbita pepo, Cucurbita maxima, Cucurbita 

moschata, crop dispersal, crop history, plant iconography 

ABSTRACT. The genus Cucurbita (pumpkin, squash, gourd) is native to the 

Americas and diffused to other continents subsequent to European contact in 

1492. For quite some time, the earliest images of this genus in Europe that were 

known to cucurbit specialists were two illustrations of C. pepo pumpkins that 

were published in Fuchs’ De Historia Stirpium, 1542, exactly 50 years after 

Columbus’ first voyage. Other images considerably antedating these have come 

to light recently as the result of the publication of two books in which are 

reproduced highly realistic botanical and horticultural color paintings from the 

first two decades of the 16 th century. One of these images appears in the prayer 

book of Anne de Bretagne, Queen of France. Completed by 1508, this painting 

shows a living branch bearing flowers and fruits, which we interpret as being a 

gourd of Cucurbita pepo subsp. texana. Other images appear in festoons painted 

on the ceiling of the Villa Farnesina in Rome, completed by 1518. These lifelike 

color depictions include pumpkins and gourds of C. maxima, C. pepo, and, 

possibly, C. moschata. 

he genus Cucurbita L. (pumpkins, squash, and some gourds) 

(Cucurbitaceae) is native to the Americas (Gray and Trumbull, 

1883; Whitaker, 1947). Pumpkins and squash were dispersed to 

other continents by transoceanic voyagers at the turn of the 16 th 

T 

century and have become a familiar and important vegetable crop in 

many countries. Whilst C. pepo L. is by far the most widely distributed 

and economically important species of the genus, C. maxima 

Duchesne and C. moschata Duchesne are also economically important 

in various regions. 

Plant iconography is the most unequivocal tool for assessing the 

historical presence of botanical taxa in a particular region; this is 

especially the case for the Cucurbitaceae (Eisendrath, 1961). The first 


known images of Cucurbita outside of the Americas had long been 

believed to be two illustrations of pumpkins of C. pepo that appeared 

in De Historia Stirpium (Fuchs, 1542), a half-century after Columbus’ 

first voyage to the New World. Indeed, the earliest unequivocal 

depiction of C. maxima appeared the botanical herbal of L’Obel 

(1576) and the earliest generally accepted illustration of C. moschata 

is in the tome of Rheede tot Draakenstein (1688). Several recent 

publications have brought to light the existence of even earlier 

depictions of Cucurbita in Europe. One of these depictions is in the 

personally painted prayer book that belonged to Anne de Bretagne, 

Queen of France and Duchess of Brittany (1477–1514) (Bilimoff, 

2001). With time, this book was to become known as the Grandes 

Heures d’Anne de Bretagne. Other depictions are in the festoons that 

surround frescoes decorating ceilings of a fabulous early Renaissance 

residence in Rome (Caneva, 1992). This building was later to become 

known as the Villa Farnesina. The objective of this paper is to describe 

and discuss these depictions. 

The Grandes Heures 

The Grandes Heures d’Anne de Bretagne was compiled and 

illustrated between 1503 and 1508 (Bilimoff, 2001). At the request of 

Queen Anne, Jean Bourdichon (1457–1521) of Touraine, who was 

official court painter to four successive French kings, richly adorned 

an Horae ad usum romanum, that is, a prayer book, for the Queen’s 

personal use (Camus, 1894; Bilimoff, 2001). On many of the 30 × 

19cm pages, he painted fairly accurately a part of a plant and, on or 

next to it, several small animals, mostly insects. Well over 300 plant 

species are represented in the book, drawn from live specimens found 

in the fields, woods, and royal gardens at Tours and Blois in the Loire 

Valley. Most of the paintings are vertically oriented 165 × 45mm 

rectangles positioned on the outside margins of the prayers, whilst 

other paintings surround or bracket the prayers. Each painting is 

labeled with a Latin name and a French vernacular name of the plant. 

Catalogued as Ms. Latin 9474 at the Bibliothèque Nationale de 

France, the book can be browsed on-line in its entirety at 

, by clicking on 

Recherche, typing Latin 9474 in the small window Manuscrits, 

clicking on Chercher and then on Images. 

The most outstanding feature of this book is the lifelike 

representation of so many species of living plants and animals, even 

though these are not always biologically accurate (Camus, 1894). The 

relative sizes of the various plant parts and the animals, the shapes of 


the leaves, and the colors of the flowers and the animals sometimes 

succumbed to fantasy in order to enhance the artistic effects. 

Nonetheless, the paintings are accurate enough to allow positive 

identification of most of the plants to genus and species. Each of the 

paintings is labeled with a Latin name and a French name. 

Used by Queen Anne until her death, this book remained within 

the collection of precious documents of the French kings. In 1722, 

King Louis XV th allowed the royal botanist, Antoine de Jussieu (1686– 

1758), to critically analyze the illustrations of plants in the royal 

collection. Jussieu considered the plant images in the Grandes Heures 

to be much more realistic than those of earlier documents but he 

considered many of the Latin plant labels in the Grandes Heures to be 

inappropriate. Therefore, he prepared a catalogue, in which he 

tabulated the Latin and French labels of the plants in the Grandes 

Heures next to Latin designations used in major botanical works such 

as those of L’Obel, the Bauhin brothers, and Tournefort, as well as the 

contemporary French common names. Joseph Decaisne (1807–1882), 

the Belgian botanist, also examined the book and catalogued the 

plants, apparently without knowing that Jussieu had done so a century 

earlier. Camus (1894) compared the identifications of the plants by 

Jussieu and by Decaisne and, noticing they were not always the same, 

he offered his own identifications of the plants. Bilimoff (2001), in her 

book Promenade dans des Jardins Disparus, reproduced in color, 

some whole and some in part, the paintings in the Grandes Heures. 

This book, through which we became aware of the existence of these 

images, also has a catalogue of its own, listing the names from the 

Grandes Heures alongside Latin and contemporary French names. 

Four cucurbits are depicted in the Grandes Heures. One is bryony, 

Bryonia dioica Jacq., which grows wild but is not cultivated. Another, 

bearing the Latin name Cucumer and the French name Concombres, is 

indeed cucumber, Cucumis sativus L. Another cucurbit, bearing the 

Latin name Cucurbita and the French name Quegourdes, is bottle 

gourd, Lagenaria siceraria (Molina) Standley. These three cucurbits 

are of Old World origin. The fourth cucurbit image bears the Latin 

name Colloqui[n]tida and the French label Quegourdes de turquie. 

Jussieu, Decaisne, Camus, and Bilimoff agreed that this image was 

modeled from a plant of the New World genus Cucurbita, but the 

identification of the species has been controversial and to our 

knowledge no cucurbit specialists have examined the image previously 

and positively identified it to that taxonomic level. 


The Image of Cucurbita in the Grandes Heures 

The image of Quegourdes de turquie in the Grandes Heures 

d’Anne de Bretagne is bracket-shaped with a golden background 

(Figure 1) (Paris et al., 2006). It depicts a branch with three pistillate 

flowers at anthesis that closely resemble those of Cucurbita pepo. The 

large orange-yellow corollas are moderately flaring, fused at the base 

and parted, as petals, near the apex. The corollas are 6-parted, rather 

than the typical 5-parted, but 6-parted and even 7-parted corollas do 

occur occasionally in C. pepo (Duchesne, 1786). The calyx is short 

and its sepals are thin, tapering to a point at the apex, like an awl. The 

ovaries are inferior and pyriform. A fourth pistillate flower, one day 

prior to anthesis, is depicted near the apex of the branch. Six leaf 

laminae and parts of three petioles are depicted. The leaf laminae are 

acute, serrated, and deeply indented, as in C. pepo, but their shape is 7parted 

rather than the usual more-or-less 5-parted. The two pyriform 

fruits are connected to the stem by long, narrow peduncles. No detail is 

evident for the petioles, stem, or peduncles, and no tendrils are 

depicted. On and next to the plant are depicted a snail and four 

arthropods: a caterpillar, a fly, a moth, and a beetle, all in lifelike 

color. 

The two pyriform fruits of the Quegourdes de turquie appear to be 

similar to gourds of C. pepo (Figure 1). Upon closer examination, the 

fruits of the Quegourdes de turquie can be seen to bear longitudinal 

stripes: broad, light yellow-green ones alternating with narrow light 

blue-green ones. The striped color pattern is quite common among the 

pyriform gourds, wild and ornamental, of C. pepo, although most often 

the broad stripes are dark green (Paris, 2000). On the mature fruits of 

such pyriform gourds, the broad dark green stripes alternate with 

narrow ivory white stripes; when the fruits are immature, the narrow 

stripes are light blue-green, as shown in the image. The green 

coloration of both the broad and the narrow stripes suggests that the 

fruits were immature. 

The image is labeled at the bottom with the French vernacular 

name Quegourdes de turquie (Figure 1). The French word quegourdes 

apparently had been used during that era, first for bottle gourds and 

then extended to include other gourds. Quegourdes was to become 

cougourdes or gourdes, epithets applied to forms of Lagenaria 

(Duchesne, 1786). During the 16 th century, the adjective “Turkish” 

implied an exotic origin. For example, one of the two C. pepo 

pumpkins appearing in the herbal of Fuchs (1542) is labeled 

“Türckisch Cucumer” whilst maize (Zea mays L.) is labeled 

“Türckisch Korn”; both of these species, of course, are native 


American plants. Therefore, this particular plant, the Quegourdes de 

turquie, was recognized in the Grandes Heures as being new or 

foreign, de turquie, distinguishing it from the Lagenaria gourds. 

As has been asserted concerning the identity of the cucurbits 

illustrated in the botanical herbals of the Renaissance (Eisendrath, 

1961), the identification of the plants illustrated in the Grandes Heures 

d’Anne de Bretagne must take into account the artistic style of the 

illustrator. Therefore, comparison of the illustration of Quegourdes de 

turquie with that of each of the two other cultivated species of 

cucurbits offers valuable assistance in interpretation. The illustration 

of Lagenaria siceraria (Figure 1) in the Grandes Heures accurately 

depicts the plant as having rounded leaf laminae and large white 

flowers, but it too lacks detail of the stem. The illustration of Cucumis 

sativus (Figure 1) also lacks details of the stems and petioles. 

Moreover, the leaf laminae are depicted as deeply lobed, whilst in 

reality those of C. sativus are angular but shallowly pentalobate. The 

peduncles of the cucumber fruits are depicted as originating separately 

Fig. 1. The paintings of Cucurbita pepo subsp. texana, Lagenaria siceraria, and 

Cucumis sativus in the Grandes Heures d’Anne de Bretagne (1503–1508). 


Fig. 2. A painting of Cucurbita maxima from the Loggia of Cupid and Psyche 

ceiling of the Villa Farnesina (1515–1518). 

on the main stem rather than in leaf axils; they are very long, narrow, 

and smooth. However, C. sativus peduncles are typically spiculate and 

not so long, and furthermore originate in leaf axils. The long, narrow, 

smooth peduncles not originating in leaf axils are in common, though, 

with the illustration of Quegourdes de turquie. So it would seem that 

these anomalous characteristics were stylistic of the artist.The striping 

pattern of the fruits of Quegourdes de turquie is inconsistent with the 

possibility that this depiction is of the cultivated species Cucurbita 

moschata, C. argyrosperma Huber, or C. ficifolia Bouché. C. maxima 

fruits can exhibit narrow light-colored stripes, but this species has 

rounded, rather than angular leaves. Wild Cucurbita taxa invariably 

have round fruits, with the exception of C. pepo subsp. texana, which 

bears fruits that are most often oviform to globose, with pyriform 

variants being fairly common (Erwin, 1938; Bailey, 1943; Andres, 

1987). Whilst on most fruits the broad stripes are dark, intense green, 

fruits having light-colored broad stripes or that are even entirely lightcolored 

or ivory white have also been observed (Bailey, 1937; Decker- 

Walters et al., 1993). Quegourdes de turquie, therefore, is a 

representation of a pyriform gourd of C. pepo subsp. texana. 

The Villa Farnesina 

The festoons on ceilings in the Villa Farnesina were painted by 

Giovanni Martini da Udine (1487–1564) from 1515 to early 1518. 

These festoons and the frescoes that they surround, designed by 

Raphael Sanzio (1483–1520), were painted for Agostino Chigi, an 

extremely wealthy Sienese banker, in this, his new home on the west 


ank of the Tiber in Rome. Today, the Villa Farnesina is open to 

tourists and a complete photographic study of the decorations was 

recently published by Frommel (2003). Identifications of the fruits and 

plants in the festoons were offered in the book by Caneva (1992), 

through which we became aware of the existence of these paintings. 

Remarkably, within the festoons are the first European images of Zea 

mays (Janick and Caneva, 2005). 

The depictions in the festoons are in lifelike color and highly 

accurate, to the point of showing blemishes and diseases on the fruits. 

Most of the species have more than one fruit depicted. The variation 

among fruits of the same species is a mirror on intraspecific genetic 

diversity. Analysis of the maize images has suggested that in some 

cases prototypes were copied, and, while some artistic license was 

used, the portraits nonetheless do appear to be quite accurate and can 

be considered true representations of the flowers and fruits that were 

probably harvested from the gardens that at the time surrounded the 

Villa Farnesina (Janick and Caneva, 2005). The images in the festoons 

of the Villa Farnesina led to the painting of similar images, beginning 

in 1517, in the Vatican, the Villa Medici in Rome, and the Villa d’Este 

in Tivoli. 

The Images of Cucurbita in the Villa Farnesina 

The festoons in the Villa Farnesina contain seven images of fruits 

of Cucurbita maxima (Janick and Paris, 2006). These are from three 

cultivars, two of which appear to be representative of the market class 

of show pumpkins, although they are not nearly so large as those that 

are customarily encountered today. One of the show-pumpkin cultivars 

has intense-orange fruits, which are shallowly furrowed and have a 

somewhat protruding stylar end surrounded by a narrow but large 

circular scar (Figure 2). The other show-pumpkin cultivar has graywhite 

fruits with shallow furrows and a small stylar scar. The third C. 

maxima is a smaller pumpkin, greenish gray and oblate. 

The festoons contain 15 images of fruits of Cucurbita pepo. All but 

three of these are of small, oviform to pyriform, striped fruits, C. pepo 

subsp. texana. Gourds of this appearance occur in cultivation and in 

the wild. If cultivated, they are referred to the Oviform, Smooth- 

Rinded Group (Paris, 2001). Two of the remaining fruits occur close 

together in the festoons. They appear to be larger than the gourds, but 

are nonetheless modest in size. They are striped with two shades of 

green and apparently immature. Their oblate shape indicates that they 

are of the Pumpkin Group, which belongs to C. pepo subsp. pepo. The 

remaining fruit is a large pumpkin. It is oval, slightly furrowed, and 


mostly light orange-yellow though partly gray-green, indicating a fruit 

that has just barely attained ripeness. The identity of this fruit is not 

entirely certain. While the coloration is more consistent with C. pepo, 

the lobing of the fruit is more reminiscent of C. moschata. A lobed 

pumpkin growing on a plant having leaf laminae like those of C. pepo 

was illustrated in the herbal of Tabernaemontanus (1591). 

Discussion and Conclusion 

The image of Cucurbita in the Grandes Heures (1503–1508) and 

most of the images of Cucurbita fruits in the Villa Farnesina (1515– 

1518) are small, pyriform or oviform striped gourds of C. pepo subsp. 

texana. These inedible gourds are most often cultivated today for 

ornament, but may have been grown for some other purposes in the 

past. Often, they are indistinguishable phenotypically from wild C. 

pepo subsp. texana gourds, which are native to the southern, 

southeastern, and central U.S.A. Wild plants of this subspecies have 

been encountered most often in Texas, on the floodplains of several 

creeks and rivers that enter the Gulf of Mexico, and including two 

coastal counties (Erwin, 1938; Nee, 1990; Decker-Walters et al., 

1993). It is possible that these early 16 th -century images of C. pepo 

subsp. texana were based on offspring of plants found growing wild 

along the Gulf Coast of what is now the United States. Europeans first 

entered the Gulf of Mexico no later than 1498 (Mollat, 2005). 

Significantly, no edible fruits of C. pepo subsp. texana are represented 

in the Grandes Heures or the Villa Farnesina. 

Remarkably, the depictions in the Villa Farnesina of Cucurbita 

maxima and C. pepo indicate that several types of both species were 

introduced into Europe within 25 years of the first voyage of 

Columbus. The images of C. maxima and of C. pepo subsp. pepo in 

the Villa Farnesina are pumpkins and, by definition, esculents, serving 

as evidence of European contact with American horticulture. 


Andres, T. C. 1987. Cucurbita fraterna, the closest wild relative and progenitor of C. 

pepo. Cucurbit Genet. Coop. Rep. 10:69–71. 

Bailey, L. H. 1937. The garden of gourds. Macmillan, New York. 

Bailey, L. H. 1943. Species of Cucurbita. Gentes Herbarum. 6:266–322. 

Bilimoff, M. 2001. Promenade dans des jardins disparus: les plantes au moyen age, 

d’après les Grandes Heures d’Anne de Bretagne. Editions Ouest-France, Rennes. 

Camus, G. 1894. Les noms des plantes du livre d’Heures d’Anne de Bretagne. J. 

Botanique 8:325–335, 345–352, 366–375, 396–401. 

Caneva, G. 1992. Il mondo di cerere nella loggia di Psiche. Fratelli Palombi, Rome. 


Decker-Walters, D. S., T. W. Walters, C. W. Cowan, and B. D. Smith. 1993. 

Isozymic characterization of wild populations of Cucurbita pepo. J. Ethnobiol. 

13:55–72. 

Duchesne, A. N. 1786. Essai sur l’histoire naturelle des courges. Panckoucke, Paris. 

Eisendrath, E. R. 1961. Portraits of plants. a limited study of the “icones”. Ann. 

Missouri Bot. Gard. 48:291–327. 

Erwin, A. T. 1938. An interesting Texas cucurbit. Iowa St. Coll. J. Sci. 12:253–261. 

Frommel, C. L. 2003. La Villa Farnesina a Roma. Franco Cosimo Panini, Modena. 


Gray, A. and J. H. Trumbull. 1883. Review of DeCandolle’s origin of cultivated 

plants. Amer. J. Sci. 25:370–379. 

Janick, J. and G. Caneva. 2005. The first images of maize in Europe. Maydica. 

50:71–80. 

Janick, J. and H. S. Paris. 2006. The cucurbit images (1515–1518) of the Villa 


L’Obel, M. de. 1576. Sev stirpium historia. Plantin, Antwerp. 

Mollat, M. 2005. Les explorateurs du XIII e au XVI e siècle: premiers regards sur des 

mondes nouveaux. Editions du comité de trauvaux historiques et scientifiques, 

Paris. 

Nee, M. 1990. The domestication of Cucurbita (Cucurbitaceae). Econ. Bot. 44(3, 

suppl.):56–68. 

Paris, H. S. 2000. Gene for broad, contiguous stripes in cocozelle squash (Cucurbita 

pepo). Euphytica. 115:191–196. 

Paris, H. S. 2001. History of the cultivar-groups of Cucurbita pepo. Hort. Revs. 

25:71–170, 4 pl. 

Paris, H. S., M.-C. Daunay, M. Pitrat, and J. Janick. 2006. First known image of 

Cucurbita in Europe, 1503–1508. Ann. Bot. 98:41–47. 

Rheede tot Draakenstein, H. A. van. 1688. Hortus indicus malabaricus 8:3 p. 2. 

Tabernaemontanus, D. J. T. 1591. Kraeuter Buch. Basse, Frankfurt-on-Main. 

Whitaker, T. W. 1947. American origin of the cultivated cucurbits. Ann. Missouri 

Bot. Gard. 34:101–111. 


POLYPHYLETIC ORIGIN OF CULTIVATED 

MELON INFERRED FROM ANALYSIS OF ITS 

CHLOROPLAST GENOME 

K. Tanaka, K. Fukunaga 

Research Institute for Humanity and Nature, 457-7 Motoyama, 

Kamigamo, Kita-ku, Kyoto, Japan 

Y. Akashi, H. Nishida, K. Kato 

Faculty of Agriculture, Okayama University, 1-1-1 Tsushima, Naka, 

Okayama, Japan 

M. T. Khaing 

Vegetable and Fruit Research and Development Center (VFRDC), Yemon, 

Indaingpo, Hlecuts, Yangon, Myanmar. 

ADDITIONAL INDEX WORDS. ccSSR, Cucumis melo, PS-ID 

ABSTRACT. To discover the origin and differentiation of cultivated melon, the plastid 

type of 245 melon accessions was assessed using PS-ID sequence analysis. European 

melon groups with large seeds (≥9.0mm length) and East Asian melon groups with 

small seeds (

Decker-Walters, 1997). During the long history of cultivation and 

transmission, melon has been diversified and different types of melon 

have been established in various parts of the world. They have been 

classified into seven groups by Munger and Robinson (1991). Genetic 

relationships and differentiation among these groups have been 

investigated by the analysis of morphological characters and molecular 

markers such as ISSR, SSR, and RAPD. East Asian melon of the group 

Conomon, whose seed length is shorter than 8.5mm, proved to be 

genetically differentiated from European and American melon, Groups 

Cantalupensis and Inodorus, with seeds longer than 9.0mm (Fujishita et 

al., 1993; Stepansky et al., 1999; Mliki et al., 2001; Monforte et al., 2003; 

Nakata et al., 2005). The analysis of isozyme, AFLP, and RAPD markers 

of eastern and southern Asian melons can also be differentiated by 

seed-size groups rather than geographical groups (Akashi et al., 2002; 

Yashiro et al., 2005; K. Tanaka et al., unpublished). However, the 

evolutionary process that resulted in the difference in seed size among the 

western and the eastern melon and the origin of the Indian small-seed 

melon remains to be investigated. 

Molecular markers of chloroplast DNA (cpDNA) have advantages for 

the analysis of genetic relationship among plant species or varieties, since 

they have a low rate of nucleotide substitution and show maternal 

inheritance, which lowers the impact of intermolecular recombination 

(Clegg et al., 1994). In the chloroplast genome, the linker sequence 

between the genes rpl16 and rpl14 proved to be polymorphic among plant 

species, and Nakamura et al. (1998) proposed to designate it as the plastid 

subtype ID sequence (PS-ID). PS-ID was also polymorphic within species 

and could be applicable to the identification of subspecies japonica and 

indica of rice (Ishikawa et al., 2002). Another marker of the chloroplast 

genome, the consensus chloroplast SSR marker (ccSSR), applicable to 

Cucurbitaceae crops, has been developed by Chung et al. (2003), who 

have suggested its potential utility for taxonomic and phylogenetic 

analyses. 

In this study, therefore, chloroplast genome type was determined for 

245 melon accessions from various parts of the world by the analysis of 

PS-ID sequence and four ccSSR markers. Based on varietal and 

geographical variation of cytoplasmic genotype, genetic differentiation in 

cultivated melon and the origin of small-seed-type melon predominately 

cultivated in Asia are discussed. 



PLANT MATERIALS. A total of 245 accessions of cultivated melon 

(Cucumis melo L.) were examined in this study. They were selected to 

represent five groups of C. melo from wide geographic areas, and were 

classified into small (

Table 1. Classification of 245 melon accessions based on the analysis of PS-ID, ccSSR7 and seed length. 

Variety/Area Countory / 

Cultivar group 

No. of 

PS-ID type 

ccSSR7 (bp) 

accessions Large seed Small seed Large seed Small seed 

T A NA T A NA 331 336 331 336 

Group cantalupensis European cantaloup 

England house type 

American field type 

Japan breeding line 

4 

5 

8 

12 

4 

5 

8 

12 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

3 

- 

4 

5 

5 

12 

- 

- 

- 

- 

- 

- 

- 

- 

Group inodorus Honeydew 6 6 - - - - - 6 - - - 

Hmi-Gua 5 5 - - - - - - 5 - - 

Group flexuosus 

Group conomon 

Spain•Russia 

India•Indonesia 

China 

6 

6 

16 

6 

1 

- 

- 

4 

- 

- 

- 

- 

- 

- 

- 

- 

1 

16 

- 

- 

- 

5 

- 

- 

1 

5 

- 

- 

- 

- 

- 

1 

16 

Korea 4 - - - 1 3 - - - - 4 

Japan 10 - - - - 10 - - - - 10 

Group agrestis Africa North 1 - - - - - 1 - - - 1 

Center 6 - - - - - 6 - - - 6 

South 1 - - - - 1 - - - - 1 

unknown 1 - - - - 1 - - - - 1 

Iran 2 - - - - 2 - - - - 2 

Pakistan 6 - - - - 6 - - - - 6 

India 5 - - - - 5 - - - - 5 

Maldives 1 - - - - 1 - - - - 1 

South America 

Nepal•Bangladesh 

Korea•Japan 

Bolivia 

6 

6 

1 

- 

- 

1 

- 

- 

- 

- 

- 

- 

- 

- 

- 

6 

6 

- 

- 

- 

- 

- 

- 

1 

- 

- 

- 

- 

- 

- 

6 

6 

- 

Europe 2 - - - 1 1 - - - - 2 

Africa North 8 8 - - - - - 2 6 - - 

Center 6 1 1 1 - - 3 - 3 - 3 

South 9 1 - - - 5 3 1 - - 8 

unknown 1 - 1 - - - - - 1 - - 

West•Central Asia Turkey 

Syria 

5 

2 

3 

2 

2 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

- 

5 

2 

- 

- 

- 

- 

Lebanon 4 2 2 - - - - - 4 - - 

Iraq 2 1 - - - 1 - - 1 - 1 

Iran 3 3 - - - - - 2 1 - - 

Afghanistan 5 4 1 - - - - 1 4 - - 

Uzbekistan 1 - 1 - - - - - 1 - - 

South Asia Pakistan 1 1 - - - - - - 1 - - 

India West 

North 

7 

9 

3 

3 

2 

2 

- 

- 

1 

2 

1 

2 

- 

- 

- 

- 

5 

5 

- 

- 

2 

4 

Center 8 2 1 - - 5 - - 3 - 5 

South 7 4 - - 1 2 - 2 2 - 3 

East 12 3 4 - 1 4 - - 7 - 5 

Maldives 2 - - - - 2 - - - - 2 

Nepal 3 - - - 1 2 - - - - 3 

Bangladesh 8 - 2 - - 6 - - 2 - 6 

Myanmer 8 - 2 - - 6 - - 2 - 5 

Southeast Asia 14 2 1 - 1 10 - 1 2 - 11 

245 91 26 1 9 105 13 24 94 - 126 


PS-ID sequences were compared among 24 accessions of various 

groups, and PS-ID sequences were compared among 24 accessions of 

various groups, and two SSR polymorphisms (SSR1; (T)n, SSR2; (T)n) 

and one SNP at S2 (A/T, 425th base from common primer A) were 

detected. Single nucleotide polymorphism (SNP) types of 245 accessions 


Table 2. Four types of chloroplast genome revealed by the analysis of PS-ID sequence 

of 18 melon accessions. 

Name/ Country/ variety/Aria Seed PS-ID SNPs SSR (T) n 

accession No. Cultivar group size type S1 S2 S3 SSR1 SSR2 

PI 505602 Zambia Ssp. melo S NA C T C 16 15 

PI 436533 Senegal Ssp. melo S NA C T C 15 13 

940108 Senegal Group agrestis S NA C T C 15 13 

PI 436534 Senegal Group agrestis S NA C T C 15 13 

PI 482398 Zimbabwe Ssp. melo S NA C T G 16 14 

PI 185111 Ghana Group agrestis S NA C T G 15 13 

940111 Sudan Group agrestis S NA C T G 16 15 

Cam-84-3 Cameroon S NA C T G 16 15 

PI 169379-1 Turkey Ssp. melo L T A T G 15 14 

PI 525105 Egypt Ssp. melo L T A T G 15 14 

PI 164585 India Tamil Nadu L T A T G 14 14 

PI 124096 India Andra Pradesh S T A T G 15 14 

Earls' Favourite England house type Group cantalupensis L T A T G 14 9 

Kinpyo Japan Group conomon S A A A G 12 13 

PI 482411 Zimbabwe Ssp. melo S A A A G 11 13 

PI 126054 Afghanistan Ssp. melo L A A A G 11 13 

PI 536480 Maldives Ssp. melo S A A A G 11 13 

PI 116666 India Pun Punjab L A A A G 11 13 

Arabidopsis thaliana (NC_000932) C T G 7 7 

of melon from various parts of the world were determined by dCAPS 

analysis. One hundred accessions were proved to be of T-type and 131 of 

A-type (Table 1); PCR amplification was unsuccessful in 14 accessions 

(NA-type). For the characterization of NA-type, PCR product amplified 

by Psid1F and Psid1R primers were sequenced, and additional SNPs were 

detected at S1 (C/A) and S3 (C/G) in NA-type (Table 2). NA-type proved 

to be of T-type at S2. Furthermore, since S1 is included in the sequence 

complementary to dCAPS forward primer, no amplification by dCAPS 

primer was caused by this SNP. According to the result of dCAPS 

analysis, all of the 46 large-seed accessions classified as Group 

Cantalupensis and Group Inodorus were of T-type, while 34 of 35 

small-seed accessions belonging to Group Agrestis and Group Conomon 

were of A-type. This result clearly indicated that chloroplast genome type 

was different between areas of origin and between small- and large-seed 

types. 

The SNP type of landraces from Africa and Asia is rather diversified, 

and differs among geographical groups even in Africa. T-type with large 

seed was frequent in the northern part of Africa, while A-type with small 

seed was frequent in the southern part. NA-type was commonly found in 

central and southern parts of Africa, where wild species of Cucumis were 

abundant. Furthermore, the sequence of NA-type (C-T-G) at S1, S2, and 

S3 was the same as that of Arabidopsis thaliana and Nicotiana tabacum. 

These results suggest that NA-type could be the primitive type of 


cultivated melon, and that the T-type has evolved by single nucleotide 

substitution from C to A at S1. A-type seems to have originated from 

T-type by single nucleotide substitution from T to A at S2, or from 

NA-type by double nucleotide substitutions from C to A at S1 and from T 

to A at S2. Another NA-type (C-T-C) should appear by single nucleotide 

substitution from G to C at S3. Taking the geographical distribution of 

each PS-ID type and seed length of each accession into consideration, the 

results suggest that large-seed T-type and small-seed A-type were 

independently established in the northern and southern parts of Africa, 

respectively. Genetic difference among melon accessions from the 

northern and southern parts of Africa detected by RAPD analysis (Mliki et 

al., 2001; Akashi et al., 2006) could be explained by the difference in their 

origin. 

In Asia, NA-type was not found; T-type with large seed was 

predominant in West and Central Asia. Indian melon was diversified and 

both T- and A-types were found in large-seed type as well as in 

small-seed type. The existence of recombinant types, that is, A-type with 

large seed and T-type with small seed, could be explained by the genetic 

interchange that has occurred in India. Based on these results, we propose 

the following hypothesis about the transmission route of small- and 

large-seed types (Figure 1): A-type with small seed was transmitted from 

South Africa to India by the so-called sea route, and the prototype of 

Group Conomon vars. makuwa and conomon originated from small-seed 

melon under wet conditions in East India. Another type of melon 

introduced from North Africa to Europe and India via the Middle East 

(Robinson and Decker-Walters 1997) was the large-seed T-type. After the 

establishment of the two types in India, the interchange between them 

resulted in differentiation into several varieties, enhancing India’s genetic 

diversity. India is considered as the secondary center of diversity in melon 

(Whitaker and Davis 1962). The result obtained by the analysis of nuclear 

genome (Akashi et al., 2006) also supports this hypothesis. 

Among the four ccSSR markers analyzed, ccSSR7 proved to be 

polymorphic by the deletion of 5bp (ATATT), and its size polymorphism 

was surveyed for 245 accessions of melon. Most of the melon accessions 

possessed this 5bp, and amplicon size was 336bp. The deletion of 5bp, the 

amplicon size being 331bp, was found in 24 accessions of large-seed 

melon whose PS-ID sequence was of T-type (Table 1). The deletion type 

was found in accessions of Group Inodorus (Honeydew and Russian or 

Spanish winter melon) and landraces from North Africa and West and 

Central Asia. Therefore, it is likely that the deletion occurred in the 

population of T-type with large seed and that Group Inodorus has evolved 


from such deletion type. However, ccSSR7 of ‘Hami Gua’, cultivated in 

the western part of China, is without this deletion, though this melon is 

also classified as Group Inodorus. Further study is necessary to discover 

the origin of ‘Hami Gua’. Deletion type found in the American field type 

should be ascribable to the use of Honeydew-type cultivars as 

cross-parents for breeding. 

By the analysis of PS-ID sequence, we could hypothesize that T-type 

melon with large seed and A-type melon with small seed has been 

polyphyletically differentiated from NA-type melon originating in the 

central part of Africa. Differentiation into several groups or varieties 

within T-type melon with large seed could be partially discovered by the 

analysis of ccSSR marker. Wild species of Cucumis should be analyzed, 

using cytoplasmic markers, to understand the origin of cultivated melon. 

Large-seed type 

T / 336bp type 

Group Inodorus 



T / 336bp & 331bp types 

Small-seed type 

NA type 


NA, A types 







interchange 


A type 


A type 

Fig. 1. Polyphyletic origin and transmission route of cultivated melon, revealed by 

the analysis of chloroplast genome. White and black arrows indicate the 

transmission route of the large- and small-seed type, respectively. PS-ID type (T, A, 

NA) and size (bp) of ccSSR7 are also indicated. 


Akashi, Y., N. Fukuda, T. Wako, M. Masuda, and K. Kato. 2002. Genetic variation and 

phylogenetic relationships in East and South Asian melons, Cucumis melo L., based 

on the analysis of five isozymes. Euphytica 125:385–396. 

Akashi, Y., K. Tanaka, H. Nishida, S. S. Yi, T. T. Chou, and K. Kato. 2006. Genetic 

diversity and phylogenetic relationship among melon germplasm from Africa and 

Asia revealed by RAPD analysis. Cucurbitaceae 2006. 

Chung, S., D. S. Decker-Walters, and J. E. Staub. 2003. Genetic relationships within the 

Cucurbitaceae as assessed by consensus chloroplast simple sequence repeats 

(ccSSR) marker and sequence analyses. Can. J. Bot. 81:1–19. 


Clegg, M. T., B. S. Gaut, G. H. Learn, Jr., and B. R. Morton. 1994. Rates and patterns of 

chloroplast DNA evolution. Proc. Natl. Acad. Sci. 91:6795–6801. 

Fujishita, N., H. Furukawa, and S. Morii. 1993. Distribution of three genotypes for 

bitterness of F1 immature fruit in Cucumis melo. Jpn. J. Breed. 43(Suppl.2):206 (in 

Japanese). 

Ishikawa, R., Y.-I. Sato, T. Tang, and I. Nakamura. 2002. Different maternal origins of 

Japanese lowland and upland rice populations. Theor. Appl. Genet. 104:976–980. 

Kimura, M. 1980. A simple method for estimating evolutionary rates of base substitutions 

through comparative studies of nucleotide sequences. J. Mol. Evol. 16:111–120. 

Mliki, A., J. E. Staub, Z. Sun, and A. Ghorbel. 2001. Genetic diversity in melon (Cucumis 

melo L.): an evaluation of African germplasm. Genet. Res. Crop. Evol. 48:587–597. 

Monforte, A. J., J. Garcia-Mas, and P. Arus. 2003. Genetic variability in melon based on 

microsatellite variation. Plant Breed. 122:153–157. 

Munger, H. M. and R. W. Robinson. 1991. Nomenclature of Cucumis melo L. Cucurbit 

Genet. Coop. Rpt. 14:43–44. 

Murray, G. C. and W. F. Thompson. 1980. Rapid isolation of high molecular weight DNA. 

Nuc. Acid Res. 8:4321–4325. 

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proposal for identifying the short ID sequence which addresses the plastid subtype of 

higher plants. Breed. Sci. 47:385–388. 

Nakata, E., J. E. Staub, and A. I. López-Sesé. 2005. Genetic diversity in Japanese melon 

cultivars (Cucumis melo L.) as assessed by random amplified polymorphic DNA and 

simple sequence repeat markers. Genet. Res. Crop. Evol. 52:405–419. 

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the genetic analysis of single nucleotide polymorphisms: experimental applications in 

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York. 

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22:4673–4680. 

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Interscience, New York. 

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RESISTANCE OF CITRULLUS LANATUS VAR. 

CITROIDES GERMPLASM TO ROOT-KNOT 

NEMATODES 

Judy A. Thies and Amnon Levi 

U.S. Vegetable Laboratory, USDA, ARS, Charleston, SC 

ADDITIONAL INDEX WORDS. Citrullus colocynthis, Citrullus lanatus var. lanatus, 

Meloidogyne arenaria, Meloidogyne incognita, nematode resistance, root-knot 

nematode, watermelon 

ABSTRACT. Root-knot nematodes (Meloidogyne spp.) cause extensive damage to 

watermelon [Citrullus lanatus (Thunb.) Matsum. & Nakai var. lanatus] and rootknot 

nematode-resistant watermelon cultivars are not available. Twenty-nine 

accessions from the U.S. Plant Introduction (USPI) collection of Citrullus spp. were 

evaluated for resistance to the southern root-knot nematode [Meloidogyne incognita 

(Kofoid & White) Chitwood Race 3] in greenhouse tests. The Plant Introductions 

(PI) evaluated in these studies included: 25 Citrullus lanatus (Thunb.) Matsum. & 

Nakai var. citroides (L. H. Bailey) Mansf., 1 C. lanatus var. lanatus, and 3 Citrullus 

colocynthis (L.) Schrad. Twenty-three of the C. lanatus var. citroides PI and the C. 

lanatus var. lanatus PI had been previously identified as moderately resistant to M. 

arenaria Race 1. In general, the C. lanatus var. citroides PI exhibited low to 

moderate resistance to M. incognita Race 3 and the C. lanatus var. lanatus and C. 

colocynthis PI were susceptible. The C. lanatus var. citroides PI 482303 was the most 

resistant PI with gall index (GI) = 2.97 and reproductive index (RI) = 0.34 (1 = no 

galling; 5 = 26 to 38% root system galled; 9 = 81 to 100% root system galled). 

Significant genetic variability was observed within C. lanatus var. citroides for 

reaction to M. incognita, and several C. lanatus var. citroides PI may be useful 

sources of resistance to root-knot nematodes. 

R 

oot-knot nematodes (Meloidogyne spp.) cause significant damage 

to watermelon throughout the southern U.S. (Sumner and Johnson, 

1973; Thies, 1996; Thomason and McKinney, 1959; Winstead and 

Riggs, 1959), and increase the severity of Fusarium wilt in watermelon 

fields (Sumner and Johnson, 1973). Several reports describe the reactions 

of cultivated watermelon to root-knot nematodes. Winstead and Riggs 

(1959) evaluated 78 watermelon cultivars and five breeding lines, and all 

were susceptible to root-knot nematode. In Puerto Rico, 10 watermelon 

cultivars were evaluated and found susceptible to M. incognita (Montalvo 

and Esnard, 1994). Thomason and McKinney (1959) reported that 

‘Striped Klondike’ watermelon was susceptible to M. incognita acrita and 

M. javanica. 


Root-knot nematodes are chiefly managed in watermelon by 

fumigation with methyl bromide or other fumigant nematicides. 

Watermelon and melon (Cucumis melo L.) account for approximately 6% 

of methyl bromide used for preplant soil treatments in vegetable crops 

worldwide (USDA, 1993). According to the Montreal Protocol and the 

U.S. Clean Air Act, methyl bromide was phased out 1 January 2005 (Rich 

and Olson, 2004; U. S. Environmental Protection Agency, 2000); 

however, under the U.S. nomination for critical-use exemption program, 

growers continue to use specified allocations (U.S. Environmental 

Protection Agency, 2006). The use of methyl bromide in watermelon may 

be underestimated because watermelon is often grown as the second crop 

in double-crop systems. In the double-crop system, soil fumigation prior 

to planting the first crop also greatly benefits the second crop by 

preventing build-up of root-knot nematodes and disease pathogens in the 

soil. In Florida and Georgia, annual yield losses of 15 to 20% have been 

predicted for watermelon due to the imminent loss of methyl bromide for 

preplant soil fumigation (Lynch and Carpenter, 1999). Alternative 

methods to methyl bromide are urgently needed for managing root-knot 

nematodes in watermelon. Root-knot-nematode-resistant cultivars would 

provide the most economical and environmentally friendly alternative for 

managing root-knot nematodes in watermelon. 

In an earlier study, we identified moderate resistance to M. arenaria 

Race 1 among C. lanatus var. citroides PI (Thies and Levi, 2003). The 

objective of this study was to evaluate selected C. lanatus var. citroides 

PI, previously shown to be moderately resistant to M. arenaria Race 1, for 

reaction to M. incognita Race 3. 


NEMATODE INOCULUM. Meloidogyne incognita Race 3 was cultured 

on ‘Rutgers’ tomato (Lycopersicon esculentum Mill.) in isolated 

greenhouse benches. Egg inocula were extracted from tomato roots using 

0.5% sodium hypochlorite (Hussey and Barker, 1973). 

CITRULLUS GERMPLASM. Twenty-five PI of C. lanatus var. citroides, 

one PI of C. lanatus var. lanatus, and three PI of C. colocynthis from the 

U.S. PI Citrullus germplasm collection were evaluated for resistance to M. 

incognita Race 3 in greenhouse tests. The PI were selected based on their 

reactions to M. arenaria Race 1 in earlier greenhouse studies (Thies and 

Levi, 2003). ‘Charleston Gray’, ‘Crimson Sweet’, and ‘Dixie Lee’ (C. 

lanatus var. lanatus) were included as susceptible control cultivars in all 

tests. 


EXPERIMENTAL DESIGN. The experimental design was a randomized 

complete block with five plants per replicate. The experiment was 

conducted twice with four replicates in the first experiment and three 

replicates in the second experiment. 

GREENHOUSE EVALUATIONS. Seeds of each watermelon entry were 

sown in trays containing 50 0.2-L cells (one seed per cell) filled with a 

commercial potting medium in the greenhouse. When seedlings were at 

the first-true-leaf stage, approximately 3mL distilled water containing 

approximately 2,500 eggs of M. incognita Race 3 was pipetted into the 

rhizosphere soil of each plant. Two and five weeks after sowing, plants 

were fertilized with one-half strength 20N-20P-16K water-soluble 

fertilizer. Greenhouse temperatures ranged from 26 to 31˚C during the 

experiments. Eight weeks after inoculation, shoots of all plants were 

clipped at the crown, and roots were removed from each cell and carefully 

washed. The root system of each plant was placed in a 15% solution of 

McCormick’s red food color (Thies et al., 2002) for approximately 15 min 

to stain the egg masses. Then the root system was rinsed under tap water 

and evaluated for galling severity and egg-mass production using a 1 to 9 

scale where 1 = 0; 2 = 1 to 3%; 3 = 4 to 12%; 4 = 13 to 25%; 5 = 26 to 

38%; 6 = 39 to 50%; 7 = 51 to 65%; 8 = 66 to 80%; and 9 = 81 to 100% of 

the root system galled or covered with egg masses, respectively (Thies and 

Fery, 1998). Ratings of 1 to 2.9 = high resistance; 3.0 to 4.0 = moderate 

resistance; 4.1 to 4.9 = low resistance; 5.0 to 6.9 = susceptible; and 7.0 to 

9.0 = highly susceptible (Thies and Levi, 2003). Then the entire root 

system of all plants of each genotype in a replicate were cut into 1- to 2cm 

pieces, total root weight was recorded, and root-knot nematode eggs 

were extracted from the root sample using 1.0% NaOCl (Hussey and 

Barker, 1973). Eggs were counted with a stereomicroscope. Nematode 

reproduction was assessed by calculating the reproductive index (RI) in 

which RI = Pf/Pi, where Pi = the initial inoculum level and Pf = final egg 

recovery (Sasser et al., 1984). Eggs per g fresh root and nematode 

reproductive index data were transformed by log10 (x+1) before analysis. 

Data were analyzed using the GLM procedure of SAS for Windows, v.8.0, 

and the means were separated using Fisher’s Protected Least Significant 

Difference Test. 


In general, the C. lanatus var. citroides PI exhibited higher resistance 

to M. incognita than the watermelon cultivars C. lanatus var. lanatus PI 

and C. colocynthis PI. The C. lanatus var. citroides root systems had 


minimal to moderate galling (Table 1). The C. lanatus var. citroides PI 

had more fibrous roots and fewer, smaller galls than the C. colocynthis PI, 

the C. lanatus var. lanatus PI, and the control watermelon cultivars. The 

C. lanatus var. lanatus PI was susceptible and the PI of the desert species 

C. colocynthis were highly susceptible to M. incognita Race 3. Results of 

Experiments 1 and 2 were similar; results of the second experiment are 

shown. 

Table 1. Gall indices, egg-mass indices, numbers of Meloidogyne 

incognita Race 3 eggs per g fresh root, and reproductive indices for 

selected accessions of Citrullus lanatus var. citroides, C. lanatus var. 

lanatus, and C. colocynthis from the U.S. Citrullus spp. Plant Introduction 

(PI) Collection, and control cultivars inoculated with M. incognita Race 3 

in a replicated greenhouse test. a 

Accession Gall 

(PI No.) index b 

Citrullus lanatus var. citroides 

Egg-mass 

index b 

Eggs/g fresh 

root c 

Reproductive 

index c 

482303 2.97 a d 

2.11 a 1,535 a-c 0.34 ab 

482307 3.22 a 2.13 a 889 a 0.29 ab 

270563 3.00 a 2.50 a 4,151 a-g 0.59 a-d 

482379 3.28 a 2.52 a 5,577 b-g 1.08 b-f 

482338 3.39 ab 2.11 a 1,221 ab 0.24 a 

532624 3.53 ab 3.03 a 6,869 c-h 1.59 d-g 

482326 3.40 ab 2.67 a 2,967 a-e 0.55 a-d 

482319 3.47 ab 2.80 a 2,863 a-e 0.97 b-f 

482324 3.58 ab 2.61 a 3,963 a-g 0.55 a-d 

189225 3.67 ab 2.80 a 16,508 f-h 2.17 d-g 

255137 3.25 ab 2.32 a 3,145 a-f 0.82 b-g 

482333 3.67 3.00 a 4,045 a-g 0.89 b-g 

482342 3.69 ab 2.88 a 3,607 a-g 0.75 a-g 

482301 3.83 a-c 2.60 a 2,299 a-d 0.37 a-d 

244019 3.87 a-c 3.07 a 3,954 a-g 0.69 a-f 

500331 3.93 a-c 2.93 a 4,902 b-g 0.61 a-e 

512854 3.90 a-c 3.40 ab 2,225 a-d 0.61 a-e 

482259 4.00 a 3.40 ab 4,749 b-g 1.60 e-i 

542119 4.07 a-c 3.80 a-c 6,191 b-h 1.46 e-i 

244017 4.07 a-c 2.67 a 4,006 a-g 0.57 a-e 

244018 4.11 a-c 2.70 a 12,873 e-h 1.96 f-i 

485583 4.25 a-c 2.93 a 4,193 a-g 0.80 b-g 

248774 4.50 a-d 3.78 a-c 6,343 b-h 1.12 d-i 

542114 5.38 b-e 5.25 b-d 29,020 hi 5.56 jk 

532666 6.52 e-g 5.12 b-d 18,789 gh 3.15 i-k 

288316 --- --- --- --- 


(Table 1, 

continued) 

Accession 

(PI No.) 

Gall 

index b 

Egg-mass 

index b 

Eggs/g fresh 

root c 

Reproductive 

index c 

Citrullus lanatus var. lanatus 

459074 4.82 a-e 2.97 a 10,278 d-h 2.96 h-k 

C. colocynthis 

525082 5.75 c-f 3.07 a 4,080 a-g 1.18 e-i 

386015 7.67 gh 7.67 e 100,577 i 8.45 k 

432337 8.42 h 5.93 de 8,327 d-h 1.62 e-i 

C. lanatus var. lanatus controls 

Charleston Gray 7.53 f-h 6.87 de 9,658 d-h 5.37 jk 

Crimson Sweet 6.25 d-g 5.25 b-d 5,530 b-g 1.08 d-i 

Dixie Lee 6.09 d-g 5.49 cd 3,497 a-f 0.57 a-e 

a Means of 3 replicates of 5 plants per replicate (n=15). 

b 1 to 9 scale where 1 = no galling or visible egg masses present; 2 = 1% to 3%; 3 

= 4% to 10%; 4 = 11% to 25%; 5 =26% to 35%; 6 =36% to 50%; 7 = 51% to 65%; 8 = 

66% to 80%; and 9 = 81 to 100% of root system galled or covered with egg masses, 

respectively. 

c Data were log10(x+1) transformed before analysis. Nontransformed data are shown. 

d Mean separation within columns by Fisher’s Protected Least Significant Difference Test, 

P < 0.05. 

The C. lanatus var. citroides PI 482303 exhibited high resistance to M. 

incognita Race 3; GI = 2.97 with 1,535 M. incognita eggs per g fresh root 

and RI = 0.34 (Table 1). Likewise, 21 additional C. lanatus var. citroides 

PI exhibited low to moderate resistance to M. incognita Race 3, based on 

root gall severity (GI ranges: 3.00 to 4.25). Some of these PI had 

relatively high numbers of eggs per g fresh root (>5,000) and RI>1.0. For 

example, PI 189225 supported 16,508 eggs of M. incognita per g fresh 

root and RI = 2.17. The PI 248774 and 532666 exhibited susceptible 

reactions to M. arenaria Race 1 in previous tests (Thies and Levi, 2003) 

and also were susceptible to M. incognita Race 3 in the current study. The 

PI 542114 exhibited low resistance to M. arenaria Race 1 in a previous 

unreplicated test (Thies and Levi, 2003), but was moderately susceptible, 

based on nematode reproduction, to M. incognita in the present study. 

PI 459074 was the only one of 156 C. lanatus var. lanatus PI 

evaluated that exhibited any resistance to M. arenaria Race 1 (Thies and 

Levi, 2003). However, in this study, PI 459074 was susceptible to M. 

incognita Race 3, based on both root-gall severity and nematode 

reproduction. The three C. lanatus var. lanatus watermelon cultivars 

(‘Crimson Sweet’, ‘Dixie Lee’, and ‘Charleston Gray’) were also 

susceptible to M. incognita Race 3, as in a previous study with M arenaria 


Race 1 (Thies and Levi, 2003). The GI ranged from 6.09 to 7.53 for 

‘Crimson Sweet,’ ‘Dixie Lee’, and ‘Charleston Gray’; numbers of eggs 

per g fresh root ranged from 3,497 to 9,658 and RI ranged from 0.57 to 

5.37. 

The three C. colocynthis PI were highly susceptible to M. incognita 

Race 3, similar to their reactions to M. arenaria Race 1 in prior tests 

(Thies and Levi, 2003). The root-gall severity indices for the C. 

colocynthis accessions ranged from 5.75 to 8.42; numbers of eggs per g 

fresh root ranged from 4,080 to 100,577; and RI ranged from 1.18 to 8.45. 

In general, the C. lanatus var. citroides PI exhibited low to moderate 

resistance to M. incognita. The C. lanatus var. citroides PI 482303 was 

the most resistant with GI of 2.97 and the RI was 0.34. These results 

demonstrate that there is significant genetic variability within C. lanatus 

var. citroides for reaction to M. incognita, and several C. lanatus var. 

citroides PI may provide sources of resistance to root-knot nematodes for 

the development of resistant watermelon cultivars. 


Hussey, R. S. and K. R. Barker. 1973. A comparison of methods of collecting inocula of 

Meloidogyne spp., including a new technique. Plant Dis. Rep. 57:1025–1028. 

Lynch, L. and J. Carpenter. 1999. The economic impacts of banning methyl bromide: 

where do we need more research? 1999 Annual Research Conference on Methyl 

Bromide Alternatives and Emissions Reductions. 

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Montalvo, A. E. and J. Esnard. 1994. Reaction of ten cultivars of watermelon (Citrullus 

lanatus) to a Puerto Rican population of Meloidogyne incognita. Suppl. J. Nematol. 

26(4S):640–643. 

Rich, J. R., and S. M. Olson. 2004. Influence of MI-gene resistance and soil fumigant 

application in first crop tomato on root-galling and yield in a succeeding cantaloupe 

crop. Nematropica. 34:103–108. 

Sasser, J. N., C. C. Carter, and K. M. Hartman. 1984. Standardization of host suitability 

studies and reporting of resistance to root-knot nematodes. Crop Nematode 

Resistance Control Project, N.C. State Univ., U.S. Agency for Intl. Dev., Raleigh, 

NC. 

Sumner, D. R. and A. W. Johnson. 1973. Effect of root-knot nematodes on Fusarium 

wilt of watermelon. Phytopathology. 63:857–861. 

Thies, J. A. 1996. Diseases caused by nematodes, p. 56–58. In: T. A. Zitter, D. L. 

Hopkins, and C. E. Thomas (eds.). Compendium of cucurbit diseases. APS Press, 

St. Paul, MN. 

Thies, J. A. and A. Levi. 2003. Resistance of watermelon germplasm to the peanut rootknot 

nematode. HortSci. 38:1417–1421. 


Thies, J. A. and R. L. Fery. 1998. Modified expression of the N gene for southern rootknot 

nematode resistance in pepper at high soil temperatures. J. Amer. Soc. Hort. 

Sci. 123:1012–1015. 

Thies, J. A., S. B. Merrill, and E. L. Corley, Jr. 2002. Red food coloring stain: new, 

safer procedures for staining nematodes in roots and egg masses on root surfaces. J. 

Nematol. 34:179–181. 

Thomason, I. J. and H. E. McKinney. 1959. Reaction of some Cucurbitaceae to rootknot 

nematodes (Meloidogyne spp.). Plant Dis. Rep. 43:448–450. 

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ozone: incorporation of Clean Air Act amendments for reductions in Class I, Group 

VI controlled substances. Fed. Reg. 65 (No. 229):70795–70804. 

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United States Department of Agriculture. 1993. USDA workshop on alternatives for 

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FACTORS INFLUENCING A CUCUMBER 

FRUIT SUSCEPTIBILITY TO INFECTION BY 

PHYTOPHTHORA CAPSICI 

Kaori Ando and Rebecca Grumet 

Plant Breeding and Genetics Program Michigan State University, 

A291-A Department of Horticulture PSSB, East Lansing, MI 48824 

ADDITIONAL INDEX WORDS. Age-related resistance, fruit rot, epidermal 

ABSTRACT. Phytophthora capsici-induced fruit rot causes severe losses in 

cucumber production. Our earlier studies showed that cucumber fruit are most 

susceptible when very young, but become resistant as the fruit stop elongating. 

In the field, infection occurs primarily at the blossom end. To determine 

whether preferential infection of the blossom end was due to position-related 

differences, or was a result of earlier contact with inoculum-containing soil, 

greenhouse-grown cucumber fruit ranging in age from 6–14 days 

postpollination (dpp) were inoculated with P. capsici at the stem and blossom 

ends. The youngest fruits supported sporulation on both ends. As fruit age 

increased, the stem end became less susceptible sooner, suggesting a 

developmental gradient within the fruit influencing susceptibility. We tested 

whether the increase in resistance is due to changes in surface or mesocarp 

properties by inoculating peeled fruit or epidermal sections from 7- or 14-dpp 

fruit that were placed onto intact 7- or 14-dpp fruit. Peeled fruits rapidly 

became infected. Peel sections from 7-dpp, but not 14-dpp, fruit supported 

sporulation. Intact, underlying 7-dpp fruit were protected if covered with 14dpp, 

but not 7-dpp, peels. These results indicate that the epidermal section plays 

a role in the age-related resistance. 

I 

n recent years, Phytophthora capsici has become an increasingly 

severe disease of a wide range of vegetable crops, including 

cucumber, where it can cause devastating yield losses (Hausbeck 

and Lamour, 2004). Cucumber fruit infected by P. capsici typically 

develop a depressed fruit surface with a water-soaked appearance 

followed by white powdery mycelium covering the affected region. 

Field observations show that cucumber fruit are susceptible to P. 

capsici, while roots or crowns are much less susceptible. Fields will 

frequently look healthy at time of harvest as judged by vegetative 

growth, but the fruits can be heavily diseased (Hausbeck and Lamour, 

2004). Fruit are usually located under the canopy where the 

This research was supported in part by the Pickle Seed Research Fund and MSU- 

GREEEN. We would like to thank Drs. Mary Hausbeck and Amanda Gevens for 

helpful advice and providing the P. capsici isolate OP97. We also thank Dr. Hua 

Zhang for helpful suggestions and Seminis Vegetable Seed Inc. for providing seeds. 


environment is moist, warm, and close to the pathogen in the soil, thus 

favoring conditions for disease development. Trellis and plant 

architecture studies have indicated that removal of fruit from the soil 

surface can reduce disease incidence (Ando and Grumet, 2006). 

Genetic resistance would be the optimal method of disease control. 

Unfortunately, screening of a diverse collection of the cucumber 

germplasm accessions for fruit resistance to P. capsici has not 

identified any resistant genotypes to date (Gevens et al., 2006). In the 

course of screening, however, we observed an effect of fruit age on 

susceptibility (Gevens et al., 2006). ‘Vlaspik’ fruits used as susceptible 

controls did not become uniformly infected. It appeared that smaller, 

younger fruit were more susceptible, while larger, older fruit were less 

frequently infected. When hand-pollinated fruit of known ages were 

tested, most fruit younger than 10 days postpollination (dpp) exhibited 

sporulation at 4 days postinoculation (dpi), whereas fruit at 14dpp 

generally remained symptom-free (Gevens et al., 2006). Fruit of 

intermediate ages (10 to 14dpp) often exhibited water-soaked regions 

without sporulation. The effect of fruit age was observed for several 

different genotypes. Field-grown cucumbers (cv. Straight Eight), 

harvested to test a range of fruit sizes, exhibited the same trend as 

observed with greenhouse-grown fruit. These results indicated that 

there are changes in the degree of susceptibility among fruit of 

different ages in both greenhouse and field conditions, and that the 

age-related decrease in susceptibility is not genotype specific. 

Interestingly, the transition from susceptible to resistant appeared 

to coincide with the transition from the period of rapid fruit elongation 

to the period of increased fruit diameter (approximately 12cm length, 

3cm width). Cucumber fruit rapidly elongate longitudinally until they 

reach mature size; elongation then ceases, while width of the fruit 

continues to grow (Wehner and Saltveit, 1983). Physiological changes 

associated with fruit growth and development might explain changing 

susceptibility among fruit of different ages. 

Further examination of P. capsici-infected cucumber fruit in the 

field led to the observation that field-grown cucumbers are typically 

covered with mycelium on the blossom end of the fruit rather than the 

stem end (Figure1A). We speculated that this was due either to the 

tendency of the blossom end to come into contact with the inoculumcontaining 

soil sooner than the stem end, or that there is a 

susceptibility gradation within the fruit such that the blossom end is 

more susceptible than the stem end. In these experiments we sought to 

determine the nature of the difference in occurrence of disease at the 

blossom and stem ends and also to further examine the basis for age- 


elated increased resistance by asking whether it is due to changes in 

surface or mesocarp properties. 


FRUIT POSITION EXPERIMENTS. ‘Vlaspik’ fruits were grown in the 

MSU Research greenhouse, East Lansing, MI, during spring 2004. 

Fruits were hand-pollinated over a period of days to allow for 

simultaneous harvest of fruit ranging in age from 6 to 14dpp. 

Harvested fruit were screened for P. capsici resistance according to the 

methods of Gevens et al. (2006). Two 6-mm-diameter plugs of isolate 

OP97 were placed approximately 2cm from the stem and blossom ends 

of each fruit, and covered with an inverted lidless 1.5-ml 

microcentrifuge tube held in place with petroleum jelly (Figure 1B). 

Inoculated fruit were incubated at room temperature in sealed 

aluminum trays, and were scored for symptoms (0 = no symptoms, 1 = 

water soaked, and 2 = sporulation) at 4dpi. 

EPIDERMAL SECTION EXPERIMENTS. Field-grown (MSU 

Horticulture Research Farm, East Lansing, MI, summer 2004) 

oversized ‘Vlaspik’ fruits of unknown age were harvested, washed 

with distilled water, and air-dried. Fruits were peeled with a 

conventional peeler to remove a section of fruit surface of 

approximately 1.5cm x 23cm, 0.2cm from the center of the fruit. 

Peeled and unpeeled fruits were then inoculated with P. capsici 

mycelium as described above and evaluated for symptoms at 4dpi. 

In a second set of experiments, greenhouse-grown, hand-pollinated 

fruit-surface sections (1.6cm diameter and 0.1cm thick) were removed 

from 7 or 14dpp with a cork bore and a petit knife. Surface sections of 

7-dpp fruit were placed on 7- and 14-dpp fruit, and 14-dpp surface 

sections were placed on 7- and 14-dpp fruit. The underlying tissue was 

left intact. P. capsici mycelia were placed on top of the transplanted 

fruit-surface sections. Inoculated fruits were evaluated for symptoms 

at 4dpi on both the fruit-surface section and underlying intact fruit. 


EFFECT OF FRUIT POSITION. P. capsici-infected cucumber fruit in the 

field are typically covered with mycelium on the blossom end of the 

fruit rather than the stem end. To determine whether this phenomenon 

is due to a greater tendency of the blossom end of the fruit to touch the 

soil, or if there is a difference in susceptibility due to position on the 

fruit, hand-pollinated greenhouse-grown fruit (ranging from 6 to 

14dpp) were inoculated with P. capsici mycelium approximately 2cm 

from the blossom end and the stem end (Figure 1B). 


Fig. 1. Typical symptoms of P. capsici in the field: (A) Mycelium covering the 

blossom end of fruits. (B) Mycelium inoculation of the stem and blossom ends of 

6- to 14-dpp fruit. The picture was taken at 4dpi. Arrows indicate blossom end 

of fruit. (C) Greenhouse-grown, hand-pollinated ‘Vlaspik’ inoculated on stem 

and blossom ends of fruit age ranging from 6 to 14dpp. (D) Symptoms of fruit 

ranging from 6 to 14dpp evaluated at 4dpi (0 = no symptoms, 1 = water soaking, 

and 2 = sporulation). Each point is the mean of a minimum of 10 fruits ± S. E. 

When fruit were very young (6dpp), both the blossom end and the 

stem end sporulated (Figure 1 C, D). However, as fruit develop, the 

stem end becomes less susceptible earlier than the blossom end, so that 

sporulation occurs on the blossom end, but not the stem end (e.g., at 8– 

12dpp). At 14dpp the stem end did not develop symptoms, whereas the 

blossom end sometimes developed water-soaked regions. These results 

suggest a developmental gradient within the fruit with the stem end 

maturing more rapidly than the blossom end. 

These results indicate that there are differences in susceptibility 

within a fruit. Fruit-growth analyses performed by Wehner and 

Salveit (1983) showed that the blossom end continues growing after 

the stem end has stopped elongating, and that the differential transition 

in growth phase occurs in the 7–14-dpp time period. This positional 

difference in growth rate is consistent with the observations that the 

blossom end is more susceptible than the stem end, and with our 


previous studies (Gevens et al., 2006) showing that the degree of P. 

capsici susceptibility decreases as the fruit matures beyond the stage of 

rapid elongation. 

ROLE OF FRUIT SURFACE. Preliminary tests were conducted to 

examine whether the age-related decrease in susceptibility is 

associated with the fruit surface, mesocarp, or both. The peeled fruits 

all sporulated, whereas intact control fruit did not sporulate at 4dpi, 

indicating that the fruit surface is an important factor for the resistance 

of older fruits to infection by P. capsici (Figure 2). However, these 

experiments do not allow us to rule out possible effects of wounding 

due to peeling. 

To further study the role of the epidermis using intact (nonwounded) 

fruit, epidermal exchanges were made between fruits at 7 and 14dpp (i.e., 

pieces of epidermis from one fruit were placed on top of a second, intact 

fruit). The 7-dpp fruit-surface pieces sporulated regardless of fruit age 

underneath (mean disease rating = 2 ± 0) (Figure 3). Fruit-surface 

sections from 14-dpp fruit generally did not sporulate regardless of fruit 

age underneath; however, approximately 50% developed water-soaked 

symptoms (mean disease rating = 1 ± 0.3). The fruit underneath the 7dpp 

fruit-surface pieces showed susceptibility similar to the fruit-age 

study; 7-dpp fruit generally sporulated while 14-dpp fruit generally did 

not, even in the presence of sporulating 7-dpp fruit-surface sections. 

However, when 14-dpp fruit-surface pieces were inoculated, the 

underlying 7-dpp fruit did not sporulate, indicating that the 14-dpp fruitsurface 

sections protected the underlying 7-dpp fruit. These results 

suggest that 14-dpp fruit-surface sections possess properties that inhibit 

P. capsici infection. 


Fig. 2. Greenhouse-grown ‘Vlaspik’ were inoculated with P. capsici mycelium 

after peeling. (A) 0dpi, (B) 4dpi (arrows indicate sporulation), and (C) 

summary of fruit symptoms at 4 dpi. Each value is the mean of 7 fruit ± S. E. 

This study suggests that properties of the epidermal sections could 

be the source for the susceptibility difference associated with fruit 

development. This difference could be due to cuticle properties, as was 

observed to be a physical barrier to P. capsici infection in New 

Mexican-type pepper (Capsicum annuum) (Biles et al., 1993). 

Another possibility is a difference in frequency of stomata, since 

Smith et al. (1979) reported that stomata are almost absent from the 

stem end compared to the blossom end, and are less frequent in larger 

(older) fruits relative to younger fruit. Whether the P. capsici 

sporangia enter primarily through cucumber fruit stomata remains to 

be determined. Other possibilities include changes in cell-wall 

properties, production of defense compounds, or induced resistance 

mechanisms. 


Disease score: 0-no symptoms, 1-water 

soaked, 2-sporulation 

2 

1 

0 

7 on 7 7 on 14 14 on 7 14 on 14 

Fig. 3. Response to P. capsici inoculation for the 7- or 14-dpp isolated epidermis 

section and intact 7- or 14-dpp fruit underneath. Left bar is the epidermal 

section and right bar is intact fruit underneath the peel. The open bar is 7 dpp 

and the dotted bar is 14 dpp. The P. capsici mycelium plug was placed on the 

peel. Each point is the mean of a minimum of 4 fruits ± S. E. 

In conclusion, these results demonstrate that there is a gradation of 

susceptibility within the fruit such that the stem end of the fruit 

becomes less susceptible to P. capsici sooner than the blossom end. 

This difference in susceptibility could be due to position-related 

differences in fruit development between the stem and blossom end, as 

older fruit are less susceptible than younger fruit, and the stem end 

completes the elongation phase sooner than the blossom end. The 

increased resistance appears to be associated, at least in part, with 

epidermal properties. Further experiments are required to determine 

the properties responsible for the change in susceptibility that occurs 

as fruit develop. 


Ando, K. and R. Grumet. 2006. Evaluation of altered cucumber plant architecture as 

a means to reduce Phytophthora capsici disease incidence on cucumber fruit. J. 

Am. Soc. Hort. Sci. (In press.) 

Biles, C. L., M. M. Wall, M. Waugh, and H. Palmer. 1993. Relationship of 

Phytophthora fruit rot to fruit maturation and cuticle thickness of New 

Mexican–type peppers. Phytopathology. 83:607–611. 

Gevens, A. J., K. Ando, K. Lamour, R. Grumet, and M. K. Hausbeck. 2006. 

Development of a detached cucumber fruit assay to screen for resistance and 

effect of fruit age on susceptibility to infection by Phytophthora capsici. Plant 

Dis. (In press.) 


Hausbeck, M. and K. Lamour. 2004. Phytophthora capsici on vegetable crops: 

research progress and management challenges. Plant Dis. 88(12):1292–1302. 

Smith, K. R., H. P. Fleming, C. G. van Dyke, and R. L. Lower. 1979. Scanning 

electron microscopy of the surface of pickling cucumber fruit. J. Am. Soc. Hort. 

Sci. 104(4):528–533. 

Wehner, T. C. and M. E. Saltveit. 1983. Photographic analysis of cucumber fruit 

elongation. J. Am. Soc. Hort. Sci. 108(4):465–468. 


EARLY PROTECTION AGAINST ROOT-KNOT 

NEMATODES THROUGH NEMATICIDAL 

SEED COATING PROVIDES SEASON-LONG 

BENEFITS FOR CUCUMBERS 

J. O. Becker and J. Smith Becker 

Department of Nematology, University of California, Riverside, CA 

92521, 

H. V. Morton 

VIVA, 1212 Heathrow Drive, Greensboro, NC 27410 

D. Hofer 

Syngenta Crop Protection AG, WRO-1004 7.10, CH-4002, Basel, CH. 

ADDITIONAL INDEX WORDS. Meloidogyne incognita, abamectin, nematicides, 

oxamyl, fosthiazate 

ABSTRACT. Seed coating of cucumber with the microbial-derived 

abamectin at rates of 0.1 to 0.3mg a.i./seed resulted in excellent earlyseason 

seedling protection against root-knot nematodes (Meloidogyne 

incognita) and their detrimental effects on root development. Ten days 

after seeding in root-knot nematode-infested sandy soil, cucumber 

seedlings derived from abamectin-coated seeds contained 

approximately 3–4 times fewer juveniles in their root systems than 

nontreated check plants. The reduction in nematode attack resulted in a 

doubling of the total root length at Day 12 of the greenhouse trial. In a 

Southern California field trial in root-knot nematode-infested sandy 

loam, abamectin seed coating on fresh market cucumber (cv. Kahina) at 

0.3mg a.i./seed (approximately 20g/ha) resulted in growth improvement 

after the first four weeks over the nontreated control . These results 

were similar to the soil-applied nematicides oxamyl and fosthiazate. 

Abamectin seed coating resulted in increased numbers of fruit and 

higher yields compared to the nontreated control and equal to 

carbamate and organophosphate nematicides. The seed coating offers a 

dramatic reduction in nematicide application rate per ha, which is a 

milestone in reduced-risk farming. Nematicidal seed coating promises 

to be a valuable new tool for economically feasible and ecologically 

sensible plant protection. 

R 

oot-knot nematodes (Meloidogyne spp.) are major pests in 

cucurbits, especially in warmer climates. In US cucurbit 

production, estimated yield losses due to plant-parasitic 

nematodes exceed 4% (Koenning et al., 1999). Young seedlings are 

particularly susceptible to the effects of nematode attack and the 

consequent morphological and physiological changes associated with 

the host-parasite relationship. Under favorable environmental 


conditions, high population densities of infectious second-stage 

Meloidogyne juveniles at seeding result in massive invasion of roots 

just behind their tips. Fine secondary roots are often aborted under 

such attack. The establishment of permanent feeding sites, the socalled 

giant cells, lead to nematode-induced changes in root tissues 

that ultimately manifest in root galls. The lack of useful crop resistance 

in cucurbits against root-knot nematodes has lead to the use of 

nematicides as the almost exclusive nematode management tool in 

commercial production. However, in recent years the use of many of 

these compounds has been severely restricted by regulatory action. 

The relatively high application rates are a concern for both the user 

and potential environmental pollution. In addition, the high cost for the 

nematicides and their application has become prohibitive for many 

growers. Some of the contact nematicides have been investigated as 

potential seed treatments (Truelove et al., 1977; Brown, 1984; 

Rodriguez-Kabána and Weaver, 1987; Orion and Shlevin, 1989). But 

issues related to crop tolerance and low efficacy in comparison to high 

soil application rates, as well as lack of interest of the agrochemical 

industry, have hindered their further development. The most potent 

nematicide known has never been developed against plant parasitic 

nematodes. Abamectin is composed of macrocyclic lactones (≥ 80% 

avermectin B1a and ≤ 20% avermectin B1b) that are metabolites of the 

actinomycete Streptomyces avermitilis. Insecticidal and antihelmintic 

activity (Putter et al., 1981) led to its development in antiparasite drugs 

as well as foliar insecticides. Despite some encouraging results by 

low-volume chemigation (Garabedian and Van Gundy, 1983), the 

efficacy of abamectin against root-knot nematodes in other field trials 

was considered insufficient (Nordmeyer and Dickson, 1985). We have 

recently reported on the potential of abamectin as an effective seed 

coating for early- season protection against plant-parasitic nematodes 

(Becker et al., 2003, 2004). This report will further strengthen our 

notion that seed-delivered plant protection will achieve similar benefits 

for the grower while reducing the cost on production and unintentional 

impacts on the environment. 

Materials And Methods 

GREENHOUSE EXPERIMENTS. All cucumber seeds were coated by 

Syngenta Crop Protection. Initially, abamectin was evaluated in 

greenhouse trials using seed-coating loading rates on cucumber cv. 

Mondeo of 0.0, 0.1, and 0.3 mg a.i./seed. The seeds were additionally 

treated with the fungicide thiram (1.6g a.i./kg seed). The trials were 

conducted in soil infested with root-knot nematodes (Meloidogyne 


incognita Race 1). Root-knot inoculum was raised during the previous 

two months on tomato plants in the greenhouse. Nematode eggs were 

harvested by a bleach/sieving extraction (Hussey and Barker, 1973) 

and hatched on Baerman funnels at 26˚C for 3 days. Steam-pasteurized 

river bottom sand (90% sand, 1% silt, 8% clay, 0.2% OM, pH 7.5) was 

infested with 500 J2/100cm 3 soil and filled into 250-cm 3 cups. Each 

cup was seeded with one cucumber seed. The cups were arranged in 

the greenhouse in a randomized complete block design with six 

replications per treatment at 25˚C +/- 2˚C and ambient lighting. 

Irrigation was applied daily as needed. After 12 days, the seedlings 

were carefully removed from the cups and washed free of debris with a 

water spray. The roots were floated in a transparent tray with a 

shallow water layer. They were scanned at 300dpi with an HP flatbed 

scanner and analyzed for total root length with MacRhizo V3.10B 

software (Régent Instruments Inc., Quebec, Canada). 

Pots were filled with 500-cm 3 steam-pasteurized river bottom sand 

infested at planting with 5,000 eggs of M. incognita Race 1. 

Abamectin seed coatings were evaluated at loading rates of 0.0, 0.03, 

0.1, and 0.3 mg a.i./seed. The cucumber (cv. Straight Eight) seeds 

were additionally treated with the fungicide thiram (1.6g a.i./kg seed). 

Each pot was seeded with one seed. Fenamiphos was used as a 

nematicide check and drenched onto the soil at 3mg a.i./L to waterholding 

capacity. The pots were incubated in the greenhouse at ca. 

24˚C +/- 3˚C and ambient lighting. Irrigation and fertilization was 

applied as needed. Five weeks after seeding, root galling was rated on 

a scale of 0–10 (Zeck, 1971). 

FIELD TRIAL. A cucumber trial was conducted at the University of 

California South Coast Research and Extension Center, Irvine, CA, 

during August/September. The soil was a San Emigdio sandy loam 

(12.5% sand, 75.4% silt, 12% clay, 0.45% organic matter, and pH 7.2). 

The field was previously cropped to root-knot nematode-susceptible 

tomatoes. At seeding, the plot site was fairly uniformly infested (73 

J2/cm 3 ) with M. incognita Race 1. Fresh market cucumber seeds (cv. 

Kahina) were coated with abamectin (0.3 mg a.i./seed) and thiram 

(1.6g a.i./kg seed) or with only the fungicides that were used for the 

check and the soil nematicide treatments. Oxamyl and fosthiazate were 

band-applied at 2.03kg a.i./ha and thoroughly incorporated into the top 

10cm soil. The trial was designed as a randomized complete block 

with five replications on 10m x 0.68m beds. Weed and insect control 

as well as fertilization and irrigation were applied using drip tubing 

with 2-L/hr emitters spaced at 0.3m. These agricultural operations 

were conducted according to local standards. The trial was harvested 

after eight and nine weeks and both fruit number and weight per pot 


were taken. Root galling and number of J2/100cm 3 soil at harvest were 

determined. All data were subject to ANOVA and, if appropriate, 

means separation with Fisher’s LSD (SuperANOVA, Abacus, 

Berkeley, CA). 

Results And Discussion 

GREENHOUSE EXPERIMENTS. The first impact of an intense rootknot 

nematode attack on cucumber seedlings is stunting and death of 

many small secondary roots. Galling is more easily recognized and 

more often reported than the reduction in root length, and its 

consequence on seedling development is equally important. Abamectin 

seed coating mitigated this impact and increased the total root length 

by 75 and 92% with 0.1 and 0.3mg a.i./seed, respectively (Figure 1). 

There was no difference in rate response. in another greenhouse trial at 

five weeks after seeding. At this time, the abamectin seed coatings 

reduced the disease expression by 1 to 3 rating classes depending on 

the nematicide seed loading rate (Figure 2). While even the lowest rate 

of 0.03mg a.i./seed reduced galling, the higher rates were more 

effective. A soil-drench application of fenamiphos was the most 

effective. 

The effect of the nematicidal seed coating on root galling was 

assessed in another greenhouse trial at 5 weeks after seeding. At this 

time, the abamectin seed coatings reduced the disease expression by 1 

to 3 rating classes depending on the nematicide seed loading rate 

140 

120 

100 

80 

60 

40 

20 

0 

check aba 0.1 

treatments 

aba 0.3 

Fig.1. Effect of abamectin seed coatings on root length of cucumber seedlings 

after 12 days in root-knot nematode-infested sandy soil. Bar indicated standard 

error (P = 0.05). 


(Fig.2). While even the lowest rate of 0.03 mg a.i./seed reduced galling, 

the higher rates were more effective. A soil drench application of 

fenamiphos was the most effective. 

FIELD TRIAL. The trial was conducted under a high initial rootknot 

nematode-population density. All nematicide treatments resulted 

in more vigorous growth that became obvious about three–four weeks 

after seeding in comparison to the check. At the first harvest, eight 

weeks after seeding, the number of fruits in all nematicide treatments 

was increased by 40–50% compared to the nontreated check (Figure 3). 

This resulted in fruit weight increases that exceeded the check by more 

than 100% (Figure 4). There was no difference among treatments in 

the second harvest (Figures 3 and 4). This may be an indication that 

the presumably larger and healthier root systems in the nematicide 

treatments provided more feeding sites for the nematodes. The 

increasing root-knot nematode population started to affect the treated 

plants by diminishing the root-size advantage. At termination of the 

trial, there were no differences in galling or number of J2 among the 

treatments (data not shown). 

root galling (scale 0-10) 

6 

5 

4 

3 

2 

1 

0 

check 

0.03 mg aba 

0.1 mg aba 

Fig. 2. Effect of abamectin seed coating (aba) on cucumber root galling (0–10 

scale) in M. incognita-infested sandy soil five weeks after seeding. Fenamiphos 

was applied as a soil drench. Bars indicate standard error (P = 0.05). 


0.3 mg aba 

fenamiphos 3 ppm

160 

140 

120 

100 

80 

60 

40 

20 

0 

check abamectin oxamyl fosthiazate 

1. harvest 

2. harvest 

Fig. 3. Yield comparison of abamectin seed coating (0.3mg a.i./seed) to soil 

nematicides (each 2.03kg a.i./ha) in a cucumber field trial in M. incognitainfested 

sandy loam. Bars indicate standard error (P = 0.05). 

These results challenge earlier opinions that predicted a limited 

value of nematicidal seed coatings due to their inability to provide the 

soil-population reduction necessary for season-long control 

(Thomason, 1987). The presented data show that under high nematode 

disease pressure a significant reduction in root loss can be avoided 

with an effective protection by a seed-delivered nematicide. Seed 

treatments are ideally positioned to mitigate nematode attack against 

the young root system. In addition, the protection of the area around 

the seed might last much longer than that achieved by a soil-applied 

nematicide. Although this protection might not be sufficient for longseason 

crops or in situations were multiple nematode generations 

occur, it has been shown for a number of crops that the first couple of 

weeks determine a large part of the yield potential (Seinhorst, 1995; 

Ploeg and Phillips, 2001). Equally important, the huge reduction in 

pesticide-application rates as a consequence of seed-coating 

technology must be considered a major breakthrough in crop 

protection against plant-parasitic nematodes and a significant 

contribution to reduced-risk farming. 


fruit weight (%) 

250 

200 

150 

100 

50 

0 

check abamectin oxamyl fosthiazate 

1. harvest 

2. harvest 

Fig. 4. Yield comparison of abamectin seed coating (0.3mg a.i./seed) to soil 

nematicides (each 2.03kg a.i./ha) in a cucumber field trial in M. incognitainfested 

sandy loam. Bars indicate standard error (P = 0.05). 


Becker, J. O., B. Slaats, and D. Hofer. 2004. Cucumber seed coating with abamectin 

guards against early root damage by root-knot nematodes. Phytopathology. 

94:S149. 

Becker, J. O., H. V. Morton, and D. Hofer. 2003. Seedling protection against plant 

parasitic nematodes by abamectin seed dressing. 8th International Congress of 

Plant Pathology. 2:251. 

Brown, R. H. 1984. Cereal cyst nematode and its chemical control in Australia. Plant 

Dis. 68:922–928. 

Garabedian, S. and S. D. Van Gundy. 1983. Use of avermectins for the control of 

Meloidogyne incognita on tomatoes. J. Nematol. 13:503–510. 

Hussey, R. S., and K. Barker. 1973. A comparison of methods of collecting inocula 

of Meloidogyne spp., including a new technique. Plant Dis. Rep. 57:1025–1028. 

Koenning, S. R., C. Overstreet, J. W. Noling, P. A. Donald, J. O. Becker, and B. A. 

Fortnum. 1999. Survey of crop losses in response to phytoparasitic nematodes in 

the United States for 1994. J. Nematol. 31: 587618. 

Nordmeyer, D. and D. Dickson. 1985. Management of Meloidogyne javanica, M. 

arenaria, M. incognita on flue cured tobacco with organophosphate, carbamate, 

and avermectin nematicides. Plant Dis. 69:67–69. 

Orion, D. and E. Shlevin.1989. Nematicide seed dressing for cyst and lesion 

nematode control in wheat. J. Nematol. 21(4S): 629-631. 

Ploeg, A. T. and M. S. Phillips. 2001. Damage to melon (Cucumis melo L.) cv. 

Durango by Meloidogyne incognita in Southern California. Nematology. 3:151– 

157. 

Putter, I., J. G. MacConnell, F. A. Preiser, A. A. Haidri, S. S. Ristich, and R. A. 

Dybas. 1981. Avermectins: novel insecticides, acaricides, and nematicides form 

a soil microorganism. Experientia. 37:963–964. 

Rodriguez-Kabana, R. and C. F. Weaver. 1987. Nematicide seed treatments for 

control of soybean nematodes. Nematropica. 17:79–93. 


Seinhorst, J. W. 1995. The effect of delay of attack of oats by Heterodera avenae on 

the relation between initial nematode density and plant growth, plant weight, 

water consumption and dry matter content. Nematologica. 41:487–504. 

Thomason, I. 1987. Challenges facing nematology: environmental risks with 

nematicides and the need for new approaches, p. 469–476. In: J. Veech, D. 

Dickson, and E. O. Painter (eds.). Vista on nematology. Printing Co., DeLeon 

Springs, FL. 

Truelove, B., R. Rodriguez-Kabana, and P. S. King. 1977. Seed treatment as a means 

of preventing nematode damage to crop plants. J. Nematol. 9:326–330. 

Zeck, W. M. 1971. A rating scheme for field evaluation of root-knot nematode 

infestations. Pflanzenschutz-Nachrichten, Bayer AG. 24:141–144. 


THE DOWNY MILDEW EPIDEMIC OF 2004 

AND 2005 IN THE EASTERN UNITED 

STATES 

Susan J. Colucci 1 , Todd C. Wehner 2 , and Gerald J. Holmes 1 

1 Department of Plant Pathology, North Carolina State University, 

Raleigh, NC 27695-7616 

2 Department of Horticultural Science, North Carolina State 

University, Raleigh, NC 27695-7609 

ADDITIONAL INDEX WORDS. Pseudoperonospora cubensis, Cucumis sativus, 

cucumber, disease resistance, pathology 

ABSTRACT. Downy mildew of cucurbits is caused by Pseudoperonospora 

cubensis (Berk. & M. A. Curtis) Rostovtsev. It has always been considered one 

of the most important diseases of cucumber worldwide. In the United States, 

successful breeding efforts in the 1950s and 1960s were responsible for 

effectively controlling the disease in cucumber. In May 2004 downy mildew 

struck cucumber early and severely, resulting in huge economic losses 

(approximately 40% of the crop or $20 million) to growers in North Carolina, 

Delaware, and Maryland. In 2005 the disease spread to Michigan and Ontario, 

Canada, where it was equally virulent on cucumber, but due to a late arrival, 

spared most of the acreage. Forecasting efforts at North Carolina State 

University, which began in 1998, were temporarily suspended in 2004 due to 

lack of funding, but resumed in 2005 due to a renewed interest in the disease. 

This paper puts the epidemics of 2004 and 2005 into historical perspective with 

regard to breeding and forecasting efforts and outlines future efforts to combat 

this disease. 

D 

owny mildew of cucurbits is caused by Pseudoperonospora 

cubensis (Berk. & M. A. Curtis) Rostovtsev. P. cubensis is an 

obligate parasite and requires living host tissue to survive. 

Oospores are extremely rare, reported only on cucumber, and their role 

in reproduction and survival is uncertain (Waterhouse, 1981; Lange et 

al., 1987). As a result, the pathogen must travel from locations where 

a frost sufficient to kill a host plant does not occur. The pathogen can 

overwinter as active mycelium on wild or cultivated cucurbits in areas 

that experience mild winters, such as southern Florida (Bains and 

Jhooty, 1976). Infection is initiated by sporangia, which are 

transported via air currents and travel from local or distant sources. 

Moisture, as leaf wetness and high relative humidity, is required for 

sporangia to release zoospores and for zoospore movement (Cohen, 

1981; Lange et al., 1987; Palti and Cohen, 1980). 


Downy mildew threatens warm and humid cucurbit production 

areas worldwide and has been reported on species in the genus 

Cucumis in 70 countries (Cohen, 1981; Palti, 1974; Thomas, 1996). 

Historically, in the eastern United States, the disease frequently occurs 

on squash, pumpkin, and melons in late summer and fall. The disease 

also occurs on watermelon, although less commonly than on squash, 

pumpkin, and melons, and is most common in Florida. In each of 

these crops, fungicides are necessary to control the disease. In 

contrast, downy mildew on cucumber has been successfully controlled 

through the use of resistant cultivars. Although the disease has 

occurred on cucumber during the last several decades, its presence was 

inconspicuous and did not result in obvious crop losses. 

In 2004 the story on cucumber changed dramatically. A new 

pathogen more virulent than the previous emerged and downy mildewresistant 

cultivars were not sufficient to prevent severe economic 

losses. The disease ravaged cucumber-growing areas of North 

Carolina, Delaware, Maryland, and Virginia in 2004. In 2005, the 

disease continued its path of destruction in Florida, then moved 

northward reaching Michigan and southwestern Ontario, Canada. As 

in 2004, the disease was quite severe on cucumber, but owing to its 

late arrival, most of the Michigan/Ontario, Canada, crop was spared. 

While the timing of the epidemic was different each year, it was clear 

that the pathogen was similar in virulence to that which was initially 

found on cucumber in North Carolina in May of 2004. 

This paper provides a brief account of historical breeding efforts 

and disease forecasting to control cucurbit downy mildew, and 

summarizes the epidemics of 2004 and 2005 by tracing the dates and 

locations of disease occurrence and giving estimates of economic 

losses. 

Breeding for Resistance 

Breeding efforts in the 1940s led to the release in 1948 of cultivar 

Palmetto with moderate resistance to downy mildew. In 1966, cultivar 

Poinsett was released. This cultivar proved highly resistant to downy 

mildew. Early research on resistance of cucumbers to downy mildew 

showed that at least one single recessive gene, dm, controlled 

resistance in ‘Poinsett’ (Van Vliet and Meysing, 1977). Since 

‘Poinsett’s release, this disease resistance has been bred into most of 

the popular cultivars used today. 

As cucurbit breeding progressed through the twentieth century, a 

review by Pierce and Wehner indicated that several genes are actually 

involved in downy mildew resistance (1990). In 1992, Doruchowski 


and Lakowska-Ryk reported that three recessive genes, dm-1, dm-2, 

and dm-3 control resistance in Wisconsin-4783. In addition, a single 

dominant gene, Dm-3, and two complementary genes, Dm-1 and Dm- 

2, control susceptibility of cultivar SMR-18 (Doruchowskin and 

Lakowska-Ryk, 1992). 

New cultivars incorporated these genetic factors and were doing a 

magnificent job of controlling downy mildew on cucumbers for 

several decades. Because of this successful breeding effort, downy 

mildew was no longer a threat to cucumber production in the United 

States. In most cases, this resistance controlled the disease without the 

use of fungicides. 

Disease-Forecasting Efforts 

Downy mildew was the most important disease of cucumber in the 

1940s in the United States. It was during this time that J. G. Horsfall 

at the Connecticut Agricultural Experiment Station and C. J. Nusbaum 

of Clemson University in South Carolina began reporting the presence 

and spread of the disease in the eastern United States from Florida to 

Massachusetts. Nusbaum noted that the disease occurred nearly every 

year, and that it progressed northward from Florida. He used a 

network of cooperators in 11 states along the eastern seaboard to track 

progress of the disease. Nusbaum also reported that the disease 

usually didn’t reach South Carolina until late May at the very earliest. 

In addition, he noted the sporadic nature of P. cubensis and that the 

disease was destructive in only four out of eight years (Nusbaum, 

1944). 

While downy mildew has not been a problem on cucumbers since 

the late 1960s, it is present nearly every year on other cucurbits 

(squash, pumpkin, melon, and watermelon) in the United States. In 

1997 Holmes and Main at North Carolina State University developed a 

forecasting system to predict the spread and development of cucurbit 

downy mildew. The system is made available to growers via a Web 

site: . The system is 

based on the tobacco blue mold (Peronospora tabacina) forecasting 

system created by C. E. Main (Main et al., 2001). Forecasting downy 

mildew began in 1998 and continued for the next six years. The Web 

site reported the progression of downy mildew on all commercially 

grown cucurbits. However, in 2004 forecasts ceased due to lack of 

funding. 


2004 Epidemic 

Interestingly, 2004 marked a turning point for downy mildew. In 

North Carolina, the spring crop was looking unusually good. Rainfall 

and temperatures were such that growers were expecting very high 

yields. In mid-May a grower in Sampson County, North Carolina, 

reported a blighted area in his cucumber field that had occurred so 

rapidly that it was initially thought to be herbicide injury. The 

problem was soon diagnosed as downy mildew. Two things were 

particularly important about this event: (1) the disease had reached the 

state earlier than had ever been reported before; and (2) it was highly 

virulent on cucumber. Within days the presence of downy mildew was 

reported in six counties in eastern North Carolina. Within a few weeks 

the disease was present in nearly every field of cucumbers throughout 

eastern North Carolina (Holmes et al., 2006). 

By the first week in July the disease had spread to Accomack 

County, Virginia, and soon thereafter to cucumber fields in Delaware. 

Nearly all of the pickling-cucumber acreage on the Eastern Shore of 

Delaware was affected. Early August brought P. cubensis spores to 

more counties throughout North Carolina and also north to 

Connecticut and Suffolk County, New York. As the fall cucumber 

crop was planted in North Carolina, even more devastation was 

apparent. Some growers experienced as much as 95% yield loss. 

Many fields were abandoned without harvesting. 

In an effort to provide adequate control options, an emergency 

fungicide performance trial was established in 2004 in the heart of the 

affected area (Sampson County, NC) during the worst part of the 

epidemic (late summer). The trial consisted of 15 treatments and 

included old standards, new materials, and several products that 

growers had speculated about, but had little or no data to support. 

Treatments were applied at 5- to 7-day intervals for a total of 11 

applications. Disease was detected 10 days after plant emergence and, 

in the nontreated plots, prevented a single fruit from being produced. 

Many other fungicides failed to control the disease, but four products 

provided excellent disease control: Tanos (fenamidone + cymoxanil), 

Previcur Flex (propamocarb), Ranman (cyazofamid), and Gavel 

(zoxamide + mancozeb). These products mixed with protectants such 

as Bravo (chlorothalonil) and mancozeb now form the backbone of 

successful downy mildew-control programs throughout the eastern 

U.S. 

In North Carolina the 2004 spring and fall cucumber crops were 

devastated. An estimated 40% crop loss for a total of $16 million was 

the result. Loss estimates are based on economic value of the crop as 


eported by the National Agricultural Statistics Service (Quick Stats, 

2006) (Fig.1) as well as the cost of fungicide applications (ranging $12 

to $86/hectare/application). Additional economic repercussions were 

felt from lost markets, increased commodity prices, and dramatic 

changes in the demand for labor and machinery. A similar level of 

disease (i.e., 40% crop loss) occurred in Delaware, Maryland, and 

Virginia, with losses estimated at $4 million. 

Later in the year, the disease began to make its way southward. By 

November the disease was present in Charleston county South 

Carolina on cucumber. By mid-December the South Florida 

Vegetable Pest and Disease Hotline reported that the University of 

Florida/IFAS Plant Pathology Clinic in Immokalee had received more 

cucumber downy mildew samples than ever before. 

This news was of particular interest since southern Florida 

represents the starting point of the disease for areas northward. In this 

case, it looked as if it was the end point of the 2004 epidemic. If the 

disease was present on cucumber, it meant that the disease had gone 

south and would likely go north again leading to a possible repeat of 

2004. During the months of January through April, Florida reported 

downy mildew in all areas where cucumbers were grown. Fields were 

described with 100% disease incidence. Major yield decreases and 

field abandonment was reported (economic loss data are not available). 

Many growers were calling the epidemic the worst case of downy 

mildew they could ever remember. 

2005 Epidemic 

When North Carolina’s 2005 season began, growers hoped that the 

epidemic of 2004 was a fluke that would not repeat. Florida’s 

experience with the disease suggested that the pathogen was still 

virulent on cucumber and would remain so in 2005. The bigger 

question was when and where would it strike. 

With interest in downy mildew rekindled, forecasting resumed in 

March. In addition, based on the 2004 field trial results, a 

management program where Tanos + mancozeb is alternated with 

Previcur Flex + Bravo on a 5- to 7-day interval was recommended for 

2005. In early June, two fields with downy mildew were reported in 

Georgia. Oddly, the next report of the disease came from New Jersey, 

far to the north of the Georgia outbreaks with hundreds of miles of 

cucurbits in between. Sources reported that the New Jersey outbreak 

involved a field where transplants, purchased in Florida, were used. 

New Jersey also reported downy mildew in several of its cucumbergrowing 

counties in early July. By the middle of July, Maryland had 


eported widespread infection of their cucumber crop. In Delaware, 

Maryland, and Virginia growers reduced acreage by about 20% and 

when P. cubensis arrived in early July, many simply stopped planting 

altogether (Ed Kee, personal communication). 

It was also at this time that P. cubensis showed up in North 

Carolina. Fortunately, the timing was such that the spring crop was 

spared, but the summer crop was just beginning. Once again, 

cucumber was very susceptible and fungicides were necessary to 

prevent severe economic losses. 

On 5 August, an outbreak was reported in Michigan, the top 

cucumber-producing state in the U.S., whose average production is 

approximately 16,000ha of cucumbers annually (Figure 1). 

Eventually, 15 counties throughout Michigan reported the disease on 

cucumber. Downy mildew had not been confirmed in Michigan for at 

least fifteen years and had not been a problem there in the past (Mary 

Hausbeck, personal communication). 

In Ohio, the disease was again mistaken for herbicide injury; 

however, pumpkins planted nearby did not exhibit any symptoms. 

(This same observation of host-specificity for cucumber but not 

pumpkin or squash has been made in many other locations.) 

Reports from Ontario, Canada, were also received. In 2005, in 

Ontario, 1,983ha of cucumbers and gherkins were harvested (Ontario 

Ministry of Agriculture, 2006). Fortunately, the disease did not show 

up until late in the season for the Ontario growers. All of the acreage 

still in production during that time had at least some level of downy 

mildew present in their fields. The industry overall did not suffer; 

however, many smaller growers were devastated. Ontario had had 

incidence of downy mildew previously, but not at such high levels. 

Smuckers (a commercial processor) reported that much of the 

production was affected. About 12 acres were completely abandoned 

and 12 acres were used for relish for a total loss of about a $30,000 

Canadian ($27,095 US) (George Pape, personal communication). 

Determining Economic Losses 

Reports of downy mildew on cucurbit crops were compiled 

throughout the 2004 and 2005 growing seasons. Information was 

gathered through personal communication via email, telephone, and 

personal interviews with commercial and independent growers, 

researchers, and agricultural extension agents throughout the eastern 

United States and Ontario, Canada. In 2004, there was personal 

communication with 18 individuals, and approximately 30 individuals 

in 2005. These include many key industry representatives who are 


intimately familiar with production across several states. We consider 

our estimates to be fair and unexaggerated. 

Hectares (x 1000) 

20 

18 

16 

14 

12 

10 

8 

6 

4 

2 

0 

North 

Carolina 

Fresh 

Processed 

Value 

Michigan Florida Maryland New 

Jersey 

Ontario, 

CAN 

Fig. 1. Cucumber production and value in the eastern U.S. and southwestern 

Ontario, Canada. 

Summary of Forecasts 

In 2004, 14 states representing over 20 counties had reported 

downy mildew. Of these, 7 states had confirmed it on cucumber. By 

the end of 2005, 18 states, representing more than 62 counties, plus 

Ontario, Canada, reported downy mildew (nearly all on cucumber). 

There were approximately 320,000 hits to the forecasting Web site, an 

average of 1350 hits per day. This is almost a threefold increase over 

previous years. 

Summary 

The events of 2004 and 2005 strongly suggest the development or 

introduction of a variant of P. cubensis previously not present in the 

U.S. The virulence on previously resistant cucumber cultivars is 

unprecedented in the last 40 years. Breeding programs in all the major 

seed companies are again focused on developing cultivars for the 

United States with resistance to downy mildew. Growers should still 

plant resistant cultivars since they hold up better than susceptible 

cultivars when attacked by the new variant. However, resistance is no 


120 

100 

80 

60 

40 

20 

0 

Value (x million USD)

longer sufficient to prevent foliar damage, so fungicides are now 

needed in addition to the resistance genes. Meanwhile, the fungicide 

performance trials in 2004 and 2005 and grower reports have 

confirmed excellent disease control following the Tanos + mancozeb 

alternated with Previcur Flex + Bravo (chlorothalonil) on a 5- to 7-day 

interval program. 

That this new variant of P. cubensis has spread to all of the major 

cucumber-producing areas of the eastern United States and 

southwestern Ontario, Canada, within two years is further evidence of 

the method of dispersal for this pathogen (first reported by Nusbaum 

in the mid-1940s) and the potential to forecast its movement. 

Current research efforts are focused on determining the pathotype 

using the methods described by Lebeda (2003) and Thomas et al. 

(1987) and on determining fungicide sensitivity. The latter arises from 

reports of control failures of several popular fungicides. Research 

efforts are also focused on validating the cucurbit downy mildewforecasting 

system that serves as a valuable decision-making tool for 

growers. One thing that will drastically reduce the value of forecasting 

is the existence of overwintering sites for the pathogen in greenhouses 

in areas where the pathogen cannot overwinter, or the long-distance 

transport of infected transplants from infested areas to noninfested 

areas. 


Bains, S. S. and J. S. Jhooty. 1976. Over wintering of Pseudoperonospora cubensis 

causing downy mildew of muskmelon. Indian Phytopathology. 29:213–214. 

Cohen, Y. 1981. Downy mildew of cucurbits, p. 341–345. In: D. M. Spencer (ed). 

The downy mildews. Academic Press, New York. 

Doruchowski, R. W. and E. Lakowska-Ryk. 1992. Inheritance of resistance to 

downy mildew (Pseudoperonospora cubensis Berk. & Curt.) in Cucumis 

sativus. Proc. 5th Eucarpia Symposium, 27–31 July, Poland:132–138. 

Holmes, G., Wehner, T., and Thornton, A. 2006. An old enemy re-emerges. 

American Vegetable Grower. February. 

Lange, L., U. Eden, and L. W. Olson. 1987. Internal mycelium of 

Pseudoperonospora cubensis, the causal agent of cucurbit downy mildew. 

Nordic J. Bot. 8(5):505–510. 

Lebeda, A. and Widrlechner. 2003. A set of Cucurbitaceae taxa for the 

differentiation of Pseudoperonospora cubensis pathotypes. J. Plant Dis. & Prot. 

110(4):337–349. 

Main, C. E., T. Keever, G. J. Holmes, and J. M. Davis. 2001. Forecasting long-range 

transport of downy mildew spores and plant disease epidemics. APSnet Feature 

Story. Apr 25 through May 31, 2001. 

. 

Nusbaum, C. J. 1944. The seasonal spread and development of cucurbit downy 

mildew in the Atlantic coastal states. Plant Dis. Rep. 28:82–85. 


Ontario Ministry of Agriculture. 2006. Food and Rural Affairs. Ontario Agriculture 

and Food Statistics, Ontario Processing Vegetable Growers and Statistics 

Canada: Fruit and Vegetable Survey. Online. 

Palti, J. 1974. The significance of pronounced divergences in the distribution of 

Pseudoperonospora cubensis on its crop hosts. Phytoparasitica. 2:109–115. 

Palti, J. and Y. Cohen. 1980. Downy mildew of cucurbits (Pseudoperonospora 

cubensis): the fungus and its hosts, distribution, epidemiology and control. 

Phytoparasitica. 8(2):109–147. 

Pierce, L. K. and T. C. Wehner. 1990. Review of genes and linkage groups in 

cucumber. HortSci. 25:605–615. 

Quick Stats. 2006. National Agriculture Statistics Service-United States Department 

of Agriculture. On-line. 

Thomas, C. E., T. Inaba, and Y. Cohen. 1987. Physiological specialization in 

Pseudoperonospora cubensis. Phytopathology. 77:1621–1624. 

Thomas, C. E. 1996. Downy mildew, p. 25–27 In: T. A. Zitter, D. L. Hopkins, and 

C. E. Thomas (eds.). Compendium of cucurbit diseases. APS Press, St. Paul, 

MN. 

Van Vliet, G. J. A. and W. D. Meysing. 1977. Relation in the inheritance of 

resistance to Pseudoperonospora cubensis Rost. and Sphaerotheca fuliginea 

Poll. in cucumber (Cucumis sativus L.). Euphytica. 26, 793–796. 

Waterhouse, G. M. and M. P. Brothers. 1981. The taxonomy of Pseudoperonospora. 

Mycolog. Papers. 148:1–28. 


WATERMELON RESISTANCE TO POWDERY 

MILDEW RACE 1 AND 

RACE 2 

Angela R. Davis 

USDA, ARS, South Central Agricultural Research Lab, Lane, OK 

74555 

Antonia Tetteh and Todd Wehner 

North Carolina State University, Raleigh, NC 27695-7609 

Amnon Levi 

USDA, ARS, U. S. Vegetable Laboratory, Charleston, SC 29414 

Michel Pitrat 

INRA - Génétique et Amélioration des Fruits et Légumes, Montfavet, 

France 

ADDITIONAL INDEX WORDS. Citrullus lanatus, Podosphaera xanthii, 

Sphaerotheca fuliginea, resistance 

ABSTRACT. Powdery mildew is an emerging disease of watermelon in the 

United States. To date, Race 1 and Race 2 of Podosphaera xanthii have been 

detected on watermelon. In this study, the entire available U.S. Plant 

Introduction collection of watermelon (Citrullus lanatus [Thunb.] Matsum. & 

Nakai) was evaluated for resistance to P. xanthii Race 1 and Race 2 in six to 

nine replications of one plant per accession in greenhouse studies. Eight of the 

1,573 accessions were resistant to Race 1, and five accessions were resistant to 

Race 2. The majority of the resistant lines for Race 1 came from plants collected 

from Zimbabwe, while those resistant to Race 2 came from Iran, Egypt, and 

Zambia, along with one of unknown origin. 

I 

n the past six years, powdery mildew has been noted as a disease 

of watermelon in the United States (McGrath, 2001b; Davis et al., 

2001). Previously, powdery mildew affected other cucurbit crops, 

but was not a significant problem on watermelon (Robinson and 

Provvidenti, 1975). In recent years, research on watermelon powdery 

mildew has increased as crop damage from this disease has increased 

in South Carolina, Georgia, Florida, Oklahoma, Texas, Maryland, New 

York, Arizona, and California (McGrath, 2001b; Davis et al., 2001). 

This disease decreases plant canopy and can reduce yields through 

decreased fruit size and number of fruit per plant (McGrath and 

Thomas, 1996). The reduced canopy may also result in sunscald of the 

remaining fruit, making it unmarketable. Detection of powdery mildew 

can be difficult because the presence of the pathogen is less apparent 

on watermelon than on pumpkin and squash. 


Two genera, Podosphaera xanthii (syn. Sphaerotheca fuliginea 

auct. p.p.) and Golovinomyces cichoracearum (formerly Erysiphe 

cichoracearum DC), are considered the predominant fungi that incite 

powdery mildew in cucurbits. These two organisms differ in virulence 

against cucurbit species and in their sensitivity to fungicides (Bertrand, 

1991; Epinat et al., 1993; McGrath, 2001a and 2001b). P. xanthii has 

frequently been reported based on differential reactions of 10 melon 

(Cucumis melo) genotypes (Pitrat et al., 1998; McCreight et al., 1987), 

but 28 races have been described on a larger set of 32 melon genotypes 

(McCreight, 2006). 

Powdery mildew has been controlled on pumpkin by using 

resistant cultivars and fungicide applications (Keinath, 2001). 

Unfortunately, resistance of P. xanthii to certain fungicides has been 

detected, and application of fungicides to the undersides of leaves is 

difficult and often requires the use of systemic materials to achieve 

adequate control of the disease (McGrath and Thomas, 1996). We 

reported resistance in watermelon to Race 1 P. xanthii after screening 

100 Plant Introduction (PI) accessions in field studies (Davis et al., 

2001). These experiments led to the release of a watermelon breeding 

line (PI 525088-PMR) that has resistance to Race 1 P. xanthii (Davis 

et al., 2005). The inheritance of resistance in that line to Race 1 was 

multigenic (Davis et al., 2002), and was independent from resistance 

to P. xanthii Race 2. In the current study, we evaluated the available 

U.S. PI accessions of Citrullus sp. from the USDA, ARS, Southern 

Regional Plant Introduction Station, Griffin, GA, for Race 1 and Race 

2 P. xanthii resistance. 

We would like to thank Amy Helms, Anthony Dillard, and Tammy Ellington for 

technical assistance. Some melon differentials were kindly supplied by Dr. Claude 

Thomas. This work was partially supported by the Cucurbit Crop Germplasm 

Committee and the National Germplasm System, USDA, ARS. Mention of trade 

names or commercial products in this article is solely for the purpose of providing 

specific information and does not imply recommendation or endorsement by the U.S. 

Department of Agriculture. All programs and services of the U.S. Department of 

Agriculture are offered on a nondiscriminatory basis without regard to race, color, 

national origin, religion, sex, age, marital status, or handicap. The article cited was 

prepared by a USDA employee as part of his/her official duties. Copyright protection 

under U.S. copyright law is not available for such works. Accordingly, there is no 

copyright to transfer. The fact that the private publication in which the article 

appears is itself copyrighted does not affect the material of the U.S. Government, 

which can be freely reproduced by the public. 



RACE 1. One seed of each of the available PI accessions was 

planted in Speedling flats containing Redi-earth growth medium 

(SunGro, Vancouver, BC, Canada) in six completely randomized 

greenhouse experiments. Seedlings were inoculated with the Lane, 

OK, isolate of P. xanthii maintained on greenhouse-grown 

watermelon. Inoculation was carried out by brushing an infected leaf 

onto each seedling at the two-leaf stage of growth. Ratings were taken 

when 50% of the leaf surface areas of susceptible differentials 

demonstrated symptoms. Three ratings were made of each plant (stem, 

upper side of the leaves, and cotyledons) using the Horsfall and Barratt 

(1945) method. The plants were then classified as resistant, 

intermediate, or susceptible according to the mean of three ratings: 

≤6% = resistant; >6% and ≤12% = intermediate; and >12% = 

susceptible. Some plants were missing cotyledons at rating and thus 

the mean rating of leaves and stems were used. The following melon 

differentials were included in all experiments to determine the race of 

P. xanthii present: ‘Delicious 51’, ‘Edisto 47’, ‘Iran H’, ‘MR-1’, 

‘Nantais Oblong’, PI 124112, PI 414723, ‘PMR 45’, ‘PMR 5’, ‘PMR 

6’, ‘WMR 29’, and ‘TopMark’. Race 1 was the only powdery mildew 

found in these experiments. 

RACE 2. Seeds were planted in 100-mm diameter pots containing 

4P Fafard mix growing medium (Fafard, Agawam, MA) in a 

randomized complete block design in nine greenhouse experiments. At 

the first-true-leaf stage, seedlings were inoculated with P. xanthii Race 

2 isolate that had been maintained on melon (Cucumis melo) ‘PMR 

45’ and Gray Zucchini (Cucurbita pepo) in the greenhouses in 

Charleston, SC. Inoculation was done with a spore suspension 

prepared by washing six mildewed leaves with a spray of distilled 

water and making the volume up to 1L. Each seedling was sprayed 

with a 2-ml spore suspension once each week for six weeks. Plants 

were maintained at 24 to 32 o C, with a relative humidity of 60 to 80%. 

Spreader plants were placed between rows as an additional source of 

powdery mildew infection (Sivapalan, 1993; Ziv and Zitter, 1992). 

Disease ratings were collected two weeks after the first and two weeks 

after the last inoculation on the leaves and stem separately using a 

rating scale of 0 (no disease) to 9 (plant was fully covered with mildew 

and had turned yellow). The plants were then classified as resistant, 

intermediate, or susceptible according to the mean of three ratings: 

5.5 susceptible. ‘PMR 45’, 

which is susceptible to Race 2, was included to increase the inoculum 

of Race 2 for use in the studies. The melon differentials mentioned 


above were included in all experiments to determine which race of P. 

xanthii was present. Race 2 was the only powdery mildew found in 

these experiments. 


RACE 1 RESISTANCE. In each of the six randomized experiments, 

we saw a range of symptoms from none detectable to 97% coverage 

with sporulation in all replications. The majority of the PI lines (92%) 

had mean detectable infection covering 12 to 50% of the plant surface 

(data not shown). The 50 most Race 1-resistant PI lines are listed in 

Table 1. They were given a numerical ranking, with 1 being the most 

resistant. Only 8 PI lines were ranked as resistant, with an average 

percent sporulation of 6% or less. In our judgment, all 50 of these lines 

had commercially significant resistance to the disease. The average 

sporulation for PI 189318, which was ranked as number 50, was less 

than 12% coverage. 

Table 1. Ranking of the 50 watermelon PI lines most resistant to 

Race 1 P. xanthii. 

PI 

PI 

Rank 

line Resistance Rank line Resistance 

1 Grif5601 R 27 P179881 I 

2 PI482255 R 28 P179885 I 

3 PI388770 R 29 P184800 I 

4 PI482362 R 30 PI71749 I 

5 PI381750 R 31 PI296334 I 

6 PI459074 R 32 PI381731 I 

7 PI386015 R 33 PI386025 I 

8 PI482248 R 34 PI482251 I 

9 PI381742 I 35 PI482278 I 

10 PI532738 I 36 PI482295 I 

11 PI500331 I 37 PI482302 I 

12 PI508443 I 38 PI482355 I 

13 PI 60008 I 39 PI500332 I 

14 PI525082 I 40 PI512343 I 

15 PI482264 I 41 PI525088 I 

16 PI482258 I 42 PI482312 I 

17 PI217938 I 43 PI482259 I 

18 PI250145 I 44 PI482308 I 

19 PI482333 I 45 PI482380 I 

20 PI526233 I 46 PI500334 I 

21 PI482313 I 47 PI270545 I 

22 PI482328 I 48 PI482268 I 

23 P 500323 I 49 Grif5602 1 

24 PI505585 I 50 P189318 I 

25 P169241 I 

26 P179239 I 

R = resistant; I = intermediate resistance. 


Many of the PI lines were heterogeneous for resistance, which 

reduced their ranking; individuals in one accession ranged from 6% to 

94% sporulation coverage. We kept the most-resistant plants from each 

replicate for self-pollination and retesting. Out of 1,573 PI lines tested, 

only 13 had no data due to lack of germination or early death of 

seedlings. The most Race 1-susceptible PI lines are listed in Table 2, 

with 1 being the highest severity of infectivity. All PI lines in Table 2 

were rated as susceptible. 

There was an unexpectedly high percentage of Praecitrullus 

fistulosus (12%) and Citrullus colocynthis (8%) in the 50 accessions 

most resistant to Race 1; these two species comprise

soaking of the stem and petioles. Of all the USDA accessions, fewer 

than 1% were found to be resistant. No data were obtained for 

theremaining percentage. Out of 1,573 PI lines tested, only 11 had no 

data due to poor germination or early death of seedlings. 

RACE 2 RESISTANCE. A range of symptoms on the watermelon 

accessions was observed in each of the nine replications. These 

symptoms varied from no detectable powdery mildew to complete 

(100%) mildew coverage; few chlorotic spots to complete yellowing 

of entire leaves; little necrosis with some curling of leaf margins to 

necrosis of the entire plant; and no water soaking to extensive water 

soaking of the stem and petioles. Of all the USDA accessions, less 

than 1% were found to be resistant. No data were obtained for the 

remaining percentage. Out of 1,573 PI lines tested, only 11 had no data 

due to poor germination or early death of seedlings. 

Table 3. Ranking of the 50 watermelon PI lines most resistant to 


PI 

PI 

Rank line Resistance Rank line Resistance 

1 PI386015 R 27 PI482307 I 

2 PI632755 R 28 PI388770 I 

3 PI525082 R 29 PI482326 I 

4 PI307608 R 30 PI386019 I 

5 PI500354 R 31 Grif14202 I 

6 PI346082 R 32 PI482246 I 

7 PI560010 R 33 PI500331 I 

8 PI482286 R 34 PI269365 I 

9 PI494531 I 35 PI482278 I 

10 PI482333 I 36 PI186489 I 

11 PI386021 I 37 PI595203 I 

12 PI500334 I 38 PI381752 I 

13 PI386024 I 39 PI512828 I 

14 PI482361 I 40 PI386026 I 

15 PI500329 I 41 PI532722 I 

16 PI386016 I 42 PI482324 I 

17 PI482302 I 43 PI482308 I 

18 PI326516 I 44 PI560003 I 

19 PI560020 I 45 PI482288 I 

20 PI500332 I 46 PI482301 I 

21 PI500303 I 47 PI482299 I 

22 PI540911 I 48 PI381720 I 

23 PI500312 I 49 PI482338 I 

24 PI251244 I 50 PI381750 I 

25 PI482319 I 

26 PI482355 I 

R = resistant; I = intermediate resistance. 


Table 4. Ranking of the 50 watermelon PI lines most susceptible to 


Rank PI line Rank PI line Rank PI line Rank PI line 

1 PI357751 14 PI370427 27 PI370434 40 PI368526 

2 PI369220 15 PI169297 28 PI508442 41 PI211582 

3 PI357745 16 PI175652 29 PI278058 42 PI368506 

4 PI368519 17 PI271132 30 PI357752 43 PI277980 

5 PI512355 18 PI169284 31 PI370428 44 PI368521 

6 PI357737 19 PI537277 32 PI512360 45 PI178873 

7 PI536449 20 PI176907 33 PI271308 46 PI278003 

8 PI172792 21 PI278038 34 PI379235 47 PI379239 

9 PI176917 22 PI278049 35 PI379229 48 PI538888 

10 PI593373 23 PI174105 36 PI271987 49 PI593363 

11 PI357686 24 PI269677 37 PI271771 50 PI278034 

12 PI512361 25 PI176923 38 PI600903 

13 PI490380 26 PI357666 39 PI368496 

Table 5. Ranking of 25 cultivars for Race 2 P. xanthii resistance. 

Rank Cultivar Rank Cultivar 

1 Tastigold 14 Giza 

2 Blacklee 15 Rhode Island 

3 Florida Favorite 16 Sugarlee 

4 Chubby Gray 17 Black Boy 

5 Black Diamond Yellow Belly 18 Golden 

6 Graybelle 19 Tendersweet 

7 Tendergold 20 Picnic 

8 Black Diamond Yellow Fish 21 Charleston Gray 

9 Big Crimson 22 Sugarloaf 

10 Crimson Sweet 23 Hopi Red Flesh 

11 Perola 24 Early Arizona 

12 Desert King 25 Moon & Stars 

13 Carolina Cross 18 

Table 3 shows the 50 most-resistant PI lines in rank order. PI 

482299 had an average rating of 3.3. As expected, many of the 

watermelon accessions were heterogeneous for expression of 

resistance, with a mean rating from 2 to 7 within a PI accession over 

the replications. The most-resistant accessions and most- susceptible 

accessions were identified for retesting and self-pollination. Many of 

the resistant PI accessions came from Zimbabwe as in the Race-1 test, 

and there were many from Zambia, Iran, and India. PI 269677, 

reported susceptible in 1975 (Robinson and Provvidenti, 1975), was 

susceptible to P. xanthii Race 2 in our test. Eleven of the PI accessions 

demonstrated resistance or intermediate resistance to both Race 1 and 

Race 2 P. xanthii. Table 4 lists the 50 PI lines most susceptible to 


Race 2 P. xanthii; all had a rating of susceptible. Table 5 shows the 

ranking of 25 cultivars used as checks for the PI screening study. All 

watermelon cultivars were susceptible, with ‘Tastigold’ showing less 

susceptibility than the other cultivars to Race 2. 

The experiments reported here evaluated watermelon germplasm 

for resistance to P. xanthii Races 1 and 2 in greenhouse experiments. 

The 50 most resistant and most susceptible will be retested to verify 

our results. 

Since we were using cantaloupe differentials to determine the race 

present in these studies and we do not currently have differentials of 

Citrullus sp., we can state only that the races detected show the same 

infectivity on the differentials as Race 1 and Race 2 P. xanthii of 

Cucumis melo. We are currently developing watermelon lines with 

homogeneous expression for infectivity to these two isolates of P. 

xanthii; once produced, we will likely find that the P. xanthii strain 

that affects watermelon will differ from that which infects cantaloupe. 


Bertrand, F. 1991. Les Oidiums des Cucurbitacees: maintien en culture pure, étude 

de leur variabilité et de la sensibilité chez le melon. PhD Diss., Université of 

Paris XI, Orsay, France. 

Davis, A. R., B. D. Bruton, S. D. Pair, and C. E. Thomas. 2001. Powdery mildew: an 

emerging disease of watermelon in the United States. Cucurbit Gen. Coop. Rpt. 

24:42–48. 

Davis, A. R., C. E. Thomas, A. Levi, B. D. Bruton, and S. D. Pair. 2002. 

Watermelon resistance to powdery mildew race 1, p. 192–198. In: D. N. 

Maynard (ed.). Cucurbitaceae ’02. ASHS Press, Alexandria, VA. 

Davis, A. R., T. Wehner, A. Levi, and M. Pitrat. 2005. Notice of release of PI 

525088-PMR: a watermelon line useful for introducing race 1 powdery mildew 

resistance into watermelon lines. USDA-ARS Germplasm Release. 

Epinat, C., M. Pitrat, and F. Bertrand. 1993. Genetic analysis of resistance of five 

melon lines to powdery mildews. Euphytica. 65:135–144. 

Horsfall, J. G. and R. W. Barratt. 1945. An improved grading system for measuring 

plant disease. Phytopathology. 35:655(Abstr.). 

Keinath, A. P. 2001. Powdery mildew-resistant cultivars. SC Pumpkin News. 5:1. 

McCreight, J. D., M. Pitrat, C. E. Thomas, A. N. Kishaba, and G. W. Bohn. 1987. 

Powdery mildew resistance genes in muskmelon. J. Amer. Soc. Hort. Sci. 

112:156–160. 


melon cultigens and Podosphaera xanthii races 1 and 2. J. Amer. Soc. Hort. Sci. 

131:59–65. 

McGrath, M. 2001a. Fungicide resistance in cucurbit powdery mildew: experiences 

and challenges. Plant Dis. 85:236–245. 

McGrath, M. T. 2001b. Distribution of cucurbit powdery mildew races 1 and 2 on 

watermelon and muskmelon. Phytopath. 91:S197(Abstr.). 

McGrath, M. T. and C. E. Thomas. 1996. Powdery mildew, p. 28–30. In: T. A. 

Zitter, D. L. Hopkins, and C. E. Thomas (eds.). Compendium of cucurbit 



Pitrat, M., C. Dogimont, and M. Bardin. 1998. Resistance to fungal diseases of 

foliage in melon, p. 167–173. In: J. D. McCreight (ed.). Cucurbitaceae’98. 

ASHS Press, Alexandria, VA. 

Robinson, R. W. and R. Provvidenti. 1975. Susceptibility to powdery mildew in 

Citrullus lanatus (Thunb.) Matsum. & Nakai. J. Amer. Soc. Hort. Sci. 100:328– 

330. 

Sivapalan, A. 1993. Effect of water on germination of powdery mildew conidia. 

Mycol. Res. 97:71–76. 

Ziv, O. and T. A. Zitter. 1992. Effects of bicarbonates and film-forming polymers on 

cucurbit foliar diseases. Plant Dis. 76:513–517. 


DEVELOPMENT OF THE FUNGICIDE 

MANDIPROPAMID IN THE UNITED 

STATES FOR CONTROL OF DOWNY MILDEW 

OF CUCURBITS 

Tyler L. Harp, Donald R. Tory, and Paul J. Kuhn 

Syngenta Crop Protection, Inc., Vero Beach, FL 

ADDITIONAL INDEX WORDS. Oomycete, Pseudoperonospora cubensis, cucumber, 

cantaloupe, disease control, nonionic surfactant, NIS 

ABSTRACT. Mandipropamid 250 SC is a new fungicide developed by Syngenta 

for control of foliar oomycete pathogens, including cucurbit downy mildew. On 

cucurbits, mandipropamid provides outstanding preventive control of downy 

mildew when applied at rates of 100 to 150GA/ha on a 7- to 10-day application 

interval. The use of a nonionic surfactant at a rate of 0.125% v:v in mixture 

with mandipropamid significantly improves efficacy against cucurbit downy 

mildew, and should be included for optimal performance. The results presented 

here, along with fungicide-resistance management practices, formed the basis 

for use recommendations described in labeling for mandipropamid. 

D 

owny mildew caused by the oomycete fungus 

Pseudoperonospora cubensis (Berk. & M.A. Curtis) 

Rostovzev is an important foliar disease of cucurbits, and the 

last two to three seasons have witnessed serious epidemics in Florida 

and other cucurbit-producing states on the east coast of the United 

States. In 2004, grower losses to the disease were estimated to be ca. 

40% in North Carolina, Virginia, Delaware, and Maryland (Gerald 

Holmes, personal communication). 

Integrated pest management of the disease is based in part on hostplant 

resistance and on cultural practices such as spacing plants widely 

enough to reduce canopy density and avoiding overhead irrigation. 

However, effective control relies on the use of fungicide programs that 

include both conventional protectant and systemic products. 

At the most recent British Crop Protection Council International 

Congress in Glasgow, Scotland, Huggenberger, Lamberth, Iwanzik, 

and Knauf-Beiter (2005) introduced mandipropamid (Figure 1), a new 

fungicide from Syngenta that is highly effective in the control of 

We would like to thank colleagues working with Syngenta in Greensboro, NC (Jim 

Frank, Allison Tally, and David Laird) and Basel, Switzerland (Fritz Huggenberger) 

for reviewing the manuscript and for their helpful advice during the studies described 

here. 


oomycete diseases such as downy mildews. A submission to the EPA 

was made in March 2006, with registration anticipated in time for 

launch early in 2008. Mandipropamid will be available as a solo 

product and also in premixes with a range of partner fungicides. This 

paper describes biological studies conducted in the US to develop use 

recommendations for mandipropamid on downy mildew of cucurbits. 

Cl 

O 

O 

H 

N 

Fig. 1. Chemical structure of mandipropamid 


PLANTS. All of the biological evaluations reported here were 

conducted in field trials at Vero Beach Research Center, Vero Beach, 

Florida, using either cucumber (Cucumis sativus L. cv. Straight Eight) 

or cantaloupe (Cucumis melo L. cv. Athena), and naturally occurring 

epidemics of downy mildew. Trials were laid out in a randomized 

block design with four replicates per treatment. Cucumbers were 

direct-seeded on bare ground, while cantaloupes were transplanted into 

plastic mulch with drip irrigation. Plots consisted of double rows, 

typically 6–9m in length. Local agronomic practices were employed 

for weed control, insect control, and fertility. 

FUNGICIDES. Mandipropamid (NOA446510) was a 250g/L 

(2.08lb/gal) suspension concentrate (SC), which will be the 

commercial solo formulation. Commercial standards and surfactants 

were obtained through Syngenta or purchased locally. Applications, at 

a spray volume of 375L/ha and a pressure of 345kPa, were made from 

a boom mounted on the front end of a spray ram. Unless stated 

otherwise, applications were initiated at the onset of visible disease 

symptoms in the foliage. 

DISEASE ASSESSMENT. Levels of disease were evaluated as % 

severity of the leaf area infected. For determination of statistical 

significance, data were analyzed by analysis of variance (p = 0.05). 

For comparison of disease development over time, disease levels were 

expressed as Area Under Disease Progress Curve (AUDPC) values. 


O 

O


EFFICACY. In numerous field trials in the US and elsewhere, 

mandipropamid at rates of 100–150GA/ha has provided excellent 

control of downy mildew on cucurbits (Figure 2). 

Mandipropamid Untreated 

Fig. 2. Control of downy mildew on cucumber (‘Straight Eight’) from 

mandipropamid (150GA/ha) + 0.125% (v:v) NIS (Activator 90). 

In the trial illustrated in Figure 3, the first application was made 

when disease symptoms were first observed in older leaves. By 20 

days after the final application, disease severity in untreated checks 

was 46%. By contrast, in plots receiving a program of mandipropamid 

(150GA/ha) alternated with Bravo ® Weather Stik ® (1262GA/ha), 

severity was less than 5%, indicating greater than 90% disease control. 

A range of commercial standards each alternated with Bravo ® Weather 

Stik ® and representing diverse modes of action, also provided good 

control of downy mildew, statistically equivalent to mandipropamid, 

though numerically slightly inferior. 

EFFECT OF RATE AND APPLICATION INTERVAL ON EFFICACY. In 

order to evaluate the effect of rate and application interval on efficacy, 

a trial was undertaken on cucumber in which sprays of mandipropamid 

were applied at intervals of 7, 10, or 14 days with three rates of 

mandipropamid (100, 125, and 150GA/ha) tested for each (Figure 4). 

Disease control was observed in all treatment régimes, and for each 

application interval a dose-response effect was observed, with 

progressively improved control with increasing rate of product. There 


was also a clear trend toward better efficacy with shorter application 

intervals. In practice, the application interval and rates used will 

depend on a number of factors, including susceptibility of the cultivar, 

level of disease at first application, and environmental conditions 

during growth of the crop. 

Disease Severity (%) 

50 

45 

40 

35 

30 

25 

20 

15 

10 

5 

0 

check check 

a 

mandipropamid mandipropamid (150 (150 GA/Ha) GA/Ha) 

b 

Cabrio Cabrio (150 (150 GA/Ha) GA/Ha) 

b 

Ranman Ranman (80GA/Ha) (80GA/Ha) 

b 

b 

Tanos Tanos (280 (280 GA/Ha) GA/Ha) 

b 

Gavel Gavel (1261 (1261 GA/Ha) GA/Ha) 

Fig. 3. Protectant disease control from mandipropamid in comparison with 

commercial standards. For each fungicide program, there were a total of three 

applications on a 7–10-day interval. The second application in each case was 

Bravo Weather Stik (1262GA/ha). The trial was conducted on cucumber var. 

‘Straight Eight’, and the data shown are for evaluations taken at 20 days after 

the last application. Mandipropamid and Ranman were mixed with 0.125% 

Activator 90 or 0.06% (v:v) Silwet L77, respectively. 

EFFECT OF NONIONIC SURFACTANT ON EFFICACY. The effect on 

disease control from the addition of a nonionic surfactant (NIS) to 

spray solutions was evaluated in trials on cucumber (data not shown) 

and cantaloupe (Figure 5). In this case, the first spray was made when 

disease severity was ca. 15%, therefore representing a curative 

application. Mandipropamid (150GA/ha) was applied solo or with 


NIS (Activator 90) to final concentrations of 0.06, 0.125, or 0.25% 

(v:v), and disease progress monitored periodically over the following 

five weeks. 

Mandipropamid alone had good effect, with ca. 70% control at the 

final rating, by which time severity in the untreated check was 

approaching 100%. However, efficacy was significantly (p = 0.05) 

better in treatments containing NIS at all rates, but especially at 0.125 

and 0.25% (Figure 5). Careful observation of the disease progress 

revealed the development of lesions on initially asymptomatic leaves 

that expanded after the first application timing in the untreated checks 

and, to a lesser extent, in plants treated with mandipropamid alone. 

AUDPC 

2000 

1800 

1600 

1400 

1200 

1000 

800 

600 

400 

200 

0 

Untreated 7 10 14 

Application Interval (days) 

Untreated 

100 GA/Ha 

125 GA/Ha 

150 GA/Ha 

Fig. 4. Effect of application interval on protectant disease control from 

mandipropamid. Values for Area Under Disease Progress Curve (AUDPC) are 

based on five assessments of disease severity. At the time of the last evaluation, 

disease severity in the untreated check was ca. 71%. All mandipropamid 

applications included NIS at 0.125% (v:v). The trial was conducted on 

cucumber var. ‘Straight Eight’. 

By contrast, lesions did not develop in the corresponding leaves of 

plants treated with mandipropamid + NIS. Consequently, in the latter 

treatments, overall disease severities either remained similar to those 

present at the time of the first application, or even decreased slightly 

due to the contribution of newly developing, healthy foliage (Figure 5). 


These observations suggest that the inclusion of NIS in the spray 

mixture may boost the preventive activity of the fungicide on the 

younger leaves where no subsequent symptoms were observed, or 

possibly enhance any curative effect that mandipropamid exhibits, so 

reducing inoculum production on and spread from older infected 

leaves. Regardless of the basis for the beneficial effect, the addition of 

an adjuvant certainly optimizes the level of downy mildew control 

provided by mandipropamid. Consequently, inclusion of an adjuvant 

is strongly recommended especially in circumstances where the first 

application is made after disease onset and where conditions are 

conducive to the development of an epidemic. 

Disease severity (%) 

100 

90 

80 

70 

60 

50 

40 

30 

20 

10 

0 

Nov 11 

Nov 18 

Nov 24 

Dec 2 

Dec 9 

Assessment Dates (2004) 

Check 

Mandipropamid 

+NIS (0.06%) 

+NIS (0.125%) 

+NIS (0.25%) 

Fig. 5. Effect of NIS (Activator 90) on protectant disease control from 

mandipropamid. Mandipropamid (150GA/ha) was applied alone or with NIS at 

0.06, 0.125, or 0.25% (v:v). The trial was conducted on cantaloupe var. 

‘Athena’, with the applications made on Nov 10, Nov 14, Nov 19, and Nov 26, 

2004. 


Huggenberger, F., C. Lamberth, W. Iwanzik, and G. Knauf-Beiter. 2005. 

Mandipropamid a new fungicide against oomycete pathogens. Proc. BCPC 

Congress Crop Sci. & Tech. 1:93–98. 


Dec 16

INTEGRATING CULTURAL AND CHEMICAL 

STRATEGIES TO CONTROL 

PHYTOPHTHORA CAPSICI AND LIMIT ITS 

SPREAD 

M. K. Hausbeck, A. J. Gevens, and B. Cortright 

Department of Plant Pathology, 

Michigan State University, E. Lansing, MI 48824 

ADDITIONAL INDEX WORDS. Fruit rot, irrigation baiting, fungicide efficacy, soil 

fumigation 

ABSTRACT. Phytophthora root, crown, and fruit rot caused by the oomycete 

Phytophthora capsici Leonian causes significant disease on cucurbits and other 

vegetables nationwide. We investigated the efficacy of fungicide and fumigant 

programs, the use of surface water for irrigation, and susceptibility of snap 

beans to P. capsici. All fungicides tested in three efficacy trials provided 

significant disease control when compared to the untreated. In one trial, 

fungicides were evaluated with different sprayer types, and air-assisted 

applications provided better fruit protection than conventional sprays. All but 

one of the seven fumigant regimes compared provided significant control. The 

consistent detection of P. capsici in surface water used for irrigation suggests 

that this is an important means of pathogen dissemination. With the recent 

identification of P. capsici on beans, rotating P. capsici-susceptible crops with 

beans is no longer recommended. Results of this study can be used to develop 

an overall management scheme for limiting P. capsici on susceptible crops in 

Michigan. 

M 

ichigan has over 22,900 hectares of cucurbits that are 

susceptible to root, crown, and fruit rot caused by the 

soilborne pathogen Phytophthora capsici. P. capsici has 

two mating types that allow for the production of long-term survival 

spores (oospores) and genetic recombination that can result in 

fungicide resistance. Oospores can survive in soil for up to 10 years 

without a susceptible crop, and both mating types have been found in 

every sampled field in Michigan (Lamour and Hausbeck, 2000; 2001). 

P. capsici is favored by rain and warm temperatures, which occur 

during the Michigan growing season. The pathogen has recently been 

detected in Michigan irrigation ponds and other surface water sources 

(Gevens and Hausbeck, 2004; Hausbeck and Lamour, 2004). 

Movement of other Phytophthora spp. via irrigation water has been 

documented and aboveground water sources may play a role in the 

long-distance movement of P. capsici (Jung and Blaschke, 2004; 

Ouedmans, 1999; Yamak, et al., 2002). The most effective control 


measure that growers have available is to avoid planting in infested 

soil. Crop rotation is difficult as infested acreage and urban pressure is 

increasing across vegetable-production areas. Raised plant beds can 

be helpful as they reduce saturated soil conditions (Hausbeck and 

Lamour, 2004). At one time, the systemic fungicide, mefenoxam 

(Ridomil, Ultra Flourish) was widely effective. However, repeated use 

of this fungicide and genetic adaptation of P. capsici has resulted in 

resistant pathogen populations in many Michigan fields (Lamour and 

Hausbeck, 2000). Once resistance occurs, the use of mefenoxam does 

not offer the needed control and alternative fungicides should be used. 

The fungicides Acrobat (dimethomorph), Gavel (mancozeb + 

zoxamide), and Tanos 50DF (famoxadone + cymoxanil) provide 

growers with alternatives to mefenoxam (Hausbeck and Lamour, 

2004). 

A combined approach of all available cultural and chemical control 

techniques is necessary to manage this pathogen and limit its spread. 

The objectives of this research were to evaluate the role of cultural 

practices, such as irrigating cucurbits with surface water and rotating 

cucurbits with snap bean, and the efficacy of fungicides and fumigants 

in managing disease caused by P. capsici. 


FUNGICIDE STUDIES. Two trials were conducted on a farm with 

sandy loam soil using registered and unregistered fungicides (Table 1). 

The farm has a history of P. capsici, and in the year prior, had been 

planted to cucumber. Plots were 183m long with nine rows per plot, 

76cm between rows and 7.6cm between plants. Treatments were 

replicated in a random order. Fungicide treatments were applied with 

a conventional boom sprayer with XR8003 nozzles spaced 51cm apart, 

operating at 415kPa and delivering 281l/ha. Sprays were applied 

when the oldest fruits on the vine were 2.5, 7.6, and 12.7cm in size. In 

Trial 2, a fourth spray was made three days after the third application. 

The number of infected fruit that came across the transfer belt of the 

harvester was recorded for a pass of three rows by 183m (418m 2 ). 

Fruit were collected from each treatment strip and stored for five days 

under ambient conditions prior to disease evaluation. 

A third trial was conducted on sandy loam soil with a history of P. 

capsici. The plot was 274m long with 9 rows per plot, 76cm between 

rows and 7.6cm between plants. Fungicide treatments were applied 

with a conventional boom sprayer or an air-assisted sprayer. The 

conventional sprayer had XR8003 nozzles spaced 51cm apart, 

operated at 415kPa and delivered 281l/ha. The air-assisted sprayer 


Table 1. Products used in fungicide trials. 

Product Active ingredient Company Crops on label 

Acrobat 50WP dimethomorph BASF cucurbits 

Forum 500SC dimethomorph BASF cucurbits 

Gavel 75DF mancozeb + Dow cucumber, melon, 

zoxamide 

summer squash 

Kocide 2000 54DF copper hydroxide DuPont cucurbits 

Maestro 80DF captan Arysta no 

ManKocide 61DF mancozeb + copper DuPont cucumber, melon, 

hydroxide 

summer squash 

Manzate 75DF mancozeb DuPont cucumber, melon, 

summer squash 

Ridomil Gold MZ mefenoxam + Syngenta cucumber, melon, 

mancozeb 

summer/winter squash 

Tanos 50DF famoxadone + 

cymoxanil 

DuPont cucurbits 

had 4 Proptec nozzles spaced 152cm apart and delivered 94l/ha. 

Sprays were applied when the oldest fruits on the vine were 2.5, 7.6, 

and 12.7cm in size. During harvest the number of infected fruit that 

came across the transfer belt of the mechanical harvester was recorded 

for a pass of 3 rows by 274m (626m 2 ). Three bulk bins of fruit were 

collected at harvest from each treatment strip and stored for four days 

under ambient conditions prior to disease evaluation. 

FUMIGANT STUDIES. Studies conducted on P. capsicisusceptible 

crops (tomato, eggplant, pepper, zucchini, winter 

squash, melon, and watermelon) compared the efficacy of 

registered fumigants for the control of P. capsici and evaluated 

their potential as replacement products for methyl bromide. 

Two trials were conducted in fields with severe P. capsici 

disease pressure. Treatments of methyl bromide/chloropicrin, 

chloropicrin alone (100%), and Telone C-35 TM (1,3dichloropropene/ 

chloropicrin) were applied using standard 

gas-injection knives 25.4-30.5cm below the soil and then 

covered with plastic mulch. Applications of Vapam TM (metam 

sodium) and K-Pam TM (metam potassium) were made via drip 

tapes installed under the plastic mulch. In 2003, each product 

was applied alone. For 2004, K-Pam TM was tested alone and in 

combination with chloropicrin. Each crop was planted after the 

appropriate period of off-gassing had expired for each 

treatment. Plots were rated for the number of plants killed by 

P. capsici. 


SURFACE WATER STUDIES. Surface water sources in 

Michigan used for irrigating cucurbit crops were monitored for 

the presence of P. capsici during 2001 and the pathogen was 

recovered from irrigation ponds on two farms. Additional 

water sites including a creek, a river, naturally fed ponds, 

ponds fed by wells, a culvert, and two ditches were monitored 

for P. capsici during 2002 to 2005. The pathogen was baited 

from the irrigation sources using green pears and cucumber 

fruits. Studies are ongoing in 2006. 

ROTATIONAL BEAN STUDIES. From 2003 through 2005, 

several bean (snap and yellow wax) fields in Michigan were 

diagnosed with disease caused by P. capsici. In all situations, 

the beans were being used as a rotational crop to either pickling 

cucumber or zucchini. Infected bean plants exhibited watersoaked 

lesions, necrosis, and wilt. In some instances, pods 

were blighted. Diseased tissues were excised, surface 

sterilized, and plated onto antibiotic-amended V-8 agar. 

Isolates of P. capsici collected from beans were tested for 

pathogenicity on cucumber fruit and other bean types. 


FUNGICIDE STUDIES. In Trial 1, excessive rain (38.1cm total) 

occurred during the 12 days between the first and final fungicide 

sprays. The soil remained wet throughout the trial and disease 

developed uniformly across all untreated plots. All treatments 

significantly reduced the amount of infected pickling cucumber fruits 

at the time of harvest (Table 2). Significant differences were not 

observed among treatments when assessing postharvest fruit rot. 

In Trial 2, a driving rainstorm between the second and third 

fungicide applications provided 2.5cm of rain. All treatments 

significantly reduced the amount of infected fruits at the time of 

harvest (Table 3). All treatments of Maestro 80DF (both 

rates)+Kocide 2000 54DF applied either alone or in rotation with 

Acrobat 50WP+Kocide 2000 54DF were very effective in limiting P. 

capsici infection to fewer than 16 fruit per pass of the harvester. There 

were no significant differences among the treatments for the number of 

infected pickling cucumber fruits evaluated after five days of storage. 

In Trial 3, all fungicides limited disease compared to the control 

(Table 4). The least amount of disease occurred when an air-assisted 

sprayer was used. This may be due to the ability of the air-assisted 

sprayer to force the fungicide into the plant canopy and reach the fruit. 

The results of this trial indicate the need for good coverage when 


Table 2. Efficacy of fungicides for Phytophthora fruit rot of pickling 

cucumbers in 2004. 

Phytophthorainfected 

fruit 

Treatment and rate/ha (fruit size when treated) 

At 

harvest 

(number) 

After 5 

days 

storage 

(%) 

Untreated......................................................................................1,092.5 b * Forum 500SC 0.5L+Kocide 2000 54DF 1.7kg (2.5, 7.6, 

33.0 

12.7cm) ........................................................................................ 

Forum 500SC 0.5L+Kocide 2000 54DF 1.7kg 

6.5 a 11.5 

+ Manzate 75DF 2.2kg (2.5, 7.6, 12.7cm) ................................ 

Gavel 75DF 2.2kg+Kocide 2000 54DF 1.7kg (2.5cm) 

12.5 a 11.5 

Acrobat 50WP 0.4kg+Kocide 2000 54DF 1.7kg (7.6, 12.7cm) 10.5 

Gavel 75DF 2.2kg+Kocide 2000 54DF 1.7kg (2.5, 7.6cm) 

a 24.0 

Acrobat 50WP 0.4kg+Kocide 2000 54DF 1.7kg (12.7cm)....... 

Tanos 50DF 0.6kg+Kocide 2000 54DF 1.7kg 

+Manzate 75DF 2.3kg (2.5cm) 


11.5 a 17.0 





1.0 a 10.0 




Gavel 75DF 2.2 kg+Kocide 2000 54DF 1.7kg (7.6cm) 

y a 36.0 

Acrobat 50WP 0.4kg (12.7cm) .............................................. 

Tanos 50DF 0.7kg+ManKocide 61DF 2.2kg (2.5cm) 


2.0 a 3.5 


Ridomil Gold MZ 2.8kg+Kocide 2000 54DF 1.7kg 

10.0 a 7.5 

(2.5, 7.6, 12.7cm) ...................................................................... 

Maestro 80DF 6.7kg+Kocide 2000 54DF 1.7kg (2.5, 7.6, 

10.0 a 31.0 

12.7cm) ........................................................................................ 37.0 a 5.5 

* 

Column means with a letter in common or no letter are not significantly different 

(SNK; P = 0.05). 

applying fungicides to control fruit rot. Plant-row spacing may need to 

be increased to prevent an especially dense canopy from forming, 

thereby increasing the potential of a fungicide spray reaching the 

underlying fruit. 

FUMIGANT STUDIES. Melon and watermelon plants were 

especially susceptible to P. capsici. In 2004, applications of both rates 


%P 

40 

35 

30 

25 

20 

15 

10 

5 

0 

b 

Untreated 

a 

Vapam 704l/ha 

2003 

a 

MBr/pic 

Combined data for: 

Tomato 

Eggplant 

Pepper 

Zucchini 

Winter Squash 

Melon 

Watermelon 

a 

Telone C35 

Fig. 1. Fumigant evaluation for P. capsici, 2003 (left) and 2004 (right). 

30 

25 

20 

15 

10 

of K-Pam TM applied either alone or in combination with chloropicrin 

were very effective in controlling P. capsici in both melon and 

watermelon (Table 5). Applications of methyl bromide/chloropicrin 

and chloropicrin alone also significantly limited disease. 

Combining the data of plant death (%) for all crops in this trial showed 

that significant disease control was achieved for all treatments in 2003 

(Figure 1). In 2004, both rates of K-Pam TM applied alone or in 

combination with chloropicrin were very effective in limiting P. 

capsici (Figure 1). Applications of methyl bromide/chloropicrin and 

chloropicrin alone were also significantly better than the untreated. 

Telone C-35 TM allowed more disease in 2003 compared with 2004. 

SURFACE WATER STUDIES. Phytophthora capsici was frequently 

detected in a river, creek, and a naturally fed pond. All water sources 

monitored were located near crops infected with P. capsici and/or 

fields with a history of disease. Using surface water that may be 

contaminated with P. capsici to irrigate healthy crops must be avoided 

to limit pathogen spread. 

ROTATIONAL BEAN STUDIES. All P. capsici isolates from beans 

caused disease on cucumber fruit, and resulted in dark, water-soaked 

lesions with pathogen sporulation. Isolates of P. capsici from snap 

beans caused disease on 12 bean cultivars that represented four 

Phaseolus species (lunatus, vulgaris, vulgaris var. humilis, and 

vulgaris var. vulgaris) and one Glycine species (max). The use of 

beans as a rotational crop with P. capsici-susceptible hosts is not 

recommended. 

5 

0 

c 

Untreated 

bc 

Telone C35 35 327 

ab 

Chloropicrin 234 l/ha 

2004 

ab 

MBr/Pic 392 kg/ha 

a 

K-Pam 280 l/ha 

a 

K-Pam 561 l/ha 

Combined data for: 

Tomato 

Eggplant 

Pepper 

Zucchini 

Winter Squash 

Melon 

Watermelon 


a 

K-Pam 

a 

K-Pam High

Table 3. Efficacy of fungicides for Phytophthora fruit rot of pickling 

cucumbers in 2004. 

Phytophthorainfected 

fruit 

After 5 

days 

At harvest storage 

Treatment and rate/ha (fruit size when treated) (number) (%) 

Untreated............................................................................ 83.3 b * Acrobat 50WP 0.4kg +Kocide 2000 54DF 1.7kg 

4.4 

(2.5, 7.6, 12.7cm) ............................................................ 29.3 

Maestro 80DF 4.5kg+Kocide 2000 54DF 1.7kg 

a 0.2 

(2.5, 7.6, 12.7cm) ............................................................ 15.7 


a 0.8 

(2.5, 7.6, 12.7cm) ............................................................ 

Tanos 50DF 0.8 kg+Kocide 2000 54DF 1.7kg 

5.0 a 0.0 

(2.5, 7.6, 12.7cm) ............................................................ 30.3 

Gavel 75DF 2.2 kg+Kocide 2000 54DF 1.7kg 

a 2.3 

(2.5, 7.6, 12.7cm) ............................................................ 20.0 

Gavel 75DF 2.2kg+Kocide 2000 54DF 1.7kg 

(2.5cm) Tanos 50DF 0.8kg+Kocide 2000 54DF 1.7kg 

(7.6cm) Acrobat 50WP 0.4kg+Kocide 2000 54DF 

a 0.2 

1.7kg (12.7cm) ................................................................ 28.0 


(2.5, 12.7cm) Acrobat 50WP 0.4kg+Kocide 2000 

a 0.0 

54DF 1.7kg (7.6cm)........................................................ 7.7 a 0.0 

* 

Column means with a letter in common are not significantly different (SNK; P = 

0.05). 

In summary, our studies indicate that managing P. capsici on 

susceptible crops requires attention to cultural and chemical practices 

at all stages of crop production. A recommended disease program 

includes: (1) selecting a site without P. capsici infestation or preplant 

fumigation of infested sites, (2) irrigating crops with water from wells 

or well-fed ponds, (3) applying effective fungicides and thoroughly 

covering foliage and fruit, and (4) choosing appropriate rotational 

crop(s) to limit pathogen buildup in the soil. 


Table 4. Evaluation of fungicides and sprayers to manage 

Phytophthora fruit rot on pickling cucumbers in 2004. 

Phytophthora-infected 

fruit 

Spray regime, treatment and rate/ha 

At harvest 

(number) * 

After 5 days 

storage (%) 

Air assisted sprayer 

Acrobat 50WP 0.4kg+Kocide 2000 54WG 1.7kg ....... 16.3 a ** Conventional boom sprayer 

5.9 a 

Acrobat 50WP 0.4kg+Kocide 2000 54WG 1.7kg ....... 89.3 b 27.5 bc 

Gavel 80WG 2.2kg+Kocide 2000 54WG 1.7kg.......... 70.0 ab 27.4 bc 

Untreated ........................................................................ 178.0 c 37.4 c 

* 

Number of infected fruit crossing the harvest belt over a 3-row x 274-m plot. 

** 

Column means with a letter in common are not significantly different (SNK; P = 

0.05). 

Table 5. Evaluation of fumigants for P. capsici of cucurbits in 2004. 

Plant death (%) y 

Treatment 

Rate/ 

ha 

Application 

method z Melon 

Watermelon 

Untreated ............................................ – – 97.8 b x 37.7 b 

Methyl bromide/chloropicrin (67/33) . 392kg Shank 22.2 a 11.1 ab 

Telone C35 ......................................... 327L Shank 66.7 b 36.7 b 

Chloropicrin 

234L Shank 

K-Pam................................................. 280L Drip 0.0 a 0.0 a 

Chloropicrin 

234L Shank 

K-Pam................................................. 561L Drip 0.0 a 0.0 a 

Chloropicrin........................................ 234L Shank 20.0 a 33.3 b 

K-Pam................................................. 561L Drip 3.3 a 8.8 a 

K-Pam................................................. 280L Drip 8.8 a 0.0 a 

z 

Materials were applied either at time of bed formation using swept-back knives or 

preplant through drip tape. 

y 

Percentage of plants killed by disease out of nine original plants. 

x 

Column means with no letter or a letter in common are not significantly different, 

SNK, P = 0.05. 


Gevens, A. J. and M. K. Hausbeck. 2004. Characterization and distribution of 

Phytophthora capsici from irrigation water near Michigan cucurbit fields: a first 

report of P. capsici in irrigation water in Michigan. Phytopathology. 94:S157. 

Hausbeck, M. K. and K. H. Lamour. 2004. Phytophthora capsici on vegetable crops: 

research progress and management challenges. Plant Dis. 88:1292–1303. 


Jung, T. and M. Blaschke. 2004. Phytophthora root and collar rot of alders in 

Bavaria: distribution, modes of spread, and possible management strategies. 

Plant Path. 53:197–208. 

Lamour, K. H. and M. K. Hausbeck. 2000. Mefenoxam insensitivity and the sexual 

stage of Phytophthora capsici in Michigan cucurbit fields. Phytopathology. 

90:396–400. 

Lamour, K. H. and M. K. Hausbeck. 2001. Investigating the spatiotemporal genetic 

structure of Phytophthora capsici in Michigan. Phytopathology. 91:973–980. 

Ouedmans, P. V. 1999. Phytophthora species associated with cranberry root rot and 

surface irrigation water in New Jersey. Plant Dis. 83:251–258. 

Yamak, F., T. L. Peever, G. G. Grove, and R. J. Boal. 2002. Occurrence and 

identification of Phytophthora spp. pathogenic to pear fruit in irrigation water in 

the Wenatchee River valley of Washington State. Phytopathology. 92:1210– 

1217. 


TREATMENTS TO PREVENT SEED 

TRANSMISSION OF BACTERIAL FRUIT 

BLOTCH OF WATERMELON 

D. L. Hopkins and C. M. Thompson 

University of Florida, Mid-Florida Research and Education Center, 

2725 Binion Road, Apopka, FL 32703-8504, USA 

ADDITIONAL INDEX WORDS. Citrullus lanatus, Acidovorax avenae subsp. citrulli, 

hot-water treatment, chemical treatments, fermentation of seeds 

ABSTRACT. Contaminated seed lots have been the primary way in which 

bacterial fruit blotch (BFB) (Acidovorax avenae subsp. citrulli [Schaad et al.] 

Willems et al.) has been introduced into watermelon (Citrullus lanatus [Thunb.] 

Matsum. & Nakai) fields or transplant houses. In this study, various wet-seed 

treatments were evaluated for the elimination of A. avenae subsp. citrulli from 

watermelon seeds. Peroxyacetic acid (PA), hydrochloric acid (HCl), and acetic 

acid were all effective in eliminating seed transmission of BFB; however, HCl 

can adversely affect seed quality. Hot-water treatments at 55 o C for 2 h and at 

60 o C for 1 h eliminated seed transmission, but the 60 o C treatment also reduced 

seed germination. Natural fermentation in watermelon pulp for 16–24 h 

eliminated seed transmission in most but not all seed lots. With 16-h 

fermentations containing Lactobacillus brevis or L. plantarum additives, seed 

transmission of BFB was prevented in all of our seed lots tested. Shorter 

fermentations were ineffective and the effect of 16-h fermentation on triploid 

seed quality is unknown. 

B 

acterial fruit blotch (BFB) of watermelon ( Citrullus lanatus), 

caused by Acidovorax avenae subsp. citrulli, was first 

observed in commercial production areas in the United States 

in the spring of 1989 (Latin and Rane, 1990; Somodi et al., 1991), but 

had been observed in Australia and Guam earlier (Wall and Santos, 

1988). BFB on watermelon has occurred somewhere every year since 

1989 (Maynard and Hopkins, 1999; D. L. Hopkins, unpublished), 

occasionally resulting in losses of more than 90% of the total 

marketable fruit in a field. 

The causal agent of BFB causes disease and can be seed-borne in 

various cucurbits (Hopkins and Thompson, 2002: Kucarek et al., 1993; 

Rane and Latin, 1992). Contaminated seed lots of cucurbits have been 

the primary way in which A. avenae subsp. citrulli has been introduced 

into cucurbit fields or transplant houses (Hopkins and Thompson, 

Financial support for this research came in part from USDA/CSREES award number 

2003-34135-14077. 


2002). The most effective control of BFB of cucurbits, is the 

exclusion of the bacterium (Latin, 1996). The intensive efforts of the 

cucurbit seed and transplant industry to produce seeds and transplants 

free of A. avenae subsp. citrulli have reduced the incidence of BFB 

over the last few seasons, but the disease still occurs every year. There 

is a need for more consistent, efficient methods of producing cucurbit 

seed free of the bacterium. 

Fermentation and hydrochloric acid (HCI) treatments are effective 

in removing the bacterium from seed, but may adversely affect seed 

quality (Hopkins et al., 1996). Peroxyacetic acid (PA) can be very 

effective when properly used with good quality control (Hopkins et al., 

2003). These strategies have been used successfully as wet-seed 

treatments at seed harvest; however, there is a need for a treatment to 

clean up contaminated seed lots already dried and stored. There 

remains a need for seed treatments that can efficaciously eradicate 

seed-borne A. avenae subsp. citrulli while ensuring the safety of the 

worker and the environment, and not reducing any measurable 

parameters of seed quality. The objective of this study was to evaluate 

various chemical, biological, and hot-water seed treatments for the 

elimination of A. avenae subsp. citrulli from wet seeds of watermelon. 


PRODUCTION OF INFESTED WATERMELON SEEDS. In the fall of 

2003, as well as in the spring and fall of 2004 and 2005, BFB-infected 

‘Charleston Gray’ watermelon seeds were produced on the MREC 

research farm in Apopka. In most seasons, the bacterium occurred 

naturally in the seed-production areas. To assure infected seed, fruit 

also were inoculated with strain WFB89-1, which has given 

reproducible symptoms on watermelon. For inoculation of fruit in the 

field, A. avenae subsp. citrulli was grown on nutrient agar for 48 h, 

washed from the agar surface with sterile, deionized water, and 

adjusted to a suspension of approximately 10 6 colony-forming 

units/ml. Inoculations were done 14–21 days prior to fruit maturation 

by misting the upper rind surface with the bacterial suspension. Seeds 

were collected by hand from mature fruit with symptoms and were 

bulked. The seeds were divided into batches of approximately 2,000 

seeds in 3L of watermelon juice and pulp and placed in 9-L buckets 

for treatment. All treatments were initiated within 3 h of the 

completion of seed harvesting. 

CHEMICAL SEED TREATMENTS. Seeds were washed thoroughly 

with tap water on screens to remove all watermelon tissue and juice 

prior to treatment. Chemical treatments were applied for 15 or 30 


minutes to approximately 2,000 seeds each in a 2-L treatment solution. 

The solutions were stirred periodically throughout the treatments. 

Treatments with 10,000μg/ml (1%) HCl and 1600μg/ml PA (Tsunami 

100; 15% PA and 11% hydrogen peroxide; Ecolab Inc., Mendota 

Heights, MN) were included in the tests because they had been 

effective seed treatments in previous studies (Hopkins et al., 1996, 

2003). Other chemicals evaluated included 10,000μg/ml acetic acid, 

10,000μg/ml NaOCl, 2,700μg/ml hydrogen dioxide (1:100 Zerotol; 

BioSafe Systems, Gastonbury, CT), and 667μg/ml quaternary 

ammonia compounds (1:300 Physan 20; Maril Products Inc., Tustin, 

CA). After treatment, seeds were rinsed in two changes of water, air 

dried on screens for 48 h on the greenhouse bench at temperatures 

ranging from 26 o C at night to 40 o C in the afternoon, and stored in a 

seed-storage room. 

For the greenhouse assay of seed transmission of BFB to seedlings, 

seeds were planted in 28 x 52cm plastic trays filled with commercial 

potting mix. Four replications of 250 or more seeds were planted from 

each treatment. Watering was done carefully on the surface of the 

potting mix with a hose, avoiding contact with the seedlings. 

Greenhouse temperatures ranged from 26 o C to 40 o C. Disease 

incidence evaluations were made 9–10 days after planting. Percent 

seed transmission was calculated based on the number of emerged 

seedlings. Analyses of variance were performed for all experiments 

and means were compared by Duncan’s multiple range test (P=0.05). 

Percentage seed transmission data were analyzed after transformation 

to arc sine √x. 

HOT-WATER SEED TREATMENTS. Seeds were washed thoroughly 

with tap water on screens to remove all watermelon tissue and juice 

prior to hot-water treatment. Approximately 2,000 seeds per treatment 

were submerged in a hot-water bath at the desired temperature for the 

specified time. Seeds were stirred regularly throughout the treatments. 

Treatment temperatures ranged from 45 o C to 60 o C and treatment times 

ranged from 0.5 h to 2 h. After treatment, seeds were air dried on 

screens for 48 h on the greenhouse bench at temperatures ranging from 

26 o C at night to 40 o C in the afternoon and stored in a seed-storage 

room. The greenhouse assay was as described above for the chemical 

treatments. 

FERMENTATION SEED TREATMENTS. Fermentation of watermelon 

seeds in the pulp from the fruit for 24 to 48 h was found to be an 

effective way of eliminating seed transmission of bacterial fruit blotch 

(Hopkins, et al., 1996). The problem with this long fermentation 

treatment was its effect on seed quality, especially with triploid seed. 


For fermentation treatments, seeds were divided into batches of 

approximately 2,000 seeds in 3L watermelon juice and pulp and 

placed in 9-L buckets for treatment. Bacterial fermentation additives 

included Lactobacillus brevis, L. plantarum, and Bacillus subtilus. 

Both 5- and 16-h fermentations were evaluated. After fermenting at 

air temperatures of 23 o C to 33 o C with periodic stirring, seeds were 

washed thoroughly to remove watermelon tissue, air dried on screens 

for 48 h on the greenhouse bench at temperatures ranging from 26 o C at 

night to 40 o C in the afternoon, and stored in a seed-storage room. The 

greenhouse assay was as described above for the chemical treatments. 


CHEMICAL SEED TREATMENTS. As in previous studies (Hopkins 

et al., 1996, 2003), PA and HCl were effective in eliminating A. 

avenae subsp. citrulli from watermelon seed, whether applied for 15 

min or 30 min (Table 1). The 0.1% seed transmission in the PA 

treatment of watermelon seeds in the fall of 2003 may have been the 

result of cross-contamination in the grow-out rather than a failure of 

the seed treatment. Acetic acid at 1% concentration was also very 

effective in eliminating seed transmission of BFB. Sodium 

hypochlorite at 1% reduced the seed transmission of BFB, but did not 

completely eliminate it. Zerotol and Physan were ineffective as seed 

treatments for bacterial fruit blotch. Both PA and acetic acid appear to 

be good options to use in the control of seed transmission of BFB. 

HOT-WATER SEED TREATMENTS. In the spring and fall of 2004, 

none of the hot-water treatments of infected watermelon seeds 

eliminated seed transmission of BFB (Table 2). At 50 o C, the 1-h 

treatment was more effective than 0.5 h. Based on these results, longer 

treatment times and higher temperatures were investigated in 2005. In 

both the spring and fall of 2005, a 2-h treatment at 55 o C eliminated 

seed transmission of BFB in watermelon without affecting seed 

germination. One-hour treatment at 60 o C eliminated BFB transmission, 

but also reduced seed germination. While hot-water treatment did 

eliminate seed transmission without adversely affecting seed 

germination, the margin for error is apparently very small. Increasing 

the temperature by only 5 o C resulted in reduced seed germination. 

FERMENTATION SEED TREATMENTS. Three-hour fermentation 

was not sufficient to eliminate seed transmission in watermelon 

regardless of the bacterial additive (data not shown). Five-h 

fermentation with or without added bacteria was variable in efficacy, 

reducing seed transmission but not eliminating it (Table 3). With 16-h 

fermentation, L. brevis and L. plantarum additives eliminated seed 


Table 1. Elimination of seed transmission of bacterial fruit blotch 

(BFB) in watermelon with various chemical seed treatments. 

Seed treatment 

% seed transmission in seed 

from: a 

Exposure 

time (min) Fall03 Spring04 

Untreated - 5.5 c 11.2 b 

1% HCl 15 0 a 0 a 

1600μg/ml PA b 15 0 a 0 a 

1% acetic acid 15 0 a 0 a 

1600μg/ml PA 30 0.1 a 0 a 

1% acetic acid 30 0 a - 

1% Na0Cl 30 1.0 b 0.3 a 

2700μg/ml 

hydrogen dioxide 30 5.5 c - 

667μg/ml 

quaternary ammonia 

products 30 6.8 c - 

a The percent seed transmission of BFB was determined by a greenhouse grow-out of 

the seedlings and represents the percentage of seedlings (germinated seeds) that were 

symptomatic. Means in columns followed by the same letter are not significantly 

different (P=0.05) by Duncan’s multiple range test. Data were analyzed after 

transformation to arc sine √x. 

b PA = peroxyacetic acid. 

transmission of BFB in watermelon. Natural fermentation in the 

watermelon pulp for 16–24 h eliminated most but not always all seed 

transmission. There did not seem to be any benefit from combining L. 

plantarum and L. brevi. 


Table 2. Elimination of seed transmission of bacterial fruit blotch 

(BFB) in watermelon with hot-water treatments. 

% seed transmission in seed from: a 

Hot-water 

treatments Spring04 Fall04 Spring05 Fall05 

Untreated 8.3 bc 4.3 b 2.1 b 

(94) b 

1.8 bc (91) 

45 o C for 2 h 5.0 ab - - - 

50 o C for 0.5 h 11.8 cd 2.3 ab - - 

50 o C for 1 h 3.6 a - - - 

50 o C for 2 h - - - 2.2 c (91) 

55 o C for 0.5 h 15.1 d 1.0 a - - 

55 o C for 1 h - - 0.2 a (99) 1.4 b (95) 

55 o C for 2 h - - 0 a (94) 0 a (90) 

60 o C for 1 h - - - 0 a (80) 

a 

The percent seed transmission of BFB was determined by a greenhouse grow-out of 





b 

The number in parenthesis is the percent seed germination. 

There was an apparent benefit from adding L. brevis or L. 

plantarum to the seed fermentation mixture and, overall, L. plantarum 

appeared to be the most effective bacterial additive. Fermentation 

continues to be an effective way of eliminating seed transmission of 

BFB in watermelon; however, even with the bacterial additives, it 

remained necessary to ferment overnight, which may be detrimental to 

triploid seed quality. Combining a 5-h fermentation with a bacterial 

additive and chemical treatment may provide the best chance of 

eliminating A. avenae subsp. citrulli from watermelon seeds. 


Table 3. Effect of various bacterial additives on elimination of seed 

transmission of bacterial fruit blotch (BFB) in watermelon with 

fermentation. 

% seed transmission in seed from: a 

Fermentation Fall03 Spring04 Fall04 Spring05 Fall05 

None 5.5 b 8.3 b 4.3 c 2.1 b 1.8 b 

Natural, 5 h - - 0.1 ab - - 

Natural, 16 h 0 a - - 0 a - 

Lactobacillus 

brevis, 5 h 

- - 0.3 b 0.1 a - 

L. brevis, 16 h - 0 a 0 a - - 

L. plantarum, 5 h - - 0.1 ab 0.1 a 0.4 a 

L. plantarum, 16 h - 0 a 0 a 0 a - 

Bacillus subtilus, 5 

h 

- - - 0 a 0.3 a 

B. subtilus, 16 h - - - 0.1 a - 

L. plantarum + L. 

brevi, 5 h 

L. plantarum, 5 h 

+ 1600μg/ml PA, 

30 min 

- - - 0.1 a - 

- - 0 a - - 

a The percent seed transmission of BFB was determined by a greenhouse grow-out of 






Hopkins, D. L. and C. M. Thompson. 2002. Seed transmission of Acidovorax 

avenae subsp. citrulli in cucurbits. HortSci. 37:924–926. 

Hopkins, D. L., C. M. Thompson, J. Hilgren, and B. Lovic. 2003. Wet seed 

treatment with peroxyacetic acid for the control of bacterial fruit blotch and 

other seedborne diseases of watermelon. Plant Dis. 87:1495–1499. 


Hopkins, D. L., J. D. Cucuzza, and J. C. Watterson. 1996. Wet seed treatments for 

the control of bacterial fruit blotch of watermelon. Plant Dis. 80:529–532. 

Kucharek, T., Y. Perez, and C. Hodge. 1993. Transmission of the watermelon fruit 

blotch bacterium from infested seed to seedlings. Phytopathology. 

83:466(Abstr.). 

Latin, R. X. 1996. Bacterial fruit blotch, p.34–35. In: T. A. Zitter, D. L. Hopkins, 

and C. E. Thomas (eds.). Compendium of cucurbit diseases. APS Press, St. 

Paul, MN. 

Latin, R. X. and K. K. Rane. 1990. Bacterial fruit blotch of watermelon in Indiana. 

Plant Dis. 74:331. 

Maynard, D. N. and D. L. Hopkins. 1999. Watermelon fruit disorders. HortTech. 

9:155–161. 

Rane, K. K. and R. X. Latin. 1992. Bacterial fruit blotch of watermelon: 

Association of the pathogen with seed. Plant Dis. 76:509–512. 

Somodi, G. C., J. B. Jones, D. L. Hopkins, R. E. Stall, T. A. Kucharek, N. C. Hodge, 

and J. C. Watterson. 1991. Occurrence of a bacterial watermelon fruit blotch in 

Florida. Plant Dis. 75:1053–1056. 

Wall, G. C. and V. M. Santos. 1988. A new bacterial disease of watermelon in the 

Mariana Islands. Phytopathology. 78:1605(Abstr.). 


IDENTIFICATION AND SURVEY OF 

CUCURBIT POWDERY MILDEW RACES IN 

CZECH POPULATIONS 

A. Lebeda and B. Sedláková 


Botany, Šlechtitelů 11, 783 71, Olomouc, Czech Republic 

ADDITIONAL INDEX WORDS. Golovinomyces cichoracearum, Podosphaera 

xanthii, Cucumis melo, differential genotypes, pathogenicity variation, race 

identification 

ABSTRACT. Physiological races of two cucurbit powdery mildew species 

[Golovinomyces cichoracearum s.l. (Gc) and Podosphaera xanthii (Px)], were 

identified by using 11 differential genotypes of Cucumis melo. Altogether, 89 

different races (63 Gc, 26 Px) were recorded in the studied set of 218 isolates 

(172 Gc, 46 Px) collected in the Czech Republic in the years 2000–2004. Isolates 

virulent to C. melo (line MR-1) and avirulent to C. melo (Iran H) were found as 

well as both powdery mildew species. These data demonstrate the existence of 

new and until now unknown virulence/avirulence and susceptibility/resistance 

factors in pathogen populations and differential host genotypes. Repeated 

occurrence of some races (28 Gc, 8 Px) was noted during the period of 

investigation. Races S of Gc and F of Px were virulent on all C. melo 

differentials. The races with medium and high virulence prevailed in cucurbit 

powdery mildew (CPM) populations. The most frequent hosts were Cucurbita 

pepo and C. maxima for both CPMs with the highest number of detected races. 

A remarkable shift toward increased virulence was observed for both Gc and 

Px over a five-year period. 

P 

owdery mildew is the major cause of losses in production in 

cucurbits worldwide (Cohen et al., 2004; Jahn et al., 2002; 

McCreight, 2006; Vakalounakis et al., 1994). In Central Europe, 

the disease is caused by two obligate biotrophic ectoparasites: 

Golovinomyces cichoracearum s.l. (Gc) (syn. Erysiphe cichoracearum 

s.l.) and Podosphaera xanthii (Px) (syn. Sphaerotheca fuliginea) (Jahn 

et al., 2002). These two species induce identical symptoms; however, 

they can be distinguished easily under light microscopy (Braun et al., 

2002). The two species differ in host range, ecological requirements, 

geographic distribution (Lebeda, 1983; Lebeda et al., 2006; Sitterly, 

This research was supported by grants MSM 6198959215, MSM 153100010, and 

QD 1357; and by the National Programme of Genepool Conservation of 

Microorganisms and Small Animals of Economic Importance. 


1978), and response to fungicides (McGrath, 2001; Sedláková and 

Lebeda, 2004a, b). Broad pathogenic variation is represented by the 

existence of different pathotypes and races (Bertrand, 1991; Bertrand 

et al., 1992; Jahn et al., 2002; Vakalounakis and Klironomou, 1995). 

Twenty-two races of Px and two races of Gc have been described on 

melons (Cohen et al., 2004; Hosoya et al., 2000; McCreight, 2006; 

Pitrat et al., 1998); however, recent results suggest that even more 

pathotypes and races exist (Lebeda and Sedláková, 2004; Lebeda et 

al., 2004, 2006). 

Long-lasting study of CPMs virulence variation is one of the 

priorities of our laboratory. The aim of the current paper is to 

summarize the recent knowledge about physiological races identified 

in cucurbit powdery mildew (CPM) populations in the Czech Republic 

in the years 2000–2004. The main purpose of our work was to study in 

more detail the virulence variation of CPM populations as a part of the 

complex research of population biology, ecology, genetics, and spatial 

and temporal dynamics. 


PATHOGEN COLLECTING, IDENTIFICATION, ISOLATION, 

MULTIPLICATION, AND MAINTENANCE OF ISOLATES. Samples of 

powdery-mildew-infected cucurbit plants were obtained during 

collecting expeditions in the territory of the Czech Republic in the 

years 2000–2004. Before isolation, the samples were microscopically 

examined in a 3% KOH solution (Lebeda, 1983). Isolates with a 

mixture of powdery mildew species were excluded. Conidia of pure 

cultures were transferred by tapping onto primary leaves of the highly 

susceptible cucumber (Cucumis sativus) ‘Stela F1’ (Lebeda, 1986). A 

total of 218 (172 Gc, 46 Px) isolates were used for race determination. 

Isolates were multiplied and cultured in plastic boxes (24°C/18°C 

day/night) for 12h. 

DETERMINATION OF RACES. Altogether, 218 isolates of Gc and Px 

were used for screening: 3 (2 Gc,1 Px) from 2000; 34 (27 Gc, 7 Px) 

from 2001; 52 (45 Gc, 7 Px) from 2002; 67 (45 Gc, 22 Px) from 2003; 

and 62 (53 Gc, 9 Px) from 2004. Race determination was made by a 

leaf-disc method (Bertrand et al., 1992; Lebeda, 1986). Races were 

identified by using 11 differential genotypes of Cucumis melo (‘Iran 

H’, ‘Védrantais’, ‘Solartur’, ‘PMR 45’, ‘WMR 29’, ‘Edisto 47’, PI 

414723, ‘PMR 5’, PI 124112, ‘MR-1’, and ‘Nantais Oblong’) (Pitrat et 

al.,1998; Bardin et al., 1999). Each genotype was represented by three 

leaf discs (15mm in diameter) in three replicates (one replicate = one 

plant). Discs from true leaves (2–3-leaf stage) of cucumber and 


muskmelon plants were used for screening. Discs were inoculated by 

tapping leaf discs with 3–4-day-old sporulating mycelia of CPMs and 

then incubated under the conditions described above. Evaluations were 

conducted 6–14 days after inoculation by using a 0–4 scale (Lebeda, 

1984). Differential genotypes with little or no sporulation (degree of 

infection [DI] = 0–1) were considered as resistant (R); genotypes with 

DI = 2–4 were scored as susceptible (S). The degree of virulence of 

CPM isolates was expressed by the number of differentials (from 1 to 

11) infected by the individual isolate. 


In total, 89 races (63 Gc and 26 Px) were determined in the studied 

set of isolates in the years 2000–2004 (Tables 1 and 2). Obtained 

results showed that Czech CPM populations are very heterogeneous 

and markedly different in comparison with those of some western and 

southern European countries (Pitrat et al., 1998) and other parts of the 

world (Cohen et al., 2004; Hosoya et al., 2000; McCreight, 2006). All 

recorded Gc races and 23 Px races have not yet been described in 

Europe and elsewhere. Nevertheless, occurrence of three Px known 

races (1, 2US, 3) was confirmed in the Czech Republic. Seven isolates 

of Gc and five isolates of Px were avirulent to C. melo ‘Iran H’, which 

is consistent with our previous observations (Křístková et al., 2004). 

Until now, ‘Iran H’ was considered to be susceptible to all races 

(McCreight, 2006). Our results indicate that even in ‘Iran H’ there 

exist as yet unidentified race-specific resistance factors to both CPM 

species. Repeated occurrence of some races (28 Gc, 8 Px) was noted 

during the study period. The most frequently occurring Gc races were 

S (28x), Y (14x), e (13x), and P (8x). The most frequent Px race was B 

(7x). Races S of Gc and F of Px were virulent on all C. melo 

differential genotypes (Tables 1 and 2). These results and our previous 

observations (Křístková et al., 2004) indicate that, in Czech pathogen 

populations, races exist that are able to overcome the resistance of C. 

melo lines MR-1 and PI 124112. 

Virulence data for Gc and Px isolates are summarized in Table 3. 

Gc isolates were in the range of medium (32%) and high (58%) 

virulence and were classified into 31 and 27 races, respectively. In the 

case of Px, isolates of medium virulence were the most frequent (61%) 

and were classified into 16 races. As was found in preliminary studies 

(Lebeda and Sedláková, 2004; Lebeda et al., 2004), Czech CPM 

populations exhibited a temporal shift to higher virulence. Occurrence 

of Gc and Px races on different cucurbit hosts is shown in Table 4. The 

most frequently infected host plants were Cucurbita pepo and C. 


Table 1. Races of Golovinomyces cichoracearum identified in the 

Czech Republic in the years 2000–2004. 

Differential genotype of Cucumis melo a / 

Reaction patterns of G. cichoracearum isolates 

IrH Véd Sol P45 W29 E47 PI41 P5 PI12 MR1 Nobl Race 

R R S R R R R R R R R N1 1 

R S S S R R R R R R S f 1 

R S S S R S R R R R S T R 1 

R S S S R S S R R R S T 1 

R S S S S R R S R R S A 1 

R S S S S S S S R R R B 1 

R S S S S S S S S R S C 1 

S R R S S S R R S R S D 1 

S R S R S R S R R R R E 1 

S R S S R R R R R R S N2 1 

S S R R S S S R R R R U 1 

S S S R R R R R R R S e 13 

S S S R R R R S R R S g 6 

S S S R R R S R R R S N3 2 

S S S R R R S S R R S h 5 

S S S R R S R R R R S ch 4 

S S S R R S S R R R S N4 3 

S S S R R S S S R R S i 1 

S S S R R S S S S R S N5 1 

S S S R S R R R R R S N6 1 

S S S R S S S S S R S b 1 

S S S S R R R R R R S N7 2 

S S S S R R R S R R R j 1 

S S S S R R R S R R S d R 1 

S S S S R R S R R R R F R 2 

S S S S R R S R R R S F 1 

S S S S R R S S R R R k 1 

S S S S R R S S R R S d 3 

S S S S R S R R R R S I R 2 

S S S S R S R R R S S l 1 

S S S S R S R S R R S I 1 

S S S S R S R S S R R H 1 

S S S S R S S R R R S N8 2 

S S S S R S S R S R R G 2 

S S S S R S S R S R S u 1 

S S S S R S S S R R R N9 1 

S S S S R S S S R R S n 3 

S S S S R S S S S R R o 6 

S S S S R S S S S R S s 3 

S S S S R S S S S S S N11 2 

No 

IS* 


(Table 1, continued) 


Reaction patterns of G. cichoracearum isolates 


S S S S S R S R R R S K 2 

S S S S S R S S R R R J R 2 

S S S S S R S S R R S J 1 

S S S S S R S S S R S J S 1 

S S S S S R S S S S S a 2 

S S S S S S R R R R S L R 1 

S S S S S S R R R S S v 1 

S S S S S S R R S R S M 1 

S S S S S S R S R R S L 1 

S S S S S S R S S R R w 1 

S S S S S S S R R R R O R 1 

S S S S S S S R R R S N 4 

S S S S S S S R R S S x 1 

S S S S S S S R S R R c 1 

S S S S S S S R S R S N S 4 

S S S S S S S R S S R X 1 

S S S S S S S S R R R O 3 

S S S S S S S S R R S P 8 

S S S S S S S S R S S V 4 

S S S S S S S S S R R R 4 

S S S S S S S S S R S Y 14 

S S S S S S S S S S R R S 2 

S S S S S S S S S S S S 28 

*IS = isolates; R = resistant reaction; S = susceptible reaction. 

a Genotypes of Cucumis melo: IrH (Iran H), Véd (Védrantais), Sol (Solartur), P45 

(PMR 45), W29 (WMR 29), E47 (Edisto 47), PI41 (PI 414 723), P5 (PMR 5), PI12 

(PI 124 112), MR1 (MR-1), Nobl (Nantais oblong). 

No. 

IS* 

maxima. On these hosts both CPM species with the highest number of 

races were detected. Cucumis sativus was the species least frequently 

infected by CPMs (Lebeda and Sedláková, 2004; Lebeda et al., 2004, 

2006) (Table 4). Cucumis melo, Cucurbita foetidissima, and Lagenaria 

siceraria were very rare hosts of CPMs, and only one isolate of Px 

from each host was obtained (Table 4). 


Table 2. Races of Podosphaera xanthii identified in the Czech 

Republic in the years 2000–2004. 


Reaction patterns of P. xanthii isolates 


R S S R R R R R R R S A 1 

R S S R R S R R R R S D R 1 

R S S R R S S R R R S D 1 

R S S S R R S R R S S CH 1 

R S S S R R S S R R S R1 1 

S R S R R R S R R R S I 1 

S S S R R R R R R R S 1 4 

S S S R R R R S R R S K 2 

S S S R R R S R R R S B 7 

S S S R R R S R S S S C 1 

S S S R R R S S R R S R2 1 

S S S R R S R S R R S R3 1 

S S S R R S R R R R S J 1 

S S S R R S S R R R S B R 4 

S S S R R S S S R R S L 1 

S S S R S S S R R R S M 1 

S S S S R R S S R R S 3 1 

S S S S R S R R R R S N 2 

S S S S R S R S R R S O 1 

S S S S R S S S R R S E 3 

S S S S R S S S S R S P 1 

S S S S R S S S R S S R4 1 

S S S S R S S S S S S H 1 

S S S S het S S R R R S 2US 2 

S S S S S S S S R R S G 4 

S S S S S S S S S S S F 1 

*IS = isolates; het = heterogenous reaction; R = resistant reaction; S = susceptible 

reaction. 

a Genotypes of Cucumis melo: IrH (Iran H), Véd (Védrantais), Sol (Solartur), P45 

(PMR 45), W29 (WMR 29), E47 (Edisto 47), PI41(PI 414 723), P5 (PMR 5), PI12 

( PI 124 112), MR1 (MR-1), Nobl (Nantais oblong). 

No. 

IS* 


Table 3. Virulence of Golovinomyces cichoracearum and 

Podosphaera xanthii isolates in the years 2000–2004. 

Category of isolate* 

No. of 

isolates 

% of all 

isolates 

No. of 

races 

Golovinomyces cichoracearum 

Low virulence (1–4) 17 10 5 

Medium virulence (5–7) 55 32 31 

High virulence (8–11) 100 58 27 

Total 

Podosphaera xanthii 

172 100 63 

Low virulence (1–4) 7 15 4 

Medium virulence (5–7) 28 61 16 

High virulence (8–11) 11 24 6 

Total 46 100 26 

*1–11 = Number of infected differentials. 

Table 4. Frequency of Golovinomyces cichoracearum and 

Podosphaera xanthii races on different host species of Cucurbitaceae 

in the years 2000–2004. 

G. cichoracearum P. xanthii 

Host species 

No. of 

isolates 

No. of 

races 

No. of 

isolates 

No. of 

races 

Cucumis sativus 13 9 5 5 

Cucumis melo - - 1 1 

Cucurbita pepo 104 50 24 18 

Cucurbita maxima 


55 27 14 10 

foetidissima - - 1 1 

Lagenaria siceraria - - 1 1 


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INDIVIDUAL AND POPULATION ASPECTS OF 

INTERACTIONS BETWEEN CUCURBITS AND 

PSEUDOPERONOSPORA CUBENSIS: 

PATHOTYPES AND RACES 

A. Lebeda 1, M.P. Widrlechner 2 , and J. Urban 1 

1 Palacký University in Olomouc, Faculty of Science, Department of 

Botany, Šlechtitelů 11, 783 71 Olomouc, Czech Republic 

2 USDA-Agricultural Research Service, North Central Regional Plant 

Introduction Station, Iowa State University, Departments of Agronomy 

and Horticulture, Ames, Iowa 50011-1170 

ADDITIONAL INDEX WORDS. Cucurbitaceae, cucurbit downy mildew, hostpathogen 

specificity, race-specific resistance, pathogenicity, genetic structure 

ABSTRACT. This paper reviews the current state of knowledge regarding 

variation in interactions between Cucurbitaceae and Pseudoperonospora 

cubensis as a backdrop for the development and use of systems to characterize 

pathogenicity at the individual and population levels. Host-parasite specificity 

and interactions between Cucurbitaceae and P. cubensis exhibit significant 

variation on both the individual and population level. However, our 

phytopathological and genetic knowledge of the interactions between individual 

P. cubensis isolates and a broad range of accessions of most important genera of 

cultivated cucurbits (e.g., Cucumis, Cucurbita, Citrullus) remains limited. 

Recently, an improved differential set of cucurbit accessions was developed to 

characterize pathogenic variability (pathotypes) among P. cubensis isolates 

(Lebeda and Widrlechner, 2003). That set included 12 genotypes from six 

genera (Benincasa, Citrullus, Cucumis, Cucurbita, Lagenaria, and Luffa) and is 

now being used for pathotype differenatiation. Nevertheless, we have reasons to 

believe that these differentials are incomplete. Data on patterns of pathogenic 

variation are available only for certain countries (e.g., the Czech Republic, 

India, Israel, Japan, and the USA), but even those studies are typically based on 

individual isolates, not populations. Some highlights and future trends 

regarding the development of new differential sets are suggested. For example, 

recent research in the Czech Republic demonstrated how data at the population 

level can contribute to elucidating temporal and spatial pathogen distribution 

and dynamics, as well as to clarifying host-pathogen interactions. It is also 

important to consider the practical application of these data in resistance 

breeding and disease management. We conclude by proposing some ideas to 

promote research and collaboration on these topics. 

This research was supported by grants MSM 6198959215 and MSM 153100010, QD 

1357; and by the National Programme of Genepool Conservation of Microorganisms 

and Small Animals of Economic Importance of the Czech Republic. The assistance 

of Dr. Charles Block in locating references is much appreciated. 


P 

seudoperonospora cubensis (cucurbit downy mildew) is one of 

the most important foliar pathogens infecting cucurbits (Palti 

and Cohen, 1980; Thomas, 1996; Lebeda and Widrlechner, 

2003). It is widely distributed throughout the world and can inflict 

major production losses in both open-field and protected culture 

(Lebeda and Widrlechner, 2004). 

There are about 60 cucurbit species known as hosts of P. cubensis 

(Palti and Cohen, 1980; Lebeda, 1990, 1992a, 1999; Lebeda and 

Widrlechner, 2003). However, most of the published information on 

its host range and specificity originates from Asia and the USA (Bains 

and Sharma, 1986; Cohen et al., 2003; Thomas et al., 1987), with 

relatively little information obtained from Europe (Lebeda, 1999; 

Lebeda and Gadasová, 2002; Lebeda and Urban, 2004a,b, 2006) or 

other parts of the world. Although our understanding of intraspecific 

variation and genetics in the Cucurbitaceae -Pseudoperonospora 

cubensis pathosystem is rather limited, given the damage that this 

pathogen can cause, it is extremely important from both the theoretical 

and practical perspectives (Lebeda and Widrlechner, 2003, 2004). This 

information is crucial to the development of effective differential sets 

of Cucurbitaceae for the characterization of P. cubensis variability 

(Lebeda and Widrlechner, 2003). 

Pseudoperonospora cubensis is characterized by large variation in 

pathogenicity, which was recently reviewed by Lebeda and 

Widrlechner (2003). Pathogenicity of P. cubensis, on the basis of 

individual isolates, has been characterized in India (Bains and Sharma, 

1986), Israel (Cohen et al., 2003; Thomas et al., 1987), Japan and the 

USA (Thomas et al., 1987), and recently in the Czech Republic 

(Lebeda, 1991, 1999; Lebeda and Gadasová, 2002; Lebeda and Urban, 

2004a,b, 2006), leaving much of its geographic range unstudied. These 

studies solely defined pathotypes (Lebeda and Widrlechner, 2003), not 

races. The main reason for this situation is the lack of differentials to 

distinguish among races on the most important Cucurbitaceae host 

taxa (e.g., Cucumis, Cucurbita, Citrullus). Recently, the first detailed 

studies to focus on the variation and dynamics of pathogenicity of P. 

cubensis at the population level were conducted in the Czech Republic 

(Lebeda and Urban, 2004a,b, 2006). However, comparable studies are 

needed from other parts of Europe, the USA, and Asia. 

The aims of this paper are to review and critically consider the 

current state of knowledge on host-parasite (Cucurbitaceae-P. 

cubensis) specificity and interactions and to suggest future directions 

for basic and applied research in this area. Our primary focus is on the 

characterization and differentiation of P. cubensis pathogenic 

variability, at both the pathotype and race level. 


General Aspects of Host-Pathogen Specificity and 

Variation from the Viewpoint of Characterization of 

P. cubensis Pathogenicity 

In host plant-downy mildew interactions, there is often a very clear 

expression of reaction, i.e., susceptibility (+) or resistance (-). This 

binary system can serve as a simple classification system for 

pathotypes or races, based on the reaction patterns of differential host 

genera, species, and/or genotypes (Lebeda and Jendrulek, 1987). Each 

differential-host genotype may differ by presence of resistance alleles, 

which interact with corresponding pathogen avirulence genes. In 

investigating downy mildew pathogenic variability, the following 

issues are of importance (Lebeda & Schwinn, 1994): (1) Pathogen and 

host phenotypes (host range, virulence, and specificity); (2) pathogen 

and host genetics; and (3) genetic models to explain host-pathogen 

interactions. 

The most recent review of P. cubensis variation and its 

Cucurbitaceae host genera was presented by Lebeda and Widrlechner 

(2003). Data presented therein clearly demonstrated the existence of 

distinct physiological forms of P. cubensis, i.e., pathotypes and races 

(Table 1). On the basis of an initial set of differentials (Thomas et al., 

1987; Table 2) and new experimental data, the authors develepod an 

improved set of Cucurbitaceae differentials for more detailed 

determination of pathotypes (Table 3), including the creation of a 

tetrade coding system for precise pathotype description (Table 4) 

(Lebeda and Widrlechner, 2003). That review also outlined the most 

important reasons for a more detailed examination of host and 

pathogen variation from the viewpoint of development of specific 

differential sets for P. cubensis. 

Host-Plant Resistance Variation and Possibilities 

for the Development of Race Differential Sets for 

the Determination of P. cubensis 

There are at least three candidate genera (Cucumis, Cucurbita, and 

Citrullus) for the development of differential sets for race 

differentiation of P. cubensis. All these genera are known as natural 

hosts of P. cubensis (Lebeda and Widrlechner, 2003; Palti and Cohen, 

1980). 

CUCUMIS. The genus Cucumis is rather diverse, encompassing 32 

species (Kirkbride, 1993). Of these, in addition to the two 

economically important species, cucumber (C. sativus L.) and melon 

(C. melo L.), two additional species, C. anguria L. (“West Indian 

gherkin”) and C. metuliferus E. Meyer ex Naudin (“African horned 

cucumber” or “jelly melon”), are also commercially exploited for fruit 


production (Baird and Thieret, 1988; Morton, 1987). Other wild 

species originating mostly from arid and/or semi-arid regions of Africa 

are cultivated as ornamental plants, e.g., C. dipsaceus Ehrenberg ex 

Spach (“hedgehog gourd”) and C. myriocarpus Naudin (“gooseberry 

gourd”) (Kirkbride, 1993). A comprehensive review of recent 

knowledge about Cucumis germplasm and its genetic variation can be 

consulted for more information (Lebeda et al., 2006). 

Table 1. Survey of data on the occurrence of pathotype- and racespecificity 

in interactions between key Cucurbitaceae host genera and 

P. cubensis. 

Hostparasite 

specificity Pathotype Race 

Data 

Data 

Host taxon availability References availability References 

Cucumis 

3-6,8,9,12, 

1, 4-6,8,9,11sativus 

-? 13 -? 13,14 

Cucumis 

2,3,5,6,9,12, 

melo 

Cucumis 

+ 2,3,5,6,7,12,13 + 13 

spp. 


+ 3,5,9 -? 3,5,9 

pepo 


+ 6,7,9,10,12,13 + 6,7,9,10,12,13 

maxima 


+ 6,9,10,13 + 6,9,10,13 

foetidissima 


+ 10 + 10 

spp. 

Citrullus 

+ 15 + 15 

lanatus + 6,9,12,13 + 6,9,12,13 

- = pathotype- or race-specificity absent; + = pathotype- or race-specificity present; 

? = data not available or confirmed. 

References (for full references see Literature Cited): 

(1) Horejsi et al. (2000); (2) Lebeda (1991); (3) Lebeda (1992a); (4) Lebeda (1992b); 

(5) Lebeda (1999); (6) Lebeda and Gadasová (2002); (7) Lebeda and Kristkova 

(1993, 2000); (8) Lebeda and Prasil (1994); (9) Lebeda and Widrlechner (2003); (10) 

Lebeda and Widrlechner (2004); (11) Shetty et al. (2002); (12) Thomas (1982); (13) 

Thomas et al. (1987); (14) Wehner and Shetty (1997); (15) Wessel-Beaver (1993). 


Critical analysis of the scientific literature demonstrated (Lebeda 

and Widrlechner, 2003) that available C. sativus germplasm accessions 

and recent commercial cucumber cultivars do not possess effective 

sources of resistance, and experimentally verified, race-specific 

interactions are unknown. It was concluded that C. sativus genotypes 

could be used in differential sets only as a susceptible control (Tables 

1–3). Nevertheless, it was suggested to continue investigations of this 

host-parasite interaction because there may still be opportunities to 

discover race-specific resistance in this species (Lebeda and 

Widrlechner, 2003). 

The natural range of C. melo has yet to be determined conclusively 

(Lebeda et al., 2006). Prior to its domestication, it may have been 

limited to Africa or may have reached the Near East or perhaps farther 

east into Asia (Bates and Robinson, 1995). The center of diversity and 

perhaps of the origin of the principal melons of world commerce (i.e., 

the C. melo Inodorus and Cantalupensis groups) is located in the Near 

East and adjacent central Asian regions (Jeffrey, 1980). In contrast to 

C. sativus, recent reports characterize C. melo as displaying 

considerable intraspecific variation at various levels, i.e., 

morphological, resistance, genetic, and molecular (Pitrat et al., 2000; 

Stepansky et al., 1999). Cucumis melo is the only species in the genus 

where the phenomenon of pathotype- and race-specificity of P. 

cubensis is relatively well known (Lebeda and Widrlechner, 2003). 

Recent, more detailed research on about 100 accessions of C. melo 

maintained in the US National Plant Germplasm System’s collection 

held at the USDA-ARS North Central Regional Plant Introduction 

Station in Ames, Iowa, is yielding new information on host-pathogen 

interactions (i.e., identifying various and unknown pathotype-specific 

reaction patterns) (Lebeda and Widrlechner, unpublished data). 

Among the 100 accessions, susceptible responses were most 

common, but there were also very clear and diverse race-specific 

reaction patterns, giving an opportunity for the development of a 

differential set composed solely of C. melo accessions, resembling the 

system developed for race differentiation of cucurbit powdery mildew 

(Bardin et al., 1999; McCreight, 2006; Pitrat et al., 1998). 

Beyond the cultivated Cucumis species, there are about 30 wild 

Cucumis species, mostly native to Africa (Kirkbride, 1993). However, 

Lebeda and Widrlechner (2003) concluded that wild Cucumis species 

are unlikely to haveplayed an important role in the differentiation of P. 

cubensis pathotypes and races. More research is needed in this area. 

CUCURBITA. The genus Cucurbita is native exclusively to the New 

World, but has been widely cultivated in the Old World since the 


1500s (Paris, 1989, 2001a,b). It is not closely related to other cucurbit 

genera (Merrick, 1995). 

The genus is now thought to consist of between 12 and 15 species, 

with 5 regularly cultivated (Lira-Saade, 1995; Sanjur et al., 2002). The 

current state of knowledge about the origin, evolution, and genetic 

variation of domesticated Cucurbita species was recently reviewed 

(Lebeda et al., 2006). 

Host-parasite specificity among Cucurbita species and various P. 

cubensis isolates is a complex phenomenon deserving closer analysis 

(Lebeda and Křístková, 1993; Lebeda and Widrlechner, 2003). To 

date, fairly limited evaluation of Cucurbita species for resistance to P. 

cubensis has been conducted (Lebeda and Widrlechner, 2003, 2004). 

Only a few papers describing the variation of interactions between 

Cucurbita spp. and P. cubensis, and their specificity, are available. An 

evaluation of the responses of 60 cultivars of C. pepo to three 

pathotypes of P. cubensis suggested that host-parasite specificity was 

controlled by race-specific resistance factors (Lebeda and Křístková, 

1993). 

Table 2. Differential set of Cucurbitaceae and differentiation of P. 

cubensis pathotypes (adapted from Thomas et al., 1987). 

Groups of P. cubensis isolates (country) 

M1, M2 

(Japan) 

Host plant C1 C2 83, 85 C T 

differential taxon (Japan) (Japan) (Israel) (US) (US) 


C. melo var. 

+ + + + + 

reticulatus* 

C. melo var. 

+ + + + + 

conomon 

C. melo var. 

- + + + + 

acidulus - - + + + 

Citrullus lanatus - - - + + 

Cucurbita spp. 

Pathotype 

- - - - + 

designation 1 2 3 4 5 

* also known as C. melo var. cantalupensis Naudin. 

- = incompatible to slightly compatible response; + = highly compatible response. 


Pathotype-specificity has also been described for C. maxima and 

C. moschata (Bains and Sharma, 1986; Thomas et al., 1987). More 

recently, a study was initiated to evaluate a set of wild and weedy 

Cucurbita populations (97 accessions representing 10 species and 14 

taxa), representing a wide phylogenetic cross-section of the genus with 

relationships to all five domesticated species, for their responses to 

diverse P. cubensis pathotypes (altogether 11 isolates representing 9 

pathotypes) (Lebeda and Widrlechner, 2004). That study reported 

extensive variation in the responses of these Cucurbita accessions to 

11 isolates of P. cubensis. In total, 57 different reaction patterns were 

recorded with 13 accessions completely resistant, 12 accessions 

completely susceptible, and 32 accessions expressing pathotype/racespecific 

patterns. These data demonstrated that most of the studied 

taxa displayed pathotype- and/or race-specificity. The most variable 

taxa included C. argyrosperma, C. foetidissima, C. okeechobensis, and 

C. pepo (Lebeda and Widrlechner, 2004). The screening of about 50 

accessions of different C. pepo morphotypes for resistance to different 

P. cubensis pathotypes, as a basis for more detailed understanding of 

this host-pathogen interaction, is now underway (Lebeda and Paris, 

unpublished results). Collectively, these data can serve as a strong 

foundation for the future development of an improved differential set 

of Cucurbita spp. for race determination of P. cubensis. 

CITRULLUS. Four species of Citrullus (C. lanatus [syn. C. 

vulgaris], C. colocynthis, C. ecirrhosus, and C. rehmii) have been 

generally recognized (Lebeda et al., 2006). Two species (C. lanatus 

and C. colocynthis) are widely distributed, and the others are restricted 

to the desert regions of Namibia (Jarret and Newman, 2000; Levi et 

al., 2000). Citrullus lanatus and C. colocynthis are natural hosts of P. 

cubensis (Palti and Cohen, 1980). The first clear pathotype/racespecific 

reaction pattern between Citrullus spp. and P. cubensis was 

identified in C. lanatus (Thomas et al., 1987). This initial report was 

verified by inoculation with some European isolates (Lebeda and 

Gadasová, 2002; Lebeda and Urban, 2004a,b, 2006). Reactionspecificity 

has not been identified in other Citrullus species. A broader 

screening of Citrullus species germplasm collections should be 

conducted to obtain information about patterns of variation for 

resistance (Lebeda and Widrlechner, 2003). 

As noted above, there are at least three candidate groups of 

cucurbitaceous host plants (Cucumis, Cucurbita, and Citrullus) that 

could be used for the development of race differential sets for P. 

cubensis. Of course, to accomplish this end we need more information 

about the phenotypic expression of host-pathogen variation, 

mechanisms responsible for resistance, and their genetic bases. 


Table 3. Differential set of cucurbit taxa for determination of P. 

cubensis pathotypes (adapted from Lebeda and Widrlechner, 2003). 

Cucurbitaceae 

Accession 

number 

Cultivar 

name 

Dif. Differential 

No. Value Taxon 

Donor EVIGEZ 

H39- Marketer 

1 1 Cucumis sativus 

0121 430 

C. melo subsp. PI H40- Ananas 

2 2 melo 

292008 1117 Yoqneám 

3 4 

4 8 

5 1 

6 2 

C. melo subsp. 

agrestis var. 

conomon 

C. melo subsp. 

agrestis var. 

acidulous 

Cucurbita pepo 

subsp. pepo * 

C. pepo subsp. 

ovifera var. texana 

CUM 

238/1974 

PI 

200819 

PI 

171622 

PI 

614687 

H40- 

0625 

H40- 

0611 

H42- 

0117 

H42- 

0130 

Baj-Gua 

Dolmalik 

C. pepo var. PI H42- 

7 4 fraterna ** 532355 0136 

H42- 

8 8 Cucurbita maxima 

0137 

H37- 

Goliáš 

9 1 Citrullus lanatus 

0008 

H15- 

Malali 

10 2 Benincasa hispida BEN 485 0001 

H63- 

11 4 Luffa cylindrical 

0010 

Lagenaria 

H63- 

12 8 siceraria 

0009 

EVIGEZ - Czech genebank number. 

*taxonomy of Cucurbita species follows recent correspondence with J.H. Wiersema 

(USDA-ARS, Beltsville, USA). 

**originally described as Cucurbita fraterna (Lebeda and Gadasová, 2002). 


Variation in the Pathogenicity of 

Pseudoperonospora cubensis 

INDIVIDUAL LEVEL (PATHOTYPE AND RACE DETERMINATION). 

Variability in pathogenicity (virulence) has been described in several 

downy mildew isolates at the level of pathotype or race (Table 5). 

When there is clear expression of susceptibility or resistance, 

classification of pathotypes and races can be based on the patterns of 

these reactions in differential hosts (for pathotypes, mostly at the 

generic level; for races, mostly at the specific level) (Lebeda and 

Schwinn, 1994). However, when relatively good information on host 

variation for specific resistance is available (i.e., a validated genetic 

model explaining host-pathogen interactions), then there is an 

opportunity to move from solely the taxonomic classification of 

pathogen races to the concept of virulence phenotypes, i.e., presence or 

absence of specific virulence/avirulence factors in individual pathogen 

isolates (Lebeda and Schwinn, 1994). For pathotypes, this translates 

into the presence or absence of specific disease phenotypes for each 

differential genotype (e.g., Table 3). 

We can now describe very precisely the pathogenicity of 

individual isolates as a pathotype (as a unique tetrade code [Lebeda 

and Widrlechner, 2003]) (Table 4) and characterize the structure of 

pathogen population at the individual level (Lebeda and Urban, 

2004a,b, 2006). This approach is more understandable and efficient 

than the previous system of pathotype description (Cohen et al., 2003; 

Thomas et al., 1987). 

The development of a comparable system for race differentiation is 

clearly needed, at least for Cucumis melo, the most economically 

important Cucurbita species, and Citrullus lanatus. This system can 

serve as a foundation for future developments in resistance breeding 

and the effective deployment of various resistance alleles and loci for 

coordinated control of pathogen populations. Two control strategies 

that could function as models for P. cubensis management are the use 

of carefully selected multilines or cultivar mixtures (Mundt, 2002) and 

the development of cultivars with pyramided resistance alleles (Castro 

et al., 2003), which may be facilitated through advances in markerassisted 

selection (Servin et al., 2004). 

POPULATION STUDIES. The development of differentiation systems 

provides an opportunity for the more exact description of pathotypes 

or races of P. cubensis and is creating the basis for the application of 

population biology and genetic studies, and for comparative 

investigations of virulence variation in distinct pathogen populations 

(Lebeda, 1982). The recent system of description of P. cubensis 

pathotypes (Lebeda and Widrlechner, 2003) is providing a good base 


Table 4. Examples of different P. cubensis pathotypes (expressed in 

tetrade codes; see Lebeda and Widrlechner, 2003) collected in the 

Czech Republic in 2001–2004. 

Level of 

pathogenicity/ 

Year/no. of isolates 

pathotype 

Low 

pathogenicity 

(PF = 1–4) 

2001 2002 2003 2004 

3.0.12. 1 0 0 0 

11.0.8. 

Medium 

pathogenicity 

(PF = 5–8) 

1 0 0 0 

15.2.10. 1 5 0 0 

15.10.10. 

High 

pathogenicity 

(PF = 9–12) 

1 10 0 0 

15.14.10. 1 8 4 2 

15.14.11. 1 0 11 4 

15.14.14. 4 6 6 3 

15.15.11. 0 0 2 10 

15.15.14. 2 1 6 1 

15.15.15. 1 0 6 2 

PF = pathogenicity factor (for explanation see Population Studies heading). 

for such studies on the level of frequencies of “pathogenicity factors” 

(each hypothetical pathogenicity factor [PF] is able to overcome 

resistance of one differential genotype [see Table 3]). PF frequencies 

(0–100%, or 0.0–1.0) can express very clearly the pathogen population 

structure (Figure 1), and their spatial and temporal changes (Lebeda 

and Urban, 2004a,b, 2006). Elaboration of this system on the level of 

races (i.e., specific virulences to specific genotypes of host species), 

could bring a new methodological approach for this plant pathosystem, 

i.e., investigations of the genetic structure of virulence of P. cubensis 

populations. This approach has been very beneficial for practical 

breeding and cultivar use in some other vegetable crops, such as lettuce 

(Lebeda, 1981; Lebeda and Zinkernagel, 2003). 

We are confident that similar benefits could be gained for cucurbit 

breeding and research. Detailed virulence surveys on the local, 


national, and international levels are very important for elucidating 

pathogen population structure and behaviour (Lebeda, 1982), including 

the understanding of pathogen virulence tactics, strategy, and evolution 

(McDonald and Linde, 2002). 

Frequency (%) 

Fig. 1. Frequency of pathogenicity factors (see Table 3) in P. cubensis popula- 

tions in the Czech Republic in the years 2001–2004. 

Table 5. Survey of recent data on the determination of P. cubensis 

pathotypes and races in various countries. 

Pathogenicity 

category 

Pathotype Race 

Data 

References 

Data 

References 

Country 

100 

80 

60 

40 

20 

0 

1 2 3 4 5 6 7 8 9 10 11 12 

Pathogenicity factor 

avail. 

avail. 

China ? +? 10 

Czech Republic + 5,6,7,8,9 + 5,6,7,8,9 

Bulgaria +? 1 +? 1 

India + 2 +? 2,10 

Israel + 3,11 + 3,11 

Japan + 11 +? 11 

Poland ? +? 10 

USA + 10 + 4,10,11 

Others (FR, NL, SP) * + 6 + 6,8 

- = pathotype or race absent; + = pathotype or race present; ? = data not available or 

not experimentally confirmed; * only one isolate determined. 

References (for full references see Literature Cited): 

(1) Angelov et al. (2000); (2) Bains and Sharma (1986); (3) Cohen et al. (2003); (4) 

Horejsi et al. (2000); (5) Lebeda (1999); (6) Lebeda and Gadasová (2002); (7) 

Lebeda and Urban (2004a,b, 2006); (8) Lebeda and Widrlechner (2003); (9) Lebeda 

and Widrlechner (2004); (10) Shetty et al. (2002); (11) Thomas et al. (1987). 


2001 

2002 

2003 

2004

Conclusions and Recommendations 

In conclusion, most of the summary comments and 

recommendations made by Lebeda and Widrlechner (2003) remain 

timely today: 

1. It is evident that P. cubensis is an extremely variable 

pathogen from the viewpoint of host specificity/pathogenicity 

and virulence. 

2. Pathogenicity must be clearly described, first at the 

pathotype and race levels, and then at the level of virulence 

phenotypes. 

3. To this end, internationally recognized and accepted 

differential host genotypes (at the level of genera and species 

for pathotype differentiation) and differential host lines (at the 

species level within a single genus for race differentiation) 

must be defined, conserved, and made widely available to the 

research and breeding community. 

4. There must be a clear and standardized system for P. 

cubensis pathotype description (now available [Lebeda and 

Widrlechner, 2003]), and for race determination (under 

development for Cucumis melo and Cucurbita spp. [Lebeda et 

al., unpublished]). 

5. Differential host lines for race differentiation must be 

sufficiently genetically characterized (i.e., genetics of racespecific 

resistance [race-specific resistance genes and/or 

factors]). 

6. The above-mentioned systems should be incorporated into 

basic research (e.g., host-pathogen interactions, resistance 

mechanisms, population genetic studies, genetics of virulence, 

etc.), and also broadly applied in practical resistance breeding 

of the world’s most important cucurbit crops. 


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EVALUATING ZUCCHINI YELLOW MOSAIC 

VIRUS RESISTANCE IN WATERMELON 

Kai-Shu Ling and Amnon Levi 

USDA-ARS, U.S. Vegetable Laboratory, Charleston, SC 29414 

Nihat Guner and Todd C. Wehner 

Department of Horticultural Science, North Carolina State University, 

Raleigh, NC 27695 

ADDITIONAL INDEX WORDS. Citrullus, inheritance, recessive gene, SRAP 

ABSTRACT. Zucchini yellow mosaic virus (ZYMV) resistance was previously 

identified in Citrullus lanatus var. lanatus U.S. Plant Introduction (PI) 595203, 

and inheritance studies indicated that the ZYMV resistance is conferred by a 

single recessive gene. In this study we examined the inheritance of resistance to 

the original ZYMV Florida strain (ZYMV-FL) using F1, F2, and reciprocal BC1 

populations derived from PI 595203 and the watermelon cultivar ‘New 

Hampshire Midget’ (NHM). The results confirmed that PI 595203 is resistant 

to ZYMV-FL and the resistance was conferred by a single recessive gene. A 

study was initiated to identify DNA markers closely linked to the gene 

conferring the ZYMV resistance. Genomic DNA was isolated from the parents 

(PI 595203 and NHM) as well as their F1, F2, and BC1 plants. Two hundred and 

seventy sequence-related amplified polymorphism (SRAP) markers were tested 

for polymorphism between the parents (PI 595203 and NHM). Of these, 135 

markers appeared to be polymorphic, and are being used (with the mapping 

BC1 and F2 populations) for constructing a genetic linkage map and locating the 

ZYMV-resistance gene locus. 

Z 

ucchini yellow mosaic virus (ZYMV), Watermelon mosaic virus 

(WMV), Papaya ringspot virus watermelon strain (PRSV-W), 

and Cucumber mosaic virus (CMV) are considered major 

viruses of cucurbits. Provvidenti et al. (1984) identified two major 

ZYMV strains in the United States and showed that although ZYMV- 

CT incites more severe disease symptoms in watermelon, its 

distribution is limited to the Northeast. On the other hand, ZYMV-FL 

is the most prevalent strain in cucurbit crops in North America. 

Therefore it is important to first develop watermelon cultivars that are 

resistant to ZYMV-FL. Provvidenti (1991) identified four watermelon 

(C. lanatus) landraces (PI 482322, PI 482299, PI 482261, and PI 

482308) that are resistant to ZYMV-FL. Inheritance studies indicated 

that a single recessive gene (zym) is conferring ZYMV-FL resistance 

in PI 482261 (Provvidenti, 1991). Boyhan et al. (1992) was the first to 

identify cv. Egun (apparently PI 595203) with strong resistance to 

ZYMV-FL. Lecoq et al. (1998) also identified other PI accessions 


with resistance to ZYMV. Guner (2004) reevaluated a large PI 

collection of watermelon for ZYMV resistance and identified a few 

more PIs (including PI 595203). Xu et al. (2004) examined ZYMV- 

China Strain (ZYMV-CH) and WMV resistance in watermelon (PI 

595203) and determined that the resistance to ZYMV-CH is also 

conferred by a single recessive gene (zym-CH). The objective of this 

study is to evaluate the inheritance of resistance to ZYMV-FL in 

watermelon, and identify molecular markers closely linked to the 

resistance gene. These markers will be useful in marker-assisted 

selection (MAS) to incorporate the resistance into watermelon 

cultivars. 


HOST PLANT AND GENETIC MATERIALS. F1, F2, and reciprocal 

BC1 populations, derived from a cross between PI 595203 (ZYMVresistant) 

and cv. New Hampshire Midget (NHM) (ZYMVsusceptible), 

were developed by Guner and Wehner at North Carolina 

State University (Guner, 2004). Seeds were germinated in an insectfree 

greenhouse with temperature of 18–30C and 14–16 hours of 

natural lighting period at the U. S. Vegetable Laboratory in 

Charleston, SC. The two youngest leaves were collected from young 

seedlings (4–5 leaf stage) for DNA isolation. Plants were then 

mechanically inoculated with the ZYMV-FL culture. 

VIRUS ISOLATE AND INOCULATION. The ZYMV-FL culture is 

derived from the original ZYMV-FL strain isolated by Provvidenti 

(1984). The virus was propagated and maintained on Gray zucchini 

squash. Virus inoculum was prepared by macerating virus-infected 

leaves (1:5 w/v) in 0.01M phosphate buffered saline, pH 7.4, with 

mortar and pestle. Seedlings were inoculated by lightly dusting the 

leaves with carborundum then mechanical rubbing with acotton Q-tip 

soaked in the virus inoculum. Application involved several circular 

motions until the entire leaf was covered. Excess carborundum was 

rinsed with water and the inoculated seedlings were placed under the 

shade for a few hours to minimize direct sunlight damage to the newly 

inoculated leaves. Three weeks after inoculation, plants were 

evaluated for virus symptoms (Figure 1). Virus disease severity was 

rated as: 0 = no symptoms; 1= slight mosaic on leaves; 2 = mosaic 

patches and/or necrotic spots on leaves; 3 = leaves near apical 

meristem are slightly deformed, with yellow color and reduced leaf 

size; 4 = apical meristem has deformed shape and with mosaic 

appearance; 5 = extensive mosaic appearance and severe leaf 

deformation, or plant is dead (Xu et al., 2004). 


ELISA. Enzyme linked immunosorbent assay (ELISA) was 

performed according to the manufacturer’s instructions (BioReba, 

Switzerland). Microtiter plates were first coated with 1μg/ml of 

ZYMV antibody, and virus particles were trapped after incubating the 

tissue extract on the coated plates. Leaf extract was prepared by 

processing the collected leaf tissue with a tissue homogenizer Homex- 

6 (BioReba) in tissue extraction buffer (1:20 w/v). The alkaline 

phosphatase conjugated antibody to ZYMV was then added to the 

plate. Finally, yellow color developed in positive samples due to 

enzyme-substrate hydrolysis was measured with an ELISA reader. A 

sample with absorbance value of at least twice the mean health 

controls (OD at 405nm) is regarded as positive. 

Fig. 1: Comparison of the resistant PI 595203 (left) and the susceptible cv. New 

Hampshire Midget (right) to Zucchini yellow mosaic virus infection. 

SEQUENCE-RELATED AMPLIFICATION POLYMORPHISM. Genomic 

DNA was isolated from the parent plants (PI 595203 and NHM), and 

from their F1, F2, and BC1 plants as described by Levi et al. (2004). 

Two hundred and seventy sequence-related amplified polymorphism 

(SRAP) markers were tested for polymorphism between the parents 

(PI 595203 and NHM) as described by Levi et al. (2006). 



All 19 F1 plants from the cross between NHM and PI 595203 were 

susceptible, indicating that the ZYMV-FL resistance in PI 595203 is 

controlled by a recessive gene. The F2, BC1R, and BC1S segregation 

data were tested against the expected ratio for a single recessive gene 

and to confirm the observations by Provvidenti (1991) and Xu et al. 

(2004). Interestingly, the F2 segregation data (71S:10R) did not 

support the expected 3:1 (susceptible: resistance) ratio (Table 1). The 

cause for this skewed characterization toward susceptibility to ZYMV- 

FL in the F2 population was not determined. However, BC1S 

population was all susceptible and BC1R population was segregating 

in 1:1 (69S:61R) ratio supporting a single recessive gene. 

Of the 270 SRAP primer pairs tested, 135 produced polymorphism 

between the parents PI 595203 and NHM. These polymorphic markers 

are being used with mapping BC1 and F2 populations for constructing 

a genetic linkage map and locating the ZYMV-resistance gene locus. 

Table 1. Single locus goodness-of-fit-test for ZYMV-FL resistance in 

watermelon (‘New Hampshire Midget’ x PI 595203). 

Generation Total Susceptible Resistance Expected ratio X 2 df P-value 

NHM 12 12 0 

PI 595203 11 0 11 

F1 19 19 0 

F2 81 71 10 3:1 7.78 1 0.005 

BC1R 130 69 61 1:1 0.49 1 0.5 

BC1S 20 20 0 

This study confirmed that a single recessive gene confers 

resistance to ZYMV-FL in PI 595203. The slight deviation from the 

expected 3:1 ratio in the F2 population may be a result of preferential 

segregation that may occur in wide crosses between Citrullus PIs and 

watermelon cultivars (Levi et al. 2006). The preliminary SRAP 

analysis revealed sufficient polymorphism between NHM and PI 

595203 that might be useful in identifying a DNA marker linked to the 

ZYMV-resistance gene locus. The marker will be useful in markerassisted 

selection (MAS) to incorporate the resistance into watermelon 

cultivars. 



Boyhan, G., J. D. Norton, B. J. Jacobsen, and B. R. Abrahams. 1992. Evaluation of 

watermelon and related germplasm for resistance to Zucchini yellow mosaic 

virus. Plant Dis. 76:251–252. 

Guner, N. 2004. Papaya ringspot virus watermelon strain and Zucchini yellow 

mosaic virus resistance in watermelon. PhD Diss., Department of Horticultural 

Sciences, North Carolina State Univ., Raleigh, NC. 

Lecoq, H., G. Wisler, and M. Pitrat. 1998. Cucurbit viruses: the classics and the 

emerging, p. 126–142. In: J. D. McCreight (ed.). Cucurbitaceae 1998: 

evaluation and enhancement of cucurbit germplasm. ASHS Press, Alexandria, 

VA. 

Levi, A., C. E. Thomas, M. Newman, O. U. K. Reddy, X. Zhang, and Y. Xu. 2004. 

ISSR and AFLP markers sufficiently differentiated among American 

watermelon cultivars with limited genetic diversity. J. Amer. Soc. Hort. Sci. 

129:553–558. 

Levi, A., C. E. Thomas, T. Trebitsh, A. Salman, J. King, J. Karalius, M. Newman, O. 

U. K. Reddy, Y. Xu, and X. Zhang. 2006. An extended linkage map for 

watermelon based on SRAP, AFLP, SSR, ISSR and RAPD markers. J. Amer. 

Soc. Hort. Sci. (In press.) 

Provvidenti, R. 1991. Inheritance of resistance to the Florida strain of Zucchini 

yellow mosaic virus in watermelon. HortSci. 26:407–408. 

Provvidenti, R., D. Gonsalves, and H. S. Humaydan. 1984. Occurrence of Zucchini 

yellow mosaic virus in cucurbits from Connecticut, New York, Florida, and 

California. Plant Dis. 68:443–446. 

Xu, Y., D. Kang, Z. Shi, H. Shen, and T. Wehner, T. 2004. Inheritance of resistance 

to Zucchini yellow mosaic virus and Watermelon mosaic virus in watermelon. 

J. Hered. 95:498–502. 


OCCURRENCE OF FUNGICIDE RESISTANCE 

IN PODOSPHAERA XANTHII AND IMPACT 

ON CONTROLLING CUCURBIT POWDERY 

MILDEW IN NEW YORK 

Margaret Tuttle McGrath 

Department of Plant Pathology, Cornell University 

Long Island Horticultural Research and Extension Center 

3059 Sound Avenue, Riverhead, New York 11901-1098 

ADDITIONAL INDEX WORDS. QoI fungicides, Strobilurin fungicides, DMI 

fungicides, pumpkin 

ABSTRACT. Frequency of QoI-resistant strains of Podosphaera xanthii 

(Castagne) U. Braun & N. Shishkoff was low in commercial production fields at 

the start of powdery mildew development in 2003 (detected in one of five fields), 

but increased greatly to 61–100% in all fields, and was associated with poor 

disease control. Resistant strains were found in nontreated fields at the end of 

the season. Frequency was high at the start of powdery mildew development in 

2004 (32–91%). Strains moderately insensitive to DMI fungicides were detected 

in all fields in 2003 (1–25%) and in 2004 (15–84%) before these fungicides were 

used but the DMIs Nova and Procure provided excellent control when 

evaluated in 2005 where DMI-insensitive strains were present, indicating that 

DMI resistance has not reached a degree that control is affected. Programs 

with QoI, DMI, and protectant fungicides were effective, but not as effective as 

the new fungicide Quintec applied alone. 

P 

owdery mildew is a common disease of cucurbits under field 

and greenhouse conditions in most areas of the world. 

Management is needed to avoid loss in fruit quantity and market 

quality. Mobile fungicides (those with systemic, translaminar, or 

volatile activity) are needed to control powdery mildew on the 

underside of leaves where this disease typically develops best. 

Unfortunately these fungicides tend to be at risk for resistance 

development due to their single-site mode of action. And the cucurbit 

powdery mildew fungus, Podosphaera (sect. Sphaerotheca) xanthii 

(Castagne) U. Braun & N. Shishkoff, has a history of developing 

resistance, beginning with benomyl in the late 1960s, which was one 

of the first cases of fungicide resistance (McGrath, 2001). 

Application of fungicides continues to be the principal practice for 

managing powdery mildew in cucurbit crops, but successful control is 

challenged by development of resistance to key fungicides (McGrath 

2001). While there are varieties with genetic resistance to this disease, 

an integrated program is recommended to reduce selection pressure for 


pathogen strains able to overcome the genetic resistance in the plant as 

well as fungicide resistance. Powdery mildew is the most common 

disease occurring every year throughout the U.S. The pathogen 

develops best on the lower surface (underside) of leaves, thus a 

successful management program necessitates controlling the pathogen 

on the lower as well as the upper surface. It is difficult to deliver 

fungicide directly to the lower surface, even with new nozzle types and 

air-assist sprayers. Consequently, an important component of 

fungicide programs has been fungicides able to move to the lower leaf 

surface. Most of these fungicides are systemic (e.g., Topsin M, 

Nova) or have translaminar activity (e.g., Amistar, Cabrio, 

Flint, Quadris). Some, notably the new fungicide Quintec 

(Fungicide Resistance Action Committee [FRAC] Group 13), have 

high volatility, enabling redistribution from upper to lower leaf 

surfaces. 

Unfortunately, these fungicides effective on lower leaf surfaces 

have been prone to resistance development due to their single-site 

mode of action. Additionally, the cucurbit powdery mildew fungus has 

demonstrated ability to evolve new strains resistant to these fungicides. 

Presence of resistant strains has been associated with control failure. 

With some fungicides, including MBC (methyl benzimidazole 

carbamate) fungicides, aka benzimidazoles (e.g., Topsin M) (FRAC 

Group 1), and QoI (quinone outside inhibiting) fungicides, aka 

strobilurins (e.g., Amistar, Cabrio, Flint, Quadris) (FRAC Group 11), 

this change renders the pathogen strain completely resistant to the 

fungicide (qualitative resistance). With other fungicides, including the 

DMI (demethylation inhibiting) fungicides (Bayleton, Nova, and 

Procure) (FRAC Group 3), pathogen strains exhibit a range in 

fungicide sensitivity depending on the number of genetic changes they 

possess that affect the fungicide’s ability to function (quantitative 

resistance). 

During the 1990s, grower complaints of inadequate control of 

powdery mildew in New York, as well as elsewhere in the U.S., were 

found to be due to resistance to Bayleton (DMI) and Benlate (MBC), 

the only mobile fungicides registered at that time (McGrath et al., 

1996). For a few years after detection of Bayleton resistance, initial 

resistance frequency was found to be sufficiently low that one 

application of this fungicide was effective; however, the proportion of 

the pathogen population that was resistant subsequently increased 

greatly, thus additional applications were ineffective (McGrath, 1996). 

This information combined with efficacy data from fungicide 

evaluation experiments provided the necessary support to obtain 


emergency registration of Quadris and Nova, a new DMI, in some 

states in 1998 and 1999, respectively. These fungicides received full 

federal registration in the U.S. in 1999 and 2000, respectively. Nova 

was effective while Bayleton was not because resistance to the DMIs 

is quantitative and Nova is inherently more active. DMI resistance did 

have some impact on Nova efficacy: the lowest labeled rate was no 

longer as effective as higher rates. Growers were encouraged to use 

high rates to obtain good control as well as to manage further 

development of DMI resistance. Strains of P. xanthii fully resistant to 

Bayleton but controlled by high rates of Nova were called moderately 

insensitive to DMIs. 

The recommendation to growers was to apply Quadris in 

alternation with Nova plus a protectant fungicide on a seven-day 

schedule, beginning before powdery mildew started to develop or, 

preferably, after reaching the action threshold of 1 leaf with symptoms 

out of 50 older leaves examined. Initially QoIs were thought to have 

medium resistance risk and resistance would be quantitative. After 

reports of rapid development of qualitative resistance that rendered 

QoIs ineffective elsewhere in the world, the recommended fungicide 

program was modified to include a protectant with the QoI. Protectants 

are a valuable component of the program because they have low 

resistance risk and control strains resistant to the mobile fungicides. 

QoI resistance was detected in NY and elsewhere in the U.S. in 

2002 when QoIs failed in fungicide-efficacy experiments (McGrath 

and Shishkoff, 2003a). This was three years after federal registration 

and the fourth year that QoIs were used by growers in some states. 

Resistance was detected in the geographically separated east and west 

regions. In the eastern U.S., P. xanthii is thought to survive over 

winter in southern Florida and then move northward with the 

successive cropping seasons. Winds do not move in a southward 

direction often enough to provide the pathogen a means to move back 

down the coast in the fall. It could survive north of Florida as 

ascomata (cleistothecia), but these are not thought to play an important 

role considering cropping practices. 

In 2002 fungicide sensitivity was determined for isolates of P. 

xanthii collected at crop maturity from fungicide-efficacy experiments 

in Georgia (GA), North Carolina (NC) and NY. The maximum 

concentration tolerated by most of the 73 isolates (89%) was either 0.5 

or 100mg/ml of the QoI trifloxystrobin, indicating that resistance was 

qualitative. Four of 9 NY isolates, 19 of 21 GA isolates, and 13 of 15 

NC isolates from plants treated weekly with a QoI fungicide were able 

to grow well on leaf disks treated with 100mg/ml trifloxystrobin. In 

the NY fungicide-efficacy experiment, after two applications, Quadris 


used alone was as effective as Quadris alternated with Nova plus 

sulfur; but at the assessment made eight days later following two more 

applications, powdery mildew was as severe on Quadris-treated leaves 

as on nontreated leaves (McGrath and Shishkoff, 2003a, 2003c). 

Surprisingly, many isolates resistant to QoIs were moderately 

insensitive to DMIs, an unrelated fungicide group: 56% of the NY 

isolates and 90% of the NC isolates. No isolate was found that was 

resistant to QoIs and sensitive to DMIs. This situation constrains 

managing QoI and DMI resistance (McGrath and Shishkoff, 2003b). 

Research was conducted in 2003 to 2005 in NY to (1) examine 

occurrence of fungicide-resistant strains in commercial production 

fields before mobile fungicides for powdery mildew were applied to 

these crops, and (2) examine the impact of fungicide use on resistance 

and of resistance on fungicide efficacy. These goals were achieved by 

monitoring resistance in commercial fields using a seedling bioassay, 

examining powdery mildew control, and assessing fungicide efficacy 

and resistance occurrence in replicated experiments. Preliminary 

reports of the individual experiments have been published and contain 

additional detail on methods and results (McGrath, 2004a, 2004b, 

2005a, 2005b; McGrath and Davey, 2006). 


An in-field seedling bioassay was used in 2003 and 2004 to 

determine the fungicide sensitivity of the powdery mildew fungal 

pathogen population in cucurbit fields at commercial farms and at the 

Cornell University Long Island Horticultural Research and Extension 

Center (LIHREC) in Suffolk County, NY. 

The seedling bioassay entailed placing fungicide-treated seedlings 

in a field of cucurbits with powdery mildew. Summer squash 

seedlings were grown in a growth chamber. Their growing point and 

unexpanded leaves were removed just before treatment. Seedlings 

varied in size from one to nine true leaves. Treatments were no 

fungicide, QoI fungicide (50mg/L trifloxystrobin formulated as Flint), 

DMI fungicide (20mg/L myclobutanil; Nova), and a combination of 

the QoI and DMI fungicides. Seedlings were dipped in the fungicide 

solutions, then allowed to dry overnight before setting in a cucurbit 

crop in groups of four plants with the four treatments. There were two 

to seven groups per field. After being in fields for 4 to 22 hours, 

seedlings were kept in a greenhouse until symptoms of powdery 

mildew were visible, which took at least one week. Then severity 

(percent tissue with symptoms) was visually estimated for each leaf on 

a 0 to 100% continuous scale. Frequency of resistant pathogen strains 


in a field was estimated by calculating the ratio of severity on 

fungicide-treated plants relative to nontreated plants for each group, 

then determining the field average. 

In 2003, three early crops of zucchini and yellow summer squash 

that had not been sprayed with QoI or DMI fungicides were identified 

for the first bioassay. Two early plantings of pumpkin that had not 

been treated with fungicides were also selected for this bioassay 

because powdery mildew was observed in these plants in late July at 

the same time symptoms were first observed in the squash plants. 

Another bioassay was conducted about one month later on 31 August 

in six commercial pumpkin fields and a winter squash experiment at 

LIHREC after QoI and/or DMI fungicides were used in these fields. 

Powdery mildew severity was assessed in some of these fields before 

the bioassay was conducted and again at the time of the bioassay. A 

third assay conducted on 25 September included pumpkin fields where 

no QoI or DMI fungicides were used. 

In 2004, the seedling assay was conducted in seven fields at six 

farms on 29 July. Due to the high frequency of resistance the assay 

was not conducted again. 

Efficacy of fungicides used alone plus combined in fungicide 

programs and impact of fungicide use on fungicide resistance were 

assessed through three replicated experiments conducted with 

pumpkin at LIHREC in 2003 to 2005. Most treatments were initiated 

after the IPM threshold of one leaf with powdery mildew symptoms of 

50 old leaves examined was reached. Upper and lower surfaces of 5 to 

25 leaves in each plot were examined weekly for powdery mildew. 

Average severity for the entire canopy was calculated from the 

individual leaf assessments. Area under the disease progress curve 

(AUDPC) was calculated for severity over all assessment dates to 

obtain a summation value for the entire epidemic. Fungicides were 

applied weekly with a tractor-mounted boom sprayer equipped with 

D5-25 hollow cone nozzles spaced 17 inches apart that delivered 85– 

100gpa at 100–110psi. Fungicide sensitivity was determined for 

isolates collected from select plots using a leaf disk assay (McGrath, 

1996). 


In 2003, QoI resistance was detected in one of four fields at the 

start of powdery mildew development before QoIs had been applied 

(Table 1). QoI resistance was not detected in a research field. 

Moderate resistance to DMIs was detected in all fields (at a frequency 

of 1–25%). One month later, QoI resistance was detected at a high 


level (61–100%) in all six commercial fields examined, including a 

field where only DMI fungicides had been used, plus the research site. 

Moderately DMI-insensitive strains were also more common (13– 

56%). Control of powdery mildew in commercial pumpkin fields was 

poor. The lower surface of most leaves was at least 50% covered by 

powdery mildew and many leaves had died prematurely by 31 August. 

Control appeared to be only slightly better in plots in a fungicideefficacy 

experiment where a fungicide program recommended to 

growers (QoI plus sulfur alternated with DMI plus sulfur on a weekly 

schedule) was applied (Table 2). Another bioassay was conducted in 

2003 to determine how widespread QoI resistance had become. It was 

detected in three fields where neither QoI nor DMI fungicides had 

been used. Two were organic production fields. Typically in these 

bioassays powdery mildew severity on plants treated with a DMI was 

similar to severity on plants treated with both a DMI and a QoI, 

indicating that most strains moderately insensitive to DMIs were also 

resistant to QoIs. 

Several fungicide programs were evaluated in 2003 (Table 2). On 

8 September after five weekly applications to pumpkin (7 Aug to 6 

Sept), the QoI Flint applied in alternation with sulfur was providing 

poor control (37% on upper leaf surfaces and 0% on lower surfaces). 

Control was improved by applying sulfur every week and applying on 

alternate weeks a DMI fungicide; using Nova provided 84% and 48% 

control on upper and lower leaf surfaces, respectively; the DMI 

Procure provided a similar level of control (89% and 34%). Control 

was not improved significantly by applying Flint and Procure more 

than once in a fungicide program with weekly applications of sulfur, 

most likely due to high frequency of resistant strains. Flint plus sulfur 

applied Week 1 followed by Procure plus sulfur Week 2, then sulfur 

alone Weeks 3 to 5 provided 68% and 24% control. Sulfur applied 

alone Weeks 3 to 5 did contribute to control; where only Week 1 and 2 

applications of Flint, Procure, and sulfur were made, powdery mildew 

on 8 September, 25 days after the last application, was as severe as 

where no fungicides were applied. In comparison, 95% and 91% 

control was obtained with Quintec (quinoxyfen) applied on the same 

dates. Strains moderately insensitive to DMIs and resistant to QoIs 

were present. 

In 2004, QoI resistance was detected in all seven fields at the start 

of powdery mildew development (frequency of 32–91%) (Table 3). 

QoIs had been applied only in one field. Moderate insensitivity to 

DMIs was detected in all fields (15–84%). Although MBC fungicides 

are now very rarely used for cucurbit disease control, resistant strains 

of P. xanthii were common (31–91%). Fungicide programs with a QoI 


Table 1. Proportion of cucurbit powdery mildew fungal population 

estimated to be moderately insensitive to DMI fungicides and 

proportion resistant to QoI fungicides based on results from a 

fungicide-sensitivity seedling bioassay in 2003. 

DMI moderately 

insensitive isolates 

(%) z 

QoI-resistant 

isolates 

(%) z 

Site 7/27 8/31 9/25 7/27 8/31 9/25 

1 

33 61 

2 1 13 0 100 

Cornell LIHREC 16 56 0 100 

3 (no QoIs) 44 5 83 88 

4 6 56 61 91 

5 12 1 89 89 

6 25 0 

7 35 1 97 97 

8 (Organic) 1 2 

9 (Organic) 1 56 

10 (no QoIs or 

DMIs) 

17 1 0 38 

z 

blank indicates bioassay not conducted at that site on that date. 

(Flint or Pristine) and a DMI (Nova or Procure) were not as effective as 

applying Quintec weekly even when Quintec was in two of the six 

applications (Table 2). When efficacy of products as well as programs 

was examined in 2005, the DMIs were more effective than Pristine, 

which was equal in efficacy to Endura (FRAC Group 7), which 

contains the second active ingredient in Pristine. Therefore, strains 

moderately insensitive to DMIs are controlled by Nova and Procure, 

and QoIs no longer appear to be effective due to resistance. 

As a result of QoI and DMI resistance, the cost of controlling 

powdery mildew plus other foliar diseases with fungicides has doubled 

due to the need to apply DMIs at a high rate, to include a protectant 

fungicide with each application, and to include additional fungicides in 

the control program. QoIs have broader spectrum activity than other 

mobile fungicides effective for powdery mildew. 


Table 2. Control of powdery mildew achieved on upper and lower leaf 

surfaces of pumpkin with fungicide programs evaluated in replicated 

experiments conducted in 2003 to 2005. Control based on AUDPC 

values relative to nontreated control. 

Powdery mildew control (%) 

Year Treatment (week of application) Upper Lower 

2003 Flint (1,3,5) alternated with sulfur (2,4) 63 c 25 b 

2003 Flint + sulfur (1,3,5), Nova + sulfur (2,4) 91 e 59 de 

2003 Flint + sulfur (1,3,5), Procure + sulfur (2,4) 93 e 56 de 

2003 Flint + S + Nova (1,3,5), Procure + S (2,4) 92 e 68 e 

2003 Flint + S (1), Procure + S (2), S (3–5) 85 de 44 cd 

2003 Flint + S (1), Procure + S (2) 33 b 37 bc 

2003 Quintec (1–5) 93 e 86 f 

2004 Pristine + sulfur (1,3,5), Nova + S (2,4), S (6) 95 cd 87 cd 

2004 Pristine + sulfur (1), Quintec + S (2), Nova + 

S (3), Quintec + S (4), Pristine + S (5), S (6) 

97 cd 92 d 

2004 Procure + sulfur (1,3,5), Flint + S (2,4), S (6) 92 c 83 c 

2004 Procure + sulfur (1), Quintec + S (2), Procure 

+ S (3), Quintec + S (4), Procure + S (5), S(6) 

97 cd 88 cd 

2004 Procure + sulfur (1), Quintec + S (2), Quintec 

+ S (3), Procure + S (4), Procure + S (5), S (6) 

95 cd 98 e 

2004 Quintec (1–6) 99 d 98 e 

2005 Bravo Ultrex(1–5) 91 bcde 44 b 

2005 Pristine (1–5) 94 cde 46 b 

2005 Endura (1–5) 87 bcd 56 bc 

2005 Nova (1–5) 91 bcde 82 ef 

2005 Procure 50WS (1–5) 98 e 93 f 

2005 Procure 480SC (1–5) 96 cde 76 def 

2005 Procure 480SC (1,3,5), Quintec (2,4) 91 bcde 88 ef 

2005 Procure 480SC (1,3,5), Pristine (2,4) 89 bcde 75 cdef 

2005 Nova (1,4,5), Pristine (2,4) 78 b 60 bcd 

2005 Pristine (1), Quintec (2), Nova (3), 

Pristine(4), Quintec (5), S (1–5) 

97 de 71 cde 

2005 Quintec (1), Pristine (2), Quintec (3), Nova 

(4), Pristine (5), S(1–5) 

96 cde 74 cdef 

2005 Quintec (1), Pristine (2), Quintec (3), Nova 

(4), Quintec (5), S (2X premildew,1–5) 

98 e 87 ef 

S = sulfur. Treatments in year with a letter in common are not significantly different. 


Table 3. Proportion of cucurbit powdery mildew fungal population 

estimated to be moderately insensitive to DMI fungicides and 

proportion resistant to QoI or MBC fungicides based on results from a 

fungicide-sensitivity seedling bioassay in 2004. 

Resistant isolates (%) on July 29, 2004 

QoI + 

Site Crop QoI DMI DMI MBC 

1 Pumpkin 

Summer 

39 20 1 36 

2 squash 

91 57 59 91 

2 Pumpkin 

Summer 

56 26 34 84 

3 squash 

Summer 

88 78 64 75 

4 squash 

87 84 53 90 

5 Pumpkin 66 15 24 73 

6 Pumpkin 32 31 50 31 


McGrath, M. T. 1996. Increased resistance to triadimefon and to benomyl in 

Sphaerotheca fuliginea populations following fungicide usage over one season. 

Plant Dis. 80:633–639. 

McGrath, M. T. 2001. Fungicide resistance in cucurbit powdery mildew: 

experiences and challenges. Plant Dis. 85(3):236–245. 

McGrath, M. T. 2004a. Evaluation of fungicide programs for managing powdery 

mildew of pumpkin, 2003. Fungicide & Nematicide Tests. 59:V056. 

McGrath, M. T. 2004b. Seasonal dynamics of resistance to QoI and DMI fungicides 

in Podosphaera xanthii and impact on control of cucurbit powdery mildew. Res. 

Pest Mgmt. 13(2). 

. 

McGrath, M. T. 2005a. Evaluation of fungicide programs for managing pathogen 

resistance and powdery mildew of pumpkin, 2004. Fungicide & Nematicide 

Tests. 60:V049. 

McGrath, M. T. 2005b. Occurrence of resistance to QoI, DMI, and MBC fungicides 

in Podosphaera xanthii in 2004 and implication for controlling cucurbit 

powdery mildew. Res. Pest Mgmt. 14(2). 

. 

McGrath, M. T. and Davey, J. F. 2006. Evaluation of fungicide programs for 

managing powdery mildew of pumpkin, 2005. Fungicide & Nematicide Tests. 

61:V038 

McGrath, M. T. and Shishkoff, N. 2003a. Evaluation of fungicide programs for 

managing powdery mildew of pumpkin, 2002. Fungicide & Nematicide Tests. 

58:V023. 


McGrath, M. T. and Shishkoff, N. 2003b. First report of the cucurbit powdery 

mildew fungus (Podosphaera xanthii) resistant to strobilurin fungicides in the 

United States. Plant Dis. 87:1007. 

McGrath, M. T. and Shishkoff, N. 2003c. Resistance to strobilurin fungicides in 

Podosphaera xanthii associated with reduced control of cucurbit powdery 

mildew in research fields in the Eastern United States. Res. Pest Mgmt. 12(2). 

. 

McGrath, M. T, Staniszewska, H., Shishkoff, N., and Casella, G. 1996. Fungicide 

sensitivity of Sphaerotheca fuliginea populations in the United States. Plant 

Dis. 80:697–703. 


FRUIT YIELD, QUALITY PARAMETERS, AND 

POWDERY MILDEW (SPHAEROTHECA 

FULIGINEA) SUSCEPTIBILITY OF 

SPECIALTY MELON (CUCUMIS MELO L.) 

CULTIVARS GROWN IN A PASSIVELY 

VENTILATED GREENHOUSE 

J. M. Mitchell, D. J. Cantliffe, and S. A. Sargent 

University of Florida, IFAS, Horticultural Sciences Dept., Gainesville, 

FL 32611 

L. E. Datnoff 

University of Florida, IFAS, Plant Pathology Dept., Gainesville, FL 

32611 

P. J. Stoffella 

University of Florida, IFAS, Horticultural Sciences Dept., Indian 

River Research and Education Center, Ft. Pierce, FL 34945 

ADDITIONAL INDEX WORDS. Galia, muskmelon, protected agriculture, disease 

progress curves 

ABSTRACT. Like other cucurbits, many specialty melons (Cucumis melo L.) are 

susceptible to powdery mildew (Sphaerotheca fuliginea). Although greenhouse 

production decreases the risk for some diseases, greenhouses provide an optimal 

environment for the development of powdery mildew. Eighteen specialty melon 

cultivars were grown during fall 2005 in a passively ventilated greenhouse in 

Citra, FL, and rated for their fruit yield, fruit-quality characteristics, and 

susceptibility to powdery mildew. Disease-severity ratings (DSR) based on the 

percentage of leaf area infected were taken once per week from 47 days after 

transplanting (DAT) until the final harvest. Plants were sprayed with potassium 

bicarbonate (Milstop) once per week, beginning at first sign of disease (after the 

first rating), to suppress mildew infection. Area under disease progress curve 

(AUDPC) values were calculated from the weekly DSR ratings. Yield as number 

of fruit per plant was similar for all cultivars. Although three cultivars with 

100% DSR had among the lowest yields, powdery mildew severity could not be 

correlated with yield or fruit quality for any of the cultivars. The Charentaistype 

cultivars were the most susceptible to powdery mildew and had the lowest 

yields; two Galia-type cultivars had favorable yields and quality and were less 

susceptible to powdery mildew. 

S 

pecialty melons have been gaining in popularity throughout the 

United States (Jett, 2005a; Rangarajan and Ingall, 2003; 

Schultheis and Jester. 2004; Shaw et al., 2001; Simon et al., 

1993; Smith and Bartolo, 2005). These melons are extremely 

attractive and come with diverse tastes, colors and aromas (Simon et 

al., 1993). A few of these melons include the yellow-netted, green- 


fleshed, highly aromatic and sweet ‘Galia’ (Cucumis melo L. var. 

reticulatus Ser.)—a favorite throughout the Mediterranean and Europe 

with prices averaging $2 to $4 each (Karchi, 2000; Rodriguez et al., 

2002). A French favorite is the ‘Charentais’ (Cucumis melo L. var. 

cantalupenis Naud.), which has a smooth exterior with green sutures, a 

bright orange flesh, and a rich honey flavor and aroma (Goldman, 

2002; Rangarajan and Ingall, 2003). Another distinct type, named for 

both its bright yellow exterior and the island where it first became 

popular, Canary (Cucumis melo L. var. inodorus Naud.) has a smooth, 

and a sugary, white flesh (Goldman, 2002; Anon., 2004). These 

melons are an ideal high-value crop for protected culture (Cantliffe et. 

al., 2003). 

Unfortunately, the high temperatures (25–35°C), high humidity 

(90%), and dense plant growth of a greenhouse melon crop provide the 

optimum conditions for powdery mildew (Sphaerotheca fuliginea) 

disease development (Elad et. al., 1996; Jett, 2005b; Pottorff, 2005). 

Sphaerotheca fuliginea is the most common and aggressive powdery 

mildew fungus of cucurbits (McGrath, 1997). It is easily recognized 

by its white or gray talcum powder-like spots or patches (Pottorff, 

2005). Powdery mildew can be a devastating disease because a severe 

epidemic can reduce photosynthesis, increase respiration and 

transpiration, impair vegetative and fruit growth, and ultimately reduce 

yields (Agrios, 2005). Because of this potential for yield reduction, 

even in a protected environment, information on how powdery mildew 

affects specialty melons’ fruit yield and quality parameters in a 

greenhouse environment would be useful to growers and plant 

breeders. For this study, 18 different cultivars were grown in a 

passively ventilated greenhouse to ascertain any relationships between 

the incidence of powdery mildew and melon fruit yields and quality. 


Seeds of 18 melon cultivars (Table 1) were sown in polystyrene 

trays (Model 128A, Speedling, Bushnell, FL) on 22 July 2005. The 

growing medium used was a commercial fine-grade mixture (Premier 

ProMix FPX, Quakertown, PA). Seedlings were grown at the 

University of Florida, Gainesville campus in a growth chamber 

(Controlled Env. Ltd., Winnipeg, Manitoba, Canada) at temperatures 

of 28 ° C (day) and 22 ° C (night) with 12-hour daily artificial lighting. 

When cotyledons were fully expanded, seedlings were fertilized once 

per week with a solution of 100ppm each of 20N: 20P: 20K (Peters 

Professional All Purpose Plant Food, Spectrum Group, St. Louis, MO). 


Table 1. Specialty melon (Cucumis melo) cultivars, fall 2005. 

Diseasetolerance 

Cultivar Type 

listing z Seed source y 

Kamila x 

Galia N.R. Outstanding Seed 

Co. 

Gallicum Galia PM1,2 Seigers Seed Co. 

(Seminis variety) 

Gala Galia PM2 D. Palmer Seed Co. 

GVS 125 Galia N.R. Golden Valley Seed 



Melon 96-Nestor Galia PM Hazera Genetics 

Melon 6003- 

Elario 

Galia PM Hazera Genetics 

Melon 6004 Galia N.R. Hazera Genetics 

Angel Mediterranean N.R. Known-You Seed 

Amy Canary/ 

Mediterranean 

N.R. Known-You Seed 

Galia F1 (H) Galia N.T. Hazera Genetics 

Vedrantais Charantais N.R. Technisem 

Vicar Galia PM1 Rogers/Syngenta 

Galileo Galia PM1 Rogers/Syngenta 

z 

Disease tolerance to powdery mildew (S. fuliginea) as listed by suppliers: PM1,2 = 

tolerance to race 1 or 2; N.R.= not reported; N.T.= no tolerance. 

y 

Seeds were kindly donated by these suppliers. 

x 

‘Kamila’ was advertised as a ‘Galia-type’, but it was found to be a Japanese-type. 

Seedlings were transplanted on 18 August 2005 when they had 

three true leaves in a passively ventilated, high-roof, sawtooth 

greenhouse (TOP greenhouses, Ltd., Barkan, Israel) located at the UF 

Plant Science Research Education Unit, Citra, FL. Commercial 

greenhouse production techniques and nutrient requirements for 

producing ‘Galia’ melons hydroponically were used according to 

guidelines of Shaw et al. (2001). Plants were pollinated by bumble 

bees (Bombus impatiens, Natupol, Koppert Biological Systems, Inc., 

Romulus, MI). 


Table 2. Final AUDPC and disease severity ratings for specialty 

melon (Cucumis melo) cultivars, fall 2005. 

Cultivar 

AUDPC z 

Disease-severity 

rating y (%) 

Vedrantais 3182 x 100 

Charantais 2910 100 

Gala 2262 100 

Kamila 1173 80 

Galia (T) 1143 83 

Amy 1116 78 

GVS 206 970 55 

GVS 125 794 48 

GVS 205 786 54 

Gallicum 594 50 

Galia (H) 534 49 

Angel 478 53 

Girlie 444 42 

Vicar 431 38 

Melon 6004 236 28 

Melon 6003-Elario 127 14 

Galileo 87 15 

Melon 96-Nestor 42 3 

LSD (0.05) 516* 9* 

z 

AUDPC = area under disease progress curve. 

y 

Disease severity ratings (0–100%) were the mean of three leaves per plant and 

recorded as percent leaf area infected. 

x 

Means separated using Fisher’s Least Significant Difference (P

Systems, Inc., Romulus, MI) were released on 8 Sept. 2005. 

Neoseiulus californicus (Biotactics Inc., Perris, CA) predatory mites 

were used to control the two-spotted spider mite (Tetranychus urticae). 

N. californicus (3000) were released on 8 Sept., 6,000 on 28 Sept., and 

10,000 on 5 Oct. 2005. Erotmocerus eremicus (ERCAL, Koppert 

Biological Systems, Inc., Romulus, MI) were released to control 

whitefly (Bemisia tabaci biotype B). E. eremicus (500) were released 

on 8 Sept. and 7,000 on 5 Oct. 2005. Orius insidiosus (THRIPOR-I, 

Koppert Biological Systems, Inc., Romulus, MI), a predatory bug for 

control of flower thrips (Frankliniella occidentalis), were released on 

8 Sept. (500 O. insidiosus) and again on 5 Oct. 2005. 

Disease ratings were recorded at first sign of disease symptoms. 

Ratings were taken on each of the five plants per plot. Plants were 

divided into three sections—lower, middle and upper—and ratings 

were taken as a percentage of leaf area infected per one sample leaf 

per section once per week until the end of the crop production (seven 

ratings total). Mean of three sample leaves represents the disease 

severity rating (DSR). In order to keep the crop alive, plants were 

sprayed with potassium bicarbonate (Milstop, BioWorks Inc., Fairport, 

NY), a foliar fungicide that suppresses powdery mildew, once a week 

directly after the ratings. No other chemical fungicides were used. 

Final area under disease progress curves (AUDPC) were generated by 

the method of Shaner and Finney (1967) from the weekly DSR. 

AUDPC indicates the total amount of disease that occurred throughout 

the crop production period whereas DSR reflects the amount of disease 

present at a given time interval only. 

Fruits were harvested at full-slip stage. First harvest began on 17 

Oct. 2005 and ended on 21 Nov. 2005. Harvest data collected 

included mean number of fruit per plant, weight, flesh thickness, 

soluble solids content (SSC), and firmness. All postharvest parameters 

were measured on the day of harvest. 

After fruits were weighed, a 2.5-cm slice was taken from the 

equatorial region of each fruit and flesh thickness, firmness, and 

soluble solids content measured. A caliper (Digimatic Mycal, 

Mitutoyo, Japan) was used to measure flesh thickness from peel to 

cavity. A firmness reading was taken at two equidistant points on the 

equatorial region of each fruit slice using an Instron Universal Testing 

Instrument (Model 4411-C8009, Canton, MA). The Instron was fitted 

with a 50-kg load cell and an 11-mm convex probe with a crosshead 

speed of 50mm/min. Firmness data were expressed as the maximum 

force (Newton) attained during deformation. Soluble solids content 

(°Brix) (SSC) was measured with a temperature-compensating, 

handheld refractometer (Model 10430, Reichert Scientific Instrument, 


Buffalo, NY) from three samples taken from the equatorial slice. The 

means from five plants in each plot were subjected to an analysis of 

variance (ANOVA) by the Statistical Analysis System (SAS Institute, 

Version 9, Cary, NC). Cultivar means were subjected to Fisher’s least 

significant difference (LSD 0.05). 


Powdery mildew was first observed 47 days after transplanting. 

After seven weeks of ratings, cultivars ‘Vedrantais’, ‘Charentais’ and 

‘Gala’ had the highest final AUDPC and the final DSR for each was 

100% (Table 2). Final DSR was over 50% for ‘Angel,’ ‘GVS-205,’ 

‘GVS-206,’ ‘Amy,’ ‘Kamila’, and ‘Galia (T)’; and from 25 to 50% for 

‘Melon 6004,’ ‘Vicar,’ ‘Girlie’ ‘GVS-125’, ‘Gallicum’, and ‘Galia 

(H).’ Cultivars ‘Galileo’ and ‘Elario’ had a final DSR of less than 20% 

and ‘Nestor’ was the lowest with 3%. Cultivars ‘Charentais’ and 

‘Vedrantais’ are Charentais-type melons, which are highly susceptible 

to powdery mildew (Jett, 2005a) and were at 100% DSR by Week 6. 

‘Gala’ is a Galia-type that is also highly susceptible to powdery 

mildew. Also interesting was the difference between the two true 

‘Galia’ cultivars, ‘Galia (Hazera)’ and ‘Galia (Technisem).’ ‘Galia 

(H)’ had a final DSR rating 49%, which was significantly lower than 

‘Galia (T)’—83%. Also, soluble solids content (°Brix) was higher for 

‘Galia (H)’ (11.5°Brix) than ‘Galia (T)’ (9.6°Brix); and ‘Galia (H)’ 

was a firmer fruit (18N) than ‘Galia (T)’ (8N) (Table 3), yet yields 

were the same. Thus, although fruit quality was potentially reduced by 

powdery mildew in this case, yields were not. The AUDPC was lower 

for ‘Galia (H)’ than ‘Galia (T)’, which may have helped contribute to 

the better fruit quality of ‘Galia (H).’ 

The number of marketable fruit per plant was similar among all 

cultivars. Each cultivar averaged only one to two fruit (data not 

shown). Fruit numbers per plant were low due to poor bumble bee 

activity. Previous greenhouse melon cultivar trials averaged 2.2 to 3.6 

fruits per plant (Shaw et. al., 2001). Mean fruit weight per plant was 

different among cultivars. Average individual fruit weight ranged 

from 0.7kg (‘Charentais’) to 1.9kg (‘Girlie’) (Table 3). Several 

cultivars, ‘Angel,’ ‘Vedrantais,’ ‘GVS 206,’ ‘Nestor,’ ‘Gala’, and 

‘Charantais’, had significantly lower yields (less than 40, 000kg · ha -1 ) 

than ‘Galileo,’ ‘Elario,’ ‘Kamila’ and ‘Girlie’ (more than 70,000 kg · 

ha -1 ); however, the final DSR varied from 100 to 3% on the lowest 

yielding cultivars. Soluble solids content also seemed to have no 

relationship to DSR or yield since ‘Gala’ had a final DSR of 100 and 


Table 3. Marketable yields and fruit quality of screened specialty 

melons (Cucumis melo), fall 2005. 

Fruit Fruit 

weight number 

per per Fruit 

plant square yield 

Cultivar (kg) meter (kg · ha -1 Flesh Fruit Soluble 

thickness firmness solids 

) (mm) (N) (°Brix) 

Vedrantais 0.9 Z 4.1 34,063 27 2 8.5 

Charentais 0.7 5.6 39,644 24 12 11.0 

Gala 1.5 2.5 39,158 35 17 11.8 

Kamila 1.5 5.2 76,272 35 31 15.0 

Galia (T) 1.6 3.5 55,526 32 8 9.6 

Amy 1.1 4.1 45,646 28 12 14.3 

GVS 206 1.4 2.6 36,396 33 24 9.4 

GVS 125 1.1 4.0 45,139 31 22 10.3 

GVS 205 1.4 3.9 53,674 34 17 9.6 

Gallicum 1.3 5.2 65,163 33 13 10.1 

Galia (H) 1.3 3.8 50,456 33 18 11.7 

Angel 1.1 2.9 30,851 30 12 16.3 

Girlie 1.9 3.9 72,609 34 16 11.5 

Vicar 

Melon 

1.2 4.8 59,990 33 30 11.7 

6004 

Melon 

6003- 

1.7 3.7 64,576 38 20 8.9 

Elario 1.7 4.6 76,917 41 20 8.8 

Galileo 

Melon 96- 

1.5 5.5 81,499 36 32 12.4 

Nestor 1.1 3.6 38,940 32 23 10.3 

z 

LSD (0.05) 0.3* 2.0 28,615* 4* 7* 1.3* 

Z 

Means separated using Fisher’s Least Significant Difference (P

SSC were ‘Angel’ (16.3°Brix) and ‘Kamila’ (15°Brix), which also had 

a DSR >50% but still maintained Brix. However, the lowest SSC was 

found for ‘Vedrantais’ (8.4°Brix, 100 DSR), ‘Elario’ (8.8°Brix, 14 

DSR), and ‘Melon 6004’ (8.9°Brix, 28 DSR). Charentais melons may 

reach 14 to 16°Brix (Rangarajan and Ingall, 2003), whereas in 

this study they were below this at 8.4°Brix (‘Vedrantais’) and 

11.0°Brix (‘Charentais’). 

The Charentais-type cultivars in this trial (‘Charentais’ and 

‘Vedrantais’) were the most susceptible to powdery mildew (final 

DSR=100%), had the lowest yields and smallest fruit size, and are not 

recommended for greenhouse production. The Canary and 

Mediterranean types had less flavor and quality than the Galia-type 

melons. The cultivar ‘Kamila’ also had a high SSC and was 

advertised as a Galia-type; however, it did not slip, netting and flesh 

were not similar to ‘Galia’, and it had little flavor. Of the Galia-type 

cultivars, ‘Galileo’ and ‘Vicar’ had high yields, SSC and fruit quality 

that were considered good, and were less susceptible to powdery 

mildew. 


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Extension. 20 April, 2006. . 

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greenhouses in the Southeastern United States. Acta Hort. 626:483–485. 

Elad, Y., N. Malathrakis, and A. Dik. 1996. Biological control of Botrytis-incited 

diseases and powdery mildews in greenhouse crops. Crop Protection. 

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McGrath, M. 1997. Powdery mildew of cucurbits. Fact Sheet Page: 732.30 Date: 2- 

1997. Cooperative Extension, New York State, Cornell Univ. 17 Nov., 2005. 

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Rodriguez, J., N. Shaw, and D. Cantliffe. 2002. Production of Galia-type muskmelon 

using a passive ventilated greenhouse and soilless culture, p. 365–372. 

Cucurbitaceae 2002. ASHS Press, Alexandria, VA. 

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for market potential. 10 April, 2006. 

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of slow-mildewing resistance in Knox wheat. Phytopathology. 67:1051–1056. 

Shaw, N. and D.J. Cantliffe. 2003. Hydroponically produced mini-cucumber with 

improved powdery mildew resistance. Proc. Fla. State Hort. Soc. 116:58–62. 

Shaw, N., D. J. Cantliffe, and B. S. Taylor. 2001. Hydroponically produced 'Galia’ 

muskmelon—what’s the secret? Proc. Fla. State Hort. Soc. 114:288–293. 

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area. 10 April, 2006. . 


INTEGRATION OF BIOLOGICAL CONTROL 

AND PLASTIC MULCHES TO MANAGE 

WATERMELON MOSAIC VIRUS IN SQUASH 

John F. Murphy and Micky D. Eubanks 

Department of Entomology & Plant Pathology, Auburn University, AL 

36849 

ADDITIONAL INDEX WORDS. Plant virus, WMV, vegetable, Cucurbita pepo, 

PGPR 

ABSTRACT. A study was undertaken to evaluate the combined effects of 

reflective plastic mulch and a commercially available biological control 

treatment to reduce the incidence and resulting disease-related yield losses 

caused by an aphid-borne plant virus in squash. Squash plants grown on silveron-black 

mulch did not have reduced incidence of Watermelon mosaic virus 

(WMV) relative to plants grown on black mulch. In contrast, squash plants 

grown on a highly reflective silver mulch had significantly less WMV incidence 

relative to those grown on silver-on-black and black mulches. The silver-onblack 

mulch treatment led to greater squash fruit yields relative to the other 

mulch treatments. The biological control treatment, BioYield TM , did not reduce 

virus incidence or result in increased fruit yields relative to nontreated control 

plants. 

P 

lant viruses are difficult to manage when resistant varieties are 

not available. The application of management strategies 

becomes more complicated and typically less effective when the 

viruses of concern are transmitted by aphids in a nonpersistent manner. 

This manner of transmission leads to very rapid acquisition of virus 

from infected plants and transmission to healthy plants. This rapid 

transmission process essentially eliminates the effectiveness of 

insecticides as a means to manage virus by way of its vector. 

Cucurbit crops are vulnerable to infection by several viruses in the 

genus Potyvirus, the most frequent being: Papaya ringspot virus 

(PRSV), Watermelon mosaic virus (WMV), and Zucchini yellow 

mosaic virus (ZYMV). Cucumber mosaic virus (CMV, genus 

Cucumovirus) also poses a serious threat to cucurbit crops since it too 

is transmitted by aphids in a nonpersistent manner (Zitter et al., 1996). 

Plant growth-promoting rhizobacteria (PGPR) have been used in 

previous studies to induce resistance in cucumber plants against CMV 

(Raupach et al., 1996) and in tomato plants against CMV and Tomato 

mottle virus (genus Begomovirus) (Murphy et al., 2003; Murphy et al., 

2000). In cucumber and particularly in tomato, the PGPR treatment 

resulted in an enhancement of plant growth. Protection against CMV 


infection was observed in the form of a complete lack of symptoms or 

mild symptoms with reduced virus accumulation in systemically 

infected leaves, or interference in systemic infection. 

Although protection against virus infection occurred in response to 

treatment with PGPR, it varied in level of protection under greenhouse 

conditions and even more so in the field. In the project described in 

this report, efforts were made to integrate a commercially available 

PGPR product with different types of plastic mulch in an effort to 

reduce the incidence of aphid-borne plant viruses in squash. 


Two field trials were performed in 2005 at the E.V. Smith 

Agricultural Research and Education Center, Shorter, AL.: one trial in 

the spring (referred to as Spring trial) and the second trial in the fall 

(referred to as Fall trial). 

The objective was to evaluate silver-based reflective versus 

nonreflective plastic mulch in conjunction with the biological control 

treatment BioYield TM . The field design was a split-plot with mulch as 

the whole-plot treatment and plant treatment (i.e., BioYield TM and 

nontreated control) as the subplot treatment. For the Spring trial, two 

mulch treatments were used: black, nonreflective mulch and silver-onblack 

reflective mulch. For the Fall trial, three mulch treatments were 

used: black, nonreflective mulch, silver-on-black reflective mulch, 

and a highly reflective silver mulch. 

Within each mulch treatment, a BioYield TM and a nontreated 

control treatment were arranged in a randomized complete block with 

four replications consisting of 12 plants in each. BioYield TM was a 

commercial product marketed by Gustafson, Inc. and consisted of two 

PGPR strains, GB03 and IN937a, Bacillis subtilis and B. 

amolyliquefaciens, respectively, and the carrier chitosan. Each PGPR 

strain was formulated as endospores at 4.0 x 10 10 colony-forming units 

(CFU)/liter. BioYield TM was mixed with soil-less growth medium 

(Speedling Inc., Busnell, FL) at a ratio of 1:40 (v/v) to achieve a 

bacterial density of 10 9 CFU/litre medium. The medium was placed 

into Styrofoam trays (Speedling Inc.,) containing 72 cavities per tray. 

To produce transplants, squash seeds (Cucurbita pepo L cv 

‘Dixie’) were individually placed into each cavity, and given 2–3 

weeks to germinate and grow in a temperature-controlled greenhouse 

at the Auburn University Plant Science Research Complex, Auburn, 

AL. At the time of transplant, seedlings were transported to the 

appropriate field location and planted 30cm apart on raised beds (20cm 

high by 84cm wide). Prior to bed preparation, P205 fertilizer was 


applied as a broadcast at 2.5Kg per 30.5m of row. All beds were 

fumigated with methyl bromide (335Kg/ha of 67% methyl bromide + 

33% chloropicrin) before being covered with polyethylene plastic tarp 

(mulch). After bedding, 15-0-30 fertilizer was banded onto the bed 

shoulders at 9.5Kg per 30.5m of row. 

Disease evaluations included visual and serological assessments. 

Plants in each treatment were monitored routinely for virus symptoms 

such as mosaic patterns on leaves and leaf deformation. Foliar tissues 

were collected from each plant at the midway point to harvest (Spring 

trial) and once fruit harvest was underway (Spring and Fall trials). 

Each sample was wrapped in a dampened paper towel, placed in a ziplock 

bag and transported to Auburn University for analysis. Virus was 

detected using Agdia, Inc. (Elkhart, IN) enzyme-linked 

immunosorbent assay (ELISA) kits specific to CMV, PRSV, WMV, or 

ZYMV and performed according to the manufacturer’s instructions. 

Squash fruit was harvested and graded as marketable and total 

(marketable and nonmarketable), and each grade measured by number 

and weight of fruit. 

The data were analyzed with analysis of variance (SAS, Cary, NC) 

and for significant ANOVA’s treatment means separated using LSD 

mean separation tests. Significant interactions were tested at P = 0.05. 

Results 

SPRING TRIAL. Two viruses were detected among infected squash 

plants in the Spring trial, CMV and WMV, with CMV incidence being 

very low (~2.5%) but with high incidence of WMV (~66%). The low 

incidence of CMV made analysis difficult and, therefore, data 

presentations will focus on WMV. 

For the Spring trial, there was no difference in the incidence of 

WMV infection among plants grown on black versus silver-on-black 

mulches (P < 0.05; although significantly less WMV occurred on 

silver-on-black mulch at P < 0.10). Similarly, no difference in WMV 

incidence occurred among squash plants treated with BioYield TM 

versus the nontreated control (P < 0.05) and no mulch x plant 

treatment interaction occurred. 

Squash marketable yields, both in number of fruit and weight, were 

significantly greater for plants grown on silver-on-black mulch than 

for plants grown on black mulch (P < 0.05). For both marketable fruit 

number and weight, there was no significant difference among plants 

treated with BioYield TM versus the nontreated controls. The same 

trend occurred for total fruit number, i.e., significantly more fruit for 

plants grown in silver-on-black mulch with no difference due to plant 


treatment; however, total fruit weight did not differ among mulch or 

plant treatments. 

FALL TRIAL. As observed in the Spring trial, CMV incidence was 

low with a high incidence of WMV. Significantly fewer squash plants 

grown on silver mulch were infected with WMV than on silver-onblack 

or black mulch (P < 0.05). No difference in WMV incidence 

was observed for plants treated with BioYield TM versus those in the 

nontreated control treatment. 

All parameters for squash yield, marketable number and weight, 

and total number and weight resulted in significantly greater yields for 

plants grown on silver-on-black mulch than the other mulches with no 

difference occurring for BioYield TM versus nontreated control plants. 

Discussion 

The goal of this research was to reduce aphid-borne plant virus 

incidence in squash and thereby reduce yield losses due to virus 

infection. The four viruses examined represent commonly occurring 

viruses that, due to their mode of transmission, are difficult to manage 

without availability of resistant varieties. WMV was the predominant 

virus in both trials with a low incidence of CMV and no PRSV or 

ZYMV detected. 

Two methods were used to reduce virus incidence: reflective 

plastic mulch and a commercially available PGPR treatment shown 

previously to enhance plant growth. The reflective mulch was 

intended to delay the introduction of virus into the crop by deterring 

aphids from flying onto the plants. The PGPR treatment was intended 

to induce systemic resistance to virus infection and enhance plant 

growth, the latter of which was expected to shorten the window of 

time when plants are small and more vulnerable to development of 

severe disease symptoms if infected by a plant virus during this early 

stage of growth. 

The use of silver-on-black plastic mulch did not reduce WMV 

incidence in either trial; however, silver mulch had significantly fewer 

WMV-infected plants than the two other mulch treatments in the Fall 

trial. Based on a simple visual assessment, the reflective properties of 

the silver mulch were far greater than those of the silver-on-black; the 

greater reflective property of the silver mulch may have deterred 

aphids from entering the crop, thereby leading to a reduction in WMV 

incidence. Yet there is a possibility that the effect on plant growth 

from silver-on-black is not only better than that of silver but that the 

highly reflective nature of the silver mulch has a negative effect. This 


could be important since it might offset the positive effects of reduced 

virus incidence. 

Silver reflective mulch, also referred to as UV-reflective mulch, 

has been shown to reduce insect densities on plants and/or virus 

incidence relative to other mulches (Diaz-Perez et al., 2003; 

Loebenstein et al., 1975; Momol et al., 2004; Summers et al., 2004; 

Summers, et al., 1995; Wilson, 1999; Wyman et al., 1979). Silver 

mulches deterred aphids and delayed the introduction of aphid-borne 

viruses in spring- and fall-grown zucchini squash crops (Summers et 

al., 1995). There was a corresponding effect on squash fruit yields 

with an approximately 70% greater yield for plants grown on silver 

mulch than those grown on nonmulched plots. Wyman et al. (1979) 

described a similar effect of reflective mulch on reducing aphid 

numbers on summer squash plants, with a corresponding reduction in 

the incidence of WMV. 

The BioYield TM treatment did not lead to any apparent increase in 

plant growth relative to plants in the nontreated control treatment, nor 

to any reduction in virus incidence. The lack of an increase in fruit 

yield for BioYield TM -treated squash plants further supports the 

observation that the PGPR treatment did not enhance plant growth and 

development. The treatment of tomato plants with BioYield TM led to 

significant enhancement of plant growth with significant reductions in 

CMV infection when tested in greenhouse conditions (Murphy et al., 

2003). The tomato plants responded to inoculation with CMV in a 

similar manner to older, more mature plants, which suggested the 

protection afforded the BioYield TM -treated plants may have been a 

form of induced mature plant resistance rather than induced systemic 

resistance. 

Our hypothesis at the onset of this work was that the integration of 

the insect-deterring properties of the reflective mulch along with 

enhanced plant growth induced by the PGPR treatment would combine 

to have a highly significant effect on reduction of virus incidence and 

corresponding fruit yields. Silver-reflective mulch was effective at 

reducing WMV incidence; this was dependent on the reflective 

properties of the mulch, i.e., higher levels of reflectance are necessary 

for effective deterrence of aphid vectors. 

There was no evidence that the BioYield TM treatment resulted in 

enhanced plant growth, development, or yield increase relative to the 

nontreated control. Likewise, there was no evidence of protection 

against virus infection, whether from induced systemic resistance or 

induced mature plant resistance, for plants treated with BioYield TM . 

Previous studies using PGPR-based treatments suggested that the 

protection afforded plants against infection by CMV did not occur 


when a virus in the genus Potyvirus was used as inoculum (e.g., J. F. 

Murphy, unpublished data). This may explain, in part, the lack of 

protection observed for BioYield TM -treated plants against the 

potyvirus, WMV. The possibility that a PGPR-based treatment may 

protect plants against CMV but not a virus in the genus Potyvirus is 

intriguing and should be pursued in future studies. 


Diaz-Perez, J. C., K. D. Batal, D. Granberry, D. Bertrand, D. Giddings, and 

H. Pappu. 2003. Growth and yield of tomato on plastic film mulches as 

affected by tomato spotted wilt virus. HortSci. 38:395–399. 

Loebenstein, G., M. Alper, S. Levy, D. Palevitch, and E. Menagem. 1975. 

Protecting peppers from aphid-borne viruses with aluminum foil and 

plastic mulch. Phytoparasitica. 3:43–53. 

Momol, M. T., S. M. Olson, J. E. Funderburk, J. Stavisky, and J. J. Marois. 

2004. Integrated management of tomato spotted wilt on field-grown 

tomatoes. Plant Dis. 88:882–890. 

Murphy, J. F., G. W. Zehnder, D. J. Schuster, E. J. Sikora, J. E. Polston, and 

J. W. Kloepper. 2000. Plant growth-promoting rhizobacterial mediated 

protection in tomato against Tomato mottle virus. Plant Dis. 84:779– 

784. 

Murphy, J. F., M. S. Reddy, C. M. Ryu, J. W. Kloepper, and R. Li. 2003. 

Rhizobacteria-mediated growth promotion of tomato leads to protection 

against Cucumber mosaic virus. Phytopathology. 93:1301–1307. 

Raupach, G. S., L. Liu, J. F. Murphy, S. Tuzun, and J.W. Kloepper. 1996. 

Induced systemic resistance against cucumber mosaic cucumovirus using 

plant growth-promoting rhizobacteria (PGPR). Plant Dis. 80:891–894. 

Summers, C. G., J. J. Stapleton, A. S. Newton, R. A. Duncan, and D. Hart. 

1995. Comparison of sprayable and film mulches in delaying the onset of 

aphid-transmitted virus diseases in zucchini squash. Plant Dis. 79:1126– 

1131. 

Summers, C. G., J. P. Mitchell, and J. J. Stapleton. 2004. Management of 

aphid-borne viruses and Bemisia argentifolii (Homptera: Aleyrodidae) in 

zucchini squash by using UV reflective plastic and wheat straw mulches. 

Env. Entomol. 33:1447–1457. 

Wilson, C. R. 1999. The potential of reflective-mulching in combination with 

insecticide sprays for control of aphid-borne viruses of iris and tulip in 

Tasmania. Ann. Appl. Biol. 134:293–297. 

Wyman, J. A., N. C. Toscano, K. Kido, H. Johnson, and K. S. Mayberry. 

1979. Effects of mulching on spread of aphid-transmitted watermelon 

mosaic virus to summer squash. J. Econ. Entomol. 72:139–143. 

Zitter, T. A., D. L. Hopkins, and C. E. Thomas. 1996. Compendium of 

cucurbit diseases. APS Press, St. Paul, MN. 


BACTERIAL LEAF SPOT (XANTHOMONAS 

CAMPESTRIS PV. CUCURBITAE) AS A 

FACTOR IN CUCURBIT PRODUCTION AND 

EVALUATION OF SEED TREATMENTS FOR 

CONTROL IN NATURALLY INFESTED SEEDS 

Zahide Özdemir 

Department of Plant Protection, Faculty of Agriculture, 

Adnan Menderes University, Aydın, 09100, Turkey 

Thomas A. Zitter 

Department of Plant Pathology, Cornell University, Ithaca, NY 14853 

ADDITIONAL INDEX WORDS. Cucurbitaceae, Cucurbita pepo, pumpkin, kabutiá 

squash 

ABSTRACT. Pumpkin (Cucurbita pepo cv. New Rocket) seeds naturally infested 

with Xanthomonas campestris pv. cucurbitae, the pathogen responsible for 

bacterial leaf spot disease of winter squash, pumpkin, gourd, and cucumbers, 

were treated with four different chemicals to determine the most effective 

treatment to eliminate bacteria from the infested seeds. The seed treatments 

included 3% hydrogen peroxide, 1% peroxyacetic acid, 1% sodium 

hypochlorite, copper hydroxide plus mancozeb (0.36g + 0.27 g/100ml of water, 

respectively), and sterile distilled water as control. Seeds were treated in 

aqueous solutions of chemicals for 15 min. Treated seeds were washed in sterile 

cold saline solution and dilution platings were made onto XCS agar. 

Treatments with copper plus mancozeb, 1% peroxyacetic acid, and 1% sodium 

hypochlorite were effective in eliminating the pathogen on seed. Hydrogen 

peroxide was less effective, resulting in a 90–92% reduction in bacteria 

recovered from seed in the first experiment. In a second experiment, no 

bacteria were recovered following treatment. 

B 

acterial leaf spot of cucurbits, caused by the bacterium 

Xanthomonas campestris pv. cucurbitae (Xcc), was first 

described in New York on Hubbard squash (Bryan, 1926). 

The disease was subsequently reported on other winter squash, 

pumpkins, summer squash, cucumber, and gourds. Foliar symptoms 

are common on pumpkin and winter squash, and lesions on fruit can 

be severe enough to result in a total loss. The disease occurs 

sporadically, and literature on its epidemiology is currently limited 

(Zitter et al., 1996). Recently, bacterial leaf spot has been observed on 

pumpkins (Cucurbita pepo), causing reduced fruit quality in the U.S. 

(Latin, 1996; Zitter et al., 1996), and it continues to be an issue in crop 

production in several midwestern states. In Uruguay, the disease was 

observed in both 2005 and 2006 in kabutiá winter squash (C. moschata 


x C. maxima), and has spread into neighboring butternut squash (C. 

moschata) fields (T. A. Zitter, unpublished data). Since growers also 

use locally produced seed saved from two pollinator species (C. 

moschata and C. pepo), it is not known which seed may have 

introduced the pathogen and may be perpetuating the problem. The 

pathogen appears to be short-lived in the soil (Bryan, 1930; Vincent- 

Sealy and Brathwaite, 1982) and infested seed is the primary source of 

inoculum (McLean, 1958; Moffett and Wood, 1979). 

Chemical seed treatments were evaluated by Moffett and Wood 

(1979) on winter squash (C. maxima cv. Queensland Blue) seeds 

collected from naturally Xcc-infected fruits. One percent sodium 

hypochlorite with 1% nonionic spreader sticker and hot-water 

treatment at 54˚C and 56˚C for 30 min reduced the incidence of the 

disease in the field, but the pathogen was not eliminated from the seed. 

Hydrochloric acid treatment (31.5% w/w) at 1:20 diluted concentration 

eradicated the pathogen from seed and no disease was observed in the 

field; however, seed germination was adversely affected. Treatment of 

cucumber seeds with 10% Clorox ® (sodium hypochlorite) for 1, 10, or 

15 min, with 1:1000 diluted mercuric chloride solution for 10 min, or 

with 400ppm streptomycin sulfate for 10 min, also reduced seed 

infection, but hot-water treatment at 55˚C for 25–30 min was not 

effective (Vincent-Sealy and Brathwaite, 1982). 

In this study, several chemical seed treatments were evaluated for 

the elimination of Xcc from naturally infested pumpkin seeds and their 

effects on seed germination were noted. 


CHEMICAL SEED TREATMENTS. Chemical treatments tested were 

hydrogen peroxide (Fisher Scientific) at 3%; copper hydroxide 

(Kocide 101, Griffin LLC, Valdosta, GA) plus mancozeb (Dithane DF, 

Griffin LLC) at 0.36g + 0.27g, respectively, per 100ml of sterile 

distilled water; peroxyacetic acid at 1% (BioSide HS 5.2%, Enviro- 

Tech, Modesto, CA); and sodium hypochlorite at 1%. Sterile distilled 

water was used as control. The experiment was repeated twice. 

APPLICATION OF THE TREATMENTS. Approximately 500 seeds 

weighing 71.2g were used for each treatment. Seeds were added to 

500-ml sterile flasks with 200ml of the prepared chemical solutions. 

Seeds were shaken vigorously (250rpm) at room temperature (RT) for 

15 min. Treated seeds were drained and thoroughly rinsed with sterile 

distilled water. Seeds were placed in surface-sterilized plastic boxes 

containing sterile filter paper and incubated overnight at 4˚C. 

Incubated seeds were put in 200ml of 0.85% cold sterile saline 


solution with one drop of Tween 20 in 500-ml sterile flasks for each 

treatment and were shaken vigorously (250rpm) for 3 hrs at RT. 

Dilutions of 10 -1 and 10 -2 of the slurry for each treatment were 

prepared in 0.85% sterile sodium chloride. From each sample, 50μl of 

the slurry was plated in duplicate for the undiluted solutions and 

dilutions onto Xanthomonas campestris semiselective medium (XCS) 

(Williford and Schaad, 1984). Plates were incubated at 27–29˚C and 

observed over 5–6 days for bacterial growth, and the number of 

colonies counted for each plate. Suspected colonies of Xcc were 

sampled for identification by plating onto yeast extract-dextrose- 

CaC03 medium (YDC), by Biolog carbohydrate utilization plates, and 

by pathogenicity testing. YDC (Wilson et al., 1967) is a nonselective 

medium for the genus Xanthomonas, and was used to identify 

suspected colonies from XCS medium. 

IDENTIFICATION OF SUSPECTED COLONIES ON YDC MEDIUM BY 

BIOLOG AND PATHOGENICITY TESTS. On XCS medium, suspected 

colonies of Xcc were distinguishable in 3–5 days. The colony 

morphology of Xcc was previously observed on XCS medium using 

Xcc LMG 690, type strain for this pathogen. All isolated colonies on 

YDC were additionally tested on Biolog GN2 carbohydrate utilization 

plates. 

Pathogenicity of the suspected Xcc colonies was confirmed on 

Blue Hubbard winter squash (C. maxima). Leaves of 5-week-old 

plants were infiltrated with bacteria using a sterile needleless syringe. 

Inoculum was prepared from 48-hr-old pure colonies grown on YDC 

medium. Inoculum was standardized at OD600=0.3 

spectrophotometrically. Xcc LMG 690 was used as the positive 

control, and both Pseudomonas fluorescens LMG 5849 and sterile 

water were used as negative controls. Two leaves were used for each 

isolate and controls. Inoculated plants were incubated for 24–48 hrs in 

a mist chamber for symptom development. Plants were later 

transferred to the greenhouse for disease development. Pathogenicity 

was determined by the expansion of necrotic lesions and appearance of 

greasy pinpoint spots as observed on the bottom surface of the leaves. 

EFFECTS OF SEED TREATMENTS ON GERMINATION. One hundred 

and ten seeds were counted out for each treatment and placed in 600ml 

sterile beakers. Seeds were covered with 50ml of the chemical 

solutions for each treatment and were soaked for 15 min at RT, with 

occasional mixing of the solutions. Soaked seeds were drained, 

thoroughly rinsed with sterile water, transferred to blotter papers, and 

covered with another layer of blotter paper. Germination was based on 

emergence of the radicle from the seed. Seeds were observed for 5–6 


days for maximal germination and the blotter paper was remoisturized 

as needed. The number of germinated seeds was counted for each 

treatment and the percentage of germination calculated. Germination 

assays were repeated three times. 

DATA ANALYSIS. Seed germination data were analyzed by twoway 

analysis of variance (ANOVA) model by using MINITAB (State 

College, PA) statistical software. 

Results 

EVALUATION OF THE SEED TREATMENTS. Preliminary studies 

were performed for the detection of Xcc in order to confirm the 

presence of the bacteria on naturally infested seeds and to select the 

semiselective media for detection of the pathogen. The pathogen was 

detected only once out of five trials from the seed lot (ca. 10,000 

seeds) examined. The performance of YDC, semiselective media SX, 

and XCS, as recommended by STA Laboratories (personal 

communication, Longmont, CO), was evaluated. XCS medium 

provided a higher recovery rate of Xcc colonies than either YDC or SX 

media. When plating on XCS medium, a known strain of Xcc was 

used to facilitate the distinction of its colony morphology from that of 

saprophytes. On XCS, suspected colonies of Xcc were observed in 3– 

5 days. The suspected colonies were round, almost colorless, and 

translucent; they later became darker, shiny with smooth edges, 

domed, and yellow brown. Saprophytic bacterial growth was reduced 

on XCS medium as compared to growth on YDC medium. Detection 

of Xcc on SX medium was very difficult because many saprophytic 

bacteria covered the medium before the growth of the Xcc colonies 

began. Overall, recovery of the bacteria was low and it required 9 

days for growth of the suspected colonies. 

No bacteria were detected from the copper plus mancozeb, 

peroxyacetic acid, and sodium hypochlorite treatments in undiluted 

and diluted plates of XCS medium in either experiment (Table 1). In 

the first experiment, Xcc colonies were detected in the 3% hydrogen 

peroxide treatment. The number of colonies detected in this treatment 

was 8% of those found on the water control on 1:10 dilution colony 

counts (Table 1). Hydrogen peroxide reduced the number of bacteria 

by 90–92% of those found on the water control of undiluted and 1:10 

diluted colony counts, respectively. In the second experiment, the 

number of colonies detected in the water control was one-tenth of that 

found in the first experiment. No Xcc colonies were detected in seed 

that had been subjected to any of the chemical treatments. Saprophytic 


colonies were not eliminated when the 3% hydrogen peroxide 

treatment was used. 

IDENTIFICATION OF SUSPECT COLONIES ON YDC BY BIOLOG AND 

PATHOGENICITY TESTS. In the first experiment, 15 suspect colonies 

plated on XCS medium were isolated to YDC medium. Six of these 

colonies were from hydrogen peroxide-treated seed and 9 were from 

water-control plates on XCS medium. In the second experiment, 16 

suspect colonies from water-control plates on XCS were transferred to 

YDC. All isolated colonies on YDC medium were identical in colony 

morphology to the positive control Xcc LMG 690. Colonies on YDC 

medium appeared within two days, were distinctive in colony 

morphology, and were shiny, yellow, and mucoid, with round, smooth 

edges. Biolog carbohydrate utilization profiles of the isolates 

were identified at the genus level as Xanthomonas, confirming the 

identity of the isolates identified on YDC. 

Pathogenicity of 21 isolated colonies from both experiments was 

tested on Blue Hubbard winter squash. All the isolates were 

pathogenic (data not shown). Xcc LMG 690 gave positive symptoms 

as described above. 

EFFECTS OF SEED TREATMENTS ON SEED GERMINATION. Data 

analysis showed no statistically significant differences among the 

treatments on seed germination as compared with the water control 

(data not shown). Germination rates were 92% to 98% for all 

treatments. Sodium hypochlorite-treated seeds tended to germinate 

more slowly than the others, but these differences were not apparent 

when the final readings were made. Peroxyacetic acid-treated seeds 

germinated faster than seeds from the other treatments; however, the 

treated seeds had a bleached appearance and weaker radicle 

development in two of the three assays. Copper hydroxide-plusmancozeb-treated 

seeds looked healthy, except for showing weak 

radicle growth in one of the assays. Hydrogen peroxide-treated seeds 

showed no adverse effects on germination. 

Discussion 

Elimination of Xcc from naturally infested pumpkin seeds by 

chemical seed treatments with copper hydroxide plus mancozeb, 

peroxyacetic acid, and sodium hypochlorite was successful as 

determined by dilution plating of seed washings. In this study, 

chemicals were applied in fixed concentrations and time. Pernezny et 

al. (2002) used different concentrations and application times of 

sodium hypochlorite, hydrogen peroxide, and copper hydroxide plus 

mancozeb, and included some other chemicals including the antibiotic 


streptomycin as chemical seed treatments on artificially inoculated 

lettuce seeds to control X. campestris pv. vitians, which causes 

bacterial leaf spot of lettuce. In their study, treatments with 3% and 

5% hydrogen peroxide for 5 and 15 min eliminated bacteria; however, 

the 5% hydrogen peroxide treatment also reduced seed germination 

significantly. Sodium hypochlorite treatment of seeds at 1% 

concentration for 15 min reduced bacterial infestation to as low as 2%. 

Copper hydroxide plus mancozeb treatment significantly reduced the 

seed infestation to 2% or less when applied for 5 and 15 min and at 

three different concentrations: 0.18g copper hydroxide plus 0.14 

mancozeb; 0.24g copper hydroxide plus 0.18g mancozeb; and 0.36g 

copper hydroxide plus 0.27g mancozeb /100 ml of water. 

Table 1. Evaluation of seed treatments for elimination of 

Xanthomonas campestris pv. cucurbitae (Xcc) from naturally infected 

pumpkin seeds. 

Experiment Treatment 

Conc. of Xcc 

(cfu/ml) a 

I. Water control 5000 

Hydrogen peroxide, 3% 

Copper hydroxide + 

mancozeb (0.36g + 0.27 

400 

g/100ml) 0 

Peroxyacetic acid, 1% 0 

Sodium hypochlorite, 1% 0 

II Water control 600 

Hydrogen peroxide, 3% 

Copper hydroxide + 

mancozeb (0.36g + 0.27 

0 

g/100ml) 0 

Peroxyacetic acid, 1% 0 

Sodium hypochlorite, 1% 0 

a 

Colony-forming units per ml (cfu/ml) was calculated by average colony number on 

duplicate 1:10 dilution plates x dilution factor x 20[1000µl (1ml)/50µl of (seed 

slurry)]. 

In our study, seed treatments of sodium hypochlorite at 1%, copper 

hydroxide at 0.36g plus 0.27g mancozeb /100 ml of water, and 

peroxyacetic acid at 1% for 15 min eliminated Xcc on naturally 

infested pumpkin seeds. The 3% hydrogen peroxide treatment did not 

eliminate bacteria, but reduced the infestation by 90–92% as compared 


to those on water-control plates in the first experiment (Table 1). In 

the second experiment, the 3% hydrogen peroxide treatment 

eliminated the pathogen, although recovery of the bacteria in watercontrol 

plates was only 10% of that recovered in the first experiment. 

The differences with recovery of bacteria observed could be due to the 

overgrowth of the plates by saprophytic bacteria or to the 

heterogeneous infestation of the seed lot tested. In preliminary 

experiments for detection of Xcc from this naturally infested seed lot, 

bacteria were detected in only one of five trials conducted. In the one 

successful trial, a high number of Xcc colonies were detected. 

Franken et al. (1991) evaluated agar plating assays for detection of 

X. campestris pv. campestris from naturally contaminated crucifer 

seeds. They tested the efficacy of a centrifugation step prior to plating 

the seed washings, and concluded that this step should be omitted 

since no increase in recovery of the bacteria was obtained. They 

suggested that, theoretically, the centrifugation step could be useful if 

bacterial counts on seeds are low, as low as 1cfu of X. campestris pv. 

campestris in the undiluted seed washing of 50μl plated onto a 9-cm 

Petri dish, and with less than 250cfu of saprophytes present in the 

same plate. However, they indicated that they had not found seed lots 

that met these requirements. They also realized that seed lots could 

have varying levels of saprophytic bacteria depending on the growing 

area and season in which the seed was produced, and that the numbers 

of X. campestris pv. campestris would also vary. 

Roberts et al. (2002) tested several nondestructive methods of 

detection of bacteria for seeds stored in a germplasm collection. They 

used artificially inoculated and naturally infected seeds of several 

crops. Naturally infested pea seeds were soaked in sterile tap water for 

three incubation periods for recovery of Pseudomonas syringae pv. 

pisi, but results were inconclusive due to high sample-to-sample 

variability in naturally infected seed lots. 

Peroxyacetic acid, a peroxygen compound and oxidizing agent, is 

used mainly in the food industry as a disinfectant (Block, 1991). 

Recently, Hopkins et al. (2001) evaluated this oxidizing agent for 

seed-borne eradication of Acidovorax avenae subsp. citrulli, the causal 

agent of bacterial fruit blotch of watermelon. Peroxyacetic acid at 

concentrations of 1600ppm and higher eliminated the seed 

transmission of bacteria to the seedlings in greenhouse grow-out tests, 

with no adverse effects on germination observed. In the current study, 

peroxyacetic acid at 1% concentration eliminated Xcc from infected 

seeds, also with no adverse effect on germination. However, slightly 

weaker root growth was observed in two of the three germination 


assays. This most probably occurred from residual chemical left on 

the seed coats due to inadequate rinsing. Seed coats also appeared 

bleached in all assays. None of the chemical treatments we tested 

adversely affected seed germination; however, seedling vigor was 

slightly reduced with peroxyacetic acid and for one assay with copper 

hydroxide plus mancozeb. Seeds treated with sodium hypochlorite 

tended to germinate more slowly than the others. In order to evaluate 

the effects of seed treatment on seedling vigor and stand appearance, 

grow-out tests using sterilized soils in a temperature-controlled 

greenhouse would be required. Fatmi et al. (1991) treated tomato 

seeds with acidified cupric acetate to control Clavibacter 

michiganensis subsp. michiganensis and found that the adverse effects 

of this chemical on seed germination were eliminated by sowing 

treated seeds in sterilized soil. Germination blotter tests, however, 

showed a significant reduction in germination. Fatmi et al. suggested 

that this reduction could be due to inadequate dispersal of acidic cupric 

acetate residues, whereas the binding of these residues to soil particles 

would enhance seed germination. 

The hydrogen peroxide treatment in our study did not affect 

seedling vigor and germination. This chemical has been used for 

promotion of seed germination for certain crops such as tomatoes and 

legumes. It may promote germination by providing the rapid release of 

oxygen as a respiration stimulant (Copeland and McDonald, 2001) or 

by oxidation of germination inhibitors as in treatment of Zinnia 

elegans seeds (Ogawa and Iwabuchi, 2001). Compared to peroxyacetic 

acid, hydrogen peroxide is relatively inefficient in eliminating bacteria, 

possibly because it has fewer bactericidal properties (Baldry, 1983). 

Baldry compared the bactericidal, fungicidal, and sporicidal properties 

of the two chemicals and found that peroxyacetic acid had strong 

bactericidal properties. Hydrogen peroxide had poorer bactericidal 

properties but was more effective as a sporocide. Hydrogen peroxide 

can easily be decomposed by enzymes such as catalase or peroxidase 

and by heat to harmless end products of oxygen and water, whereas 

peroxyacetic acid is not degraded by these enzymes (Block, 1991). 


Baldry, M. G. C. 1983. Bactericidal, fungicidal and sporicidal properties of 

hydrogen peroxide and peracetic acid. J. Appl. Bacteriol. 54:417–423. 

Block, S. 1991. Disinfection, sterilization and preservation, 4 th ed. Lea and Febiger, 

Malvern, Pa. 

Bryan, M. K. 1926. Bacterial leaf spot on Hubbard squash. Science 63:165. 

Bryan, M. K. 1930. Bacterial leaf spot of squash. J. Agric. Res. 40:385–391. 


Copeland, O. L. and McDonald, M. B. 2001. Principles of seed science and 

technology, 4 th ed. Kluwer Academic Publishers, Norwell, MA. 

Fatmi, M., N. W. Schaad, and H. A. Bolkan. 1991. Seed treatments for eradicating 

Clavibacter michiganensis subsp. michiganensis from naturally infected tomato 

seeds. Plant Dis. 75:383–385. 

Franken, A. A. J. M., C. Van Zeijl, J. G. P. M. Van Bilsen, A. Neuvel, R. De Vogel, 

Y. Van Wingerden, Y. E. Birnbaum, J. Van Hateren, and P. S. Van Der 

Zouwen.1991. Evaluation of a plating assay for Xanthomonas campestris pv. 

campestris. Seed Sci. Tech. 19:215–226. 

Hopkins, D., C. Thompson, J. Hilgren, and B. Lovic. 2001. Wet seed treatment with 

peroxyacetic acid for the control of bacterial fruit blotch of watermelon. 

Phytopathology. 91:S40 (Abstr.). 

Latin, R. 1996. Bacterial spot hits the pumpkin patch. Am. Veg. Grower. 44–46. 

McLean, D. M. 1958. A seed-borne bacterial cotyledon spot of squash. Plant Dis. 

Rep. 42:425–426. 

Moffett, M. L., and B. A. Wood. 1979. Seed treatment for bacterial spot of 

pumpkin. Plant Dis. Rep. 63:537–539. 

Ogawa, K., and M. Iwabuchi. 2001. A mechanism for promoting the germination of 

Zinnia elegans seeds by hydrogen peroxide. Plant Cell Physiol. 42:286–291. 

Pernezny, K., R. Nagata, R. N. Raid, J. Collins, and A. Carroll. 2002. Investigation 

of seed treatments for management of bacterial leaf spot of lettuce. Plant Dis. 

86:151–155. 

Roberts, S. J., J. Brough, and S. Chakrabarty. 2002. Non-destructive seed testing for 

bacterial pathogens in germplasm material. Seed Sci. Tech.. 30:69–85. 

Vincent-Sealy, L., and W. D. Brathwaite. 1982. Bacterial leaf spot of cucumber 

in Trinidad. Trop. Agricl. 59:287–288. 

Williford, R. E., and N. W. Schaad. 1984. Agar medium for selective isolation of 

Xanthomonas campestris pv. carotae from carrot seeds. Phytopathology. 

74:1142. 

Wilson, E. E., F. M. Zeitoun, and D. L. Frederickson. 1967. Bacterial phloem 

canker, a new disease of Persian walnut trees. Phytopathology. 57:618–621. 

Zitter, T. A., D. L. Hopkins, and C. E. Thomas. 1996. Compendium of cucurbit 



DETERMINING DENSITY OF 

PHYTOPHTHORA CAPSICI OOSPORES IN 

SOIL 

C. Pavon and M. Babadoost 

Department of Crop Sciences, University of Illinois, 

1102 S. Goodwin Avenue, Urbana, IL, USA, 61801 

ADDITIONAL INDEX WORDS. Cucurbit, Cucurbita moschata, Cucurbita pepo 

ABSTRACT. A sucrose-centrifugation method was developed to extract oospores 

of Phytophthora capsici from soil. The relationship between the number of 

oospores recovered from soil and the number of oospores incorporated into the 

soil was Ŷ= -0.75 + 1.21X -0.02X 2 (R 2 =0.98), where Ŷ = log10 of the number of 

oospores recovered from soil and X = log10 of the number of oospores in soil. 

The oospores were germinated after treating with 0.1% KMnO4 solution for 10 

min. Using a sucrose-centrifugation method, oospores of P. capsici were 

successfully extracted from soil samples collected from commercial squash 

fields. A real-time quantitative polymerase chain reaction (QPCR) protocol was 

developed to assay the density of P. capsici oospores in soil. PCR-inhibition was 

avoided by extracting oospores from soil using the sucrose-centrifugation 

method. The relationship between the amount of DNA measured and the 

number of oospores of P. capsici included in the test was Ŷ = - 3.57 - 0.54X + 

0.30X 2 (R 2 = 0.93), where Ŷ = log10 (ng of P. capsici DNA), X = log10 (number of 

oospores). 

P 

hytophthora blight of cucurbits, caused by Phytophthora capsici 

Leonian, is one of the most serious threats to production of 

cucurbits, pepper, and eggplant worldwide. P. capsici is a soilborne 

oomycete that infects more than 50 plant species in 15 families 

(Erwin and Ribeiro. 1996; Tian and Babadoost, 2004). It can infect 

plants at any stage of growth, causing seedling damping-off, crown 

rot, foliage blight, and fruit rot. Phytophthora blight can cause yield 

losses of up to 100% in cucurbits and pepper fields (Babadoost and 

Islam, 2003; Hausbeck and Lamour, 2004). At present, there is no 

long-term sustainable solution for disease management, however, a 

combination of cultural practices, fungicide application, and genetic 

resistance can be used to minimize the damages caused by P. capsici 

to vegetable crops (Babadoost and Islam, 2003; Hausbeck and 

Lamour, 2004). 

P. capsici is a heterothallic organism in which two compatible 

mating types, designated as A1 and A2, are needed for sexual 

reproduction. The sexual spore of P. capsici is the oospore, which is 

the primary source of inoculum and the overwintering propagule in the 

soil (Erwin and Ribeiro. 1996). A reliable method for quantifying P. 


capsici oospores in soil is needed to assess survival of the pathogen 

and the potential for disease development in the field. Methods used 

for assessing density of P. capsici in soil have been primarily dilution 

plating and baiting assays but, these methods are time-consuming and 

the accuracy of the assays is questionable (Erwin and Ribeiro, 1996; 

Silvar et al., 2005). 

Sucrose-centrifugation and sieving is basically a method t\hat 

separate particles based on their density and size. This method has 

been used to extract propagules of mycorrhizal fungi (Smith and 

Skipper, 1979), teliospores of Tilletia species (Babadoost and Mathre, 

1998), and nematodes (Jenkins, 1964) from soil. 

Silvar et al. (2005) used a polymerase chain reaction (PCR)-based 

method to detect P. capsici in soil. The protocol they used was for 

detecting the pathogen only and the method cannot determine quantity 

of P. capsici oospores in soil. Also, they reported the presence of PCR 

inhibitory factors in the DNA extracts for molecular detection of plant 

pathogens in soil (Van de Graaf, 2003). Real-time quantitative 

polymerase chain reaction (QPCR) is a relatively new molecular 

technique that has been used to quantify nematodes (Cao et al., 2005), 

viruses (Delanoy et al., 2003), bacteria (Bach et al., 2003), and fungal 

plant pathogens (Hayden et al., 2004: Silvar et al., 2005). 

The objective of this study was to develop a reliable QPCR method 

to quantify P. capsici oospores in soil. 


IN VITRO OOSPORE PRODUCTION. For in vitro production of 

oospores of P. capsici, two plugs of opposite mating types (A1 and 

A2) of the pathogen were grown in 40-ml aliquots of V8-CaCO3 

medium in 250-ml Erlenmeyer flasks at 24°C for 2 mo in darkness. 

Then, oospores were harvested, blending the culture at full speed for 

90s in a Hamilton Beach blender (model 52250, Southern Pines, NC). 

The suspension in the blender was passed through 68- and 38-µm 

metal sieves and the filtrate was collected. The filtrate then was passed 

through a 20-µm Spectra/Mesh nylon filter (Spectrum, Houston, TX). 

The oospores collected on the mesh were washed into a beaker and the 

number of oospores was determined using a spore-counting chamber 

(Hauser Scientific, Horsham, PA). 

OOSPORE EXTRACTION FROM SOIL. Five agricultural soils, 

including a sandy loam, a silt clay, and three silt loam, collected from 

various locations in Illinois, were used in this study to develop the 

procedure for extraction of oospores of P. capsici from soil. The 

calculations were based on the air-dried weight for all of the soils. 


Samples of the five soil types were air dried at room temperature 

on a laboratory bench for 14 days and passed through a 2-mm sieve. 

Predetermined quantities of oospores were added to soil samples and 

thoroughly mixed. The artificially infested soil samples were estimated 

to have10 1 , 10 2 , 10 3 , 10 4 , and 10 5 oospores of P. capsici per 10g of airdried 

soil. 

Each 10-g infested soil sample was suspended in 400ml tap water 

with two drops of Tween-20 and shaken for 15 min. The soil 

suspension was passed through nested 106-, 63-, and 38-µm metal 

sieves. The material caught on the 38-µm sieve was washed using a 

sprinkler with a gentle stream of water and the filtrate was collected. 

This suspension (approximately 2L) was then passed through a 20-µm 

mesh filter. The materials caught on the 20-µm mesh were washed into 

two 50-ml centrifuge tubes and spun for 4 min (900 x g) using a 

bench-top centrifuge. The supernatant was discarded and the pellet 

was suspended in 30ml of 1.6M sucrose solution. This suspension was 

centrifuged for 45s (190 x g) and the supernatant was passed through 

the 20-µm mesh. The pellet was resuspended in the sucrose solution 

and centrifuged again (45s, 190 x g). The procedure was repeated six 

times to maximize oospore recovery from soil. The materials caught 

on the 20-µm mesh were washed into a 50-ml centrifuge tube and spun 

for 4 min (900 x g). The pellet was resuspended in 0.5 to 1.5ml of 

distilled water, depending on the original number of oospores added to 

the soil, and the number of oospores was determined using a sporecounting 

chamber. 

The oospore recovery at each inoculum level for each of the five 

artificially infested soils was determined using four replicates of 10-g 

soil sample and with four oospore counts per replicate (a total of 16 

spore counts for each inoculum level for each soil type). 

EXTRACTION OF OOSPORES FROM FIELD SOIL. Eight commercial 

fields in three counties, with a history of Phytophthora blight, were 

sampled to test the soil for presence of P. capsici oospores. In each 

field, 20 subsamples of soil were taken from 0–20cm deep from 

approximately 0.4ha area using a soil probe. The subsamples from 

each field were mixed thoroughly and a 1-kg sample was taken. Four 

replicates of 10-g soil samples from each field were processed using 

the procedure described above, to extract and enumerate oospores of 

P. capsici. 

OOSPORE GERMINATION. Extracted oospores from soil were 

germinated to determine their viability. The oospores were plated onto 

the semiselective medium PARP in Petri plates (50 oospores per 

plate). The effect of potassium permanganate (KMnO4) treatment on 

oospore germination was evaluated by suspending oospores in 0.02, 


0.04, 0.1, and 0.2% of KMnO4 solution in water for 10 min. After the 

treatment, oospores were washed three times with sterile-distilled 

water and plated onto PARP medium. The plates were incubated at 

24°C under fluorescent light for 4 days and the percentage of 

germinated spores was determined. Four replica plates of oospore 

germination were included for each treatment. Single colonies of 

germinated oospores from commercial fields were transferred to lima 

bean agar in Petri plates and P. capsici colonies were identified from 

colonies of other Phytophthora and Pythium species based on 

sporangial morphology. 

REAL-TIME QPCR QUANTIFICATION OF OOSPORES. P. capsici 

DNA was extracted from the oospores using a protocol based on 

FastDNA kit (Qbiogene, Inc., Carlsbad, CA), which was modified for 

removal of PCR inhibitors by Malvick and Grunden (2005). The 

QPCR assays were conducted in a 96-well plate format with the ABI 

PRISM 7000 Sequence Detection System instrument and software (PE 

Applied Biosystems, Foster City, CA). P. capsici primers were: 

forward, 5’-GGA ACC GTA TCA ACC CTT TTA GTT G-3’; reverse, 

5’-CGC CCG GAC CGA AGT C-3’; and probe, 5’-6FAM-TCT TGT 

ACC CTA TCA TGG CG-MGBNFQ-3’. The manufacturer’s 

instructions were followed, except that 25-µl reaction mixtures were 

used instead of 50µl. Thermal cycling conditions consisted of 10 min 

at 95°C followed by 40 cycles of 15 s at 95°C and 1 min at 60°C, in 

addition to a 2-min preincubation at 50°C. 

The modified QPCR procedure was used to detect P. capsici 

oospores in soil samples collected from the eight commercial fields. 

First, oospores were extracted using the sucrose-centrifugation 

method. Then the extracted oospores were tested to determine the 

quantity of P. capsici oospores. 


IN VITRO OOSPORE PRODUCTION. Both mating types of P. capsici 

(A1 and A2) were identified among the isolates tested. The three 

pairings used for oospore production yielded almost the same amount 

of oospores per plate. The number of oospores per plate after 2 mo 

ranged from 4.41 x 10 5 to 6.72 x 10 5 (mean 5.56 x 10 5 ) oospores per 

plate. 

OOSPORE EXTRACTION FROM SOIL. Oospores of P. capsici were 

successfully recovered from all five artificially infested soil types. 

Overall, 50.9% of oospores incorporated into the soil were recovered. 

There was no significant difference in oospore recovery among the soil 

types. The relationship between the number of oospores recovered 


from soil and the number of oospores incorporated into the soil was 

Ŷ= -0.75 + 1.21X -0.02X 2 (R 2 =0.98), where Ŷ = log10 of the number 

of oospores recovered from soil and X = log10 of the number of 

oospores in soil (Figure 1). The average recovery rates of P. capsici 

oospores from the soils were 25.5 (13.5-37.8), 43.7 (33.0-54.4), 53.6 

(42.9-64.4), 60.3 (49.5-71.0), and 72.1% (61.3-82.8%) from soil 

samples containing 10 1 , 10 2 , 10 3 , 10 4 , and 10 5 oospores per 10g, 

respectively. There was no significant difference in oospore recovery 

between soil samples containing 10 4 and 10 5 oospores per 10g. 

Percentage of oospores recovered from soil samples with 10 1 oospores 

per 10g was significantly lower than the percentage of oospores 

recovered from the samples with 10 2 , 10 3 , 10 4 , and 10 5 oospores per 

10g. Similarly, percentage of oospores recovered from soil 

_ = - 0.75 + 1.21X - 0.02X 2 

R 2 = 0.98 

Fig. 1. Relationship between number of oospores recovered from soil and 

number of oospores incorporated into soil. 

samples with 10 2 oospores per 10g soil was significantly lower than 

those of soil samples with 10 4 and 10 5 oospores per 10g soil. In 

addition, the percentage of oospores recovered from soil samples with 

10 3 oospores per 10g was not significantly different than the 

percentage of oospores recovered from samples with 10 2 , and 10 4 

oospores per 10g. 


OOSPORES IN COMMERCIAL FIELDS. We extracted oospores from 

soil samples collected from all eight commercial fields in Illinois. The 

number of oospores recovered from commercial fields ranged from 

694 to 2,467 per 10g of soil. There was no significant difference in 

diameter of oospores recovered from different fields. Number of P. 

capsici oospores extracted from commercial fields ranged from 79 to 

529 per 10g soil. The rate of P. capsici oospores of the total number of 

oospores extracted from soil samples from commercial fields ranged 

from 9.2% to 18.5% (mean 14%). 

OOSPORE GERMINATION. Germination rates of oospores produced 

in vitro with the pretreatment with 0, 0.02, 0.04, 0.1, and 0.2 solution 

of KMnO4 were 15.2, 29.8, 27.6, 44.5, and 40.7%, respectively. The 

rates of oospore germination for 0.1 and 0.2% solution of KMnO4 

were not significantly different. But the rates of oospore germination 

after treating with either 0.1 or 0.2% solution of KMnO4 were 

significantly higher than those of treatments with 0.02 and 0.04% 

KMnO4 solutions. Therefore, for germination of oospores extracted 

from soil samples from commercial fields, we used a 0.1% KMnO4 

solution and oospores were treated for 10 min. Germination rates of 

oospores extracted from commercial fields ranged from 18.8% to 

51.1% (mean 36.8%). In addition to P. capsici, P. sojae and Pythium 

spp. were identified in the culture plates. 

REAL-TIME PCR QUANTIFICATION OF OOSPORES. The 

relationship between the number of oospores and P. capsici DNA 

quantity was Ŷ = -3.57 - 0.54X - 0.30X 2 (R 2 = 0.93), where Ŷ = log10 

(ng of P. capsici DNA), X = log10 (number of oospores) (Figure 2). 

According to this model, the DNA quantities corresponding to 10 1 , 

10 1.5 , 10 2 , 10 2.5 , 10 3 , 10 3.5 , 10 4 , 10 4.5 and 10 5 oospores were 1.4 × 10 -4 , 

1.9 × 10 -4 , 3.5 × 10 -4 , 9.0 × 10 -4 , 3.2 × 10 -3 , 1.6 × 10 -2 , 1.5 × 10 -1 , 1.2 × 

10 0 , and 1.7 × 10 1 ng, respectively. 

Using the QPCR procedure, we detected P. capsici in all field soil 

samples tested. There was no PCR-inhibition in the DNA extracts 

from oospores extracted from soil using the sieving-sucrosecentrifugation 

method, although some soil particles and organic matter 

were associated with the oospores. PCR inhibition, however, was 

found in the DNA extraction directly from soil. 

Using the sieving-centrifugation with sucrose solution method, we 

were able to recover oospores of P. capsici from soil with a spore 

density as low as 10 spores per g of soil. The results showed that the 

method can be used to determine density of P. capsici oospores in 

fields with different soil types. There were, however, some difficulties 

in the identification of the oospores extracted from naturally infested 

commercial field soils, because oospores of other Phytophthora and 


Pythium species were co-extracted by the procedure. These difficulties 

were overcome by germinating and identifying oospores based on 

morphological characteristics of sporangia. Thus, the sieving-sucrosecentrifugation 

procedure can be used to estimate P. capsici oospores in 

soil in areas where Phytophthora blight of vegetables is a problem, or 

any other area suspected of having P. capsici. 

_ = -3.57 - 0.54X + 0.30X 2 

R 2 = 0.93 

Fig. 2. Relationship between quantity of DNA and number of oospores used in 

quantitative polymerase chain reaction tests. 

Difficulty in the in vitro germination of oospores of Phytophthora 

species has been reported. These difficulties have been overcome by 

pretreatment of oospores with KMnO4 solution. For example, 

germination of oospores of P. parasitica was improved by a 

pretreatment with 0.25% solution of KMnO4 for 20 min. Similarly, 

germination of oospores of P. megasperma was enhanced by a 

pretreatment of 0.05% of KMnO4 for 10 min. Similarly, we increased 

percent of germination of P. capsici oospores from about 10% to 50% 

by pretreatment with 0.1% KMnO4 solution. 

QPCR is useful for detecting and quantifying nonculturable and 

slow-growing organisms. Some reports indicated that P. capsici can be 

outgrown by closely related and fast-growing Pythium spp. The QPCR 

procedure used in this study is a reliable method for quantifying P. 

capsici oospores in soil. The procedure detected P. capsici in all field 

soil samples tested. This QPCR protocol is a fast, accurate, and 

sensitive method for quantifying P. capsici oospores in soil. Also, the 

combination of sieving-centrifugation and QPCR is effective in 

eliminating PCR inhibitors that affect PCR tests in direct assays from 

soil samples. 



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CHARACTERISTICS OF DOUBLE-HAPLOID 

CUCUMBER (CUCUMIS SATIVUS L.) LINES 

RESISTANT TO DOWNY MILDEW 

(PSEUDOPERONOSPORA CUBENSIS [BERK. 

ET CURT.] ROSTOVZEV) 

J. Sztangret-Wiśniewska 

Plant Breeding and Acclimatization Institute, Mlochow Research Center, 

19 Platanowa Str., 05-831 Mlochow, Poland 

T. Gałecka, A. Korzeniewska, L. Marzec, G. Kołakowska, U. 

Piskurewicz, M. Śmiech, and K. Niemirowicz-Szczytt 

Department of Plant Genetics, Breeding, and Biotechnology, Warsaw 

Agricultural University, Nowoursynowska St., 02-776 Warszawa, Poland 

ADDITIONAL INDEX WORDS. DH lines, diploidization, haploid embryo rescue, 

irradiated pollen 

ABSTRACT. Downy mildew caused by Pseudoperonospora cubensis (Berk et Curt.) 

Rostovzev is one of the most important diseases of field-grown cucumbers. A 

study was designed to determine the feasibility of obtaining double-haploid (DH) 

lines from cucumber hybrids tolerant to Pseudoperonospora cubensis that would 

result in creation of diverse, genetically fixed lines that are homozygous for 

resistance to downy mildew. On average, 20% of embryos generated in two 

experiments were converted to haploid plants. Utilization of two diploidization 

methods, direct regeneration and colchicine treatment, resulted in the doubling of 

these haploids (53% and 33% conversion rate, respectively). This is the first report 

on DH cucumber lines tolerant to downy mildew. 

E 

fficient generation of haploid (H) and double-haploid (DH) lines 

in cultivated plants can significantly contribute to breeding 

efforts. DH cucumber lines characterized by useful agronomic 

characteristics could be used as inbred lines in hybrid production. This 

study was undertaken to test the feasibility of production of DH lines that 

are resistant to downy mildew, one of the most important diseases of 

cultivated cucumber. 

Two methods of cucumber haploid production are known. The first 

method utilizes an in vitro culture of either ovules or ovaries. The second 

method, called haploid parthenogenesis, consists of pollination with 

irradiated pollen followed by haploid embryo rescue and in vitro culture 

This research was partially supported by a grant from AWRSP – Poland. The authors 

thank E. Gniazdowska and J. Szewczyk for careful technical assistance. 


(Gemes-Juhasz et al., 2002; Truong-Andre, 1988; Niemirowicz-Szczytt 

and Dumas de Vaulx, 1989; Sauton, 1989; Przyborowski and 

Niemirowicz-Szczytt, 1994; Caglar and Abak, 1996). 

In 1990 the Dutch seed company Nunhems Zaden BV (Haelen, The 

Netherlands) patented a method for the ovule-culture method (patent NP- 

374755); however, no data were provided on the efficacy and application 

of this invention. More recently, a similar approach was reported by 

Gemes-Juhasz et al. (2002). In the latter, longitudinally sliced 

unpollinated ovaries were harvested six hours before anthesis of the 

hermaphrodite flower and cultured in vitro on various media at 24°, 28°, 

and 35°C. The best result was regeneration of plants from 7.1% of 

approximately 150 ovaries, of which 87.7% were haploids. This result 

was obtained by culturing the ovaries at 35 o C after 2–4 days of treatment 

on induction medium containing thidiazuron (TDZ). No information was 

reported on morphology and number of DH plants obtained in that study. 

The second approach, the embryo-rescue method, has given in 

general better results than ovary culture (Truong-Andre, 1988; 

Niemirowicz-Szczytt and Dumas de Vaulx, 1989; Sauton, 1989; 

Przyborowski and Niemirowicz-Szczytt, 1994; Caglar and Abak, 1996). 

In this method, pollination with an irradiated pollen resulted in direct 

formation of haploid embryos that developed into haploid plants. For 

instance, Przyborowski and Niemirowicz-Szczytt (1994) reported that an 

average of 1.2 to 3.1 embryos per fruit were obtained in six cucumber 

genotypes and approximately 50% of the excised haploid embryos 

developed into plants. 

Since cucumber haploid plants are sterile (Przyborowski and 

Niemirowicz-Szczytt, 1994), their use in plant breeding requires 

chromosome doubling. Two methods for chromosome doubling are in 

use. The first method involves colchicine treatment of the apical 

meristem (Nikolova and Niemirowicz-Szczytt, 1996), and the second 

method employs direct regeneration of plants from young haploid leaf 

tissue (Faris et al., 2000). The method of repeated treatment of the 

meristems with colchicine solution resulted in generation of 20.9% 

diploid plants. The method of direct regeneration of plants resulted in 

29.5% of diploid plants. 

The process of H and DH plant production is both time- and laborconsuming. 

Therefore, it is important to estimate at a relatively early 

stage of employment of this technology the potential breeding value (i.e., 

presence of valuable characteristics, such as resistance to downy mildew) 

of the obtained plants. Moreover, it is important to be able to recover DH 

plants from genetically diverse material. To evaluate the efficacy of DH 

production in different genotypes, a study was designed to determine 


whether DH cucumber plants could be obtained effectively from three 

cucumber hybrids resistant to Pseudoperonospora cubensis. 


HAPLOID PLANT PRODUCTION. The haploid embryo production 

utilized the process of haploid parthenogenesis (Przyborowski and 

Niemirowicz-Szczytt, 1994). Female flowers of three commercial 

hybrids resistant to Pseudoperonospora cubensis, ‘Krak’, ‘Frykas’, and 

‘Izyd’, and one susceptible hybrid, ‘Polan’, were pollinated with pollen 

irradiated at 0.3kGy gamma rays, cobalt source, 0.80Gy/min at the 

Department of Food Safety and Public Health, Warsaw Agricultural 

University. Flowers of 40 plants of each cultivar grown at a Warsaw 

Agricultural University greenhouse at the Wolica Experimental Station 

were used in the pollination. Three to five weeks after pollination, 

presence of individual haploid embryos was detected using a 

stereomicroscope. The embryos were removed aseptically from the 

ovaries and transferred to E20A medium (Sauton and Dumas de Vaulx, 

1987). Embryos that developed into plants were maintained on MS 

medium (Murashige and Skoog, 1962). The experiment was conducted 

during the months of May and June and then August and September, 

1999. 

DIPLOIDIZATION METHODS. Two methods of diploidization were 

used. One method consisted of direct regeneration of plants from young 

haploid leaf tissue and the second method was a colchicine treatment of 

apical meristems of H plants. Direct regeneration from leaf explants of 

haploid plants obtained in the spring of 1999 (May and June) was 

conducted in the autumn (September–November) of the same year, 

whereas the haploids obtained in the summer/autumn of 1999 (August– 

September) were used in regeneration during winter/spring of 2000 

(January–April). 

DIRECT-REGENERATION METHOD. In vitro-grown haploid plants 

that were about 10cm were used in the direct-regeneration method. The 

first and second leaves, closest to the apical shoot meristem and between 

0.50 to 0.75cm 2 , were cut into 2- to 3-mm 2 squares (explants), placed on 

a modified Murashige and Skoog (MS) medium (Burza and Malepszy, 

1995), and incubated in the dark at 25±2 o C for 11 to 15 days. Next, the 

explants were transferred to a hormone-free medium (1/2 MS 

macroelements, complete MS microelements, complete MS vitamins), 

and cultured in a 16-h photoperiod (54μmol m -2 s -1 ) until shoots were 

formed on the cut edge of the explant. The explants and the shoots were 

transferred to a fresh medium every 15 days. To induce rooting, three to 

five shoots detached from the explant-tissue shoots were placed 


aseptically in 0.33-dm -3 glass jars containing 1/2 MS medium containing 

1mg /dm -3 indole-3-acetic acid (IAA). Rooted plants were grown in 0.6dm 

-3 pots filled with universal substrate for cucumber (Hollas, Pasłęk, 

Poland) under greenhouse conditions (23/25°C, 16/h day, 1000μmol m -2 

s -1 ). 

The ploidy level of actively growing young plants was estimated by 

flow-cytometry according to Galbraith et al. (1983). In brief, two weeks 

after transplanting to soil, two–three top leaves were collected, the nuclei 

were extracted and stained with 4’,6-diamidino-2-phenylindole (DAPI) 

for flow-cytometry measurements using a PARTEC model ANII 

cytometer (Partec GmbH, Münster, Germany). 

MERISTEM COLCHICINE TREATMENT. All regenerated H plants were 

subjected to colchicine treatment according to Nikolova and 

Niemirowicz-Szczytt (1996). In this method H plants were maintained 

aseptically in glass jars on 1/2 MS medium. Each plant was clonally 

propagated by cutting the stem into segments, each segment containing 

one leaf. After 10 to15 days each cutting produced four to five leaves 

and roots. Colchicine solution (0.1% in water) was applied to apical 

meristems of actively growing plants by placing a 50-μL drop on the 

meristem. At least 15 clones of each H plant were treated. There were 

three successive applications with a 24-h interval between treatments. 

This method resulted in the death of approximately 50% of the treated 

plants. New shoots developed from the main and auxiliary meristems of 

the surviving plants after three to four weeks. These shoots were 

removed and transferred to jars containing 1/2 MS medium and 1mg 

/dm -3 IAA for root induction. The ploidy level of these shoots was 

estimated by flow-cytometry as described above. 

PHENOTYPIC OBSERVATIONS OF DH LINES. Visual observations of 

DH lines were made on 20 plants per line grown at the Warsaw 

Agriculture University experimental field nursery. Four-week-old 

seedlings were transplanted to the field on May 25, 2001, with 50 x 120cm 

spacing between plants. Plants were grown in a randomized block 

design with four replicates of 5 plants each. They were evaluated for the 

following traits: sex expression (female or monoecious), growth type 

(vine, intermediate, dwarf), leaf color (A = dark green, B = dark 

green/light green, C = light green [Green group 135, R.H.S Colour Chart, 

The Royal Horticultural Society London, Flower Council of Holland, 

Leiden, 1995]), and susceptibility to downy mildew. The evaluation of 

plant infection was made in the field on August 15–20, 2001. Symptoms 

were assessed using a scale from 0 to 9 (0 = no visible symptoms; 1 = 

several necrotic spots on leaves; 3 = 25% of infected leaves; 5 = 50% 

infected leaves; 7 = 75% infected leaves; and 9 = 100% infected leaves, 

leaf decay), according to Jenkins and Wehner (1983) and Doruchowski 


and Łąkowska-Ryk (1992). In each class, a disease index (DI) was 

calculated by averaging the scores for each DH line according to 

Happstadius et al. (2003). A one-way analysis of variance for DI was 

performed using the Statgraphics Plus 4.1 (Manugistics, Rockville, MD) 

statistical software package. 

Results 

CUCUMBER HAPLOID DEVELOPMENT. Fruit developed from about 

50% of pollinated flowers and numerous haploid embryos and plants 

were obtained in four hybrid varieties as a result of pollination with 

irradiated pollen (Table 1). 

Typically, 2–3 fruit per plant for a total of 119 fruit were obtained 

from pollination of 40 plants in each cultivar. The number of embryos 

that developed from seeds within five weeks after pollination ranged 

between 70 and 116 per cultivar. In total, the number of embryos 

developed from the four cultivars was similar in each season (356 in 

spring and 333 in summer/autumn). Average number of embryos that 

developed into plants varied from experiment to experiment and from 

cultivar to cultivar, ranging between 7.8 and 41.9%. Haploid plants were 

obtained in each genetic background; the results were consistent over the 

two years. Using cytometric evaluation, all plants were classified as 

monohaploids (n = 7). These plants were maintained in vitro and 

vegetatively propagated to provide material for chromosome doubling. 

CHROMOSOME DOUBLING. Chromosome doubling was 

accomplished using two methods applied consecutively: (1) direct 

regeneration from young leaf explants (Tables 2 and 3), and (2) 

colchicine treatment of the apical meristem (Tables 4 and 5). 

Not all H plants developed juvenile leaves that were suitable for 

direct regeneration during the first six months when the juvenile leaves 

were collected (96.3% and 42.4% in the first and the second experiment, 

respectively). 

The results of plant regeneration in five genetically different haploid 

clones are shown in Table 3. The number of regenerated plants ranged 

between 2 and 45, while the cumulative percentage of diploids in these 

clones was 53.3%. 

A summary of results of six experiments with colchicine treatment is 

shown in Table 4. Out of a total of 245 plants, 132 plants (53.9%) 

developed new shoots after colchicine treatment. Ploidy estimation on 

rooted cuttings revealed that 32.6% were diploid, 57.6% were haploid, 

and 9.8% were either mixoploid and/or chimeras. Table 5 shows the 

results of colchicine treatment on five haploid clones, represented by 15– 

27 plants. The mean percentage of shoot recovery after colchicine 


treatment for all five clones was 61.5%, while the diploid recovery was 

25.0%. Not all haploid clones could be “diploidized” (e.g., Clone no. 2). 

Table 1. Cucumber haploid embryo and plant development from four F1 

donor cultivars in two environments. 

a. Spring 1999 

Embryos 

regener- 

F1 donor cultivar 

Fruits 

(no.) 

Embryos 

(no.) 

Plants 

(no.) 

ated into 

plants (%) 

Polan 

132 116 9 7.8 

Krak 

102 76 16 21.1 

Frykas 

127 79 15 19.0 

Izyd 

120 85 14 16.5 

Total 481 356 54 15.2 

b. Summer/autumn 1999 

Polan 

123 92 20 21.7 

Krak 

100 74 31 41.9 

Frykas 

130 70 25 35.7 

Izyd 

117 97 9 9.3 

Total 470 333 85 25.5 

Table 2. Direct regeneration of plants from young leaf explants of 

cucumber haploid plants in two environments. 

a. 1999 

Total no. Explant Regenerating 

F1 donor haploid donor plants donor plants 

cultivar 

Polan 

Krak 

Frykas 

Izyd 

plants No. % No. % 

9 

16 

15 

14 

9 

15 

14 

14 

100.0 

93.7 

93.3 

100.0 

6 

4 

4 

6 

66.7 

25.0 

26.7 

42.9 

Total 54 52 96.3 20 37.0 

b. 1999/2000 

Polan 

Krak 

Frykas 

Izyd 

20 

31 

25 

9 

9 

9 

11 

7 

45.0 

29.0 

44.0 

77.8 

3 

3 

12 

5 

15.0 

9.7 

48.0 

55.5 

Total 85 36 42.4 23 27.1 


Table 3. Ploidy level and number of plants regenerated from leaf 

explants of five haploid cucumber clones. 

Haploid plant 

No. 

regenerated 

Ploidy level of plants* 

plants 2n 1n 

Mixoploids 

and 

chimeras 

1 2 2 0 0 

2 45 16 17 12 

3 29 11 15 3 

4 13 11 2 0 

5 31 24 6 1 

Total 120 64 40 16 

(53.3%) (33.3%) (13.4%) 

*Ploidy determined using a flow-cytometric procedure as described by Galbraith et al. 

(1983). 

Table 4. The number and ploidy of plants recovered from colchicine 

treatment of the apical meristem. 

Exp. 

no. 

No. 

treated 

haploid 

plants 

Shoot 

Recovery 

No. % 

No. plants according to 

ploidy level* 

Mixoploids 

2n 1n and chimeras 

1 45 20 44.4 8 9 3 

2 30 12 40.0 6 3 3 

3 33 24 72.7 5 13 6 

4 42 36 85.7 11 25 0 

5 25 14 56.0 3 11 0 

6 70 26 37.1 10 15 1 

Total 245 132 53.9 43 76 13 

(32.6%) (57.6%) (9.8%) 

*Ploidy determined using a flow-cytometric procedure as described by Galbraith et al. 

(1983). 

CHARACTERISTICS OF DH LINES. Table 6 presents data from DH 

lines derived from the three hybrid cultivars tolerant to downy mildew 

and the susceptible control hybrid ‘Polan’. In each line plants were 

morphologically homogenous. 

Most numerous fertile DH plants were obtained from the cultivar 

‘Izyd’. Because the group of 14 lines derived from the cultivar ‘Izyd’ 

was the largest, it represents the majority of the morphological 


Table 5. Number of cucumber plants obtained from colchicine treatment 

from five haploid clones. 

Shoot No. plants according 

Hap- No. treated recovery 

to ploidy level 

loid 

clone 

haploid 

plants No. % 2n 1n Mixoploids 

and chimeras 

1 21 15 71.4 7 6 2 

2 17 6 35.3 0 3 3 

3 24 18 75.0 3 12 3 

4 27 20 74.0 5 14 1 

5 15 5 33.3 1 2 2 

Total 104 64 61.5 16 37 11 

(25.0%) (57.8%) (17.2%) 

* Ploidy determined using a flow-cytometric procedure as described by Galbraith et al. 

(1983). 

Table 6. Phenotype characteristics of cucumber DH (10 plants per line), 

grown in the open field in 2001. 

Suscepti- 

DH line 

Growth Leaf bility to downy 

denomination Sex type color mildew DI 

F-1 100 F V C 5.90* 

F-2 310 F V C 4.25 

F-3 311 F V C 4.40 

I-1 18 M V C 8.50* 

I-2 89 F V B 8.80* 

I-3 114 M V B 8.75* 

I-4 150 M V B 8.75* 

I-5 228 F I A 8.75* 

I-6 243 F I B 3.85 

I-7 275 M V A 2.65* 

I-8 280 M V B 8.65* 

I-9 290 M V B 6.00* 

I-10 321 F V A 6.95* 

I-11 502 F I A 2.65* 

I-12 503 F I A 2.60* 

I-13 504 F I A 4.95* 

I-14 506 M V B 8.70* 

K-1 25 F V A 4.55 

K-2 228 F V A 4.55 

P-1 356 F V C 8.45* 

Izyd F1 (standard) F V B 4.15 

Sex expression: F = female, M = monoecious; Growth type: V = vine, I = intermediate, 

D = dwarf; Leaf color: A = dark green, B = dark green/light green, C = light green. 


observations that were made. The lines within this group differed in sex 

expression, growth type, leaf color, and susceptibility to downy mildew 

(disease ratings between 2.6 and 8.8). The cultivar ‘Izyd’ is gynoecious 

with indeterminate (vine) growth, has a leaf color rating of “B,” and is 

tolerant to downy mildew (Table 6). Out of the 14 DH lines derived from 

‘Izyd’, 3 lines were more tolerant to downy mildew than the original 

cultivar. Increased resistance was not observed among the DH lines 

developed from the other two resistant varieties. 

Discussion 

In this study we demonstrated a complete procedure for cucumber 

DH line production based on previous partial investigations 

(Przyborowski and Niemirowicz-Szczytt, 1994; Nikolova and 

Niemirowicz-Szczytt, 1996; Faris et al., 2000). 

The frequency of embryo development was comparable in the two 

experiments performed in this study (about 0.7 embryo per fruit). This 

efficiency is not considered high when compared to the efficiency of 1.2 

to 3.1 embryos per fruit published earlier (Przyborowski and 

Niemirowicz-Szczytt, 1994). However, the results presented here are 

unique in the fact that embryos and haploid plants were obtained from 

each of the four tested varieties. 

As reported here, the frequency of plant regeneration, calculated by 

dividing the total number of plants by the number of ovaries that 

produced embryos, ranges from 0.07% to 0.31%. Frequency of 0.0 to 

7.1% was reported by Gemes-Juhasz et al. (2002); however, in that study 

the plantlets were obtained through ovary culture. 

The results presented here are similar to those observed in 

muskmelon (Cucumis melo L.), which has been studied to a greater 

extent than cucumber (Sauton and Dumas de Vaulx, 1987; Cuny et al., 

1993; Ficcadenti et al., 1995; Abak et al., 1996; Ficcadenti et al., 1999, 

Lotfi et al., 2003). 

It was reported that ovary culture results in fewer plants and in the 

increase in the percentage of mixoploids as compared to haploid embryo 

rescue (Ficcadenti et al., 1999; Lotfi et al., 2003). In contrast, pollination 

with irradiated pollen, embryo rescue, and subsequent in vitro culture 

was reported to produce stable muskmelon haploid plants (Sauton and 

Dumas de Vaulx, 1987; Cuny et al., 1993; Ficcadenti et al., 1995; Abak 

et al., 1996). The latter method clearly produces more desirable results. 

Haploid plant production is only the first step in DH production. 

Cucumber H embryos do not double spontaneously during in vitro 

culture, and efficient methods of chromosome doubling are needed. 

Colchicine treatment, the technique most frequently used for 


chromosome doubling, has been applied to cucumber DH production 

previously (Nikolova and Niemirowicz-Szczytt, 1996). The most 

effective procedure (28% of diploids) proved to be colchicine treatment 

of young plants grown in vitro. The results of the current study are 

similar to the previously published data, but indicate that not every 

haploid clone is amenable to doubling. 

Another drawback of the colchicine treatment is the death of 38.5% 

to 46.1% of plants (Tables 4 and 5). A complementary haploid-doubling 

procedure of direct regeneration from haploid leaf explants was applied 

to augment the low recovery of diploids (25.0 to 32.6%) resulting from 

the application of colchicine (Tables 4 and 5). 

Direct regeneration from leaf tissue allows the production of both 

haploid and diploid plants (Faris et al., 2000), and has the additional 

benefit of multiplying the haploid plants, which in turn provides more 

plant material for colchicine treatment. However, only a portion of the 

haploid plants (27.1 to 37.0%) was amenable to in vitro regeneration 

(Table 2). Furthermore, only some (53.3%) of the regenerated plants 

were diploid. This result confirms a similar observation made earlier in 

our laboratory (Faris et al., 2000). 

The number of H plants obtained from H embryos in both 

experiments (Table 1) was relatively high, with 20.2% embryos 

developing into plants. However, the outcome of the two methods of 

chromosome doubling is not satisfactory. Only 20 DH plants (14.4 % of 

all haploid plants, Table 2) produced viable seeds. This frequency is low 

as compared to the frequency reported for Triticum aestivum (60–81%) 

(Lefebvre and Devaux, 1996) and Hordeum vulgare (64%) (Furusho et 

al., 1999). 

The reason for the low fertility and consequently low seed set is 

unclear. It is possible that the diploid plants are either partial chimeras or 

even low-level mixoploids. Fertility problems were also reported by 

Gemes-Juhasz et al. (2002). 

There is an additional difficulty in achieving self-pollination in DH 

plants, namely, there is a need for the induction of male flowers in 

otherwise female plants by the application of silver nitrate. In our 

experience induction of male flowers is especially difficult in plants 

derived from in vitro culture. 

It appears that the difficulty of diploidization of cucumber and melon 

plants is the main reason for insufficient production of DH lines. For 

example, the recent report by Lotfi et al. (2003) describes a spontaneous 

appearance of two DH and one mixoploid melon plant in an in vitro 

culture of a total of 175 haploid plants, whereas 10 diploid and 100 

mixoploid plants were obtained after treating 167 shoots with colchicine. 


In order to be useful in breeding, DH lines must be produced in large 

quantities. It appears that the most serious obstacle to cucumber DH line 

production occurs at the stage of haploid doubling (Tables 2, 3, 4, and 5). 

Such limitation was not reported in Triticum aestivum (Lefebvre and 

Devaux, 1996; Inagaki et al., 1998) or Hodeum vulgare (Furusho et al., 

1999). In the studies cited above, the colchicine treatment gave better 

results than in cucumber. 

Relatively facile production of numerous DH lines (65 to 97), as 

described in wheat (Inagaki et al., 1998), allows evaluation of agronomic 

characteristics and comparisons with lines produced by conventional 

reproductive methods. 

Although the number of the DH lines obtained in our study is rather 

small, this is the first reported characterization of cucumber DH lines. 

Moreover, 3 DH lines showed significantly lower susceptibility to 

downy mildew than their donor variety. This characteristic, together with 

a high fruit yield of several other DH lines (data not published), as well 

as the possibility of early detection of downy mildew resistance using 

RAPD markers, may prove to be useful in cucumber breeding. 


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NATURALLY OCCURRING STRAINS OF 

BIPARTITE BEGOMOVIRUSES AFFECT 

SOME MEMBERS OF CUCURBITACEAE 

FAMILY INSIDE AND OUTSIDE THE COTTON 

ZONE IN PAKISTAN 

M. Tahir and M. S. Haider 

School of Biological Sciences, University of the Punjab, Lahore, 

Pakistan 

ADDITIONAL INDEX WORDS. Luffa cylindrica, Momordica charantia, Cucurbita 

pepo, Tomato leaf curl New Delhi virus, Pumpkin yellow mosaic Lucknow virus, 

Bemisia tabaci (Gennadius) 

ABSTRACT. Young symptomatic leaves of Luffa cylindrica, Momordica charantia, 

and Cucurbita pepo were collected from vegetable fields from both the cotton 

zone (Multan) and noncotton zone (Lahore) in Pakistan. Total DNA was 

extracted by the CTAB method. For each of the six samples, PCR products of 

expected size (i.e., 2.8kb for both full-length DNA A and DNA B) were amplified 

by using specific primers designed from published sequences of ToLCNDV. The 

PCR-amplified products were cloned, sequenced, and analyzed. The partial 

DNA A sequences obtained from M. charantia (Lahore) and L. cylindrica 

(Lahore and Multan) showed the highest sequence homology (91% to 98% over 

a stretch of 486 to 728 nucleotides) to Tomato leaf curl New Delhi virus 

(ToLCNDV). The partial DNA A sequence obtained from Cucurbita pepo 

(Lahore) showed the highest sequence homology (97% over a stretch of 503 

nucleotides) to Pumpkin yellow mosaic Lucknow virus (PYMLkV). The partial 

DNA B sequences obtained from M. charantia (Multan) and L. cylindrica 

(Lahore and Multan) had the highest level of sequence identity (88% to 94% 

over a stretch of 447 to 606 nucleotides) to ToLCNDV. However, the sequence 

for DNA A component of M. charantia (Multan) and Cucurbita pepo (Multan) 

and for DNA B component of M. charantia (Lahore) and Cucurbita pepo 

(Lahore and Multan both) have yet to be determined. 

B 

egomoviruses (Family Geminiviridae) are single-stranded 

DNA viruses that infect dicotyledonous plants, are transmitted 

by a single species of whitefly [Bemisia tabaci (Gennadius)], 

and typically have genomes consisting of two components (Rybicki et 

al., 2000), referred to as DNA A and DNA B, both of which are 

essential for virus proliferation. The components share a region of high 

sequence identity known as the “common region” that contains motifs 

required for the control of gene expression and replication, notably 

conserved reiterated motifs and a putative stem-loop structure 

containing the highly conserved nonanucleotide motif (TAATATTAC) 

that functions in the initiation of rolling circle replication. 


The genome component designated DNA A encodes all virus 

functions required for DNA replication (Stanley, 1983; Hanley- 

Bowdoin et al., 1999), as well as the coat protein, which plays an 

essential role in insect transmission (Briddon et al., 1998; Azzam et 

al., 1994). 

The DNA B component encodes two genes involved in virus 

movement within plants (Noueiry et al., 1994) and their products are 

symptoms determinants for the bipartite begomoviruses (Klinkenberg 

and Stanley, 1990; von Arnim and Stanley, 1992). 

However, a small number of monopartite begomoviruses, for 

instance Tomato yellow leaf curl virus (TYLCV), have been identified 

that lack the second component. For these viruses, all viral products 

required for replication, gene expression, whitefly transmission, and 

systemic infection are encoded on a single component (a homolog of 

the DNA A component of the bipartite begomoviruses; Navot et al., 

1991; Rojas et al., 2001). 

Satellite molecules have been identified for several monopartite 

begomoviruses. These molecules, called DNA β, are approximately 

1350nts in length (approximately half that of the genomes of their 

helper viruses) and are unrelated in sequence to their helper viruses; 

they require their helper viruses for replication, movement in plants, 

and insect transmission. DNA β satellites affect the replication of their 

helper begomoviruses and alter the symptoms induced in some host 

plants (Saunders et al., 2000; Briddon et al., 2001). 

A B 

Fig. 1. (A) L. cylindrica (mosaic); (B) Bitter gourd (yellow vein). 


The characteristic symptoms include leaf curling, vein thickening, 

yellow leaf curling, vein yellowing, mosaic, and, in some cases, 

yellow blotch and stunting (Briddon and Markham, 2001, Harrison, et 

al., 1997). 

Geminiviruses cause several of the world’s most serious viral 

diseases of crop plants in tropical and subtropical regions, e.g., cassava 

in Africa and India, tomato, legumes, and cucurbits worldwide, and 

cotton in Pakistan. 

Occurrence of whitefly-transmitted diseases in plants, particularly in 

vegetables, ornamental plants, and agricultural and economic crops, 

presents a challenge for plant scientists concerned with yield and 

quality in plant production. In recent years, the role of whitefly and 

begomoviruses both in yield and quality has been recognized. During 

the last decade the output of cotton from Pakistan has been seriously 

compromised due to an epidemic of Cotton leaf curl disease (CLCuD), 

with losses between 1992 and 1997 estimated at US$5 billion. 

Disease incidence on cucurbits is quite high; it ranges from 60– 

90% depending on disease severity. It is hard to find a field without 

begomovirus infection over a distance of hundreds of kilometres. The 

present study aimed at the analysis of the genetic variability of 

cucurbit viruses within each location and for two distant locations in 

Pakistan: Multan (cotton zone) and Lahore (noncotton zone). 


SAMPLE COLLECTION. Young leaves of Luffa cylindrica, 

Momordica charantia, and Cucurbita pepo showing typical 

begomovirus-like symptoms and symptomless leaves were collected 

from both cotton-growing (Multan) and non-cotton-growing (Lahore) 

regions separated by 350Km. Leaf samples were kept separately in 

plastic bags and stored at -80 о C in a freezer. Total DNA from both 

healthy and infected leaves was isolated by the cetyl trimethyl 

ammonium bromide (CTAB) method (Doyle and Doyle, 1987) by 

using 1g of leaf tissues. 

POLYMERASE CHAIN REACTION. To amplify full-length DNA A 

and B, specific primers were designed from published sequences of 

ToLCNDV. PCR was performed in volumes of 50μl containing 

1.5mM MgCl2, 200μM dNTPs, 2.5μM each of primers, 100ng of 

Total DNA, and 2.5U of Taq polymerase (Fermentas). Reactions were 

carried out in an Applied Biosystems 2720 thermal cycler programmed 

for 30 cycles of 1 min at 94 о C, 1 min at 55 о C, and 3 mins at 72 о C. 

Amplified PCR products were detected by 1% agarose gel 

electrophoresis using DNA ladder Mix (Fermentas) as marker. 


LIGATION AND TRANSFORMATION. Amplified products were 

ligated into pTZ57R/T (Fermentas) cloning vector according to the 

supplier’s instructions and transformed in DH5α strain of E. coli. Blue 

and white selection was made by spreading the transformed cells onto 

the surface of LB medium agar plates (0.5% NaCl, 0.5% yeast extract, 

1% trypton, and 1.5% agar) containing 130μg/ml of IPTG, 270μg/ml 

of X-gal, and 100μg/ml of ampicillin. Plates were incubated at 37 о C 

for 14–16 hours. 

The white colonies were selected and reinoculated in LB broth 

(0.5% NaCl, 0.5% yeast extract, and 1% trypton) for small-scale 

preparation and restricted analysis was done by HindIII, EcoRI, and 

BamHI restriction endonucleases. Sequencing was performed by 

CEQ 8000 Genetic Analysis System (Beckman and Coulter 

sequencer). 


The objectives of this study were to confirm the relation between 

the observed diseases in local vegetable fields and the presence of 

begomoviruses, to have an idea about the distribution of the problem 

around the country, and to determine the genetic diversity of 

begomoviruses infecting cucurbits. This study also aimed to find out if 

cucurbits could function as reservoir hosts of the begomoviruses 

infecting other vegetables, or vice versa. 

Cucurbit vegetables showing begomovirus-like symptoms 

collected from two distant locations (the cotton-growing area of 

Multan and the non-cotton-growing area of Lahore) from Pakistan 

produced PCR products of expected size (i.e., 2.8kb each for DNA A 

and DNA B), and there was no amplification from any of the healthy 

plant samples. The PCR products were detected on 1% agarose gel 

using DNA ladder as marker (Figure 2). 

The partial sequences obtained from virus isolates were compared 

with sequences from other begomoviruses available in databases. The 

presence of begomoviruses, in the symptomatic plant species 

evaluated was positive for nearly all the samples tested. There is about 

the same level of variability when comparing sequence identities (%) 

between all begomovirus isolates from different cucurbit plant species 

as there is within a single plant species. 

The partial DNA A sequences obtained from M. charantia 

(Lahore) and L. cylindrica (Lahore and Multan) showed the highest 

sequence homology (91% to 97% over a stretch of 486 to 728 

nucleotides) to Tomato leaf curl New Delhi virus (ToLCNDV). The 

partial DNA A sequence obtained from Cucurbita pepo (Lahore) 


1. M. charantia Lhr, A 

2. M. charantia, Lhr, B 

3. M. charantia Mn, A 

4. Luffa, Mn, A 

5. Luffa, Mn, B 

6. Luffa, Lhr, A 

7. DNA ladder 

8. C. pepo Lhr, A 

9. C. pepo Lhr, B 

Mn = Multan 

Lhr= Lahore 

A = DNA A 

B= DNA B 

Fig 2. Ethidium bromide stained 1% agarose gel. Samples from Lane 1–9 are 

described on the right-hand side of figure. 

showed the highest sequence homology (97% over a stretch of 503 

nucleotides) to Pumpkin yellow mosaic Lucknow virus (PYMLkV). 

The partial DNA B sequences obtained from M. charantia (Multan) 

and L. cylindrica (Lahore and Multan) had the highest level of 

sequence identity (88% to 94% over a stretch of 447 to 606 

nucleotides) to ToLCNDV (Table 1). However, the sequence for the 

DNA A component of M. charantia (Multan) and Cucurbita pepo 

(Multan) and the DNA B component of M. charantia (Lahore) and 

Cucurbita pepo (Lahore and Multan both) have yet to be determined. 

The results of these studies on cucurbits showed that begomovirus 

diseases are widespread in Pakistan as in many countries of the world. 

Begomoviruses were detected in all six samples collected from two 

distant locations. According to the comparisons and their partial 

sequence analysis, they were arranged in two groups in relation to the 

previously described begomoviruses. They were found to be widely 

distributed (present in almost every part of Asia, especially Pakistan) 

and belonged to at least two species, i.e., ToLCNDV and PYMLkV. 

ToLCNDV covers quite a broad host range from diverse plant families 

including Compositeae (Haider et al., 2005) and Solanaceae (Hussain 

et al., 2004). This indicates that ToLCNDV is a potential threat to 

vegetables and to the cotton industry in Pakistan. The results suggest 

the existence of ToLCNDV on Luffa cylindrica and M. charantia and 

of PYMLkV on Cucurbita pepo under natural conditions in two 

regions of Pakistan. 


Table 1. Percentage blast homology of the sequences from investigated 

sample. 

Blast 

Sample 

Size 

homology 

name Location Genome (kb) Virus (%) 

M. charantia Multan A 2.8 ToLCNDV 95 

M. charantia Lahore A 2.8 ToLCNDV 91 

L. cylindrica Lahore A 2.8 ToLCNDV 91 

L. cylindrica Lahore A 2.8 ToLCNDV 95 

C. pepo Lahore A 2.8 PYMLkV 97 

M. charantia Multan B 2.8 ToLCNDV 88 

L. cylindrica Lahore B 2.8 ToLCNDV 94 


Azzam, O., J. Frazer, D. de la Rose, J. S. Beaver, P. Ahiquist, and D. P. Maxwell. 

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EFFECT OF FUNGICIDE CHEMISTRY AND 

CULTIVAR ON THE DEVELOPMENT OF 

CUCURBIT POWDERY MILDEW ON PUMPKIN 

IN NEW JERSEY 

Christian A. Wyenandt 

Rutgers University, Rutgers Cooperative Research and Extension 

Rutgers Agricultural Research and Extension Center 

121 Northville Road, Bridgeton, New Jersey 08302 

ADDITIONAL INDEX WORDS. Podosphaera xanthii, fungicide resistance, FRAC 

groups 

ABSTRACt. A trial was conducted in 2005 at the Rutgers Agricultural Research 

and Extension Center in southern New Jersey to determine if resistance to 

commonly used fungicides would develop in cucurbit powdery mildew. Five 

fungicide application programs were applied to two pumpkin cultivars every 7 

to 10 days season-long: (1) mancozeb (Manzate) + sulfur alternated with 

Maneb + fixed copper (Champ) (protectant fungicides only); (2) chlorothalonil 

(Bravo) + myclobutanil (Nova) alternated with azoxystrobin (Amistar); (3) 

myclobutanil + Maneb alternated with famoxadone+cymoxanil (Tanos); (4) 

chlorothalonil + myclobutanil alternated with myclobutanil; and (5) 

chlorothanolil + azoxystrobin alternated with azoxystrobin. Area under the 

disease progress curve (AUDPC) values were significantly higher when 

azoxystrobin was applied weekly. AUDPC values were 1067.0 for ‘Howden’ and 

1245.0 for ‘Magic Lantern’ compared to 625.6 in ‘Howden’ and 489.2 in ‘Magic 

Lantern’ when azoxystrobin was alternated weekly with a myclobutanil. 

AUDPC was lowest when myclobutanil was applied weekly. 

O 

ver 2,400 hectares of cucurbit crops are grown annually for 

commercial wholesale or roadside markets in New Jersey. 

Powdery mildew caused by Podosphaera xanthii (Castagne) 

U. Braun & N. Shishkoff (also known as Sphaerotheca fusca (Fr.) S. 

Blumer and S. fuliginea (Schlechtend.:Fr.) Pollacci) is one of the most 

destructive diseases of pumpkin and other cucurbit crops in the United 

States. Without proper fungicide applications, powdery mildew can 

cause 100% premature defoliation leading to sunscald injury to fruit 

and reduced yield. Fungicide applications are the principle practice in 

most cucurbit crops for managing powdery mildew (McGrath, 2001). 

In recent years new fungicide chemistries, such as the QoIs (Fungicide 

Resistance Action Committee [FRAC] Group 11) have been 

introduced to the market for the control of powdery mildew (Kuck and 

Mehl, 2003). These new chemistries, although highly effective at 

controlling powdery mildew, have a high risk for the development of 


esistance by the pathogen (McGrath, 2001). Powdery mildew 

resistance to FRAC Group 11 (QoI) as well as to DMIs (FRAC Group 

3) fungicides has been reported in other states and other countries 

(McGrath, 2001). In recent years, cucurbit growers in northern New 

Jersey have stated that some of the fungicides (i.e., DMIs and QoIs) 

commonly used in recommended powdery mildew control programs 

have not been as effective as in previous years. This noticeable 

reduction in control may be the result of resistance development in 

commercial fields where QoI and DMI fungicides are being used 

extensively for cucurbit powdery mildew control in New Jersey. 

In 2005, a fungicide trial was conducted at Rutgers Agricultural 

Research and Extension Center (RAREC) in Bridgeton (Cumberland 

County), New Jersey, to determine if P. xanthii would develop 

resistance to either a DMI and/or QoI fungicide chemistry. 


Five blocks, each consisting of 2 rows on 3.1m centers, of 

pumpkin cvs. Howden and Magic Lantern were established 22 June. 

Prior to pumpkin establishment, and on 20 April 2005, Sudangrass was 

seeded at 68 kg/ha between each block to provide a windbreak and 

prevent fungicide drift between blocks. Each block consisted of five 

replications each of sprayed (fungicide treatment) or unsprayed plots 

(7.6m long) with in-row breaks of 3.1m between plots. Five different 

fungicide programs consisting of (1) 2.24kg/ha Manzate + 2.24kg/ha 

sulfur alternated with 2.24kg/ha Maneb + 1.51L/ha Champ (protectant 

fungicides only); (2) 3.5L/ha Bravo + 0.34kg/ha Nova alternated with 

0.34kg/ha Amistar (standard program, FRAC Groups 3 and 11); (3) 

0.34kg/ha Nova + 2.24kg/ha Maneb alternated with 0.55kg/ha Tanos 

(FRAC Groups 11 + 27); (4) 3.5L/ha Bravo + 0.34kg/ha Nova 

alternated with 0.34kg/ha Nova (FRAC Group 3 weekly); and (5) 

3.5L/ha Bravo + 0.34kg/ha Amistar alternated with 0.34kg/ha Amistar 

(FRAC Group 11 weekly) were applied season-long starting on 27 

July and repeated every 7 to 10 days (10 total applications/block). 

Fungicide was applied using a tractor-mounted CO2-assisted 3.1m 

boom sprayer (R&D Sprayers, Opelousa, LA) with flat fan nozzles 

(TeeJet 8002VS) on 0.51m centers with a delivery rate of 412L/ha at 

58psi. All sprayed and unsprayed plots in each block were rated on a 

weekly basis on a scale of 0.0 to 1.0 (0.05 increments) for percentage 

of foliage with powdery mildew symptoms. At the end of the season 

arcsine transformed AUPDC values were calculated for all plots. On 3 

Oct five pumpkin leaves from each plot were visually rated for the 


percentage of leaf surface (top and bottom) with symptoms of powdery 

mildew. On 17 Oct all fruit were harvested and weighed. 


The effects of fungicide chemistry and pumpkin cultivar on the 

development of cucurbit powdery mildew were studied in southern 

New Jersey in 2005. Powdery mildew was most severe when a Group 

11 fungicide was applied weekly to either a fully susceptible 

‘Howden’ or powdery mildew-tolerant ‘Magic Lantern’ (Figure 1). 

Powdery mildew severity was lower when a Group 11 fungicide was 

alternated with a Group 3 fungicide weekly, and lowest when a Group 

3 fungicide was applied weekly (Figure 1). At the end of the growing 

season, powdery mildew severity was visually 

Fig. 1. Effects of fungicide program and cultivar on the development of 

powdery mildew on pumpkin in southern New Jersey in 2005. 

rated on the top and bottom sides of leaves of the fully susceptible 

‘Howden’ or the powdery mildew-tolerant ‘Magic Lantern’. Powdery 

mildew was highest on the bottom side of leaves when a FRAC Group 

11 fungicide was applied weekly (71% in ‘Howden’ and 66% in 

‘Magic Lantern’) compared to the protectant fungicide program (48% 

in ‘Howden’; 38% in ‘Magic Lantern’) and standard program (Group 


3 alt. Group 11) (27% in ‘Howden’; 26% in ‘Magic Lantern’). When a 

Group 3 fungicide was applied weekly, powdery mildew was only 2% 

on the bottom side of leaves in both ‘Howden’ and ‘Magic Lantern’. 

AUDPC values were significantly higher in ‘Howden’ and ‘Magic 

Lantern’ when Group 11 fungicide was applied weekly. AUDPC 

values were 1067.0 for ‘Howden’ and 1245.0 for ‘Magic Lantern’ 

compared to 625.6 in ‘Howden’ and 489.2 in ‘Magic Lantern’ when 

Group 11 fungicide was alternated weekly with a Group 3 fungicide. 

AUDPC was lowest when Group 3 fungicide was applied weekly, 

362.7 in ‘Howden’ and 321.6 in ‘Magic Lantern’ (Table 1). In general, 

pumpkin yield (tons/ha) was higher in the powdery mildew-tolerant 

‘Magic Lantern’ compared to the susceptible ‘Howden’. The 

respective yields (tons/ha) for ‘Howden’ and ‘Magic Lantern’ were 

15.4 and 19.6 for the Group 11/weekly program; 12.5 and 20.7 for 

Group 3/weekly; 9.3 and 13.6 for Group 3 alternated with Group 11 

(standard); 9.8 and 15.0 in protectant fungicides only; and 9.3 and 13.6 

in Group 3 alternated with Groups 11 + 27. 

This research suggests that cucurbit powdery mildew developed 

resistance to the FRAC Group 11 fungicide azoxystrobin when applied 

on a weekly basis during the production season, and that a resistant 

powdery mildew population is most likely to develop on the bottom 

side of the leaf surface when protectant fungicides, such as 

chlorothalonil (FRAC Group M5), are included in fungicide program. 

Importantly, from a control standpoint, once QoI resistance develops 

to one strobilurin chemistry, the pathogen is expected to develop 

cross-resistance to other QoIs, even if those chemistries haven’t been 

applied. Powdery mildew severity was lower when a FRAC Group 11 

fungicide was alternated weekly with a FRAC Group 3 fungicide, 

suggesting that the alternation of these chemistries is effective in 

slowing the development of a resistant population. Powdery mildew 

severity was lowest when a FRAC Group 3 fungicide was applied 

weekly, suggesting that in the case of this study, the use of a Group 3 

fungicide was not selecting for a resistant population. 


Table 1. Fungicide program (rate per hectare), FRAC grouping(s), and 

AUDPC values for cucurbit mildew development in pumpkin cvs. 

Howden and Magic Lantern at the Rutgers Agricultural Research and 

Extension Center (RAREC) in southern New Jersey in 2005. 

Fungicide FRAC 

AUDPC value 

program 

3.5L/ha Bravo 

+ 0.34kg/ha 

Amistar alt. 

0.34kg/ha 

Amistar 

groups(s) Howden Magic Lantern 

Group 11 

weekly 1067.0 1245.0 

2.24kg/ha 

Manzate + 2.24 

kg/ha sulfur alt. 

2.24kg /ha 

Maneb + 1.51 

L/ha Champ Group M only 910.3 796.1 

0.34kg/ha Nova 

+ 2.24kg/ha 

Maneb alt. 

0.55kg/ha 

Tanos 

3.5L/ha Bravo 

+ 0.34kg/ha 

Nova alt 

0.34kg/ha 

Amistar 

Group 3 alt 

Group 11+27 858.3 912.2 

Group 3 alt 

Group 11 625.6 489.2 

3.5L/ha Bravo 

+ 0.34kg/ha 

Nova alt 

0.34kg/ha Nova Group 3 weekly 362.7 321.6 

LSD (p = 0.05) 162.9 215.4 


Kuck, K.-H. and A. Mehl. 2003. Trifloxystrobin: resistance risk and resistance 

management. Pflanzenschutz-Nachrichten Bayer. 56:313–325. 

McGrath, M. T. 2001. Fungicide resistance in cucurbit powdery mildew: experiences 



SURVEY FOR CUCURBIT VIRUSES IN 

COMMERCIAL FIELDS AND EVALUATION OF 

VIRUS-RESISTANT SUMMER SQUASH 

BREEDING LINES IN NEW JERSEY 

Christian A. Wyenandt 

Rutgers University, Rutgers Cooperative Research and Extension 

Michelle Infante-Casella 

Gloucester County Agricultural Agent 

Melvin R. Henninger 

Specialist in Vegetable Crops 

Richard Buckley 

Coordinator, Plant Diagnostic and Nematode Detection Service 

Resource Center 

Sabrina Tirpak 

Principle Lab Technician, Plant Diagnostic and Nematode Detection 

Service Resource Center 

Kristian E. Holmstrom 

IPM Research Project Coordinator II 

Peter J. Nitzsche 

Morris County Agricultural Agent 

William H. Tietjen 

Warren County Agricultural Agent 

Raymond J. Samulis 

Burlington County Agricultural Agent 

Wesley L. Kline 

Cumberland County Agricultural Agent 

Win Cowgill 

Hunterdon County Agricultural Agent 

Joseph R. Heckman 

Extension Specialist in Soil Fertility, Rutgers Cooperative Research 

and Extension, Rutgers University, 88 Lipman Drive, 

New Brunswick, NJ 08901 

ADDITIONAL INDEX WORDS. ELISA, mosaic virus 

ABSTRACT. A survey of commercial fields was carried out to determine which 

viruses were most prevalent in cucurbit production areas of New Jersey in 2005. 

Concurrently, 22 summer squash breeding lines and named cultivars were 

sampled for virus infection. In total, 22 summer squash breeding lines and 

cultivars and 37 virus-infected cucurbits from commercial plantings from eight 

counties in New Jersey were tested for CMV (Cucumber mosaic virus), WMV-2 

(Watermelon mosaic virus 2), PRSV (Papaya ring spot virus), and ZYMV 

(Zucchini yellow mosaic virus). In total, 85% of the samples tested positive for 


WMV, 33% tested positive for ZYMV, 12% tested positive for CMV, and 5% 

tested positive for PRSV. Thirty-five percent of the samples tested positive for 

two viruses and 2% tested positive for three viruses. Of the 22 squash breeding 

lines and cultivars, only 2, ‘Payroll’ and ‘Patriot II’, tested negative for all four 

viruses during this survey. 

O 

ver 2,400 hectares of cucurbit crops are grown annually for 

commercial wholesale and roadside markets in New Jersey. 

New summer squash cultivars are regularly released that 

contain resistance packages for important cucurbit viruses. These 

resistance packages may include resistance and/or tolerance to one or 

more cucurbit viruses, such as Watermelon mosaic virus (WMV 2), 

Cucumber mosaic virus (CMV), Zucchini yellow mosaic virus 

(ZYMV), and/or Papaya ring spot virus (PRSV). Cucurbit growers in 

New Jersey rely on summer squash cultivars with virus-resistance 

packages along with resistance and/or tolerance to other important 

diseases, such as powdery or downy mildew, to reduce the chances for 

significant losses. The objective of this study was to survey 

commercial fields to determine which viruses were most prevalent in 

cucurbit production areas of New Jersey in 2005. Concurrently, 22 

summer squash breeding lines and named cultivars with and without 

resistance/tolerance packages were sampled for virus infection. 


During the summer of 2005, commercial cucurbit fields from eight 

counties in New Jersey were surveyed for symptoms of virus infection. 

Infected plant tissue was collected by county agricultural agents and 

sent to the Plant Diagnostic Laboratory in New Brunswick, New 

Jersey, for virus identification. At the Plant Diagnostic Laboratory, 

commercially available Agdia, DAS ELISA, alkaline phosphatase 

label, PathoScreen Test Kits for CMV, WMV, ZYMV, and PRSV 

detection were used for the testing protocol. Symptomatic plant 

material was selected from each sample. Most samples were leaf 

tissue, but some fruit and stems were tested. A 1:10 ratio (0.5g to 5ml) 

solution of tissue and extraction buffer was placed in Agdia sample 

extraction pouches and ground using Agdia’s tissue homogenizer 

attached to a 9-volt drill press. The buffer/tissue solution was 

subsequently loaded into sample wells alongside positive and negative 

controls supplied with the test kit. The test plates were incubated for 

two hours at room temperature. After incubation the plates were 

washed with PBST. An enzyme conjugate was added and the plates 

were incubated again at room temperature for two hours. After 


incubation, as before, the plates were washed with PBST. PNP buffer 

(100ul) was added to the plates and then incubated for another hour. 

Lastly, sodium hydroxide (50ul) was added to each well to stop the 

reaction. The wells were examined and color change was interpreted 

visually. Wells in which color developed indicated positive results. 

Individual wells in which there was no significant color development 

indicated negative results. 

Results 

In total, 22 summer squash breeding lines and cultivars and 37 

virus-infected cucurbit plantings from eight counties were tested for 

CMV, WMV-2, PRSV, and ZYMV in 2005 (Table 1). In the 

breeding-line and cultivar evaluation, symptoms of virus infection first 

appeared on 8 Sept. in ‘Sunray’, which tested positive for WMV-2. Of 

the 22 squash breeding lines and cultivars, only 2, ‘Payroll’ and 

‘Patriot II’, tested negative for all four viruses, although ‘Payroll’ 

developed virus-like symptoms during this survey (Tables 2 and 3). 

Only ‘Patriot II’, ‘Liberator III’, ‘Seminis Exp.’, and ‘Conqueror III’ 

showed no symptoms of virus infection on foliage or fruit. However, 

‘Liberator III’, ‘Conqueror III’, and ‘Seminis Exp.’ tested positive for 

CMV. Although ‘Payroll’ developed virus-like symptoms, it tested 

negative for all four viruses (Table 3). ‘Independence II’ showed 

symptoms of virus-like infection on developing fruit only. In total, 

85% of the samples collected from commercial fields and from a 

breeding-line and cultivar evaluation tested positive for WMV-2, 33% 

tested positive for ZYMV, 12% tested positive for CMV, and 5% 

tested positive for PRSV. Thirty-five percent of the samples tested 

positive for two viruses and 2% tested positive for three viruses. 

Watermelon mosaic virus was the most commonly identified virus in 

cucurbit crops in New Jersey in 2005. Overall, 35 % of the samples 

tested positive for two viruses. Because of this, cucurbit growers in 

New Jersey, as well as elsewhere, should continue to plant cultivars 

with resistance packages for one or more of the important viruses in 

cucurbits. 


Table 1. Host, county, and ELISA results for commercial cucurbit fields 

sampled for Cucumber mosaic virus (CMV), Watermelon mosaic virus 

(WMV), Papaya ring spot virus (PRSV), and Zucchini mosaic virus 

(ZYMV) in New Jersey in 2005. 

Host County CMV PRSV WMV2 ZYMV 

Pumpkin Cumberland - - + + 

Pumpkin Cumberland - - + - 






Pumpkin Morris - - + - 


Pumpkin Warren - - + + 

Pumpkin Warren - - + - 

Pumpkin Warren - - + - 



Pumpkin Sussex - - + - 



Pumpkin Middlesex - - + - 

Pumpkin Warren - - - + 

Pumpkin Hunterdon - - + + 

Zucchini Hunterdon - - + - 

Pumpkin Burlington - - + - 

Pumpkin Burlington - - + + 

Pumpkin Burlington - + + +W 

Pumpkin Burlington - + + +w 

Pumpkin Burlington - + + +w 




Gourd Burlington - - + - 



Squash Burlington - - + +w 

Yellow 

squash Burlington - - + +w 




Zucchini Cumberland - - + - 

Yellow 

squash Cumberland - - + - 

(+) = positive for virus, (-) = negative for virus, (+w) = weak positive. 


Table 2. ELISA test results for yellow summer squash breeding line(s) 

and cultivars evaluated at the Rutgers Agricultural Research and 

Extension Center, Bridgeton, New Jersey, in 2005. 

Company/ 

Cultivar CMV PRSV WMV2 ZYMV 

Resistance 

package 

Seminis 

‘General Patton’ 

- - + - CMV, WMV 

(PY) 

Seminis 

+ - - - CMV, WMVII, 

experimental 

ZYMV 

(trans) 

Seminis ‘Patriot 

II’ 

Seminis 

‘Liberator III’ 

Seminis 

Conquerer III’ 

Seminis 

‘Sunray’ 

Harris Moran 

‘Cougar’ 

Harris Moran 

‘Lioness’ 

- - - - WMII, ZYMV 

(trans) 

+W - - CMV, 

WMVII, 

ZYMV (trans) 

+ - - - CMV, 

WMVII, 

ZYMV 

(trans), PRSV 

(conv) 

- - + - CMV, 

WMVII, (PY) 

- - + - CMV, 

WMVII, (PY), 

PRSV, ZYMV 

(T) 

- - + - CMV, 

WMVII, 

PRSV (IR), 

ZYMV (T) 



Table 3. ELISA test results for zucchini breeding lines and cultivars 

evaluated at the Rutgers Agricultural Research and Extension Center, 

Bridgeton, New Jersey, in 2005. 

Resistance 

Company/cultivar CMV PRSV WMV2 ZYMV 

Harris Moran/ 

‘Tigress’ 

Harris Moran/ 

‘Leopard’ 

package 

- - - + ZYMV, 

WMVII (T) 

+W - + - WMV, 

PRSV (IR) 

Harris Moran/‘Lynx’ - - + + WMVII, 

ZYMV, 

PRSV (T) 

Harris Moran/ 

‘Wildcat’ 

- - +W - 

Sieger/experimental - - + - 

Sieger/ experimental - - + - 

Sieger/ experimental - - + +W 

Sieger/ experimental - - + - 

Sieger/‘Payroll’ - - - - WMVII, 

ZYMV 

(T)(trans) 

Sieger/‘Revenue’ - - + - CMV, 

WMVII, 

ZYMV 

(T)(trans) 

Sieger/‘Independence 

II’ 

- - +W - WMVII, 

ZYMV 

(R)(trans) 

Sieger/‘Justice III’ - - + + CMV, 

WMVII, 

ZYMV 

(R)(trans) 

Seminis/‘Senator’ - - + + none 

Seminis/ 

experimental 

+ - - - CMV, 

WMVII, 

ZYMV 

(trans) 



A RAPID HEXANE-FREE METHOD FOR 

ANALYZING TOTAL CAROTENOID CONTENT 

IN CANARY YELLOW-FLESHED 

WATERMELON 

Angela R. Davis, Julie Collins, Wayne W. Fish, 

Charles Webber III, and Penelope Perkins-Veazie 

USDA, ARS, South Central Agriculture Research Laboratory, 

P.O. Box 159, Lane, OK 74555 

Y. Tadmor 

Newe Ya’ar Research Center, ARO, Israel 

ADDITIONAL INDEX WORDS. Citrullus lanatus, light-absorption method, puree 

absorbance method, carotenoid quantification 

ABSTRACT. Lycopene is the predominant carotenoid in red watermelon 

(Citrullus lanatus [Thunb. ] Matsum. and Nakai) and pro-lycopene is the 

predominant carotenoid in most orange watermelon. However, yellow 

watermelons contain many different carotenoids, all in low to trace amounts. 

Since carotenoids have antioxidant properties and potential health benefits, 

selecting varieties with high concentrations of these valuable pigments is 

important for breeding lines. Unfortunately, current methods to assay total 

carotenoid content are time consuming and require hazardous organic solvents. 

This report describes a rapid and reliable light-absorption method to assay total 

carotenoid content for yellow watermelon that does not require organic 

solvents. Light absorption of 67 watermelon flesh purees was measured with a 

diode array xenon flash spectrophotometer that can measure actual light 

absorption from opaque samples; results were compared with a hexane 

extraction method. The puree absorbance method gave a linear relationship 

(R 2 =0.85) to total carotenoid content and was independent of watermelon 

variety within the total carotenoid concentration range measured (0μg/g to 

7μg/g fresh weight). 

C 

arotenoids have been linked to lower risk of myocardial 

infarction (Kohlmeier et al., 1997), may possess anticancer 

properties (Gerster, 1997), and promote healthy eye function 

(Rodriguez-Carmona et al., 2006; Giovannucci, 1999; Simon, 1997; 

Stahl and Sies, 1996). These attributes may be in part responsible for 

increased purchase of products containing these compounds (McBride, 

1999; National Watermelon Promotion Board, 1999). Red-fleshed 

watermelons contain high quantities of lycopene, a red-pigmented 

carotenoid with powerful antioxidant properties (Di Mascio et al., 

1989, Tomes et al., 1963). Orange watermelons typically contain high 

amounts of pro-lycopene, similar to the tomato “tangerine” mutation 

(Isaacson et al., 2002), and, in some watermelon varieties, β-carotene 


and ζ-carotene (Tadmor et al., 2004, 2005; Tomes and Johnson, 1965; 

Perkins-Veazie et al., unpublished data). Yellow watermelons are 

divided into two major types: canary yellow and salmon yellow 

(Henderson et al., 1998). The salmon yellow is a “tangerine type” 

watermelon that contains small amounts of pro-lycopene, whereas the 

canary yellow contains multiple carotenoids all in low or trace 

amounts (Tadmor et al., 2004, 2005; Perkins-Veazie and King, 

unpublished data). 

Carotenoid content varies greatly among watermelon cultivars and 

production environments (Perkins-Veazie et al., 2001), but yellow 

watermelon seems to be consistently low in all of these potent 

antioxidants. Total carotenoid content was tested for ‘Early 

Moonbeam’, a canary yellow watermelon (Henderson et al., 1998); 

only trace amounts of carotenoids were detected (Tadmor et al., 2004, 

2005). Tadmor and others suggest that this may be similar to tomatoes, 

where a nonfunctioning phytoene synthase gene results in the 

accumulation of very low levels of carotenoids in yellow-fleshed fruits 

(Camara, 1993). Because carotenoids are valued as phytonutrients, a 

quick, reliable screening method for carotenoid content is needed for 

germplasm evaluations. Conventional spectrophotometric or HPLC 

assays to quantify total carotenoids utilize organic solvents to extract 

and solubilize these compounds from tissue (Adsule and Dan, 1979; 

Beerh and Siddappa, 1959; Sadler et al., 1990). These methods are 

time consuming, require the use and disposal of hazardous organic 

solvents, and do not work well for the more hydrophilic carotenoids. A 

simple, inexpensive, and reliable method to determine total carotenoid 

content without organic solvents is highly desirable for the watermelon 

industry. One such approach is a modified method from Davis et al. 

(2003), which utilizes a diode array xenon flash spectrophotometer to 

measure absorbed visible color and correlate these values to lycopene 

We would like to thank Amy Helms, Buddy Faulkenberry, Anthony Dillard, and 

Bryan Deak for providing valuable technical support, and Pat Bischof and Gordon 

Leggett of Hunter Associates Laboratory, Inc., for technical assistance. Mention of 

trade names or commercial products in this article is solely for the purpose of 

providing specific information and does not imply recommendation or endorsement 

by the U.S. Department of Agriculture. All programs and services of the U.S. 

Department of Agriculture are offered on a nondiscriminatory basis without regard to 

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content. In this report, we demonstrate that watermelon puree 

evaluated with a diode array xenon flash spectrophotometer is a novel 

and fast method for accurately quantifying total carotenoid content in 

yellow watermelon. This method overcomes detection problems with 

carotenoid levels and missing hydrophilic carotenoids. 


WATERMELON FRUIT. Watermelons ranging from yellow to offwhite 

were grown at Lane, OK, in 2005. Four open-pollinated varieties 

(‘Early Moonbeam’, ‘Yellow Baby’, ‘Yellow Doll’, and ‘Cream of 

Saskatchewan’), and five Plant Introduction (PI) lines (271773, 

271769, 299378, 314655, 494531) were evaluated. Uncut watermelons 

were stored at room temperature. Heart tissue was removed within 

one week of harvest. 

SAMPLE PREPARATION. All steps from the time watermelons were 

cut lengthwise were performed in subdued lighting at room 

temperature, unless otherwise stated. The heart tissue was collected cut 

into chunks approximately 3cm 3 or smaller. Samples were pureed 

immediately or after storage for several months at -80 o C. Tissue 

(~30g) was homogenized using a Brinkmann Polytron Homogenizer 

(Brinkmann Instruments, Inc., Westbury, NY) with a 20mm O.D. 

blade to produce a uniform slurry with particles smaller then 3mm 3 . 

Samples were not allowed to heat or froth. 

LOW-VOLUME HEXANE EXTRACTION METHOD. The low-volume 

hexane extraction method, performed as described by Fish et al. 

(2002), was used to measure total carotenoid content of watermelon 

purees. Briefly, approximately 0.6g samples were weighed from each 

puree into two 40-mL amber screw-top vials containing 5mL of 0.05% 

(w/v) BHT in acetone, 5mL of 95% ethanol, and 10mL of hexane. 

Purees were stirred on a magnetic stirring plate during sampling. 

Samples were placed on ice on an orbital shaker at 180rpm for 15 min. 

After shaking, 3mL of deionized water were added and samples 

shaken for an additional 5 min on ice. The vials were left at room 

temperature for 5 min to allow for phase separation. The absorbance of 

the upper, hexane layer was measured at 450nm in a 1-cm path length 

quartz cuvette blanked with hexane. Total carotenoid content of 

watermelon was calculated based on sample weight using the 

absorbance at 450nm (Fish et al., 2002). A comparison was made 

between the hexane extraction, methanol extractions, and ethanol 

extractions for each variety and PI used to ensure the hexane method 

was adequately extracting all carotenoids. Additionally, the aqueous 

phase of the hexane extraction was scanned to ensure all carotenoids 


were extracted into the hexane layer. This step was deemed necessary 

since the carotenoid profile of all yellow watermelons is not known 

and it was necessary to determine that water-soluble carotenoids were 

not missed. 

PUREE ABSORBANCE METHOD. The UltraScan XE (Hunter 

Associates Laboratory, Inc., Reston, VA) is a diode array xenon flash 

colorimeter/spectrophotometer with a wavelength range from 360 to 

750nm that reads both reflectance and transmittance. All tristimulus 

integrations are based on a triangular bandpass of 10nm and a 

wavelength interval of 10nm. The instrument sensor uses a specially 

coated plastic integrating sphere (6-in. diameter) that diffuses the light 

from the xenon light source. The light source illuminates and is 

transmitted through the sample. A lens located at an angle of 8 o 

perpendicular to the sample surface collects the transmitted light and 

directs it to a diffraction grating that separates the light into its 

component wavelengths. The intensities of these component 

wavelengths of transmitted light are then measured by two 

polychromators, each with 40-element diode array detectors. 

The instrument was standardized per company specifications and 

blanked on an empty cuvette. Watermelon puree was mixed well to 

keep separation to a minimum; about 20mL of the sample were 

immediately poured into a 1-cm, 20- mL SR101A cuvette (Spectrocell, 

Oreland, PA). The sample was scanned in the transmittance (TTRAN) 

mode under the following settings: the large reflectance port (1.00”), 

Illuminant at D65, MI Illuminant Fcw, and observer 10 o . Absorbance 

at 670nm was subtracted from absorbance at the maximum absorbance 

(430nm) to adjust for light scatter. The low-volume hexane analysis 

and the Hunter UltraScan XE readings were performed on the same 

day. 

STATISTICAL ANALYSIS. Linear least square regression analyses 

were performed using the statistical component of Microsoft ® Excel 

2002 SP-2 software (Redmond, WA). 


ABSORBANCE SPECTRAL MEASUREMENTS OF WATERMELON- 

TISSUE PUREE. Previous attempts at reliably correlating reflectance 

parameters to carotenoid content of watermelon with handheld 

colorimeters were unsuccessful (Perkins-Veazie et al., 2001). Use of 

tissue puree can overcome this limitation as well as provide access to 

light absorption to provide a reliable quantification method. The 

Hunter UltraScan XE subjects samples to light intensities that are 

orders of magnitude greater than those of analytical 


spectrophotometers with quartz halogen lamps. The UltraScan XE also 

has sphere collectors that collect scattered as well as nonscattered 

light. This potentially allows reliable spectral measurements on 

translucent samples that scatter light. The spectra for yellow 

watermelon exhibit apparent absorption maxima of 410, 430, and 

480nm (data not shown). 

ABSORBANCE BEHAVIOR OF WATERMELON PUREE AS RELATED 

TO TOTAL CAROTENOID CONTENT. Based on the spectral results 

obtained from the UltraScan XE, we investigated the possibility of 

employing absorbance measurements of watermelon puree at 430nm 

as a means to estimate total carotenoid content of watermelon tissue. 

Since many purified carotenoids are not soluble in an aqueous phase, 

and because the carotenoid profile of yellow watermelon is not fully 

known, a standard curve with purified carotenoids could not be 

performed. Therefore, visible absorbance of watermelon puree using 

the UltraScan XE was correlated with total carotenoid content 

determined by absorption measurement of organic solvent extraction. 

Flesh of tissue from 47 yellow to off-white watermelons from four 

varieties and four PIs were used in the study. The absorbance at 

430nm measured for each puree (adjusted for light scatter by 

subtracting the absorbance at 670nm) was plotted against its total 

carotenoid content as measured by hexane extraction (Figure 1). 

Hexane method total carotenoid estimate 

(ug/g fresh tissue) 

7 

6 

5 

4 

3 

2 

1 

y = 2.9993x - 0.5497 

R 2 = 0.8509 

0 

0 0.5 1 1.5 2 2.5 

Puree absorbance (A430nm-A670nm) 

Fig. 1. Forty-seven yellow watermelon heart tissue purees (4 varieties and 4 PI 

lines) were analyzed with the UltraScan XE. Absorbance is plotted versus total 

carotenoid content as determined by hexane extraction. The absorbance at 

430nm is adjusted for scatter by subtracting the absorbance at 670nm. The R 2 

value and the linear least squares fit equation are given in the figure. 


The scatter-adjusted absorbance at 430nm appears to obey Beer’s 

law with respect to total carotenoid content. The absorbance reading is 

linearly correlated with total carotenoid content, and the linear least 

squares fit to the data yields the equation: y = 2.9993x - 0.5497 with 

an R 2 value of 0.85 (p< 0.05). 

USE OF WATERMELON-PUREE ABSORBANCE TO QUANTIFY TOTAL 

CAROTENOID CONTENT. To validate the equation obtained in Figure 1 

as a predictive equation for total carotenoid content in watermelon 

tissue, the scatter-adjusted absorbances (430nm - 670nm) of 20 

additional watermelon purees (two hybrid varieties and three PI lines) 

were measured. Then, each value was inserted into the linear equation 

generated by Figure 1 to estimate the total carotenoid content of the 

tissue. Predicted values were plotted against the total carotenoid values 

estimated by hexane extraction (Figure 2). A linear relationship was 

obtained between the estimates by the two methods with an R 2 of 0.85. 

The equation for the linear least squares fit to the data, y = 0.9875x - 

0.0337, differs only slightly from that expected for an ideal fit, y = x. 

Hexane method total carotenoid content 


4 

3.5 

3 

2.5 

2 

1.5 

1 

0.5 

y = 0.9875x - 0.0337 

R 2 = 0.8476 

0 

-0.5 0.5 1.5 2.5 3.5 

-0.5 

Predicted lycopene content from puree absorbance method 


Fig. 2. Total carotenoid content determined for 20 individual yellow 

watermelons (3 varieties and 2 PI lines) by the puree absorbance method as 

compared with the determinations by the low-volume hexane assay. The 

adjusted absorbance at 430nm (i.e., 430 nm - 670nm) for each watermelon 

puree was converted to a total carotenoid content with the use of the linear least 

squares equation of Fig. 1 (y = 2.9993x - 0.5497). The R 2 value and the linear 

least squares fit equation are given in the figure. The hatched line indicates a 

perfect predictive model, i.e., y = x. 


To achieve the desired level of reliability in the puree absorbance 

procedure, one must: (1) maintain subdued light, since light degrades 

carotenoids, (2) thoroughly homogenize the tissue, and (3) mix the 

puree before reading the samples. 

This paper details a simple, rapid, and cost-effective method to 

quantify total carotenoids in flesh of canary yellow watermelons. We 

evaluated 67 watermelon samples (four hybrid varieties and five PI 

lines) with total carotenoid contents ranging from 0μg/g to 7μg/g fresh 

weight. The puree absorbance method gave a linear relationship to 

carotenoid content and is independent of watermelon variety. This 

method offers an improvement to the conventional method by 

reducing sample-processing time by at least half and requires no 

hazardous reagents. It is important to note that this equation may have 

to be altered for yellow fruit that have a higher total carotenoid than 

tested here (> 7ug/g). Until this germplasm is found, if it indeed exists, 

this method is a good screening tool to search for high-carotenoidcontaining 

yellow-watermelon germplasm. 


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QUANTITATIVE TRAIT LOCI ASSOCIATED 

WITH SUSCEPTIBILITY TO POSTHARVEST 

PHYSIOLOGICAL DISORDERS AND DECAY 

OF MELON FRUIT 

J. P. Fernández-Trujillo, J. A. Martínez, J. Obando, C. Miranda 

Technical University of Cartagena (ETSIA) and Institute of Plant 

Biotechnology, Cartagena, Spain 

A. J. Monforte, I. Eduardo, and P. Arús 

Laboratori de Genética Molecular Vegetal CSIC-IRTA, Cabrils, Spain 

ADDITIONAL INDEX WORDS. Cucumis melo, fruit quality, near isogenic lines, 

chilling injury, overripening, water-soaking texture, necrosis of placental tissue 

ABSTRACT. Melon (Cucumis melo L.) is a perishable fruit that requires 

refrigeration to extend its shelf life. A genomic library of near isogenic lines 

(NILs) derived from a cross between the Spanish cultivar ‘Piel de Sapo’ (PS) 

and an exotic Korean accession (PI 161375), each of them with a single 

introgression from PI 161375 into the PS background, was used to detect 

quantitative trait loci (QTLs). The analyzed polygenes included those affecting 

the susceptibility of fruit to physiological disorders and associated decay after 

long-term storage (35d at 8 o C and 75%RH). One QTL improved overall fruit 

flavor after refrigeration while another reduced total losses compared with PS. 

However, most of the QTLs identified had a detrimental effect on melon fruit 

quality compared with PS because they increased flesh susceptibility to watersoaking 

texture, overripening, and flavor loss. Other QTLs were associated 

with decreased susceptibility to chilling injury or Fusarium sp. decay, or 

increased susceptibility to Stemphylium sp. decay. The association of two QTLs 

to necrosis of the placental tissue at harvest was confirmed after the postharvest 

phase. Hollow flesh disorder was associated with three QTLs. 

P 

hysiological disorders, including chilling injury (CI), are a 

serious problem for melon exporters, because most commercial 

melon varieties are sensitive to CI (Tatsumi and Murata, 1981). 

Other disorders such as water-soaking (du Chatenet et al., 2000) and 

overripening are also important problems for the melon-processing 

This work was funded by grants 00620/PI/04 (Fundación Séneca, Región de Murcia), 

AGL2003-09175-C02-01, and AGL2003-09175-C02-02 from the Spanish Ministry 

of Education and Science and FEDER (EU). JO is grateful to the Spanish Ministry of 

Foreign Affairs for an MAE-AECI fellowship, and IE to the Spanish Ministry of 

Education and Science for an FPI grant. The parental line of PS melon was provided 

by Semillas Fitó S. A. (Barcelona, Spain) and plastic liners by Plásticos del Segura S. 

L. (Murcia, Spain). 


industry, and for when attempts are made to promote whole or freshcut 

melon consumption in the marketplace, as in fast-food or 

conventional restaurants (Fernández-Trujllo, 2006). Typical CI 

symptoms in melon are pitting and scald, which are usually colonized 

in a few days by saprophytic or necrotrophic fungi (Miccolis and 

Saltveit, 1995; Xu et al., 1990). Sensitivity to some disorders is 

strongly dependent on cultivar, storage time, and temperature 

(Miccolis and Saltveit, 1995). 

Wild or exotic germplasm can be used to improve input or output 

traits (i.e., fruit yield and quality, respectively) if selected genomic 

regions are introgressed into elite genetic backgrounds (Bernacchi et 

al., 1998; Fernández-Trujillo et al., 2005; Schauer et al., 2006). 

Approaches to identify the quantitative trait loci (QTLs) responsible 

for low-temperature responses in the introgressed region of tomato 

have been conducted with near isogenic lines (NILs) by Oyanedel et al. 

(2001). A genomic library of NILs derived from a cross between the 

Spanish cultivar ‘Piel de Sapo’ (PS) and an exotic Korean accession 

(PI 161375) has been recently developed (Eduardo et al., 2005). NIL 

genomic libraries facilitate the genetic dissection of complex traits 

(Zamir 2001). The NIL melon genomic library has been used to 

identify QTLs affecting fruit-quality traits during refrigerated storage. 


Twenty-seven NILs derived from a cross between the Spanish 

cultivar ‘Piel de Sapo’ (PS) and the exotic Korean accession 

‘Shongwan Charmi’ PI 161375 (SC) were used. Each of the NILs had 

a single introgression from SC into the PS background covering the 

whole genome of the SC (Eduardo et al., 2005). Ten plants for each 

NIL and SC and 50 plants for the PS were grown in a completely 

randomized design in Torre Pacheco (Murcia, Spain) using standard 

growing practices under Mediterranean summer conditions 

(Fernández-Trujillo et al., 2005). Every single plant of the NIL or 

parentals was considered a replication. 

Fruit from the previous experiment were used for the storage 

experiment. At least two fruit of 23 replications of PS and at least 5 

replications of eight NILs with introgression of SC located in six 

linkage groups (LG) (II, IV, V, VIII, IX, and X) were tested. At least 

two fruit from 3 to 5 replications of eight additional NILs with 

introgressions from SC located in seven LG (I, III, IV, V, VIII, IX, and 

XII) were also analyzed. Fruit were stored, distributed randomly in the 

cold chamber, for 35d at 8±0.5ºC and 75±3% relative humidity (RH), 

covered with plastic liners (Plásticos del Segura S.L., Murcia, Spain). 


The skin CI symptoms of PI161375 were also inspected after one, two, 

or three weeks at 8 o C. After this time, the stored fruits were examined 

for CI, decay, loss of whole-fruit finger texture, internal overripening, 

disorders, and flavor. 

Typical symptoms of CI were pitting, surface pitting, skin scald, 

and watery breakdown as a result of decay by secondary infection of 

microorganisms as reported by Tatsumi and Murata (1981). The 

degree of rind CI was evaluated subjectively. Fruit were divided into 

five classes depending on the skin-surface area affected by CI: 0 = 

absent; 1 = very slight, 0–5%; 2 = slight, 5–10%; 3 = moderate, 10– 

25%; 4 = severe, 25–50% (Fernández-Trujillo and Artés, 1998; 

Miccolis and Saltveit, 1995). Moderately to severely injured fruit was 

considered unmarketable since these disorders affected more than 20% 

of the epidermis surface in a double cut parallel to the longitudinal 

diameter. In the case of water-soaking texture (glassy texture, 

vitrescence), moderate to severely injured fruit were not of commercial 

quality since these disorders affected more than 20% of the epidermis 

surface. A hedonic test after storage scored whole-fruit hardness 

(finger texture), flesh overripening, and loss of fruit flavor by using a 

five-degree scale as follows: 1 = poor, soft, complete loss of flavor; 2 

= insipid and nonsweet, or senescent aroma; 3 = fair texture, slightly 

sweet and aromatic flavor; 4 = good, firm, balance between sweetness 

and flavor; 5 = very firm, excellent. 

Decay caused by necrotrophic fungi and other rots was recorded 

and considered as losses according to Blancard et al. (1995). The fungi 

were recorded with digital pictures and later identified by light 

microscopy. When only sterile mycelium appeared, melon fruits were 

subjected to additional storage (around one week at 22±2ºC with 

>90%RH) in order to facilitate the emergence of the conidiophores and 

conidia. In cases of doubt (due, for example, to the absence of 

conidiophores and conidia), potato dextrose agar culture and other 

techniques for stimulating the reproductive hyphae, such as cutting the 

mycelium, were applied. Decay-causing fungi were identified up to 

genus level by using the complete manual of imperfect fungi of 

Barnett and Hunter (1999). No index of severity was applied and fruits 

were classified according to the absence or presence of decay. 

The effect of the SC introgressions was studied by an analysis of 

variance (ANOVA) of the data untransformed or transformed into 

their respective arc sin or the arc sin of the square root of the data. The 

data transformation was performed to follow a normal distribution 

according to a normal probability plot. Mean NIL values were 

compared with the control genotype PS using the Dunnet contrast with 

Type-I error α ≤ 0.05 calculated by JMP 5.1.2 (SAS Institute Inc., 


Cary, NC). The number of QTLs was estimated assuming that there 

was only one QTL per introgression. When two NILs with overlapping 

introgressions showed significant effects, the QTL was considered to 

be located in the overlapping region. The effects of the QTLs located 

in NILs with significant effects are reported as relative to the PS mean: 

QTL effect = 

( NILmean − PSmean) 

100 * 

PSmean 


PI161375 showed slight CI symptoms after one week at 8 o C and 

moderate CI symptoms after two weeks at 8 o C, while PS showed no 

CI symptoms even after two–three weeks at 8 o C. CI developed mostly 

on fruit with netted skin, which was later colonized by Alternaria sp. 

and Cladosporium sp., generally growing together at 8 o C, as has been 

previously reported (Snowdon, 1991). 

One QTL located in LG VIII reduced total losses due to lower 

levels of CI and associated decay than observed in the PS control 

(Table 1). The main rots that developed after one month of storage at 

8 o C were Alternaria, Cladosporium, Stemphylium, and Fusarium sp., 

and, to a lesser extent, Botrytis sp. These necrotrophic fungi developed 

mainly on scald and pitting caused by CI. In some NILs the above 

fungi developed in cracks and CI associated to netting, and in the 

peduncle. No QTLs were found for susceptibility to Alternaria sp. or 

Cladosporium sp., or other occasional rots such as Aspergillus sp. or 

Botrytis sp. In agreement with Snowdon (1991), Fusarium sp. was 

found mainly in the peduncle. This rot was not specific to CI fruit 

because it also grew in fruit stored at 10 o C (data not shown). One QTL 

in LG VIII was responsible for a higher incidence of Stemphylium sp. 

in the NILs than in PS while another QTL in LG VIII showed the 

opposite trend for Fusarium sp. rot (Table 1). Other QTLs located in 

LGs IV, VIII, and IX may have reduced the sensitivity to CI (P

absent in PS. One QTL in LG X also had some influence in the 

development of the water-soaked texture associated with late ripening. 

In fact, harvesting fruits from every NIL with the QTL in LG X in the 

right state of maturity was difficult because the abscission layer beside 

the peduncle was not defined. 

Table 1. Analyzed disorders, decay, and flavor after storage for 35d at 

8 o C in the parent control ‘Piel de Sapo’ (PS) and quantitative trait loci 

(QTLs) detected with near isogenic line (NIL) genomic library. PS 

column indicates the mean for this genotype, the first number of the 

QTL indicates the linkage group number of the genetic map of melon 

where the QTL was mapped, the range of QTL effects and and P was 

the significance of the Dunnett test. 

PS 

Quality trait mean QTL Range of QTL effects relative to PS P 

Total losses 87% tl8.1 -86%

Four to 10% of fruits in NILs with introgressions in LGs III, VIII, 

and IX were affected by slight symptoms of hollow flesh disorder 

probably due to fruit senescence (Figure 1A). This disorder has not 

received attention in melons, but in cucumber or watermelon one that 

may be related (pillowy fruit disorder) has been associated with 

environmental and postharvest stress (Navazio and Staub, 1994). At 

least two QTLs (n2.3 and n9.2) located in LGs II and IX at harvest 

were responsible for necrosis of the placental tissue and surrounding 

flesh tissue (Figure 1B). The necrosis was also detected in another 

experiment analyzing fruit at harvest (data not shown), and was 

confirmed after the postharvest phase. The NILs with QTLs n2.3 and 

n9.2 had 100 and 60% of the fruit affected by the necrosis disorder, 

respectively. 

A B 

Fig. 1. Internal disorders in near isogenic lines (NILs) of melons after storage 

for 35d at 8 o C. (A) Necrosis of the placental tissue and adjacent areas in 

Linkage Group IX (QTL n9.2). (B) Hollow flesh disorder located in a NIL with 

a PI161375 introgression in LG IX. 

It is possible to identify the QTLs involved in the detrimental 

effects on melon fruit during refrigerated storage by using NILs, but to 

get good results a minimum number of replicates (five to eight) and 

fruit per replicate (to give at least thirty to forty fruit per NIL) are 

required to confirm these results. QTL identification can be 

implemented in breeding programs to develop new varieties suitable 

for long-term storage with improved flavor and reduced susceptibility 

to internal disorders and CI. The effect of these introgressions should 

be evaluated in a range of elite genetic backgrounds, to assess the 


suitability of using them as sources of new genetic variability in 

modern breeding programs. 


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POSTHARVEST CHARACTERIZATION OF A 

CUSHAW SQUASH BREEDING LINE 

L. Fernando Grajeda-García and Sergio Garza-Ortega 

Departamento de Agricultura y Ganadería, Universidad de Sonora, 

Rosales y Blvd. Encinas, Hermosillo, Sonora, México 83000 

Alberto Sánchez-Estrada and Rosalba Troncoso-Rojas 

Dirección de Tecnología de Alimentos de Origen Vegetal, Centro de 

Investigación en Alimentación y Desarrollo, Carr. a La Victoria km 

0.6. Hermosillo, Sonora, México 83000 

ADDITIONAL INDEX WORDS. Winter squash, firmness, soluble solids content, 

respiratory rate, weight loss 

ABSTRACT. Work was conducted to study the postharvest characteristics of 

Cushaw squash (Cucurbita argyrosperma) mature fruit stored at 20ºC and 80% 

relative humidity for 98 days. Respiratory rate and ethylene production rate 

fluctuated from 32.28 to 79.19mg CO2.kg -1 hr -1 and from 0 to 3.09µL C2H4 .kg - 

1 hr -1 , respectively. The soluble solids content was 10% (highest value) after 56 

days of storage and the average fructose, glucose, and sucrose was 0.66, 2.02, 

and 2.16mg.mL -1 , respectively. The highest weight loss was obtained after 98 

days of storage and the fruit firmness changed from 155N to 55N after 77 days 

of storage. Fruit density decreased from 0.72 at the beginning of the experiment 

to 0.6 after 98 days. Titratable acidity ranged from 0.018 to 0.041% and pH 

values from 7.4 to 7.84. We conclude that cushaw squash fruit showed the 

typical physiological behavior of other mature winter squash, but weight losses 

in storage may be significant. 

C 

ushaw squash, Cucurbita argyrosperma Huber, has been 

cultivated in Mexico, its domestication site, for at least 7000 

years. Contrary to the more widely grown species of winter 

squash, C. pepo L., C. maxima Duchesne, and C. moschata Duchesne, 

the information related to C. argyrosperma culture and management is 

very limited, particularly concerning fruit quality and postharvest 

behavior. Landraces of Cushaw squash that are grown in northwest 

Mexico, usually under rain-fed conditions, are taxonomically 

Cucurbita argyrosperma subsp. argyrosperma var. callicarpa. Fruits 

collected in the mountain region of this part of the country have a pale 

yellow to orange flesh color and an average weight of 2.5kg per fruit 

(Merrick, 1991). 

The authors are thankful to MSI F. Alfonso Aguilar-Valenzuela for his assistance in 

graph construction. 


In mature squash, high soluble solids content (SSC) is an indicator 

of good fruit quality. Nineteen days after harvest, fruits of cushaw 

squash breeding lines, hybrids, and landraces had SSCs ranging from 4 

to 7.5% for a spring crop and from 3.6 to 10.4% for a fall crop (Nuñez- 

Grajeda and Garza-Ortega, 2005). Using the same equipment for 

measuring SSC, Garza-Ortega et al. (2002) observed that fruits of 

‘Waltham Butternut’ (C. moschata), after one month of storage at 15 

to 22°C, had a higher SSC, 13.1%. 

The SSC of the kabocha (C. maxima) squash ‘Delica’ was 13.6% 

when measured 60 days after flowering plus 3 weeks of simulated 

refrigerated shipment at 12–14ºC (Harvey et al., 1997). In Australia, 

kabocha cultivars grown in different sites and seasons had SSCs that 

fluctuated from 5.7 to 13.3% at harvest time (Morgan and Midmore, 

2003). In butternut-type germplasm, the SSC increased from 2.6% at 

harvest to 9.4% after a period of storage of 60 days at 20ºC (Morales- 

Munguía et al., 2004). 

Winter squash fruits have a long postharvest life but weight losses 

in storage may be significant. The internal appearance of ‘Butternut’ 

squash began to deteriorate when fruits reached a weight loss of 15% 

(Francis and Thomson, 1965). This cultivar showed weight losses of 

15.8, 17.4, and 15% when stored at room temperature after 63, 70, and 

84 days, respectively, in different years (Holmes, 1951). Weight loss 

of ‘Delica’ reached 11.3% when stored at 20ºC and 75%RH for 12 

weeks (Arvayo-Ortiz et al., 1994). 

Fruit firmness of ‘Delica’ at harvest time was 81N but increased to 

100N and then slightly decreased to 94N after two and three months of 

storage at 12ºC and 80–85%RH, respectively (Ratnayake et al., 1999). 

The firmness of butternut fruit increased after 15 days of storage at 

20ºC and 70%RH but decreased after 45 days of storage, reaching a 

value of 51N; in this work, the average density of fruits at harvest was 

1.13 but decreased to 1.06 after 60 days of storage (Morales-Munguía 

et al., 2004). 

The objective of the present work was to study quality attributes 

and the physiological behavior of mature fruit from a true-to-type 

cushaw squash breeding line after harvest. 


FRUIT MATERIAL. C. argyrosperma A-43, a breeding line that 

was selected for having uniform fruit size, shape, color, and tolerance 

to Squash leaf curl virus, was used in this experiment. In the fall 

season of 2002, A-43 had an average fruit weight of 2.1kg (0.8 to 4.6) 

and a seed weight per fruit of 84.2g. The soluble solids content and 


flesh color measured 19 days after harvest was 7.4% and 7.3Y 

respectively (results not published). Seeds were directly sown into 

moist soil on August 14, 2002, and on September 24 the first female 

flowers appeared. Young fruits were removed on October 5, the date 

on which all plants had female flowers and set fruits uniformly 

afterwards. Mature fruits were harvested on November 29, so that 

most fruits had been on the vines for 55 days. After harvesting, fruits 

were stored in a laboratory at room temperature (15–24°C) and 

40%RH for five days. Fruits were then sorted, selecting those of 

uniform size and free of physical damage, washed with chlorinated 

water (250μL/L), and air-dried at room temperature. A group of 168 

fruits was stored for 98 days at 20°C and 80%RH and evaluated every 

1, 2, or 3 weeks. Evaluations consisted of measuring respiration, 

ethylene production, SSC, sugar content, weight loss, firmness, 

density, pH, and titratable acidity. 

RESPIRATION AND ETHYLENE PRODUCTION. Respiratory rate (RR) 

and ethylene-production rate (EPR) were determined (Troncoso-Rojas 

et al., 2005) by using six fruits that were weighed and placed 

individually in 6-L glass containers and kept at 20°C. Every week, the 

containers were covered with a plastic cap fitted with a septum. After 

1 h, 1-mL samples from the headspace were withdrawn and injected 

into a Varian 3400 cx gas chromatograph. RR and EPR were reported 

as mg CO2kg -1 .hr -1 and µL C2H4kg -1 .hr -1 , respectively. 

WEIGHT LOSS. In order to estimate weight losses, 12 fruits were 

individually marked, weighed in a digital balance (Mettler Pe Mod. 

2000), and stored at 20°C and 80%RH. Each fruit was weighed every 

week for 12 weeks and weight loss was calculated as a percentage. 

DENSITY. The volume of fruit was measured by water 

displacement in a graduated cylinder and expressed as milliliters (mL), 

and weight was measured in grams (g). Fruit density was calculated by 

dividing weight by volume. 

FIRMNESS. Six fruits were longitudinally cut in two pieces, for 

testing each piece with a Chatillon penetrometer model DFG-50 

equipped with a 10-mm plunger (J. Chatillon and Sons, NY). Firmness 

was expressed as the force in Newtons (N) needed to penetrate the 

mesocarp tissue. 

PERCENTAGE OF TOTAL SOLUBLE SOLIDS (TSS) AND SUGARS. The 

total soluble solids content was determined in fruit juice using an 

ABBE Leica Mark II refractometer model 10459 (American Optical 

Co.) expressing the TSS as a percentage. The sugar content was 

determined after every 2 or 3 weeks of storage at 20°C by a 

chromatographic method using an HPLC equipped with a refractive 

index detector (López-Hernández et al., 1994). Quantification was 


done with a standard curve of glucose with different concentrations. 

Sugar concentration is expressed as mg.mL -1 of tissue. 

TITRATABLE ACIDITY AND PH. An automatic Mettler DL21 tritator 

standardized at pH 4 was used to determine both parameters. The 

TA% is reported using citric acid as standard. 

STATISTICAL ANALYSIS. ANOVA were performed based in a 

completely randomized design. When significant differences were 

found, (P

EPR had values from 0 to 3.09µL kg -1 .hr -1 (Figure 1). During the first 

43 days of storage, EP was low (0 to 0.84µL C2H4kg -1 .hr -1 ) but a sharp 

increase was observed at day 59 (3.09µL C2H4kg -1 .hr -1 ). Other than 

this peak, the EPR was low. It is known that winter squash produce 

only trace amounts of ethylene, but wounding greatly increases the 

production (Brecht, 2004). 

SOLUBLE SOLIDS AND SUGAR CONTENT. The soluble solids content 

(SSC) reached a maximum value of 10% in fruits stored for 56 days 

and then decreased significantly at 98 days of storage (Figure 2). The 

same pattern for SSC changes after harvest was reported for both 

kabocha and butternut squash (Harvey et al., 1997; Morales-Munguía 

et al., 2004). However, the SSC of cushaw squash was lower than the 

SSC of kabocha squash measured at harvest (Morgan and Midmore, 

2003) and that of ‘Waltham Butternut’ measured 4 weeks after harvest 

(Garza-Ortega et al., 2002). The average fructose, glucose, and sucrose 

concentrations for the six sample dates were 0.66, 2.02, and 

2.16mg.mL -1 , respectively, increasing similarly to the SSC at 56 days 

of storage and decreasing afterwards (Figure 2). The fructose plus 

glucose levels increased slightly after 35 and 56 days of storage and 

then decreased at 98 days. In kabocha squash, the fructose plus 

glucose levels decreased in fruit picked 60 days after flowering and 

stored for 3 weeks at 12ºC (Harvey et al., 1997). 

Fig. 2. Changes in soluble solids concentration (SSC%) and sugar content in 

Cushaw squash fruit during storage at 20°C. Each value is the mean of 24 

replicates ± standard deviation. 


WEIGHT LOSS. As expected, the weight loss increased as storage 

time increased, reaching a maximum of 18.2% after 98 days of 

storage. At this point fruits showed yellowing of the skin but had no 

signs of shrivelling. However, fruits that were stored at 4°C for 3 

weeks and then at 20°C for 6 weeks showed depressions of the skin, 

mold development, and dehydration of the seed cavity (results not 

shown). Cushaw squash fruits usually become mummified when 

stored for long periods of time, sometimes keeping their original 

shape. The weight loss in this study was higher than the loss reported 

for kabocha and butternut squash (Arvayo-Ortiz et al., 1994; Holmes, 

1951). 

FRUIT FIRMNESS AND DENSITY. Fruit firmness at the beginning of 

the experiment was 115N, decreased slightly after 35 and 56 days, but 

then markedly decreased to 55N after 77 days of storage (Figure 3). 

However, firmness increased again, reaching 138.6N, after 98 days of 

storage. Initial firmness in this experiment was higher than the one 

reported for butternut squash (Morales-Munguía et al., 2004). The 

firmness of cushaw squash measured through the skin was similar to 

that found for kabocha (Ratnayake et al., 1999) after 6 weeks of 

storage, but the cushaw squash fruits were firmer after 12 weeks of 

storage to that observed previously. 

Fig. 3. Changes in firmness and density in Cushaw fruit during storage 

at 20°C. Each value is the mean of 24 replicates for firmness and 6 

replicates for density ± standard deviation. 


Fruit density measured at the beginning of the storage period was 

0.72, but significantly decreased to 0.6 after 98 days of storage (Figure 

3). The density of butternut fruit changed from an initial value of 1.13 

to 1.06 after 60 days of storage (Morales-Munguía et al., 2004). The 

large seed cavity of these more rounded cushaw fruits is most likely 

the reason for the lower density. 

TITRATABLE ACIDITY AND PH. Titratable acidity (TA) ranged 

from 0.018 to 0.041%, being higher at the beginning of the experiment 

but significantly decreasing after 35 days of storage. The pH values of 

the cushaw squash fruit ranged from 7.40 to 7.84, with no changes 

during the storage period at 20°C. These results agree with those 

reported by Alosi et al. (1988), who found pH values in phloem 

exudates of Cucurbita moschata from 7.5 to 7.8. 

Under the conditions of this study, cushaw squash fruits showed 

the typical physiological behavior of a mature winter squash, 

characterized by low ethylene production and low respiratory rate. The 

high weight loss indicates that further study is required, using lower 

storage temperatures and relative humidity combinations in order to 

prolong storage life. Cushaw squash fruits might have a favorable 

response to modified atmosphere storage. In regions where the fruit 

flesh is the main part of the fruit used for food, breeding programs 

should target higher soluble solids content and selection of fruits with 

smaller seed cavities and thus higher densities. 


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phloem exudate from Cucurbita fruit by dilution, glutathione, and glutathione 

reductase. Plant Physiol. 86:1089–1094. 

Arvayo-Ortiz, R. M., S. Garza-Ortega, and E. M. Yahia. 1994. Postharvest response 

of winter squash to hot-water treatment, temperature, and length of storage. 

HortTech. 4:253–255. 

Brecht, J. K. 2004. Pumpkins and winter squash. In: The commercial storage of 

fruits, vegetables, and florist and nursery stocks. USDA Agriculture Handbook 

66. . 

Francis, F. J. and C. L. Thomson. 1965. Optimum storage conditions for Butternut 

squash. Proc. Amer. Soc. Hort. Sci. 86:441–456. 

Garza-Ortega, S., A. Serrano-Esquer, and J. K. Brown. 2002. Yield, quality, and 

SLCV and SSL reactions of Cucurbita moschata Duchesne lines and hybrids 

evaluated in Sonora, México, p. 109–115. In: D. N. Maynard (ed.). 

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changes during the development and storage of buttercup squash. New Zealand 

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Holmes, A. D. 1951. Factors that affect the storage life of butternut squashes. Food 

Tech. 5:372–373. 


López-Hernández, J., M. J. González-Castro, M. E. Vazquez-Blanco, M. L. 

Vazquez-Oderiz, and J. Simal-Lozano. 1994. HPLC determination of sugars and 

starch in green beans. J. Food Sci. 59:1048–1049. 

Merrick, L. C. 1991. Systematics, evolution, and ethnobotany of a domesticated 

squash, Cucurbita argyrosperma. PhD Diss., Cornell Univ., Ithaca, NY. 

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(Cucurbita moschata Duchesne). Biotecnia. 6:43–52. 

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squash (Cucurbita argyrosperma Huber) lines, hybrids, and landraces in spring 

versus fall culture in Sonora, Mexico. HortSci. 40:1108(Abstr.). 

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J. Crop & Hort. Sci. 27:133–143. 

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Tiznado-Hernández. 2005. Effect of benzyl isothiocyanate on tomato fruit 

infection development by Alternaria alternata. J. Sci. & Food Agric. 85:1427– 

1434. 


HARVEST PERIOD AND STORAGE AFFECT 

BIOMASS PARTITIONING AND ATTRIBUTES 

OF EATING QUALITY IN ACORN SQUASH 

(CUCURBITA PEPO) 

J. Brent Loy 

University of New Hampshire, Department of Plant Biology, 

Durham, NH 

ADDITIONAL INDEX WORDS: Biomass allocation, Brix levels, embryo growth 

ABSTRACT. The effects of harvest date and storage on eating quality and 

biomass partitioning in acorn squash (Cucurbita pepo) were evaluated in two 

years. In 2003, ‘Table Ace’ and NH1605 were harvested at 30–35, 40–45, or 50– 

55 days after pollination (DAP), with or without storage for 20 days at 14 o C. In 

2005, NH1634, NH1635, NH1636, and ‘Tip Top’ were evaluated 25, 35, and 45 

DAP, with or without 10-day storage at 21 o C. In 2003, peak mesocarp dry 

weight (DW) occurred at 30 DAP, but Brix levels for good eating quality were 

not attained until 50 DAP. In 2005, the NH hybrids attained peak DWs 25 

DAP; ‘Tip Top’ at 35 DAP. Brix levels were low (5.9 to 7.2) across all cultivars 

25 DAP; 8.1 –10.9 35 DAP; and 9.7–13.4 45 DAP. Following 10-day storage at 

21 o C, o Brix reached acceptable (11+) levels 35 and 45 DAP in all NH hybrids, 

and 45 DAP in ‘Tip Top’. Embryos were 8–19mg DW 25 DAP and grew linearly 

until 55 DAP (45 DAP + 10-d storage). Reallocation of assimilates from 

mesocarp to embryos during storage significantly increased the proportion of 

seed to total DW biomass in NH1636 and ‘Tip Top’ 25 35 DAP. 

A 

corn squash, Cucurbita pepo L. subsp. texana (Scheele) Filov 

Acorn Group is one of the three most popular classes of 

squash consumed in North America. Based on evaluation of 

numerous samples of acorn squash from supermarkets and from 

cultivars grown at the University of New Hampshire research farms, 

eating quality appears to be inconsistent and generally poor. This is in 

contrast to buttercup/kabocha cultivars of C. maxima Duchesne, which 

generally exhibit uniformly good quality because of the high 

percentage of dry weight (DW) and sugar content. Kabocha squash 

are an important export crop in New Zealand, and this has led to 

extensive studies on culture (Buwalda and Freeman, 1986a, b), harvest 

time (Harvey and Grant, 1992; Harvey et al., 1997), and establishment 

of quality standards (Harvey et al., 1997). Such is not the case for 

acorn squash. Mesocarp DW in acorn squash is usually in the range of 

12 to 20% (Culpepper and Moon, 1945; J. B. Loy, unpublished data), 

considerably below that of kabocha squash (20 to 30%). An additional 

problem is that fruit of most modern acorn squash cultivars reach near 


full size and assume mature fruit pigmentation within two weeks from 

pollination, and may appear mature and thus be harvested well ahead 

of peak mesocarp DW (30 days), before acceptable Brix levels are 

attained, and considerably before seed maturation at 50 to 55 DAP 

(Loy, 2004). The objectives of the current studies were (1) to examine 

the relationship between harvest date and physiological factors 

affecting eating quality in acorn squash, and (2) to quantify the effect 

of harvest date and subsequent storage on allocation of biomass 

between mesocarp tissue and seeds. 


Field studies in 2003 and 2005 were conducted at the Woodman 

Horticultural Research Farm in Durham, NH. The soil type was a 

Charlton fine sandy loam (Inceptisol). In 2003, the experimental 

design was a split plot with two black-green acorn cultivars, ‘Table 

Ace’ (semibush) and NH1605 (bush), as main plots and six treatments 

as subplots. The six treatments were stored or nonstored fruit 

harvested at 30 to 35, 40 to 45, and 50 to 55 days after pollination 

(DAP). Fruit were tagged within two days of anthesis, and treatments 

were assigned randomly to plants within plots. For the three storage 

treatments, fruit were stored for 20 days at 14 o C. Raised beds, covered 

with 1.5-mil black polyethylene mulch and provided with 8-mil drip 

tape (T-tape with 12-inch emitter spacing), were spaced 1.8m on 

center. Each plot was seeded on 10 June 2003, and consisted of 10 

plants spaced 0.6m apart. Fertilizer at 56kg.ha -1 N and K2O was 

broadcast preplant, prior to bed formation. Additional soluble 

fertilizer was applied weekly as a 20-2-20 formulation at 5.6kg.ha -1 N 

and K2O through fertigation, beginning at one week prior to flowering, 

and continuing for five weeks. One application of imidacloprid 

applied through drip irrigation shortly after seeding was the only insect 

control. The fungicides chlorothalonil, azoxystrobin, and mycobutanil 

were applied as recommended in the 2002–2003 New England 

Vegetable Management Guide to control foliar diseases. 

The 2005 study consisted of four PMT F1 hybrids: ‘Tip Top’ 

(semibush; Johnny’s Selected Seeds, Albion, ME), NH1634 (bush), with 

black-green exterior pigmentation, and two semibush hybrids, NH1635 

and NH1636, with yellow and green striped exterior pigmentation 

similar to that of ‘Sweet Dumpling’. Plots were seeded on 03 June, 

2005. Each hybrid was grown as a separate experimental unit (row), 

with five replications and six treatments randomized within each plot of 

20 plants spaced 1.5m apart. The six treatments were harvests at 25, 35, 

or 45 DAP, with or without storage for 10 days at 21 o C. All fruits were 


tagged at or near the pollination date, and treatments were assigned 

randomly to different fruits. Cultural conditions and pest control were 

the same as for the 2003 study. 

In 2003, data were taken on fruit number per plant, fruit fresh 

weight (FW), mesocarp FW, DW, and o Brix, and seed/placental FW 

and DW. DW of mesocarp tissue was determined by multiplying FW 

x % DW of two core samples taken from the middle of fruit. Samples 

were dried for 48h at 60 o C in a VWR Model 1350FM drying oven. 

Subjective ratings (1 to 5 scale) were made by the investigator and 

research technician on perceived dryness, sweetness, texture, and 

overall eating quality of all squash. In 2005, data were taken on fruit 

FW, mesocarp FW and DW, seed/placental FW and DW, and seed 

coat and embryo FW and DW (20-seed sample). These data were used 

to generate data on biomass partitioning between seeds and mesocarp. 


FRUIT YIELD AND BIOMASS PARTITIONING IN 2003. ‘Table Ace’ 

had significantly larger fruit than NH1605, but about the same number 

of fruit per plant, so produced higher FW yields than NH1605 (Table 

1). ‘Table Ace’ has a semibush growth habit, and leaf-canopy cover 

was nearly complete at time for fruit set, whereas NH1605 is a bush 

hybrid, and the leaf canopy did not completely fill in rows spaced 1.8m 

apart. In preliminary studies in 2003 on plant population density with 

NH1605, decreases in within-row spacing from 0.6 to 0.3m increased 

fruit yields by about 20% without affecting fruit size or quality (J. B. 

Loy, unpublished data). 

Table 1. Comparison of fresh weight (FW) yield components in ‘Table 

Ace’ and NH1605 acorn squash in 2003, across all treatments. 

Mean 

fruit 

Cultivar FW (kg) z 

Mesocarp 

FW per 

fruit z 

Mean 

fruit 

no. z 

Means fruit 

FW per 

plant (kg) z 

Table Ace 0.78 a 0.67 a 3.75 a 2.92 a 

NH1605 0.56 b 0.45 a 3.92 a 2.19 b 

z 

Means within columns followed by the same letter are not significant by analysis of 

variance, P = 0.05. 

Total fruit DW per plant, total mesocarp DW per fruit, total 

mesocarp DW per plant, and total seed DW per fruit were not 

significantly different between cultivars (Table 2). The lack of 

significant differences between cultivars for DW as compared to FW 


Table 2. Comparison of dry weight (DW) yield components in ‘Table 

Ace’ and NH1605 acorn squash in 2003, across all treatments. 

Ratio seed 

Fruit Fruit DW Mesocarp DW total 

Cultivar DW (g) plant (g) DW fruit DW 

Table Ace 121.2 a 454.5 a 324.0 a 28.7 a 

NH1605 105.2 a 412.3 a 304.7 a 26.1 a 

z 

Means within rows followed by the same letter are not significantly different at 

P = 0.05 according to analysis of variance. 

y 

Seed DW includes placental mesocarp tissue surrounding seed. 

attributes of yield was due to the higher percentage of DW of 

mesocarp tissue in NH1605 (17.8%) as compared to ‘Table Ace’ 

(13.6) across all treatment comparisons. The proportion of biomass 

allocated to seeds and placental tissue was similar for both hybrids, 

amounting to slightly more than one fourth of the total fruit biomass. 

As elucidated in detail by Harvey et al. (1997), in kabocha squash 

grown in New Zealand, sugar levels and dry-matter content of the 

mesocarp are the overriding factors in determining eating quality of 

squash. Sugar levels and perceived sweetness of squash were highly 

correlated with measurements of o Brix with a refractometer. Squash 

with high dry matter (% DW) have a high starch content, and much of 

the starch is converted to sugars during the maturation of squash or 

during storage following harvest (Phillips, 1946; Schales and Isenberg, 

1963). A smooth pasty texture produced by a high starch content 

together with high Brix levels following conversion of some of the 

starch to sugar contributes to high eating quality in squash. For 

kabocha squash, 20% DW and 11 o Brix have been suggested as the 

minimum standards for acceptable eating quality at point of 

consumption (Harvey et al., 1997). For shipping and long term 

storage, % DW should be higher than 20%, so as to account for 

biomass loss due to respiration. 

In the present study, time of harvest did not significantly affect 

mesocarp % DW prior to storage (Table 3). Previous studies showed 

that maximum DW occurs at about 30 DAP in Cucurbita pepo squash 

(Culpepper and Moon, 1945), the time of first harvest in the present 

study. A significant reduction in mesocarp biomass during storage 

was expected, however, due to respiratory losses (Irving et al., 1997) 

and remobilization of assimilates to developing seeds (Loy, 2004). 

Storage did not have a consistent effect of % DW, possibly because of 

excessive error variance with only six replications, but also because 

moisture loss in storage increases % DW. However, using paired 


comparisons of nonstored to stored fruit, significant decreases in % 

DW following storage were obtained for ‘Table Ace’ harvested at 30 

DAP, and NH1605 harvested at 40 DAP. 

Time of harvest had a strong impact on o Brix (Table 3). Brix 

levels increased linearly over time of harvest for both cultivars. 

However, because ‘Table Ace’ had low dry biomass, the increase in 

o Brix from 6.0 at 30 DAP to 8.4 at 50 DAP was not sufficient to 

provide acceptable eating quality. In NH1605, o Brix increased from 

6.6 at 30 DAP to 8.8 at 40 DAP and to 11.2 at 50 DAP, the latter level 

being sufficient for good eating quality. Brix levels increased 

significantly during all three storage periods for ‘Table Ace’. 

However, the highest Brix levels, 10 to 10.5 attained at 40 and 50 DAP 

in stored ‘Table Ace’, largely depleted starch reserves, resulting in 

squash with a fibrous, watery texture that did not have acceptable 

eating quality. On the other hand, acceptable Brix levels were attained 

during all three storage periods with NH1605, and sufficient starch 

remained following storage so that texture remained good. 

Table 3. Dry weight and o Brix in ‘Table Ace’ (TA) and NH1605 

acorn squash harvested at 30, 40 and 50 DAP, with or without storage 

for 20 days at 14 o C. 

Harvest periods z 

Cultivar/storage 30 DAP 40 DAP 50 DAP 

Mesocarp % DW 

TA nonstored 14.4 a 14.7 a 13.6 a 

TA stored 10.9 a 14.8 a 13.6 a 

NH1605 nonstored 17.3 a 19.5 a 17.8 a 

NH1605 stored 17.7 a 16.3 a 17.2 a 

Mesocarp o Brix 

TA nonstored 6.1 d 7.3 cd 8.7 b 

TA stored 7.6 bc 10.6 a 10.0 a 

NH1605 nonstored 6.9 c 8.9 b 11.3 a 

NH1605 stored 11.5 a 11.0 a 11.6 a 

z Harvest periods of 30 to 35, 40 to 45 and 50 to 55 days after pollination(DAP). 

y Means for the 6 treatments within each hybrid followed by same letter were not 

significantly different at P = 0.05 by Duncans’ New Multiple Range Test. 

Texture, along with overall eating quality of squash cooked in a 

microwave, was subjectively evaluated by the author and a technician 

on a scale of 1–5: 1 = watery/fibrous; 2 = moist/fibrous; 3 = 

grainy/moist; 4 = smooth/moist; 5 = smooth/dry. For Treatment 6 (50 

DAP harvest + 20-day storage), the average texture rating for 16 


samples of ‘Table Ace’ was 2.4, and for 24 samples of NH1605 was 3.9. 

Evaluation of eating quality on a 1 to 5 scale (1 = poor; 2 = fair; 3 = 

good; 4 = very good; 5 = excellent) was similar, with ‘Table Ace’ 

averaging 1.9 for Treatment 6 and NH1605 averaging 4.3. 

2005 BIOMASS STUDY. Among the four hybrids, ‘Tip Top’ had the 

largest fruit (792g), followed by the ‘Sweet Dumpling’ type hybrids, 

NH1635 (638g) and NH1636 (604g), and the acorn hybrid NH1634 at 

559g (Table 4). Between 81 to 85% of fruit biomass was allocated to 

mesocarp tissue among the four hybrid cultivars. 

The primary aim of this study was to quantify the effect of 

remobilization of assimilates from mesocarp tissue to embryos (seed 

fill) on quality parameters (% DW and soluble solids) of the edible 

mesocarp. However, variation in fruit size and % DW of mesocarp 

tissue both within and among treatments precluded a precise 

determination of this relationship. Nonetheless, it is possible to show 

the magnitude o f the effect of remobilization by following increases 

in embryo DW biomass, and changes in the proportion of seed DW 

before and after storage of squash harvested at different time periods 

(Table 5). 

Table 4. Comparison of fruit biomass components among four C. pepo 

hybrids grown in 2005, and harvested at 25, 35, and 45 days after 

pollination (DAP). 

Cucurbita pepo F1 hybrids 

Fruit biomass 

components NH1634 NH1635 NH1636 Tip Top 

Fruit FW 559 638 604 792 

Fruit DW 111 126 123 139 

Mesocarp DW 91 107 101 112 

% Meso. DW/Total DW 82 85 82 81 

Mean seed number 276 247 219 260 

Reallocation of assimilates from mesocarp tissue to seeds varied 

considerably among cultivars, primarily because of variation in seed 

fill (embryo DW), but also due to differences in seed numbers. 

Changes in biomass distribution during storage were most evident in 

‘Tip Top’ and NH1636 because these cultivars had the largest seeds. 

With both of these cultivars, fruit harvested at 25 and 35 DAP showed 

significant increases in the proportion of seed DW biomass to total 

biomass following 10 days of storage at 21 o C. The increases were 


greatest, 52% in NH1636 and 67% in ‘Tip Top’, in fruit harvested at 

35 DAP. 

In nonstored fruit, increases in embryo biomass between 25 and 45 

DAP were nearly linear in ‘Tip Top’, but were more curvilinear in 

NH1634 and 1636 (Table 5). Seed fill was poor and reached a plateau 

at 35 DAP in NH1635. Increases in embryo biomass during 10-day 

intervals in stored fruit were comparable to those in fruit remaining on 

the plant. Moreover, fruit harvested at 45 DAP and stored continued 

to show large increases in embryo biomass, corroborating previous 

studies in C. pepo (Vining and Loy, 1998) showing that substantial 

seed fill may occur up to 55 DAP. 

Table 5. Changes in mesocarp % DW, o Brix, ratios of seed DW to 

total fruit DW, and increases in embryo biomass before and after 10 

days storage at 21 o C following harvest at 25, 35, or 45 days after 


25 DAP 35 DAP 45 DAP 

Treat- 

ments 

NS 

S 

Ratio seed/total DW 

NH1634 0.18a 0.23a 0.15a 0.20a 0.18a 0.23a 

NH1635 0.15a 0.21a 0.12a 0.16a 0.16a 0.19a 

NH1636 0.18a 0.23bc 0.21c 0.32a 0.29ab 0.24bc 

Tip Top 0.19ab 0.26a 0.15b 0.25a 0.19ab 0.25a 

20 Embryo DW (mg) y 

NH1634 8.6a 31.1b 31.3b 38.8b 36.2b 57.1c 

NH1635 11.3a 24.9a 28.4a 33.4a 25.5a 28.5a 

NH1636 19.1c 45.5b .42.0b 68.6a 48.5b 76.9a 

Tip Top 16.0d 54.9c 48.8c 74.3b 73.7b 87.5a 

20 Seed coat DW (mg) y 

NH1634 30.4a 22.7bc 26.3ab 23.2bc 22.8bc 22.5c 

NH1635 35.1a 23.5c 27.9b 23.8c 25.2bc 21.7c 

NH1636 39.7a 28.6b 29.2b 27.5b 27.9b 27.3b 

Tip Top 31.6a 28.0bc 29.6ab 26.6bc 27.3bc 25.4c 

Embryo DW per fruit (g) x 

NH1634 2.6 9.4 9.5 11.8 9.9 15.6 

NH1635 2.9 6.4 7.5 8.8 7.7 8.6 

NH1636 5.0 12.0 9.9 16.1 10.0 15.9 

Tip Top 4.6 15.6 15.6 23.8 16.7 19.8 

z Harvest and storage treatments within the same cultivar having the same letter are 

not significantly different at P = 0.05 by Duncan’s New Multiple Range Test. 

y Biomass of 20 embryo or seed coat samples. 

x Embryo DW per fruit determined by embryo DW x seed number per fruit. 

NS 


S 

NS 

S

The largest increase in total embryo biomass per fruit during 

storage was 11g in fruit of ‘Tip Top’ harvested at 25 DAP. In ‘Tip 

Top’, the mesocarp DW averaged 80g for fruit harvested at 25 DAP 

and stored. The 11g of embryo growth represents a fairly large sink 

for remobilization of assimilates from the mesocarp because in terms 

of energy equivalents, embryos, composed chiefly of lipids and 

protein, contain about twice those of mesocarp tissue (Loy, 2004). 

In squash harvested after seed maturation is complete (50 to 55 DAP), 

there is a steady loss of moisture during storage (Hopp et al., 1960; 

Phillips, 1946; Schales and Isenberg, 1963), along with a steady 

decline in dry biomass due to respiration of sugars (Hopp et al., 1960; 

Phillips, 1946; Irving et al., 1997). In squash harvested prematurely, 

respiratory biomass losses are compounded by additional losses, 

primarily of starch reserves, because of remobilization of mesocarp 

assimilates to developing seed. The effect on eating quality may not 

be readily evident in squash with relatively high dry matter content, 

but can be quite substantial in cultivars having low starch levels at 

time of harvest. 

Changes in seed-coat biomass were also evident (Table 5). Seedcoat 

biomass was greatest at 25 DAP, and decreased about 25% to 

30% within 10 days to stationary levels throughout the rest of seed 

development. Previous studies in seed pumpkins (Stuart and Loy, 

1988; Vining and Loy, 1998) showed that seed-coat DW peaks at 20 to 

25 DAP and then decreases, indicating that the seed coat may function 

as a transient storage organ, capable of providing assimilates to the 

developing embryo. 

COMPONENTS OF EATING QUALITY. Compared to the 2003 study, 

mesocarp % DW was relatively high for all of the cultivars in the 2005 

study (Table 6). Harvest time and storage did not have a statistically 

significant effect on mesocarp DW, with the exception of a significant 

drop in % DW during storage of ‘Tip Top’ fruit harvested at 25 DAP. 

It also appeared that mesocarp DW may not have reached maximum 

levels in ‘Tip Top’ at 25 DAP as compared to the other cultivars. 

Soluble solids levels, on the other hand, were markedly affected by 

harvest date and subsequent storage (Table 6). Fruit harvested at 25 

DAP had low Brix levels. Storage for 10 days increased o Brix, but in 

most cases the levels were not sufficient (11 or greater) for good eating 

quality. Brix levels increased at 35 DAP, and approached reasonable 

levels for good eating quality in all of the NH experimental hybrids. 

Brix levels were further elevated during storage, but were still 

insufficient in ‘Tip Top’ for good eating quality. At 45 DAP all 

experimental hybrids exhibited high Brix levels with or without 

storage, and ‘Tip Top’ attained 12.1 o Brix after 10 days of storage. 


Table 6. Changes in mesocarp % DW and o Brix before and after 10 

days storage at 21 o C, following harvest at 25, 35 or 45 days after 


25 DAP z 

35 DAP z 

45 DAP z 

Treatments NS S NS S NS S 

Mesocarp %DW 

NH1634 20.3a 18.0a 20.7a 20.5a 21.3a 21.1a 

NH1635 19.2a 16.9a 22.6a 20.6a 20.3a. 20.1a 

NH1636 20.3a 19.0a 20.8a 16.4a 21.3a 19.3a 

Tip Top 16.9a 12.6b 19.3a 16.7ab 19.8a 17.5a 

Mesocarp o Brix 

NH1634 7.2c 10.3b 9.6b 13.8a 12.8a 13.0a 

NH1635 6.8d 8.4c 10.9b 13.3a 13.4a 14.4a 

NH16346 6.8c 10.6b 10.2b 10.9b 12.8ab 13.9a 

Tip Top 5.9d 7.1cd 8.1c 9.7b 9.7b 12.1a 

z Harvest and storage treatments within the same cultivar having the same letter are 

not significantly different at P = 0.05 by Duncan’s New Multiple Range Test. 

Conclusions 

As noted in previous studies (Culpepper and Moon, 1945) and as 

illustrated in the present study, cultivars of Cucurbita pepo squash 

may vary considerably in soluble solids content and mesocarp DW, 

primary parameters contributing to eating quality. Premature harvest 

can markedly affect eating quality because peak dry matter is not 

attained until 25 to 30 DAP, and even in cultivars having sufficient dry 

matter, adequate o Brix are often not attained until after the fruit are 

fully mature in terms of seed maturation (50 to 55 DAP). Cultivars 

with low mesocarp DW are impacted more adversely than those with 

high DW by early harvest because conversion of starch to sugar and 

remobilization of sugars to seeds depletes starch below a threshold 

level necessary for an acceptable smooth, pasty texture. Acorn 

cultivars of C. pepo are particularly prone to being harvested immature 

because the fruit of most cultivars attain near full size and mature fruit 

rind coloration within two weeks of fruit set. The popularity among 

growers of cultivars with poor eating quality, along with negligence in 

harvesting squash at the proper time, negatively impacts consumer 

demand for a potentially excellent vegetable crop. New cultivars with 

higher dry matter should help to alleviate this problem. 



Buwalda, J. G. and R. E. Freeman. 1986a. Growth and development of hybrid squash 

(Cucurbita maxima L.) in the field. Proc. Agron. Soc. New Zealand. 16:7–11. 

Buwalda, J. G. and R. E. Freeman. 1986b. Hybrid squash: responses to nitrogen, 

potassium, and phosphorus fertilizers on a soil of moderate fertility. New 

Zealand J. Exp. Agr. 14:339–345. 

Culpepper, C. W. and H. H. Moon. 1945. Differences in the composition of the fruits 

of Cucurbita varieties at different ages in relation to culinary use. J. Agr. Res. 

71:111.136. 

Harvey, W. J. and D. G. Grant. 1992. Effect of maturity on the sensory quality of 

buttercup squash. Proc. Agron. Soc. New Zealand. 22:25–30. 

Harvey, W. J., D. G. Grant, and J. P. Lammerink. 1997. Effect of maturity on the 

sensory changes during development and storage of buttercup squash. New 

Zealand J. Crop & Hort. Sci. 25:341–351. 

Hopp, R. J., S. B. Merrow, and E. M. Elbert. 1960. Varietal differences and storage 

changes in β-carotene content of six varieties of squashes. Proc. Amer. Soc. 

Hort. Sci. 76:568–576. 

Irving, D. E., P. L. Hurst, and J. S. Ragg. 1997. Changes in carbohydrates and 

carbohydrate metabolizing enzymes during development, maturation, and 

ripening of buttercup squash (Cucurbita maxima D. ‘Delica’). J. Amer. Soc. 

Hort. Sci. 122:310–314. 

Loy, J. B. 2004. Morpho-physiological aspects of productivity and quality in squash 

and pumpkins (Cucurbita spp.). Crit. Rev. Plant Sci. 23:337–363. 

Phillips, T. G. 1946. Changes in composition of squash during storage. Plant Physiol. 

21:533–541. 

Schales, F. D. and F. M. Isenberg. 1963. The effect of curing and storage on 

chemical composition and taste acceptability of winter squash. Proc. Amer. Soc. 

Hort. Sci. 83:667–674. 

Stuart, S. G. and J. B. Loy. 1988. Changes in testa composition during seed 

development in Cucurbita pepo L. Plant Physiol. (Life Sci. Adv.). 7:191–195. 

Vining, K. J. and J. B. Loy. 1998. Seed development and seed fill in hull-less seeded 

cultigens In: J. D. McCreight (ed.). Cucurbitaceae 98: evaluation and 

enhancement of cucurbit germplasm. ASHS Press, Alexandria, VA.of pumpkin 

(Cucurbita pepo L.), p. 64–69. 


RIPENING CHANGES IN MINIWATERMELON 

FRUIT 

Penelope Perkins-Veazie and Julie K. Collins 

South Central Agricultural Research Laboratory 

USDA-ARS, Lane, OK 74555 

Donald J. Huber 

Horticulture Department, 

University of Florida, Gainesville, Florida 32611 

Niels Maness 

Department of Horticulture, Oklahoma State University 

Stillwater, OK 74074 

ADDITIONAL INDEX WORDS. Citrullus lanatus, lycopene, beta-carotene, pectin, 

cell wall 

ABSTRACT. In this study, seedless and seeded miniwatermelons (Citrullus 

lanatus) of ripe, underripe, and overripe stages were evaluated for quality 

characteristics and changes in carotenoids and pectins. Similar to seeded, large 

watermelon, minimelon weight, pH, and soluble solids content increased in fruit 

between underripe and ripe stages, while firmness decreased. Phytofluene, 

total, trans-, and cis- lycopene, and total carotenoid content increased as fruit 

changed from underripe to ripe stages. Beta-carotene content increased in 

overripe fruit compared to ripe fruit and may explain the orange tint that is 

seen in overripe watermelons. The proportion of large molecular weight pectins 

increased as minimelons became fully ripe, then overripe. Among cultivars, 

‘Xite’ had firmer flesh, thicker rind, and more lycopene than ‘Minipool’ or 

‘Valdoria’. ‘Minipool’ had fewer large molecular weight pectins while 

‘Valdoria’ had more beta-carotene and large molecular weight pectins 

compared to ‘Xite’. 

I 

n the U.S., the market has shifted from seeded to seedless 

watermelons, and from large (>10kg) to small fruit (4–10kg) fruit. 

A new introduction to the market is the miniwatermelon. These 

fruit are round and weigh less than 4kg. In addition, several of these 

selections, especially the seedless types, feature extremely firm, almost 

crunchy flesh. 

Little is known about the ripening events in watermelon fruit. 

Corey and Schlimme (1988) found that seeded watermelons exhibited 

a slight loss in soluble solids content and an increase in pH as fruit 

progressed from under- to overripe stages. At about 10 days 

postanthesis, lycopene accumulation is initiated in the locular area of 

the fruit (P. Perkins-Veazie, unpublished), while sugar accumulation 

occurs well before full color formation (Elmstrom and Davis, 1981). 


As fruit become overripe, cell-wall and membrane breakdown occur, 

yielding a mealy or grainy texture as cell wall fragments aggregate. 

Elkashif and Huber (1988) found that cell-wall changes in seeded 

‘Charleston Gray’ fruit shifted from large molecular weight to small 

molecular weights as fruit went from ripe to overripe stages. Although 

several of the minimelon cultivars exhibit firm flesh and high soluble 

solids and lycopene content, the ripening characteristics of 

miniwatermelons is unknown. 

Table 1. Indicators of watermelon ripeness (subjective and objective). 

Indicator Description 

Color Underripe is pink in locule first, then pink all 

over; overripe has orange cast; color expands 

into rind (rind looks thinner). 

Lycopene Underripe has 20 to 50% less, depending on 

variety and days from full ripeness; overripe 

will have slightly more, the same, or 10% less. 

pH Increases slightly from under to overripe. 

Soluble solids content (SSC) 

Difficult to separate ripe from overripe; can 

sometimes see higher SSC in locule compared 

to heart in slightly underripe; locations are 

equal in ripe; heart slightly higher in overripe. 

Texture (finger/mouth feel) Mealy in overripe; slimy in very overripe. 

Ripe fruit may be crunchy (‘Xite’) or crisp 

Flavor (taste) 

(‘Minipool’). 

Underripe has a ‘green’ or cucumber like 

flavor; overripe has pumpkin flavor. 


PLANT MATERIAL. Miniwatermelons were planted in Bixby, OK, 

in 2005, following recommended cultural practices for Oklahoma 

(Bolin and Brandenberger, 1999). Three cultivars were used: 

‘Minipool’ (seeded, crisp texture), ‘Valdoria’ (seedless, crisp texture), 

and ‘Xite’ (Hazera 6007) (seedless, crunchy texture, high lycopene). 

Fruit were harvested at underripe (7 to 10 days before ripe), ripe, and 

overripe (2–7 days after ripeness) stages using planting date, ground 

spot, and tendril browning as ripeness guides. Fruit were transported 

to Lane, OK, washed, weighed, measured for diameter and length, and 

cut into halves from stem end to blossom end. Fruit were classified by 


ipeness stage using subjective and objective measurements (Table 1). 

Rind thickness was measured at four points (stem and blossom ends 

and each side) with digital calipers. Firmness of flesh was measured at 

corresponding locations in the heart (center of watermelon) and in the 

interlocular tissues using a handheld Wagner gauge (2 to 25N) and flat 

(8mm) probe. Percent soluble solids content was measured on heart 

and locular samples using a digital refractometer. Ripeness of each 

fruit was determined using the indicators listed in Table 1. Three to 

four fruit per ripeness stage and cultivar were sampled. Samples of 

heart tissue were frozen at -80˚C for lycopene, carotenoid, and pectin 

analysis. 

FRUIT ANALYSIS. For sugar, carotenoid, and cell-wall analysis, 

three melons per ripeness stage were selected from each of the three 

cultivars. Lycopene and carotenoid content of frozen samples was 

determined within four months of harvest by spectrophotometry, 

scanning colorimetry, and HPLC using established methods (Fish et 

al., 2002; Davis et al., 2003; Craft, 2001). Ethanol insoluble powders 

were extracted from about 200g of fresh tissue per fruit using the 

methods of Karakurt and Huber (2002). Molecular size of chelatorsoluble 

polyuronides was determined using a Sepharose CL-2B-300 

gel column (Chun and Huber, 2000). The experiment was designed as 

a factorial arrangement and data were analyzed using ANOVA and 

general linear means model (SAS, v.9.0, Cary, NC). A significant 

interaction of cultivar and ripeness stage was seen only in β-carotene 

and in percent polyuronic acids. Main-effect means were separated by 

LSD, P

the minimelons, averaging 80mg/kg in ripe fruit. Phytofluene 

decreased in overripe fruit, while cis and trans lycopene were not 

significantly different in ripe and overripe fruit. Only beta-carotene 

increased significantly in overripe watermelon. As a percent of total 

carotenoids, beta-carotene increased from 2 to 4 to 6% in underripe, 

ripe, and overripe fruit, respectively. This increase may explain the 

distinct orange tint that appeared in overripe fruit. 

Among cultivars, ‘Xite’ had the thickest rind, firmest flesh, and 

highest lycopene content (Table 3). Beta-carotene was higher in 

‘Valdoria’, especially in overripe fruit (5.6 vs. 3.3 and 3.8mg/kg for 

‘Valdoria’, ‘Minipool’, and ‘Xite’, respectively; data not shown). 

mg/kg carotenoid 

100 

80 

60 

40 

20 

0 

A 

b a a b a a b a a 

translycopene 

total lycopene total 

carotenoids 

Fig. 1. Changes in the carotenoids trans-lycoepne, total lycopene, and total 

carotenoids (A), and beta-carotene, phytofluene, and cis-lycopene (B) with 

ripening of minimelons. 

Pectins (measured as chelator-soluble uronic acids) from the 

minimelons underwent a distinct downshift in molecular size during 

ripening, largely evident as a disappearance of polymers excluded 

from the molecular sieve gel (Figure 2). With overripening, 

proportionally greater quantities of low-molecular size polymers were 

evident, as seen also in pectins from ‘Charleston Gray’, a large, 

seeded watermelon cultivar (Elkashif and Huber 1988). Mao et al. 

(2004) reported a loss of membrane integrity and subsequent increase 

in lipoxygenase activity in watermelon gassed with ethylene. Similar 

changes in lipoxygenase activity may occur in watermelon as they pass 

from ripe to overripe stages, leading to a loss of cell integrity and 

changes in carotenoid and polyuronic profiles. 

Among cultivars, ‘Valdoria’ had the largest percent and ‘Minipool’ 

the smallest percent of large molecular weight uronic acids in ripe fruit 

(Figure 3). These results fail to explain the crunchy eating texture of 

‘Xite’ compared the more crisp texture of the other cultivars. The 

mg/kg carotenoid 

10 

8 

6 

4 

2 

0 

B 

b a a 

b a a 

cis-lycopene phytofluene _-carotene 

underripe 

ripe 

c b a 

overripe 


Table 2. Changes in ripening watermelons averaged over 3 minimelon 

cultivars. 

Averages of Xite, Valdoria, and Minipool 

Ripeness stage Underripe Ripe Overripe 

Weight (kg) 2.4b 3.4a 2.9ab 

Length (cm) 17.1a 19.2a 18.8a 

Diameter (cm) 16.2a 17.9a 17.4a 

Rind thickness (mm) 13.6a 12.4a 12.2a 

Firmness (N) 19.6a 14.6b 13.0b 

SSC (%) 8.1b 11.1a 11.2a 

pH 5.2b 5.8a 6.0a 

Total lycopene x mg/kg 30.6b 80.4a 70.7a 

Values represent means of 12–28 fruit; means separated by LSD, P

A 

C 

Fig. 2. Changes in pectin fragment size (% total uronic acids) in underripe (A), 

ripe (B), and overripe (C) miniwatermelons. Pectin fragment size decreases with 

volume. 

A Minipool 

B 

Xite 

C 

Fig. 3. Changes in pectin fragment size in ‘Minipool’ (seeded, crisp) (A), ‘Xite’ 

(seedless, crunchy) (B), and ‘Valdoria’ (seedless, crisp) (C) miniwatermelons. 

Pectin fragment size decreases with volume. 

B 

Valdoria 



Bolin, P. and Brandenberger, L. 1999. Cucurbit integrated crop management. 

Chun, J. P. and D. J. Huber. 2000. Firmness, ultrastructure, and polygalacturonase 

activity in tomato fruit expressing an antisense gene for the ß-subunit protein. J. 

Plant Physiol.121:1273–1279 

Corey, K. A. and D. V.Schlimme. 1988. Relationship of rind gloss and groundspot 

color to flesh quality of watermelon fruits during maturation. Sci. Hort. 34:211– 

218. 

Craft, N. 2001. Chromatographic techniques for carotenoid separation. Curr. 

Prot. Food Anal. Chem. F2.3.1–F2.3.15. Don’t understand coent about cited in 

text-Corey and Schlimme or Bolin and Brandenberger? 

Davis,A. R., W. Fish, and P. Perkins-Veazie. 2003. A rapid hexane-free method for 

analyzing lycopene content in watermelon. J. Food Sci. 68:328–332. 

Elkashif, M. E. and D. J. Huber. 1989. Enzymic hydrolysis of placental cell wall 

pectins and cell separation in watermleon (Citrullus lanatus) fruits exposed to 

ethylene. J. Amer. Soc. Hort. Sci. 114:81–85. 

Elmstrom, G. W. and P. L. Davis. 1981. Sugars in developing and mature fruits of 

several watermelon cultivars. J. Amer. Soc. Hort. Sci. 106:330–333. 

Fish, W., P. Perkins-Veazie, and J. K.Collins. 2002. A quantitative assay for 

lycopene that utilizes reduced volumes of organic solvents. J. Food 

Composition Anal. 15:309–317. 

Karakurt, Y. and D. J. Huber. 2002. Cell wall-degrading enzymes and pectin 

solubility and depolymerization in immature and ripe watermelon (Citrullus 

lanatus) fruit in response to exogenous ethylene. Physiol. Plant. 116:398–405. 

Mao, L-C., Y. Karakurt, and D. J. Huber. 2004. Incidence of water-soaking and 

phospholipid catabolism in ripe watermelon (Citrullus lanatus) fruit: induction 

by ethylene and prophylactic effects of 1-methylcyclopropene. Postharvest Biol. 

Tech. 33:1–9. 


CHANGES IN CAROTENOID CONTENT 

DURING PROCESSING OF WATERMELON 

FOR JUICE CONCENTRATES 

Penelope Perkins-Veazie and J. K. Collins 

USDA-ARS, South Central Agricultural Research Laboratory 

Lane, OK 74555 

M. Siddiq and K. Dolan 

Department of Food Science and Human Nutrition 

Michigan State University, East Lansing, MI 48824 

ADDITIONAL INDEX WORDS. Citrullus lanatus, processing, lycopene, betacarotene 

ABSTRACT. Watermelons (Citrullus lanatus) are used primarily as a fresh 

product in the U.S. There is interest in developing value-added products for 

additional markets and for use of cosmetically damaged fruit. In this study, 

watermelons were processed into juice concentrates, using a series of heat and 

treatment applications. Application of heat (50ºC) and a pectinase slightly 

increased juice yield from fruit macerate. Pasteurization had little or no effect 

on carotenoid content of most juices. Application of heat up to 40 or 50ºC 

slightly increased lycopene content of juice concentrates, while concentrating 

the juice to 42ºBrix increased lycopene content fivefold but reduced betacarotene 

content by 40 to 50%. Results indicate that watermelon juice exhibits 

lycopene stability with application of short or longer durations of heat 

treatment. 

I 

n the U.S., watermelon is consumed mostly as a fresh fruit. Fresh 

watermelon juice is popular in summer months, but there is little 

commercialization of the processed product. Challenges in 

developing a shelf-stable watermelon juice product include stabilizing 

This work was supported in part by grants from the National Watermelon Promotion 

Board and the USDA Rural Development Value-Added Producer Grants Program. 

Mention of trade names or commercial products in this article is solely for the 

purpose of providing specific information and does not imply recommendation or 

endorsement by the U.S. Department of Agriculture. All programs and services of 

the U.S. Department of Agriculture are offered on a nondiscriminatory basis without 

regard to race, color, national origin, religion, sex, age, marital status, or handicap. 

The article cited was prepared by a USDA employee as part of his/her official duties. 

Copyright protection under U.S. copyright law is not available for such works. 

Accordingly, there is no copyright to transfer. The fact that the private publication in 

which the article appears is itself copyrighted does not affect the material of the U.S. 



color and flavor (J. K. Collins, unpublished data). Additionally, as 

fresh watermelon contains large amounts of lycopene, a juice naturally 

high in lycopene and other carotenoids is desirable. 

Generally, during tomato fruit processing, lycopene is stabilized 

with heat application, and beta-carotene is lost (Takeoka et al., 2001; 

Abushita et al., 2000). Changes in watermelon carotenoids during 

food processing and pasteurization steps are unknown. This project 

was undertaken to determine carotenoid content in processed 

watermelon. 


PROCESSING STEPS. Seedless watermelons were obtained from 

local markets in East Lansing, MI. Fruit were sanitized using a 5-min 

dip in SCJ Fruit and Vegetable Wash (SC Johnson Professional, 

Sturtevant, WI.) (100µl/L available chlorine), peeled and cut by hand, 

and macerated with a propeller-type blender. Macerate was subjected 

to no heat, heat (50ºC in steam-jacketed kettle), or heat + enzyme 

treatments (50ºC plus pectinase [Crystalzyme]). Macerates were then 

filtered through a nylon cloth and stored at -20ºC (Figure 1). Batch 

pasteurization was done by placing 1-L juice samples into heated 

water in double-jacketed steam kettles, 15 sec at 85ºC. Juice was 

concentrated to 42ºBrix using a lab-scale vacuum evaporator at 40 or 

50ºC. 

POMACE 

MACERATE 

SIEVE 

JUICE 

+HEAT 

+ PASTUERIZATION 

+HEAT (40 or 50ºC) 

CONCENTRATE (42ºBrix) 

Fig. 1. Food-processing steps of watermelon to develop juices and juice 

concentrates. 


LYCOPENE AND CAROTENOID MEASUREMENT. Duplicate 

watermelon samples were pureed using a tissue homogenizer and a 0.2 

to 0.5g sample was extracted with hexane:ethanol:acetone (2:2:1 v/v), 

following the methods of Sadler et al. (1990) and a modified 

micromethod developed by Fish et al. (2002). Pomace samples were 

diluted 1:3 w/w with distilled deionized water prior to 

homogenization, and sonicated for 10 min to aid lycopene release from 

fibers. Concentrates were diluted 2:1 w/w with ddi water prior to 

homogenization. High performance liquid chromatography (HPLC) 

was used to determine carotenoid profiles of each sample in triplicate 

using a modified method of Craft (2001), as outlined in Perkins- 

Veazie et al. (2006). Tomato lycopene and carrot beta-carotene from 

Sigma (St. Louis, MO) and synthetic trans-lycopene from Lycovit 

(BASF) were used to verify spectra and calculate lycopene 

concentrations. 


Juice yield from watermelon was 55–57% on a per melon basis, 

and 82–86% of the flesh, with 5 to 8% of the flesh left as pomace 

(Table 1). Addition of pectinase and heat increased juice yields by 4% 

and reduced amounts of pomace by 3%. Lycopene and total 

carotenoid recovery after juicing was 61 to 66% of the original 

lycopene content of the macerate (Table 2). Heating and adding a 

pectinase to the watermelon macerate prior to filtering the juice had 

little effect on the amount of lycopene or carotenoids recovered in the 

juice, but greatly increased the amount of lycopene, beta-carotene, and 

total carotenoids in the pomace (Table 2). Although percent yield of 

pomace was less than 10% of the beginning amount of flesh, 

significant amounts of carotenoids appeared to remain bound to the 

fibers in the pomace. 

In the second experiment, use of pasteurization had no effect on 

individual carotenoids or on total carotenoid content of the juice 

(Table 3). Use of heat and pectinase in initial juice extraction slightly 

reduced lycopene, beta-carotene, and total carotenoid contents. When 

juice was concentrated to 42ºBrix, using 40 or 50ºC heat, all 

carotenoids were concentrated (Table 4). However, about 50% of 

beta-carotene was lost during heating. Isomerization of trans-lycopene 

to cis-lycopene was not found during pasteurization or heating (about 

4mg/kg cis-lycopene in all treatments). In tomato, exposure to heat 

during processing stabilizes lycopene, but can also cause losses of 9– 

28% of total lycopene with losses from lycopene oxidation or 

isomerization (Re et al., 2002; Takeoka et al., 2001; Stahl and 


Sies, 1992). Abushita et al. (2000) found that heating raw tomatoes 

followed by dehydration into paste stabilized lycopene but decreased 

beta-carotene by 30%, much of which was due to isomerization. With 

watermelon, an increase to 9mg/kg beta-carotene was expected due to 

concentration to 42ºBrix (Table 4). The 50% lower yield of betacarotene 

indicates a significant loss of this carotenoid during heating at 

40 or 50ºC. 

Table 1. Yield of juice and pomace. 

% Juice yield (per Juice yield Pomace yield 

Macerate melon basis) (%w/w, flesh (%w/w, flesh 

treatment 

basis) 

basis) 

Cold (no heat) 54.7 82.5 8.0 

Heat (50ºC) 

Heat (50ºC) + 

55.5 83.8 5.2 

enzyme 57.2 86.2 5.0 

Table 2. Changes in carotenoids after processing of watermelon flesh 

into juice and loss/gain in carotenoids from macerate to juice and 

pomace. 

Product 

Macerate Juice Pomace 

Heat 

(50ºC) 

+ 

Enzyme 

Heat 

(50ºC) 

+ 

Enzyme 

Heat 

(50ºC) 

+ 

Enzyme 

Quality 

component 

Soluble 

Cold 

Cold 

Cold 

solids (%) 

8.3 8.7 8.5 8.9 8.5 8.9 

pH 

Total 

5.4 5.3 5.7 5.6 5.2 5.2 

lycopene 

Beta- 

36.4+1.3 36.0+1.9 24.1+0.6 21.6+1.1 122.8+3.5 176.1+3.2 

carotene 

Total 

1.4+0.7 1.2+0.1 0.9+0 0.8+0.8 4.2+0.2 5.3+0.02 

carotenoids 38.6+1.3 37.2+2.0 26.8+0.6 22.3+1.1 129.0+1.0 181.4+3.2 

% loss/gain 

Total 

Macerate to juice Macerate to pomace 

lycopene 

Beta- 

- - -33.8 -40.0 +237.4 +389.2 

carotene 

Total 

- - -35.7 -33.3 +200.0 +341.7 

carotenoids - - -30.6 -40.0 +234.2 +387.6 

Means of duplicate samples, +standard deviation. Gain/loss is calculated from juice 

minus macerate value/macerate value for cold and heat, respectively. 


Table 3. Changes in carotenoids (mg/kg fresh wt) of watermelon juice 

subjected to pasteurization. 

Unpasteurized 

Pasteurized juice 

Macerate 

treatment 

None 

(cold) 

Total 

lycopene 

22.1+ 

0.1 

Betacarotene 

1.8+ 

0.1 

juice 

Total 

carotenoids 

26.5+ 

0.1 

Heat 

(50ºC) + 20.8+ 1.5+ 24.0+ 

enzyme 0.3 0.1 0.3 

Means of duplicate samples, +standard deviation. 

Total 

lycopene 

22.7+ 

0.7 

20.3+ 

0.1 

(85ºC, 15 sec) 

Beta- Total 

carotene carotenoids 

1.8+ 27.0+ 

0.1 0.8 

1.4+ 

0.01 

23.6+ 

0.01 

In the second experiment, use of pasteurization had no effect on 

individual carotenoids or on total carotenoid content of the juice 

(Table 3). Use of heat and pectinase in initial juice extraction slightly 

reduced lycopene, beta-carotene, and total carotenoid contents. When 

juice was concentrated to 42ºBrix, using 40 or 50ºC heat, all 

carotenoids were concentrated (Table 4). However, about 50% of 

beta-carotene was lost during heating. Isomerization of trans-lycopene 

to cis-lycopene was not found during pasteurization or heating (about 

4mg/kg cis-lycopene in all treatments). In tomato, exposure to heat 

during processing stabilizes lycopene, but can also cause losses of 9– 

28% of total lycopene with losses from lycopene oxidation or 

isomerization (Re et al., 2002; Takeoka et al., 2001; Stahl and Sies, 

1992). Abushita et al. (2000) found that heating raw tomatoes 

followed by dehydration into paste stabilized lycopene but decreased 

beta-carotene by 30%, much of which was due to isomerization. With 

watermelon, an increase to 9mg/kg beta-carotene was expected due to 

concentration to 42ºBrix (Table 4). The 50% lower yield of beta- 

carotene indicates a significant loss of this carotenoid during heating at 

40 or 50ºC. 

Table 4. Effects of heating juice to 40 or 50ºC on carotenoid content 

(mg/kg) of 42ºBrix unpasteurized juice concentrate. 

Beta- Total 

Temperature Treatment Total lycopene carotene carotenoids 

40ºC 

Cold macerate 


91.9±2.1 3.6±0.1 95.5±2.2 

enzyme 

111.2±6.3 4.6±0.01 119.0±6.4 

50ºC 

Cold macerate 


109.5±3.0 4.8±0.1 117.6±3.2 

enzyme 

94.8±1.7 3.6±0.1 98.4±1.8 


Conclusions 

Recovery of lycopene from a juice product was approximately 

60% of the original fruit. Methods using heat and pectinase enzymes 

slightly increased juice recovery but reduced recovery of lycopene and 

total carotenoids. Heat pasteurization had no effect on lycopene loss 

but may have helped stabilize the color of the juice during storage. 

Watermelon juice products as a source of lycopene and beta-carotene 

were successfully developed. 


Abushita, A. A., H. G. Daood, and P. A. Biacs. 2000. Change in carotenoids and 

antioxidant vitamins in tomato as a function of varietal and technological 

factors. J. Agri. Food Chem. 48:2075–2081. 


Prot. Food Anal. Chem. F2.3.1–F2.3.15. 

Fish, W., P. Perkins-Veazie, and J. K. Collins. 2002. A quantitative assay for 

lycopene that utilizes reduced volumes of organic solvents. J. Food Comp. 

Anal. 15:309–317. 

Perkins-Veazie, P., J. K.Collins, A. R. Davis, and W. Roberts. 2006. Carotenoid 

content of 50 watermelon cultivars. J. Ag. Food Chem. 54:2593–2597. 

Re, R., P. M. Bramley, and C. Rice-Evans. 2002. Effects of food processing on 

flavonoids and lycopene status in a Mediterranean tomato variety. Free Radical 

Res. 36:803–810. 

Sadler, G., J. Davis, and D. Dezman. 1990. Rapid extraction of lycopene and b- 

carotene from reconstituted tomato paste and pink grapefruit homogenates. J. 

Food Sci. 55:1460–1461. 

Stahl, W. and H. Sies. 1992. Uptake of lycopene and its geometrical isomers is 

greater from heat-processed than from unprocessed tomato juice in humans. J. 

Nutr. 122:2161–2166. 

Takeoka, G. R., L. Dao, S. Flessa, D. M. Gillespie, W. T. Jewell, B. Huebner, 

D. Bertow, and S. E. Ebeler. 2001. Processing effects on lycopene content and 

antioxidant activity of tomatoes. J. Agric. Food Chem. 49:3713–3717. 


VARIATION IN CAROTENOIDS AMONG 

MINIWATERMELONS PRODUCED IN FOUR 

LOCATIONS IN THE EASTERN U.S. 

Penelope Perkins-Veazie and Julie K. Collins 

USDA-ARS, South Central Agricultural Research Laboratory, 

Hwy 3W, Lane, OK 74555 

Richard L. Hassell 

Clemson University, Coastal Research and Education Center, 

2700 Davannah Highway, Charleston, SC 29414 

Donald N. Maynard 

Gulf Coast Research and Education Center, University of Florida, 

14625 CR 672, Wimanma, FL 33598 

Jonathan Schultheis 

North Carolina State University, Dept. Horticultural Science, 

Box 7609, Raleigh, NC 27695-7609 

Bill Jester 

North Carolina State University, Dept. Horticultural Science, Specialty 

Crops Program, Cunningham Research Station, 202 Cunningham Rd., 

Kinston, NC 28501 

Steve Olson 

North Florida Research and Education Center, University of Florida, 

155 Research Rd., Quincy, FL 32351 

ADDITIONAL INDEX WORDS. Citrullus lanatus, lycopene, beta-carotene, 

environmental effects 

ABSTRACT. Eighteen miniwatermelon (Citrullus lanatus) cultivars or lines 

(cultigens), all representing mini size (less than 4kg), were grown in South and 

North Florida, South Carolina, and North Carolina. The watermelon was 

produced at each location using plasticulture (black plastic mulch and drip 

irrigation) at an in-row spacing of 0.46m and row center spacing of 2.7m. 

Fertilization and pest-management practices were varied according to 

recommendations for each state. Germplasm had the greatest effect on total 

lycopene content. A strong geographical production effect on total lycopene was 

detected, with fruit from the two Florida locations having 10 to 15mg/kg more 

lycopene than fruit from the other two locations. Two minimelon cultigens had 

unusually high beta-carotene levels. On average, minimelons have high amounts 

of lycopene, over 100mg/kg in some cultigens, and production environment can 

significantly affect lycopene content. 

S 

eedless watermelons usually contain more lycopene than seeded 

cultivars, and irrigation and growing season (winter/summer) can 

slightly affect lycopene content (Leskovar et al., 2004; Perkins- 

Veazie et al., 2001; 2006). Miniwatermelons have been marketed in the 


U.S. for less than five years. These fruit are characterized by their 

rounded shape, size (less than 4kg), high soluble solids content (SSC); 

they are usually seedless. A number of minimelons are now available 

from seed producers. The purpose of this study was to determine the 

total lycopene content among minimelon cultivars, and to determine if 

production environment affected lycopene content. 


PLANT MATERIAL AND PRODUCTION PRACTICES. Eighteen 

miniwatermelon cultivars and cultigens were grown in three replicated 

blocks at four locations in the eastern U.S.: Bradenton, FL; Quincy, FL; 

Kinston, NC; and Charleston, SC. Standard field cultural practices for 

each state/location were used for the growing season. Ten plants were 

planted in one replication. Three replications were used per location. 

Plots with missing plants were replanted approximately seven days after 

planting to achieve 100% stand in most cases. Spacing between row 

middles was 2.7m while in-row spacing was 46cm. Pollenizer plants of 

SP-1 were interplanted in the plots at plants 1, 4, and 7. Field conditions 

at each location are given in Table 1. 

Fruits were harvested when ripe, with most subsequent harvests 

being made on a weekly basis. Fruits were graded for shape and disease 

defects according to USDA grading standards. Watermelons were cut 

from blossom to stem end and through the ground spot. Tissue from the 

center of the fruit (heart and locular area) was removed and placed in 

Ziploc bags held on ice. Samples were quickly frozen at -20°C, 

shipped to Lane, OK, and held at-80°C until analyzed. The total number 

of watermelons sampled per cultivar and location ranged from 6 to 12. 

Five miniwatermelon cultivars were selected for carotenoid-profile 


Board and the Oklahoma Center for the Advancement of Science and Technology 

(AP02(2)-i05). We thank the companies listed in Table 1 for providing seed. We thank 

Shelia Magby, Sheli Magby, Toni Magby, and Melissa O’Hern for their technical 

assistance. Mention of trade names or commercial products in this article is solely for 

the purpose of providing specific information and does not imply recommendation or 

endorsement by the U.S. Department of Agriculture. All programs and services of the 

U.S. Department of Agriculture are offered on a nondiscriminatory basis without 

regard to race, color, national origin, religion, sex, age, marital status, or handicap. The 

article cited was prepared by a USDA employee as part of his/her official duties. 

Copyright protection under U.S. copyright law is not available for such works. 

Accordingly, there is no copyright to transfer. The fact that the private publication in 

which the article appears is itself copyrighted does not affect the material of the U.S. 



Table 1. Field conditions at each location. 

NC 

(Kinston) 

SC 

(Charleston) 

N. FL 

(Quincy) 

S. FL 

(Bradenton) 

Latitude/longitude (º) 77.5/35 80/32.8 85/30.5 82.8/26.2 

Soil type Norfolk 

sandy 

loam 

Final fertility 

(NPK kg/ha) 

Temperature (ºC) 

(max/min) 30 days 

from 10% bloom 

Yauhannah 

loamy fine 

sand 

Dothan 

loamy fine 

sand 

Eau Gallie 

fine sand 

84-45-168 120-80-120 164-22-136 167-45-167 

30/21 32/22 32/17 29/16 

Harvest dates 7/12–8/26 6/20–7/6 5/20–7/20 5/30–6/3 

determination by high performance liquid chromatography (HPLC). 

These cultivars were selected as representatives of each seed company 

used in the variety trial. For HPLC analysis, five replicates, consisting 

of one to three fruit, were used from each variety and location. 

LYCOPENE, SSC, AND PH MEASUREMENT. Frozen watermelon 

tissue (40g per melon) was pureed using a tissue homogenizer. The pH 

of purees was taken using an Orion 8950 electrode and Orion pH meter. 

SSC was measured by placing about 0.5ml of puree on a digital 

refractometer (Atago PR 100). A 0.2- to 0.5-g sample of puree was 

extracted with hexane:ethanol:acetone (2:2:1 v/v), following the 

methods of Sadler et al. (1990) and a modified micromethod developed 

by Fish et al., (2002). One extraction was sufficient to completely 

remove color. The maximum absorbance of lycopene in hexane is at 

471nm, with an additional absorption peak at 503nm. The 503-nm 

wavelength was used to prevent measuring beta-carotene absorption, 

which can occur at 471nm. HPLC was used to check lycopene 

concentrations of five samples from each study, using the method of 

Craft (2001). Tomato lycopene from Sigma (St. Louis, MO) and 

synthetic trans lycopene from Lycovit (BASF) were used to verify 

spectra and calculate lycopene concentrations. Lycopene was also 

measured using a scanning colorimetric method with a Hunter XE xenon 

colorimeter (Hunter Associates, Reston,VA). In this method, watermelon 

puree was placed in a cuvette and absorbance was measured at 

560 and 700nm (Davis et al., 2003). Absorbance was plotted against 

lycopene values extracted with hexane to generate the slope, which is 

then used to calculate lycopene in the puree (total 

lycopene=39.5*[abs560-abs700]. 


Data from the completely randomized block design were subjected 

to analysis of variance (SAS v. 8.2, Cary, NC) using a general linear 

means model. When significant interactions were found between main 

effects, means were separated using REGWQ, P

with more vine resources enhancing carbohydrate allocation and 

carotenoid synthesis. 

Carotenoid profiles of the five minimelon varieties show strong 

similarities between ‘Xite’ and ‘Mohican’ (Table 3). These fruit were 

high in lycopene and total carotenoids. ‘Precious Petite’ fruit, although 

visibly red without an orange tint, had unusually high amounts of betacarotene. 

These levels of beta-carotene are higher than those found in 

orange-tinted overripe red-fleshed watermelon. It may be desirable to 

enhance beta-carotene without affecting visible color as beta-carotene is 

a vitamin A precursor, and is necessary for healthy eyesight. 

Watermelon currently have 10% vitamin A in a serving size (considered 

a ‘good’ source); increasing vitamin A to a consistent 20% per serving 

would improve the nutritional labeling to the “excellent” level. 

Phytofluene content was higher in the high lycopene cultivars ‘Xite’ and 

‘Mohican’, most likely an indicator of increased lycopene synthesis. 

Soluble solids content and pH are indicators of ripeness, and the high 

average for the four locations indicates full fruit ripeness (Table 3). 

Conclusions 

All seedless miniwatermelons tested exceeded 50 mg/kg lycopene, 

and would provide the suggested lycopene intake of 25 mg/day in a 2cup 

serving (~ 500g). Five of the miniwatermelons had enhanced 

lycopene content when produced in Florida compared to the Carolinas. 

This geographic difference was unexpected and indicates germplasm 

sensitivity to production environment. 

Table 3. Carotenoid profiles (mg/kg), SSC, and pH of puree from five 

minimelon cultivars averaged over the four geographic locations. 

Total CisBeta- Total 

lycolycocaroPhytocaro- SSC 

Cultivar penepenetenefluenetenoids (%) pH 

Xite 96.2a 8.1a 4.0b 7.4a 108.8a 12.6a 6.2 

Mohican 92.5a 12.0a 3.4b 6.3b 102.2a 11.7b 6.1 

Petite 

Treat 

62.8b 7.8a 4.4b 5.6b 68.2b 11.8b 6.2 

Precious 

Petite 

68.2b 9.7a 6.1a 5.9b 70.7b 12.3a 6.0 

Vanessa 71.5b 8.9a 3.2b 4.3c 79.6b 12.1ab 5.9 

Means separated within columns by REGWQ, P



Prot. in Food Anal. Chem. F2.3.1–F2.3.15. 

Davis, A. R., W. Fish, and P. Perkins-Veazie. 2003. A rapid hexane-free method for 

analyzing lycopene content in watermelon. J. Food Sci. 68:328–332. 



Comp. Anal. 15:309–317. 

Leskovar, D., H. Bang, K. Crosby, N. Maness, A. Franco, and P. Perkins-Veazie. 2004. 

Lycopene, carbohydrates, ascorbic acid and yield components of 

diploid and triploid watermelon cultivars are affected by limited irrigation. J. 

Hort. Sci. Biotech. 79:75–81. 


content differs among red-fleshed watermelon cultivars. J. Sci. Food Agri. 

81:983–987. 

Perkins-Veazie, P., J. K. Collins, A. Davis, W. and Roberts. 2006. Carotenoid 

content of 50 watermelon cultivars. J. Agric. Food Chem. 54:2593-2597. 



Food Sci. 55:1460–1461. 


WATERMELON CAROTENOID CONTENT IN 

RESPONSE TO GERMPLASM, MATURITY, 

AND STORAGE 

Penelope Perkins-Veazie, Julie K. Collins, and Angela Davis 

U.S. Dept. Agriculture, Agricultural Research Service 

South Central Agricultural Research Laboratory, 


Niels Maness 

Department of Horticulture 

Oklahoma State University, Stillwater, OK 74074 

Warren Roberts 

Oklahoma State University 

Wes Watkins Research and Extension Center, 


ADDITIONAL INDEX WORDS. Citrullus lanatus, carotenoids, shelf life, triploid 

melon, seedless, genotype 

ABSTRACT. Watermelon (Citrullus lanatus) contain high amounts of lycopene, 

the pigment that imparts red color to some fruits. Lycopene is a highly efficient 

oxygen radical scavenger and has been implicated in human studies as 

providing protection against cardiovascular disease and some cancers, 

particularly that of the prostate. Over the last six years, we have evaluated 

4,000 fruit from 80 cultivars to determine the effects of germplasm, ripeness, 

and storage on lycopene content. Watermelon lycopene is most affected by fruit 

maturity and germplasm; fruit about 7 days from full ripeness had 10–20% less 

lycopene. Postharvest storage of whole watermelon at room temperature 

enhanced lycopene content while fresh-cut watermelon lost about 10%, 

probably from membrane breakdown and lycopene oxidation. The results from 

these studies indicate that watermelon lycopene is highly dependent on 

germplasm and ripeness stage. Lycopene content is relatively stable in fresh-cut 

fruit and can be influenced by storage temperature in intact watermelon. 

A 

diet rich in fruits and vegetables has been linked to reduced 

incidence of some chronic diseases such as atherosclerosis and 

cancers (Joshipura et al., 2001; Steinmetz and Potter, 1996). 

The reasons for this relationship appear to be multifaceted and may 

include natural plant phytochemicals that act as electron scavengers in 

photosynthesis, and/or detoxification agents. 

Lycopene, a red pigment of the carotenoid class found in only a 

few fruits and vegetables, is a powerful singlet oxygen scavenger and 

highly effective antioxidant (Gerster, 1997). A high dietary intake of 

tomatoes, rich in lycopene content, is associated with a lower risk of 

certain cancers, primarily of the prostate (Giovannucci, 2002). 


Watermelon and tomatoes are the most familiar sources of lycopene in 

the Western diet, containing on average 46 and 31mg lycopene/kg 

fresh weight, respectively (USDA National Nutrient Database for 

Standard Reference, Release 18, 2005). 

Most watermelon sold in the U.S. is consumed fresh, with 50 to 

80% of this sold as seedless (National Watermelon Promotion Board, 

2005, personal communication). Much of the fresh market fruit is 

shipped around the U.S., and shelf life of intact fruit is expected to be 

7 to 21 days (Rushing et al., 2001). Minimally processed (cut or 

cubed) watermelon represents about 20–30% of watermelon sales in 

the U.S, but can be as high as 80% of watermelon sales in other 

countries. The objectives of our work over the last six years have been 

to determine the genotype, germplasm, and ripeness effects on 

lycopene, and to determine the stability of lycopene in stored and cut 

(minimally processed) watermelon. 


PLANT MATERIAL. Experiments were conducted at the South 

Central Agricultural Laboratory in Lane, OK. Types of melons 

sampled included seeded heirloom (such as ‘Black Diamond’); seeded 

hybrid (such as ‘Summer Flavor 800’); seedless (such as ‘Tri-X-313’); 

seeded pollinators (such as ‘Minipool’), and seedless miniwatermelons 

(such as ‘Extazy’). About 4,000 watermelons have been sampled from 

1999 to 2005. About 10 to 20 fruit per cultivar per year were sampled 

to determine the relative lycopene content. Fruit were cut transversely 

through the ground spot and watermelon center and 100–300g tissue 

was sampled from heart and locular areas and held at 

-80 o C until analyzed. 


Board, the Initiative for Future Agricultural Farming Systems (IFAFS Agreement 

No. 00-521021-9635), and the Oklahoma Center for the Advancement of Science 

and Technology (AP02(2)-i05). Mention of trade names or commercial products in 

this article is solely for the purpose of providing specific information and does not 

imply recommendation or endorsement by the U.S. Department of Agriculture. All 



age, marital status, or handicap. The article cited was prepared by a USDA employee 

as part of his/her official duties. Copyright protection under U.S. copyright law is not 

available for such works. Accordingly, there is no copyright to transfer. The fact that 

the private publication in which the article appears is itself copyrighted does not 

affect the material of the U.S. Government, which can be freely reproduced by the 

public. 


GERMPLASM, GENOTYPE, AND RIPENESS STUDIES. Six cultivars 

were selected for ripeness studies, using 10 to 20 watermelons per 

underripe, ripe, and overripe stage. Underripe melons were about 7 

days from fully ripe and overripe about 7 days past ideal ripeness, as 

judged by flesh color (pale red in underripe to orange in overripe), 

texture (firm or mealy/slimy), odor/taste (green taste in underripe and 

pumpkinlike in overripe), and sweetness (at least 9% soluble solids 

content for fully ripe). More details on analysis of these melons can be 

found in Perkins-Veazie et al. (2001, 2006). 

STORAGE AND FRESH-CUT STUDIES. For storage studies, 10 uncut 

melons per cultivar and temperature treatment of ripe fruit of seedless 

and hybrid seeded cultivars (‘Sugar Shack’ and ‘Summer Flavor 800’) 

were placed at 5, 13, or 21°C for 0 or 14 days, tissue was sampled as 

described above, and lycopene and soluble solids analyzed as 

described below. To determine effects of minimal processing on 

lycopene stability, 20 melons each of the above were cut in half and 

cubes of 3cm 3 flesh placed in sealed plastic boxes held at 2°C for 10 

days in darkness. The same melon was used to fill one box replicate 

for 0 and 10 days (Perkins-Veazie and Collins, 2004). 

LYCOPENE AND SOLUBLE SOLIDS CONTENT MEASUREMENT. 

Frozen watermelon tissue (40g per melon) was pureed using a tissue 

homogenizer. Soluble solids content was determined by placing 0.5ml 

puree on a digital refractometer (Atago PR100). 

A 0.2- to 0.5-g sample was extracted with hexane:ethanol:acetone 

(2:2:1 v/v), following the methods of Sadler et al. (1990) and a 

modified micromethod developed by Fish et al., (2002). One 

extraction was sufficient to completely remove color. Absorbance of 

hexane extracts was measured at 503nm by spectrophotometer 

(Shimazdu UV160). Lycopene purees were placed in cuvettes and also 

measured using a scanning colorimetric method with a Hunter XE 

xenon colorimeter (Hunter Associates, Reston, VA) (Davis et al., 

2003). Absorbance at 560nm was plotted against lycopene values 

extracted with hexane to generate the slope, which is then used to 

calculate lycopene in the puree (total lycopene=39.5*[abs560-abs750]. 

High performance liquid chromatography (HPLC) was used to 

determine carotenoid profiles of cultivar subsets in triplicate using a 

modified method of Craft (2001), as outlined in Perkins-Veazie et al. 

(2006). Tomato lycopene and carrot beta-carotene from Sigma (St. 

Louis, MO) and synthetic trans-lycopene from Lycovit (BASF) were 

used to verify spectra and calculate lycopene concentrations. 


STATISTICS. All studies were designed as randomized complete 

block or factorial experiments. Data were subjected to ANOVA and a 

general linear model was used (SAS, v. 9.0, Cary, NC). Means were 

separated by REGWQ, P12 32.5 to 69.2c 0.1 to 3.0 

Hybrid, seeded 

Minipollenizer, 

6 to 12 45.5 to 92.7ab 0.8 to 9.3 

seeded 4 45.5 to 100.5ab 0.8 to 7.4 

(minimelon)

Table 2. Range of total lycopene content among ripeness stages of 

watermelon; underripe is about 7 days from full ripeness and overripe 

is 7 days past ideal ripeness. 

Total lycopene content (mg/kg) 

Stage 

Genotype 

Heirloom seeded 

Underripe Ripe Overripe 

(Black Diamond) 

Hybrid seeded (Summer 

26.0b 35.7a 32.8a 

Flavor 800; Dumara) 49.2b 55.8a 57.3a 

Seedless (Tri-X-313) 

Pollenizer, 

51.2b 65.5a 60.4ab 

seeded (Minipool) 

Seedless minimelon 

48.0ab 63.8a 53.4ab 

(Xite; Valdoria) 72.5b 90.3a 94.6a 

Means separated within row and genotype by REGWQ, P

(64 to 60mg/kg) (Perkins-Veazie et al., 2004). This loss may have 

been from degradation of cut surfaces exposed to air during storage, 

with oxidation of lycopene, or from chilling injury. Soluble solids 

content slightly decreased (0.5 to 1%) for most samples after storage 

(data not shown). 

Conclusions 

Red-fleshed watermelon consistently has large amounts of 

lycopene per unit fresh weight, although amounts are dependent on the 

germplasm and ripeness of the fruit. Holding uncut fruit at room 

temperature increased lycopene and beta-carotene contents. 



Prot. Food Anal. Chem. F2.3.1–F2.3.15. 

Davis, A. R., W. Fish, and P. Perkins-Veazie. 2003. A rapid hexane-free method 




Comp. Anal. 15:309–317. 

Gerster, H. 1997. The potential role of lycopene for human health. J. Amer. 

Coll. Nutr. 16:109–126. 

Giovannucci, E. 2002. A review of epidemiologic studies of tomatoes, lycopene, 

and prostate cancer. Exp. Biol. Med. (Maywood). 227:852–859. 

Joshipura, K. J., F. B. Hu, and J. E. Manson. 2001. The effect of fruit and vegetable 

intake on risk for coronary heart disease. Ann. Intern. Med. 134:1106–1114. 

Perkins-Veazie, P. and J. K. Collins. 2004. Flesh quality and lycopene stability of 

fresh-cut watermelon. Postharv. Biol. Tech. 31:159–166. 

Perkins-Veazie, P., J. K. Collins, A. Davis, and W. Roberts. 2006. Carotenoid 

content of 50 watermelon cultivars. J. Agric. Food Chem. 54:2593–2597. 


content differs among red-fleshed watermelon cultivars. J. Sci. Food Agric. 

81:983–987. 

Rushing, J. W., J. M. Fonseca, and A. P. Keinath. 2001. Harvesting and 

postharvest handling. In: D. N. Maynard (ed.). Watermelons: characteristics, 

production, and marketing. ASHS Press, Alexandria, VA. 



Food Sci. 55:1460–1461. 

Showalter, R. K. 1960. Watermelon color as affected by maturity and storage. 

Proc. Fla. State Hort. Soc. 73:289–293. 

Showalter, R. K., S. A. Harmon, B. B. Brantely, D. W. Newson, and J. F. Pittman. 

1955. 

Changes in Congo watermelons after harvest. Proc. Assoc. South. Agric. 

Wrkrs. 52:136–137. 

Steinmetz, K. A., and J. D. Potter. 1996. Vegetables, fruit, and cancer prevention: 

a review. J. Amer. Diet. Assoc. 96:1027–39. 

USDA National Nutrient Database for Standard Reference Release 18. 2005. 

.

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