entire symposium - Plant Management Network

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Proceedings of the Meeting 

Editors: Jackie K. Burns, James H. Graham, and Tim R. Gottwald 

General Editor: Barbara Thompson 

Organizing Committee: 

Jackie Burns (Chair) – University of Florida/IFAS – jkbu@ufl.edu 

Wayne Dixon – FDACS/DPI – dixonw@doacs.state.fl.us 

Tim Gottwald – USDA/ARS – Tim.Gottwald@ars.usda.gov 

Tim Spann – University of Florida/IFAS – spann@ufl.edu 

Jim Graham – University of Florida/IFAS – jhgraham@ufl.edu 

Research Program Committee: 

Elizabeth Baldwin – USDA/ARS 

Renato Bassanezi – Fundecitrus 

Bill Dawson – University of Florida/IFAS 

Megan Dewdney – University of Florida/IFAS 

MaryLou Polek – California Citrus Research Board 

Industry Organizing Committee: 

Bobby Barben – Citrus Research & 

Development Foundation 

Dan Gunter – Citrus Research & 

Development Foundation 

IRCHLB Proceedings Jan 2011: www.plantmanagementnetwork.org 

David Hall – USDA/ARS 

Mike Irey – U.S. Sugar 

John DaGraça – Texas A&M University 

Michael Rogers – University of Florida/IFAS 

Juliano Ayres – Fundecitrus 

Ray Prewett – Texas Citrus Mutual 

Mike Sparks – Florida Citrus Mutual 

Conference Contacts: 

Clark Baxley – Florida Citrus Mutual – clarkb@flcitrusmutual.com 

Christen Johnson – University of Florida/IFAS – chris29@ufl.edu 

Kevin Metheny – Florida Citrus Mutual – kevinm@flcitrusmutual.com 

Jewel Letchworth – Florida Citrus Mutual – jewell@flcitrusmutual.com 

IRCHLB Proceedings Compilation Copyright © 2011 Plant Management Network 

Orlando, Florida January 2011

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Sponsors 

The Organizing Committee extends its sincere thanks to all who assisted with the conference. 

Special appreciation is extended to Clark Baxley, Kevin Metheny, Mike Sparks, and the staff of 

Florida Citrus Mutual for providing expert service and outstanding support for the 

2 nd International Research Conference on Huanglongbing. We also thank Dr. Earl Taylor and 

Dr. Gavin Poole for their logistical and audio-visual contributions during the meeting. 

IRCHLB Proceedings Jan 2011: www.plantmanagementnetwork.org

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Conference Organizers 


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The 2 nd International Research Conference on 

Huanglongbing 

January 10-14, 2011 

Index of Presentations: 

No. Presentation and Authors Page 

Session 1: Pathogen Genomics, Bioinformatics, Phylogenetics, and Culturing – 

John Hartung, Moderator 

1.1 Genomic Comparisons of the Ca. Liberibacter asiaticus Chromosome 17 

with Other Members of the Rhizobiales – Hartung, J.S., Shao, J., 

Kuykendall, L.D. 

1.2 Genetic and Functional Characterization of the znu Operon in the 18 

Intracellular Citrus Pathogen, Candidatus Liberibacter asiaticus – 

Vahling, C.M., Benyon, L.S., Duan, Y.-P. 

1.3 Comparison of the Ca. Liberibacter asiaticus Genome with a Draft Ca. 19 

L. americanus Genome Reveals Similar Prophage with Likely 

Pathogenicity Factors – Zhang, S.J., Wulff, N.A., Flores-Cruz, Z., Zhou, 

L.J., Kang, B.-K., Fleites, L.A., Gooch, M.D., Davis, M.J., Duan, Y.-P., 

Gabriel, D.W. 

1.4 Analysis of Candidatus Liberibacter americanus Genome – Wulff, N.A., 20 

Zhang, S.J., Ayres, A.J., Bové, J.M., Gabriel, D.W. 

1.5 Population Genetics Analysis of Candidatus Liberibacter asiaticus from 21 

Multiple Continents – Glynn, J.M., Bai, Y., Chen, C., Duan, Y.-P., 

Civerolo, E.L., Lin, H. 

1.6 Phylogenetic Analysis of Asian Candidatus Liberibacter asiaticus; Asian 22 

Common Strains Are Distributed in Northeast India, Papua New 

Guinea, and Timor-Leste – Miyata, S., Kato, H., Tomimura, K., Davis, 

R., Smith, M.W., Weinert, M., Iwanami, T. 

1.7 Bioinformatic Analysis of Genome Sequence Data for Ca. Liberibacter 23 

asiaticus – Lindeberg, M., Saha, S. 

1.8 Genetic Diversity of Candidatus Liberibacter asiaticus Isolates from 24 

Paraná State, Brazil – Meneguim, L., Marques, V.V., Murata, M.M., 

Barreto, T.P., Vasquez-Souza, G.V., Villas-Boas, L.A., Paccola-Meirelles, 

L.D., Leite, R.P., Jr. 

1.9 Analysis of Endophytic Bacterial Diversity from Huanglongbing 

Pathogen-Infected Citrus Tissues – Wang, A., Yin, Y., Li, Y., Li, J., 

Xian, J., Wang, Z. 

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1.10 Evolving Diversity of Candidatus Liberibacter asiaticus Revealed by 31 

Comparative Analysis of Two Intragenic Tandem Repeat Genes – 

Zhou, L.J., Powell, C.A., Hoffman, M., Li, W.B., Fan, G.-C., Liu, B., Duan, 

Y.-P. 

1.11 In vitro Culture of the Fastidious Bacteria Candidatus Liberibacter 32 

asiaticus in Association with Insect Feeder Cells – Fontaine-Bodin, L., 

Fabre, S., Gatineau, F., Dollet, M. 

1.12 Preliminary Report of Cultivation of Candidatus Liberibacter asiaticus 33 

from Citrus Tissue with Huanglongbing – Xie, P., Yin, Y., Li, Y., Li, J., 

Wang, Z. 

1.13 Characterization of Highly Mosaic Genomic Loci of Candidatus 34 

Liberibacter asiaticus in Southern China and Florida – Wang, X.F., 

Zhou, C.Y., Deng, X., Su, H.N., Chen, J. 

1.14 Further Evidence That U.S. and Chinese Populations of Candidatus 38 

Liberibacter asiaticus are Different – Deng, X., Liu, R., Zhang, P., Chen, 

J. 

Session 2: Asian Citrus Psyllid Biology and Genomics – David Hall, Moderator 

2.1 Phylogeographic and Population Genetic Studies Uncover Two 40 

Founding Events in Asian Citrus Psyllid Populations Collected in the 

Americas – de León, J.H., Sétamou, M., Gastaminza, G.A., Buenahora, J., 

Cáceres, S., Yamamoto, P.T., Logarzo, G.A., Stañgret, C.R.W. 

2.2 Alteration of Microbiome of Bactericera cockerelli and Diaphorina citri 41 

Based on Candidatus Liberibacter sp. Infection – Hail, D., Hunter, W.B., 

Bextine, B.R. 

2.3 Oral Uptake of dsRNA Increases Mortality in Diet Fed Psyllids – 42 

Shatters, R.G., Jr., Powell, C.A., Borovsky, D. 

2.4 The Psyllid Feeding Process: Composition and Biosynthetic Inhibition 43 

of the Salivary Sheath – Shatters, R.G., Jr. 

2.5 A New Method for Short-Term Rearing of Psyllid Adults and Nymphs 44 

on Detached Citrus Leaves and Young Terminal Shoots – Ammar, 

E.-D., Hall, D.G. 

2.6 Comparative Analysis of Asian Citrus Psyllid and Potato Psyllid 45 

Antennae – Arras, J., Hunter, W.B., Bextine, B.R. 

2.7 The Emerging Psyllid Genome: RNA-Interference and Insect Biology – 46 

Hunter, W.B., Bextine, B.R., Shatters, R.G., Jr., Reese, J., Shelby, K.S., 

Hall, D.G. 

2.8 Bacterial Population Diversity in Diaphorina citri: Analysis by PCR- 47 

DGGE and RFLP Methodology – Wang, Z., Tian, S., Liu, T., Yin, Y. 

Session 3: Asian Citrus Psyllid Ecology and Transmission – Lukasz Stelinski, Moderator 

3.1 Antennal Responses of Diaphorina citri to Host Plant Volatiles 

Recorded Using a Coupled Gas Chromatograph Electroantennogram 

Detector System – Robbins, P.S., Alessandro, R.T., Lapointe, S.L. 

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3.2 Population Dynamics of the Asian Citrus Psyllid and Potential 50 

Generations in Northern Sinaloa, Mexico – Cortez-Mondaca, E., López- 

Arroyo, J.I., Pérez-Márquez, J., González, V.M. 

3.3 Localization of Candidatus Liberibacter asiaticus in Dissected Organs of 51 

Its Psyllid Vector Diaphorina citri Using Fluorescent in situ 

Hybridization and Quantitative PCR – Ammar, E.-D., Shatters, R.G., Jr., 

Hall, D.G. 

3.4 Interactions of the Asian Citrus Psyllid, Diaphorina citri, with 52 

Candidatus Liberibacter asiaticus – Pelz-Stelinski, K.S., Rogers, M.E. 

3.5 Seasonal Changes in Numbers of Asian Citrus Psyllids Carrying 53 

Candidatus Liberibacter asiaticus – Ebert, T.A., Brlansky, R.H., Rogers, 

M.E. 

3.6 Understanding Diaphorina citri-Candidatus Liberibacter asiaticus 54 

Interactions and D. citri Behavior for Managing Huanglongbing (HLB) 

in Florida – Mann, R.S., Pelz-Stelinski, K.S., Rouseff, R.L., Stelinski, L.L. 

3.7 Effects of Soil-Applied and Foliar-Applied Insecticides on Asian Citrus 55 

Psyllid (Diaphorina citri) Feeding Behavior and Their Possible 

Implication for HLB Transmission – Serikawa, R.H., Okuma, D.M., 

Backus, E.A., Rogers, M.E. 

3.8 Effect of Insecticides and Mineral Oil on Probing Behavior of 56 

Diaphorina citri Kuwayama (Hemiptera: Psyllidae) in Citrus – de 

Miranda, M.P., Felippe, M.R., Garcia, R.B., Yamamoto, P.T., Lopes, 

J.R.S. 

3.9 A New Detached-Leaf Assay Method to Test the Inoculativity of 57 

Psyllids with Candidatus Liberibacter asiaticus Associated with 

Huanglongbing Disease – Ammar, E.-D., Walter, A., Hall, D.G. 

3.10 Preliminary Study of Comparative Acquisition of Candidatus 59 

Liberibacter asiaticus and Ca. L. americanus by Diaphorina citri Under 

Different Temperatures – Barbosa, J.C., Eckstein, B., Belasque, J., Jr., 

Bergamin Filho, A. 

3.11 Host Range of Diaphorina citri Kuwayama and Leuronota fagarae on 60 

Citrus and Zanthoxylum spp. – Russell, D.N., Halbert, S.E., Roberts, 

P.D. 

3.12 Abundance of Diaphorina citri (Hemiptera: Psyllidae) in Orange 61 

Jasmine and Backyard Citrus of Yucatán, Mexico – Lozano-Contreras, 

M., Jasso-Argumedo, J., Morales-Koyoc, D., Jasso-Laucirica, T., 

González-Hernández, A., López-Arroyo, J.I. 

3.13 Difference of Gender and Effect of Photoperiod on Asian Citrus Psyllid 62 

Feeding Behavior – Okuma, D.M., Serikawa, R.H., Rogers, M.E. 

3.14 Seasonal Abundance of Diaphorina citri (Hemiptera: Psyllidae) and 63 

Natural Enemies in Citrus Groves of Yucatán, Mexico – Jasso- 

Argumedo, J., Lozano-Contreras, M., Barroso-Aké, H., López-Arroyo, J.I. 

3.15 Host Plants of Psyllids in South Texas – Thomas, D.B. 64 


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Session 4: Survey, Detection and Diagnosis – John da Graça, Moderator 

4.1 Development and Reactivity of Polyclonal Antibodies Based on OMP 67 

Sequences of Candidatus Liberibacter asiaticus – Coletta-Filho, H.D., 

Peroni, L.A., De Souza, A.A., Takita, M.A., Stach-Machado, D.R. 

4.2 Development of Single-Chain Antibody Fragments (scFVs) Against 68 

Candidatus Liberibacter asiaticus by Phage Display – Yuan, Q., Jordan, 

R., Brlansky, R.H., Minenkova, O., Hartung, J.S. 

4.3 Highly Sensitive Detection by Real-Time PCR Targeting the Multiple 69 

Tandem Repeats of Two Prophage Region Genes of the Citrus 

Huanglongbing Disease Bacterium, Candidatus Liberibacter asiaticus – 

Morgan, J.K., Zhou, L.J., Shatters, R.G., Jr., Manjunath, K.L., Duan, Y.-P. 

4.4 Comparison of Different Extraction and Assay Protocols in Different 70 

Laboratories to Develop a Standardized Assay for Detection of 

Huanglongbing-Associated Bacteria from Psyllids – Manjunath, K.L., 

Irey, M.S., Ramadugu, C., Lee, R.F., Levesque, C.S., Brady, B., Polek, 

M.L., Lin, H., Civerolo, E.L., Afunian, M., Vidalakis, G. 

4.5 Assessment of Various Spectroscopic Techniques for Detection of HLB 71 

– Poole, G.H., Hawkins, S.A., Windham, W.R., Heitschmidt, J., Albano, 

J.P., Park, B., Lawrence, K.C., Gottwald, T.R. 

4.6 Seasonal Variability in HLB Testing Data in Plant and Psyllid Samples 72 

in Florida – Irey, M.S., Gast, T., Cote, J., Gadea, P., Santiago, O., 

Briefman, L., Graham, J.H. 

4.7 Survey to Estimate the Rate of HLB Infection in Florida Citrus Groves 73 

– Irey, M.S., Morris, R.A., Estes, M. 

4.8 Two Survey Protocols to Detect Newly Introduced HLB and Other 74 

Exotic Pathogens and Pests – Gottwald, T.R., Riley, T.D., Irey, M.S., 

Parnell, S.R., Hall, D.G. 

4.9 Distribution of Candidatus Liberibacter Americanus and Ca. L. 75 

asiaticus in Foliage of Naturally Infected Citrus Trees – Sousa, M.C., 

Lemos, M.V.F., Frare, G.F., Santos, M.A., Lopes, S.A. 

4.10 A Perspective on the Activities of Texas HLB Diagnostic Laboratory – 76 

Kunta, M., da Graça, J.V., Sétamou, M., Skaria, M. 

4.11 Two New Real-Time PCR-Based Surveillance Systems for Candidatus 77 

Liberibacter Species Detection – Lin, H., Bai, Y., Civerolo, E.L. 

4.12 Detection of Candidatus Liberibacter solanacearum in Potato Psyllid 78 

Isolated from Sticky Traps – Kwok, K., Levesque, C.S., Manjunath, K.L., 

Irey, M.S., Polek, M.L. 

4.13 Detection of Candidatus Liberibacter asiaticus (Las) on Yellow Sticky 80 

Traps by Real-Time PCR – Irey, M.S., Gadea, P., Hall, D.G. 

4.14 Validation of the Starch-Iodine Reaction for Field Pre-Diagnosis of 

Huanglongbing in Citrus of México – Loredo-Salazar, R.X., Uribe- 

Bustamante, A., Rodríguez-Quibrera, C.G., Curtí-Díaz, S.A., Alanís- 

Martínez, E.I., Velázquez-Monreal, J.J., López-Arroyo, J.I. 

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4.15 Detecting HLB Using NIR Remote Sensing – Gonzalez-Mora, J., Dima, 82 

C.S., Irey, M.S., Ehsani, R. 

4.16 Isothermal Detection of Huanglongbing in Psyllids and Citrus Tree 83 

Samples – Russell, P.F., McGowen, N., Bohannon, R. 

4.17 Assessment of Candidatus Liberibacter asiaticus in the Psyllids, 87 

Diaphorina citri Collected from Murraya paniculata in Thailand – 

Jantasorn, A., Duan, Y.-P., Hoffman, M., Zhang, S., Puttamuk, T., 

Thaveechai, N. 

4.18 Liberibacter Reservoirs in Cities and Villages in the State of São Paulo, 88 

Brazil – Lopes, S.A., Frare, G.F., Camargo, L.E.A., Wulff, N.A., Teixeira, 

D.C., Bassanezi, R.B., Beattie, G.A.C., Ayres, A.J. 

4.19 Pictorial Gallery of Foliar HLB Symptoms on Various Citrus Varieties 89 

and Citrus Relatives – Robl, D.J., Riley, T.D., Gomez, H. 

Session 5: Economics, Fruit Quality, and Crop Loss – Mike Irey, Moderator 

5.1 Evaluation of Chemical Flavor Compounds in Orange Juice from 91 

Multiple Harvests of Hamlin and Valencia Fruit from HLB- 

Symptomatic Versus Healthy Trees – Baldwin, E., Bai, J., Dea, S., 

Plotto, A., Manthey, J., Rouseff, R.L., Irey, M.S. 

5.2 Evaluation of Bitterness Caused by Huanglongbing Disease in Orange 92 

Juice – Dea, S., Plotto, A., Manthey, J., Baldwin, E., Irey, M.S. 

5.3 Sensory Evaluation of Juice Made with Fruit from Huanglongbing 93 

(HLB) Affected Trees – Plotto, A., Valim, F., Rouseff, R.L., Dea, S., 

Manthey, J., Narciso, J., Bai, J., Irey, M.S., Baldwin, E. 

5.4 Economic Considerations to Treating HLB with the Standard Protocol 94 

or an Enhanced Foliar Nutritional Program – Morris, R.A., Muraro, 

R.P. 

5.5 When Should a Grower with HLB Stop Removing Trees? – Irey, M.S. 95 

5.6 Use of Electronic Sensor Technology to Discriminate between Juices 96 

from Huanglongbing Infected and Healthy Orange Trees – Bai, J., 

Dea, S., Plotto, A., Baldwin, E., Irey, M.S. 

5.7 A Regional Epidemiological Approach for Yield Loss Estimates Due to 97 

Candidatus Liberibacter Under Different Risk Scenarios – 

Mora-Aguilera, G., Acevedo, G., López-Arroyo, J.I., Velázquez-Monreal, 

J.J., Gómez, R., Robles-González, M.M., Salcedo, D. 

Session 6: Epidemiology – Tim Gottwald, Moderator 

6.1 Five Years of Experience with Huanglongbing in Florida: Current 99 

Assessment; How Did We Get Here? – Halbert, S.E., Manjunath, K.L., 

Ramadugu, C., Lee, R.F. 

6.2 Designing Sampling Schemes to Maximize the Probability of Early 

Detection of a Huanglongbing Outbreak – Parnell, S.R., Gottwald, T.R., 

van den Bosch, F. 

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6.3 Candidatus Liberibacter africanus Subspecies capensis on Calodendrum 101 

capense in South Africa – Phahladira, M.N.B., Viljoen, R., Pietersen, G. 

6.4 Distribution of Psyllids Positive for Candidatus Liberibacter asiaticus in 102 

Citrus Groves in Southwest Florida – Halbert, S.E., Manjunath, K.L., 

Ramadugu, C., Mears, P., Lee, R.F. 

6.5 Seasonal Prevalence of Citrus Huanglongbing (Candidatus Liberibacter 103 

asiaticus) in a Central Florida Sweet Orange Grove – Parkunan, V., 

Wang, N.-Y., Ebert, T.A., Rogers, M.E., Dewdney, M.M. 

6.6 A Mathematical Model for Transmission of HLB by Psyllids – Chiyaka, 104 

C., Singer, B., Halbert, S.E., van Bruggen, A.H.C. 

6.7 Potential Spread of Huanglongbing Through Soil – Nunes da Rocha, U., 105 

Dickstein, E.R., van Bruggen, A.H.C. 

Session 7: International Citrus Industries, Regulation, and Grower Experiences – 

MaryLou Polek, Moderator 

7.1 Laws, Huanglongbing Management, and the Current Status of the 107 

Disease in São Paulo, Brazil – Belasque, J., Jr., Ayres, A.J., Barbosa, J.C., 

Massari, C.A., Bové, J.M. 

7.2 Distribution of Citrus Huanglongbing in the Dominican Republic – 108 

Matos, L., Hilf, M.E., Cayetano, X., Feliz, A., Puello, H., Méndez, F., 

Borbón, J., Folimonova, S.Y. 

7.3 Citrus Huanglongbing in Cuba: Current Situation, Management, and 109 

Main Research – López, D., Luis, M., Collazo, C., Batista, L., Peña, I., 

González, C., Pérez, J.L., Zamora, V., Borroto, A., Pérez, D., Alonso, E., 

Acosta, I., Llauger, R., Casín, J.C. 

7.4 Spreading and Symptoms of Huanglongbing in Mexican Lime Groves 110 

in the State of Colima, Mexico – Robles-González, M.M., Velázquez- 

Monreal, J.J., Manzanilla Ramirez, M.A., Orozco Santos, M., Flores 

Virgen, R., Medina Urrutia, V.M., Carrillo Medrano, S.H. 

7.5 The Asian Citrus Psyllid/Huanglongbing Detection, Treatment, and 111 

Regulatory Program in California – Galindo, T. 

7.6 Detection and Reporting of Asian Citrus Psyllid and Huanglongbing in 112 

Commercial Citrus Within California: An Industry Program – Taylor, 

B.J., Polek, M.L., Batkin, T. 

7.7 Citrus Health Research Forum: A National Research Effort – Polek, 113 

M.L., Wisler, G. 

7.8 The Identification and Distribution of Citrus Greening Disease in 114 

Jamaica – Oberheim, A.P., Brown, S.E., McLaughlin, W.A. 

7.9 Fitting a Spatial Analysis Grid for Research on Huanglongbing in 

Mexico – Aldama-Aguilera, C., Olvera-Vargas, L.A., Galindo-Mendoza, 

M.G. 

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Session 8: Host-Pathogen Interactions – Bill Dawson, Moderator 

8.1 Examination of Stages of the HLB Disease Development in Citrus Trees 117 

– Folimonova, S.Y., Achor, D.S., Hilf, M.E. 

8.2 New Defense Response Insights of Sweet Orange Infected with Two 118 

Candidatus Liberibacter Species – Mafra, V.S., Martins, P.K., Locali- 

Fabris, E.C., Ribeiro-Alves, M., Francisco, C.S., Freitas-Astúa, J., Kishi, 

L.T., Machado, M.A. 

8.3 Differential Expression of Potential Virulence Genes of Candidatus 119 

Liberibacter asiaticus in Infected Plants and Psyllids – Sreedharan, A., 

Wei, S., Wang, N.-Y. 

8.4 Metabolome Analysis of Tolerant and Susceptible Citrus Varieties in 120 

Response to Infection with Candidatus Liberibacter asiaticus – 

Albrecht, U., Skogerson, K., Bowman, K.D., Fiehn, O. 

8.5 Deep Transcriptome Profiling of Citrus Fruit in Response to 124 

Huanglongbing Disease – Martinelli, F., Uratsu, S.L., Albrecht, U., 

Reagan, R.L., Leicht, E., D’Souza, R., Bowman, K.D., Dandekar, A.M. 

8.6 Carbohydrate Metabolism and Related Gene Expression Changes in 125 

Huanglongbing-Affected Sweet Orange – Chen, C., Fan, J., Yu, Q., 

Brlansky, R.H., Li, Z.-G., Gmitter, F.G., Jr. 

8.7 Analysis of Colonization of Citrus Seeds by Ca. Liberibacter asiaticus 126 

and Its Possible Role in Seed Transmission – Hilf, M.E. 

8.8 Natural Transmission of Huanglongbing Caused by Candidatus 127 

Liberibacter americanus and Ca. L. asiaticus and with Two Different 

Sources of Inoculum Plants (Citrus sinensis or Murraya exotica) – 

Gasparoto, M.C.G., Bassanezi, R.B., Amorim, L., Montesino, L.H., 

Lourenço, S.A., Wulff, N.A., Bergamin Filho, A. 

8.9 Callose Predominates over Phloem Protein 2 in Phloem Plugging of 128 

Trees Affected with Huanglongbing – Albrigo, L.G., Achor, D.S. 

8.10 Influence of Huanglongbing (HLB) on the Composition of Citrus Juices 129 

and Mature Leaves – Cancalon, P.F., Bryan, C., Haun, C., Zhang, J. 

8.11 Gene Expression in Citrus sinensis Fruit Tissues Harvested from 131 

Huanglongbing-Infected Trees – Liao, H.-L., Burns, J.K. 

8.12 Expression Profiling of Host Response of Citrus to Candidatus 132 

Liberibacter asiaticus Infection – Aritua, V., Wang, N.-Y. 

8.13 Arabidopsis Responses to the HLB-Relative Candidatus Liberibacter 133 

psyllaurous – Patne, S., Manjunath, K.L., Roose, M.L. 

8.14 Comparative Studies of the Endophytic Microbial Community 134 

Structures in Huanglongbing-Infected and Non-Infected Citrus Plants – 

Zheng X.-F., Liu, B., Ruan, C.-Q., Lin, Y.-Z., Xiao, R.-F., Zhu, Y.-J., Fan, 

G.-C., Cai, Z.J., Duan, Y.-P. 

8.15 HLB Influences the Diversity, Structure, and Function of the Bacterial 

Community Associated with Citrus – Trivedi, P., Wang, N.-Y. 

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8.16 Functional Studies of Putative Effectors of Candidatus Liberibacter 136 

asiaticus Using Citrus Tristeza Virus Vector – Hajeri, S., Duan, Y.-P., 

Gowda, S. 

8.17 First Report of a New Host (Pithecellobium lucidum Benth) of the 137 

Citrus Huanglongbing Bacterium, Candidatus Liberibacter asiaticus – 

Fan, G.-C., Cai, Z.J., Weng, Q.Y., Ke, C., Liu, B., Zhou, L.J., Duan, Y.-P. 

8.18 Citrus Seed Grafting: A Simple Technology for Testing Seed 138 

Transmission of Citrus Greening/HLB and of Other Pathogenic Agents 

– Bar-Joseph, M., Robertson, C., Hilf, M.E., Dawson, W.O. 

8.19 Lack of Transmission of HLB by Citrus Seed – Graham, J.H., Johnson, 139 

E.G., Bright, D.B., Irey, M.S. 

8.20 Visualization of Ca. Liberibacter asiaticus in Immature Citrus Seed 140 

Coats by Fluorescent In Situ Hybridization (FISH) of 16S rRNA – Hilf, 

M.E. 

8.21 Rapid, Sensitive, and Non-Radioactive Tissue-Blot Diagnostic Method 141 

for the Detection of Citrus Greening Disease (HLB) – Gowda, S., 

Nageswara Rao, N., Miyata, S., Ghosh, D.K., Irey, M.S., Rogers, M.E., 

Garnsey, S.M. 

Session 9: Asian Citrus Psyllid Management – Michael Rogers, Moderator 

9.1 A Database for Analysis of Diaphorina citri Population Monitoring Data 143 

from Commercial Groves – Gast, T., Irey, M.S., Hou, H. 

9.2 RNAi Strategy in Citrus Trees to Reduce Hemipteran Pests: Psyllids 148 

and Leafhoppers – Hunter, W.B., Glick, E., Bextine, B.R., Paldi, N. 

9.3 Application of Insecticidal Sprays to Citrus in Winter Provides 149 

Significant Reduction in Asian Citrus Psyllid Diaphorina citri 

Populations and Opportunity for Additional Suppression Through 

Conservative and Augmentative Biological Control – Qureshi, J.A., 

Stansly, P.A. 

9.4 Studies on Imidacloprid and Management of ACP in California – 150 

Byrne, F., Morse, J.G., Bethke, J. 

9.5 Selection and Dosage of Insecticides for the Control of the Asian Citrus 151 

Psyllid in the Citrus Groves of Mexico – López-Arroyo, J.I., Díaz- 

Zorrilla, U., Hernández-Fuentes, L.M., Cortez-Mondaca, E., Robles- 

González, M.M., Villanueva-Jiménez, J.A., Cabrera-Mireles, H., Loera- 

Gallardo, J., Jasso-Argumedo, J., Curtí-Díaz, S.A. 

9.6 Asian Citrus Psyllid (ACP) Control: Potential Use of Systemic 152 

Insecticides in Citrus Bearing Trees – Yamamoto, P.T., de Miranda, 

M.P., Felippe, M.R. 

9.7 Insecticide Resistance and Susceptibility of Uninfected and Candidatus 

Liberibacter asiaticus-Infected Asian Citrus Psyllid in Florida – Tiwari, 

S., Rogers, M.E., Stelinski, L.L. 

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9.8 Development of Area-Wide Asian Citrus Psyllid Management 154 

Strategies in Texas – Bartels, D.W., Sétamou, M., Ciomperlik, M.A., da 

Graça, J.V. 

9.9 Asian Citrus Psyllid Management Strategies for California Citrus 155 

Growing Regions – Grafton-Cardwell, E.E., Morse J.G., Taylor, B.J. 

9.10 Area-Wide Management of Asian Citrus Psyllid in Southwest Florida – 156 

Stansly, P.A., Arevalo, H.A., Zekri, M., Hamel, R. 

9.11 Evaluation of Low Volume Sprayers Used in Citrus Psyllid Control 157 

Applications – Hoffmann, C., Fritz, B., Martin, D., Atwood, R., Hurner, T., 

Ledebuhr, M., Tandy, M., Jackson, J.L., Wisler, G., Polek, M.L. 

9.12 Identification of Parasitoids and Haplotypes of Tamarixia radiata 158 

(Waterston) (Hymenoptera: Eulophidae) from Diaphorina citri in 

Yucatán, México – González-Hernández, A., Jasso-Argumedo, J., Cruz- 

García, R., Lozano-Contreras, M., López-Arroyo, J.I., Villanueva-Segura, 

O.K. 

9.13 Host Specificity Testing of Tamarixia radiata for the Classical Biological 159 

Control of Asian Citrus Psyllid, Diaphorina citri, in California – 

Pandey, R.R., Hoddle, M.S. 

9.14 Predators in Non-Commercial Citrus and Preliminary Evaluation of 160 

Their Potential Against the Asian Citrus Psyllid in Texas – 

Pfannenstiel, R.S., Unruh, T.R. 

9.15 Suitability of Diaphorina citri, Toxoptera citricida, and Aphis spiraecola 161 

as Prey for Hippodamia convergens – Qureshi, J.A., Stansly, P.A. 

9.16 Molecular Analysis of Tamarixia radiata from America Uncovers 162 

Extensive Haplotype Variation: Multiple Groups? – de León, J.H., 

Gastaminza, G.A., Sétamou, M., Cáceres, S., Kanga, L.H.B., Buenahora, J., 

Parra, J.R., Logarzo, G.A., Stañgret, C.R.W. 

9.17 Molecular Characterization of a New Entomopathogenic Fungus Isaria 163 

poprawskii: A Potential Biocontrol Agent for Diaphorina citri. 

Development of Isaria-Specific Molecular Markers – de León, J.H., 

Cabanillas, H.E., Humber, R.A., Murray, K.D., Moran, P., Jones, W.A. 

9.18 RNAi – Evaluating Injection into Citrus Trees and Grapevine to Target 164 

Psyllids and Leafhoppers – Hunter, W.B., Stover, E., Glick, E., Bextine, 

B.R., Paldi, N. 

9.19 Using Novel Photonic Fence Technology to Protect Foundation Block 165 

and Nursery Stock from Asian Citrus Psyllid – Johanson, E., Patt, J., 

Mullen, E., Rutschman, P., Pegram, N. 

9.20 Development of a Diaphorina citri-Specific Molecular Diagnostic 166 

Marker for Gut Content Examinations – de León, J.H., Thomas, D.B., 

Sétamou, M., Hagler, J.R. 

9.21 Development of a Pathogen Dispenser to Control Asian Citrus Psyllid 

(ACP) in Residential Citrus – Patt, J., Jackson, M., Dunlap, C., Meikle, 

W., Adamczyk, J. 

167 


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9.22 Producing New Flush at Will in Citrus to Study ACP-Plant Interactions 168 

– Malik, N.S.A., Brockington, J., Pérez, J.L., Mangan, R.L. 

9.23 Thresholds for Vector Control in Young Citrus Treated for Symptoms 169 

of HLB with a Nutrient/SAR Package – Monzó, C., Arevalo, H.A., 

Stansly, P.A. 

9.24 Experimental Release Rate Analysis of Volatile Compounds from Wax- 170 

Based Dispensers – Neuman, R.D., Mills, D.R., Shelton, A.B. 

9.25 Vegetation Canopy Airflow Modeling for Airborne Dispersion of 171 

DMDS – Shelton, A.B., Neuman, R.D. 

9.26 Methods and Systems to Deliver Volatile Compounds for Biological 172 

Control Strategies – Neuman, R.D., Shelton, A.B., Zee, R.H. 

Session 10: HLB Management – Tim Spann, Moderator 

10.1 Trunk Injection of Copper Sulfate Pentahydrate (Magna-Bon) Affects 174 

Expression of HLB – Graham, J.H., Irey, M.S., Miele, F. 

10.2 Chemical Compounds Effective Against the Citrus Huanglongbing 175 

Bacterium, Candidatus Liberibacter asiaticus In Planta – Zhang, M.-Q., 

Powell, C.A., Zhou, L.J., He, Z., Stover, E., Duan, Y.-P. 

10.3 Regional HLB Management on the Effectiveness of Local Strategies of 176 

Inoculum Reduction and Vector Control – Bassanezi, R.B., Yamamoto, 

P.T., Montesino, L.H., Gottwald, T.R., Amorim, L., Bergamin Filho, A. 

10.4 The Theory of Managing Huanglongbing with Plant Nutrition and Real 177 

World Success in Florida – Spann, T.M., Rouse, R.E., Schumann, A.W. 

10.5 Nutritional Treatments: Inconsequential Effect on HLB Control and 178 

Promote Area-Wide Titer Increase and Disease Spread – Gottwald, 

T.R., Irey, M.S., Graham, J.H., Wood, B. 

10.6 Nutritional Approaches for Management of Huanglongbing (Citrus 179 

Greening) in China – Xia, Y., Sequeira, R. 

10.7 First Steps Towards Rescuing Las-Infected Citrus Germplasm – 180 

McCollum, G., Stover, E. 

10.8 Screening Chemical Compounds Against Citrus Huanglongbing Using 181 

an Optimized Grafting System from Candidatus Liberibacter asiaticus- 

Infected Citrus Scions – Zhang, M.-Q., Duan, Y.-P., Powell, C.A. 

10.9 Discovery of Antimicrobial Small Molecules Against Candidatus 182 

Liberibacter asiaticus by Screening Novel SecA Inhibitors Using 

Structure Based Design – Akula, N., Wang, N.-Y. 

10.10 The Low Pressure Trunk Injection System: A Technology to Fight 184 

Against HLB – Tomas, J. 

10.11 Does Systemic Acquired Resistance (SAR) Control HLB Disease 185 

Development? – Graham, J.H., Myers, M.E., Irey, M.S., Gottwald, T.R. 

10.12 Use of Growth-Priming Agents to Extend the Growth of HLB-Affected 

Citrus – He, Z., Zhang, M.-Q., Viana, E., Merlin, T., Duan, Y.-P., Stoffella, 

P.J., Liptay, A., Powell, C.A. 

186 


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10.13 Evaluation of Foliar Zinc and Manganese for Control of HLB or 187 

Associated Symptom Development – Johnson, E.G., Irey, M.S., Gast, T., 

Bright, D.B., Graham, J.H. 

10.14 Role of Nutritional and Insecticidal Treatments in Mitigation of HLB: 188 

Main Effects and Interactions – Stansly, P.A., Arevalo, H.A., Rouse, R.E. 

10.15 Use of Horticultural Practices in Citriculture to Survive Huanglongbing 189 

– Stuchi, E.S., Girardi, E.A. 

10.16 Critical Control Point (CCP) Analysis to Build a Model System for 191 

Measuring Citrus Propagation Risk Mitigations. II. Sampling and 

Monitoring – Brown, L.G., Jones, E.M., Hartzog, H.M. 

10.17 The Need of an Epidemio-Surveillance Network to Prevent 192 

Huanglongbing Arrival in the South Mediterranean Basin – Dollet, M., 

Aubert, B., Imbert, E., Gatineau, F. 

10.18 Presence of Candidatus Liberibacter asiaticus in Diaphorina citri 193 

Kuwayama Collected from Plants for Sale in Florida – Halbert, S.E., 

Manjunath, K.L., Ramadugu, C., Lee, R.F. 

10.19 A Model System for Studying Huanglongbing – Manjunath, K.L., 194 

Ramadugu, C., Kund, G., Trumble, J., Lee, R.F. 

Session 11: Host Tolerance and Resistance – Fred Gmitter, Moderator 

11.1 Incidence of Huanglongbing on Several Sweet Orange Cultivars 196 

Budded onto Different Rootstocks at the Citrus Experimental Station 

(EECB), Bebedouro, São Paulo, Brazil – Stuchi, E.S., Reiff, E.T., 

Sempionato, O.R., Girardi, E.A., Parolin, L.G., Toledo, D.A. 

11.2 Host Preference and Suitability of Native North American Rutaceae for 197 

the Development of the Asian Citrus Psyllid, Diaphorina citri 

Kuwayama – Sandoval, J.L., II, Sétamou, M., da Graça, J.V. 

11.3 Progress Using Transgenic Approaches and Biotechnology-Facilitated 198 

Conventional Breeding to Develop Genetic Resistance/Tolerance to 

HLB in Commercial Citrus – Grosser, J.W., Dutt, M., Shohael, A., 

Barthe, G.A. 

11.4 Promoter Regulation of the β-Glucuronidase (GUS) Gene and 199 

Antimicrobial Peptide D4E1 in a Citrus Rootstock – Benyon, L.S., 

Stover, E., Bowman, K., McCollum, G., Niedz, R. 

11.5 Responses of Transgenic Hamlin Sweet Orange Plants Expressing the 200 

attacin A Gene to Candidatus Liberibacter asiaticus Infection – Felipe, 

R.T.A., Mourão-Filho, F.A., Pereira, E.V., Jr., Lopes, S.A., Sousa, M.C., 

Mendes, B.M.J. 

11.6 Screening Antimicrobial Peptides In-Vitro for Use in Developing 

Huanglongbing and Citrus Canker Resistant Transgenic Citrus – 

Stover, E., Stange, R., McCollum, G., Jaynes, J. 

201 


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11.7 Response of Citrus Transgenic Plants Expressing STX IA Gene to 202 

Candidatus Liberibacter asiaticus – Marques, V.V., Bagio, T.Z., 

Sugahara, V.H., Vasquez-Souza, G.V., Grange, L., Meneguim, L., 

Kobayashi, A.K., Bespalhok, J., Pereira, L.F.P., Vieira, L.G.E., Leite, R.P., 

Jr. 

11.8 Rootstocks and Pruning Effects on Huanglongbing Incidence on Tahiti 204 

Limes in Bebedouro, Northern São Paulo State, Brazil – Stuchi, E.S., 

Reiff, E.T., Sempionato, O.R., Cantuarias-Avilés, T., Girardi, E.A., Parolin, 

L.G., Toledo, D.A. 

11.9 Candidatus Liberibacter asiaticus (CLas) Titer in Field HLB-Exposed 205 

Commercial Citrus Cultivars – Stover, E., McCollum, G. 

11.10 Host Response of Different Citrus Genotypes and Relatives to 206 

Candidatus Liberibacter asiaticus Infection – Boscariol-Camargo, R.L., 

Cristofani-Yaly, M., Malosso, A., Coletta-Filho, H.D., Machado, M.A. 

11.11 Candidatus Liberibacter asiaticus (CLas) Titer in Poncirus trifoliata and 207 

P. trifoliata Hybrids: Inferences on Components of HLB Resistance – 

Stover, E., Shatters, R.G., Jr., McCollum, G., Hall, D.G., Duan, Y.-P. 

11.12 The Role of Salicylic Acid and Systemic Acquired Resistance in the 208 

Response of Citrus to HLB – Khalaf, A., Febres, V.J., Brlansky, R.H., 

Gmitter, F.G., Jr., Moore, G.A. 

11.13 Observations of Citrus × Poncirus Hybrid Tolerance to Infection with 209 

Candidatus Liberibacter asiaticus – Bowman, K.D., Albrecht, U. 

11.14 Performance of a Phage Gene in Transgenic Citrus Resistant to Citrus 210 

Greening – Jiang, Y., Perazzo, G., Septer, A., Kress, R., Gabriel, D.W. 

11.15 Genome Sequences of Haploid Clementine Mandarin and Diploid Sweet 211 

Orange – Gmitter, F.G., Jr. 

11.16 Exploring Metabolic Profiles of Plant Tissue with Increased or 212 

Decreased Susceptibility – Malik, N.S.A., Pérez, J.L., Brockington, J., 

Mangan, R.L. 

Take Home Messages: What Can Be Implemented Now or in the Near Future? 213 

HLB Pathology Lessons – M. Dewdney / T. Schubert 214 

Entomology Lessons – L.L. Stelinski / M. Sétamou 219 

Horticulture Lessons – C. Oswalt / E. Stover 229 

Addenda 

A1 Author Index 235 

A2 List of Participants 246 

A3 Meeting Agenda 265 

A4 Session 9 Addendum – Extended Papers 9.24 & 9.25 280 


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Session 1: 

Pathogen Genomics, 

Bioinformatics, 

Phylogenetics, and 

Culturing 


P a g e | 17 

1.1 Genomic Comparisons of the Ca. Liberibacter asiaticus Chromosome with Other 

Members of the Rhizobiales 

Hartung, J.S., Shao, J., Kuykendall, L.D. Molecular Plant Pathology Laboratory, United States 

Department of Agriculture, Agricultural Research Service, Beltsville, MD, USA 

Ca. Liberibacter asiaticus is a member of the Rhizobiales, as are the nitrogen fixing 

Sinorhizobium meliloti and Bradyrhizobium japonicum, the plant pathogen Agrobacterium 

tumefaciens, and the facultative intracellular mammalian pathogen Bartonella henselae. 

Comparisons of chromosomal gene content identified 52 clusters of conserved orthologous genes 

found in all five metabolically diverse species. The intracellular pathogens, Ca. Liberibacter 

asiaticus and Bartonella henselae, have drastically reduced genomes with low content of guanine 

and cytosine. The Ca. Liberibacter asiaticus genome has 319 coding sequences not found on the 

chromosomes of the other members of the Rhizobiales from a total of 1136 coding sequences. 

Ca. Liberibacter asiaticus has all 100 COGs previously found in all bacteria to support basic 

physiological functions, but also has representatives of 10 COGs that have been lost in other 

intracellular pathogens (Merhej et al., 2009) including a hemolysin, an ATP dependent Clp 

protease, an ABC transport permease, and a flagellar motor component. These COGS, as well as 

six proteins uniquely shared by Ca. Liberibacter asiaticus and Bartonella henselae, may 

condition host cells for intracellular colonization or facilitate transmission by insects. InvA, 

shared with Bartonella henselae and believed to prevent apoptosis of cells infected by Rickettsia 

prowazeckii (Gaywee et al., 2002), is also present. Only two chromosomal protein encoding 

genes were uniquely shared by Ca. Liberibacter asiaticus and Agrobacterium tumefaciens, the 

other plant pathogen in the study. These genes may also be important to the host-pathogen 

interaction. Five and twelve chromosomal genes were uniquely shared between Ca. Liberibacter 

asiaticus and Sinorhizobium meliloti and Bradyrhizobium japonicum, respectively. 

References 

Gaywee, J., Xu, W., Radulovic, S., Bessman, M.J., Azad, A.F. 2002. The Rickettsia prowazekii 

invasions gene homlog (invA) encodes a nudix hydrolase active on adenosine (5’)- 

pentaphospho-(5’)-adenosine. Molecular and Cellular Proteomics 1:179-185. 

Merhej, V. et al. 2009. Massive comparative analysis reveals convergent evolution of specialized 

bacteria. Biology Direct 4(13). 


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1.2 Genetic and Functional Characterization of the znu Operon in the Intracellular Citrus 

Pathogen, Candidatus Liberibacter asiaticus 

Vahling, C.M., Benyon, L.S., Duan, Y.-P. USDA-ARS-USHRL, Fort Pierce, FL, USA 

The uptake of zinc by Candidatus Liberibacter asiaticus (Las) may act as a virulence mechanism 

in its citrus hosts because symptoms of citrus huanglongbing often mimic those with zinc 

deficiencies. Annotation of the Las genome has revealed two putative operons, approximately 

191 kb apart, each encoding for a high-affinity zinc uptake system. The operons contained either 

three or four genes consisting of znuACB or znuACBB, respectively. In this uptake system, 

ZnuA is predicted to function as a periplasmic metallochaperone, ZnuB as a membrane 

permease, and ZnuC as the ATPase subunit of the ATP-binding cassette transporter. Several 

motifs that are characteristic of these proteins, such as the Walker A and B motifs within ZnuC, 

have been identified in the Las encoded homologue. Interestingly, neither proteins homologous 

to those known to regulate this operon (zur) nor its regulatory elements (Zur box) have been 

identified in Las. To understand the role of these two operons in the fastidious Las bacterium, 

each individual gene was placed under an inducible promoter, transformed into the Escherichia 

coli BW25113 strain with the corresponding gene of interest knocked out, and analyzed in a 

complementation assay. Results from the assays have demonstrated that not all of the Las 

encoded genes appear to be able to functionally complement the E. coli knock-out strains. 

Because of the essential nature of zinc within the cell, this system may be crucial for the 

pathogen’s survival. Thus, disruption of this system may provide a possible mechanism for 

eliminating the bacterium. 


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1.3 Comparison of the Ca. Liberibacter asiaticus Genome with a Draft Ca. L. americanus 

Genome Reveals Similar Prophage with Likely Pathogenicity Factors 

Zhang, S.J. 1 , Wulff, N.A. 2 , Flores-Cruz, Z. 1 , Zhou, L.J. 3 , Kang, B.-K. 4 , Fleites, L.A. 1 , Gooch, 

M.D. 5 , Davis, M.J. 6 , Duan, Y.-P. 7 , Gabriel, D.W. 1 

1 Plant Pathology Dept., University of Florida, Gainesville, FL, USA 

2 Departamento Científico, Fundecitrus, Araraquara, SP, Brazil 

3 UF-IFAS Indian River Research and Education Center, Fort Pierce, FL, USA 

4 Microbiology and Cell Science Dept., University of Florida, Gainesville, FL, USA 

5 Division of Plant Industry, Florida Department of Agriculture and Consumer Services, 

Gainesville, FL, USA 

6 UF-IFAS Citrus Research and Education Center, Lake Alfred, FL, USA 

7 U.S. Horticultural Research Laboratory, USDA-ARS, Fort Pierce, FL, USA 

Huanglongbing is a lethal disease of citrus caused by psyllid transmitted, phloem limited, 

α-Proteobacteria: Ca. L. asiaticus (Las), Ca. L. americanus (Lam), and Ca. L. africanus (Laf). 

Genomic sequencing of curated Las strain UF506 revealed two largely homologous, circular 

phage genomes, SC1 and SC2, which were found tandemly integrated in the UF506 chromosome 

as prophages in all hosts tested. SC1 carried suspected lytic cycle genes, was found in lytic cycle 

DNA forms only in planta, and its genes were highly induced only in planta and particularly in 

periwinkle. Phage particles associated with Las were found in the phloem of infected periwinkles 

by transmission electron microscopy. SC2 appeared to lack lytic cycle genes and replicated as an 

excision plasmid in psyllids and in planta. SC2 encoded putative adhesin and peroxidase genes 

that had not previously been identified in Las and which may be involved in lysogenic 

conversion (pathogenicity). Shotgun sequencing of Lam strain São Paulo is ca. 98% complete, 

with a total of 1,203,790 bps of sequence in 355 contigs with average length of 3.4 kb to date. 

Lam has a high level of synteny with Las, including 80% of the likely proteins encoded by SC1 

and SC2. Overall, DNA sequence similarity is surprisingly low. SC1 encodes one putative 

colicin and SC2 encodes a putative colicin immunity protein; neither has been found in Lam, 

which is being supplanted by Las as the predominant HLB species in Brazil. It is possible that 

Las colicin kills Lam, indicating a potential control strategy. 

Acknowledgement 

Funding: Florida Citrus Advanced Technology Program and USDA APHIS. 


P a g e | 20 

1.4 Analysis of Candidatus Liberibacter americanus Genome 

Wulff, N.A. 1 , Zhang, S.J. 2 , Ayres, A.J. 1 , Bové, J.M. 3 , Gabriel, D.W. 2 

1 Fundecitrus, Araraquara, SP, Brazil 

2 Plant Pathology Dept., University of Florida, Gainesville, FL, USA 

3 INRA and U. Bordeaux 2, Villenave d’Ornon, France 

Candidatus Liberibacter asiaticus (Las), Ca. L. americanus (Lam) and a 16Sr group IX 

phytoplasma are individually associated with huanglongbing (HLB) symptoms in Brazil. 

Whereas Lam was the prevalent species in 2004 when HLB was first reported (Teixeira et al., 

2005), since 2006 Las has been the dominant species and is now present in larger areas than Lam 

(Coletta-Filho et al., 2007; Lopes et al., 2007, 2009a). The reason for this shift might be related 

to the fact that Las tolerates higher temperatures and reaches on average 10 times higher titer in 

citrus trees than Lam (Lopes et al., 2009a,b), favoring its psyllid transmission from diseased to 

healthy trees and spread to areas with wider ranges in temperature. Under favorable conditions 

and, despite the lower titers, Lam-infected trees usually express more severe symptoms than 

Las-infected trees. A preliminary comparative analysis of Lam and Las genomes has been 

carried out in order to possibly define the factors underlying such distinctions. Due to the 

inability to continuously grow Liberibacter in culture media, a pulse field strategy was employed 

to obtain Lam chromosomal DNA from infected periwinkles (Wulff et al., 2009). The DNA was 

amplified with phi29 and subjected to further purification using cesium chloride density gradient 

centrifugation in the presence of bisbenzimide. DNA with % GC lower than 50 was collected 

and subjected to 454 pyrosequencing. A draft genome of Lam was obtained and the similarities 

and differences against Las (Duan et al., 2009) at the structural level, including ribosomal operon 

architecture, prophage number, and genome synteny, will be presented. 


Financial Support: CRDF (Grant #65) and FAPESP (2010/16820-9). 

References 

Coletta-Filho et al. 2007. Proceedings of the XVII Conference of the International Organization 

of Citrus Virologists (IOCV), Adana, Turkey, p. 124. 

Duan et al. 2009. Molecular Plant-Microbe Interactions 22:1011-1020. 

Lopes et al. 2007. Proc. Workshop sobre Epidemiologia de Doenças de Plantas. Campos do 

Jordão, São Paulo, Brazil, p. 69-76. 

Lopes et al. 2009a. Phytopathology 99:301-306. 

Lopes et al. 2009b. Plant Disease 93:257-262. 

Teixeira et al. 2005. Molecular and Cellular Probes 19:173-179. 

Wulff et al. 2009. International Journal of Systematic and Evolutionary Microbiology 59:1984- 

1991. 


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1.5 Population Genetics Analysis of Candidatus Liberibacter asiaticus from Multiple 

Continents 

Glynn, J.M. 1 , Bai, Y. 2 , Chen, C. 3 , Duan, Y.-P. 4 , Civerolo, E.L. 1 , Lin, H. 1 

1 USDA-ARS-SJVASC-CDPG, Parlier, CA, USA 

2 Guangxi Academy of Agricultural Sciences, Nanning, Guangxi, China 

3 Guangxi Citrus Research Institute, Guilin, China 

4 USDA-ARS-HRL-SPPR, Fort Pierce, FL, USA 

Huanglongbing (HLB) is currently the most destructive citrus disease in the world and has 

caused enormous economic losses in the citrus industry. In the United States (U.S.), HLB is 

typically associated with the presence of a fastidious phloem-limited bacterium named 

Candidatus Liberibacter asiaticus (Las), though other Liberibacter species have also been 

associated with HLB. Unfortunately, very little is known regarding the population characteristics 

of Las and epidemiology of HLB. The recent release of the Las genome sequence has allowed 

for the development of molecular markers to better understand the origins, transmission, and 

population dynamics of the HLB-associated bacterium. From a starting pool of more than 

100 putative simple sequence repeats (SSRs), we developed a panel of 7 polymorphic molecular 

markers for Las. Using this panel of markers, we obtained complete genotypic profiles for nearly 

300 Las isolates from the US, China, India, Brazil, Cambodia, Vietnam, Taiwan, Thailand, and 

Japan. Analysis of these profiles identified two distinct clonal complexes of Las associated with 

HLB and indicates that at least two separate introduction events associated with the occurrence 

and distribution of HLB in Florida citrus. The panel of markers we introduce here should be 

useful for future population genetic and molecular epidemiologic studies of HLB. 

References 

Feil, E.J., Li, B.C., Aanensen, D.M., Hanage, W.P., Spratt, B.G. 2004. eBURST: inferring 

patterns of evolutionary descent among clusters of related bacterial genotypes from 

multilocus sequence typing data. Journal of Bacteriology 186:1518-1530. 

Lin, H., Civerolo, E.L., Hu, R., Barros, S., Francis, M., Walker, M.A. 2005. Multilocus simple 

sequence repeat markers for differentiating strains and evaluating genetic diversity of 

Xylella fastidiosa. Applied and Environmental Microbiology 71:4888-4892. 


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1.6 Phylogenetic Analysis of Asian Candidatus Liberibacter asiaticus; Asian Common 

Strains Are Distributed in Northeast India, Papua New Guinea, and Timor-Leste 

Miyata, S. 1 , Kato, H. 1 , Tomimura, K. 1 , Davis, R. 2 , Smith, M.W. 3 , Weinert, M. 4 , Iwanami, T. 1 

1 Natl Inst Fruit Tree Sci, NARO, Japan 

2 NAQS, AQIS, Australia 

3 Bundaberg Res St, DEEDI, Australia 

4 Cent Trop Agric, DEEDI, Australia 

Gram-negative bacterium Candidatus Liberibacter asiaticus causes citrus greening disease 

(huanglongbing) – with a history of new introduction and spread through several major citrus 

producing areas worldwide. Especially in Asia, it was reported since the early 19th century, and 

citrus production in many Asian countries is still overwhelmed by this destructive disease. The 

Indian isolates of Ca. L. asiaticus had been reported from several areas in India; however, their 

16S rDNA sequences were different from other Asian isolates, e.g., Japanese, Taiwanese, 

Indonesian, Vietnamese, or Chinese ones. Thus, we investigated the phylogenetic relationships 

among these Asian and Northeast Indian isolates. Ca. L. asiaticus was detected by PCR using 

primers specific for nusG-rplK genes and 16S rDNA in symptomatic leaves collected from 

Northeast India, Papua New Guinea (PNG), and Timor-Leste (East Timor). Phylogenetic 

analysis with 16S rDNA sequences and single nucleotide polymorphisms (SNPs) of omp gene 

region was conducted. It was revealed that the Northeast Indian isolates were genetically closer 

to Asian common isolates from Japan, Taiwan, and Vietnam than to Indian isolates previously 

reported. This suggests that the Asian common strains of Ca. L. asiaticus are distributed also in 

Northeast India. Further phylogenetic analyses were also conducted using other genomic regions, 

and new results will be presented. 


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1.7 Bioinformatic Analysis of Genome Sequence Data for Ca. Liberibacter asiaticus 

Lindeberg, M., Saha, S. Department of Plant Pathology and Plant-Microbe Biology, Cornell 

University, Ithaca, NY, USA 

Availability of genome sequence data for Ca. Liberibacter asiaticus (Las) (Duan et al., 2009), 

believed to be the cause of huanglongbing, provides the opportunity to use sequence analysis to 

gain insights into mechanisms of adaptation to host and vector. To enable the research 

community to easily view the Las genome and its components features, a GBrowse genome 

viewer for Las has been installed for use via the CG-HLB Genome Resources Website 

(http://www.citrusgreening.org/HLB-GBrowse.html). The genome viewer is searchable by gene 

name, locus tag, feature type, or coordinates. Las genome characterization provided within the 

viewer includes information on protein functional domains, subcellular locations, and predicted 

signal peptide or lipopeptide cleavage sites, indicative of proteins located on the bacterial 

surface. Locations of repeat sequences of use for designing primers for strain detection are 

provided, as are operon predictions. Features are hyperlinked to outside databases or more 

detailed information pages as appropriate, with links to in-depth protein characterizations 

generated by the Grishin lab similarly provided. Links to various additional online resources and 

genome analysis tools can be found at the Citrus Greening-HLB Genome Resources Website 

(http://citrusgreening.org). 

Reference 

Duan, Y.-P., Zhou, L., Hall, D.G., Li, W., Doddapaneni, H., Lin, H., Liu, L., Vahling, C.M., 

Gabriel, D.W., Williams, K.P., Dickerman, A., Sun, Y., Gottwald, T. 2009. Complete 

genome sequence of citrus huanglongbing bacterium Candidatus Liberibacter asiaticus 

obtained through metagenomics. Molecular Plant-Microbe Interactions 22:1011-1020. 


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1.8 Genetic Diversity of Candidatus Liberibacter asiaticus Isolates from Paraná State, 

Brazil 

Meneguim, L. 1 , Marques, V.V. 2 , Murata, M.M. 1,2 , Barreto, T.P. 2 , Vasquez-Souza, G.V. 2 , 

Villas-Boas, L.A. 1 , Paccola-Meirelles, L.D. 1 , Leite, R.P., Jr. 2 

1 UEL, Universidade Estadual de Londrina, Londrina, Brazil 

2 IAPAR, Instituto Agronômico do Paraná, Londrina, Brazil 

Huanglongbing (HLB) is one of the most devastating diseases for citrus production around the 

world. The disease was first reported in Paraná State in 2006. In Brazil, Candidatus Liberibacter 

asiaticus and Candidatus Liberibacter americanus have been associated with HLB. However, 

only Ca. Liberibacter asiaticus has been reported in Paraná. In this study, we investigated the 

genetic diversity of Ca. Liberibacter asiaticus present in Paraná State by RFLP analyses and 

sequencing of the β-operon ribosomal protein genes. For RFLP analyses, DNA fragments were 

amplified with the primers HP1/LP1c for omp gene from 40 isolates of the bacterium. PCR 

products were digested with the restriction enzymes ApoI, HinfI, and TaqI. The primers A2/J5 

were used to amplify a fragment of 703 bp of the β-operon ribosomal protein genes from 

21 isolates. The amplified fragments were sequenced and a region of 402 bp was used for 

comparisons. In the PCR-RFLP analysis, no genetic diversity was observed among the isolates. 

The partial sequences of the β-operon ribosomal Ca. Liberibacter asiaticus protein genes were 

identical to each other. When compared with known Ca. Liberibacter spp. sequences, the 

homology was higher for Ca. Liberibacter asiaticus (100%) than for Candidatus Liberibacter 

africanus (84%) or Candidatus Liberibacter solanacearum (80%). These results indicate a low 

variability among the isolates Ca. Liberibacter asiaticus present in Paraná State. 

Introduction 

In Brazil, the disease was first reported in São Paulo State in 2004 (Coletta-Filho et al., 2004). In 

Paraná State, the disease was observed for the first time in 2006. Two species of Ca. Liberibacter 

have been associated with HLB, Candidatus Liberibacter asiaticus and Candidatus Liberibacter 

americanus (Coletta-Filho et al., 2004; Teixeira et al., 2005). However, only Ca. Liberibacter 

asiaticus has been reported in Paraná. Four years after the first report, the disease is present in 

citrus orchards of more than 54 counties of the north and northwest regions of the State of 

Paraná. 

Assessment of the genetic diversity may provide a framework for understanding the 

population structure and dynamics of plant pathogenic bacteria. The omp gene encoding an outer 

membrane protein (omp) has been used for studying Ca. Liberibacter asiaticus diversity on the 

basis of phenotypic traits (Bastianel et al., 2005). 

In this study, we investigated the genetic diversity of Ca. Liberibacter asiaticus present in 

Paraná State by restriction fragment length polymorphisms (RFLPs) analyses using the omp 

locus and sequencing of the β-operon ribosomal protein genes. 


P a g e | 25 

Materials and Methods 

Leaf samples were collected from HLB-infected citrus trees in different orchards of Paraná State 

(Table 1). DNA was extracted from leaf midribs by using the cetyl trimethyl ammonium bromide 

(CTAB) method (Murray and Thompson, 1980). 

PCR reactions were carried out in a 40 ml reaction mixture containing 1X PCR buffer, 

2.5 mM MgCl 2 , 0.2 mM dNTPs, 0.2 mM of each primer, 1 U of Taq DNA polymerase 

(Invitrogen), and 2 ml of DNA. Amplification products were evaluated by electrophoresis in 1% 

agarose gels and visualized by staining with ethidium bromide. 

For RFLP analyses, the entire omp gene of Ca. Liberibacter asiaticus from 40 isolates was 

amplified with the primers HP1/LP1c (Table 1). PCR products were digested with the restriction 

enzymes ApoI, HinfI and TaqI, and the DNA fragments were separated by 2% agarose gel 

electrophoresis by using standard procedures. 

The primers A2/J5 were used to amplify a fragment of 703 bp of the β-operon ribosomal 

protein genes from 21 isolates (Table 1) (Hocquellet et al., 1999). 

PCR amplicons were sequenced directly in both orientations by using the BigDye 

Terminator v3.1 Cycle Sequencing Kit in a 3130X1 Genetic Analyzer (Applied Biosystems, 

Inc.) by using the same primers used for PCR. DNA sequences of 402 bp were used for 

comparisons with the current GenBank database by using the BLASTn network service available 

in the National Center for Biotechnology Information. 

Results 

Primers HP1 and LP1c were used to amplify the omp gene from 40 isolates (Table 1). The PCR 

products (approximate size of 2.4 kb) were digested with three distinct enzymes, ApoI, HinfI, and 

TaqI. In the PCR-RFLP analysis, a single profile of restriction was obtained for each restriction 

enzyme (Fig. 1). No genetic diversity was observed among the isolates of Ca. Liberibacter 

asiaticus used in this study, based on the omp gene. 

DNA sequences of 420 bp were used for comparisons among the HLB isolates from Paraná 

State and with those of the current GenBank. The partial sequences of the β-operon ribosomal of 

Ca. Liberibacter asiaticus protein genes were identical to each other (Fig. 2). The phylogenetic 

tree also showed that all isolates were identical independent of their geographical origins 

(Fig. 2). When compared with known Ca. Liberibacter spp. sequences, the homology was higher 

for Ca. Liberibacter asiaticus (100%) than for Ca. Liberibacter africanus (84%) or 

Ca. Liberibacter solanacearum (80%) (Fig. 2). 


P a g e | 26 

Conclusion 

These results indicate no variability among the isolates Ca. Liberibacter asiaticus was present in 

Paraná State respective to the genes investigated. 

References 

Bastianel, C., Garnier-Semancik, M., Renaudin, J., Bové, J.M., Eveillard, S. 2005. Diversity of 

Candidatus Liberibacter asiaticus, based on the omp gene sequence. Applied and 

Environmental Microbiology 71:6473-6478. 

Coletta-Filho, H.D., Targon, M.L.P.N., Takita, M.A., De Negri, J.D., Pompeu, J., Jr., Amaral, 

A.M., Muller, G.W., Machado, M.A. 2004. First report of the causal agent of 

huanglongbing (Candidatus Liberibacter asiaticus) in Brazil. Plant Disease 88:1382. 

Hocquellet, A., Toorawa, P., Bové, J.M., Garnier, M. 1999. Detection and identification of the 

two Candidatus Liberibacter species associated with citrus huanglongbing by PCR 

amplification of ribosomal protein genes of the β operon. Molecular and Cellular Probes 

13:373-379. 

Murray, M.G., Thompson, W.F. 1980. Rapid isolation of high molecular weight plant DNA. 

Nucleic Acids Research 8:4321-4325. 

Teixeira, D.C., Ayres, A.J., Kitajima, E.W., Tanaka, F.A.O., Danet, J.L., Jagoueix-Eveillard, S., 

Saillard, C., Bové, J.M. 2005. First report of a huanglongbing-like disease of citrus in Sao 

Paulo State, Brazil, and association of a new liberibacter species, Candidatus Liberibacter 

americanus, with the disease. Plant Disease 89:107. 

Table 1. Isolates of Candidatus Liberibacter asiaticus of different regions of Paraná State, 

Brazil, used in this study. 

Sweet orange 

Years of 

Isolate Origin 

variety 

planting 

1 Altônia/NW a n.d. b n.d. 

2 Altônia/NW n.d. n.d. 

3 Paranavaí/NW Pera n.d. 

8 Paraíso do Norte/NW n.d. n.d. 




16 Alto araná/NW Pera 2002 

18 Rolândia/N Iapar 73 1997 


46 Pitangueiras/N Pera n.d. 

76 Cambé/N Valência 2003 

109 Sabáudia/N Valência n.d. 


P a g e | 27 



117 Pitangueiras/N Valência n.d. 

119 Alto Paraná/NW n.d. n.d. 

139 Pitangueiras/N Pera 2002 

147 Cambé/N Pera 2003 

153 Sabáudia/N Valência 1997 

157 Rolândia/N Pera 1997 

175 Pitangueiras/N Valência 1996 

207 Cambé/N Folha Murcha 2002 

232 Bandeirantes/N Pera 2006 

233 Sabaudia/N n.d. n.d. 

251 Santo Antonio do Paraíso/NW Iapar 73 2004 

253 Prado Ferreira/N Pera 2004 

258 Sabáudia/N Folha Murcha 1996 

259 Sabáudia/N Iapar 73 1996 

262 Astorga/N Pera 2005 

266 Cambé/N Pera 2004 

268 Rolândia/N Pera Rio 2002 


293 Prado Ferreira/N Pera 2007 

302 Cornélio Procópio/N Pera 2004 

311 Rolândia/N Valência 2003 

312 Rolândia/N Pera n.d. 

323 Arapongas/N Folha Murcha n.d. 

330 Bela Vista do Paraiso/N Pera 2006 

350 Rolandia/N n.d. n.d. 

a NW, Northwest region; N, North region; b n.d., not determined. 


P a g e | 28 

M 1 2 3 4 5 6 7 8 9 10 11 12 13 nd M M 1 2 3 4 5 6 7 8 9 10 11 12 13 M nd 

A 

B 

M 1 2 3 4 5 6 7 8 9 10 11 12 13 nd M 

C 

C 

Fig. 1. ApoI (A), HinfI (B), and TaqI (C) restriction profile analyses of PCR-amplified DNAs 

from citrus infected with Ca. Liberibacter asiaticus of different regions of Paraná State, Brazil. 

M, 1kb DNA ladder; 1, 10 (Paranavaí); 2, 16 (Alto Paraná); 3, 76 (Cambé); 4, 119 (Alto Paraná); 

5, 147 (Cambé); 6, 157 (Rolândia); 7, 175 (Pitangueiras); 8, 207 (Cambé); 9, 232 (Bandeirantes); 

10, 233 (Sabáudia); 11, 251 (Santo Antonio do Paraíso); 12, 258 (Sabáudia); 13, 259 (Sabáudia); 

nd, not digested. 


P a g e | 29 

Fig. 2. Phylogenetic trees based on β-operon ribosomal gene sequence of Ca. Liberibacter spp., 

using MEGA version 2.1. GenBank accession numbers are given in parentheses. 


P a g e | 30 

1.9 Analysis of Endophytic Bacterial Diversity from Huanglongbing Pathogen-Infected 

Citrus Tissues 

Wang, A. 1 , Yin, Y. 1 , Li, Y. 1 , Li, J. 1 , Xian, J. 2 , Wang, Z. 1 

1 Bioengineering College, Chongqing University, Chongqing, 400030, Chongqing, China 

2 Lucky Team Biotech Development (Hepu) Ltd., Co. Hepu, Beihai, 536128, Guangxi, China 

The endophytic bacterial diversity in huanglongbing pathogen-infected and healthy citrus plant 

tissues of stem, leave midrib, bark, and roots from Hepu, Guangxi municipality were 

investigated to decipher the interactions of the HLB pathogen with companion microbial 

populations. Classical morphological, physiological, and chemotaxonomic characteristics 

methodology, and 16S rRNA and PCR-RFLP analysis methods were applied. Using traditional 

isolation and 16S rDNA amplicon methods, 10 genera of bacteria were identified from 

21 culturable bacterial populations. The sequence analyses using the NCBI GenBank database 

showed that they belonged to 10 different genera of bacteria. The dominant bacterial population 

in healthy citrus belonged to Bacillus sp., Pseudomonas sp., and Kocuria sp.; while in phloem 

tissue of infected 10-year-old citrus, Bacillus sp., Pseudomonas sp., Kocuria halotolerans, and 

Microbacterium sp. were found. Two 16Sr DNA clone libraries of endophytic bacteria were 

constructed and 29 restriction endonuclease types were detected; 10 genera of bacteria and 

11 genera of uncultured bacteria were identified. The dominant bacterial population in libraries 

belonged to Dyella sp., Pseudomonas sp., Serratia sp., and unculturable bacteria. The density 

and species of endophytic bacteria were remarkably different compared to diseased and healthy 

trees. By traditional cultivation, 16S rDNA sequencing, and PCR-RFLP analysis, the diversity of 

populations and species of endophytic bacteria mainly in citrus phloem tissue were determined to 

be different in infected and healthy citrus plants, while some of the functional bacterial isolates 

including Microbacterium sp., Curtobacterium sp., etc., were gram-positive bacteria belonging 

to the genus Microbacteriaceae, Actinomycetales. They are most likely associated with the 

huanglongbing causal agent currently still under investigation. 


This work was granted by the National Science Foundation of China, in NSFC No. 30971875. 

Keywords: Huanglongbing, endophytic bacteria, 16S rDNA, isolation culture, RFLP, 

biodiversity 


P a g e | 31 

1.10 Evolving Diversity of Candidatus Liberibacter asiaticus Revealed by Comparative 

Analysis of Two Intragenic Tandem Repeat Genes 

Zhou, L.J. 1,2 , Powell, C.A. 1 , Hoffman, M. 2 , Li, W.B. 3 , Fan, G.-C. 4 , Liu, B. 4 , Duan, Y.-P. 2 


2 USDA-ARS-USHRL, Fort Pierce, FL, USA 

3 USDA-APHIS_PPQ_CPHST, Beltsville, MD, USA 

4 Citrus Huanglongbing Research Center, Fujian Academy of Agricultural Sciences, Fuzhou, 

Fujian 350002, China 

Candidatus Liberibacter asiaticus (Las) is the most prevalent species of citrus huanglongbing 

bacteria in the world. Two related and hyper variable genes (hyvI and hyvII) were identified in 

prophage regions of the psy62 Las genome. The hyvI contains up to 12 nearly identical tandem 

repeats (NITR, 132 bp) and 4 partial repeats, while the hyvII contains up to 2 NITR and 4 partial 

repeats, and shares homology with hyvI. Frequent deletions or insertions of these repeats within 

the hyvI/II genes were observed, but none of which disrupted the open reading frames. 

Sequencing analysis of the hyvI/hyvII genes from 35 Las DNA samples collected globally 

revealed sequence conservation within individual repeats but extensive variation regarding repeat 

numbers, their rearrangement, and the sequences flanking the repeat region. These differences 

are found not only in samples with distinct geographical origins but also from a single origin and 

even from a single Las-infected sample. The Florida isolates contain both hyvI and hyvII, while 

all other global isolates contain only one of the two. Interclade assignments of the putative 

HyvI/II proteins from Florida isolates with other global isolates in the phylogenic trees imply a 

strong potential of multiple populations existing in the Las bacteria in the world, and a 

multi-source introduction of the Las bacterium into Florida. On the other hand, the high copy 

number of these multiple nearly identical repeats provides a significant advantage for developing 

more sensitive diagnosis tools. 


P a g e | 32 

1.11 In vitro Culture of the Fastidious Bacteria Candidatus Liberibacter asiaticus in 

Association with Insect Feeder Cells 

Fontaine-Bodin, L., Fabre, S., Gatineau, F., Dollet, M. CIRAD-BIOS, Etiology Wilts, 

TA A 29/F, Campus International de Baillarguet, 34398 Montpellier cedex 5, France 

Ca. Liberibacter asiaticus (LAS) is vectored by psyllids and is able to proliferate inside the 

insect. We therefore hypothesize that insect cells could act like feeder cells, providing nutrients 

in a continuous way and a favorable environment to the bacteria. Various insect cell lines and 

sources of LAS inoculum were tested in an empirical way to select for a suitable cell line and to 

establish a protocol for primo-cultures. LAS presence in the inoculated cell cultures was checked 

by direct PCR and confirmed by sequencing of the amplicons. Nine different cell lines were 

tested from Mamestra, Spodoptera, Drosophila, Aedes, and Diaphorina insects. Mamestra and 

Spodoptera cell lines were not suitable for LAS growth. One Aedes and two Drosophila cell 

lines sustained Liberibacter survival and growth. Diaphorina cell lines were recently received 

and are under investigation regarding their capacity to maintain the bacteria. To reach higher 

bacterial concentrations, we analyzed metabolic pathways potentially encoded by the Ca. 

Liberibacter genome to define limiting factors and/or growth inhibitors, and we complemented 

the primo-cultures with various additives (sugars, vitamins, etc.). A culture of LAS was obtained 

with Aedes cells as feeder cells. This culture has been continuously growing for 9 months and 

16 successive transfers. We are currently working on axenization of this culture. By adding 

selected additives in our LAS/Aedes co-cultures, we increased the yield of LAS (~1.10 7 cells/ml), 

and we are currently looking into the best ways to maintain/increase this concentration and to 

stock those LAS cultures. 


P a g e | 33 

1.12 Preliminary Report of Cultivation of Candidatus Liberibacter asiaticus from Citrus 

Tissue with Huanglongbing 

Xie, P., Yin, Y., Li, Y., Li, J., Wang, Z. Genetic Engineering Research Center, Bioengineering 

College of Chongqing University, Chongqing, 400030, P. R. China 

Huanglongbing (HLB) is an extremely devastating disease of citrus caused by the 

phloem-limited bacteria Candidatus Liberibacter spp., which has not been grown in sub-culture 

continually. In this paper, we attempted to cultivate Ca. L. asiaticus using media formulations 

developed in response to the growth of other bacteria that may appear to be related to the 

Liberibacter. A co-cultivation medium named Las-VAE has been designed and used to 

successfully cultivate Ca. L. asiaticus in liquid culture consisting of citrus vascular tissue 

extracts, growth factors, and auto QS inducers of bacterial population. By supplying known 

quantities of endophytes isolated from several citrus varieties and common periwinkle phloem 

tissue in China into liquid co-cultivation system with Las in a facultative anaerobic plastic box, 

the preliminary results indicated that several endophytic bacteria such as Microbacterium 

laevaniformans and Curtobacterium sp. facilitate the growth of the HLB pathogen, which was 

detected by SGI-qPCR in culture. Culture solution, positive for Ca. L. asiaticus by a 16S-rDNA 

quantitative polymerase chain reaction (qPCR) assay, indicated that the quantity of pathogen 

increased with the maximum about 50 times that of the initial culture mixture. 




P a g e | 34 

1.13 Characterization of Highly Mosaic Genomic Loci of Candidatus Liberibacter asiaticus 

in Southern China and Florida 

Wang, X.F. 1 , Zhou, C.Y. 1* , Deng, X. 2 , Su, H.N. 1 , Chen, J. 3* 

1 Citrus Research Institute, Southwest University, Chongqing 400712, China 

2 Citrus HLB Research Center, South China Agricultural University, Guangzhou 510642, China 

3 San Joaquin Valley Agricultural Sciences Center, USDA-ARS, Parlier, CA, USA 

*Corresponding authors 

Introduction 

Huanglongbing (HLB) is a destructive disease of citrus production worldwide. The disease was 

first noted in the Chaoshan area in Guangdong Province of the people’s republic of China in the 

late 1800s (Lin, 1956) and is currently distributed in 10 citrus producing provinces in south 

China. HLB is now established in Florida of the United States (Halbert, 2005) and poses a great 

challenge to the local citrus industry there. In both China and U.S., Candidatus Liberibacter 

asiaticus is regarded as the putative pathogen of HLB. Identification and characterization of 

unique genomic loci in Ca. L. asiaticus is currently a critical and most practical means to 

evaluate this unculturable bacterium (Chen et al., 2010; Kotoh et al., 2011; Tomimura et al., 

2009). In this study, we report our observation of a mosaic locus of prophage origin in the 

genome of Ca. L. asiaticus. PCR analyses using a primer set flanking this genomic locus 

revealed eight electrophoretic types (E-types) of Ca. L. asiaticus strains from China and U.S. 

Sequence analysis showed that the insertion/deletion events were responsible for the amplicon 

variations. 


HLB symptomatic citrus leaves were collected from nine provinces in China and Florida in U.S. 

between 2007 and 2010 (Table 1). Primer set Lap5650f /Lap5650r (5’-TCT GTG ATG CCG 

TTT GTA GG-3’/5’-CCA AAT CAG CCA GCT CAA AT-3’) flanking the chromosomal region 

of CLIBASIA_05640 to CLIBASIA_05650 was selected for further studies. PCR products were 

cloned with pGEM T-easy vector and sequenced using BigDye Terminator v3.1 Cycle 

Sequencing Kit in a 3130×1 Genetic Analyzer. Multiple sequence alignments were performed 

using the ClustalW (Ver.1.74) program with the default parameters. Manual adjustment was 

performed when appropriate. Nine DNA sequences of various Ca. L. asiaticus strains generated 

in this study have been deposited in GenBank with accession numbers JF412691 to JF412699. 

Results and Discussion 

A total of 262 HLB samples that were positive with primer set OI1/OI2c and ITSAf/ITSAr were 

analyzed (Table 1). PCR amplification with primer set Lap5650f/Lap5650r produced eight 

electrophoretic types, designated as E-type A to H. Each E-type was composed of one or more of 

five DNA amplicons, designated as P1 to P5 (Fig. 1). In silico analysis identified only a single 


P a g e | 35 

DNA region of 797 bp in the genome of Ca. L. asiaticus strain psy62 that matched E-type C 

(Fig. 1, Table 1). E-type C was found in 3 (4.1%) out of the 74 Florida HLB samples. Other 

E-types detected in Florida were A, G, and H. E-type G was predominant (82.4%), followed by 

E-type A (10.4%) and E-type H (4.1%) (Table 1). Six E-types (A, B, C, D, E, and F) were found 

in the 188 samples from China (Fig. 2). E-type A was the most frequent (71.3%), followed by 

E-type B (9.7%). It should be noted that of the six E-types in China, five (A, B, C, D, and E) 

were found in Yunnan with type B being predominant (Table 1). This is in contrast to the 

Guangdong population that has only two E-types (A and F), with type A being predominant. 

Different numbers of E-types could suggest that Ca. L. asiaticus population in Yunnan 

underwent more vigorous prophage activity than those in Guangdong. The detection of P3 only 

in Yunnan further demonstrates that the Ca. L. asiaticus population in Yunnan is more diverse. 

Information about population diversity of Ca. L. asiaticus in Yunnan is currently very limited 

and deserves more attention in the future. 

The sequences of P1, P2, P3, P4, and P5 (Fig. 1) were determined to be 797, 869, 906, 

1071, and 1143 bp, respectively. Alignment data showed that the five DNA sequences shared a 

common backbone of P1 (Fig. 2). Referenced to P1, the other four sequences were formed due to 

insertion events at position 574 and 722 (Fig. 2). P2 (869 bp) had a 72-nt direct repeat at position 

574 inside ORF CLIBASIA_05650. P3 (906 bp) had an insertion of a 109 bp fragment at 

position 722 within the annotated non-coding region. Similar to P3, P4 (1071 bp) had an 

insertion at position 722 but a fragment size of 274 bp. P5 had both the P2 and P4 type 

insertions. BLASTn search showed that P1 sequence was identical to that in strain Psy62 (Duan 

et al., 2009), and P5 was over 99% similar to those of Ca. L. asiaticus strain UF506 

(HQ377374.1), Liberibacter phage SC1 (HQ377372.1), and Liberibacter phage SC2 

(HQ377373.1) (Zhang et al., 2011). 

In summary, by identifying and analyzing a highly mosaic genomic locus, we found 

significant inter- and intra-population variations of Ca. L. asiaticus from South China and 

Florida. Our investigation shows that insertion/deletion events contribute to the DNA variations. 

Information provided in this study is potentially useful for identification, characterization, and 

understanding of the diversity of Ca. L. asiaticus populations in different geographical regions 

and various biological traits. 


Part of this research was partially supported by a California Citrus Research Board grant, MOA’s 

Public Benefit Research Foundation of China (201003067-02; 200903004-06) and Program for 

Changjiang Scholars and Innovative Research Team in University (PCSIRT, IRT0976). 

References 

Chen, J., Deng, X., Sun, X., Jones, D., Irey, M., Civerolo, E. 2010. Guangdong and Florida 

populations of Candidatus Liberibacter asiaticus distinguished by a genomic locus with 

short tandem repeats. Phytopathology 100:567-572. 


P a g e | 36 

Duan, Y., Zhou, L., Hall, D.G., Li, W., Doddapaneni, H., Lin, H., Liu, L., Vahling, C.M., 

Gabriel, D.W., Williams, K.P., Dickerman, A., Sun, Y., Gottwald, T. 2009. Complete 

genome sequence of citrus Huanglongbing bacterium, Candidatus Liberibacter asiaticus 

obtained through metagenomics. Molecular Plant-Microbe Interactions 22:1011-1020. 

Halbert, S.E. 2005. The discovery of huanglongbing in Florida. Proceedings of the 2 nd 

International Citrus Canker and Huanglongbing Research Workshop, Florida Citrus 

Mutual, Orlando, H-3. 

Kato, H., Subandiyah, S., Tomimura, K., Okuda, M., Su, H.J., Iwanami, T. 2011. Differentiation 

of Candidatus Liberibacter asiaticus isolates by variable number of tandem repeat analysis. 

Applied Environmental Microbiology [Epub ahead of print]. 

Lin, K.-H. 1956. Observations on yellow shoot of citrus. Acta Phytopathology Sin. 2:1-11. 

Tomimura, K., Miyata, S., Furuya, N., Kubota, K., Okuda, M., Subandiyah, S., Hung, T.H., Su, 

H.J., Iwanami, T. 2009. Evaluation of genetic diversity among Candidatus Liberibacter 

asiaticus isolates collected in Southeast Asia. Phytopathology 99:1062-1069. 

Zhang, S., Flores-Cruz, Z., Zhou, L., Kang, B.H., Fleites, L., Gooch, M.D., Wulff, N.A., Davis, 

M.J., Duan, Y., Gabriel, D.W. 2011. Ca. Liberibacter asiaticus carries an excision plasmid 

prophage and a chromosomally integrated prophage that becomes lytic in plant infections. 

Molecular Plant Microbe Interactions 24:458-468. 

Table 1. Frequencies of Candidatus Liberibacter asiaticus electrophoretic types (E-types) at 

different locations in China and U.S. 

Location 

E-type 

A B C D E F G H 

Total 

China 

Yunnan 6 27 6 3 1 - - - 43 

Guizhou 3 2 5 - - - - - 10 

Sichuan 2 - - - - - - - 2 

Guangxi 30 6 - - - - - - 36 

Guangdong 65 - - - - 2 - - 67 

Fujian 14 - - - - - - - 14 

Jiangxi 4 - - - - - - - 4 

Hunan 6 2 - - - - - - 8 

Zhejiang 4 - - - - - - - 4 

Total 134 37 11 3 1 2 - - 188 

Frequency 71.3 19.7 5.9 1.6 0.5 1.1 - - 

U.S. 

Florida 7 - 3 - - - 61 3 74 

Frequency 10.4 - 4.1 - - - 82.4 4.1 


P a g e | 37 

Fig. 1. Electrophoretic profiles (E-types) of representative Candidatus Liberibacter asiaticus 

strains from PCR amplification with primer set Lap5650f/Lap5650r. Lane M on the left contains 

molecular markers. Size unique amplicons are designated through P1 to P5. 

Fig. 2. Sequence comparison of five types of PCR amplicons (P1 to P5) from primer set 

Lap5650f/Lap5650r. Annotation of Candidatus Liberibacter asiaticus strain Psy62 is used as a 

reference and shown in the first row. Nucleotide positions 574 and 722 are marked. The two sites 

serve as sequence insert sites responsible for the mosaic phenomenon of the genomic locus. 


P a g e | 38 

1.14 Further Evidence That U.S. and Chinese Populations of Candidatus Liberibacter 

asiaticus are Different 

Deng, X. 1 , Liu, R. 1 , Zhang, P. 1 , Chen, J. 2 

1 South China Agricultural University, Guangzhou, PRC 

2 USDA-ARS-PWA, Parlier, CA, USA 

Citrus huanglongbing (HLB) is associated with Candidatus Liberibacter asiaticus. Many efforts 

to detect genetic variation of Ca. L. asiaticus in conserved genomic loci such as 16s rDNA have 

not been successful. We have reported a genomic locus with short tandem repeats could be used 

to separate the Ca. L. asiaticus populations of Guangdong of China and Florida of the U.S. 

(Phytopathology 100:567-572). We recently found that prophage genes are also good candidates 

to differentiate strains of Ca. L. asiaticus. Prophages are important genetic elements of bacterial 

genomes and are involved in lateral gene transfer, pathogenicity, environmental adaptations, and 

interstrain genetic variability. In this study, the sequence of a phage terminase gene was 

identified. Based on the terminase gene sequence, a set of primers was designed and used for 

PCR detection of prophage in HLB citrus samples collected from two provinces, Guangdong 

with an average altitude of 2,000 m. The 

frequency of prophage detection was 15.8% in Guangdong and 97.4% in Yunnan. However, the 

prophage gene sequences obtained from 10 Guangdong strains and 8 Yunnan strains shared 

100% similarity. It remains unclear what contributed to the prophage variation in the two 

regions. Further study showed that the prophage gene could be detected in 99% of Florida strains 

of Ca. L. asiaticus. Yet the Florida strains differed from the Chinese strains at 9 single nucleotide 

positions. 


P a g e | 39 

Session 2: 

Asian Citrus Psyllid 

Biology and Genomics 


P a g e | 40 

2.1 Phylogeographic and Population Genetic Studies Uncover Two Founding Events in 

Asian Citrus Psyllid Populations Collected in the Americas 

de León, J.H. 1 , Sétamou, M. 2 , Gastaminza, G.A. 3 , Buenahora, J. 4 , Cáceres, S. 5 , Yamamoto, 

P.T. 6 , Logarzo, G.A. 7 , Stañgret, C.R.W. 8 

1 USDA, ARS, Weslaco, TX, USA 

2 Citrus Center Texas A&M University - Kingsville, Weslaco, TX, USA 

3 Estación Experimental Agroindustrial Obispo Colombres (EEAOC), Tucumán, Argentina 

4 Instituto Nacional de Investigación Agropecuaria (INIA), Salto, Uruguay 

5 Instituto Nacional de Tecnología Agropecuaria (INTA), Bella Vista, Corrientes, Argentina 

6 Escola Superior de Agricultura ‘Luiz de Queiroz’/USP, Piracicaba, São Paulo, Brazil 

7 USDA, ARS, SABCL, Buenos Aires, Argentina 

8 Private Contractor, Itapúa, Paraguay 

A phylogeographic analysis inferred from the partial mitochondrial cytochrome oxidase subunit I 

gene (433 bp) was performed with 22 populations (n = 132) of Diaphorina citri collected in the 

Americas and one in the Pacific. Eight populations (n = 46) from four countries in South 

America, 14 (n = 76) from four countries in North America, and one from Hawaii (n = 10) were 

analyzed. Twenty-three haplotypes (hp) were identified that fell into two groups, hp1-8 were 

identified in South America (Group 1) and hp 9-23 were identified in North America and Hawaii 

(Group 2). Hp1 and 9 were present in the highest frequencies within each population and within 

their group, 81-85%. Sharing of haplotypes was not observed between the two groups. An 

analysis of molecular variance uncovered significant genetic structure (Φ CT = 0.733; P < 0.001) 

between the two groups in the Americas. A neighbor-joining phylogram and two haplotype 

networks (Parsimony Splits and Statistical Parsimony) discriminated the two groups, while both 

networks identified hp1 and 9 as the ancestral or founding haplotypes within their respective 

group. Significantly negative neutrality tests (Tajima’s D and Fu’s Fs), non-significant mismatch 

distribution parameters (SSD and HRI), and low genetic diversity levels provided evidence of 

demographic expansion within each group in the Americas. The data suggest that two founding 

events of D. citri occurred in the Americas, one in South America and one in North America. 

Furthermore, North America and Hawaii appear to share a similar source of invasion. These data 

are important to the development of biological control programs against D. citri in the Americas. 


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2.2 Alteration of Microbiome of Bactericera cockerelli and Diaphorina citri Based on 

Candidatus Liberibacter sp. Infection 

Hail, D. 1 , Hunter, W.B. 2 , Bextine, B.R. 1 

1 University of Texas, Tyler, TX, USA 

2 USDA, ARS, U.S. Horticultural Research Lab, Fort Pierce, FL, USA 

Candidatus Liberibacter species have recently been implicated as causative agents in both Zebra 

Chip (ZC) and huanglongbing (HLB), diseases of potatoes and citrus, respectively. ZC and HLB 

are limiting factors in the production of these crops and intense research is ongoing on the 

vectors, the potato psyllid (Bactericera cockerelli) and the Asian citrus psyllid (Diaphorina 

citri). An insect’s microbiome consists of the microbial flora present in the body and may serve 

as a possible avenue of biological control. In this study, the microbial flora associated with 

B. cockerelli and D. citri were evaluated. A comprehensive study of the microbiome of four life 

stages (egg, early instars, 5th instars, and adults) was determined by pyrosequencing of 16S 

ribosomal DNA using the bTEFAP methodology. The resulting sequences were compared to a 

curated high quality 16S rDNA database derived from NCBI’s GenBank. Some of the bacteria 

identified in this report are initial discoveries; species from the genera Wolbachia, Rhizobium, 

Gordonia, Mycobacterium, Xanthomonas, and many others were detected; and an assessment of 

the microbiome associated with B. cockerelli was established for each life stage. Next, microbial 

flora associated with adult B. cockerelli and D. citri (both positive and negative for Candidatus 

Liberibacter spp.) were analyzed and compared. 


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2.3 Oral Uptake of dsRNA Increases Mortality in Diet Fed Psyllids 

Shatters, R.G., Jr. 1 , Powell, C.A. 2 , Borovsky, D. 3 

1 USDA, ARS, USHRL, Fort Pierce, FL, USA 


3 FMEL, IFAS, University of Florida, Vero Beach, FL, USA 

Recent publications have shown that, in some insects, oral uptake of dsRNA targeting specific 

genes within the insect can be toxic through RNAi-induced mechanisms. This observation has 

opened a new area of research on the development of highly specific insect control strategies. 

Using an artificial diet feeding system, we have shown that feeding psyllid specific dsRNA to 

Asian citrus psyllids (Diaphorina citri) can also increase mortality. The dsRNA targeting 

specific members of three different psyllid gene families (tubulins, vacuolar ATPases, and gut 

cathepsins) was added to the artificial diets, and psyllid mortality was monitored over 6 days. 

The dsRNA targeting two specific genes (one Vacuolar ATPase and one cathepsin F) doubled 

the mortality that was observed in psyllids feeding either on standard diet or diet supplemented 

with dsRNA that was not specific to psyllids. Only dsRNA targeting two out of eight separate 

genes from the three studied gene families increased mortality. Preliminary analysis of mRNA 

transcripts to genes targeted by the dsRNA indicates a reduction in total intact transcripts in 

psyllids feeding on the cognate dsRNA, a finding that supports the induction of an RNAi 

mechanism. Research in optimizing diet for feeding trials and dosage responses will be 

presented. This is the first report of dsRNA working in psyllids and supports the possibility of 

using an RNAi-based strategy to control the Asian citrus psyllid. 


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2.4 The Psyllid Feeding Process: Composition and Biosynthetic Inhibition of the Salivary 

Sheath 

Shatters, R.G., Jr. USDA, ARS, USHRL, Fort Pierce, FL, USA 

The Asian citrus psyllid (Diaphorina citri) belongs to a group of insects that feed on plant 

vascular contents through the use of piercing mouthparts. In the piercing/probing stage of 

feeding, these insects secrete liquid saliva that solidifies immediately upon exposure. This saliva 

ultimately forms a tube that is presumed to be necessary for establishment of a successful 

feeding process. We have used an artificial diet developed to stimulate salivary sheath formation 

to study the molecular basis of this feeding process. Salivary sheaths have been successfully 

isolated from this diet and used for chemical analysis that indicates a structure composed 

primarily of carbohydrates. However, proteins are present within this sheath with 1 or 2 major 

protein bands and several less abundant bands when analyzed by SDS-PAGE. Using this diet, we 

have also identified EDTA as an inhibitor of salivary sheath formation. The addition of EDTA 

also results in changes to the composition of secreted proteins with 3 major protein bands visible 

by SDS-PAGE. Protein characterization is currently being conducted by mass spectroscopy and 

results will be discussed. This work shows that it is possible to block the psyllids’ ability to 

synthesize salivary sheaths during their probing process. The ultimate goal of this work is to 

identify salivary sheath formation inhibitors that can be used to block psyllid feeding and 

ultimately produce transgenic citrus plants that are innately resistant to the Asian citrus psyllid. 


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2.5 A New Method for Short-Term Rearing of Psyllid Adults and Nymphs on Detached 

Citrus Leaves and Young Terminal Shoots 

Ammar, E.-D., Hall, D.G. Subtropical Insects Research Unit, USDA-ARS, Fort Pierce, FL, 

USA 

Using whole citrus plants for rearing of psyllids for biological studies or for studying vector 

relations of huanglongbing disease takes considerable space, time, and other resources. We have 

developed a new and simpler method for short-term rearing of the Asian citrus psyllid 

Diaphorina citri using detached citrus leaves for psyllid adults and detached young terminal 

shoots for young nymphs. The cut petioles of young leaves or terminal shoots are immersed in 

water or moistened cotton in small microfuge tubes. Each leaf or terminal shoot is then ‘caged’ 

in a clear plastic 50 ml (or larger) tube, and the psyllids are added to it. Young D. citri adults and 

2 nd to 3 rd -instar nymphs reared on these detached leaves or terminal shoots were observed 

feeding and excreting their honey dew regularly, and the adults were observed laying eggs. 

Survival of young adults was 89, 80, and 75% after 2, 3, and 4 weeks, respectively, on detached 

leaves changed weekly. Survival and adult emergence of young nymphs were significantly 

higher in those kept for one week on the youngest fully expanded leaves on detached terminal 

shoots (78 and 55%, respectively), compared to those kept for the same period on older detached 

leaves (57 and 26%, respectively). We believe that this new short-term rearing method for 

psyllids can save time and other resources and enhance various studies on the biology, 

management, and pathogen-vector interactions of this and other psyllid species. 


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2.6 Comparative Analysis of Asian Citrus Psyllid and Potato Psyllid Antennae 

Arras, J. 1 , Hunter, W.B. 2 , Bextine, B.R. 1 

1 University of Texas, Tyler, TX, USA 

2 USDA, ARS, U.S. Horticultural Research Laboratory, Fort Pierce, FL, USA 

The comparative investigation of the morphological basis for olfactory reception in the Asian 

citrus psyllid (Diaphorina citri) and the potato/tomato psyllid (Bacterocera cockerelli 

(Hemiptera: Psyllidae) was performed using scanning electron microscopy to elucidate the 

sensory mechanisms being used by these two psyllids in host selection and mating. Different 

host plant niches are used by these psyllids. While D. citri is essentially monophagous feeding 

only from citrus and its near relatives, B. cockerelli feeds on a wide range of Solanaceous plants. 

In this study, two different antennal sensory arrays were identified, with a more complex 

arrangement occurring in D. citri over B. cockerelli. The antenna length of D. citri was 0.23 mm 

long and contained 10 segments, while B. cockerelli had antenna of 0.60 mm length with 

10 segments. In both species, apically on the sensillus terminalis there are two conspicuous 

multi-porus single-walled bristles. These were longer in B. cockerelli. Mechano- and 

chemosensory hairs appear in low numbers on all segments in both species, with higher number 

of sensillae on distal segments. Diaphorina citri coevolved with its citrus host plant in tropical 

Asian countries, thus locating the strong aromatic plants was most likely less difficult and 

females would be constrained to a specific host plant; B. cockerelli, which has fewer olfactory 

sensilla and feeds on a wider host range, may have more sensitivity to specific chemical cues to 

locate the opposite gender for mating which could occur on many different host plants. 


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2.7 The Emerging Psyllid Genome: RNA-Interference and Insect Biology 

Hunter, W.B. 1 , Bextine, B.R. 2 , Shatters, R.G., Jr. 1 , Reese, J. 3 , Shelby, K.S. 4 , Hall, D.G. 1 


2 University of Texas at Tyler, Tyler, TX, USA 

3 Reese Consulting, Inc., Alpharetta, GA, USA 

4 USDA, ARS, Columbia, MO, USA 

Psyllids are major disease vectors of many fruit tree crops, yet their genetics have remained 

poorly studied. The first genome draft of the Asian citrus psyllid (ACP), Diaphorina citri 

(Hemiptera: Psyllidae), is currently being completed for the research community. Insect 

genomics have advanced insect management by examining gene function and biological 

pathways. Invasive pests are often subjected to genetic analysis that can identify species more 

accurately for classification, thus expediting the proper application of pest management 

regulations. ACP is a highly competent vector of the phloem-inhabiting bacterium Candidatus 

Liberibacter asiaticus, associated with huanglongbing (HLB) (citrus greening disease). HLB 

threatens the U.S. citrus industry due to losses in fruit yield, palatability, and tree death. Research 

efforts underway on psyllid genomics are using this information to develop strategies to suppress 

ACP populations. Production and mining of ACP genetic libraries identified several genes that 

function in psyllid responses to stress such as temperature, insecticides, and disease (i.e., heat 

shock proteins, hsp70, hsp90, cytochrome P450’s, Glutathione-S Transferase, Cu-SOD, Toll, 

and others). Further applications into functional genomics of ACP are being applied to develop 

RNAi, gene disruption methods to more effectively suppress ACP populations and reduce the 

spread of HLB in citrus. 


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2.8 Bacterial Population Diversity in Diaphorina citri: Analysis by PCR-DGGE and RFLP 

Methodology 

Wang, Z., Tian, S., Liu, T., Yin, Y. (Key Laboratory of Gene Function and Regulation at 

Chongqing, Bioengineering College of Chongqing University, Chongqing, P. R. China, 400030) 

The internal microflora of insects is a complex micro-ecosystem, in which a large and varied 

microbial community inhabits. This community plays important roles for their hosts' growth, 

development, digestion, absorption of nutrients, and resistance to colonization by exotic 

microbes, and so on. In this paper, we examined the bacterial diversity within citrus psyllids 

(Diaphorina citri), which is a vector of Liberibacter sp., using 16S rDNA-based molecular 

approaches of denaturing gradient gel electrophoresis (DGGE) and restriction fragment length 

polymorphism (RFLP). Bacterial taxa belonging to γ-Proteobacteria, β-Proteobacteria, and α- 

Proteobacteria of Proteobacteria were obtained by RFLP, including the families 

Pseudomonadaceae, Enterobacteriaceae, Xanthomonadaceae, Burkholderiaceae, Rickettsiaceae, 

and Rhizobiaceae. Syncytium endosymbiont and secondary symbionts of Diaphorina citri can 

also be obtained by RFLP. Four different families belonging to Proteobacteria and two families 

belonging to Firmicutes were obtained by DGGE. The result by PCR-DGGE analysis revealed 

that the bacterial population of D. citri is affected by the host plant of psyllids and geographic 

location, and the host plant has more obvious impact than that of geographic location. However, 

the existence of Syncytium endosymbiont in D. citri is not affected by the host plant or 

geographic location. Both RFLP and DGGE analysis indicated that the Syncytium endosymbiont 

of D. citri is a dominant microflora, and the two methods combination can reveal the microbial 

diversity in Diaphorina citri conveniently. 



Keywords: Diaphorina citri, PCR-DGGE, RFLP, bacterial, microbial diversity 


P a g e | 48 

Session 3: 


Ecology and 

Transmission 


P a g e | 49 

3.1 Antennal Responses of Diaphorina citri to Host Plant Volatiles Recorded Using a 

Coupled Gas Chromatograph Electroantennogram Detector System 

Robbins, P.S., Alessandro, R.T., Lapointe, S.L. ARS-USDA, Fort Pierce, FL, USA 

Understanding the cues used by the Asian citrus psyllid to locate host plants and/or conspecifics 

could yield valuable tools for detection, monitoring, and control of this global pest of citrus. 

Some evidence exists from laboratory assays and olfactometer studies that this species responds 

to common plant volatiles and possibly to a female-produced sex attractant (Wenninger et al., 

2008, 2009). We recently succeeded in recording antennal responses using a gas 

chromatograph-electroantennogram detector (GC-EAD) system to distinguish 

electrophysiologically active peaks for subsequent identification and use in behavioral analyses. 

Progress in identifying compounds that elicit antennal response and the methods used to measure 

antennal responses will be presented along with plans to use GC-EAD to search for potentially 

useful semiochemicals. 

References 

Wenninger, E.J., Stelinski, L.L., Hall, D.G. 2008. Behavioral evidence for a female-produced sex 

attractant in Diaphorina citri. Entomologia Experimentalis et Applicata 128:450-459. 

Wenninger, E.J., Stelinski, L.L., Hall, D.G. 2009. Roles of olfactory cues, visual cues, and 

mating status in orientation of Diaphorina citri Kuwayama (Hemiptera: Psyllidae) to four 

different host plants. Environmental Entomology 38:225-234. 


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3.2 Population Dynamics of the Asian Citrus Psyllid and Potential Generations in 

Northern Sinaloa, Mexico 

Cortez-Mondaca, E. 1 , López-Arroyo, J.I. 2 , Pérez-Márquez, J. 1 , González, V.M. 1 

1 INIFAP, Centro Regional de Investigaciones del Noreste, Cd. Obregón, Son., México 

2 INIFAP, Centro Regional de Investigaciones del Noreste, Río Bravo, Tam., México 

cortez.edgardo@inifap.gob.mx 

The Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Psyllidae), has been present in 

northern Sinaloa, Mexico since 2005 or earlier; however, its abundance, population fluctuation, 

and the potential number of generations per year in the area were unknown. The objective of this 

study was to determine the population dynamics of D. citri in three orchards, with five citrus 

species, in northern Sinaloa, Mexico. Weekly samplings of D. citri adults were conducted with 

yellow sticky traps. We recorded the different nymphal instars in the citrus shoots of Noor and 

Oroval clementines; Fortune and Nova mandarins; Marrs Early and Navelate orange; Star Ruby 

grapefruit; and Mexican lime. The calculation of the potential number of generations was 

performed with a minimum threshold temperature of 13.5°C and thermal constant for 

development from egg to adult of 211 degree-days. The presence of D. citri in the citrus of 

northern Sinaloa, Mexico was permanent throughout the year; however, the largest population 

increases were recorded from September 2009 to January 2010, as well as during April and May 

2010. Mexican lime had the most abundant presence of the insect, whereas the mandarins were 

the least preferred (P < 0.001). Based on the degree-days accumulation, it was determined for the 

region that there is the potential for the development of 18-19 generations of D. citri during the 

year. The results have implications for the management of the pest in the region. 


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3.3 Localization of Candidatus Liberibacter asiaticus in Dissected Organs of Its Psyllid 

Vector Diaphorina citri Using Fluorescent in situ Hybridization and Quantitative PCR 

Ammar, E.-D., Shatters, R.G., Jr., Hall, D.G. Subtropical Insects Research Unit, USHRL, 

USDA-ARS, Fort Pierce, FL, USA 

Vector interactions of huanglongbing (HLB) disease with its psyllid vectors, particularly at the 

organ and cellular levels, are poorly understood. We used fluorescent in situ hybridization 

(FISH) and quantitative PCR (Q-PCR) for the localization of Candidatus Liberibacter asiaticus 

(LAS) associated with HLB in its psyllid vector Diaphorina citri. Several FISH protocols have 

been tested on hemolymph smears and dissected psyllid organs and on leaf sections from 

HLB-infected citrus plants as positive controls. LAS was detected in the hemolymph, filter 

chamber, and midgut of D. citri collected from field HLB-infected citrus trees, as well as in the 

phloem of infected leaves, but not in healthy control psyllids or leaves. Additionally, Q-PCR 

detected LAS in dissected organs of individual D. citri adults collected from HLB-infected citrus 

trees. The proportion of infected salivary glands (47%) was significantly lower than those of the 

alimentary canal (72%) or other body parts (79%). Interestingly, the relative titer of LAS, 

compared to psyllid genomic DNA in each sample, was significantly higher in both the salivary 

gland and alimentary canal compared to that in the rest of the insect body. These results provide 

the first molecular confirmation of LAS in the hemolymph, alimentary canal, and salivary glands 

of D. citri. They also strongly suggest that the salivary glands constitute a major transmission 

barrier to LAS in the psyllid vector and that LAS may replicate or accumulate in both the 

alimentary canal and salivary glands of D. citri. 


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3.4 Interactions of the Asian Citrus Psyllid, Diaphorina citri, with Candidatus Liberibacter 

asiaticus 

Pelz-Stelinski, K.S., Rogers, M.E. Entomology and Nematology Department, UF-IFAS Citrus 

Research and Education Center, Lake Alfred, FL, USA 

The Asian citrus psyllid, Diaphorina citri Kuwayama, which transmits the bacteria Candidatus 

Liberibacter asiaticus (Liberibacter), is responsible for the spread of citrus greening disease, or 

huanglongbing (HLB), throughout most of the world’s citrus-producing regions. Understanding 

the relationship between D. citri and Liberibacter is critical for the development of psyllid and 

HLB management programs. As part of a comprehensive study on this vector-pathogen 

interaction, we have conducted a series of studies investigating Liberibacter transmission and 

psyllid fitness. Acquisition was examined by caging healthy psyllid eggs or adults on infected 

sweet orange plants for 1 day to 5 weeks. Similarly, inoculation was examined by enclosing 

infected psyllids on healthy plants for 1-24 days. Real-time PCR detection assays with 

Liberibacter-specific oligonucleotides were performed on DNA extracts from psyllids and plants 

to determine whether the bacterium was present. Our findings indicate that transmission of 

Liberibacter by Florida D. citri populations occurs at a lower rate than previously reported for 

other populations of D. citri and for the African citrus psyllid, Trioza erytreae (Capoor et al., 

1974; Buitendag and von Broembsen, 1993; Pelz-Stelinski et al., 2010). Acquisition of 

Liberibacter was greatest in nymphs reared on infected plants, while a smaller percentage of 

psyllids acquired the bacterium during adult feeding. Inoculation experiments indicated that 

approximately 10% of citrus plants developed HLB within one year after exposure to a single 

infected psyllid. The results of our studies also suggested that transmission of Liberibacter is 

mediated by the effect of temperature. Liberibacter infection played an important role in the 

fitness of D. citri, resulting in greater reproductive output and decreased survival, and suggesting 

that a trade-off may exist between these life-history traits in response to Liberibacter infection. 

References 

Buitendag, C.H., von Broembsen, L.A. 1993. Living with citrus greening in South Africa. Citrus 

Journal 3:29-32. 

Capoor, S.P., Rao, D.G., Viswanath, S.M. 1974. Greening disease of citrus in the Deccan Trap 

Country and its relationship with the vector, Diaphorina citri Kuwayama. Proceedings of 

the Sixth Conference of the International Organization of Citrus Virologists. p. 43-49. 

Inoue, H., Ohnishi, J., Ito, T., Tomimura, K., Miyata, S., Iwanami, T., Ashihara, W. 2009. 

Enhanced proliferation and efficient transmission of Candidatus Liberibacter asiaticus by 

adult Diaphorina citri after acquisition feeding in the nymphal stage. Annals of Applied 

Biology 155:29-36. 

Pelz-Stelinski, K.S., Brlansky, R.H., Ebert, T.A., Rogers, M.E. 2010. Transmission parameters 

for Candidatus Liberibacter asiaticus by the Asian citrus psyllid, Diaphorina citri 

Kuwayama. Journal of Economic Entomology 103:1531-1541. 


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3.5 Seasonal Changes in Numbers of Asian Citrus Psyllids Carrying Candidatus 

Liberibacter asiaticus 

Ebert, T.A., Brlansky, R.H., Rogers, M.E. UF-IFAS Citrus Research and Education Center, 

Lake Alfred, FL, USA 

In 2008, we began collecting Asian citrus psyllids from five commercial groves in central 

Florida. Sampling in Homestead (southern Florida) began in 2009. DNA from over 

70,000 psyllids was extracted and tested for Liberibacter using RTQ-PCR methods. Seasonal 

variability was very high at all groves. Even at Homestead where all the trees were infected and 

there was no psyllid control, the fraction of psyllids carrying Liberibacter could double or 

decline by half from one month to the next. Similar variability was observed at all groves. There 

was no obvious pattern to this variability. It appears that the fraction of psyllids carrying 

Liberibacter was greater in cooler months. However, some psyllids always carried Liberibacter, 

and increases in the number of psyllids carrying Liberibacter could happen at any time. The 

grove at Arcadia was one of the places where Liberibacter was detected early in the scouting 

efforts to document the spread of this bacterium in Florida. The grove at Arcadia has an 

aggressive tree removal program and a regular scouting program. Less than 1.4% of the psyllids 

at Arcadia carry Liberibacter compared to over 15% at Lake Alfred and over 70% at Homestead. 

This may demonstrate the potential effectiveness of aggressive management programs at 

reducing the rate at which the disease can spread. 


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3.6 Understanding Diaphorina citri-Candidatus Liberibacter asiaticus Interactions and 

D. citri Behavior for Managing Huanglongbing (HLB) in Florida 

Mann, R.S., Pelz-Stelinski, K.S., Rouseff, R.L., Stelinski, L.L. UF-IFAS Citrus Research and 

Education Center, Lake Alfred, FL, USA 

We have been investigating how infection of Asian citrus psyllid (ACP) by Candidatus 

Liberibacter asiaticus (Ca. Las) affects the behavioral response of the vector to healthy versus 

diseased citrus trees. We have also investigated if Ca. Las is sexually transmitted between adult 

psyllids and if female ACP attracts males with a pheromone. Our investigations to date indicate 

that HLB-infected citrus plants are more attractive to ACP adults than healthy plants. However, 

ACP adults subsequently disperse to healthy plants and make healthy rather than diseased plants 

their final settling preference. Forced mating between Ca. Las-infected males and healthy 

females showed that Ca. Las is sexually transmitted from Ca. Las-infected male psyllids to 

healthy females but not from infected females to healthy males or among psyllids of the same 

sex. Females that acquired Ca. Las from males during mating were also able to transmit the 

bacteria transovarially. The whole body cuticular extract from female ACP adults was attractive 

to male ACP in laboratory and field bioassays suggesting that female ACP produces a 

pheromone to attract males. Field bioassays with whole body male and female ACP cuticular 

extracts suggested an aggregative function of the pheromone. Our ongoing research efforts 

include identification of chemical profiles of healthy and HLB-infected plants to determine the 

reasons for differential behavioral preferences for healthy and diseased plants, how sexual 

transmission of Ca. Las impacts spread of HLB, and identification of the putative ACP 

pheromone to improve management of the ACP and HLB complex in Florida. 


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3.7 Effects of Soil-Applied and Foliar-Applied Insecticides on Asian Citrus Psyllid 

(Diaphorina citri) Feeding Behavior and Their Possible Implication for HLB Transmission 

Serikawa, R.H. 1 , Okuma, D.M. 1 , Backus, E.A. 2 , Rogers, M.E. 1 


2 USDA, Agricultural Research Service, Parlier, CA, USA 

Application of insecticides to reduce psyllid (Diaphorina citri Kuwayama) populations is one of 

the primary methods used for ‘huanglongbing’ (HLB) management. However, it is unknown 

whether insecticide application can disrupt psyllid feeding behaviors associated with pathogen 

acquisition and inoculation before insecticide toxicity kills the insect. In this research, Electrical 

Penetration Graph (EPG) technology was used to study the feeding behaviors of D. citri and 

pathogen acquisition and inoculation in citrus under seven different insecticide treatments: 

aldicarb (Temik 15 G), chlorpyrifos (Lorsban 4 E), fenpropathrin (Danitol 2.4 EC), imidacloprid 

(Provado 1.6 F and Admire Pro 4.6 F), spinetoram (Delegate WG), and spirotetramat (Movento 

240 SC). Each insecticide was evaluated individually during 12 hours of recording. Foliar 

insecticides were fresh-applied, and soil-applied insecticides were applied 15 days prior to the 

experiments. Individual psyllids were wired and their feeding behavior recorded. Non-sequential 

feeding parameters for each treatment were compared using ANOVA. Chlorpyrifos, 

fenpropathrin, imidacloprid, and spinetoram provided the best results in disturbing the psyllid 

feeding, possibly avoiding pathogen acquisition and inoculation. In contrast, aldicarb and 

spirotetramat did not significantly disrupt psyllid feeding, possibly allowing HLB pathogen 

acquisition and inoculation. These results provide important information for improvement of 

HLB management through insecticide use. 


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3.8 Effect of Insecticides and Mineral Oil on Probing Behavior of Diaphorina citri 

Kuwayama (Hemiptera: Psyllidae) in Citrus 

de Miranda, M.P. 1 , Felippe, M.R. 1 , Garcia, R.B. 1 , Yamamoto, P.T. 2 , Lopes, J.R.S. 2 

1 Fundecitrus, Araraquara, Brazil 

2 ESALQ/Universidade de São Paulo, Piracicaba, Brazil 

This research was carried out to study the probing behavior of D. citri in citrus treated with 

insecticides and mineral oil using the Electrical Penetration Graph (EPG/DC system) technique. 

We used two application methods: spray (foliar application) and drench. Citrus sinensis 

seedlings with fully expanded leaves were sprayed with the following products and doses (ml 

commercial product/100 L water): Imidacloprid (Provado 200 SC – 20 ml); Dimethoate 

(Dimetoato 500 EC – 80 ml); λ-cyhalothrin (Karate Zeon 50 CS – 15 ml); and mineral oil (M.O.) 

(Argenfrut-RV – 1.5%). Nursery trees (C. sinensis × C. limonia) were treated by drench with the 

following products: Imidacloprid (Confidor 700 WG – 0.5 g c.p./plant) and Thiamethoxam 

(Actara 250 WG – 1 g c.p./plant). At least 15 psyllids per treatment were monitored for 6 hours 

at 1, 7, 14, and 21 days after application (DAA) for spray treatments and at 15, 35, 50, and 95 

DAA for drench applications. Insecticides and M.O. mainly affected the initial phase of probing 

(pathway). In the case of insecticides, D. citri was able to perform probes 1 DAA but it was able 

to reach the phloem only in the evaluation at 15 DAA. However, on plants treated with M.O., 

around 32% of insects were able to reach the phloem 1 DAA. With systemic insecticides, the 

main interference on probing behavior occurred during the phloem phase; phloem sap ingestion, 

as measured by duration of waveform E2, is significantly reduced (approximately 91%), and the 

insect subsequently withdraws the stylets from the plant and rarely restarts a new probe. 


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3.9 A New Detached-Leaf Assay Method to Test the Inoculativity of Psyllids with 

Candidatus Liberibacter asiaticus Associated with Huanglongbing Disease 

Ammar, E.-D., Walter, A., Hall, D.G. Subtropical Insects Research Unit, USHRL, 

USDA-ARS, Fort Pierce, FL, USA 

Nymphs of the Asian citrus psyllid (ACP) Diaphorina citri can acquire Candidatus Liberibacter 

asiaticus (LAS) from huanglongbing (HLB)-infected plants, and both nymphs and adults can 

transmit HLB to healthy citrus. Normally, however, the proportion of Las-infected ACP 

individuals, based on PCR assays, is much higher than the proportion of psyllids that are capable 

of inoculating/transmitting HLB to healthy citrus plants (Inoue et al., 2009; Pelz-Stelinski et al., 

2010). To test their inoculativity with Las, psyllids are usually fed singly or in small groups on 

citrus seedlings and the latter assayed 3-12 months later by PCR. Here, we have developed a new 

‘detached-leaf assay’ method that can potentially speed up Las-inoculativity tests on psyllids 

considerably by shortening this period to 2-3 weeks. Young adults of ACP, that had been reared 

as nymphs on HLB-infected citrus plants, were tested for inoculativity by caging them singly or 

in small groups (5 or 10 adults per leaf) in 50-ml polypropylene tubes on detached healthy 

mid-size leaves of sweet orange (Citrus sinensis (L.)) as described by Ammar and Hall (2011). 

The tubes were kept at 25ºC and 14-hour light per day. HLB-infected ACP adults were caged on 

these detached healthy leaves for 7 days before being transferred to a new healthy test leaf for 

another 7 days. One week following the end of the inoculation access feeding period, the midribs 

of the assay leaves were processed for quantitative PCR (Q-PCR) using Li primers (targeting the 

bacterial 16s gene; Li et al., 2006) as well as the more sensitive LJ900 primers (targeting a phage 

of Las; Morgan et al., 2011). At the end of the experiment, all surviving adults were also 

processed for Q-PCR using both of these primers. 

In two trials in which 10, 5, or 1 infected adult ACP per leaf were fed on detached sweet 

orange leaves for 7 days, percentages of Las-positive leaves were 40, 18.8, and 4.4%, 

respectively, using Li primers, and 60, 40.6, and 11.1%, respectively, using LJ900 primers. Ct 

values using LJ900 primers were much lower than those using Li primers. None of the leaves on 

which healthy ACP adults were similarly fed was Las-positive using either primer. Using Li 

primers, 2/7 of the positive ACP groups gave positive results in the two consecutive test weeks, 

whereas 5/7 gave positive results only in the 1 st or 2 nd week of testing. Adults tested singly, 

however, gave positive results only in the 1 st or 2 nd week. Using LJ900 primers, 6/10 ACP 

groups gave positive results in both test weeks, whereas 4/10 gave positive results only in the 1 st 

or 2 nd week. Also, adults tested singly gave positive results only in weeks 1 or 2. These results 

suggest intermittent Las-inoculation by infected ACP individuals. At the end of the above two 

trials, percentage of Las-positive psyllid adults was 77.8% using Li primers and 94.4% using 

LJ900 primers. The above results on Las-inoculativity of ACP on detached leaves, using the 

more conventional and less sensitive Li primers, are largely comparable to those reported for Las 

transmission when whole citrus seedlings are used for inoculation, e.g., 4-10% transmission by 

individual ACP were recently reported although 40-100% of the psyllids were Las-positive using 

PCR (Pelz-Stelinski et al., 2010). Our results suggest that detached citrus leaves can be used to 

test Las-inoculativity in single or small groups of ACP, which potentially can speed up this 


P a g e | 58 

process from 3-12 months when using whole citrus seedlings for inoculation, to only 2-3 weeks 

when using the detached-leaf method. Using more sensitive primers can increase the usefulness 

of this new ‘detached-leaf assay’ method. 

References 

Ammar, E.-D., Hall, D.G. 2011. A new method for short-term rearing of citrus psyllids and for 

collecting their honeydew excretions. Florida Entomologist 94:340-342. 

Inoue, H., Ohnishi, J., Ito, T., Tomimura, K., Miyata, S., Iwanami, T., Ashihara, W. 2009. 



Biology 155:29-36. 

Li, W.B., Hartung, J.S., Levy, L. 2006. Quantitative real-time PCR for detection and 

identification of Candidatus Liberibacter species associated with citrus huanglongbing. 

Journal of Microbiological Methods 66:104-115. 

Morgan, J.K., Zhou, L., Li, W., Shatters, R.G., Jr., Manjunath, K.L., Duan, Y.-P. 2011. Highly 

sensitive detection by real-time PCR targeting the multiple tandem-repeats of two prophage 

region genes of the citrus huanglongbing disease bacterium, Candidatus Liberibacter 

asiaticus. Proceedings of the 2 nd International Research Conference on Huanglongbing, 

Orlando, FL. January 10-14, 2011. 

Pelz-Stelinski, K.S., Brlansky, R.H., Ebert, T.A., Rogers, M.E. 2010. Transmission parameters 

for Candidatus Liberibacter asiaticus by Asian citrus psyllid (Hemiptera: Psyllidae). 

Journal of Economical Entomology 1031531-1031541. 


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3.10 Preliminary Study of Comparative Acquisition of Candidatus Liberibacter asiaticus 

and Ca. L. americanus by Diaphorina citri Under Different Temperatures 

Barbosa, J.C. 1 , Eckstein, B. 1 , Belasque, J., Jr. 2 , Bergamin Filho, A. 1 



In Brazil, huanglongbing (HLB) is associated with two species of bacteria, Candidatus 

Liberibacter asiaticus (Las) and Ca. L. americanus (Lam). Both species are vectored by the 

psyllid Diaphorina citri Kuwayama. Studies in Brazil have shown an increase of Las-infected 

plants when compared to Lam-infected plants. The reason behind that is unknown, but 

temperature could be influencing the transmission of Lam and Las by psyllids. The objective of 

this study was to verify the influence of temperature in the transmission of Lam and Las by 

psyllids. The transmission of Lam and Las by psyllids was analyzed under three conditions of 

day/night temperatures (20/22°C, 25/27°C, and 30/32°C) in a 12-hour photoperiod. For each 

temperature condition, groups of adult psyllids were caged on branches of Lam- or Las-infected 

citrus plants for an acquisition access period of 4 days. Therefore, for each combination of 

temperature and bacterium species, 10 psyllids were collected from infected branches and caged 

on citrus healthy plants (Citrus limonia Osbeck) for 21 days. After that, insects were collected 

and individually analyzed by PCR to assess the acquisition of Lam and Las. In this study, the 

acquisition efficiency of Lam by psyllids under 20/22°C was 10%, while acquisitions under 

25/27°C and 30/32°C were not verified. The acquisition efficiency of Las under 20/22°C and 

25/27°C was 25%, while under 30/32°C was 12.5%. These results are preliminary and further 

studies are necessary for a better understanding about the influence of temperature on the 

transmission of Ca. Liberibacter spp. 


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3.11 Host Range of Diaphorina citri Kuwayama and Leuronota fagarae on Citrus and 

Zanthoxylum spp. 

Russell, D.N. 1 , Halbert, S.E. 1 , Roberts, P.D. 2 

1 Florida Department of Agriculture and Consumer Services, Division of Plant Industry, 


2 UF-IFAS Southwest Florida Research and Education Center, Immokalee, FL, USA 

Diaphorina citri Kuwayama is one of the vectors of huanglongbing (HLB) pathogens. 

Occasional colonies of D. citri have been observed on Zanthoxylum fagara, but D. citri is not 

reported to complete development on any species of Zanthoxylum. Leuronota fagarae 

Burckhardt, described from South America, showed up in Florida in 2001 on Z. fagara. The 

objective of this study was to determine whether D. citri could develop on species of 

Zanthoxylum, and whether L. fagarae could develop on citrus or on other species of 

Zanthoxylum. We also examined whether adults could feed on the plants long enough to acquire 

or transmit the HLB pathogen. Adults were caged on plants and observed each day to determine 

viability and presence of nymphs. Results indicate that D. citri produced offspring only on citrus, 

and L. fagarae produced offspring only on Z. fagara. Diaphorina citri adults lived longer on 

citrus and Zanthoxylum clava-herculis than on Zanthoxylum coriaceum, Zanthoxylum flavum, or 

Z. fagara. However, some of the adults on each of these plants lived as long as 5 days. Leuronota 

fagarae adults lived 30 days or more on Z. coriaceum, Z. clava-herculis, Murraya paniculata, 

and Z. fagara, but average survival on citrus and Z. flavum was only 2-7.5 days. D. citri could 

feed on any of these plants long enough to transmit HLB pathogens. Any HLB infection 

probably would be a dead end, except possibly in the case of Z. fagara. Susceptibility of 

Zanthoxylum to HLB and vector capabilities of L. fagarae are under investigation. 


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3.12 Abundance of Diaphorina citri (Hemiptera: Psyllidae) in Orange Jasmine and 

Backyard Citrus of Yucatán, Mexico 

Lozano-Contreras, M. 1 , Jasso-Argumedo, J. 1 , Morales-Koyoc, D. 1 , Jasso-Laucirica, T. 1 , 

González-Hernández, A. 2 , López-Arroyo, J.I. 3 

1 INIFAP, Centro de Investigación Regional del Sureste. Mérida, Yuc., México 

2 UANL, Facultad de Ciencias Biológicas. San Nicolás de los Garza, N.L., México 

3 INIFAP-Centro Regional de Investigaciones del Noreste. Río Bravo, Tam., México 

lozano.monica@inifap.gob.mx 

Samplings of the Asian citrus psyllid (ACP) and its natural enemies were performed in 

52 localities distributed across 42 municipalities in the state of Yucatán, Mexico. Adult 

population of ACP in orange jasmine ranged from 0 to 8.56 per tap; meanwhile, in citrus, the 

ACP densities ranged from 0 to 1.4 per tap. The range of ACP population densities in young 

shoots of orange jasmine was 0-13.4; in citrus, the densities ranged from 0 to 7.2. ACP 

infestation in orange jasmine was 72.4%; in backyard citrus, the highest infestation was 34.4%. 

The natural enemies attacking D. citri were Olla v. nigrum, Cycloneda sanguinea, Ceraeochrysa 

spp., Chrysoperla spp., and Tamarixia radiata. Abundance of natural enemies was 

approximately 3:1 higher in natural jasmine than in backyard citrus. In both types of plants, the 

most abundant beneficial insect was Cycloneda sanguinea followed by Ceraeochrysa sp., Olla 

v-nigrum, and Chrysoperla sp. Parasitism by Tamarixia radiata ranged from 0 to 31% in orange 

jasmine and 0 to 4.5% in backyard citrus. 


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3.13 Difference of Gender and Effect of Photoperiod on Asian Citrus Psyllid Feeding 

Behavior 

Okuma, D.M., Serikawa, R.H., Rogers, M.E. UF-IFAS Citrus Research and Education Center, 

Lake Alfred, FL, USA 

The huanglongbing (HLB) pathogen is vectored by the Asian Citrus Psyllid, Diaphorina citri 

Kuwayama (Hemiptera: Psyllidae), through its feeding. However, detailed information regarding 

gender and photoperiod on the psyllid’s feeding behavior has never been provided. Two 

experiments were conducted using an electrical penetration graph (EPG) monitor to provide 

detailed examination of psyllid feeding behavior. In both experiments, adult psyllids 5 to 10 days 

old were selected and sexed using magnifying lens. Recordings were made for 12 hours using the 

abaxial side of a sweet orange young leaf. In the first experiment, the difference in feeding 

behaviors between female and male psyllids was examined. In the second experiment, the 

influence of photoperiod (light/dark) on psyllid feeding behavior was examined. Non-sequential 

feeding parameters were analyzed by ANOVA (PROC GLIMMIX, SAS Institute, 2001). The 

results of those studies will be presented and discussed in terms of developing a better 

understanding of D. citri feeding behavior, and further development of more efficient methods of 

monitoring and management in order to protect citrus from HLB infection. 


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3.14 Seasonal Abundance of Diaphorina citri (Hemiptera: Psyllidae) and Natural Enemies 

in Citrus Groves of Yucatán, Mexico 

Jasso-Argumedo, J. 1 , Lozano-Contreras, M. 1 , Barroso-Aké, H. 1 , López-Arroyo, J.I. 2 


2 INIFAP-Centro Regional de Investigaciones del Noreste. Río Bravo, Tam., México 

jasso.juan@inifap.gob.mx 

Diaphorina citri Kuwayama (Hemiptera: Psyllidae), the Asian citrus psyllid (ACP), has been 

present in the Peninsula of Yucatán, Mexico, since 2002; however, in spite of its distribution in 

the area and the occurrence of infective specimens in neighboring countries, the first plant 

infected by Ca. Liberibacter spp. was not found until July of 2009 in Tizimín, Yucatán, México. 

The objective of this study was to determine the seasonal abundance of D. citri in 14 groves of 

Persian lime and sweet orange. In young irrigated orange trees, the abundance of D. citri was 

constant throughout the whole year with densities in the range of 0-1.8 adults per tap; pest 

infestation ranged from 10 to 30%, though it was detected at a peak of 60%. In mature orange 

trees growing with scarce water during the dry season, the highest abundance of ACP was 

0.2 adults per tap, the average abundance during the year was 0.02 adults, and pest infestation 

ranged from 10 to 30%. In Persian lime groves, the abundance of D. citri ranged from 0 to 

1.56 adults per tap, while pest infestation was 21.05% on average per year. The most abundant 

beneficial insects were Cycloneda sanguinea followed by Chrysoperla sp. Olla v-nigrum and 

Ceraeochrysa sp. 


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3.15 Host Plants of Psyllids in South Texas 

Thomas, D.B. USDA-ARS, Kika de la Garza Subtropical Agriculture Research Center, 

Weslaco, TX, USA 

South Texas, an important citrus producing center, is home to an array of psyllid species which 

now include the Asian citrus psyllid, Diaphorina citri Kuwayama. Although psyllids typically 

breed on a narrow range of related plant species, the adults will often feed on a wider range of 

host plants (Hodkinson, 1974). To date, none of the dozen or so species native to south Texas 

have been found to breed on Citrus, but the adults of several species are commonly found in 

Citrus groves, especially Leuronota maculata (Crawford), Trioza diospyri (Ashmead), 

Heteropsylla texana (Crawford), and H. mimosae (Crawford). In addition to Citrus, L. maculata 

is also commonly encountered on torchwood, a rutaceous plant native to Texas. Although the 

breeding host for this psyllid is reported to be hackberry (Celtis spp.) (Hodkinson, 1988), a 

closely related species, L. fagarae Burkhardt, breeds on Rutaceae (Halbert and Manjunath, 

2004). In contrast, the gall and lerp forming hackberry psyllids (4 species) (see Yang and Mitter, 

1993) and willow psyllids (2 species) seem to keep to their breeding host plants as adults. This 

alternate feeding host behavior is of obvious significance because, aside from the implications 

for enhanced survival, such psyllids could spread the HLB bacterium to their regular hosts and 

thus create a reservoir of the disease agent in non-crop plants. Likewise, D. citri is often found 

on non-rutaceous plants, including hackberry. Given that the hackberry psyllid visits Citrus and 

that the citrus psyllid visits hackberry, that ubiquitous tree may be a candidate for screening as an 

HLB reservoir. The only non-rutaceous plant on which D. citri has been found breeding in Texas 

is the edible fig, Ficus carica (Moraceae). In the summer of 2010, we discovered D. citri nymphs 

on a dooryard fig tree. Fig has its own species of psyllid, Homatoma ficus (L.), but both adults 

and nymphs of that species are easily distinguished. Our colleague Jesus De Leon confirmed our 

identification of the nymphs with a molecular marker (in ref. and abstract this symposium). In a 

follow-up greenhouse experiment, we reared D. citri through to adults on potted fig plants. 

Significantly, the only other record for breeding by D. citri, on a non-rutaceous host plant, was 

for the jackfruit Artocarpus, also in the family Moraceae (Shivanker et al., 2000). Although, 

Peña et al. (2006) were unable to substantiate jackfruit as a host, the possibility of non-rutaceous 

hosts needs to be verified. Movement of D. citri among commercial Citrus and dooryard Citrus 

trees may also be of concern for disease management. In both 2009 and 2010, we saw a striking 

pattern of host usage in south Texas with spring-time peaks in dooryard populations and 

fall/winter peaks in Citrus groves. These peaks were independent of, that is, not driven by 

immature phenology that was closely related to the rainfall-flush cycle. Rather it appeared that 

there was a migration of adults from dooryards into groves in the summer with a reverse 

migration in the winter. Colleagues working with the potato psylla found large numbers of 

D. citri in potato fields in the first week of January 2009, whereas the rest of the year they were 

no more than incidental. This phenomenon requires more investigation. 


P a g e | 65 

References 

Halbert, S.E., Manjunath, K.L. 2004. Asian citrus psyllids (Sternorrhyncha: Psyllidae) and 

greening disease of Citrus: A literature review and assessment of risk in Florida. Florida 

Entomologist 87:330-353. 

Hodkinson, I.D. 1974. The biology of the Psylloidea (Hemiptera): a review. Bulletin of 

Entomological Research 64:325-339. 

Hodkinson, I.D. 1988. The nearctic psylloidea (Insecta: Hemiptera): an annotated checklist. 

Journal of Natural History 22:1174-1243. 

Peña, J.E., Mannion, C.M., Ulmer, B.J., Halbert, S.E. 2006. Jackfruit, Artocarpus heterophylus, 

is not a host of Diaphorina citri (Homoptera: Psyllidae) in Florida. Florida Entomologist 

89:412-413. 

Shivanker, V.J., Rao, C.N., Singh, S. 2000. Studies on citrus psylla Diaphorina citri Kuwayama: 

a review. Agricultural Review (India) 21:199-204. 

Yang, M.M., Mitter, C. 1993. Biosystematics of hackberry psyllids (Pachypsylla) and the 

evolution of gall and lerp formation in psyllids (Homoptera: Psylloidea): a preliminary 

report. In: Price et al. (eds.). The Ecology of Gall Forming Insects. USDA Forest Service, 

Washington, DC, p. 172-185. 


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Session 4: 

Survey, Detection and 

Diagnosis 


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4.1 Development and Reactivity of Polyclonal Antibodies Based on OMP Sequences of 

Candidatus Liberibacter asiaticus* 

Coletta-Filho, H.D. 1 , Peroni, L.A. 2 , De Souza, A.A. 1 , Takita, M.A. 1 , Stach-Machado, D.R. 2 

1 Centro de Citricultura Sylvio Moreira, IAC, Cordeiropolis, Brazil 

2 UNICAMP, Campinas, Brazil 

The use of serological approaches on Candidatus Liberibacter asiaticus (Las) research and 

diagnosis is limited by the absence of good antibodies as a consequence of uncultured 

characteristic of this bacterium. Polyclonal antibody with specific reaction against Las will be 

useful for both quick and low-cost diagnosis test, as well as immunomicroscopy studies. Here, 

we are showing results of polyclonal antibodies (PAs) produced from nine immunogenic 

synthetic peptides whose amino acid sequences were translated from Las - Outer Membrane 

Protein genomic sequences and selected based on antigenicity and hydrophilicity by 

Antogenicity plot. The antigenic peptides were synthesized by Bio-Synthesis, Inc. (Lewisville, 

TX, USA) with size ranging from 10 to 26 amino acids and were endovenously twice inoculated 

(total of 500 µg) in white rabbits. Seven days after the last injection, each rabbit was bled 

through cardiac puncture, and the serum titer and specificity were determined by indirect 

enzyme-linked immunosorbent assay (ELISA) according to Clark et al. (1986). Eight of nine 

PAs, tested with dilutions from 1:500 to 1:32,000, resulted in a linear pattern of reactivity at 

O.D. of 405 nm, by ELISA, against their antigens (10 µg/ml). So, we tested by ELISA the eight 

preselected PAs to react against citrus asiatic-HLB symptomatic leaves. Four of eight PAs 

recognized Las in the infected leaves at O.D. ranging from 0.456 to 0.669 (1:2,000 dilution) with 

no cross-reaction observed against healthy plants as well as against some bacteria species 

potentially present in citrus plants like Xanthomanas citri pathovars, Methylobacterium sp. 

(Araujo et al., 2002), and Xylella fastidosa. 


Research was supported by FCPRAC contract #061. 

References 

Araujo, W.L., Marcon, J., Maccheroni, W., Jr., van Elsas, J.D., van Vuurde, J.W.L., Azevedo, 

J.L. 2002. Diversity of endophytic bacterial populations and their interaction with Xylella 

fastidiosa in citrus plants. Applied and Environmental Microbiology 68:4906-4914. 

Clark, M.F., Lister, R.M., Bar-Joseph, M. 1986. ELISA techniques, p. 742-766. In: Weissbach, 

H., Weissbach, A. (eds.), Methods in Enzymology, vol. 118. Academic Press, New York, 

820 p. 


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4.2 Development of Single-Chain Antibody Fragments (scFVs) Against Candidatus 

Liberibacter asiaticus by Phage Display 

Yuan, Q. 1 , Jordan, R. 1 , Brlansky, R.H. 2 , Minenkova, O. 3 , Hartung, J.S. 1 

1 USDA ARS Molecular Plant Pathology Laboratory, Beltsville, MD, USA 


3 Sigma tau Pharmaceuticals, Rome, Italy 

Psyllids collected in Florida were assayed individually for Ca. Liberibacter asiaticus (CaLas) by 

q-PCR and extracts that contained >10 8 CaLas/insect were used to immunize mice. mRNA from 

spleens of immunized mice was purified and converted into a cDNA library. Antibody gene 

repertoires were PCR-amplified using 23 primers for the heavy chain variable region (V H ) and 

21 primers for the light chain variable region (V L ). The V H and V L repertoires were joined by 

overlap extension PCR and ligated into the phage vector pKM19 (Pavoni et al., 2007). Forty-five 

clones were picked at random from the library and tested by PCR. The library contained 1.3 × 

10 7 independent clones with full-length scFV inserts. Biopanning of the phage library with plant 

and insect extracts for phage expressing scFV against CaLas was not successful. Therefore, 

rabbit polyclonal antiserum raised against purified outer membrane protein (OMP) of CaLas was 

prepared. Three rounds of immunocapture were done with CaLas from extracts of psyllids using 

Dynabeads coated with sheep anti-rabbit IgG and rabbit anti-OMP serum. In each of 3 cycles of 

affinity selection with purified antigen, about 1.0 × 10 12 phage were used for panning with 1.2 × 

10 6 , 3.4 × 10 7 , and 9.2 × 10 7 phage recovered after the first, second, and third cycles, 

respectively. ELISA showed that scFVs that bound CaLas were enriched in the panned libraries, 

although the development of the ELISA plates was very slow. This could be due to low numbers 

of target CaLas even after immunocapture. Polyclonal antisera are currently being raised in 

rabbits against purified native pilus protein to substitute for OMP in the immunocapture of 

CaLas. 

Reference 

Pavoni, E., et al. 2007. New display vector reduces biological bias for expression of antibodies in 

E. coli. Gene 391:120-129. 


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4.3 Highly Sensitive Detection by Real-Time PCR Targeting the Multiple Tandem Repeats 

of Two Prophage Region Genes of the Citrus Huanglongbing Disease Bacterium, 

Candidatus Liberibacter asiaticus 

Morgan, J.K. 1 , Zhou, L.J. 2 , Shatters, R.G., Jr. 1 , Manjunath, K.L. 3 , Duan, Y.-P. 1 

1 USDA-ARS, U.S. Horticultural Research Laboratory, Fort Pierce, FL, USA 


3 USDA-ARS, National Clonal Germplasm Repository for Citrus and Dates, Riverside, CA, USA 

Candidatus Liberibacter asiaticus is the prevalent Liberibacter associated with citrus 

huanglongbing worldwide. Residing in phloem sieve cells of host plants and vectored by the 

Asian citrus psyllid (Diaphorina citri), this fastidious bacterium lives as a pathogen or symbiont. 

Uneven distribution and variation in bacterial levels within individual hosts emphasize the need 

for sensitive and reliable detection. Here, we have developed real-time PCR using SYBR Green 

1 (LJ900fr) and TaqMan ® (LJ900fpr) methods targeting near identical 132 bp multiple tandem 

repeats of two Ca. L. asiaticus prophage region genes, hyvI and hyvII. Because the copy number 

of these tandem repeats is high (up to 15 combined), targeting these improves detection of Ca. L. 

asiaticus relative to the three-copy 16S rDNA-based USDA APHIS real-time PCR standard, 

‘HLBaspr’. Relative comparative testing of both LJ900fr and LJ900fpr against HLBaspr 

indicated an average reduction in detectable threshold of approximately 9 and 3 cycles from local 

and global samples for the LJ900 methods, respectively. Additionally, multi-sample testing of 

two variable level Ca. L. asiaticus infected citrus trees (R8T1 and R8T3) indicated Ca. L. 

asiaticus positive detection within all samples by both LJ900 methods, whereas HLBaspr 

detected ~50% positive from each tree sample. Furthermore, the hyvI/hyvII repeat sequence is 

demonstrated within the Ca. L. americanus strain. Both LJ900 methods provide a significant 

increase in reliable detection of Ca. L. asiaticus within low and variable level Ca. L. asiaticus 

samples, dramatically improving the detection of the bacterium. 


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4.4 Comparison of Different Extraction and Assay Protocols in Different Laboratories to 

Develop a Standardized Assay for Detection of Huanglongbing-Associated Bacteria from 

Psyllids 

Manjunath, K.L. 1 , Irey, M.S. 2 , Ramadugu, C. 3 , Lee, R.F. 1 , Levesque, C.S. 4 , Brady, B. 4 , Polek, 

M.L. 4 , Lin, H. 5 , Civerolo, E.L. 5 , Afunian, M. 3 , Vidalakis, G. 3,6 

1 National Clonal Germplasm Repository for Citrus and Dates, USDA-ARS, Riverside, CA, USA 

2 U.S. Sugar Corporation, Clewiston, FL, USA 

3 University of California, Riverside, CA, USA 

4 Citrus Research Board, Riverside, CA, USA 

5 San Joaquin Valley Agricultural Sciences Center, USDA-ARS, Parlier, CA, USA 

6 Citrus Clonal Protection Program, Riverside, CA, USA 

Since the first detection of huanglongbing (HLB) in the Western hemisphere in 2004, the disease 

has spread to several countries. Monitoring movement of HLB and early detection, especially in 

areas where HLB has not been reported, is largely dependent on testing for HLB-associated 

bacteria (Candidatus Liberibacter spp.) in psyllid vectors. Since the HLB-associated pathogen 

can be detected in psyllids well before the onset of disease symptoms in plants, the psyllid 

testing is now used extensively in many laboratories. Since several methods of psyllid DNA 

extractions and qPCR assays are practiced, it would be desirable for reliability, 

cost-effectiveness, and accuracy to use methods that give comparable results. Field collected 

psyllids exhibit both seasonality and very low levels of infection and hence are not suitable for 

developing standardized methods. A greenhouse-maintained psyllid colony monitored for several 

months was consistently shown to contain infected psyllids and used for this study. About 

1500 single psyllids were tested simultaneously in five laboratories in Florida and California by 

employing a number of different extraction methods and tested for the HLB-associated bacteria 

by standard quantitative PCR. A total of 16 lab/extraction/qPCR combinations were conducted. 

Most of the assays resulted in a narrow range of infection of 6-8%; however, there was a range 

of infection rates of 1-10%, indicating the need for further evaluation of the methods used in 

different laboratories. The strengths and weaknesses of the different methods and minimum 

requirements for conducting a reliable psyllid HLB testing assay are discussed. 


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4.5 Assessment of Various Spectroscopic Techniques for Detection of HLB 

Poole, G.H. 1 , Hawkins, S.A. 2 , Windham, W.R. 2 , Heitschmidt, J. 2 , Albano, J.P. 1 , Park, B. 2 , 

Lawrence, K.C. 2 , Gottwald, T.R. 1 

1 USDA-ARS, Fort Pierce, FL, USA 

2 USDA-ARS, Athens, GA, USA 

Although real-time Polymerase Chain Reaction (PCR) is an accepted method of determining an 

HLB infection in citrus, this technique can be time consuming and expensive. In this study, 

several spectral techniques were tested for their ability to rapidly screen leaf samples for HLB. In 

the first trial, 179 HLB positive and negative leaf samples were collected from greenhouse- and 

farm-grown orange and grapefruit trees. In the second trial, 238 leaves were collected from 

greenhouse-grown grapefruit trees which had one of several diseases (HLB, canker, CTV, 

CLRV, or CPsV), one of several nutrient deficiencies (copper, iron, magnesium, manganese, 

zinc, or “water only”), and control samples. In both trials, the samples were individually 

analyzed with hyperspectral imaging (HSI, 400-1000 nm), fourier transform infrared-attenuated 

total reflection spectroscopy (FTIR-ATR, 700-1765 cm -1 ), near-infrared reflectance spectroscopy 

(NIRS, 450-2500 nm), and real-time PCR. The data from HSI was not promising, with multiple 

false positive and negative results in both trials. FTIR-ATR did well in trial 1 with an error rate 

under 5%, but had markedly higher false positive issues in trial 2. NIRS also did well in trial 1, 

but in trial 2 as with the FTIR-ATR analysis, the predictor models suffered from false positives. 

After combining the datasets and removing the visible portion of the spectrum (450-700 nm), the 

accuracy of the NIRS improved and was in agreement with the data from the first trial. Overall, 

there is a good possibility of using a spectral analysis of leaf tissue as an alternative, rapid, and 

inexpensive assay for HLB infection. 


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4.6 Seasonal Variability in HLB Testing Data in Plant and Psyllid Samples in Florida 

Irey, M.S. 1 , Gast, T. 2 , Cote, J. 1 , Gadea, P. 1 , Santiago, O. 2 , Briefman, L. 2 , Graham, J.H. 3 


2 Southern Gardens Citrus Corporation, Clewiston, FL, USA 


Southern Gardens Diagnostic Laboratory (SGDL) is a disease testing laboratory that has been 

testing grower samples for huanglongbing (HLB) by real-time PCR (qPCR) since October, 2006. 

Since its inception, the lab has tested over 165,000 plant samples and over 6,000 individual 

psyllid samples. Samples submitted to the laboratory come from growers throughout the state 

and are collected and delivered to the laboratory every month of the year. A database is 

maintained for all samples, and the data contained within the database has proven to be a 

valuable resource to study the seasonal and temporal differences in HLB testing results. Both the 

quantity of samples received and the results of the samples that are received can be used to draw 

conclusions as to the temporal incidence of symptoms, type of symptoms that test positive for 

HLB by qPCR, and to determine the best time of the year to sample for HLB in plants and 

psyllids. In plants, most symptomatic samples are received August through January and the 

greatest percentage of HLB positive samples is found July through January. During the less 

optimum times of year, only blotchy mottle symptoms test positive for HLB, whereas during the 

optimum time of the year, many different types of symptoms test positive. With respect to the 

testing of psyllids, more psyllids test positive for HLB during the months of October through 

May. However, the incidence of psyllids is greatest April through November. So the optimum 

time to detect HLB in psyllids may not be during the time of year when the psyllid is the most 

prevalent. 


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4.7 Survey to Estimate the Rate of HLB Infection in Florida Citrus Groves 

Irey, M.S. 1 , Morris, R.A. 2 , Estes, M. 3 



3 FDACS, DPI, Gainesville, FL, USA 

The incidence of HLB infection in Florida groves is needed to assess the magnitude of the HLB 

problem, assist in tracking disease management efforts, and to enable better forecasts of future 

infection rates, fruit production, and prices. In 2008 and 2009, two independent surveys were 

conducted to determine infection levels across the state. One survey was a collaborative project 

between IFAS, The National Agricultural Statistics Service’s Florida field office (NASS), and 

Division of Plant Industry (DPI). The second survey was conducted by U.S. Sugar Corporation 

(USSC). The IFAS/NASS/DPI surveys were based on survey forms that were mailed to 3,037 

Florida growers, whereas the USSC survey was based on GPS-based tree infection data provided 

by scouting companies and growers. For 2008, the incidence of HLB was estimated to be 1.6% 

and 2.3% for the IFAS/NASS/DPI and the USSC surveys, respectively. The infection rates were 

highest in the Southern and Indian River production regions of the state. Due to a relatively low 

response rate in 2009, the data from both surveys groups were combined. The combined surveys 

showed a statewide HLB infection rate for oranges of 6.4%. The Southern, Indian River, and the 

southern portion of the Central Regions had the highest infection rates, while most of the 

Central, Western, and Northern regions had infection rates below 1.0%. 


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4.8 Two Survey Protocols to Detect Newly Introduced HLB and Other Exotic Pathogens 

and Pests 

Gottwald, T.R. 1 , Riley, T.D. 2 , Irey, M.S. 3 , Parnell, S.R. 4 , Hall, D.G. 1 


2 USDA, APHIS, Plant Protection and Quarantine, Citrus Health Response Program, Orlando, 

FL, USA 

3 Southern Gardens, U.S. Sugar Corporation, Clewiston, FL, USA 

4 Biomathematics and Bioinformatics, Rothamsted Research, Harpenden, AL5 2JQ, UK 

Detection of initial introductions of exotic pathogens or pests is difficult because they occur in 

very low incidence. Optimal probability of eradication/mitigation depends on early detection 

prior to subsequent spread. The earlier the detection, the more likely the pathogen can be 

eliminated or its buildup can be slowed, lessening disease impact often for multiple years. 

Finding point introductions across a broad geographic landscape of mixed commercial and 

residential areas requires substantial manpower and fiscal resources. Point introductions often go 

undetected for long periods until their incidence exceeds the lower threshold of the sampling 

methods deployed. One means of ‘finding a needle in a haystack’ is to utilize epidemiological 

characteristics and knowledge of probable pathways of the pathogen/pest to parse the 

geographical area into smaller areas that can be prioritized by potential risk of introduction. Two 

survey methods for early detection are presented and both can be applied to various hosts, 

pathogens, vectors, and insect pests. The first method predicts the most likely locations in a 

given geographic area for introduction and is based on U.S. Census and international travel data, 

combined with knowledge of the pathosystem’s epidemiology. The method models and 

generates a risk index that predicts new introductions and can be deployed in areas where HLB is 

not known to exist. The second method sweeps large areas multiple times per year to estimate 

HLB incidence and ACP populations, generating a GIS-based risk index map via 

spatiotemporally modeling that can be used to target area-wide disease and vector 

control/mitigation. 


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4.9 Distribution of Candidatus Liberibacter Americanus and Ca. L. asiaticus in Foliage of 

Naturally Infected Citrus Trees 

Sousa, M.C. 1 , Lemos, M.V.F. 2 , Frare G.F. 3 , Santos, M.A. 1 , Lopes, S.A. 1 


2 FCAV/Universidade Estadual Paulista, Jaboticabal, Brazil 


Knowledge on Liberibacter distribution and titer within the foliage of a tree canopy may indicate 

the potential for that particular tree to act as a source of inoculum. Eight 5-year-old 

Valencia/Rangpur lime trees, four naturally infected with Ca. L. americanus (Lam) and four with 

Ca. L. asiaticus (Las), and exhibiting leaf mottling at the top of one of three main branches, were 

selected in an orchard near Avaré, SP. In total, 219 leaf samples (10 leaves per sample) were 

collected from 9 to 12 parts of each symptomatic branch and 9 each from two asymptomatic 

branches of each tree. The samples were processed for DNA extraction and analyzed by 

conventional (cPCR simplex and cPCR duplex), nested, and SYBR green quantitative PCR 

(qPCR). Branch positioning and sampling sites were mapped. Lam was detected in 

18 symptomatic and 3 asymptomatic samples and Las in 6 symptomatic and 6 asymptomatic 

samples. Good agreement for Liberibacter detection was obtained among all four PCR protocols 

tested. Cycle threshold values and bacterial titers (estimated in number of cells per gram of 

tissue) averaged, respectively, 30.2 and 2.45 × 10 5 for Lam and 28.1 and 5.2 × 10 6 for Las. The 

greater distribution and higher titers observed in Las-infected trees confirms why Las has spread 

much faster than Lam in Brazil. 


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4.10 A Perspective on the Activities of Texas HLB Diagnostic Laboratory 

Kunta, M., da Graça, J.V., Sétamou, M., Skaria, M. Texas A&M University-Kingsville, Citrus 

Center, Weslaco, TX, USA 

The Citrus Center diagnostic laboratory received USDA-APHIS certified status to perform 

APHIS PPQ validated diagnostic tests since August 2008. It has the capacity to test 

approximately 500 samples a day and extract DNA from 900 psyllids or 400 plants per week. 

Thus far, about 25,000 psyllid samples and 8,000 plant samples have been tested. These were 

collected by USDA-APHIS, Texas Department of Agriculture, and the Citrus Center's citrus 

commodity survey personnel. Samples that produce a Ct value less than 37 in qPCR were sent to 

the PPQ molecular diagnostic laboratory (MDL) in Beltsville, MD, for confirmation. Two citrus 

samples, from Hidalgo and Harris counties, respectively, showed high borderline Ct values; 

however, none produced an amplicon in conventional PCR tests. The results were further 

confirmed by MDL and the samples were designated as ‘inconclusive’ since positive 

conventional PCR and nucleotide sequencing information are necessary to confirm the presence 

of HLB. Moreover, re-samples from these plants showed negative results in qPCR tests. 

Forty-four orange jasmine samples yielded Ct values between 33 and 37; some of which were 

confirmed by MDL and were also designated as ‘inconclusive.’ We have performed qPCR, 

conventional PCR, nested PCR, and PCR coupled with restriction digestion on 440 orange 

jasmine leaf samples collected from 55 plants and psyllids in the vicinity of these plants. 

Twenty-four samples produced borderline Ct values, but none of them resulted in a conventional 

PCR band. 


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4.11 Two New Real-Time PCR-Based Surveillance Systems for Candidatus Liberibacter 

Species Detection 

Lin, H. 1 , Bai, Y. 2 , Civerolo, E.L. 1 

1 Crop Diseases, Pests and Genetics Unit USDA-ARS, Parlier, CA, USA 

2 Guangxi Academy of Agricultural Sciences, Nanning, Guangxi, China 

We developed two novel surveillance systems for Candidatus Liberibacter (CL) species 

detection and identification. The first system is called “single tube dual primer Taq-Man PCR” 

(STDP). The procedure involves two sequential rounds of PCR using the CL asiaticus 

species-specific outer and inner primer pairs. Different annealing temperatures between inner 

and outer pair primers allow both the first and the second rounds of PCR to be performed 

sequentially in the same closed tube. The sensitivity of the dual primer system is comparable to 

the conventional two-tube nested PCR, but the STDP eliminates the potential risk of cross 

contaminations commonly associated with conventional nested PCR. The second system is 

fluorescent dye-based real-time PCR. This diagnostic system employs a pair of Liberibacter 

universal primers flanking a sequence region homologous to the known CL species; CL asiaticus 

(Las), CL africanus (Laf), CL americanus (Lam), and CL solanacearum (Lso). This primer 

system is able to quantitatively detect all four Liberibacter species, but sequence variation among 

each species amplicon can be distinguished by performing a post PCR high resolution melting 

curve analysis (HRMCA). Both systems reported here are robust and cost-effective for reliable 

detection, quantitation, and identification of Liberibacter species in plants and insects and 

provide high throughput capabilities suitable for large-scale, year-round quarantine screening 

and epidemiological studies. 

References 

Lin, H., Chen, C., Doddapaneni, H., Duan, Y.-P., Civerolo, E.L., Bai, X.J., Zhao, X.L. 2010. 

A new diagnostic system for ultra sensitive and specific detection and quantitation of 

Candidatus Liberibacter asiaticus, the bacterium associated with citrus huanglongbing. 


Lin, H., Liao, H., Bai, Y., Civerolo, E.L. 2010. A new molecular diagnostic tool for 

quantitatively detecting and genotyping Candidatus Liberibacter species. Phytopathology 

100:S72. 


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4.12 Detection of Candidatus Liberibacter solanacearum in Potato Psyllid Isolated from 

Sticky Traps 

Kwok, K. 1 , Levesque, C.S. 1 , Manjunath, K.L. 2 , Irey, M.S. 3 , Polek, M.L. 1 

1 Citrus Research Board, Visalia/Riverside, CA, USA 

2 USDA, ARS National Germplasm Repository, Riverside, CA, USA 


The Citrus Research Board established an Operations Department in response to the 2008 

discovery of the Asian Citrus Psyllid (ACP) in California and the threat of huanglongbing (HLB) 

disease associated with this vector. The Operations Department is composed of Field, Data 

Management, and Laboratory Divisions dedicated to ACP trapping throughout the state’s 

commercial citrus, diagnostic testing of plant and insect samples using Quantitative Polymerase 

Chain Reaction (Q-PCR) to detect Liberibacter asiaticus (Las) DNA (the causative agent of HLB 

in Florida), and establishing a website for the dissemination of HLB-related information. In 

Florida, it has been determined that Las target DNA is undetectable in DNA extracted from ACP 

after 10 days on a sticky trap. Since CRB traps are serviced bimonthly, it is possible that positive 

ACP could go undetected if Las DNA is unstable in California’s climatic conditions and we 

depend on testing trapped animals for detection. Because we cannot work with the live causative 

agents of HLB in California, we have used the potato psyllid Bactericerca cockerelli infected 

with Liberibacter solanacearum (Lsol) as a surrogate system. ACP populations in Florida acquire 

Las at levels of 30-40%, unlike potato psyllids in California that acquire Lsol to very high levels, 

with essentially 100% of the population becoming infected. Based on Q-PCR, the level of 

infection in the individual psyllid is also dramatically higher. In two experiments conducted this 

spring and summer in Riverside, Lsol DNA was detected in up to 85% of dead psyllids retrieved 

after 50 days on traps. 

References 

Bonani, J.P., Fereres, A., Garzo, E., Miranda, M.P., Appezzato-Da-Gloria, B., Lopes, J.R.S. 

2010. Characterization of electrical penetration graphs of the Asian citrus psyllid, 

Diaphorina citri, in sweet orange seedlings. Entomologia Experimentalis et Applicata 

134:35-49. 

Inoue1, H., Ohnishi, J., Ito, T., Tomimura, K., Miyata, S., Iwanami. T., Ashihara, W. 2009. 



Biology 155:29-36. 

Li, W., Abad, J.A., French-Monar, R.D., Rascoe, J., Wen, A., Gudmestad, N.C., Secor, G.A., 

Lee, I., Duan, Y., Levy, L. 2009. Multiplex real-time PCR for detection, identification and 

quantification of Candidatus Liberibacter solanacearum in potato plants with zebra chip. 



P a g e | 79 

Liefting, L.W., Sutherland, P.W., Ward, L.I., Paice, K.L., Weir, B.S., Clover, G.R.R. 2009. 

A new Candidatus Liberibacter species associated with diseases of solanaceous crops. 

Plant Disease 93:208-214. 

Secor, G.A., Rivera, V.V., Abad, J.A., Clover, G.R.G., Liefting, L.W. Li, X., De Boer, S.H. 

2009. Association of Candidatus Liberibacter solanacearum with Zebra Chip Disease of 

Potato established by graft and psyllid transmission, electron microscopy, and PCR. Plant 

Disease 93:574-583. 


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4.13 Detection of Candidatus Liberibacter asiaticus (Las) on Yellow Sticky Traps by 

Real-Time PCR 

Irey, M.S. 1 , Gadea, P. 1 , Hall, D.G. 2 


2 USDA-ARS, U.S. Horticultural Research Laboratory, Fort Pierce, FL, USA 

In many areas of the world, surveys to detect the presence of citrus huanglongbing (HLB) prior 

to the discovery of symptomatic plants are carried out by the monitoring of Diaphorina citri 

(ACP) on yellow sticky traps followed by testing of the psyllids for HLB-associated bacteria 

(Las) by real-time PCR (qPCR). In most areas where psyllids are monitored on yellow sticky 

traps, the traps are left in the field for 1-2 weeks after which time the cards are brought to the 

laboratory for visual evaluation for the presence of ACP, and then, if present, the psyllids are 

removed from the cards or the cards are sent to a diagnostic laboratory for subsequent testing. 

The process from beginning to end may take up to a month, depending on the work-flow for the 

group conducting the surveys. Since the testing of psyllids is a destructive process with respect to 

the insect sample, it is hard to validate that a negative qPCR result is indeed negative because 

Las were not present, or instead due to degradation of the bacterial DNA over time on the card. 

In order to determine if time on yellow sticky traps affects qPCR results for the HLB-associated 

bacteria, Las-infected ACP were placed on yellow sticky traps and tested after defined periods of 

time by qPCR. In two separate tests, the incidence of Las-positive ACP declined with increasing 

time on the yellow sticky traps, indicating that the testing of ACP obtained from yellow sticky 

traps may not be the best method to survey for the presence of Las. 


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4.14 Validation of the Starch-Iodine Reaction for Field Pre-Diagnosis of Huanglongbing in 

Citrus of México 

Loredo-Salazar, R.X. 1 , Uribe-Bustamante, A. 1 , Rodríguez-Quibrera, C.G. 1 , Curtí-Díaz, S.A. 1 , 

Alanís-Martínez, E.I. 2 , Velázquez-Monreal, J.J. 3 , López-Arroyo, J.I. 4 

1 INIFAP, Centro de Investigación Regional del Golfo Centro, Veracruz, Ver., México 

2 SENASICA, Dirección General de Sanidad Vegetal, México, D.F. 

3 INIFAP, Centro de Investigación Regional del Pacífico Centro, Guadalajara, Jal., México 

4 INIFAP, Centro de Investigación Regional del Noreste, Río Bravo, Tam., México 

loredo.reyna@inifap.gob.mx 

The objective of this study was to validate the iodine-starch reaction for field diagnostics of 

huanglongbing (HLB) in México. We tested the method in HLB-infected Mexican lime plants, 

Tahiti lime with Wood Pocket, and with Lethal Yellowing, as well as in healthy Murcott 

tangelos. Healthy plants of Mexican and Tahiti limes were used as a control. We compared the 

staining of the chlorotic and green tissue of the same leaf. 100 leaves were used for each 

symptom or citrus species. Presence of Ca. Liberibacter was confirmed by PCR. It was found 

that: 1) The chlorotic tissue of leaves with blotchy mottle, diffuse mottle, and green islands were 

positive in 87, 75, and 70% of the tests, respectively; whereas, the green tissue of the same 

infected leaves only showed positive results in 15, 52, and 58%, in the same order. The use of 

chlorotic tissue of infected leaves in the iodine-starch reaction could yield better results in the 

detection of suspected trees infected with HLB. 2) 100% of the healthy leaves of Mexican lime 

showed very low or no staining, suggesting that it is unlikely to have a false positive. 3) The 

staining of the chlorotic tissue of leaves with Wood Pocket (similar symptoms to those caused by 

HLB) was low or absent, ruling out this disorder as a suspected infection by HLB. 4) The Tahiti 

lime leaves with lethal yellowing and healthy leaves of Murcott tangelo showed high 

concentrations of starch, discarding the method for the field diagnostic in such cases. 


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4.15 Detecting HLB Using NIR Remote Sensing 

Gonzalez-Mora, J. 1 , Dima, C.S. 1 , Irey, M.S. 2 , Ehsani, R. 3 

1 National Robotics Engineering Center, Carnegie Mellon University, Pittsburgh, PA, USA 

2 U.S. Sugar Corporation, Clewiston FL, USA 


Huanglongbing (HLB or citrus greening) is considered one of the most devastating diseases 

affecting citrus crops today. Efficient, accurate, and practical methodologies are necessary for 

detecting this disease. Remote sensing techniques have proven to be an effective approach for 

detecting stress conditions in many different crops. Compared with traditional wet chemical 

diagnosis, remote sensing methods do not require sample preparation and their subsequent 

analysis in a laboratory, resulting in fast and nondestructive techniques. In this paper, we discuss 

the use of the near-infrared reflectance to detect the huanglongbing disease in citrus trees. We 

analyze the spectral responses from an extensive collection of leaf samples to discriminate 

infected trees from healthy samples. Experimental results comparing the performance of 

different feature extraction and classification techniques are provided using data collected in a 

controlled environment and also in field conditions. 

References 

Gonzalez-Mora, J., Vallespi, C., Dima, C.S., Ehsani, R. 2010. HLB detection using hyperspectral 

radiometry. 10 th International Conference on Precision Agriculture, Denver, CO. 

Sankaran, S., Ehsani, R., Dima, C. 2010. Development of ground-based sensor system for 

automated agricultural vehicle to detect diseases in citrus plantations. 10 th International 

Conference on Precision Agriculture, Denver, CO. 


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4.16 Isothermal Detection of Huanglongbing in Psyllids and Citrus Tree Samples 

Russell, P.F., McGowen, N., Bohannon, R. Agdia, Inc., Elkhart, IN, USA 

Huanglongbing (HLB) disease is found throughout Asia, in Brazil, Mexico, the USA, and parts 

of Africa and has seriously affected citrus production in many regions. Three species of the 

causative agent Candidatus Liberibacter, which is vectored by psyllids, have been identified. 

These are Candidatus Liberibacter asiaticus, Candidatus L. americanus, and Candidatus L. 

africanus. 

We report here on two methods for detecting HLB using isothermal nucleic acid 

amplification. Specific detection and identification of the three species is possible using DNA 

purified from infected psyllids and citrus trees as well as using crude extracts prepared from the 

same samples. Plant samples tested include infected citrus trees from South Africa, Brazil, and 

the USA. 


Psyllid extracts were kindly provided by Dr. John Hartung. Citrus leaves infected with 

L. asiaticus, L. americanus, or L. africanus were provided by Dr. John Hartung, Dr. Barry 

Manicom, and Dr. Marcelo Pedreira de Miranda. 

Samples were initially processed using the Norgen Plant/Fungi DNA Isolation Kit (Catalog 

# 26200) to isolate pure DNA for use as comparison controls. Crude extracts were prepared by 

excising leaf midribs and grinding the samples at a 1:5 ratio in Extraction Buffer. Neat or diluted 

extract was then used in the AmplifyRP ® test. Psyllid DNA extracts were used neat or diluted 

serially. 

Detection of the target sequence with Agdia’s isothermal AmplifyRP ® test was done 

according to the standard protocol, using primers and probes targeted to the region surrounding 

the protein chain elongation factor. A single microliter of target sample (purified DNA or crude 

extract) is used for each reaction. Two methods for detection are available: 1) the “exo” test uses 

real-time fluorometric measurement and is complete after 12-15 minutes, and 2) the “nfo” test is 

an endpoint assay utilizing a strip to detect the amplicon and provides results in 30 minutes. 

Results 

The exo and nfo AmplifyRP ® assays were optimized using either psyllid DNA (L. asiaticus) or 

DNA isolated from infected plants (L. africanus and L. americanus). Multiple primer/probe 

combinations were tested to achieve the maximum sensitivity and specificity for each assay. 

Optimized assays were then tested using extracts prepared from infected plant tissue. 


P a g e | 84 

Figure 1, Graph A illustrates the specificity and sensitivity of the L. asiaticus exo test for 

diluted crude plant extracts. The test can detect HLB at a dilution of 1:1000 of the initial extract 

and evinces no cross-reactivity with either L. americanus or L. africanus. 

Graph D compares the detection of L. asiaticus in psyllid DNA extracts that were previously 

tested using quantitative PCR. AmplifyRP ® is able to detect HLB in samples with a wide range 

of Cq values. 

Graphs B and C show the specificity and sensitivity for the L. americanus and L. africanus exo 

tests, respectively. The L. americanus assay is sensitive down to a 1:100 dilution of crude plant 

extract. It demonstrates no cross-reactivity with either DNA from either L. asiaticus or 

L. africanus. The L. africanus assay is not completely specific, as it recognizes L. asiaticus. 

However, its sensitivity for L. africanus is 10-fold higher than it is for L. asiaticus. 

Figure 2 shows examples detection on strips of the AmplifyRP ® nfo L. asiaticus and 

L. americanus tests. Panel A demonstrates the specificity and sensitivity of the L. asiaticus test. 

Panel B provides an example of the specificity of the L. americanus test. The AmplifyRP ® nfo 

L. africanus test shows a similar cross-reactivity to L. asiaticus as the exo test, with sensitivity to 

L. africanus 10-fold greater than for L. asiaticus. 

Discussion 

Using AmplifyRP ® , we can detect all three HLB varieties with a high degree of sensitivity. 

AmplifyRP ® can also identify, either directly or through a process of elimination, those varieties. 

A summary of this can be found in Table 1. While more extensive testing needs to be done, we 

believe that AmplifyRP ® is a fast and easy method for detection of HLB, either as a primary 

screen or for confirmation. The test can be performed using crude plant extracts and produces 

results in under 30 minutes total. 

Acknowledgements 

We would like to thank the following individuals and their labs for help with this project: 

Dr. Barry Manicom, ARC-ITSC, for providing plant material infected with L. Africanus; 

Dr. John Hartung, USDA-ARS, for providing psyllid DNA extracts containing L. asiaticus and 

plant material infected with L. asiaticus, L. americanus, and L. africanus; and Dr. Marcelo 

Pedreira de Miranda, FUNDECITRUS-Brazil, for providing plant material infected with 

L. africanus. 

Reference 

Piepenburg, O., Williams, C.H., Stemple, D.L., Armes, N.A. 2006. DNA detection using 

recombination proteins. Public Library of Science Biology 4(7): e204. DOI: 

10.1371/journal.pbio.004020. 


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Fig. 1. Graph A illustrates the specificity and sensitivity of the L. asiaticus exo test for diluted 

crude plant extracts. Graph D compares the detection of L. asiaticus in psyllid DNA extracts 

that were previously tested using quantitative PCR. Graphs B and C show the specificity and 

sensitivity for the L. americanus and L. africanus exo tests, respectively. 


P a g e | 86 

A 

B 

Fig. 2. nfo detection of HLB plant samples. Panel A: L. asiaticus test on dilutions of crude 

extract. Strips 1-3 Las 1:125, 1:625, 1:3125. Strip 4 Lam neat, strip 5 Laf neat. 

Panel B: L. americanus test on 4 ng total plant DNA. Strip 1 Lam, strip 2 Laf, strip 3 Las. 

Table 1. Summary of limits of detection for HLB tests. 

Psyllid Infected Infected plant 

DNA plant total crude extract 

Test Cq 19.1-19.8 Cq 25.9-26.6 DNA exo nfo 

L. asiaticus 1:1000 1:100 ND 1:1000 1:3000 

L. africanus NA NA ND Laf 1:100 

Las 1:10 

Laf 1:250 

Las 1:25 

L. americanus NA NA 39 pg Lam 1:100 1:5 


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4.17 Assessment of Candidatus Liberibacter asiaticus in the Psyllids, Diaphorina citri 

Collected from Murraya paniculata in Thailand 

Jantasorn, A. 1 , Duan Y.-P. 2 , Hoffman, M. 2 , Zhang, S. 3 , Puttamuk, T. 1 , Thaveechai, N. 1 

1 Department of Plant Pathology, Faculty of Agriculture, Kasetsart University, Bangkok, 

Thailand, 10900 


3 UF-IFAS Tropical Research and Education Center, Homestead, FL, USA 

Orange jasmine, Murraya paniculata, is a preferred alternative host for the Asian citrus psyllid, 

the primary vector of huanglongbing (HLB) citrus greening disease (Gasparoto et al., 2010). We 

collected Asian citrus psyllids on the alternative host Murraya paniculata from 10 diverse 

geographic regions of Thailand. Total genomic DNA was extracted from individual psyllids and 

evaluated for the presence of Ca. Liberibacter asiaticus using real-time PCR (Li et al., 2006). 

Additional sampling from citrus psyllids collected from infected citrus trees in Fort Pierce, FL 

during the summer of 2010 was included for comparison. Preliminary data show evidence of 

high titers of Ca. Liberibacter asiaticus in the U.S. psyllid isolates, while titer levels in Thailand 

appear to vary by location. The results show different titer levels among individual psyllid 

isolates in each of the provinces of Thailand. Samples from Chantaburi province display average 

titer level of HLB pathogen of 21.85 Ct value. Highest titer levels were found in isolates from 

Naknon Pathom province and Samut Sakhon with Ct values of 16.12 and 22.52, respectively. 

Few HLB studies have been conducted in Thailand and the southeast regions of Asia, where 

10 to 15% of tangerine trees are lost each year to HLB disease (Bové, 2006; Roistacher, 1996). 

Our work provides a first assessment of the infection frequency of the psyllids from 

M. paniculata in Thailand. 

References 

Bové, J.M. 2006. Huanglongbing: a destructive, newly emerging, century-old disease of citrus. 

Journal of Plant Pathology 88:7-37. 

Gasparoto, M.C.G. et al. 2010. First report of Ca. Liberibacter americanus transmission from 

Murraya paniculata to sweet orange by Diaphorina. Journal of Plant Pathology 92:546. 

Li, W.B., Hartung, J.S., Levy, L. 2006. Quantitative real-time PCR for detection and 

identification of Candidatus Liberibacter species associated with citrus huanglongbing. 


Roistacher, C.N. 1996. The economics of living with citrus diseases: huanglongbing (greening) 

in Thailand, p. 279-285. In: Proceedings of the 13th Conference of the International 

Organization of Citrus Virologists, IOCV, Riverside, CA. 


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4.18 Liberibacter Reservoirs in Cities and Villages in the State of São Paulo, Brazil 

Lopes, S.A. 1 , Frare, G.F. 2 , Camargo, L.E.A. 2 , Wulff, N.A. 1 , Teixeira, D.C. 1 , Bassanezi, R.B. 1 , 

Beattie, G.A.C. 3 , Ayres, A.J. 1 



3 University of Western Sydney, New South Wales, Australia 

All commercial citrus and the orange jasmine (Murraya exotica) are hosts of Candidatus 

Liberibacter americanus (Lam) and Ca. Liberibacter asiaticus (Las). Orange jasmine and a 

variety of limes, mandarins, and sweet oranges exhibiting yellow shoots are commonly found in 

urban areas in Brazil. The incidence of orange jasmine plants infected by Liberibacter and their 

potential as source of inoculum were assessed in two surveys. Ten to twenty leaves from each 

tree were processed for conventional and qPCR analysis. Liberibacter titers were compared with 

those on HLB-positive citrus growing in backyards and commercial orchards. Liberibacters were 

detected in 91 of 786 trees distributed in 10 of 76 cities and villages, and in most citrus trees 

sampled in the city of Araraquara. The yellow shoots and/or dieback exhibited by the 

PCR-positive orange jasmine were indistinguishable from those on PCR-negative trees. Lam was 

more common in the 2005-06 (96.6%) survey and Las in the 2009 (84.8%) survey, following the 

same trend in citrus from backyards and orchards. Orange jasmine symptoms were milder and 

developed less rapidly. Lam and Las titers averaged, respectively, 4.3 and 3.0 log cells/g tissue in 

orange jasmine compared to 5.5 and 7.3 in citrus. Since in urban areas those infected trees are 

not under any insect- or tree eradication-control program, they may have an important role for 

inciting and sustaining HLB epidemics. Also, since orange jasmine is propagated in open areas 

and is freely commercialized, asymptomatic trees might be spreading Lam and Las to HLB-free 

regions. 


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4.19 Pictorial Gallery of Foliar HLB Symptoms on Various Citrus Varieties and Citrus 

Relatives 

Robl, D.J., Riley, T.D, Gomez, H. USDA-APHIS-PPQ-CHRP, FL, USA 

Since the disease was first observed in Florida (August 2005), pathologists in the Citrus Health 

Response Program (CHRP) have had the opportunity to screen thousands of field samples 

collected by CHRP technicians at various locations throughout the state. Standard operating 

procedure (SOP) requires that prior to undergoing PCR analysis each sample must first be 

screened by a pathologist. Based on foliar symptoms alone, a decision is made as to whether or 

not a sample should be forwarded to the lab. Rather than visit each office, CHRP pathologists 

have, for the most part, relied on digital photos e-mailed directly to the pathologist. This has 

worked surprisingly well, and over the years, we have amassed a large photo archive of 

symptomatic leaves. This poster is meant to provide the viewer with a sampling of classical HLB 

foliar symptoms on various citrus varieties and some citrus relatives as well. 


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Session 5: 

Economics, Fruit 

Quality, and Crop 

Loss 


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5.1 Evaluation of Chemical Flavor Compounds in Orange Juice from Multiple Harvests of 

Hamlin and Valencia Fruit from HLB-Symptomatic Versus Healthy Trees 

Baldwin, E. 1 , Bai, J. 1 , Dea, S. 1 , Plotto, A. 1 , Manthey, J. 1 , Rouseff, R.L. 2 , Irey, M.S. 3 

1 USDA-ARS, Winter Haven, FL, USA 



Fruit from HLB-affected trees are reported to be off-flavored. This study looked at flavor 

compounds in juice made from fruit harvested from 15+ trees symptomatic for HLB compared to 

healthy trees grown in the same area for multiple harvests of Hamlin (December/January 2009) 

and Valencia (April/June 2009). Fruit from HLB symptomatic trees were separated into 

asymptomatic (normal looking, HLBAS) and symptomatic (small, green, and lopsided, HLBS) 

fruit for comparison to healthy (H) fruit prior to juicing using a JBT extractor/pasteurizer. For 

Valencia, there were no differences in Brix, but HLB juices tended to have higher titratable 

acidity (TA), lower ratio (April), and higher oil (June). For Hamlin, Brix was higher in H juice 

(December), TA higher in HLBS (December), ratio lower in HLBS, and oil higher for HLBS 

juice (December). Healthy juice tended to have higher levels of sucrose and fructose, whereas 

glucose was variable, especially for HLBS juice. HLB juice had higher levels of citric acid 

(December Hamlin/April Valencia), but there were no differences for the other two 

harvests/cultivars. Malic acid tended to be lower in HLB juices. Limonin and nomilin were 

higher in HLB juices, especially HLBS, but were below reported thresholds. Gas 

chromatography-olfactometry (GC-O) research did not show differences between H and HLBAS 

juice, but observed some volatiles that were found either exclusively or at higher aroma intensity 

in either H or HLBS juices. There were more “green”/“fatty” aromas in HLBS, while there were 

more “sweet”/“fruity” components in H juices with respect to each other. These results are 

similar to those reported for 2007 and 2008 (Baldwin et al., 2010) except that with a larger 

sample size (15 trees versus 5-7 trees), there were less significant chemical differences, 

especially for HLBAS compared to H juice, due to a reduction in tree-to-tree variation. 

Reference 

Baldwin, E.A., Plotto, A., Manthey, J., McCollum, G., Bai, J., Irey, M., Cameron, R., Luzio, G. 

2010. Effect of Liberibacter infection (Huanglongbing disease) of citrus on orange fruit 

physiology and fruit/fruit juice quality: chemical and physical analyses. Journal of 

Agricultural and Food Chemistry 58:1247-1262. 


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5.2 Evaluation of Bitterness Caused by Huanglongbing Disease in Orange Juice 

Dea, S. 1 , Plotto, A. 1 , Manthey, J. 1 , Baldwin, E. 1 , Irey, M.S. 2 



As a preliminary step to understand and characterize what metabolites are responsible for the 

bitter off-favor of huanglongbing infected fruit, the thresholds of limonin, nomilin, and their 

combination in a sugar and acid matrix, as well as in healthy Valencia orange juice, were 

determined by taste panels. Food grade limonin and nomilin were added alone or in combination 

to a simple (sucrose and citric acid) or complex (sucrose, glucose, fructose, citric, and malic 

acid) matrix, or were added directly into orange juice. Thresholds were determined by taste 

panels, composed of 16 to 23 trained panelists, using a three-alternative forced choice (3-AFC) 

method (ASTM: E-679). In the simple matrix, the threshold of limonin was lower than nomilin. 

The synergetic effect of limonin and nomilin was significant in decreasing their individual 

thresholds. Interestingly, the thresholds of limonin and nomilin were lower in orange juice 

compared to the thresholds measured in the complex matrix. Our current results show that the 

threshold concentrations of limonin and nomilin when added to healthy Valencia orange juice 

are higher than the concentrations of those compounds measured in juice made with 

symptomatic HLB fruit, which was perceived bitter by a taste panel. Possibly, the lower sugar 

and higher acid content of HLB fruit decreased the threshold of those bitter compounds. 

Moreover, different concentrations of Valencia and Hamlin HLB-infected juice were blended 

into healthy juice to determine the detection and recognition thresholds. Panelists were able to 

detect the symptomatic HLB juice at different levels depending on the variety. 


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5.3 Sensory Evaluation of Juice Made with Fruit from Huanglongbing (HLB) Affected 

Trees 

Plotto, A. 1 , Valim, F. 2 , Rouseff, R.L. 3 , Dea, S. 1 , Manthey, J. 1 , Narciso, J. 1 , Bai, J. 1 , Irey, M.S. 4 , 

Baldwin, E. 1 


2 FDOC, Lake Alfred, FL, USA 



Fruit and juice from trees affected with the huanglongbing (HLB) disease are reportedly having a 

bitter or salty taste, but a full description of the flavor of such juice has not been published. 

Hamlin and Valencia oranges were harvested from groves where trees were either healthy or 

affected with HLB, and included normal looking (non-symptomatic) and symptomatic fruit 

(small, green, and lopsided). Fruit was juiced using a JBT 391 extractor and pasteurized under 

simulated commercial conditions. A sensory trained panel evaluated the juice using eight aroma, 

eight flavor, and six taste and mouth feel descriptors. Samples of each juice were analyzed for 

sugars, acids, limonoids, and volatiles. In general, there was no or little difference between juice 

from healthy and non-symptomatic HLB fruit. However, for Hamlin and Valencia harvested in 

January and April, respectively, orange and fresh (Valencia only) aromas were lower; and 

sour/fermented, peppery/musty, and paint aromas were higher in juice from HLB-symptomatic 

than healthy fruit. Orange, fruity, and fresh flavors, as well as sweetness, were lower in juice 

from HLB-symptomatic fruit; while peel oil, sour/fermented, peppery/musty, and paint flavors, 

as well as sourness, saltiness, bitterness, metallic, and tingling attributes were rated higher. There 

were little or no differences between samples due to disease status for Valencia harvested in 

June. Chemical analyses revealed higher limonin and nomilin in Hamlin and Valencia harvested 

in April, and lower Brix/TA ratio in Valencia (April), which may explain some of the differences 

in taste. 


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5.4 Economic Considerations to Treating HLB with the Standard Protocol or an Enhanced 

Foliar Nutritional Program 

Morris, R.A., Muraro, R.P. Associate Extension Scientist and Economist, Professor of Food 

and Resource Economics, UF-IFAS Citrus Research and Education Center, Lake Alfred, FL, 

USA 

From an economic viewpoint, the main consequences of HLB are increased tree mortality, 

reduced yields, and increased costs of production. In 2006, one grower in the Immokalee area 

whose grove had a relatively high HLB incidence rejected the proposition that removal of 

infected trees was the only management option. Instead of removing infected trees, he began 

using an enhanced foliar nutrient management program that included SAR compounds reported 

to activate a tree’s disease resistance mechanisms and selected micronutrients (Zn, Mn, Mg, Bo, 

and Ca). After implementing the foliar nutrient management program, the visual appearance of 

the trees improved and an above average crop has been harvested sequentially for 4 years. Two 

types of groves were analyzed; one under the standard management protocol, and the other under 

an enhanced foliar nutritional program. The two categories of groves were a mature grove with 

15 + -year-old trees and an intermediate age grove with 4- to 10-year-old trees. It was assumed 

that resets would not survive to become productive under the enhanced foliar nutritional 

program. For the mature grove, the time to switch to the enhanced foliar nutrient program was 

when annual infection rates from HLB reached 3.9% at fruit prices of $1.50 per pound solids and 

4.4% at fruit prices of $1.25 per pound solids. For the 4- to 10-year-old grove, the annual HLB 

infection rates where a foliar nutrient program was preferable were the same. 


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5.5 When Should a Grower with HLB Stop Removing Trees? 

Irey, M.S. U.S. Sugar Corporation, Clewiston, FL, USA 

HLB was first found in Florida in late 2005 and is now widely distributed throughout the 

commercial citrus growing regions of Florida. Although no hard numbers are available, survey 

data indicate that the cumulative incidence of infected trees is in the range of 8-10% statewide. 

However, the estimated incidence varies across the state with the highest infection levels 

occurring in the south and the east coast. In some areas, infection levels within individual groves 

are approaching 100%; while in many other areas of the state, infection levels within individual 

groves remain less than 1-2%. When HLB was first discovered, the citrus industry was 

reasonably united in the approach that should be taken to manage the disease. The recommended 

practices included control of the insect vector, use of disease-free planting material, and the 

removal of infected trees to lower the inoculum load. However, as infection levels began to 

increase, many growers began looking at alternative treatments, mostly nutritional in nature; and, 

the HLB management decision has come down to “Should I continue to remove trees?” or 

“Should I switch to a nutritional program?” Unfortunately, the decision is much more complex 

than this and many factors, both biologic and economic, affect the decision. Factors which affect 

the decision include tree age at the decision point, current infection levels, estimated infection 

and production estimates going forward, the time horizon being evaluated, and the cost 

differentials between the different alternatives. A spreadsheet-based model has been developed 

to help growers evaluate their individual situations. Several scenarios will be presented which 

will illustrate when each management would be the most cost effective. 


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5.6 Use of Electronic Sensor Technology to Discriminate between Juices from 

Huanglongbing Infected and Healthy Orange Trees 

Bai, J. 1 , Dea, S. 1 , Plotto, A. 1 , Baldwin, E. 1 , Irey, M.S. 2 



Orange juice squeezed from huanglongbing (HLB) infected fruit may have altered flavor in 

comparison with juice from healthy fruit. It would be useful to detect and discriminate these 

juices from regular juice in a quality control program. Electronic tongue (e-tongue) and 

electronic nose (e-nose) are devices that mimic human sense of taste and olfaction, respectively, 

based on sensor arrays and pattern recognition systems. In this study, juices from asymptomatic 

(HLBa), symptomatic (HLBs), and healthy Valencia and Hamlin fruit were analyzed using 

AlphaMOS e-nose (FOX) and e-tongue (ASTREE) over two harvests per variety. One Hamlin 

harvest included fruit from trees grown under the Maury Boyd’s (MB) nutritional and plant 

defense spray program. A blended juice series with 50, 25, 12.5, and 6.25% HLBs juice balanced 

with the healthy juice, together with 100% HLBs and healthy juices were analyzed to determine 

the influence of HLBs on juice quality. Results were compared with sensory evaluation data. 

Healthy, HLBa, and HLBs juices were separated from each other by both e-nose and e-tongue 

for all varieties and harvest times. Juices from the MB program were well separated from those 

without MB application for both HLBa and HLBs; moreover, these juices were closer to healthy 

juice than non-MB-HLB juice in discrimination distance. There were clear trends in the principle 

component analysis plots when HLBs + healthy blend juice dilution series were analyzed by 

e-tongue, but not by e-nose. Discrimination between samples by electronic sensors agreed with 

sensory data in most cases. 


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5.7 A Regional Epidemiological Approach for Yield Loss Estimates Due to Candidatus 

Liberibacter Under Different Risk Scenarios 

Mora-Aguilera, G. 1 , Acevedo, G. 1 , López-Arroyo, J.I. 2 , Velázquez-Monreal, J.J. 3 , Gómez, R. 3 , 

Robles-González, M.M. 3 , Salcedo, D. 4 

1 Colegio de Postgraduados, Instituto de Fitosanidad, Texcoco, Edo. de Méx., México 

2 INIFAP, Centro Regional de Investigaciones del Noreste. Río Bravo, Tam., México 

3 INIFAP, Centro de Investigación Regional del Pacífico Centro, Guadalajara, Jal., México 

4 Consultor Ext. IICA-México morag@colpos.mx 

Potential yield loss induced by Candidatus Liberibacter (LB) was conducted in Mexico using a 

comparative epidemiological approach as part of a governmental effort to assess the impact of 

HLB. Regional yield losses were estimated at 1-, 3-, and 5-year scenarios after the pathogen 

entrance in Mexico (Salcedo et al., 2010). The analysis was first focused on transforming 

historical spatial data from USA and Brazil to temporal progress at different regional levels 

(state, county, and field level) and modeling the resulting process. Clearly, three different 

temporal behaviors were found depending on the spatial dimension. Thus, Weibull, a flexible 

model, fit well with 0.98-0.99 R 2 values. Published fruit weight reduction data was also analyzed 

and weighted by citrus species susceptibility (Bassanezi et al., 2009). In order to use temporal 

models for predictive purposes, 23 Mexican estates were categorized on three epidemic risk 

categories using a principal component-cluster analysis based on host prevalence, climatic, and 

susceptibility indexes developed with the epidemic inductivity criterion. Host and clime 

variables, associated to two major components, comprised a 72-84% variance. Risk categories 

were associated to different temporal models based on their epidemic rate value, e.g., the highest 

risk with highest epidemic rate, to predict the proportion of counties and productive units with 

HLB. These proportions were related to scaled weight fruit reduction to estimate yield loss at 

state and national level. Multivariate analyses and modeling were performed with SAS ver. 6.10 

and yield estimates were done with MS Excel. Yield losses will be presented considering 

different citrus species and time and space scenarios. 


Project supported by SAGARPA-SENASICA PM09-4002 and CONACYT-INIFAP 

PM10-4032. 

References 

Bassanezi, R., Montesino, L., Sanches, E. 2009. Effects of huanglongbing on fruit quality of 

sweet orange cultivars in Brazil. European Journal of Plant Pathology. DOI 

10.1007/s10658-009-9506-3. 

Salcedo, D., Hinojosa, R., Mora, G., Covarrubias, I., DePaolis, F., Cintora, C., y Mora, S. 2010. 

Evaluación del impacto económico de huanglongbing (HLB) en la cadena citrícola 

Mexicana. IICA-México. 132 p. 


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Session 6: 

Epidemiology 


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6.1 Five Years of Experience with Huanglongbing in Florida: Current Assessment; How 

Did We Get Here? 

Halbert, S.E. 1 , Manjunath, K.L. 2 , Ramadugu, C. 3 , Lee, R.F. 2 




3 Department of Botany and Plant Sciences, University of California at Riverside, CA, USA 

Huanglongbing (citrus greening, HLB) was discovered in Florida in August 2005. During the 

5-year period since its discovery, both citrus acreage and production have declined, but other 

factors (natural events, market fluctuations, and development) make it difficult to determine how 

much of the loss is due to HLB. Spread of the disease in Florida has been rapid. Data indicate 

that the initial population of psyllids was free of Liberibacters. Probably, the insects encountered 

the pathogens in Miami-Dade County in 2000. DPI survey data indicate that the disease 

expanded its range approximately 36 miles per year, three times faster than estimated for Brazil 

(Gottwald, 2007). Spread was due to psyllid flight and movement on bulk citrus fruit and on 

plants for sale. Retail sales of citrus and of orange jasmine (Murraya paniculata) played a role in 

disease distribution. Florida has developed stringent regulations for propagation of citrus and 

related plants. Our data indicate these regulations are successful for production of clean plants 

and preventing further spread of HLB through plant sales, especially for trees intended for 

commercial groves. Area-wide treatments for psyllid vectors also have been successful, lowering 

the numbers of psyllids in production areas. Inoculum control (removing infected plants) has 

been more difficult for Florida. Many infected blocks have not been removed because of 

financial considerations, leaving large inoculum reservoirs. Some growers have had moderate 

success using nutrition-based programs, but it is doubtful if a replanted block could become 

productive in a high-inoculum environment. More research is needed for a long-term solution. 

Reference 

Gottwald, T.R., da Graça, J.V., Bassanezi, R.B. 2007. Citrus huanglongbing: the pathogen and 

its impact. Online. Plant Health Progress doi: 10.1094/PHP-2007-0906-01-RV. 


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6.2 Designing Sampling Schemes to Maximize the Probability of Early Detection of a 

Huanglongbing Outbreak 

Parnell, S.R. 1 , Gottwald, T.R. 2 , van den Bosch, F. 1 

1 Centre for Mathematical and Computational Biology, Rothamsted Research, Harpenden, UK 


Detecting an outbreak of huanglongbing (HLB) at low incidence is critical if control efforts are 

to be implemented in time to be effective. If detection of an outbreak occurs when epidemic 

incidence is already at even a moderate level, then little can be done to control the outbreak. 

Advances in diagnostic tools are an important facet in achieving early detection but are not 

enough alone. Even the most accurate and reliable diagnostic will not help with early detection if 

infected host trees are missed during rounds of monitoring. To achieve this, it is important to get 

the sampling design right, i.e., determine how many samples to take and from which blocks and 

trees. Here, we use mathematical modeling techniques to determine optimal sampling designs 

which maximize the probability of detecting an HLB outbreak while it is still at low incidence. 

By stochastically generating epidemics of HLB in specific host distributions, we can simulate 

monitoring programmes and determine the probability to detect an outbreak before it reaches a 

predetermined incidence. Conversely, using a Bayesian approach, we can also calculate the 

incidence an outbreak has reached when detected for the first time. We show how the probability 

of early detection declines with reduced sample size and how this varies for different sampling 

patterns. The method allows growers and regulators to make informed choices on how much 

sampling resource to commit to early detection surveillance based on factors such as the 

detection-incidence required and the amount of risk that is acceptable in achieving this. 


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6.3 Candidatus Liberibacter africanus Subspecies capensis on Calodendrum capense in 

South Africa 

Phahladira, M.N.B. 1,2 , Viljoen, R. 2 , Pietersen, G. 1,2 

1 ARC-Plant Protection Research Institute, Division of Microbiology and Plant Pathology, Private 

Bag X134, Queenswood, Pretoria 0001, South Africa 

2 Department of Microbiology and Plant Pathology, University of Pretoria, Pretoria 0002, South 

Africa 

Determining potential alternate hosts to citrus of Candidatus Liberibacter africanus (Laf) 

amongst the indigenous plants of South Africa will help to make control by disease pressure 

reduction more efficient. In this study, indigenous plants mainly of the Citrus family (Rutaceae) 

were tested to determine if they are natural hosts of the pathogen. Symptomatic and nonsymptomatic 

leaf and petiole samples of numerous indigenous Rutaceous species, primarily 

Calodendrum capense (Cape Chestnut) were collected from areas near citrus orchards, in 

gardens, in natural habitats, or from botanical gardens. The presence of Laf was tested for by 

DNA extraction and using a published conventional PCR as well as a multiplex PCR containing 

an internal control directed at a plant gene. Except for Cape Chestnut, none of the indigenous 

plants yielded Liberibacter specific bands following PCR. A number of Cape Chestnut samples, 

however, tested positive for the presence of Liberibacter DNA. Direct DNA sequencing of the 

PCR products from a number of these plants confirmed the presence of Ca. L. africanus 

subspecies capensis (LafC). A real-time PCR protocol, capable of specific detection of LafC, 

was developed. Using this, a total of 263 Cape Chestnut samples, collected from Mpumalanga, 

Limpopo, Gauteng, KwaZulu-Natal, Western Cape, and Eastern Cape were tested. LafC was 

found present in numerous trees from each region, including the Eastern Cape where Laf is 

absent. Analysis of an Eastern Cape site where Cape chestnut trees have grown in close 

proximity to citrus groves for more than 15 years suggests that LafC does not spread to citrus 

naturally, in spite of the presence of Trioza erytreae in the general area. This is based on the fact 

that 43 of 44 Cape chestnut trees were LafC infected, while 273 citrus trees, all within a 

maximum of 10 m from any given infected Cape chestnut, were negative. 


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6.4 Distribution of Psyllids Positive for Candidatus Liberibacter asiaticus in Citrus Groves 

in Southwest Florida 

Halbert, S.E. 1 , Manjunath, K.L. 2 , Ramadugu, C. 3 , Mears, P. 4 , Lee, R.F. 2 





4 Florida Department of Agriculture and Consumer Services, Division of Plant Industry, Citrus 

Health Response Program, Immokalee, FL, USA 

Florida Citrus Health Response Program inspectors collected psyllid samples from 4 trees at 

each of 7 points in 12 grove blocks in SW Florida between January and August 2009. Points 

included SE corner, mid-point of the eastern edge, NE corner, NW corner, SW corner, center 

point, and a floating point designated by the grower (center of the southern border if grower did 

not designate a point). Psyllids were counted and sent to Riverside, CA for real-time PCR 

analysis for detection of Candidatus Liberibacter asiaticus (Las). There was no clear difference 

in average numbers of insects in the samples from different points, but psyllids were most likely 

to be encountered on the eastern edges of the blocks, particularly the SE corners. Psyllids were 

most likely to be positive for Las on the eastern edges of the blocks. Several of the blocks were 

in adjacent organic and conventionally managed groves. Map plots showing the locations of 

positive psyllid samples indicated that the disease was spreading from the organic grove into the 

conventionally managed one. 


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6.5 Seasonal Prevalence of Citrus Huanglongbing (Candidatus Liberibacter asiaticus) in a 

Central Florida Sweet Orange Grove 

Parkunan, V., Wang, N.-Y., Ebert, T.A., Rogers, M.E., Dewdney, M.M. UF-IFAS Citrus 

Research and Education Center, Lake Alfred, FL, USA 

Citrus huanglongbing (HLB or greening) is the most serious citrus disease worldwide. The Asian 

species of the bacterial causal agent, Candidatus Liberibacter asiaticus (Las), is responsible for 

HLB in Florida and is transmitted by the Asian citrus psyllid (ACP), Diaphorina citri. 

Considerable gaps exist in understanding the disease transmission and epidemiology. Seasonal 

trends in Las incidence in ACP and citrus populations have been observed but they are not well 

defined. Understanding the seasonal variation of HLB is critical to develop time-bound 

management strategies for effective HLB control. An Earlygold grove in Orange county Florida 

was selected to examine seasonal prevalence of HLB over a 3-year period. Two hundred trees 

were selected from 10 consecutive rows. One leaf/tree was randomly picked every fortnight. 

Sample collection began at the end of June 2010 with collections ongoing. For qPCR detection 

of Las, the midribs of five random leaves were pooled to obtain 40 samples/date. An estimated 

Las prevalence in the branches was generated from the pools with PooledInfRate v3. 

Simultaneously, ACPs were collected from the same location to compare the seasonal prevalence 

of Las in citrus branches and ACPs. Las prevalence increased (from 0.14, 0.20, and 0.21 to 0.27) 

from June to September. The data from ACP are still being processed. Comparing the data from 

ACP and citrus trees could provide more information on seasonal variation in Las prevalence in 

both the vector and the host, and thereby enhancing our management strategies by identifying the 

essential periods for ACP control to prevent HLB transmission. 


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6.6 A Mathematical Model for Transmission of HLB by Psyllids 

Chiyaka, C., Singer, B., Halbert, S.E., van Bruggen, A.H.C. Emerging Pathogens Institute, 

University of Florida, Gainesville, FL, USA 

A mathematical model of the transmission dynamics of HLB between its psyllid vector and 

citrus host has been developed to understand and assess the mechanisms involved in pathogen 

acquisition from and transfer to host plants. Citrus flush is the basic unit. Spread is modeled from 

flush to flush within one tree. Using parameters from literature, simulations show dynamics in 

vector and host populations as the tree approaches complete infection. Simulation results of flush 

infection and HLB symptom development throughout a tree are realistic. There is widespread 

discussion about the management strategies that would curb HLB spread. Epidemiological 

research indicates that rogueing infected trees would be needed besides psyllid control. Only 

symptomatic trees testing positive for Las are removed, while infectious non-symptomatic trees 

remain. The model will be used to assess the effectiveness of different control methods such as 

rogueing of trees, removal of flush, application of nutrient solutions, use of insecticides, and 

different combinations of these strategies. Preliminary results show that the numbers of 

asymptomatic infectious flush quickly become greater than symptomatic flush, indicating that 

removal of symptomatic tissues may not be effective at controlling HLB. Although vector 

activity is essential for initial infection in a tree, it is not necessary for continued infection within 

a tree since infection can occur from flush to flush. This makes spraying psyllids less effective 

once a tree becomes infected. Migration of psyllids from tree to tree will be added to the model 

in the near future. 


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6.7 Potential Spread of Huanglongbing Through Soil 

Nunes da Rocha, U., Dickstein, E.R., van Bruggen, A.H.C. Department of Plant Pathology and 

Emerging Pathogens Institute, University of Florida, Gainesville, FL, USA 

Huanglongbing (HLB) emerged in Florida in 2005 and has spread rapidly throughout the State. 

The bacterium associated with this disease (Candidatus Liberibacter asiaticus, Las) has not been 

isolated consistently and Koch’s postulates have not been fulfilled. Las is a symbiont of the 

psyllid Diaphorina citri, which transmits the pathogen to citrus. Other potential avenues of 

transmission and spread of Las have not been investigated yet. When HLB-infected trees are 

removed, young trees are often planted in the same planting hole. Replant disease is a common 

phenomenon for other fruit trees. In 2009-2010, we tested if Las could be transmitted through 

soil. One year after planting 120 mandarin seedlings in soil collected underneath HLB-positive 

citrus trees from two groves, Las qPCR tests were positive for two symptomatic seedlings. 

Asymptomatic trees in soil from HLB-positive groves, in autoclaved soil, and in potting mix 

tested negative in Las qPCR tests. These results could mean that nonpathogenic Ca. Liberibacter 

species are common in soil and endosphere but test positive with qPCR (and that the symptoms 

were nonspecific) or that pathogenic Las is transmitted through soil besides psyllids. The 

experiment on soil transmission will be repeated; nematodes, protozoa, and root residues will be 

isolated from soil and tested for Las by qPCR. Nematode and protozoa species that test positive 

will be used in transmission tests. 


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Session 7: 

International Citrus 

Industries, 

Regulation, and 

Grower Experiences 


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7.1 Laws, Huanglongbing Management, and the Current Status of the Disease in São 

Paulo, Brazil 

Belasque, J., Jr. 1 , Ayres, A.J. 1 , Barbosa, J.C. 2 , Massari, C.A. 1 , Bové, J.M. 3 


2 FCAV/UNESP, Jaboticabal, Brazil 

3 INRA and University Victor Segalen Bordeaux 2, Bordeaux, France 

Huanglongbing (HLB) was first reported in São Paulo State (SPS), Brazil, in March 2004. 

A survey conducted in SPS in 2010 estimated that 1.87% of the total number of sweet orange 

trees in SPS were symptomatic. The main producing areas in SPS (Center and South) presented 

the highest HLB incidences (3.5% and 2.0%, respectively). In 2005, the Federal Government 

approved the first regulatory legislation, imposing, among others, the mandatory eradication of 

all HLB-affected trees. In 2006 and 2008, new regulatory rules were ordered in an effort to 

reinforce growers to adopt the recommended practices for HLB management. However, in spite 

of the rules, the regulatory agencies were not efficient and only a small number of growers 

completely followed the law. New foci were detected in areas hitherto not affected and the 

disease increased rapidly in the affected ones. With only about one-third of the citrus acreage 

being under effective HLB management, the many non-managed orchards are major handicaps 

to statewide successful management. It is estimated that 6 to 8 million trees have been eradicated 

in SPS since 2004. The recommended practices for preventive HLB management are based on (i) 

inoculum reduction by frequent removal of symptomatic trees, (ii) control of psyllid vector 

populations by insecticide treatments, and (iii) use of healthy trees from insect-proof, covered 

nurseries in the case of resets. This three-pronged, short-term system is the only one that keeps 

most of the orchard trees free of HLB until new HLB-resistant trees become available as a longterm 

solution. The current HLB status of SPS citrus farms that have adopted the three-pronged 

system during the last 6 years will be presented and discussed. 


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7.2 Distribution of Citrus Huanglongbing in the Dominican Republic 

Matos, L. 1,7 , Hilf, M.E. 2 , Cayetano, X. 1 , Feliz, A. 1,3 , Puello, H. 4 , Méndez, F. 5 , Borbón, J. 6 , 

Folimonova, S.Y. 7 

1 IDIAF-CENTA, Santo Domingo, Dominican Republic 


3 DSV-Ministerio de Agricultura, Dominican Republic 

4 Consorcio Citrícola del Este, Hato Mayor, Dominican Republic 

5 Consorcio Cítricos Dominicanos, Dominican Republic 

6 Facultad Agronomía-UASD, Universidad Autónoma de Santo Domingo, Dominican Republic 


Huanglongbing (HLB) is globally the most economically important disease of citrus. HLB was 

first found in the Dominican Republic in September 2008 in Puerto Plata Province on the north 

coast. Symptomatic trees were sampled in three different directions from the first identified point 

of infection. HLB was found in the northwest near the Haitian border approximately 70 km 

away, toward the east coast and to the south toward the middle of the country. Two positive 

samples also were found in Azua Province approximately 300 km south of Puerto Plata. One 

year after HLB was found in Puerto Plata, it was found almost simultaneously in the main central 

and eastern citrus producing areas. The presence of the bacterium (Ca. Liberibacter asiaticus) 

was confirmed in samples by polymerase chain reaction (PCR) amplification of sequence of the 

outer membrane protein gene. Out of 4,100 samples, 1,115 were positive and the disease is 

present in 13 of 32 provinces, including the three major citrus producing areas. The distribution 

of HLB indicates that factors other than psyllids could be involved in disease spread. Since the 

psyllid and citrus are present throughout the country, HLB is expected to spread to all regions. 


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7.3 Citrus Huanglongbing in Cuba: Current Situation, Management, and Main Research 

López, D. 1 , Luis, M. 2 , Collazo, C. 2 , Batista, L. 2 , Peña, I. 2 , González, C. 2 , Pérez, J.L. 2 , Zamora, 

V. 2 , Borroto, A. 1 , Pérez, D. 1 , Alonso, E. 3 , Acosta, I. 3 , Llauger, R. 2 , Casín, J.C. 4 

1 Empresa de Cítricos Ciego de Ávila, Carretera a Ceballos Km 9 ½, Ceballos, C.P 69230 Ciego 

de Ávila, Cuba. E-mail: nelson@ecca.co.cu 

2 Instituto de Investigaciones en Fruticultura Tropical, Ave. 7ma # 3005, Playa, C.P 11300, 

Ciudad de La Habana, Cuba. E-mail: despacho@iift.cu 

3 Empresa de Cítricos Victoria de Girón, Finca San José, Torriente, Matanzas, C.P 44540, Cuba. 

E-mail: irina@citrovg.cu 

4 Centro Nacional de Sanidad Vegetal, Ayuntamiento No 231, Plaza de la Revolución, C.P 

10600, Ciudad de La Habana, Cuba. E-mail: interior@sanidadvegetal.cu 

Citrus huanglongbing (HLB) was found in Cuba in 2007 and associated with Ca. L. asiaticus, 

whereas its vector, Diaphorina citri Kuwayama, was present from 1999 and has settled 

throughout the country. The presence of the pathogen was confirmed by PCR in all citrus 

commercial areas and residential sectors from 2007 to 2008. Consequently, a management 

program was established including staff training, systematic inspection, and eradication of 

symptomatic plants mainly for new plantations, vector monitoring and control using systemic 

and contact insecticides and mineral oils, and sowing with certified planting material in blocks 

isolated from possible inoculum sources. Furthermore, the national citrus development program 

was restructured towards intensive and sustainable citrus growing, which involves the search for 

new sources of revenue such as recovery of vegetative carbon from eradicated citrus trees and 

short-term production crops interspersed with citrus plantations. To understand disease evolution 

and evaluate management efficiency, epidemiologic studies are carried out in blocks of the major 

citrus areas in the country. Research related to bacterial diagnosis and characterization, vector 

biology and behavior, evolution of disease symptoms, sanitation through in vitro shoot tips 

grafting, and the influence of HLB in citrus production is conducted. Nowadays, differences are 

observed in HLB behavior from citrus areas with dissimilar epidemiological scenes and 

management strategies; nevertheless, the incidence of symptomatic plants in new plantations and 

D. citri populations has been reduced in areas with efficient eradication and vector control. These 

results indicate that a rigorous and more extensive or regional strategy, which should involve 

commercial areas and small producers with state regulations for the residential sector, will 

guarantee better results in managing the disease. 


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7.4 Spreading and Symptoms of Huanglongbing in Mexican Lime Groves in the State of 

Colima, Mexico 

Robles-González, M.M. 1 , Velázquez-Monreal, J.J. 1 , Manzanilla Ramirez, M.A. 1 , Orozco 

Santos, M. 1 , Flores Virgen, R. 2 , Medina Urrutia, V.M. 3 , Carrillo Medrano, S.H. 1 

1 INIFAP, Colima, Mexico 

2 CESAVECOL, Colima, Mexico 

3 Universidad de Guadalajara - CUCBA, Jalisco, Mexico 

Colima State, Mexico is the most important producing area of Mexican lime in the world. The 

Asian citrus psyllid (ACP), Diaphorina citri Kuwayama, reached Mexico in 2002 and was 

reported in Colima State in 2004. Huanglongbing (HLB) was first reported in Yucatan State in 

2009 where it was detected in a backyard tree. In Colima State, HLB was reported in 2010. 

Candidatus Liberibacter asiaticus has been detected in samples from HLB symptomatic Mexican 

lime trees and the ACP vector. This is a preliminary study that describes spreading of HLB in the 

first grove where it was detected in Colima State and also in the region. The size of the first 

grove where HLB was detected was 20 hectares; the total number of trees was 2,909. In April 

2010, five trees were detected showing HLB symptoms, being 0.2% of incidence, and this 

disease was confirmed by PCR. The accumulated incidence of diseased trees by visual symptoms 

were 1.0, 5.5, 15.7, 29.5, and 33.0% for the months of May, June, July, August, and September, 

respectively. In Colima State, there are 4,555 groves and 595 groves were sampled from April to 

October 2010 and HLB symptomatic trees were found in 21% of these groves. The common 

symptom that has been observed in diseased trees is an asymmetric leaf mottling, as well as color 

inversion in the fruits, and a yield reduction. 

References 

Bové, J.M. 2006. Huanglongbing: a destructive, newly-emerging, century-old disease of citrus. 

Journal of Plant Pathology 88(1):7-37. 


greening disease of citrus: a literature review and assessment of risk in Florida. Florida 

Entomologist 87(3):401-402. 

Trujillo-Arriaga, J. 2010. Situación actual, regulación y manejo del HLB en México. 2º Taller 

internacional sobre el huanglongbing y el psilido asiático de los cítricos. Mérida, Yucatán, 

México. 


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7.5 The Asian Citrus Psyllid/Huanglongbing Detection, Treatment, and Regulatory 

Program in California 

Galindo, T. California Dept. of Food and Agriculture, Van Nuys, CA, USA 

Asian citrus psyllid (ACP) was first found in California in August 2008. Since then, the 

California Department of Food and Agriculture (CDF) has partnered with the United States 

Department of Agriculture, the county agricultural commissioners, and the citrus industry to 

develop a plan to inhibit the spread of ACP and to survey for huanglongbing (HLB). The ACP 

detection system consists of government operated yellow panel traps and visual survey in urban 

areas and in areas surrounding commercial citrus, while the citrus industry conducts trapping in 

commercial groves. The ACP treatment system consists of foliar sprays of cyfluthrin and soil 

applications of imidacloprid around ACP find sites out to a 400 meter radius and is funded 

jointly by CDFA and industry. The ACP regulatory system restricts the movement of ACP host 

material within and out of designated quarantine areas extending up to 20 miles from find sites in 

order to inhibit the artificial spread of ACP. The HLB survey system consists of testing captured 

ACP for HLB and of collecting suspect plant samples for testing, and to date no HLB has been 

found. In addition, a jointly operated outreach campaign has raised awareness of these pests 

throughout the state; and finally, a state task force consisting of experts from California, Florida, 

and Texas meets periodically to review operations and to recommend any changes which could 

enhance the effectiveness of the program. 


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7.6 Detection and Reporting of Asian Citrus Psyllid and Huanglongbing in Commercial 

Citrus Within California: An Industry Program 

Taylor, B.J., Polek, M.L., Batkin, T. Citrus Research Board, Visalia, CA, USA 

The California citrus industry, through legislation and assessments, funds the work of field 

technicians to detect and report findings of Asian citrus psyllid (ACP) and huanglongbing (HLB) 

in areas of commercial citrus production. Through the use of visual observation and trapping 

protocols, field technicians place and service yellow sticky panel traps for the detection of adult 

ACP and conduct visual observations for the presence of ACP and HLB throughout California. 

Field technicians use ruggedized personal digital assistants (PDAs) and wireless technologies to 

find, locate, and report findings. Field data and laboratory results are merged into a database, 

allowing all samples to be tracked from the field through laboratory analysis. Results are 

reported to stakeholders through a web-based graphical information system interface. 


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7.7 Citrus Health Research Forum: A National Research Effort 

Polek, M.L. 1 , Wisler, G. 2 

1 California Citrus Research Board, Visalia, CA, USA 

2 USDA, ARS, GWCC-BLTSVL, Beltsville, MD, USA 

In 2007, the Citrus Health Response Plan (CHRP) was established as a national program and is a 

collaborative effort involving growers, federal and state regulatory personnel, and researchers. 

The Citrus Health Science and Technology Coordinating Group was formed as part of its 

organizational structure in December 2009. The Group’s approach to planning research is 

outcome-based, where emphasis is placed on the products of research - to manage or prevent 

HLB - even as a project is designed. This differs from evidenced-based research in which 

conclusions are drawn as data are analyzed. Three outcomes were identified: (1) how can groves 

currently affected by ACP and HLB remain productive, (2) how can the spread of ACP and HLB 

be prevented or slowed, and (3) what genetic and horticultural strategies can be pursued to keep 

groves free of ACP and HLB. These desired outcomes were used to organize the first Research 

Forum in June 2010 in Denver, CO. Participants assembled into three separate groups according 

to the outcomes and discussed what is known and what research is being conducted regarding 

that subject area. An inventory of research projects conducted within the United States and 

poster abstracts from the Denver meeting are available on the USDA APHIS web site: 

http://www.aphis.usda.gov/plant_health/cphst/ index.shtml. Notes from each of the outcome 

group breakout sessions are available on the USDA ARS web site: 

http://www.ars.usda.gov/citrusgreening/. 


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7.8 The Identification and Distribution of Citrus Greening Disease in Jamaica 

Oberheim, A.P., Brown, S.E., McLaughlin, W.A. Department of Basic Medical Sciences, 

University of the West Indies, Mona, Kinston 7, Jamaica 

Citrus huanglongbing (HLB), also known as citrus greening disease, is a devastating bacterial 

disease that affects all varieties of citrus, as well as citrus hybrids. Citrus HLB is caused by the 

Candidatus Liberibacter ssp. bacteria, of which there are three known species. These are Ca. L. 

asiaticus, Ca. L africanus, and Ca. L. americanus (Deng et al., 2008). Citrus leaf samples were 

collected island-wide from several varieties of citrus trees that appeared to be symptomatic. The 

symptoms observed were blotchy mottle and asymmetrical yellowing of the leaves, along with 

lopsided fruits and poor fruit quality. A total of 167 samples were tested for the presence of 

citrus greening using the primer pair OI1 and OI2c, of which 72% were positive. Positive 

samples were observed island-wide from all 14 parishes. The bacterium was found to be present 

in several species of citrus, including Citrus sinensis (orange), Citrus aurantium (sour orange), 

Citrus tangerine (tangerine), Citrus × paradisi (grapefruit), Citrus aurantifolia (West Indian 

lime), Citrus latifolia (Bearss lime), Citrus grandis (pommelo), and Citrus jambhiri (rough 

lemon). The HLB bacterium was also detected in the hybrids Citrus reticulata × Citrus paradisi 

(ugli) and ortanique. The most severe symptoms were observed in limes, and the least severe 

symptoms were observed in grapefruits. Select samples were cloned and sequenced, and blast 

analysis showed that the sequences were 99% similar to that of Ca L. asiaticus from Florida, 

Brazil, and Cuba. The results obtained indicate that the disease is widespread across Jamaica, 

and therefore measures such as removal and destruction of infected trees should be employed as 

a means of disease control. 

Reference 

Deng, X., Chen, J., Feng, Z., Shan, Z., Guo, H., Zhu, J, Li, H., Civerolo, E.L. 2008. 

Identification and characterization of the huanglongbing bacterium in pummelo from 

multiple locations in Guangdong, P. R. China. Plant Disease 92:513-518. 


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7.9 Fitting a Spatial Analysis Grid for Research on Huanglongbing in Mexico 

Aldama-Aguilera, C., Olvera-Vargas, L.A., Galindo-Mendoza, M.G. Sistema Nacional de 

Vigilancia Epidemiológica Fitosanitaria, Universidad Autónoma de San Luis Potosí, Sierra 

Leona N° 550, Lomas II Sección, S.L.P., Mexico 

Since the official report of huanglongbing (HLB) in Mexico in July 2009 to date, the bacterium 

has been detected in seven states. At a large spatial scale, HLB occurs in citrus plantings that are 

somewhat to highly discontinuous across most regions; many of them are solitary trees in 

backyards. This complicates determining the spatial pattern of the disease to draw conclusions 

concerning spread. The aim of this study was to generate a methodology for regional research on 

making management decisions regarding huanglongbing. Weather records from the National 

Weather Service and basic thematic mapping from INEGI were used. The information was 

stored and worked into the Geographical Information System ArcGIS 9.3, which, through 

multicriteria-operations and processed satellite images, we obtained the physical and climate 

conditions and potential risk of the bacterium and its vector, Diaphorina citri. Furthermore, we 

employed the MAXENT model (Maximum Entropy) with 19 climatic variables to extrapolate 

the autocorrelation sequences between the sites with HLB. A grid with 2381 cells, that covers all 

the country, was created; each one of the cells measures 15 minutes of latitude and 20 minutes of 

longitude. Thirty grid cells with highest risks were chosen to propose research nodes in areas 

with or without HLB in commercial citrus orchards and backyard trees. 


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Session 8: 

Host-Pathogen 

Interactions 


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8.1 Examination of Stages of the HLB Disease Development in Citrus Trees 

Folimonova, S.Y. 1 , Achor, D.S. 1 , Hilf, M.E. 2 



Citrus greening (huanglongbing, HLB) is a destructive disease of citrus, and its presence in the 

United States is a major threat to the entire citrus industry. The causal agent of HLB in Florida is 

thought to be Candidatus Liberibacter asiaticus. Understanding the early events in HLB infection 

are critical to develop effective methods to combat the disease. In order to characterize the stages 

of disease development, we conducted cytopathological studies following the progression of the 

infection in greenhouse-grown citrus trees graft-inoculated with Ca. L. asiaticus-containing 

material. An increasing degree of microscopic aberrations was observed as the disease symptoms 

progressed. The ability to observe the bacterium in the infected tissue also correlated with the 

degree of the disease progression. Large numbers of bacterial cells were found in phloem sieve 

tubes in tissue samples from pre-symptomatic young flushes. In contrast, we did not observe the 

bacteria in highly symptomatic leaf samples. Interestingly, these observations correlated with our 

recent findings made upon examination of the seed vascular bundle tissue from young immature 

and mature fruits collected from HLB-infected field trees. Large numbers of Ca. L. asiaticus 

bacteria were visualized in the sieve elements of the seed coats of the majority of seeds sampled 

from immature fruits, while no intact bacterial cells could be detected in these tissues from seeds 

of mature fruits. Thus, we can hypothesize that for a short period of time after movement of 

Ca. L. asiaticus into a growing part of an infected tree, the majority of the pathogen population is 

present as live bacteria. Later, as the symptoms develop and tissues mature, most of the bacteria 

become non-viable. 


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8.2 New Defense Response Insights of Sweet Orange Infected with Two Candidatus 

Liberibacter Species 

Mafra, V.S. 1 , Martins, P.K. 1 , Locali-Fabris, E.C. 1 , Ribeiro-Alves, M. 2 , Francisco, C.S. 1 , 

Freitas-Astúa, J. 1,3 , Kishi, L.T. 1 , Machado, M.A. 1 

1 Centro APTA Citros Sylvio Moreira-IAC, São Paulo, Brazil 

2 Fundação Oswaldo Cruz, FIOCRUZ, Rio de Janeiro, Brazil 

3 EMBRAPA Mandioca e Fruticultura Tropical, Bahia, Brazil 

Huanglongbing (HLB) is a destructive disease of citrus that is caused in Brazil by two fastidious, 

phloem-inhabiting bacteria species: Candidatus Liberibacter asiaticus and Candidatus 

Liberibacter americanus. HLB affects all known citrus species and relatives with little known 

tolerance. Upon recognition of a pathogen, plants often activate a complex series of responses 

that lead to the local and systemic induction of a broad spectrum of antimicrobial defenses. In 

order to investigate the transcriptional modulation of genes and pathways potentially involved 

with defense response of plants infected with Candidatus Liberibacter, we used our microarray 

data from sweet orange infected with CaLam and published microarray data from sweet orange 

infected with CaLas, and produced a consensus global gene expression profile. To compose the 

consensus list of genes and pathways differentially regulated, we used all genes with expression 

values two-fold, up- and down-regulated, in infected plants compared with control plants. Only 

non-redundant probe sets with Arabidopsis homologues were used. A common subset of 

47 probe sets differentially expressed was identified, comprising 30 up-regulated genes, 

9 down-regulated in both transcriptome data, and 7 that did not demonstrate the same expression 

pattern. Interestingly, we found 7 genes with transmembrane transporter activity; some of them 

are induced in response to Zn, Fe, and Cu deficiency, 4 genes involved response to bacteria, and 

several genes related to response to oxidative stress, SAR, and flavonoid metabolism. This study 

will provide new insights into gene expression modulation in HLB and direct the selection of 

potential targets for transformation. 


Financial support: INCT Citros, FCPRAC (Florida), and Embrapa-Monsanto Agreement. 


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8.3 Differential Expression of Potential Virulence Genes of Candidatus Liberibacter 

asiaticus in Infected Plants and Psyllids 

Sreedharan, A., Wei, S., Wang, N.-Y. UF-IFAS Citrus Research and Education Center, Lake 

Alfred, FL, USA 

Candidatus Liberibacter asiaticus is the causal agent of citrus greening or huanglongbing, a 

destructive disease of citrus in the United States. Citrus greening is usually transmitted through 

grafting or through feeding by the Asian citrus psyllid (Diaphorina citri). In order to identify the 

potential virulence genes, real-time quantitative PCR assays using total RNA isolated from 

infected plants and psyllids were conducted to test the expression profile of Ca. L. asiaticus. 

Gene specific primers were used to check the expression of more than 500 genes in 

Ca. L. asiaticus. The genes showing a differential expression of two-fold or more in either the 

plant or psyllid were selected and categorized into COG (Clusters of Orthologous Groups of 

proteins) functional categories. Selected genes that were overexpressed in planta were further 

studied by expression or screened on Nicotiana benthamiana plants for symptom expression, 

using transient assays. Interestingly, one orf encoding salicylate hydroxylase (sahA) was 

identified in the genome of Las. Salicylate hydroxylase is responsible for salicylic acid (SA) 

breakdown. SA is important for basal defense, hypersensitive response, and systemic acquired 

resistance. Expression assays indicate SahA is functional and can break down various 

salicylate-based substrates. In addition, constructs AS7 and AS13, expressing two hypothetical 

proteins, caused symptoms on the plants and will be further characterized using transgenic 

expression studies on Duncan grapefruit (Citrus paradisi Macf.). Since the virulence 

mechanisms of Ca. L. asiaticus is poorly understood, the results from this study will serve as the 

first step in identifying potential virulence genes involved in symptom expression and survival of 

this pathogen in planta. 


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8.4 Metabolome Analysis of Tolerant and Susceptible Citrus Varieties in Response to 

Infection with Candidatus Liberibacter asiaticus 

Albrecht, U. 1 , Skogerson, K. 2 , Bowman, K.D. 1 , Fiehn, O. 2 


2 University of California, Davis, CA, USA 

Metabolomics is a major branch of functional genomics together with transcriptomics and 

proteomics. Analysis of the metabolome, which is most directly associated with the phenotype, 

may yield important information about the mechanism of plant-pathogen interactions and may 

discover biomarkers valuable for diagnostic use. This study investigated the metabolic profile of 

citrus plants in response to infection with Candidatus Liberibacter asiaticus, the suspected causal 

agent of huanglongbing (HLB) in Florida. Leaf metabolites of infected and non-infected 

seedlings of the genotypes US-897 (Citrus reticulata Blanco × Poncirus trifoliata L. Raf.), 

tolerant to HLB, and Cleopatra mandarin, susceptible to HLB, were investigated at different 

times after graft-inoculation with the pathogen. GC-TOF-MS analysis identified 225 compounds 

that were significantly different across the study. PCA analysis resulted in distinct clustering of 

the two genotypes independent of infection (Fig. 1). Concentrations of many metabolites differed 

significantly in non-infected and infected Cleopatra seedlings, resulting in clear discrimination 

after PLS analysis upon progression of the disease (Fig. 2). Leaf symptoms of infected Cleopatra 

seedlings included chlorosis and size reduction. In contrast, fewer metabolites appeared to be 

significantly affected in US-897 seedlings upon infection, preventing a clear separation of 

metabolic profiles (Fig. 2). Most leaves of this genotype did not show any disease symptoms 

despite infection. Among the compounds most significantly increased in infected leaves of 

Cleopatra were several amino acids involved in polyamine- or alkaloid biosynthesis (Fig. 3a). 

Among the compounds found in high concentrations in US-897 leaves independent of infection 

were some with possible anti-microbial activity (Fig. 3b), which may be associated with 

tolerance to HLB observed in this genotype. 

References 

Albrecht, U., Bowman, K.D. 2011. HortScience 46:16-22. 

Fiehn, O., Wohlgemuth, G., Scholz, M., Kind, T., Lee, D.Y., Lu, Y., Moon, S., Nikolau, B.J. 

2008. Quality control for plant metabolomics: reporting MSI - compliant studies. The Plant 

Journal 53:691-704. 

Sumner, L.W., Mendes, P., Dixon, R.A. 2003. Plant metabolomics: large-scale phytochemistry 

in the functional genomics era. Phytochemistry 62:817-836. 


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PC2 (0.3%) 

US-897 

‘Cleopatra’ 

PC1 (99.2%) 

Fig. 1. Unsupervised PCA analysis of leaf metabolites shows distinct clustering of US-897 and 

Cleopatra seedlings independent of infection with Ca. Liberibacter asiaticus 


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Cleopatra 

Cleopatra 

US-897 

Fig. 2. PLS analysis of leaf metabolites shows clear separation of non-infected (red) and infected 

(green) Cleopatra profiles and overlapping of US-897 profiles 40 weeks after inoculation with 

Ca. Liberibacter asiaticus. 


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a) 

9.0E+05 

8.0E+05 

7.0E+05 

6.0E+05 

5.0E+05 

4.0E+05 

3.0E+05 

2.0E+05 

1.0E+05 

32 wai 

Ornithine 

40 wai 

0.0E+00 

b) 

2.0E+05 

1.8E+05 

Cleo Ctrl Cleo Inf US-897 Ctrl US-897 Inf Cleo Ctrl Cleo Inf US-897 Ctrl US-897 Inf 

Bin 309580 

32 wai 40 wai 

1.6E+05 

1.4E+05 

1.2E+05 

1.0E+05 

8.0E+04 

6.0E+04 

4.0E+04 

2.0E+04 

0.0E+00 

Cleo Ctrl Cleo Inf US-897 Ctrl US-897 Inf Cleo Ctrl Cleo Inf US-897 Ctrl US-897 Inf 

Fig. 3. Metabolites detected in Cleopatra and US-897 leaves infected (Inf) and non-infected 

(Ctrl) with Ca. Liberibacter asiaticus 32 and 40 weeks after inoculation (wai). 


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8.5 Deep Transcriptome Profiling of Citrus Fruit in Response to Huanglongbing Disease 

Martinelli, F. 1 , Uratsu, S.L. 1 , Albrecht, U. 2 , Reagan, R.L. 1 , Leicht, E. 3,4 , D’Souza, R. 3,4 , 

Bowman, K.D. 2 , Dandekar, A.M. 1 

1 Plant Sciences Department, University of California, Davis, CA, USA 

2 U.S. Horticultural Research Laboratory, U.S. Department of Agriculture, Agricultural Research 

Service, Fort Pierce, FL, USA 

3 Mechanical and Aeronautical Engineering Department, University of California, Davis, CA, 

USA 

4 Center for Computational Science and Engineering, University of California, Davis, CA, USA 

Huanglongbing (HLB) or citrus greening, potentially caused by Candidatus liberibacter species 

(CaLs), is considered the most destructive citrus disease worldwide. Current diagnosis requires 

PCR detection of the putative bacterial candidate but rarely detects the pathogen during early, 

asymptomatic infection stages. In this work, we used an alternative strategy based on the study 

of early host responses to infection using next-generation sequencing technologies by mining the 

deep mRNA profile at different stages of disease development with pathway and protein-protein 

network analysis, followed by quantitative real-time PCR analysis of highly regulated genes. We 

identified differentially regulated pathways and constructed networks that provide a vivid insight 

into the metabolism of fruit affected by HLB. Data mining revealed that HLB enhanced 

transcription of genes involved in the light reactions of photosynthesis and in ATP synthesis. The 

resultant oxidative stress was linked with the activation of protein degradation and misfolding. 

Transcripts for heat shock proteins were down-regulated at all stages of disease, exaggerating 

protein misfolding. HLB strongly affected pathways involved in source-sink communications 

such as sucrose and starch metabolism and hormone biosynthesis and signaling. Transcription of 

several genes involved in the biosynthesis and signal transduction of cytokinins and gibberellins 

was reduced, while that of genes involved in ethylene pathways was up-regulated. CaLs 

infection triggers a response via both the salicylic acid and jasmonic acid pathways. Transcripts 

of several members of the WRKY family of transcription factors increased greatly in response to 

CaLs infection. Significantly differentially expressed transcripts might be used as biomarkers of 

CaLs infection to improve early diagnosis at the asymptomatic stage using quantitative real-time 

PCR. 


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8.6 Carbohydrate Metabolism and Related Gene Expression Changes in 

Huanglongbing-Affected Sweet Orange 

Chen, C. 1 , Fan, J. 1,2 , Yu, Q. 1 , Brlansky, R.H. 1 , Li, Z.-G. 2 , Gmitter, F.G., Jr. 1 


2 Key Laboratory of Biorheological Science and Technology, Ministry of Education, College of 

Bioengineering, Chongqing University, Chongqing 400030, China 

Citrus huanglongbing (HLB) is caused by Candidatus Liberibacter spp., the presumed, 

uncultured pathogen. In this study, significant changes in carbohydrate metabolism in 

HLB-affected sweet orange (Citrus sinensis L. Osbeck), comparing asymptomatic and 

symptomatic tissue to their healthy counterpart, respectively, were observed. In asymptomatic 

leaves, sucrose and fructose accumulated significantly (p < 0.05) in both midribs and lobes, and 

glucose only in the midribs (greater than 5-fold); whereas, maltose levels were reduced to 64.6% 

and 86.8% of the healthy level in the midribs and foliar lobes. In symptomatic leaves, sucrose 

and glucose remained at higher levels, while no accumulation of fructose was observed; by 

contrast, the maltose content decreased to 49.6% of that in healthy leaves. Starch levels increased 

3.1- and 7.9-fold in HLB-infected asymptomatic and symptomatic leaves compared to the 

healthy level, respectively. Four-fold increase of the cell wall-bound invertase activity was 

detected in both types of infected leaves by the invertase assay. In addition, the starch breakdown 

gene expression profile suggested that the transcription of DPE2 and MEX1 were 

down-regulated. Together with the reduction of maltose, it is suggested that the impairment of 

starch breakdown may contribute to the starch accumulation in infected leaves. The imbalance of 

carbohydrate partitioning and its relation to HLB pathogenicity in citrus are discussed. 


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8.7 Analysis of Colonization of Citrus Seeds by Ca. Liberibacter asiaticus and Its Possible 

Role in Seed Transmission 

Hilf, M.E. USDA-ARS, Fort Pierce, FL, USA 

The huanglongbing disease of citrus is associated with infection by the phloem-colonizing 

bacterium Ca. Liberibacter asiaticus. Transmission by the psyllid Diaphorina citri and 

transmission by vegetative propagation of infected citrus budwood are experimentally verified 

methods of short- and long-distance dispersal of the bacterium. Vertical transmission through 

infected seed has not been verified but is a concern as a means of moving the pathogen to areas 

in which it currently is not found. To address this question of seed transmission, mature seeds 

from fruit from infected Sanguinelli sweet orange and Conner’s grapefruit trees were 

apportioned into three populations, with one group destructively sampled with analysis of the 

separated seed coats and embryo, one group germinated in soil in the greenhouse, and one group 

germinated on agar in vitro after removal of the seed coat for separate analysis. Real-time PCR 

analysis of nucleic acid extracts from separated seed coats and embryos detected bacterial DNA 

in seed coats from both varieties but not in embryos. Similarly, bacterial DNA was detected in 

seed coats from both varieties that were removed prior to germination in vitro on agar. Bacterial 

DNA was not detected in seedlings of either variety that germinated in vitro. No bacterial DNA 

was detected in seedlings of either variety that germinated from seeds planted in soil in the 

greenhouse. The data suggest that the embryo is not infected during development even though 

bacteria are present in adjacent tissue. 


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8.8 Natural Transmission of Huanglongbing Caused by Candidatus Liberibacter 

americanus and Ca. L. asiaticus and with Two Different Sources of Inoculum Plants (Citrus 

sinensis or Murraya exotica) 

Gasparoto, M.C.G. 1 , Bassanezi, R.B. 2* , Amorim, L. 1* , Montesino, L.H. 2 , Lourenço, S.A. 1 , 

Wulff, N.A. 2* , Bergamin Filho, A. 1* 

1 ESALQ/USP, Piracicaba, Brazil 


Candidatus Liberibacter asiaticus and Ca. L. americanus are associated with huanglongbing 

(HLB) in Brazil. The objective of this study was to compare the natural transmission of HLB 

caused by both Liberibacter species using two different sources of inoculum plants (SIP): Citrus 

sinensis or Murraya exotica. The experiment was carried out with different SIP, corresponding 

to the following treatments: (i) Sweet orange plants infected by Ca. L. asiaticus or Ca. L. 

americanus, (ii) only M. exotica plants infected by Ca. L. americanus, and (iii) only sweet orange 

plants infected by Ca. L. americanus. Each treatment was applied in an isolated screenhouse 

compartment. The first treatment was repeated. In all compartments, healthy sweet orange plants 

(test plants - TP) were located around the SIP. Reared Liberibacter-free Asian citrus psyllids 

were monthly confined on the SIP for 7 days and later they were released for free movement and 

rearing inside the screenhouse. Leaf samples from TP were periodically collected for 

Liberibacter detection by real-time PCR. In the first treatment, 28 months after the beginning of 

the experiment, the average incidence of TP infected by Ca. L. asiaticus (11.5%) was higher than 

the average incidence of Ca. L. americanus infected TP (0.9%). In the same period, the second 

treatment had 7.6% of Ca. L. americanus infected TP, while the third treatment did not have any 

infected TP. These results showed that Ca. L. asiaticus is better transmitted by ACP than Ca. L. 

americanus and when there is only SIP infected by Ca. L. americanus, the highest transmission is 

reached when M. exotica served as SIP. 


Financial support: Fundecitrus, FAPESP (2007/55013-9) and CRDF (NAS-7). 

*Authors granted by CNPq. 


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8.9 Callose Predominates over Phloem Protein 2 in Phloem Plugging of Trees Affected 

with Huanglongbing 

Albrigo, L.G., Achor, D.S. UF-IFAS Citrus Research and Education Center, Lake Alfred, FL, 

USA. albrigo@ufl.edu 

Phloem plugging of citrus trees affected with huanglongbing (HLB) was shown previously to 

result from two types of materials in sieve elements, callose, and a lectin of phloem protein 2 

(PP2) (Achor et al., 2009). An associated study (Etxeberria et al., 2009) found that diversion of 

sugars to starch accumulation left the root system of affected trees lacking carbohydrate reserves 

as starch. It was proposed that this plugging and sugar diversion might be responsible for the 

decline of HLB-infected trees. This study evaluated plugging in four cultivars (Flame, Valencia, 

Hamlin, and Murcott) in field sites, and two cultivars (Duncan and Valencia) in a greenhouse to 

determine if one plugging type predominated and was a better candidate for transformation or 

some other type of blocking of its production. Field and greenhouse plants affected by HLB were 

sampled and leaf tissue prepared for transmission electron microscopy. A total of 22 field and 

7 greenhouse trees were sampled to observe leaf phloem plugging types. Callose plugging 

predominated in all samples with a range of callose to PP2 of 1.8 to 13. The average ratio was 

2.7 for all field samples and 2.4 for greenhouse samples. This data suggests that callose plugging 

is more likely to cause phloem dysfunction, but PP2 plugging accounts for over 25% of the 

phloem plugs. 

References 

Achor, D.S., Etxeberria, E., Wang, N., Folimonova, S.Y., Chung, K.R., Albrigo, L.G. 2010. 

Sequence of anatomical symptom observations in citrus affected with huanglongbing 

disease. Plant Pathology Journal 9(2):56-64. 

Etxeberria, E., Gonzales, P., Dawson, W., Achor, D.S., Albrigo, L.G. 2009. Accumulation and 

distribution of abnormally high levels of starch in HLB-infected Valencia orange trees. 

Physiological and Molecular Plant Pathology 74:76-83. 


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8.10 Influence of Huanglongbing (HLB) on the Composition of Citrus Juices and Mature 

Leaves 

Cancalon, P.F., Bryan, C., Haun, C., Zhang, J. Florida Department of Citrus, Lake Alfred, FL, 

USA 

In this study, the composition of fruit and mature citrus leaves from Hamlin and Valencia orange 

and Marsh grapefruit, collected in an infected Florida grove, was followed over time during the 

2009-2010 citrus season in control, symptomatic (exhibiting symptoms), and asymptomatic trees 

(HLB infected but not showing symptoms). In most cases, the composition of components from 

control and asymptomatic samples were similar. 

As a continuation to a previous study (Cancalon et al., 2008), orange juice composition was 

further examined. Synephrine, a biological amine characteristic of immature fruit, was found to 

be consistently higher in HLB-infected fruit. The synephrine values during the season were 

69.38 ± 20.31 mg/l and 38.93 ± 9.57 mg/l for symptomatic and control Hamlin; whereas, the 

values were 50.23 ± 20.66 mg/l and 22.99 ± 5.63 mg/l for symptomatic and control Valencia, 

respectively. Limonin was also found to be higher in HLB-infected fruit. In symptomatic 

Valencia, the limonin concentration was 11.1 ± 9.4 mg/l as compared with 1.7 ± 3.5 mg/l for 

control; in Hamlin oranges, the values were 9.8 ± 4.6 mg/l and 1.3 ± 2.3 mg/l, respectively. 

Most of the study was devoted to the examination of mature leaves. Glucose, fructose, 

sucrose, and maltose concentrations were analyzed once a month during the 2009-2010 season 

by HPLC/PAD as reported previously (Cancalon, 1993). In order to determine the origin of the 

maltose, α- (EC 3.2.1.1) and β- (EC 3.2.1.2) amylase activities were also measured (Megazyme 

assay kits, Wicklow, Ireland). 

Because of large sample variations, glucose, fructose, and sucrose levels did not vary 

significantly in Hamlin, Valencia, or grapefruit, although a trend toward higher glucose and 

fructose was noted in symptomatic grapefruit leaves. Small amounts of maltose, at about two 

orders of magnitude less than the other sugars were detected. Maltose concentrations were about 

three times higher in symptomatic than in control and asymptomatic leaves. Kaplan and Guy 

(2004) postulated that a maltose accumulation may function as a solute stabilizing factor in 

response to abiotic stress. 

Both α- and β-amylase activities were detected in leaf samples. Only maltose and 

α-amylase activity were affected by the disease and were significantly higher in HLB 

symptomatic leaves. β-amylase was not found to vary with time or with any type of leaves and 

remained fairly low. No significant amounts of maltotriose, an α-amylase byproduct, could be 

found. During the season, in both Hamlin and Valencia, levels of maltose and α-amylase activity 

varied in a similar manner, indicating a possible link between the enzyme and the product. They 

peaked in April then decreased significantly until the end of the study in July. Similar changes 

occurred in grapefruit leaves but were less striking. This late decrease in maltose and amylase 

levels in the leaves may be a reaction to the removal of fruit from the trees. It should also be 


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pointed out that an increase in maltose appears to coincide with the January 2010 freezes, which 

raises the possibility that a local climate effect may not be ruled out as influencing part of the 

observed changes. 

In conclusion, high levels of maltose and α-amylase activity seem to be characteristic of 

mature HLB leaves; in this study, the potential relationship with variations in starch levels was 

not examined. The study has also revealed the difficulty of conducting research in the field, 

where many factors such as temperature, humidity, and other physiological stresses on the trees 

cannot be controlled. It is also important to note that both symptomatic and non-symptomatic 

groups are not homogeneous and that both fruit and leaves in these groups can have very 

different biochemical compositions. 

References 

Cancalon, P.F. 1993. Oligosaccharides generation in acidic sugar media. Journal of the 

Association of Official Analytical Chemists 76:584-590. 

Cancalon, P.F., Bryan, C., Haun, C., King, D. 2008. Influence of HLB on the composition of 

citrus juices. 59 th Citrus Processors= and Subtropical Technology Conference 59:29-30. 

Kaplan, F., Guy, C. 2004. β-amylase induction and the protective role of maltose during 

temperature shock. Plant Physiology 135:1674-1684. 


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8.11 Gene Expression in Citrus sinensis Fruit Tissues Harvested from 

Huanglongbing-Infected Trees 

Liao, H.-L., Burns, J.K. UF-IFAS Citrus Research and Education Center, Lake Alfred, FL, 

USA 

Global gene expression in HLB-infected fruit tissues was evaluated using an Affymetrix array 

containing 30,279 Citrus spp. ESTs. Flavedo (FF), vascular tissue (VT), and juice vesicle (JV) 

tissue of symptomatic (SY), asymptomatic (AS), and healthy (H) fruit harvested from sweet 

orange Hamlin and Valencia cultivars was investigated. Transcriptional profiles indicated the 

number of genes impacted by HLB was highest in FF and VT and least in JV. In Hamlin, over 

860 genes were changed in SYVT and SYFF compared with H tissues. In Valencia, fewer genes 

were changed in SYVT and SYFF, but approximately 50% additional genes (397 genes) were 

changed in SYJV compared with Hamlin. As in SY tissues, ASVT and ASFF were strongly 

impacted and had similar gene changes in Valencia. However, only 28 genes were altered in 

ASJV compared with HJV. The top five gene groups with high numbers of changes included 

transporters, carbohydrate metabolism, genetic information processes, phytohormone 

metabolism, and defense responses. Highest titer of the HLB bacterium was found specifically 

located in SYVT. Gene changes in SYVT were associated with host-pathogen interaction and 

seed abortion, and those in SYJV with fruit size, juice sac maturity and morphology, and juice 

quality. Similar to SY fruit, girdled fruit were small and could abscise prematurely. 

A comparison of gene expression between HLB-infected and girdled treatments will be 

presented to differentiate HLB-mediated gene changes from those related to girdling-related 

carbohydrate loss. 


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8.12 Expression Profiling of Host Response of Citrus to Candidatus Liberibacter asiaticus 

Infection 

Aritua, V., Wang, N.-Y. UF-IFAS Citrus Research and Education Center, Lake Alfred, FL, 

USA 

Huanglongbing (HLB) disease is currently the most economically damaging disease of citrus 

worldwide. Three species of Candidatus Liberibacter; Ca. Liberibacter asiaticus (Las), Ca. 

Liberibacter africanus, and Ca. Liberibacter americanus have been associated with the disease. 

These phloem-limited fastidious α-proteobacteria are vectored by Asian citrus psyllid 

(Diaphorina citri) and African citrus psyllid (Trioza erytreae). Of the three, Las has a worldwide 

distribution in citrus including Florida. Typical symptoms of HLB are mottling and chlorosis of 

leaves, yellow shoots, and lopsided fruit with green color remaining on the stylar end with 

aborted seeds and bitter taste. Many of the symptoms are similar to zinc deficiency and have 

been linked to starch accumulation. It was reported that Las infection results in significant 

differences in response between different citrus varieties or relatives, although none has been 

found to be resistant. Systemic infection studies show uneven distribution patterns of Las 

populations among different organs, tissues, and varieties of citrus. These results suggest that 

cellular response to Las is genotype dependent and may be tissue specific. Our previous study of 

gene expression in leaves showed differential gene expression patterns in leaf tissues of 

susceptible sweet orange varieties. In addition, Las infection of citrus is affected by the 

environmental conditions. In order to further understand the mechanism of citrus infection by 

Las, we are currently assessing citrus genes modulated by Las infection in 1) citrus stems and 

roots in the greenhouse and 2) citrus leaves in the citrus grove. 


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8.13 Arabidopsis Responses to the HLB-Relative Candidatus Liberibacter psyllaurous 

Patne, S. 1 , Manjunath, K.L. 2 , Roose, M.L. 1 


2 USDA-ARS, Riverside, CA, USA 

The impact of HLB disease on the citrus industry demands systems for more rapid investigation 

of HLB control strategies. To identify HLB targets using a high-throughput chemical genomics 

approach, we analyzed Arabidopsis thaliana, a tractable model system, for responses to 

Candidatus Liberibacter psyllaurous (CLps). Sequence comparisons indicate Candidatus 

Liberibacter asiaticus and Candidatus Liberibacter psyllaurous (pathogen associated with tomato 

psyllid yellows disease) are closely related species. Two widely used Arabidopsis ecotypes Col-0 

and Ler-1 were tested for Candidatus Liberibacter psyllaurous infection and disease related 

symptoms. Quantitative real-time PCR with CLps-specific primers showed that both ecotypes 

could be infected by psyllids fed on CLps-infected tomato. A higher percentage of Ler-1 than 

Col-0 plants were positive (89% vs. 23%) and Ler-1 plants had lower Ct values. CLps systemic 

infection was confirmed by testing leaf, shoot, and inflorescence samples from both ecotypes. 

Shoot and inflorescence samples showed Ct values 24-28, indicating a relatively higher titer of 

pathogen than in leaves (Ct value 31-33). Interestingly, no infected plants showed disease-related 

symptoms. Twenty genetically diverse A. thaliana ecotypes were tested to assess variation in 

resistance and disease phenotype. QPCR of leaf samples collected 11 days post-inoculation with 

psyllids showed that at least 4 out of 5 plants were CLps positive in ecotypes Fei-0 and Ler-1. 

No CLps positive plants showed disease-related symptoms. It is evident that Arabidopsis can be 

infected with CLps; however, the asymptomatic phenotype is intriguing and systematic genetic 

dissection of this response could identify potential targets useful for plant defense in citrus. 

References 

Clark, R.M., Schweikert, G., Ossowski, S., Zeller, G., Toomajian, C., Shinn, P., Warthmann, N., 

Hu, T.T., Fu, G., Hinds, D.A., Chen, H., Frazer, K.A., Huson, D.H., Schölkopf, B., 

Nordborg, M., Rätsch, G., Ecker, J.R., Weigel, D. 2007. Common sequence 

polymorphisms shaping genetic diversity in Arabidopsis thaliana. Science 317:338-342. 

Hansen, A.K., Trumble, J.T., Stouthamer, R., Paine, T.D. 2008. A new huanglongbing species, 

Candidatus Liberibacter psyllaurous, found to infect tomato and potato, is vectored by the 

psyllid Bactericera cockerelli (Sulc). Applied and Environmental Microbiology 

74:5862-5865. 

Li, W., Abad, J.A., French-Monar, R.D., Rascoe, J., Wen, A., Gudmestad, N.C., Secor, G.A., 

Lee, I.-M., Duan, Y., Levy, L. 2009. Multiplex real-time PCR for detection, identification 

and quantification of Candidatus Liberibacter solanacearum in potato plants with zebra 

chip. Journal of Microbiological Methods 78:59-65. 

Li, W., Hartung, J.S., Levy, L. 2006. Quantitative real-time PCR for detection and identification 

of Candidatus Liberibacter species associated with citrus huanglongbing. Journal of 

Microbiological Methods 66:104-115. 


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8.14 Comparative Studies of the Endophytic Microbial Community Structures in 

Huanglongbing-Infected and Non-Infected Citrus Plants 

Zheng X.-F. 1 , Liu, B. 1* , Ruan, C.-Q. 1 , Lin, Y.-Z. 1 , Xiao, R.-F. 1 , Zhu, Y.-J. 1 , Fan, G.-C. 1 , Cai, 

Z.J. 1 , Duan, Y.-P. 2 

1 Citrus Huanglongbing Research Center of Fujian Academy of Agricultural Sciences, Fuzhou, 

China 

2 U.S. Horticultural Research Laboratory USDA-ARS, Fort Pierce, FL, USA 

*Corresponding author: Prof. LIU Bo, E-mail: fzliubo@163.com 

Endophytes in both huanglongbing (HLB)-infected and non-infected citrus plants were studied 

using a phospholipid fatty acid (PLFAs) assay in conjunction with culturing. Forty isolates of 

endophytes were obtained from Citrus reticulata Blanco (cv. Hongroumiyou) by culturing on 

NA medium. Results from PLFAs assay indicated that the 40 isolates of endophytes belong to 

4 genuses, 16 species. The species and quantity of endophytes were significantly different 

between HLB-infected and non-infected plants with more endophytes in HLB-infected plants. 

The endophyte species such as Bacillus subtilis, Klebsiella pneumoniae, and B. licheniformis 

were only found in non-infected plants, while B. laevolacticus, Paenibacillus lentimorbus, 

B. atrophaeus, and Microbacterium lacticum were found in HLB-infected plants. PLFA types 

and quantity in citrus leaves varied along with different varieties and development of HLB 

infection. The varieties Lo Tangerines, Fuju, and Navel Oranges showed PLFAs from the highest 

to the least, respectively. Cluster analysis revealed community characteristics of endophytes in 

citrus leaves were more relevant to citrus variety than to the development stage of HLB 

infection. Several PLFA biomarkers were also identified as potential diagnostic markers; 

18:3 w6c (6,9,12), 11:0 and 15:03OH may be used to distinguish HLB-infected from the 

non-infected leaves of the Fuju variety, while a19:0, i15:0, 16:12OH, 10ME17:0, and i17:0 may 

be used to distinguish asymptomatic from non-infected leaves. Future applications of the PLFA 

assay in HLB microbial ecology and diagnostics are also discussed. 


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8.15 HLB Influences the Diversity, Structure, and Function of the Bacterial Community 

Associated with Citrus 

Trivedi, P., Wang, N.-Y. UF-IFAS Citrus Research and Education Center, Lake Alfred, FL, 

USA 

The diversity and stability of plant-associated bacterial communities heavily markedly influence 

soil and plant quality and ecosystem sustainability. The goal of this study is to understand how 

Candidatus Liberibacter asiaticus [Las, known to cause huanglongbing (HLB)] influences the 

structure and function of plant associated bacterial community. We have used high density 

phylogenetic microarray (Phylochip), clone library sequencing, and taxon specific quantitative 

PCR (qPCR) based analysis to provide a detailed census of bacterial communities present in 

HLB symptomatic and non-symptomatic citrus (Sagaram et al., 2009; Trivedi et al., 2010). Our 

results indicate Las to be the only causal agent of HLB in Florida (Sagaram et al., 2009) and 

reveals that HLB significantly restructures the composition of the native microbial community 

present either in leaf or roots of citrus (Sagaram et al., 2009; Trivedi et al., 2010). Cultivable 

diversity of bacteria associated with citrus was investigated, and several novel strains with the 

potential to enhance plant growth and suppress diseases were identified (Trivedi et al., 2011). 

qPCR and functional microarray “Geochip” analysis showed that HLB infection has a profound 

effect on the abundance of various genes involved in important ecological processes. Overall, 

our study shows that HLB influences the community structure and function of plant associated 

bacteria and will have serious consequences in productive capacity and sustainability of 

agro-ecosystems in the citrus groves. 

References 

Sagaram, U.S., DeAngelis, K.M., Trivedi, P., Andersen, G.L., Lu, S., Wang, N. 2009. Bacterial 

diversity analysis of HLB pathogen-infected citrus using PhyloChips and 16S rDNA clone 

library sequencing. Applied and Environmental Microbiology 75(6):1566-1574. 

Trivedi, P., Duan, Y., Wang, N. 2010. Huanglongbing, a systemic disease, restructures the 

bacterial community associated with citrus roots. Applied and Environmental Microbiology 

76(11):3427-3436. 

Trivedi, P., Spann, T., Wang, N. 2011. Isolation and characterization of beneficial bacterial 

community associated with citrus roots. Applied and Environmental Microbiology 

(Submitted). 


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8.16 Functional Studies of Putative Effectors of Candidatus Liberibacter asiaticus Using 

Citrus Tristeza Virus Vector 

Hajeri, S. 1 , Duan, Y.-P. 2 , Gowda, S. 1 


2 U. S. Horticultural Research Laboratory, USDA-ARS, Fort Pierce, FL, USA 

Citrus huanglongbing (HLB), also known as citrus greening, is one of the most destructive 

diseases of citrus worldwide. The causative agent, Candidatus Liberibacter asiaticus (CLas), is a 

fastidious, gram-negative, phloem-limited, α-proteobacterium. The pathogen is vectored by a 

psyllid, Diaphorina citri Kuwayama. Thus, CLas is both an intracellular plant pathogen and an 

insect endosymbiont. Because of its intracellular nature, there is a significant reduction in the 

genome (1.23 Mb) compared to other members of the family. It is possible that the important 

genes responsible for pathogenicity in CLas might be buried in the large amount of hypothetical 

conserved ORFs, which constitutes nearly 26% of the genome. Using bioinformatics tools, we 

have identified a number of putative effector genes based on the genome sequence of CLas. By 

the use of citrus tristeza virus (CTV) vector, we could express putative effectors (pathogenicity 

and virulence genes) of the CLas bacterium directly inside the phloem of citrus. At present, 

20 different genes encoding putative effector proteins from Las-infected citrus plants were 

amplified and cloned behind heterologous beet yellows virus CP subgenomic RNA controller 

element engineered between the CPm and CP genes in the CTV vector. The CTV virions 

containing different HLB effectors will be used to inoculate citrus plants by bark-flap 

inoculation. The resulting systemic spread and expression of the putative effectors throughout 

citrus trees will enable us to understand the role of the putative effectors in disease induction. 


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8.17 First Report of a New Host (Pithecellobium lucidum Benth) of the Citrus 

Huanglongbing Bacterium, Candidatus Liberibacter asiaticus 

Fan, G.-C. 1 , Cai, Z.J. 1 , Weng, Q.Y. 1 , Ke, C. 1 , Liu, B. 1 , Zhou, L.J. 2 , Duan, Y.-P. 2 

1 Citrus Huanglongbing Research Center of Fujian Academy of Agricultural Sciences, Fuzhou, 

China 


Most, if not all, host plants of Candidatus Liberibacter asiaticus are members of the Rutaceae 

family due to the feeding preference of its insect vector, Diaphorina citri (Halbert and 

Manjunath, 2004). A shrub of non-Rutaceae plants showing yellow shoots, mimicking the 

symptom of citrus huanglongbing (HLB), was observed in October 2008 in a citrus orchard in 

Fujian, China, where citrus plants were severely infected by HLB. The tree was identified as 

Pithecellobium lucidum Benth. Samples collected from symptomatic and asymptomatic branches 

of the tree were subjected to DNA isolation using midrib and CTAB method. Ca. L. asiaticus 

was detected by conventional PCR using 16S rDNA-based primer set (CG03F/CG05R) (Zhou 

et al., 2007) and nested PCR using β-operon-based primer sets (F1/R1 and F2/R2) (Ding et al., 

2005). Sequence analysis revealed that the PCR amplicons (ca. 800 bp and 400 bp) showed 99% 

identity with Las 16S rDNA and 100% identity with Las β-operon, respectively, at the nucleotide 

level. In addition, a few Las-like bacterial cells were observed in the sieve cells of infected 

samples as seen with electron microscope. This is the first report of P. lucidum as a naturally 

infected new host of Ca. L. asiaticus. The results of low Las bacterial titer and that the psyllid 

did not propagate in this host plant indicate that the new host is an opportunistic host of HLB. 

References 

Ding, F., Wang, G., Yi, G., Zhong, Y., Zeng J., Zhou, B. 2005. Infection of wampee and lemon 

by the citrus huanglongbing pathogen (Candidatus Liberibacter asiaticus) in China. Journal 

of Plant Pathology 87:207-212. 


greening disease of citrus: a literature review and assessment of risk in Florida. Florida 

Entomologist 87(3):330-353. 

Zhou, L.J., Gabriel, D.W., Duan, Y.-P., Halbert, S.E., Dixon, W.N. 2007. First report of dodder 

transmission of huanglongbing from naturally infected Murraya paniculata to citrus. Plant 

Disease 91(2):227-227. 


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8.18 Citrus Seed Grafting: A Simple Technology for Testing Seed Transmission of Citrus 

Greening/HLB and of Other Pathogenic Agents 

Bar-Joseph*, M., Robertson, C., Hilf, M.E., Dawson, W.O. UF-IFAS Citrus Research and 

Education Center, Lake Alfred, FL, USA 

*Formerly at the S. Tolkowsky Laboratory, ARO, Volcani Center, Bet Dagan, Israel 

Grafting plants is widely practiced for horizontal transmission of systemic pathogens. Vertical 

transmission of viruses and bacteria from infected mother plants through gametes to seedling 

plants is rare and often completely absent. Indeed it is commonly known that phloem-limited 

citrus pathogens such as citrus tristeza virus and Spiroplasma citri will not pass from the mother 

plant phloem tissues to citrus embryos and, thus, neither disease is seed transmissible. The recent 

reports on possible vertical transmission of the CLas agents from seeds collected from 

greening/HLB-infested plants to citrus seedling plants had caused considerable phytosanitary 

concern and an immediate halt on the movement of citrus seeds from infested to disease-free 

geographic regions. The most common method of testing seed transmissions is by sowing seeds 

from infected plants and testing the resulting seedlings for the presence of the infectious agents 

by symptom observations and PCR diagnosis. We have recently developed a new seed grafting 

technique that allows rapid and economical testing of seed transmission of pathogens. Using this 

method, we found neither symptoms nor positive PCR detection of Candidatus Liberibacter 

asiaticus (CLas) in Volkamer lemon or Duncan grapefruit seedlings grafted with seeds collected 

from Hamlin sweet orange or Conner’s grapefruit showing typical greening/HLB symptoms, 

whereas the similar plants side-grafted with greening/HLB-infected tissues comprising of 

mid-veins from young and small (

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8.19 Lack of Transmission of HLB by Citrus Seed 

Graham, J.H. 1 , Johnson, E.G. 1 , Bright, D.B. 1 , Irey, M.S. 2 



In Florida nurseries, rootstock seed trees are located outdoors and only protected from psyllid 

transmission of Candidatus Liberibacter asiaticus (Las) by insecticide applications. In 2008, 

a survey detected two Carrizo citrange trees as HLB+. Given the potential risk for seed 

transmission and introduction of Las into nurseries by seed from source trees, assays of seedlings 

derived from seed extracted from symptomatic fruit were begun in 2006. From 2006 to 2008, 

seed were collected from mature Pineapple sweet orange trees in Collier County and in 2009, 

from Murcott tangor trees in Hendry County, FL. For Pineapple orange, 415, 723, and 

439 seedlings and for Murcott, 332 seedlings were tested at least twice by qPCR using 16S 

primers. In 2007, a single Pineapple seedling was suspect HLB+ but upon repeated testing was 

negative. From nurseries in 2008, 290 seedlings were recovered from fruit located on 

symptomatic branches of two Carrizo trees, and in 2009, 125 seedlings were recovered from two 

trees of Swingle citrumelo, 649 from four trees of Kuharske Carrizo, 100 from one tree of 

Cleopatra mandarin, and 100 from one tree of Shekwasha mandarin. In 2008, one suspect HLB+ 

Carrizo seedling was detected, but HLB+ status was not confirmed after repeated testing. In 

2009, a single questionable PCR detection for Cleopatra mandarin was obtained. Subsequent 

detection occurred in only 75 and 33% of repeated 16S runs from two DNA extractions and the 

assay was negative using β-operon primers. Despite the occasional HLB+ test results, no plants 

have ever developed HLB symptoms, and repeated testing has never confirmed anything other 

than the transient presence of Las in seedlings grown from seed obtained from Las-infected trees. 


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8.20 Visualization of Ca. Liberibacter asiaticus in Immature Citrus Seed Coats by 

Fluorescent In Situ Hybridization (FISH) of 16S rRNA 

Hilf, M.E. USDA-ARS, 2001 South Rock Road, Fort Pierce, FL, USA 

The huanglongbing disease of citrus is associated with infection by the non-cultivable bacterium 

Ca. Liberibacter asiaticus. During the course of a study on seed transmission in sweet orange and 

grapefruit, Ca. Liberibacter asiaticus DNA was detected in nucleic acid extracts from seed coats 

but not from embryos using real-time and conventional PCR. Low crossover threshold values 

(Ct) from real-time PCR assays suggested large numbers of bacteria were present in the intact 

seed coats. As PCR does not distinguish if bacterial DNA is derived from live or dead cells and 

as this bacterium is non-cultivable, FISH specific for 16S rRNA was applied to thin sections of 

immature grapefruit seeds to provide indirect evidence of the presence and the viability of 

bacterial cells. Fluorescence is expected from bacterial cells which have a high concentration of 

ribosomes so that the copy number of the 16S rRNA is 10,000 to 100,000 per cell, indicating 

high levels of protein synthesis and hence viable, metabolically active cells. In August 2010, 

immature seeds were collected from fruit from an infected Conner’s grapefruit tree, fixed and 

imbedded in paraffin. Intense fluorescence was observed from phloem cells within the vascular 

bundle and individual bacterial cells were observed in large numbers. This data suggests that, at 

least in immature seeds, large numbers of viable cells are present in the seed coat, but not in the 

embryo. 


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8.21 Rapid, Sensitive, and Non-Radioactive Tissue-Blot Diagnostic Method for the 

Detection of Citrus Greening Disease (HLB) 

Gowda, S. 1 , Nageswara Rao, N. 1 , Miyata, S. 2 , Ghosh, D.K. 3 , Irey, M.S. 4 , Rogers, M.E. 1 , 

Garnsey, S.M. 1 


2 National Institute of Fruit Tree Science, NARO, Japan 

3 National Research Center for Citrus, ICAR, India 


Citrus huanglongbing (HLB), also known as citrus greening, is one of the most devastating 

diseases of citrus worldwide. The disease is caused by gram-negative, phloem-limited 

α-proteobacterium, Candidatus Liberibacter asiaticus, vectored by the Diaphorina citri 

Kuwayama. Citrus plants infected by the citrus greening bacterium may not show visible 

symptoms sometimes for years following infection, and non-uniform distribution within the tree 

makes the detection of the pathogen very difficult. Efficient management of HLB disease 

requires rapid and sensitive detection early in the infection followed by eradication of the source 

of pathogen and the vector. The polymerase chain reaction (PCR) based method is most 

commonly employed for screening the infected/suspected HLB plants and psyllids. This is time 

consuming, cumbersome, and not practical for screening large number of samples in the field. To 

overcome this, we have developed a simple, sensitive, non-radioactive, tissue-blot diagnostic 

method for early detection and screening of HLB disease. Digoxigenin-labeled molecular PCR 

and rioboprobes specific to Candidatus Liberibacter asiaticus sequences have been developed 

and used for the detection of HLB in plants and psyllids. 


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Session 9: 


Management 


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9.1 A Database for Analysis of Diaphorina citri Population Monitoring Data from 

Commercial Groves 

Gast, T. 1 , Irey, M.S. 2 , Hou, H. 2 

1 Southern Gardens Citrus, Clewiston, FL, USA 


The presence of the Asian Citrus Psyllid, Diaphorina citri Kuwayama (ACP), was first noted in 

the commercial orange groves of Southern Gardens Citrus Corporation (SGC) in the summer of 

2000. By the summer of 2001, ACP was well established in all areas of this 21,500 acre 

operation. Huanglongbing (HLB) or citrus greening disease, caused by the bacterium Candidatus 

Liberibacter asiaticus, was first documented at SGC on October 11, 2005. As part of its HLB 

management program, SGC began a formal program for monitoring the population of the ACP in 

September 2007. In November 2008, protocols for field scouting were revised, and a database 

for documentation and analysis of scouting results was developed. In order to determine the most 

efficient method of scouting, psyllid population data has been monitored in the field using two 

different survey methods, the tap method and monitoring of growth flushes with a hand lens. 

Psyllid population trends at two commercial groves have been documented using both methods 

for over 2 years. In addition to providing a tool for day to day management decisions, the 

database has made it possible to compare the different scouting methodologies, and to correlate 

results obtained to subsequent HLB infection levels. Use of this or similar databases will enable 

growers to predict future HLB infection levels in commercial citrus groves based on current ACP 

population data, along with other factors. This data should also help the Florida citrus industry to 

establish action thresholds for ACP populations in commercial groves. 


Data analyzed in this study was obtained from a large, commercial dataset from observations 

made by full-time, experienced HLB and ACP Scouts at SGC’s Dunwody Grove and Devil’s 

Garden Grove. A full cycle of ACP population monitoring was completed every 5 to 8 days at 

both groves in approximately 2,030 and 2,900 net planted acres, respectively. One stop to make 

observations was made for every 9 to 10 acres following one of two predetermined routes; the 

routes were customized for every Irrigation Zone, which averaged 120 acres in size. Seven out of 

ten stops were made on borders or “edges” of grove blocks. At every stop, 10 flush observations 

on 10 separate trees were made, and presence/absence of ACP adults, nymphs, and eggs 

recorded. Results were expressed in the database as percentage of flush over the entire Irrigation 

Zone with adults, nymphs, or eggs. Ten observations were then made utilizing the tap method 

pioneered for ACP by David Hall at USDA ARS Horticultural Research Lab in Fort Pierce (Hall 

et al., 2006) and Jawwad Qureshi and Phil Stansly at IFAS Southwest Florida Research and 

Education Center in Immokalee (Qureshi and Stansly, 2007). Total number of ACP adults in the 

Irrigation Zone was recorded, and results were expressed in the database as average number of 

psyllids per tap. Ten trees were observed at each stop, and the percentage of trees that had any 

amount of new flush was also documented. The percentage of stops in the Irrigation Zone in 


P a g e | 144 

which at least one ACP adult was found using the tap method was also calculated in the 

database. 

Inspection for HLB was performed on raised platforms travelling at 2 mph, or by walking, 

in the case of younger, smaller trees. Diagnosis was made using visual symptoms on leaves and 

fruit. All trees flagged as suspect for HLB infection were re-examined by one of two experienced 

“Senior Scouts.” A GPS point was recorded for locations of all trees confirmed as being infected 

by HLB using a Hammerhead XRT ruggedized computer and GeoAg Treelogger software. GPS 

data was processed using ESRI ArcGIS software. Leaf samples for PCR analysis were collected 

daily for quality control purposes. PCR analysis was carried out at the Southern Gardens 

Diagnostic Laboratory managed by Mike Irey. PCR results indicate that the HLB Scouts were 

over 95% successful in their positive visual diagnosis of HLB disease. 

The 2009 ACP population data used for correlation and regression analysis in this study 

was comprised of over 1,337,000 separate field observations, rolled into yearly averages for each 

variable for a given Irrigation Zone. The 2010 HLB inspection data used is a cumulative 

percentage for the year, derived from the total number of HLB-infected trees detected in each 

Irrigation Zone, compared to the total number of bearing trees in that Zone at the beginning of 

the year. It represents two complete cycles of inspection at the two groves studied, including 

approximately 33,500 man-hours of work, and inspection of over 1,490,000 trees. Statistical 

analysis was performed using the data analysis pack of Microsoft Excel software. 


Regarding correlations between the different ACP scouting methodologies, and the different 

variables representing ACP population levels, a very strong correlation was found between 

“Psyllids per tap” and “% stops with psyllids,” both generated by the tap method (Table 1). This 

indicates that the presence/absence of ACP adults detected by tap sampling could be a useful 

way to monitor psyllid populations, as opposed to reporting the number of adults detected, 

though we have found that the time needed to count adults detected using this method is 

minimal. Very good correlations were also found between the two methodologies, i.e., between 

“Psyllids per tap” and “% flush w/ACP adults”, and between “% stops with psyllids” and 

“% flush w/ACP adults”. Correlations between “% flush w/ACP nymphs” and the other variables 

were also significant, though not as strong. 

Scrutiny of all variables representing ACP population levels over time revealed that we had 

a lapse in ACP control at the Dunwody Grove in 2009, with significantly higher populations 

there than at the Devil’s Garden Grove, especially during the period of April to August or 

September (Fig. 1). Because our 2010 commercial HLB inspections were revealing a much 

higher HLB incidence rate at Dunwody than Devil’s Garden, we decided to examine the possible 

statistical relationships between the data generated by these two scouting programs. The best 

correlation with the 2010 HLB infection data that we found was to the 2009 “% flush w/ACP 

nymphs” data (Table 1; Fig. 2). Regression analysis shows that the 2009 “% flush w/ACP 


P a g e | 145 

nymphs” would have been a good predictor of the subsequent HLB infection at both groves 

(Fig. 3). 

Finally, recent economic models have suggested that the threshold for maintaining 

economic citrus production under the HLB management model of “inspection and elimination of 

infected trees for inoculum reduction” is around 4% to 5% HLB infection per year (Irey, 2011 

and Morris, 2011). Considering that the “tap method” is currently the most widely accepted 

method for ACP monitoring being used by Florida growers, our data suggests that maintaining 

ACP adult populations below 0.05 psyllids per tap can help to achieve that HLB infection 

threshold (Fig. 4). 

References 

Hall, D.G., Hentz, M.G., Clomperlik, M.A. 2006. A comparison of traps and stem tap sampling 

for monitoring adult Asian Citrus Psyllid (Hemiptera) in Citrus. Florida Entomologist (June 

2007) 90(2):327-334. 

Irey, M. 2011. When should a grower with HLB stop removing trees? 2 nd International Research 

Conference on Huanglongbing, Session 5: Oral Presentation 5.5. 

Morris, R.A. 2011. Economic considerations to treating HLB with the standard protocol or an 

enhanced foliar nutritional program. 2 nd International Research Conference on 

Huanglongbing, Session 5: Oral Presentation 5.4. 

Qureshi, J.A., Stansly, P.A. 2007. Integrated approaches for managing the Asian citrus psyllid 

Diaphorina citri (Homoptera: Psyllidae) in Florida. Proceedings of the Florida State 

Horticultural Society 120:110-115. 

Table 1. Correlations between 2009 ACP populations and 2010 HLB % infection. Four 

different variables representing ACP population levels, derived from two ACP scouting 

methodologies, were included in the analysis. 

HLB % 

infection 

2010 

HLB % infection 2010 1 

Psyllids 

per tap 

2009 

% stops 

w/psyllids 

2009 

% flush 

w/ACP 

adults 

2009 

% flush 

w/ACP 

nymphs 

2009 

Psyllids per tap 2009 0.51308 1 

% stops w/psyllids 2009 0.62212 0.97145 1 

% flush w/ACP adults 2009 0.49191 0.96870 0.92654 1 

% flush w/ACP nymphs 2009 0.76112 0.82578 0.87371 0.83806 1 


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Fig. 1. ACP population trends at SGC’s Dunwody Grove and Devil’s Garden Grove. From 

December 2008 to September 2009, four variables representing ACP populations are presented. 

18.0% 

Average % Flush with ACP Nymphs and % HLB Infection 2010, by Irrigation Zone 

30.0% 

16.0% 

14.0% 

25.0% 

% flush with ACP nymphs 

12.0% 

10.0% 

8.0% 

6.0% 

20.0% 

15.0% 

10.0% 

% HLB Infection 

4.0% 

2.0% 

5.0% 

0.0% 

0.0% 

201 

202 

203 

204 

205 

206 

207 

208 

209 

210 

211 

212 

213 

214 

215 

216 

217 

218 

219 

220 

221 

222 

223 

224 

225 

227 

228 

229 

230 

231 

232 

233 

234 

235 

309 

311 

312 

313 

316 

317 

318 

319 

320 

321 

322 

323 

324 

325 

326 

327 

328 

329 

330 

331 

332 

333 

% Flush w/ACP nymphs 2009 % HLB Infection 2010 

Fig. 2. Average % flush with ACP nymphs by Irrigation Zone in 2009 compared to % HLB 

infection in the same Zone in 2010. Zones 201 to 235 are Devil’s Garden Grove; 309 to 333 are 

Dunwody Grove. 


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30% 

% flush w/nymphs 2009 vs. % HLB infection 2010 

% HLB for Zone, 2010 

25% 

20% 

15% 

10% 

R 2 = 0.5886 

P-value = 0.00031 

5% 

0% 

0% 2% 4% 6% 8% 10% 12% 14% 16% 18% 

Average % flush w/nymphs for Zone, 2009 

Fig. 3. % HLB infection in 2010 as a function of yearly average of % flush with ACP nymphs 

for any given Irrigation Zone at Dunwody and Devil’s Garden Groves. 

0.400 

Average Psyllids (ACP) per Tap 2009 vs. % HLB Infection 2010 

30.0% 

Psyllids per Tap 

0.350 

0.300 

0.250 

0.200 

0.150 

0.100 

0.050 

25.0% 

20.0% 

15.0% 

10.0% 

5.0% 

% HLB Infection 

0.000 

201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 227 228 229 230 231 232 233 234 235 309 311 312 313 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 

Psyllids per Tap 2009 % HLB Infection 2010 

0.0% 

Fig. 4. Average psyllids per tap by Irrigation Zone in 2009 compared to % HLB infection in the 

same Zone in 2010. Zones 201 to 235 are Devil’s Garden Grove; 309 to 333 are Dunwody 

Grove. HLB infection threshold is red dashed line at 5.0%. 


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9.2 RNAi Strategy in Citrus Trees to Reduce Hemipteran Pests: Psyllids and Leafhoppers 

Hunter, W.B. 1 , Glick, E. 2 , Bextine, B.R. 3 , Paldi, N. 2 

1 Subtropical Insect Research Unit, USDA-ARS, USHRL, Fort Pierce, FL, USA 

2 Beeologics, Inc., LLC, Miami, FL, USA 

3 Department of Biology, University of Texas-Tyler, Tyler, TX, USA 

We demonstrate delivery of dsRNA constructs for RNAi to psyllids and leafhoppers through 

feeding on artificial diet, cuttings, and whole-plant systems (herbaceous plants, woody 

grapevine, and citrus seedlings and trees). Preliminary results indicated increased mortality by 

Asian citrus psyllid, Diaphorina citri; the potato psyllid, Bactericera cockerelli; and the 

glassy-winged sharpshooter, Homalodisca vitripennis, using species-specific dsRNA’s. 

Treatments using citrus seedlings showed that dsRNA could be introduced into whole plant 

systems. Citrus trees which are ~2.5 m tall are currently being screened for dosage titers, uptake 

time, and persistence to calculate a cost/benefit ratio and effectiveness. The citrus trees are 

6-year-old producing Mexican Limes, thus permitting examination for presence and/or 

persistence of dsRNA constructs in fruit and juice. RNA interference technology (RNAi) has 

been used successfully to silence endogenous insect genes through feeding, as shown with 

research on honey bee (Hunter et al., 2010). We propose that in the case of citrus pests 

(i.e., psyllids), specific psyllid transcripts may provide a natural specific treatment that can be 

used to reduce and suppress psyllids. RNAi strategies may one day be used across area-wide 

programs to suppress insect pests. 


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9.3 Application of Insecticidal Sprays to Citrus in Winter Provides Significant Reduction 

in Asian Citrus Psyllid Diaphorina citri Populations and Opportunity for Additional 

Suppression Through Conservative and Augmentative Biological Control 

Qureshi, J.A., Stansly, P.A. UF-IFAS, Department of Entomology and Nematology, Southwest 

Florida Research and Education Center, Immokalee, FL, USA, E-mail: jawwadq@ufl.edu 

Diaphorina citri is an economically important pest of citrus mainly because it vectors 

Candidatus Liberibacter asiaticus, causal organism of the Asian “huanglongbing” or citrus 

greening disease. Mature citrus trees in Florida are dormant in winter and produce most new 

shoots in spring, followed by sporadic growth in summer and fall. Young shoots are required for 

oviposition and nymphal development, but adults can survive and overwinter on hardened 

leaves. Therefore, foliar sprays of broad-spectrum insecticides applied to mature trees in winter 

were evaluated in a commercial citrus orchard as a tactic to reduce pest populations and 

insecticide use in spring and summer when beneficial insects are most active. Single sprays 

(AI/ha) of chlorpyrifos (2.8 kg) in January 2007 and chlorpyrifos, fenpropathrin (0.34 kg), and 

oxamyl (1.12 kg) in January 2008 reduced adult psyllids an average of 10- to 15-fold over 

5-6 months compared to untreated trees, respectively, without additional sprays. Spiders, 

lacewings, and ladybeetles were equally abundant during the growing season in both treated and 

untreated trees both years. This tactic has been adopted area-wide to manage psyllid in Florida 

and to establish Citrus Health Management Areas to reduce the spread of HLB. For additional 

suppression of psyllid, we are mass producing and releasing Tamarixia radiata, an ectoparasitoid 

of D. citri that we imported from Pakistan, South China, and North Vietnam in 2008, and an 

already established strain imported from Taiwan and South Vietnam in 1999. The repeated 

releases of these parasitoids in citrus groves are contributing to overall reduction in psyllid 

population. 


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9.4 Studies on Imidacloprid and Management of ACP in California 

Byrne, F. 1 , Morse, J.G. 1 , Bethke, J. 2 

1 Dept. of Entomology, University of California, Riverside, CA, USA 

2 University of California Cooperative Extension, San Diego, CA, USA 

ACP was recently detected in California. Thus far, the insect has been confined to backyard 

citrus and has not been detected on either nursery or field-grown citrus. HLB has not been 

detected in ACP insects sampled from find sites. Soil-applied systemic treatments with the 

neonicotinoid imidacloprid have been used for the management of ACP at find sites. In our 

research, we are evaluating possible strategies for the use of imidacloprid in commercial citrus. 

Key issues related to the use of imidacloprid are application rates and the timing of treatments to 

maximize psyllid control. In addition, concerns about the possible impacts of imidacloprid 

treatments against honey bees are being addressed. We are conducting extensive studies on the 

uptake and persistence of imidacloprid in the most important citrus-growing regions in 

California. Following treatments at different timings during a season, we monitor the 

concentrations of insecticide in leaf tissue that is most attractive to ACP; and, during the spring 

bloom, we test for the presence of imidacloprid residues in nectar sampled from flowers on 

treated trees. In addition to our work on field-grown citrus, we are also working with the citrus 

nursery industry to develop strategies for optimizing the use of imidacloprid on nursery stock. 

Imidacloprid treatments are used by nurseries as part of a quarantine mandate that requires that 

nursery stock be treated before it can be moved within the quarantine zone. 


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9.5 Selection and Dosage of Insecticides for the Control of the Asian Citrus Psyllid in the 

Citrus Groves of Mexico 

López-Arroyo, J.I. 1 , Díaz-Zorrilla, U. 2 , Hernández-Fuentes, L.M. 3 , Cortez-Mondaca, E. 4 , 

Robles-González, M.M. 3 , Villanueva-Jiménez, J.A. 5 , Cabrera-Mireles, H. 2 , Loera-Gallardo, J. 1 , 

Jasso-Argumedo, J. 6 , Curtí-Díaz, S.A. 6 

1 INIFAP, Centro de Investigación Regional del Noreste. Río Bravo, Tam., México 

2 INIFAP, Centro de Investigación Regional del Golfo Centro. Veracruz, Ver., México 

3 INIFAP, Centro de Investigación Regional del Pacífico Centro. Guadalajara, Jal., México 

4 INIFAP, Centro de Investigación Regional del Noroeste. Cd. Obregón, Son., México 

5 Colegio de Postgraduados, Campus Veracruz, Ver., México 


lopez.jose@inifap.gob.mx 

Diaphorina citri Kuwayama (Hemiptera: Psyllidae), the Asian citrus psyllid (ACP), was found in 

México in 2002 and has invaded the whole Mexican citrus industry during the past 6 years. 

Traditionally, the insect was controlled only in isolated groves where the direct damage was 

significant. Since the detection of Candidatus Liberibacter asiaticus in Yucatan, México in 2009, 

the status of ACP has changed because infective specimens could disseminate the pathogen to 

other groves in different areas of México. In order to develop a regional plan for the management 

of ACP in México, during the last 2 years, we evaluated more than 40 different insecticides in 

trials performed in different ecological areas of the Mexican citrus industry. The products 

included organophosphates, pyrethroids, neonicotinoids, mineral oils, botanicals, soaps, 

entomopathogenic fungi, etc. We selected the products that caused more than 85% mortality. In 

order to avoid the possible development of insecticide resistance in the ACP, we suggest a 

program for the use of insecticides for the control of the ACP according to its toxicological 

group. The plan includes dosages and timing for insecticide applications in citrus (grapefruit, 

mandarin, and orange), Mexican and Persian lime, as well as for young and mature trees. The 

program enforces the use of registered insecticides and presents alternative products that yield 

good control of the pest at an affordable economical cost. The program will be evaluated during 

2011 in the different citrus growing areas of México. 


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9.6 Asian Citrus Psyllid (ACP) Control: Potential Use of Systemic Insecticides in Citrus 

Bearing Trees 

Yamamoto, P.T. 1 , de Miranda, M.P. 2 , Felippe, M.R. 2 



Asian citrus psyllid (ACP) is an exotic citrus pest in Brazil that recently became very important 

as the vector of Liberibacters associated with citrus huanglongbing (HLB). Experiments were 

designed to study the efficacy of systemic insecticides to control ACP in citrus bearing trees and 

to determine the factors that influence their efficiencies. We conducted three experiments started 

at different times of the year, in which thiamethoxam and imidacloprid were tested at doses of 

1.25 g and 3.0-3.5 ml/m of plant height, respectively. In the first experiment, implemented in 

September 2009, both insecticides were effective in controlling ACP. Enzyme-linked 

immunosorbent assays (ELISA) were used to estimate the concentration of thiamethoxam in the 

plant, showing that the insecticide reached 4,000 ppb at 40 days after application in Valencia 

sweet orange on Swingle citrumelo. The concentration of thiamethoxam was higher than 400 ppb 

(concentration threshold that causes ACP mortality) from 9 to 68 days after application. The 

results of the second experiment, implemented in November 2009 in two rootstocks and soil 

types, showed lower insecticide efficiency when compared to the first experiment in Swingle 

rootstock. In Rangpur lime rootstock, the efficiency was low. The results of the third experiment, 

implemented in February 2010, showed that systemic insecticides were not effective in the 

control of ACP. The ELISA showed that the concentration of thiamethoxam was lower than 

200 ppb (concentration that causes no mortality) in the last two experiments. Factors influencing 

the absorption and translocation of systemic insecticides have not been clarified. 


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9.7 Insecticide Resistance and Susceptibility of Uninfected and Candidatus Liberibacter 

asiaticus-Infected Asian Citrus Psyllid in Florida 

Tiwari, S., Rogers, M.E., Stelinski, L.L. UF-IFAS Citrus Research and Education Center, Lake 

Alfred, FL, USA 

A 2-year field study was conducted to evaluate insecticide resistance levels in field populations 

of Asian citrus psyllid (ACP), Diaphorina citri Kuwayama. Five geographically discrete 

populations of adult ACP displayed a range of susceptibility levels against 12 tested insecticides. 

The highest level of resistance for adult ACP, as compared with a susceptible laboratory (LS) 

population, was found with imidacloprid with an LD 50 resistance ratio (RR) of 35 in one 

population. Likewise, among nymph populations, indications of resistance were observed with 

carbaryl (RR = 2.9), chlorpyriphos (RR = 3.2), imidacloprid (RR = 2.3 and 3.8), and spinetoram 

(RR = 3.0 and 5.9). Presence of varying levels of insecticide resistance in adult and nymph 

populations was potentially explained by elevated levels of three major detoxifying enzymes: 

general esterase, glutathione S-transferase, and cytochrome P 450 . These detoxifying enzymes are 

known to be up-regulated in insecticide resistant populations. Las infection significantly 

increased susceptibility of ACP adults to chlorpyriphos and spinetoram compared with 

uninfected counterparts, and infected ACP were more susceptible to insecticides in general than 

uninfected ones. Correspondingly, general esterase, glutathione S-transferase, and cytochrome 

P 450 enzyme activities were significantly lower in Las-infected than uninfected ACP. Mortality 

of both uninfected and Las-infected ACP was higher at 37° than at 20, 22, or 24°C. Across all 

temperatures tested, mean percent mortality was higher in Las-infected than uninfected ACP. 


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9.8 Development of Area-Wide Asian Citrus Psyllid Management Strategies in Texas 

Bartels, D.W. 1 , Sétamou, M. 2 , Ciomperlik, M.A. 1 , da Graça, J.V. 2 

1 USDA-APHIS-PPQ-CPHST, Mission, TX, USA 

2 Citrus Center, Texas A&M University-Kingsville, Weslaco, TX, USA 

The concept of an area-wide management approach against the Asian citrus psyllid, Diaphorina 

citri, has the potential to significantly lower the risk of huanglongbing (HLB) establishment and 

spread. Reducing the vector population has the advantage of not only lowering the risk of HLB 

spread but can make disease eradication possible in case of its accidental introduction, given it is 

detected early through ongoing survey efforts. In 2009, Texas A&M Kingsville Citrus Center 

and CPHST Mission Lab implemented a pilot project covering almost 1,500 acres of citrus to 

test area-wide management of ACP and minimize the risk of HLB in the Lower Rio Grande 

Valley, TX. Several of our objectives were to implement site-specific treatment regimes for ACP 

control in commercial citrus and refine monitoring methods for ACP populations and infestations 

pre- and post-treatment, and continue to test the efficacy of different insecticide application 

methods comprising aerial and ground application. We have documented that aerial and ground 

applications are very effective before spring flush. We saw a 93% reduction in ACP adults. The 

timing of this treatment is critical as adults need to be treated before they begin laying eggs on 

the spring flush. The most effective season-long treatment option included four foliar 

applications and soil-applied aldicarb. In 2010, we expanded the project area to cover 4,600 

acres of citrus and focused the main ACP control treatment to two dormant season sprays in 

January and November. 


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9.9 Asian Citrus Psyllid Management Strategies for California Citrus Growing Regions 

Grafton-Cardwell, E.E. 1 , Morse J.G. 1 , Taylor, B.J. 2 


2 Citrus Research Board, Visalia CA, USA 

Asian citrus psyllid (ACP) was first detected in southern California in October 2008 in the urban 

landscape. Since that time, the California Department of Food and Agriculture has conducted an 

eradication program utilizing systemic imidacloprid and foliar cyfluthrin wherever psyllids are 

detected. This program has helped to keep the psyllid from spreading into commercial citrus 

orchards; however, spread and establishment of ACP in commercial citrus is considered 

inevitable. The University of California in collaboration with the Citrus Research Board has 

conducted grower discussion sessions in five citrus growing regions of California to develop 

strategies for managing ACP once it is established in commercial citrus. In the initial phase of 

invasion, when ACP densities are low, treatments of a neonicotinoid and a pyrethroid are 

planned to potentially eradicate the pest in individual and/or neighboring orchards. When the 

psyllid becomes more widespread, then area-wide treatment programs will be necessary. Based 

on the Florida and Texas ACP management experience, ACP treatments will be directed at 

periods of flush and also at the late fall and early spring overwintering populations. The number 

of additional ACP-effective treatments needed in each region will depend on the current 

treatment program for other pests. The management program for the five citrus growing regions 

of California is discussed. 


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9.10 Area-Wide Management of Asian Citrus Psyllid in Southwest Florida 

Stansly, P.A. 1 , Arevalo, H.A. 1 , Zekri, M. 2 , Hamel, R. 3 


2 Hendry County Extension Service, LaBelle, FL, USA 

3 Gulf Citrus Growers Association, LaBelle, FL, USA 

“Dormant” sprays of broad spectrum insecticides have proven to be an effective tool for 

suppressing overwintering adult psyllid populations in Florida, reducing their entry populations 

in the spring flush and resulting in significant reductions still detectable well into the growing 

season. This tactic appears to have the greatest effect when applied area wide. Over 70,000 acres 

were sprayed at least once by air and most of the remainder by ground in southwest Florida as a 

cooperative effort during the dormant seasons of 2008-09 and 2009-10. Psyllid populations in 

late May had still not increased significantly from winter levels in treated orchards, whereas a 

17-fold increase was seen in untreated blocks in 2009. Future plans are to expand the program to 

include area-wide scouting with the objective of tracking psyllid populations throughout the 

region, pinpointing problem areas, and assisting growers in making informed decisions on a 

block-by-block basis during the growing season. 


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9.11 Evaluation of Low Volume Sprayers Used in Citrus Psyllid Control Applications 

Hoffmann, C. 1, *, Fritz, B. 1 , Martin, D. 1 , Atwood, R. 2 , Hurner, T. 3 , Ledebuhr, M. 4 , Tandy, M. 5 , 

Jackson, J.L. 6 , Wisler, G. 7 , Polek, M.L. 8 

1 USDA-ARS-Areawide Pest Management Research Unit-Aerial Application Group, 2771 F&B 

Road, College Station, TX, USA 

2 University of Florida, IFAS Extension, Tavares, FL, USA 

3 University of Florida, IFAS Extension, Sebring, FL, USA 

4 Ledebuhr Industries, Williamston, MI, USA 

5 Curtis DynaFog, Westfield, IN, USA 

6 Florida Citrus Industry Research Coordinating Council, Tavares, FL, USA 

7 USDA-ARS, National Programs, Beltsville, MD, USA 

8 Citrus Research Board, Riverside, CA, USA 

*To whom correspondence should be addressed: E-mail: clint.hoffmann@ars.usda.gov 

Citrus greening is a disease that threatens the survival and economic viability of the Florida 

Citrus Industry due to the disease’s ability to kill an infected tree. An insect called Asian citrus 

psyllid is a carrier of the bacteria that causes citrus greening; therefore, citrus growers are using 

several insecticides and sprayers to find the most effective method to control the insect. 

A number of studies involving numerous citrus sprayers and active ingredients were conducted 

to determine the droplet size generated by the different sprayers and how to adjust the sprayers to 

meet a droplet size requirement that is on many insecticide labels. In the sprayer tests, it was 

found that reductions in engine speed or increases in flow rate were required to increase droplet 

sizes to meet the product label required droplet size. As the equipment tested here represent the 

most typical application equipment used in Florida for psyllid control, these results will provide 

applicators, growers, and extension agents with general guidelines to ensure that spray systems 

are operated in a manner that complies with label restrictions. 


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9.12 Identification of Parasitoids and Haplotypes of Tamarixia radiata (Waterston) 

(Hymenoptera: Eulophidae) from Diaphorina citri in Yucatán, México 

González-Hernández, A. 1 , Jasso-Argumedo, J. 2 , Cruz-García, R. 1 , Lozano-Contreras, M. 2 , 

López-Arroyo, J.I. 3 , Villanueva-Segura, O.K. 1 

1 UANL, Facultad de Ciencias Biológicas, San Nicolás de los Garza, N.L., México 

2 INIFAP, Centro de Investigación Regional del Sureste, Mérida, Yuc., México 

3 INIFAP, Centro de Investigación Regional del Noreste, Río Bravo, Tam., México 

agonzale@fcb.uanl.mx. 

Recent studies from 2007 to 2010 have shown morphological and genetic variation in Tamarixia 

radiata (Waterston), as well as possible cryptic species. The main objective of this work is to 

identify the parasitoids associated with Diaphorina citri and to characterize the haplotypes of 

T. radiata present in Yucatán, México. During January to July of 2010, 76 samples of immature 

D. citri were collected from citrus plants and Murraya sp. from 161 different, sampled localities. 

Species identification was done through comparison of amplified sequences and those available 

at GeneBank. Phylogeny was rebuilt using the program Mega 4.1. The results showed the 

following parasitoids: Tamarixia radiata, Aprostocetus sp., Horismenus sp., Pachyneuron sp., 

and Synopeas sp. Most of the genetic diversity was found in gen COI from the identified 

specimens of T. radiata; four different haplotypes were obtained: H1, H2, H3, and H4. 

Comparing Haplotypes (H) from the current study and previous works, it was found that H1 is 

similar to H2 from Barr et al. (2010) and also similar to H7 from de León and Sétamou (2010). 

The H2 is similar to H1 from Barr et al. (2010) and also similar to H2 from de León and 

Sétamou (2010). The Haplotypes H3 and H4 are unique from México. The H1 is the most 

common; this is similar to the records found in de León and Sétamou (2010) and Barr et al. 

(2010). There is only one record of the Haplotype H4. We found one cryptic species that has not 

been reported in previous work. 


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9.13 Host Specificity Testing of Tamarixia radiata for the Classical Biological Control of 

Asian Citrus Psyllid, Diaphorina citri, in California 

Pandey, R.R., Hoddle, M.S. Department of Entomology, University of California Riverside, 

Riverside, CA, USA 

Asian citrus psyllid (ACP) vectors phytopathogenic bacteria that cause a lethal disease in citrus 

known as huanglongbing. Pest management practices largely rely on the use of insecticides to 

control ACP. However, pesticide use is not always possible, especially in the urban settings with 

backyard citrus and in commercial organic production systems. Classical biological control of 

ACP using host specific parasitoids, in particular Tamarixia radiata (Hymenoptera: Eulophidae), 

is one possible strategy for ACP control in areas where pesticide use is undesired. A strain of this 

parasitoid from the Punjab of Pakistan is of interest for use in California. Work is in progress to 

assess the environmental safety of T. radiata to native Californian psyllids and weed biocontrol 

agents. Representative non-target psyllids have been selected based on (A) native plant feeding 

psyllids that are phylogenetically related to ACP, (B) native psyllids with higher probability of 

occurrence around citrus groves, (C) native pest psyllids, (D) introduced pest psyllids, and (E) 

imported biological control agents of weeds. Laboratory-reared, late-instar ACP and non-target 

psyllid nymphs were transferred onto Murraya seedlings and respective native host plants grown 

in soil media to maintain good health of the test psyllids. The ability of T. radiata ‘Pakistani 

strain’ was tested in quarantine under choice and no-choice regimes contained within a caged 

arena. Successful completion of host specificity testing will be followed by submission of an 

Environment Assessment report for USDA-APHIS, NAPPO, and the CDFA. Demonstration of 

acceptable risk to non-target species by T. radiata to these regulatory agencies will likely result 

in permission to mass rear and release the Punjab strain of T. radiata in California for classical 

biological control of ACP. 


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9.14 Predators in Non-Commercial Citrus and Preliminary Evaluation of Their Potential 

Against the Asian Citrus Psyllid in Texas 

Pfannenstiel, R.S. 1 , Unruh, T.R. 2 

1 Beneficial Insects Research Unit, USDA-ARS, Weslaco, TX, USA 

2 YARL, USDA-ARS, Wapato, WA, USA 

Diaphorina citri Kuwayama (ACP) became established in the Lower Rio Grande Valley of 

Texas nearly 10 years ago. Although techniques have been developed that provide control in 

orchards, there are few tactics that are consistently applied to dooryard trees and abandoned 

orchards. In an effort to understand population dynamics of ACP in non-commercial citrus, the 

natural enemies present in non-commercial citrus, and their impact on ACP, we began 

a long-term sampling and evaluation program in 2010. ACP densities were estimated and natural 

enemies collected monthly at four non-commercial citrus sites in 2010. Predators were collected 

and reared if possible to confirm identity and stage. Samples of predators were collected and 

tested for feeding on ACP using a psyllid specific monoclonal antibody. Additionally, lab tests 

were conducted to determine whether predominant predators would attack and feed on adult 

ACP under laboratory conditions. Greater than 95% of all predators collected during 2010 were 

spiders. The dominant spider taxa consisted of two species of anyphaenid, a philodromid, 

a dictynid, and two salticids. Not all spiders will feed on adult ACP. 


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9.15 Suitability of Diaphorina citri, Toxoptera citricida, and Aphis spiraecola as Prey for 

Hippodamia convergens 

Qureshi, J.A., Stansly, P.A. UF-IFAS, Department of Entomology and Nematology, Southwest 

Florida Research and Education Center, Immokalee, FL, USA, E-mail: jawwadq@ufl.edu 

The convergent lady beetle, Hippodamia convergens Guérin-Méneville, is an important predator 

of soft-bodied insect pests. Some citrus producers in Florida initiated releases of commercially 

available H. convergens beetles in their groves mainly against D. citri. However, detailed 

investigations on the performance of H. convergens on diets of D. citri, T. citricida, or 

A. spiraecola were lacking. Therefore, our objective was to evaluate preference, survival, 

development, and reproduction of H. convergens on these three Homopteran pests of citrus. 

Larvae preferred D. citri over T. citricida in two-way choice tests and consumed more D. citri 

than T. citricida or A. spiraecola in no-choice tests during the first 6 hours of encounters in test 

arenas. Adults preferred T. citricida over A. spiraecola in two-way choice tests but consumed 

equal numbers of all three species in no-choice tests. Development times of larvae at 25.5 ± 

0.05°C averaged 11.5 ± 0.9 days on A. spiraecola, significantly longer than a cumulative average 

of 8.4 ± 0.4 days on other diets that were equally suitable. Larval survival and pupation times did 

not differ among diets. Females lived longer than males irrespective of diet, and there were no 

statistically significant differences among psyllid and aphid diets for fecundity or fertility of 

beetles. Thus, D. citri, T. citricida, and A. spiraecola are all suitable hosts for H. convergens. 

However, we do not yet know how these beetles will respond in the Florida citrus environment. 


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9.16 Molecular Analysis of Tamarixia radiata from America Uncovers Extensive Haplotype 

Variation: Multiple Groups? 

de León, J.H. 1 , Gastaminza, G.A. 2 , Sétamou, M. 3 , Cáceres, S. 4 , Kanga, L.H.B. 5 , Buenahora, J. 6 , 

Parra, J.R. 7 , Logarzo, G.A. 8 , Stañgret, C.R.W. 9 


2 Estación Experimental Agroindustrial Obispo Colombres (EEAOC), Tucumán, Argentina 

3 Citrus Center Texas A&M University-Kingsville, Weslaco, TX, USA 

4 Instituto Nacional de Tecnología Agropecuaria (INTA), Bella Vista, Corrientes, Argentina 

5 Florida A&M University, Tallahassee, FL, USA 

6 Instituto Nacional de Investigación Agropecuaria (INIA), Salto, Uruguay 

7 Escola Superior de Agricultura ‘Luiz de Queiroz’/USP, Piracicaba, São Paulo, Brazil 

8 USDA, ARS, SABCL, Buenos Aires, Argentina 

9 Private Contractor, Itapúa, Paraguay 

A phylogeographic analysis was performed on field-collected populations of Tamarixia radiata 

from South (Argentina, Brazil, Paraguay, and Uruguay) and North (Florida, Texas, and México) 

America along with laboratory strains from Asia (Pakistan, Southern China, and Northern 

Vietnam) by sequencing the mitochondrial cytochrome oxidase subunit 1 gene (COI) (518-bp). 

Two colonies of the same strain from Pakistan were analyzed that were maintained at two 

different locations (Pak-1 and Pak-2). Our ongoing goal is to determine whether T. radiata could 

exist as a cryptic species complex or to determine whether groups can be uncovered and 

distinguished. Extensive haplotype (hp) variation was uncovered with a total of 52 haplotypes 

out of 91 individuals, indicating that 57% of individuals carried a different haplotype. Four 

haplotypes were shared among countries and continents: hp2 in Florida, China, and Vietnam; 

hp8 in Texas, México, Argentina, Brazil, and Pak-2; hp16 in Brazil and Pak-1; and hp21 in 

Uruguay and Pak-1. Interestingly, no sharing of haplotypes was seen between the Pak-1 and 

Pak-2 stains. A NeighborNet phylogenetic network clustered the haplotypes into four groups: 

Group 1 (Florida, China, and Vietnam), Group 2 (Uruguay and Pak-1), Group 3 (Argentina, 

Brazil, Uruguay, Pak-1, and Vietnam), and Group 4 (Texas, México, Brazil, Paraguay, and 

Pak-2). Each of these haplotypes was positioned at a specific node in the network, implicitly 

suggesting that each may be an ancestral haplotype. Similar results were seen with a 95% 

confidence statistical parsimony network. Are these groups reproductively compatible? Accurate 

identifications are crucial to the success of biological control programs. 


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9.17 Molecular Characterization of a New Entomopathogenic Fungus Isaria poprawskii: 

A Potential Biocontrol Agent for Diaphorina citri. Development of Isaria-Specific 

Molecular Markers 

de León, J.H. 1 , Cabanillas, H.E. 1 , Humber, R.A. 2 , Murray, K.D. 1 , Moran, P. 1 , Jones, W.A. 3 


2 USDA, ARS, Ithaca, NY, USA 

3 USDA, ARS, Stoneville, MS, USA 

We genetically characterized Isaria poprawskii sp. nov., a new entomopathogenic fungal species 

similar to Isaria javanica (= Paecilomyces javanicus). Biological, ecological, and morphological 

data will be presented elsewhere. The fungus was discovered during natural epizootics on the 

sweet potato whitefly (Bemisia argentifolii) in the Lower Rio Grande Valley of Texas (LRGV), 

USA. Phylogenetic analyses of I. poprawskii inferred from β-tubulin (TUB2) sequence data was 

performed along with standard Isaria TUB2 sequences obtained from GenBank. Fifteen 

previously un-sequenced fungal isolates, eight of which were field collected from the LRGV, 

were also included in the analyses. I. poprawskii was shown to be closely related to I. javanica 

ex-type; however, it formed its own unique clade, thus confirming its status as a new fungal 

species. In addition, our analyses confirmed that I. poprawskii could be recovered from the fields 

of the LRGV. The results showed that both I. javanica and I. poprawskii are present in the 

LRGV in sympatry. Polymerase chain reaction-restriction fragment length polymorphism 

(PCR-RFLP) diagnostic assays reliably distinguished isolates belonging to the I. javanica and 

I. poprawskii clades. Inter-simple sequence repeat-polymerase chain reaction (ISSR-PCR) DNA 

fingerprinting also distinguished the ex-type isolates of I. javanica and I. poprawskii. PCR-RFLP 

assays were also developed toward I. fumosorosea and I. tenuipes, and the four assays were 

tested for their utility on important isolates presently being used in biocontrol programs against 

Diaphorina citri. These assays, along with the phylogenetic data, uncovered 11 misidentified 

fungal isolates. Correct identifications are critical to biological control programs. 


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9.18 RNAi - Evaluating Injection into Citrus Trees and Grapevine to Target Psyllids and 

Leafhoppers 

Hunter, W.B. 1 , Stover, E. 1 , Glick, E. 2 , Bextine, B.R. 3 , Paldi, N. 2 

1 USDA-ARS, U.S. Horticultural Research Lab, Fort Pierce, FL, USA 

2 Beeologics, Inc., LLC, Miami, FL, USA 

3 Department of Biology, University of Texas-Tyler, Tyler, TX, USA 

Methods to deliver effective dosages of dsRNA constructs into woody crops, i.e., citrus trees and 

grapevine, to induce RNA interference (RNAi) effects on targeted insect pests were evaluated. 

RNAi has been demonstrated to work in psyllids and leafhoppers. Citrus trees and grapevines 

grown in glasshouses or on an ARS research farm were treated with dsRNA, and the leaves were 

evaluated for presence and persistence over a 1- to 4-week period. Preliminary results showed 

that persistence could be extended by weekly delivery of dsRNA, or by an increase in 

concentration of a single treatment, which has also been shown in other applications of RNAi fed 

to insects (Hunter et al., 2010). While these results are promising, more research is needed to 

determine concentration levels needed within plants to trigger effective RNAi in feeding insects; 

to determine the influences of plant size; and seasonal effects. 


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9.19 Using Novel Photonic Fence Technology to Protect Foundation Block and Nursery 

Stock from Asian Citrus Psyllid 

Johanson, E. 1 , Patt, J. 2 , Mullen, E. 1 , Rutschman, P. 1 , Pegram, N. 1 

1 Intellectual Ventures Lab, Bellevue, WA, USA 

2 USDA-ARS, Weslaco, TX, USA 

Photonic Fence Technology (PFT) uses a combination of real-time image processing, optics, and 

lasers to detect, identify, track, and kill specified insects that enter the detection area. Originally 

developed to combat malaria, the system has been tested on Asian citrus psyllids (ACP). PFT 

identifies the genus and gender of an insect based on the target’s wingbeat frequency and is able 

to identify and kill insects at a range exceeding 30 m by applying a brief but lethal burst of 

photonic energy via a directed focused laser beam. The entire system can be built from relatively 

inexpensive components common in consumer electronics, including safeguards to disarm the 

system if a human enters the detection area. Recent testing shows that PFT also has the 

capability to detect, identify, and kill Asian citrus psyllids (ACP). Specifically, photonic 

exposure testing shows that ACP can be identified and eliminated at a distance of 7 m using a 

15 millisecond dose of 10 mJ of photonic energy. Next steps include optimizing this technology 

specifically to protect foundation block and nursery stock from ACP. As PFT can be adapted to 

perform non-lethal analysis of insect flight movements, future work will include research on the 

system’s capabilities in monitoring ACP presence in trucks, shipping containers, and outdoor 

citrus plantings. Initial test results indicate that PFT could become a cost-effective ACP 

management and research tool. 

References 



Hall, D.G., Hentz, G., Ciomperlik, M. 2007. A comparison of traps and stem tap sampling for 

monitoring adult Asian citrus psyllid (Hemiptera: Psyllidae) in citrus. Florida Entomologist 

90:327-334. 


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9.20 Development of a Diaphorina citri-Specific Molecular Diagnostic Marker for Gut 

Content Examinations 

de León, J.H. 1 , Thomas, D.B. 1 , Sétamou, M. 2 , Hagler, J.R. 3 


2 Citrus Center Texas A&M University-Kingsville, Weslaco, TX, USA 

3 USDA, ARS, Maricopa, AZ, USA 

The goal of the study was to develop a Diaphorina citri-specific molecular diagnostic marker to 

aid in identifying key predators to enhance biological control efforts (e.g., Tamarixia radiata, 

Isaria fungal isolates). Field studies in citrus orchards revealed that various species of lady 

beetles (Coccinellidae) were the most common and most important predators of D. citri in Texas 

(M. Sétamou, unpubl. data). The mitochondrial cytochrome oxidase subunit I gene (COI) was 

sequenced, and a specific COI marker was developed that generated a DNA fragment of 183-bp. 

The marker detected all life stages (eggs, larvae, and adults) of D. citri. Sensitivity assays 

showed the detection limit of the marker to be about 5 pg. The COI marker has been tested in 

specificity assays against 41 species of arthropods; including 10 species of Psyllidae, 10 species 

of Coccinellidae, 10 species of spiders, 3 species of green lacewings (Chrysopidae), 4 species of 

sharpshooters (Cicadellidae), an earwig (Forficulidae), and a ground beetle (Carabidae). The 

specificity tests demonstrated that the COI marker is highly specific toward D. citri, as 

cross-reactivity was not detected with any other species tested to date. The specificity of the COI 

molecular marker is critical; for example, out of the 10 Psyllidae identified in Texas, 5 are 

commonly found on traps in Texas citrus. Ash grey and convergent lady beetles tested positive 

after feeding on D. citri nymphs, demonstrating the utility of the marker for use in gut content 

analyses. The marker could aid in developing a conservation biological control program. 


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9.21 Development of a Pathogen Dispenser to Control Asian Citrus Psyllid (ACP) in 

Residential Citrus 

Patt, J. 1 , Jackson, M. 2 , Dunlap, C. 2 , Meikle, W. 1 , Adamczyk, J. 1 

1 USDA-ARS, Weslaco, TX, USA 

2 USDA-ARS, Peoria, IL, USA 

ACP dwelling in residential areas may stymie the effectiveness of area-wide management 

programs aimed at containing the spread of HLB in commercial citrus. We are developing an 

auto-disseminator (dispenser) of spores of the fungal pathogen Isaria fumosorosea (strain 3581). 

The tube-shaped dispenser, designed to be hung from backyard trees, has several features to 

optimize spore transfer to ACP. It is bright yellow and has pleats to provide multiple edges on 

which ACP can perch and crawl. The spore carrier, a powder made from pulverized cotton burrs, 

can support both blastospores and conidia, and can be dusted on the dispenser surface. In 

efficacy tests, free-flying psyllids, released from a cage in a greenhouse, were exposed to an 

array of 8 spore dispensers and 32 potted orange trees. A proportion of the psyllids settled on the 

trees after visiting the dispensers. The trees were inspected for 6 days following release and the 

number of psyllids on each tree recorded. Dead psyllids were returned to the lab where they were 

surface sterilized, placed in sterile humid chambers, and incubated at 26° ± 1°C. After 7 days, all 

remaining psyllids were collected and tested for infection. A mean of 56% (±8.2% SEM) 

(n = 3 tests) of the psyllids recovered from the trees developed mycosis, indicating the 

plausibility of using an auto-disseminator to infect ACP. Further tests will determine whether 

horizontal and secondary infections occur from ACP infected by the auto-disseminator. The 

possibility of using scent attractants is being explored. 


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9.22 Producing New Flush at Will in Citrus to Study ACP-Plant Interactions 

Malik, N.S.A., Brockington, J., Pérez, J.L., Mangan, R.L. USDA-ARS, Kika de la Garza 

Subtropical Agricultural Research Center, Weslaco, TX, USA 

Asian citrus psyllids (ACP) produce their nymphs only on new citrus leaves from where they 

multiply and possibly transmit the pathogen Candidatus Liberibacter that causes the ‘greening 

disease’. Research efforts to understand and control the lethal ‘greening disease’ require a 

constant supply of new flush to use in various studies. We have therefore tested various 

environmental and surgical treatments that could produce abundance of new flush in citrus plants 

throughout the year. Our results have shown that through environmental manipulations (i.e., by 

changing day and night temperatures, and length of induction period), we can not only produce 

new flush at will, but we could also control if the new flush will be vegetative or flowering. 

Thus, the technique may be useful for studies even on other insect interactions where insects may 

be more attracted to flowers versus vegetative shoots. The technique is also useful for nurseries 

to induce branching to enhance market value of the potted trees. In addition to environmental 

manipulations, we have also found that surgical removal of leaves prior to subjecting the plants 

to induction conditions greatly enhance the production of flush in different citrus cultivars. These 

results and the design of an inexpensive walk-in type growth chamber ($6K vs. $100K of 

commercial growth chamber) for such studies will be presented. 


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9.23 Thresholds for Vector Control in Young Citrus Treated for Symptoms of HLB with a 

Nutrient/SAR Package 

Monzó, C., Arevalo, H.A, Stansly, P.A. UF-IFAS Southwest Florida Research and Education 

Center, Immokalee, FL, USA 

Control of Diaphorina citri management is one of the basic components of HLB management, 

even for infected trees (Arevalo and Stansly, unpubl.). Foliar applications of micronutrients are a 

complementary strategy being used by many Florida growers to extend the productive life of 

infected trees. Therefore, establishment of economic thresholds for psyllid control, under 

different price scenarios, could optimize returns on investment when a nutrient/SAR package is 

being applied, even if HLB incidence is high. To accomplish this objective, two 3-year field 

experiments are being conducted in commercial orange blocks with high incidence of 

symptomatic trees. Experimental design is RCB with four replicates and four treatments: (1) No 

insecticide, (2) calendar applications, (3) nominal threshold of 0.2 psyllids/stem tap, and 

(4) nominal threshold of 0.7 psyllids/stem tap. Psyllid populations are being monitored biweekly 

by tap sampling and flushing observations. Impacts of insecticides on natural enemies are being 

evaluated by tap samples, flush observations, and suction samples. Economic thresholds will be 

calculated using the following parameters: treatment costs, fruit price, insecticide efficacy, and 

yield loss. Yield loss will be co-related with the cumulative number of ACP/tap obtained in each 

of the treatments, as well as the incidence of HLB and the average bacterial titer as estimated by 

Q-PCR. Differences in cumulative ACP/tap among treatments have become evident 4 months 

after initiating experiments, with more psyllids in treatments (1) and (4) (294.3 ± 100.3 and 

633.8 ± 331.3 cumulative ACP/tap, respectively) than in treatments (2) and (3) (58.6 ± 33.6 and 

212.5 ± 97.6 cumulative ACP/tap, respectively). 


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9.24 Experimental Release Rate Analysis of Volatile Compounds from Wax-Based 

Dispensers 

Neuman, R.D., Mills, D.R., Shelton, A.B. Auburn University, Auburn, AL, USA 

An engineering evaluation was performed to evaluate deployment strategies for dimethyl 

disulfide (DMDS) as an effective ACP repellent. An experimental apparatus was set up to 

investigate the release of volatile substances from wax-based dispenser under well-defined 

conditions of geometry, surface area, air flow, and temperature. The advantage is that the release 

rate can be modeled as a convective mass transfer process taking place in laminar boundary layer 

flow parallel to a flat surface. Therefore, the gas-phase mass-transfer coefficient and, hence, the 

release rate per unit surface area can be predicted. Release rate measurements of emulsified 

paraffin wax, with and without DMDS, show three regimes of mass loss: (a) an initial rapid loss 

linear with time, (b) a transition region, and (c) a much smaller rate of loss decreasing with time. 

Significantly, the statistically measured value of 0.87 g/h for the release rate in the constant rate 

period is found to be in excellent agreement with the theoretical calculations (0.85 g/h) of the 

mass loss of water from blank formulations. In contrast, the release rate is 0.63 g/h for 

wax-DMDS (10% w/w) formulations, a value significantly lower than that predicted (1.85 g/h). 

The release rate in wax-DMDS formulations appears to be affected by both chemical and 

structural effects, whereas the release rate in blank wax formulations appears to be only affected 

by structural effects. The release rate profiles indicate serious challenges in the deployment of 

DMDS using current wax-based formulations. Thus, for the delivery of DMDS and related 

substances, alternative deployment strategies should be developed and evaluated. 

See Addendum A4 for Extended Paper 9.24 – Page 281 - 286 


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9.25 Vegetation Canopy Airflow Modeling for Airborne Dispersion of DMDS 

Shelton, A.B., Neuman, R.D. Auburn University, Auburn, AL, USA 

A computational fluid dynamics model of airflow in and around vegetation is used to examine 

the airborne dispersion of dimethyl disulfide (DMDS) vapor within a citrus orchard under 

a narrowly scoped set of conditions. The basic airflow is governed by the Reynolds-averaged 

Navier-Stokes formulation where closure for the unresolved turbulence scales is provided by 

a traditional two-equation eddy-viscosity turbulence model. The vegetation is represented in a 

virtual manner through source terms that model the drag and turbulence length scale 

modification imparted by leaves and branches on the air. Dimethyl disulfide is treated as a 

passive scalar concentration field whose transport and distribution depend on intrinsic diffusivity 

and advection by the prevailing airflow. Numerical solution of the flow model, including the 

vegetation source terms and passive scalar evolution, is accomplished in a commercial 

computational fluid dynamics code. The simulations provide the steady state plume geometry of 

dimethyl disulfide for specified release rate and point, hedge geometry, and ambient winds. 

Results show that for 2.5 m/s winds at 10 m, a ground point release rate of 0.2 g/day provides 

good coverage at a concentration of at least 1 ppb with a relatively small local region in excess of 

the human olfactory threshold concentration of 10 ppb. 

See Addendum A4 for Extended Paper 9.25 – Page 287 - 292 


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9.26 Methods and Systems to Deliver Volatile Compounds for Biological Control Strategies 

Neuman, R.D., Shelton, A.B., Zee, R.H. Auburn University, Auburn, AL, USA 

There is great interest in natural volatile compounds having known benefits and their usage in 

the fight against the ubiquitous huanglongbing (HLB) disease. However, the methods, devices, 

and systems associated with the prior art of delivering volatile compounds are not able to meet 

the requisite demands of field applications. In this paper, we disclose a breakthrough in the art of 

controlled delivery of volatile compounds that meet the design requirements for a wide variety of 

applications and methods. The approach employed is the development of vapor delivery systems 

that utilize passive flow control nozzles uniquely designed to deliver volatile compounds in 

either open local environments or open field environments. Indeed, the methods and vapor 

delivery systems described provide (a) continuous, consistent, and sustainable rate of delivery 

over entire growing seasons, (b) controlled rate of delivery at strategic field locations which 

yields effective concentrations in open environments per application requirements, 

(c) fine-tuning of the rate of delivery to a predetermined rate for specific applications, (d) the 

means to utilize volatile compounds having vapor pressures that range over several orders of 

magnitude, and (e) deployment of advanced biocontrol configurations such as combinations of 

repellents, attractants, and immune enhancement promoters. The invention has many advantages, 

one of which is the flexibility it offers in the design and engineering of vapor delivery systems 

for biocontrol strategies. Among the benefits of biological control, especially when large-scale 

application systems are implemented, will be reduced usage of various pesticides with significant 

reduction in pesticide residues in crop products. 


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Session 10: 

HLB Management 


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10.1 Trunk Injection of Copper Sulfate Pentahydrate (Magna-Bon) Affects Expression of 

HLB 

Graham, J.H. 1 , Irey, M.S. 2 , Miele, F. 3 



3 Magna-Bon, Okeechobee, FL, USA 

HLB is caused by a phloem-limited bacterium that is unlikely to be directly contacted by 

bactericides such as copper (Cu) formulations applied to the plant surface. The objective was to 

evaluate trunk injections of soluble Cu in Magna-Bon (M-B). The trial was located in a south 

Florida grove of 5-year-old Hamlin orange trees that were healthy (PCR-), asymptomatic (PCR+ 

without symptoms), and PCR+ with mild decline. Six treatments of 24 trees consisted of 

non-treated check (NTC); 1 M-B injection @ 8,000 ppm; 1 M-B injection @ 1,000 ppm; 2 M-B 

injections @ 1,000 ppm; 6 M-B injections @ 1,000 ppm; or 6 M-B injections @ 500 ppm. Trunk 

injections of 3-5 ml per tree were made at 1- or 3-month intervals, depending on treatment. Trees 

were assayed by PCR to estimate bacterial infection and HLB canopy symptoms were visually 

assessed before and at 5 months after injections began in April 2009. All M-B treatments 

produced changes in canopy ratings that reflected positive response compared to no change for 

the NTC trees. Ct values for the NTC trees indicated positive HLB status, whereas Ct’s for M-B 

treated trees were significantly higher and indicated negative or threshold bacterial infection. 

High leaf Cu in all treatments indicated overspray of the trees with Cu bactericide. Alternatively, 

a bioassay for systemic copper activity was conducted with expanding fall flush by 

injection-infiltration of detached leaves with Xanthomonas citri subsp. citri (Xcc). Canker 

lesions were reduced in leaves of M-B treated versus NTC trees, confirming Cu from M-B is 

systemic in the tree. 


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10.2 Chemical Compounds Effective Against the Citrus Huanglongbing Bacterium, 

Candidatus Liberibacter asiaticus In Planta 

Zhang, M.-Q. 1 , Powell, C.A. 1 , Zhou, L.J. 1 , He, Z. 1 , Stover, E. 2 , Duan, Y.-P. 2 


2 USDA-ARS-USHRL, Fort Pierce, FL, USA, Yongping.duan@ars.usda.gov 

Citrus huanglongbing (HLB) is one of the most destructive diseases of citrus worldwide and is 

threatening the survival of the Florida citrus industry. Currently, there is no established cure for 

this century-old and emerging disease. As a possible control strategy for citrus HLB, therapeutic 

compounds were screened using a propagation test system with Las-infected periwinkle and 

citrus plants. The results demonstrated that the combination of penicillin and streptomycin (PS) 

was effective in eliminating or suppressing the Las bacterium and provided a therapeutically 

effective level of control for a much longer period of time than when administering either 

antibiotic separately. When treated with the PS, Las-infected periwinkle cuttings achieved 70% 

rooting versus less than 50% with other treatments. The Las bacterial titer in the infected 

periwinkle plants, as measured by quantitative real-time PCR, decreased significantly following 

root-soaking or foliar-spraying with PS. Application of the PS via trunk injection or root soaking 

also eliminated or suppressed the Las bacterium in the HLB-affected citrus plants. This may 

provide a useful tool for the management of citrus HLB and other Liberibacter-associated 

diseases. 


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10.3 Regional HLB Management on the Effectiveness of Local Strategies of Inoculum 

Reduction and Vector Control 

Bassanezi, R.B. 1 *, Yamamoto, P.T. 3 *, Montesino, L.H. 1 , Gottwald, T.R. 2 , Amorim, L. 3 *, 

Bergamin Filho, A. 3 * 


2 USDA/ARS, Fort Pierce, FL, USA 

3 ESALQ/USP, Piracicaba, Brazil 

The effectiveness of local strategies of inoculum reduction and vector control on HLB progress 

were studied in São Paulo (E1-Oct/05; E2-May/06). Regional HLB management was present for 

E1 and absent for E2. Local inoculum reduction levels for E1 were every 4, 8, and 16 weeks, and 

for E2, every 2, 4, 12, and 26 weeks. Local vector control levels for E1 were no control, program 

A (PA), and program B (PB), and for E2, no control and program C (PC). Psyllid control was 

done with two 56-day-interval applications of systemic insecticides during the rainy season, and 

in the rest of the year, with contact insecticide sprays every 28 days for PA, and every 14 days 

for PB and PC. The beginning of the HLB epidemic was delayed 10 months by regional HLB 

management but wasn’t affected by different local strategies in both experiments. After 60 and 

53 months, the HLB incidence and progress rate weren’t affected by different frequencies of 

local inoculum reduction in both experiments, and were different only in plots with and without 

local vector control in E2. Regional HLB management reduced the HLB incidence (90% less for 

E1) and progress rate (75% less for E1) in both plots with and without vector control. These 

reductions were explained by smaller psyllid population and lower frequency of infective psyllid 

in E1 than in E2. 


Financial support: Fundecitrus, FAPESP (2005/00718-2 and 2007/55013-9), CNPq 

(578049/2008-2), and CRDF (NAS-8). *Authors granted by CNPq. 


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10.4 The Theory of Managing Huanglongbing with Plant Nutrition and Real World 

Success in Florida 

Spann, T.M. 1 , Rouse, R.E. 2 , Schumann, A.W. 1 



Huanglongbing (HLB) is regarded as the most devastating disease of citrus worldwide. Countries 

infected with HLB either no longer produce citrus commercially or have had to relocate their 

industries to different geographic regions to avoid the disease. Because citrus is such an 

important part of the Florida economy, and the state is surrounded by water, neither of these 

strategies were viable options. Thus, efforts have been ongoing to find ways to maintain the 

health and productivity of existing mature trees in Florida until a long-term cure is developed. 

Our data, as well as the data of other researchers, shows that HLB-infected trees are consistently 

deficient in Ca, Mg, Mn, Zn, and B; and, in a grove setting, these nutrient deficient trees are 

more likely to be HLB-infected than the nutrient sufficient trees. It is not yet known whether 

these deficiencies are a result of decreased nutrient uptake or if the nutrients are being bound 

within the plant making them unavailable. These findings are important because mineral 

nutrients function as inhibitors, activators, and regulators of plant and pathogen physiological 

processes. For example, Ca and B are important nutrients for the development, maintenance, and 

integrity of cell walls. Plants deficient in Ca and B have higher levels of free sugars and amino 

acids in the apoplast and phloem sap – compounds needed by many pathogens and insect pests. 

Mg is important for protein synthesis through its role in the aggregation of ribosome subunits. 

Under Mg deficiency, protein synthesis stops and free amino acid content increases. 

A commercial citrus grove in Florida has been maintaining HLB-infected trees for nearly 5 years 

through an aggressive foliar nutrition and psyllid control program. These results have been 

replicated in three other locations within the state for 2 years. Overall tree health, yield, and fruit 

quality of HLB-infected trees under this management program will be discussed. 


Financial support: McKinnon Corporation, CRDF. 


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10.5 Nutritional Treatments: Inconsequential Effect on HLB Control and Promote 

Area-Wide Titer Increase and Disease Spread 

Gottwald, T.R. 1 , Irey, M.S. 2 , Graham, J.H. 3 , Wood, B. 4 


2 Southern Gardens, U.S. Sugar Corporation, Clewiston, FL, USA 


4 USDA, ARS, SE Fruit and Nut Research Lab, Byron, GA, USA 

The use of nutritional applications to control or offset the deleterious effects of huanglongbing 

(HLB) has been a topic of considerable discussion and debate. However, most reports are 

anecdotal and without sufficient statistical rigor. Even so, Florida producers are using this 

unproven method without validation in lieu of conventional control methods. The formulation 

for the nutritional program (NP) has varied considerably, but most often consists of foliar 

applications of wettable-powder micronutrients, phosphite, and methyl salicylate. A trial 

consisting of a randomized complete block design with three blocks and four replicate 

trees/block was repeated in 2009 and 2010. All trees were PCR+ for Las at the onset of the trial 

but showing only mild symptoms. This stage of infection was chosen based on claims that the 

NP maintains health and productivity of HLB-infected trees for a number of years, thereby 

extending the grove’s commercial viability. Combinations of components were compared with a 

control consisting of a standard insecticide program for psyllids. Additional treatments consisted 

of phosphite with Mn-carbonate, Mn-metalosate, Cu-metalosate, or Zn-metalosate, and injection 

treatments using soluble copper or silver mixed with a polymer. There was no significant 

difference in titer dynamics, yield (number fruit per tree, kg fruit/tree, proportion of fruit 

dropped), or quality (Brix, acid, brix:acid ratio), compared to the control. The NP did not sustain 

tree health, yield, or fruit quality. In addition, there is a major concern that because nutritional 

supplements have no effect on Las titer, their use will promote area-wide inoculum buildup and 

spread. 


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10.6 Nutritional Approaches for Management of Huanglongbing (Citrus Greening) in 

China 

Xia, Y. 1 , Sequeira, R. 2 

1 North Carolina State University, Raleigh, NC, USA 

2 USDA/APHIS, Raleigh, NC, USA 

The use of supplemental nutrition including micronutrients as a part of huanglongbing (HLB) 

management program is being adopted by a growing number of citrus growers in Florida. The 

expectation is that by adding additional nutrition, especially the micronutrients, the productive 

life of infected trees can be extended, perhaps indefinitely. Although the approach is recent in the 

United States, as is HLB itself, nutrient management has been practiced in China for many years. 

Our review of 60+ years’ history of research and field practice in China reveals no consistent 

evidence to support the notion that nutrient management can maintain productivity of 

HLB-infested trees over the long term. However, it appeared that increased nutrition, together 

with other cultural practices such as irrigation, may be able to prolong grove productivity for 

variable periods of time in certain cases. In general, under a good nutritional program plus 

optimal irrigation, mature (10 years or older) sweet oranges can sustain productivity for another 

3-5 years with symptoms; pomelos can last significantly longer. Citrus plants grown from seeds 

or high layering also appeared to perform well under a nutritional program. However, fruit 

quality from these severely infected plants was generally unsatisfactory, depending on severity 

of the disease. Currently, there is no wide-spread use of supplemental nutrition for HLB 

management as a single strategy in China. However, micronutrients are commonly used in 

commercial production to optimize plant nutritional status. 


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10.7 First Steps Towards Rescuing Las-Infected Citrus Germplasm 

McCollum, G., Stover, E. USDA, ARS, USHRL, Fort Pierce, FL, USA 

Huanglongbing (HLB) disease is having a significant impact on the USDA citrus breeding 

program as it has shown up in a number of trees that exist only in a virtually irreplaceable 

germplasm collection. It is critical that we rescue Las-budwood from elite germplasm that is 

Las+. We reasoned that by selecting budwood that tests Las-, albeit from Las+ trees, we would 

produce some propagations free of Las. At least three, and as many as six, branches from each of 

seven trees were tested for Las using standard qPCR methods. A total of 90 propagations (3 from 

each branch) were produced. Initially, 63% of the branches were Las+. Of all the propagations, 

89% survived, with no apparent difference in survival between propagations made from Las+ or 

Las- branches. Among all propagations, 29% were Las+. Among propagations made from 

branches that were Las+, 55% (18/47) tested Las+, whereas among the propagations made from 

Las- branches, 12% (3/25) were Las+, with two of these propagations originating from the same 

original branch. Average Ct value for Las+ propagations from Las+ branches was 27.9 compared 

to 36.0 for the Las+ propagations from Las- branches. So far, our data support the notion that 

testing for Las prior to propagation is an important first step in the process of rescuing Las-tissue 

from Las+ trees. Propagated trees will continue to be monitored for the appearance of HLB 

symptoms and further development of Las as determined by qPCR, to determine whether uneven 

Las distribution and selected propagation from Las+ trees will permit rescue of critical 

germplasm. 


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10.8 Screening Chemical Compounds Against Citrus Huanglongbing Using an Optimized 

Grafting System from Candidatus Liberibacter asiaticus-Infected Citrus Scions 

Zhang, M.-Q. 1 , Duan, Y.-P. 2 , Powell, C.A. 1 

1 UF-IFAS Indian River Research and Education Center, Fort Pierce, FL, USA capowell@ufl.edu 

2 USDA-ARS, U.S. Horticultural Laboratory, Fort Pierce, FL, USA 

Citrus huanglongbing (HLB) is a phloem-limited disease caused by a fastidious bacterium called 

Candidatus Liberibacter (Las). Greenhouse experiments were conducted to optimize a citrus 

system to screen chemicals for their effects on Las. The results showed that the citrus cutting 

regeneration system was not as good for screening molecules as compared to our optimized 

regeneration systems from periwinkle. Citrus lemon cuttings with HLB symptoms were used as 

scions and grafted onto Las-free grapefruit. Beginning 4 months after graft inoculation, the new 

branches had typical HLB symptoms of blotchy mottle in scions, and the rootstock had vein 

corking and yellow shoots. More than 90% of HLB-affected lemon scions survived and 80% of 

the inoculated rootstocks were infected with a Ct value of 29.7 ± 3.35 six months after grafting. 

HLB-affected lemon branches were sampled and soaked in PS (penicillin and streptomycin), 

MDL (metronidazole), and DBNPA (2,2-dibromo-3-nitrilopropionamide) solutions overnight, 

cut into two or three buds for scions, and then grafted onto the Las-free grapefruit. More than 

95% of the scions treated with PS survived and grew better than untreated controls; all new 

leaves from the scions tested negative for Las by PCR. When the HLB-affected scions were 

treated with MDL and DBNPA, less than 70% of the scions survived. Also, more than 50% of 

the new branches were infected by the HLB bacteria, with Ct values of 24.3 ± 2.74 for MDL and 

30.7 ± 1.32 for DBNPA, respectively. Other chemicals, such as kasumin, are currently being 

tested using this system. 


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10.9 Discovery of Antimicrobial Small Molecules Against Candidatus Liberibacter 

asiaticus by Screening Novel SecA Inhibitors Using Structure Based Design 

Akula, N., Wang, N.-Y. UF-IFAS Citrus Research and Education Center, Lake Alfred, FL, 

USA 

Protein secretion in bacteria is a critical and complex process. SecA is the protein translocase 

ATPase subunit and a superfamily 2 RNA helicase, involved in pre-protein translocation across 

and integration into the cellular membrane in bacteria (Hartl et al., 1990; Lill et al., 1990; 

Ulbrandt et al., 1992). Identification of small molecule inhibitors that interfere with the function 

of SecA could lead to potential antimicrobial agents. In order to find novel inhibitory structures, 

we used various computational techniques such as pharmacophore design, virtual screening, and 

molecular docking methods (Friesner et al., 2004). Based on virtual screening and docking, we 

identified ~4500 structures from millions of commercially available database (Irwin and 

Shoichet, 2005) structures. Those structures were again docked with extra-precession by using a 

glide (Friesner et al., 2006) program. We chose the top 2% of structures based on the scoring 

functions, physicochemical properties, and our chemical intuition. Further, these structures were 

energy minimized by molecular minimization studies, and we evaluated their binding energies to 

pick the best 20 structures. The selected compounds were used to perform biological activity 

studies against SecA. Among these 20 compounds, 3 were active below 10 µm. The highly 

active structures will be utilized to design a potential inhibitor compound against SecA. All 

molecular modeling studies were performed on HP ProLiant Linux system using Schrodinger 

Suite Programs (2010). An ATPase assay kit and purified SecA enzyme was used for biological 

testing. 

References 

Friesner, R.A., Banks, J.L., Murphy, R.B., Halgren, T.A., Klicic, J.J., Mainz, D.T., Repasky, 

M.P., Knoll, E.H., Shelley, M., Perry, J.K., Shaw, D.E., Francis, P., Shenkin, P.S. 2004. 

Glide: a new approach for rapid, accurate docking and scoring. 1. Method and assessment 

of docking accuracy. Journal of Medicinal Chemistry 47:1739-1749. 

Friesner, R.A., Murphy, R.B., Repasky, M.P., Frye, L.L., Greenwood, J.R., Halgren, T.A., 

Sanschagrin, P.C., Mainz, D.T. 2006. Extra precision glide: docking and scoring 

incorporating a model of hydrophobic enclosure for protein-ligand complexes. Journal of 

Medicinal Chemistry 49:6177-6196. 

Hartl, F.U., Lecker, S., Schiebel, E., Hendrick, J.P., Wickner, W. 1990. The binding cascade of 

SecB to SecA to SecY/E mediates preprotein targeting to the E. coli plasma membrane. 

Cell 63:269-279. 

Irwin, J.J., Shoichet, B.K. 2005. ZINC - a free database of commercially available compounds 

for virtual screening. Journal of Chemical Information and Modeling 45:177-182. 

Lill, R., Dowhan, W., Wickner, W. 1990. The ATPase activity of SecA is regulated by acidic 

phospholipids, SecY, and the leader and mature domains of precursor proteins. Cell 

60:271-280. 

Schrodinger Suite Programs. 2010. Schrodinger LLC, New York. 


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Ulbrandt, N.D., London, E., Oliver, D.B. 1992. Deep penetration of a portion of Escherichia coli 

SecA protein into model membranes is promoted by anionic phospholipids and by partial 

unfolding, Journal of Biological Chemistry 267:15184-15192. 


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10.10 The Low Pressure Trunk Injection System: A Technology to Fight Against HLB 

Tomas, J. Agricultural Integrated Solutions USA Corporation, 6506 SW 113 Court, Miami, FL, 

USA 

This system is a technology that has been used to control Phytophthora and Verticillium dahliae 

in Oil Palms and Avocado trees, to successfully control Red Palm Weevil at the Mediterranean 

Region, to eliminate the Thaumetopea pityocampa, and to control Witches Broom in lime trees. 

This injection is a very clean method, fast to apply, which respects environment. The method 

consists of a plastic injector that is introduced in the trunk using a drilling machine and a 

pressured injection capsule that is made with an elastic tube containing the solution. When we 

connect both elements, the pressure made by the elastic capsule (under 100 kPa) introduces the 

solution to the tree through the injector. The solution will be absorbed by the tree and enhanced 

by its evapotranspiration. This allows the product to get distributed in every part of the tree 

through the vascular system. This system guarantees that the delivery pressure will not damage 

the tree vascular system. We apply the dose in low concentrations to avoid tissue damage, and 

this allows even distribution in the tree tissues. This system got the Second Award at the Geneva 

International Inventions Summit, and we are convinced it can be used to reduce and eventually 

eradicate Diaphorina population at citrus plantations to avoid HLB disease transmission. 


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10.11 Does Systemic Acquired Resistance (SAR) Control HLB Disease Development? 

Graham, J.H. 1 , Myers, M.E. 1 , Irey, M.S. 2 , Gottwald, T.R. 3 




The objective is to evaluate soil-applied neo-nicotinoids and other SAR inducers on HLB disease 

progress in newly planted citrus trees subjected to psyllid-mediated infection or 

graft-inoculation. One-year-old Hamlin trees were planted in May 2009 and treated as follows: 

1) non-treated check (UTC), 2) foliar insecticide to control psyllids, 3) soil-applied 

imidacloprid/thiamethoxam (IMID/THIA) to induce SAR, 4) soil-applied IMID/THIA plus foliar 

insecticides, 5) graft-inoculated UTC, and 6) graft-inoculated with IMID/THIA. There were 

50 trees per treatment (5 blocks of 10 trees). In 2009, the effect of SAR inducers on HLB 

infection progress was inconclusive, perhaps attributable to the interaction of IMID/THIA with 

psyllid control that may have an uncontrolled effect on psyllid transmission. In 2010, the SAR 

inducer acibenzolar-S-methyl (ASM, Actigard 50 WP), which does not control psyllids, was 

substituted in treatments 3, 4, and 6. At 17 months after treatments began, 65 trees were PCR+ 

(22%) in the trial. Higher number of PCR+ occurred in the UTC (14), the UTC with graft 

inoculation (13), and the IMID/THIA/ASM with graft-inoculation (18). Lower number of PCR+ 

trees occurred without graft inoculation in treatments with SAR inducers (6), foliar insecticides 

(8), and foliar insecticide plus SAR inducers (6). At this time, the effect of SAR on HLB disease 

progress is minimal, which indicates a lack of promise for use of SAR inducers in HLB 

management. 


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10.12 Use of Growth-Priming Agents to Extend the Growth of HLB-Affected Citrus 

He, Z. 1 , Zhang, M.-Q. 1 , Viana, E. 1 , Merlin, T. 2 , Duan, Y.-P. 2 , Stoffella, P.J. 1 , Liptay, A. 3 , 

Powell, C.A. 1 

1 UF-IFAS Indian River Research and Education Center, Fort Pierce, FL, USA zhe@ufl.edu 

2 USDA-ARS, U.S. Horticultural Laboratory, Fort Pierce, FL, USA 

3 Stoller USA, Inc., Houston, TX, USA 

Citrus greening disease, also called huanglongbing (HLB), associated with a fastidious, 

phloem-limited bacterium (Candidatus Liberibacter asiaticus-Las), is posing a major threat to 

the citrus industry due to its rapid spread and greatly reduced marketable yields. HLB-affected 

trees characteristically suffer from a damaged root system and nutritional disorder because of 

interrupted transport of photoassimilates from shoot to the root, and mineral nutrients/water from 

root to the shoot. In this study, greenhouse experiments were conducted to examine the effects of 

growth-priming agents [including root growth enhancer, trace element combination (Zn, Cu, and 

Mn), plant growth regulators, and sugar transporter alone or in combination] on the root growth, 

leaf chlorophyll, and leaf starch of HLB-affected citrus seedlings and periwinkle plants. The 

experiments were a factorial design with six replicates, and the growth-priming agents were 

biweekly applied by soil application in liquid. Visual observation of the root systems of citrus 

seedlings was conducted in 2 months, and the measurements of leaf chlorophyll and starch 

concentrations were performed in 3 months after the treatments. Periwinkle plants were 

harvested in 4 months, and biomass yields of root and shoot were recorded and root parameters 

(length, surface area, and diameter) were determined. When applied alone, root growth enhancer 

was most effective in promoting plant growth followed by trace element combination, as 

indicated by a significant increase in shoot and root biomass, particularly root length and surface 

area, as compared with the control. Plants that received root growth enhancer had higher contents 

of leaf chlorophyll. Sugar-transporter was efficient in reducing soluble starch in the leaves of 

Las-infected periwinkle. The combination of root growth enhancer or plant growth regulators 

with sugar-transporter significantly reduced leaf soluble starch and increased leaf chlorophyll 

contents of HLB-affected citrus. Observable recovery of root growth of the HLB-affected citrus 

occurred in 2 months after the treatments. These results indicate that the growth-priming agents, 

when applied in proper combinations, may be useful for extending the growth and production of 

HLB-affected citrus. 

Keywords: Citrus greening disease, leaf starch content, plant growth-priming agents, root 

restoration 


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10.13 Evaluation of Foliar, Zinc, and Manganese for Control of HLB or Associated 

Symptom Development 

Johnson, E.G. 1 , Irey, M.S. 2 , Gast, T. 2 , Bright, D.B. 1 , Graham, J.H. 1 



Florida growers have reported that supplementary foliar nutrient applications maintain 

productivity of HLB-infected trees. However, efficacy and sustainability of the nutritional 

approach for HLB disease management has not been validated. The main cause of visible HLB 

symptoms, yield reduction, and tree decline appears to be disruption of phloem tissue, which 

blocks the flow of photosynthate and nutrients from source to sink tissue. If supplemental 

nutrition is a sustainable approach, it is expected that foliar nutrients will reduce or eliminate 

damage and plugging in citrus phloem tissue caused by the bacterium and possibly reduce spread 

or replication of the bacterium in infected trees. A greenhouse study is underway to evaluate a 

mixture of foliar nutrients representative of that used by Florida growers for HLB management. 

Infected and non-infected Hamlin trees under different combinations of nutritional treatments are 

being monitored for bacterial titer in phloem tissue and development of disease symptoms, 

including phloem cell morphological changes: plugging, necrosis, and starch accumulation. 

Initial results show no difference in the infection rate or bacterial populations in leaf midribs. 

Multiple microscopy techniques including TEM, light, and fluorescence microscopy with 

callose-specific dyes are being used to monitor phloem plugging and necrosis as the infection 

progresses. A complimentary field trial is evaluating bacterial titer, yield, and tree health in a 

south Florida Hamlin grove with a mixture of healthy, asymptomatic (PCR+), and HLB 

symptomatic trees. 


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10.14 Role of Nutritional and Insecticidal Treatments in Mitigation of HLB: Main Effects 

and Interactions 

Stansly, P.A., Arevalo, H.A., Rouse, R.E. UF-IFAS Southwest Florida Research and Education 

Center, Immokalee FL, USA 

Insecticidal control of the psyllid vector is widely considered the foundation of HLB 

management, while foliar applications of micronutrients are being touted as an effective method 

of mitigating impacts of the disease on tree health and production. Although the combination of 

these two practices is having salutatory effects in a number of Florida citrus groves, the 

individual contributions of the two main components are not known. We report preliminary 

results from a replicated factorial experiment with four treatments (nutritionals, insecticides, 

nutritionals + insecticides, and control) initiated in February 2008 on a 12-acre block of Valencia 

orange planted in 2002. The nutritional mix was applied three times a year corresponding to 

major flushes. Insecticides were applied in winter and then when psyllid density exceeded a 

predetermined threshold, maintaining a ratio of adult psyllids in treated to untreated trees of 

approximately 1:12. PCR positive trees increased from 29% in November 2008 to 95% in May 

2010 with significantly lower Ct values in insecticide-treated plots. Significantly positive effects 

on production were seen from insecticides, with highest yield from trees receiving both nutrients 

and insecticides. Preliminary results would thus indicate that the main effect of these programs is 

coming from the insecticidal component with an auxiliary role provided by corrective nutritional 

treatments. 


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10.15 Use of Horticultural Practices in Citriculture to Survive Huanglongbing 

Stuchi, E.S. 1,2 , Girardi, E.A. 1 

1 Embrapa Cassava & Fruits, Cruz das Almas, Brazil 

2 Citrus Experimental Station, Bebedouro, Brazil 

Huanglongbing (HLB) was first reported in Brazil in 2004 and had caused severe losses in the 

main producing regions, threatening the sustainability of the whole citrus chain. Current control 

strategies are based on the use of healthy nursery trees, inspection and systematic eradication of 

symptomatic plants, and chemical control of the insect vector. Research is being carried out to 

achieve HLB resistance, including genetic engineering. Horticultural practices for immediate use 

in citriculture can be evaluated to mitigate HLB effects. The following practices are discussed: 

selection of naturally occurring tolerant materials, new regions for citrus production in Brazil, 

unusual concepts for screened nursery trees’ production, use of repellent and attractive plants, 

low-input production systems, use of resistance elicitors, protected cultivation, intercropping, 

and ultra high density (UHD) plantings. Alternative production systems of screened nursery trees 

include seed-derived trees of scion varieties, intensive production systems, and the use of larger 

nursery trees. The main objective of UHD practice is to anticipate fruit bearing in order to get 

high yields until the tenth harvest. The use of UHD plantings depends on the availability of 

small-sized scion varieties, dwarfing rootstocks, viroid inoculation, and conditioning of nursery 

trees before planting. HLB threat limits the feasibility of current citriculture practices and 

demonstrates the need to join different strategies for confronting this disease: genetic advances, 

pathogen and vector control, and improved horticultural practices. No isolated strategy will 

provide a satisfactory solution. 

References 

Bar-Joseph, M. 2009. Rational management of emerging citrus greening/HLB infections - an 

open item for discussion with IOCV. Newsletter of the International Organization of Citrus 

Virologists, p. 9-14, October 2009. 

Bassanezi, R.B., Montesino, L.H., Stuchi, E.S. 2009. Effects of huanglongbing on fruit quality of 

sweet orange cultivars in Brazil. European Journal of Plant Pathology 125:565-572. 

Belasque J., Jr., Bassanezi, R.B., Yamamoto, P.T., Ayres, A.J., Tachibana, A., Violante, A.R., 

Tank, A., Jr., Di Giorgi, F., Tersi, F.E.A., Menezes, G.M., Dragone, J., Jank, R.H., Jr., 

Bové, J.M. 2010. Lessons from huanglongbing management in São Paulo State, Brazil. 




Folimonova, S.Y., Robertson, C.J., Garnsey, C.S., Gowda, S., Dawson, W.O. 2009. Examination 

of the responses of different genotypes of citrus to huanglongbing (citrus greening) under 

different conditions. Phytopathology 99:1346-1354. 


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Gmitter, F. 2009. Surveying for naturally occurring HLB field resistance. In: Citrus Greening 

Symposium 2009. Bartow, FL, USA: UF/IFAS Extension. 

http://citrusagents.ifas.ufl.edu/events/Citrus_Greening_Symposium_2009/Videos/CitrusGr 

eeningSymposium2009.htm (accessed on 01.10.2010) 

Gottwald, T.R., Irey, M., Bergamin-Filho, A., Bassanezi, R.B., Gilligan, C.A. 2008. A stochastic 

spatiotemporal analysis of the contribution of primary versus secondary spread of HLB. In: 

Gottwald, T.R., Dixon, W., Graham, J., Berger, P. (eds.). Proceedings of the International 

Research Conference on Huanglongbing, Orlando, Florida: USDA and University of 

Florida, p. 247-252. 

Rabe, E., Warrington, J., Toua, J. 1996. Spacing densities: an economic perspective. Proceedings 

of the International Society of Citriculture 2:825-831. 

Roka, F.M., Rouse, R.E., Muraro, R.P. 1997. Southwest Florida citrus yield by tree age in high 

density planting. Proceedings of the Florida State Horticultural Society 110:82-86. 

Roka, F., Muraro, R., Morris, A., Spyke, P., Morgan, K., Schumann, A., Castle, W.S., Stover, E. 

2010. Citrus production systems to survive greening - economic thresholds. Proceedings of 

the Florida State Horticultural Society 122:122-126. 

Stuchi, E.S., Donadio, L.C., Sempionato, O.R. 2003. Performance of Tahiti lime on Poncirus 

trifoliata var. monstrosa Flying Dragon in four densities. Fruits 58:13-17. 

Van Den Berg, M.A. 1994. Synopsis of strategies to reduce populations of citrus psylla, Trioza 

erytreae and the spread of greening. Fruits 49:229-234. 

Zaka, S.M., Zeng, X.N., Holford, P., Beattie, G.A.C. 2009. Repellent effect of guava leaf 

volatiles on settlement of adults of citrus psylla, Diaphorina citri Kuwayama, on citrus. 

Insect Science 17:39-45. 


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10.16 Critical Control Point (CCP) Analysis to Build a Model System for Measuring 

Citrus Propagation Risk Mitigations. II. Sampling and Monitoring 

Brown, L.G., Jones, E.M., Hartzog, H.M. USDA APHIS PPQ Center for Plant Health Science 

and Technology, Raleigh, NC, USA 

The level of sampling required for the detection and monitoring of regulated plant pests is a 

policy decision that considers, among other things, the pest risk. Pest risk analysis includes the 

evaluation of options to reduce, eliminate, or manage the risk at some level that often involves 

sampling at one or more points of mitigation in system approaches. To identify independent 

procedures for monitoring and control, a Critical Control Point system analysis is used to 

identify the hazards and points where mitigation measures can be applied in a defined system. 

Control points are monitored to detect the minimum proportion of the infestation that the 

regulator uses for detection in a designated sample population of citrus plants. The USDA Citrus 

Health Recovery Program - Regulatory Working Group (CHRP-RWG) requested a sampling and 

testing plan that would detect a prevalence of Ca. L. asiaticus (Las) infected plants in a facility 

producing citrus nursery stock (CNS). The Working Group suggested a sampling and testing 

method to detect a 1% prevalence of Las in CNS produced under a systems approach. 

Convention states that there should be no less than a 95% probability of detecting a population of 

plants with 1% prevalence of Las. After the detection level is decided, a sample number can be 

calculated. A complete citrus budwood program risk model was recently proposed by Brown 

(2008). The authors have refined the model to better illustrate risk and the monitoring of control 

points. Criteria for acceptance or rejection based on failure for each independent procedure are 

suggested. These criteria can be used to monitor the system by incorporating a risk level and 

stating the desired level of confidence. A table with sample size and the estimated cost of 

sampling and testing CNS is presented. 

Reference 

Brown, L.G. 2008. A model system for measuring citrus propagation risk mitigation based on 

Hazard Analysis and Critical Control Point (HACCP) methods. Phytopathology 98:S27. 


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10.17 The Need of an Epidemio-Surveillance Network to Prevent Huanglongbing Arrival 

in the South Mediterranean Basin 

Dollet, M. 1 , Aubert, B. 2 , Imbert, E. 3 , Gatineau, F. 1 

1 CIRAD, Department of Biological Systems, Research Unit 29 “Etiology Wilts” TA A-29/F, 

34398 Montpellier Cedex 5, France 

2 ADAC, CIRAD Consultant, Montpellier, France 

3 Department PERSYST; CIRAD, Campus International de Baillarguet, 34398 Montpellier 

Cedex 5, France 

Huanglongbing (HLB) originated in the 1900s in Asia where it is transmitted by the psyllid 

Diaphorina citri. In South Africa another form of HLB, transmitted by Trioza erytreae was 

described in the 1960s. The “African” and “Asian” forms may occasionally occur in the same 

area (Peninsula Arabia), where both species of the psyllids vectors are present, and each psyllid 

can carry both forms of the bacteria associated with HLB – Ca. L. asiaticus and Ca. L africanus. 

In the 2000s, the arrival of HLB on the American continent boosted research on HLB. HLB has 

also caused severe damage in East Africa, mainly Kenya and Tanzania. Recently, Ca. L asiaticus 

was identified in Ethiopia (Saponari et al., 2010). On the west side, in Cameroon, symptoms of 

HLB were described and T. eythreae was present. This psyllid also invaded Madeira and the 

Canary Islands 10 years ago. The Mediterranean Basin produces around 18 million tons of citrus 

yearly. Because of the importance of movements, trade, tourism, and pilgrims, Citrus production 

is in danger. The threat could come from the East (Saudi Arabia, Iran, and Yemen), the South 

(Ethiopia and Somalia), and the West (Cameroon and Canarias). CIRAD, in collaboration with 

Embrapa - Brazil, Cameroon and partners in Morocco, Tunisia, Egypt, and Turkey, will start 

actions for a prevention strategy. A training course on symptoms, psyllids identification, and 

molecular diagnosis is scheduled in early 2011 and will be the foundation of an 

epidemio-surveillance network in the South Mediterranean Basin. 

Reference 

Saponari, M., De Bac, G., Breithaupt, J., Loconsole, G., Yokomi, R.K., Catalano, L. 2010. First 

report of Candidatus Liberibacter asiaticus associated with huanglongbing in sweet orange 

in Ethiopia. Plant Disease 94:482. 


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10.18 Presence of Candidatus Liberibacter asiaticus in Diaphorina citri Kuwayama 

Collected from Plants for Sale in Florida 

Halbert, S.E. 1 , Manjunath, K.L. 2 , Ramadugu, C. 3 , Lee, R.F. 2 





Huanglongbing (HLB or greening) is a devastating disease of citrus reported from Florida in 

2005. The disease has spread to all citrus growing counties in the state. Best management 

strategies for HLB include use of clean planting material, elimination of inoculum, and excellent 

psyllid vector control. In this study, 1,186 samples of Asian citrus psyllids, Diaphorina citri 

Kuwayama, were collected from plants for sale (citrus and its relatives) in Florida over a period 

of 4 years and tested for the presence of HLB-associated Candidatus Liberibacter asiaticus (Las) 

by real-time PCR. Samples came from 44 of Florida’s 67 counties. Overall, about 9.7% of the 

1,186 tested D. citri samples collected from plants for sale were positive for Las. Among the 

psyllid samples tested, about 9% of the samples collected from citrus, 10% of the samples 

collected from Murraya paniculata, and a single sample from Bergera koenigii were positive for 

Las. Murraya paniculata is a preferred host of D. citri and is used widely as an ornamental plant. 

In 2008, Florida State regulations began requiring mandatory screened enclosures for 

propagation of citrus and related plants (including M. paniculata). After the new regulations 

came into effect, the number of psyllid finds in venues that propagate citrus (citrus propagating 

nurseries and budwood facilities) dropped substantially, indicating the efficacy of the new rules. 

Propagation and sale of M. paniculata in Florida ceased when it became necessary (and 

uneconomical) to grow the popular ornamental plant under stringent containment. 


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10.19 A Model System for Studying Huanglongbing 

Manjunath, K.L. 1 , Ramadugu, C. 2 , Kund, G. 3 , Trumble, J. 3 , Lee, R.F. 1 


2 Dept. of Botany and Plant Sciences, University of California at Riverside, CA, USA 

3 Dept. of Entomology, University of California at Riverside, CA, USA 

Since its first report from the Western hemisphere in 2004, citrus greening disease 

(huanglongbing, HLB) has caused severe damage to industries of Brazil and Florida. Both the 

vector and the disease have quickly spread into other citrus growing countries. Prolonged 

latency, lower titer of the bacteria in plants, seasonality of infection in vectors, slow disease 

progression in citrus, and several other factors make it a challenging system to conduct research 

and to develop better disease management strategies. In the present study, biological and 

molecular characterization of a bacterium (Candidatus Liberibacter psyllaurous), closely related 

to HLB associated bacteria, that causes “psyllid yellows” symptoms in tomato was used as a 

model system for research on various aspects related to management of HLB. The genome of 

Ca. L. psyllaurous was compared with that of Ca. L. asiaticus employing various techniques. 

The disease was successfully graft transmitted to healthy tomato plants. Symptom expression in 

tomatoes was shown to be temperature dependent. Symptom suppression was achieved at higher 

temperatures when plants were maintained at 36°C/30°C (16 h/8 h), but was not affected at 25°C 

or at 8°C. Results of effects of antibiotic therapy on mitigation of disease will be discussed. Host 

range studies using both graft and insect transmission of several members of Solanaceae revealed 

both resistant and susceptible plants. The suitability of this model system to screen a large 

number of chemicals, antibiotics, and transgenes for potential control of citrus HLB will be 

discussed. 


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Session 11: 

Host Tolerance and 

Resistance 


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11.1 Incidence of Huanglongbing on Several Sweet Orange Cultivars Budded onto 

Different Rootstocks at the Citrus Experimental Station (EECB), Bebedouro, São Paulo, 

Brazil 

Stuchi, E.S. 1,2 , Reiff, E.T. 2 , Sempionato, O.R. 2 , Girardi, E.A. 1 , Parolin, L.G. 2 , Toledo, D.A. 2 



Huanglongbing (HLB), caused by Candidatus Liberibacter asiaticus and Ca. L. americanus and 

vectored by Diaphorina citri Kuwayama, was first reported in 2004 in Brazil and it is currently 

widespread in São Paulo State (Belasque et al., 2010). The EECB, in partnership with Embrapa 

Cassava & Fruits, conducts a citrus improvement program aimed to select scion and rootstock 

Citrus cultivars mainly focused on the resistance or tolerance to biotic and abiotic stresses. In 

2006, the first HLB-infected tree was reported at the EECB, and since then, a program of 

inspection and removal of symptomatic trees has been applied, recording disease incidence data. 

About 200 sweet orange scion/rootstock combinations are under evaluation in trees from 4 to 

20 years of age. Main commercial rootstocks such as Rangpur lime, Swingle citrumelo, and 

Sunki and Cleopatra mandarins are considered, as well as some hybrids, mandarins, 

miscellaneous genotypes obtained from germplasm banks, and some introduced materials. Sweet 

orange cultivars and clones primarily correspond to new budlines obtained from germplasm 

banks and several old budlines from regional selections that were sanitized by shoot tip grafting 

(STG) and inoculated with the PIAC CTV strain for cross protection. Sweet orange scions 

introduced from different countries and regions of the world are also under evaluation. The 

effects of plant age, rootstock cultivar, scion maturation period, budline type, different clones of 

a given cultivar, plant localization relative to the plot borders, and the interaction of all these 

factors on HLB incidence will be discussed. 

Reference 

Belasque J., Bassanezi, R.B., Yamamoto, P.T., Ayres, A.J., Tachibana, A., Violante, A.R., Tank, 

A., Di Giorgi, F., Tersi, F.E.A., Menezes, G.M., Dragone, J., Jank, R.H., Bové, J.M. 2010. 

Lessons from huanglongbing management in São Paulo State, Brazil. Journal of Plant 

Pathology 92:285-302. 


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11.2 Host Preference and Suitability of Native North American Rutaceae for the 

Development of the Asian Citrus Psyllid, Diaphorina citri Kuwayama 

Sandoval, J.L., II, Sétamou, M., da Graça, J.V. Texas A&M University-Kingsville, Citrus 

Center, Weslaco, TX, USA 

The Asian citrus psyllid, Diaphorina citri Kuwayama, a vector of citrus huanglongbing 

(greening disease), is now present in all citrus producing states in the USA and Mexico. It can 

reproduce on several plant species in the Rutaceae family; orange jasmine (Murraya spp.) and 

curry leaf (Bergera koenigii) are amongst its favored hosts. There are several indigenous 

Rutaceae species in North America and some (e.g., Choisya spp.) are popular ornamentals. 

A study was therefore initiated to determine the suitability of some of these plants for the 

development of the psyllid using no-choice and choice experiments. D. citri was found to 

successfully colonize and reproduce on Choisya ternatea, C. arizonica, and Helietta baretata in 

no-choice tests, but reverted back to its preferred hosts, orange jasmine and curry leaf, in choice 

tests. On some of the other plant species (Amyris madrensis, A. texana, and Zanthoxylum 

fagara), adult psyllids laid eggs that hatched, but no nymphal development was recorded beyond 

the first instar. No reproduction occurred on Esenbeckia berlandieri, Ptelea trifoliate, or 

Casimiroa edulis, although adult psyllids were able to survive on these species for several days. 


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11.3 Progress Using Transgenic Approaches and Biotechnology-Facilitated Conventional 

Breeding to Develop Genetic Resistance/Tolerance to HLB in Commercial Citrus 

Grosser, J.W., Dutt, M., Shohael, A., Barthe, G.A. UF-IFAS Citrus Research and Education 

Center, Lake Alfred, FL, USA 

A primary strategy of our program to produce HLB-resistant citrus is genetic engineering to 

incorporate bacterial resistance genes not found in citrus. Antimicrobial peptides (AMPs) are 

part of the innate immune response and can be found among all classes of life, including humans. 

These peptides are usually small proteins and have an ability to associate with membranes. 

Antimicrobial peptides are also characterized by their broad spectrum antibiotic property. 

Incorporation of one or more genes encoding for antimicrobial peptides into the citrus genome 

via genetic engineering could potentially result in development of cultivars resistant to HLB 

without otherwise altering varietal integrity. We have made significant progress moving several 

AMP genes into commercial citrus cultivars, mostly grapefruits and sweet oranges, using 

standard Agrobacterium-mediated citrus transformation and an alternative protoplast/GFP citrus 

transformation developed previously in our laboratory. A second strategy being employed is to 

incorporate genes that may turn on systemic acquired resistance (SAR). We have also tested and 

identified several phloem-limited promoters that function efficiently in citrus. Genetically 

modified plants containing antimicrobial genes or SAR-induction genes driven by a 

phloem-specific Arabidopsis sucrose synthase promoter have also been produced. The targeting 

of antimicrobial gene expression using phloem specific promoters is expected to minimize the 

expression of the foreign transproteins in subsequent fruit and juice products. Hundreds of 

transgenic plants from independent transformation events have already been regenerated and 

propagated, and greenhouse and field testing (at two sites in heavy HLB-pressure areas) of these 

transgenic plants is underway. Our goal is to identify the most effective yet safe transgene(s) 

against HLB and then transform it (them) into selected high-quality sweet orange cultivars with a 

sequential range of maturity dates. We are also testing complex hybrid rootstock candidates to 

determine their affect on HLB disease establishment and severity in grafted sweet orange scions. 

A preliminary experiment showed that complex ‘tetrazyg’ rootstocks infer variable tolerance 

when grafted with HLB-infected sweet orange scion. ‘Tetrazygs’ are allotetraploid hybrids 

obtained from crosses of somatic hybrids created by protoplast fusion. The working hypothesis is 

that the identification of a rootstock that can prevent HLB bacterium replication and has a higher 

efficiency of pumping specific nutrients affected by the HLB disease complex could mitigate the 

development and spread of the disease when challenged in the field. 


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11.4 Promoter Regulation of the β-Glucuronidase (GUS) Gene and Antimicrobial Peptide 

D4E1 in a Citrus Rootstock 

Benyon, L.S., Stover, E., Bowman, K.D., McCollum, G., Niedz, R. U.S. Horticultural Research 

Laboratory, USDA-ARS, Fort Pierce, FL, USA 

Because agrochemicals and conventional breeding for disease resistance have not been sufficient 

in controlling huanglongbing (HLB), alternative strategies for sustaining citrus crops have 

attracted attention. Since no HLB resistance has been identified within cultivated citrus, 

transgenic solutions to the disease have become a focus of breeding programs. Among these 

solutions, use of antimicrobial peptides (AMPs) to enhance plant resistance is at the forefront. 

Virtually all organisms possess an innate immune response involving AMPs that can counter a 

microbial infection. At the USHRL, we have chosen to use synthetic or plant AMPs over those 

of animal origin. The first of the AMPs selected was D4E1, a 17 amino acid synthetic AMP 

which forms a β sheet (Lucca et al., 1998) and is active against Agrobacterium tumefaciens in 

poplar (Mentag et al., 2003). It is a highly active AMP with a minimum inhibitory concentration 

(MIC) less than 1 μM (Stover et al., 2008). We constructed a binary vector (pUSHRL) with an 

expression cassette containing either the β-glucuronidase (GUS) reporter gene or the D4E1 AMP 

under the control of several promoters in the trifoliate rootstock US-802. Because Candidatus 

Liberibacter, the gram-negative bacterium associated with HLB, infects only the phloem tissue 

of the plants, it may be desirable to express potential transgenes specifically in the phloem or 

companion cells. Thus, we opted to provide seven different promoters for our two genes. The 

promoters included: 2x35S from the cauliflower mosaic virus, WDV from wheat dwarf 

geminivirus, PR-1 the pathogenesis-related protein gene 1 promoter from tobacco, AtSS the 

sucrose-H+ symporter gene promoter from Arabidopsis, SSyn the sucrose synthase promoter 

from citrus, PP2 the phloem protein 2-like gene promoter from citrus, and 409S a truncated 

polyubiquitin promoter from potato. The promoter activities include constitutive (2x35S; 409S), 

inducible (PR-1), phloem-specific (AtSS, SSyn, and WDV), and Affymetrix citrus microchip 

HLB up-regulated (PP2). Seedlings are being screened for transformation efficiency, localization 

of expression, and level of transgene expression. 

References 

Lucca, A.J., de Bland, J.M., Grimm, C., Jacks, T.J., Cary, J.W., Jaynes, J.M., Cleveland, T.E., 

Walsh, T.J. 1998. Fungicidal properties, sterol binding, and proteolytic resistance of the 

synthetic peptide D4E1. Canadian Journal of Microbiology 44:514-520. 

Mentag, R., Luckevich, M., Morency, M.J., Seguin, A. 2003. Bacterial disease resistance of 

transgenic hybrid poplar expressing the synthetic antimicrobial peptide D4E1. Tree 

Physiology 23:405-411. 

Stover, E., Bowman, K., McCollum, G., Niedz, R. 2008. Developing transgenic solutions for 

HLB resistant citrus at the US Horticultural Research Laboratory. Proceedings of the 

International Research Conference on Huanglongbing, p. 374. 


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11.5 Responses of Transgenic Hamlin Sweet Orange Plants Expressing the attacin A Gene 

to Candidatus Liberibacter asiaticus Infection 

Felipe, R.T.A. 1 , Mourão-Filho, F.A. 1 , Pereira, E.V., Jr. 1 , Lopes, S.A. 2 , Sousa, M.C. 2 , Mendes, 

B.M.J. 3 



3 CENA/Universidade de São Paulo, Piracicaba, Brazil 

Genes that code for antimicrobial peptides have been used in an attempt to produce transgenic 

plants against bacterial pathogens. Herein, we show the responses of Hamlin sweet orange 

cultivar transformed with attacin A gene (attA), in 12 independent transformation events, against 

Candidatus Liberibacter asiaticus infection. Four plants of each event were inoculated, by stem 

grafting, with HLB-positive budsticks, and evaluated for symptom expression and bacterial titers 

(Log 10 Liberibacter copies per gram of leaf tissue), using SYBR green qPCR, 8 months after 

inoculation. Four non-transformed inoculated plants were used as positive controls. HLB leaf 

symptom severities and average bacterial titers of most plants did not differ from that found on 

the control. However, plants of two transformation events (8 and 11) showed lower symptom 

severities and lower bacterial titers. Current work might indicate if symptom and bacterial titers 

correlate with attA expression levels. Details of this study will be presented and the perspectives 

in HLB control with the use of transgenic plants are discussed. 


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11.6 Screening Antimicrobial Peptides In-Vitro for Use in Developing Huanglongbing and 

Citrus Canker Resistant Transgenic Citrus 

Stover, E. 1 , Stange, R. 1 , McCollum, G. 1 , Jaynes, J. 2 


2 Integrative Biosciences Program, Tuskegee University, Tuskegee, AL, USA 

Huanglongbing (HLB) and Asiatic citrus canker (ACC) seriously threaten sustainability of the 

Florida citrus industry. No HLB resistance has been identified within cultivated citrus, so 

development of resistant transgenic citrus is a high priority. With the HLB pathosystem largely 

unknown, broad-spectrum antimicrobial peptides (AMPs) are the current focus. In vitro 

assessment of minimum inhibitory concentration was conducted using Sinorhizobium meliloti 

and Agrobacterium tumefaciens as α-proteobacteria surrogates for Liberibacter (causal agent of 

HLB). Xanthomonas citri (causing ACC) was included in these analyses in the hope that HLB 

and ACC resistance can be achieved with the same AMP transgene, using non-tissue-specific 

promoters. Nineteen AMPs were initially tested from diverse sources. The most active AMPs 

included Tachyplesin 1 from horseshoe crab, SMAP-29 from sheep, and the synthetic D4E1. 

These AMPs inhibited growth of all three test bacterial species at 1 µM or less. A further 

24 synthetic AMPs were designed based on initial results, and 11 of these showed effectiveness 

at 1 µM or less across all three test bacteria. Most AMPs were comparable in effectiveness 

across the three bacterial species, but some species × AMP interactions were observed. 

Hemolytic activity was assessed by exposure of porcine erythrocytes to a range of AMP 

concentrations. Hemolysis from most AMPs at 10 µM was not significantly different from water, 

while melittin (from bee venom) was highly hemolytic. AMPs which are not of animal origin, 

that suppress bacterial growth at 1 µM or less, and show negligible hemolysis are the focus for 

further work developing transgenic citrus resistant to HLB and ACC. 


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11.7 Response of Citrus Transgenic Plants Expressing STX IA Gene to Candidatus 


Marques, V.V. 1,2 , Bagio, T.Z. 3 , Sugahara, V.H. 1 , Vasquez-Souza, G.V. 1,2 , Grange, L. 4 , 

Meneguim, L. 3 , Kobayashi, A.K. 5 , Bespalhok, J. 4 , Pereira, L.F.P. 5 , Vieira, L.G.E. 1 , Leite, R.P., 

Jr. 1 

1 IAPAR/Instituto Agronômico do Paraná, PR, Brazil 

2 FAPEAGRO/Fundação de Amparo à Pesquisa e ao Agronegócio, Londrina, PR, Brazil 

3 UEL/Universidade Estadual de Londrina, Londrina, PR, Brazil 

4 UFPR/Universidade Federal do Paraná, Curitiba, PR, Brasil 

5 EMBRAPA/Empresa Brasileira de Pesquisa Agropecuária, Brasília, DF, Brazil 

Huanglongbing (HLB) is a major threat for citrus production around the world. However, no 

resistance to this disease has been found in species of the genus Citrus or in plants of closely 

related genera. The development of transgenic plants for disease resistance in economically 

important cultivars of citrus may represent a unique and efficient strategy for control of HLB. 

Plant transformation with antibacterial peptides has been used to enhance pathogen resistance in 

different plants. Sarcotoxin IA (STX IA) is an antibacterial peptide that is secreted by a meat-fly 

Sarcophaga peregrina and belongs to the peptide group that interacts with bacterial cellular 

membrane causing an electrochemical potential loss. In this study, we evaluated transgenic sweet 

orange events transformed with the STX IA gene in regard to the HLB disease. Experiments were 

conducted at the Instituto Agronômico do Paraná – IAPAR, Londrina, PR, Brazil, under 

greenhouse conditions, by using five independent events of sweet orange cv. Pera (STX-3, 

STX-5, STX-11, STX12, and STX-13) transformed with the vector pST10 containing the STX IA 

gene under control of CaMV 35S promoter and the signal peptide from tobacco PR1a 

(Bespalhok et al., 2001). For each transformation event, five plants at the same developmental 

stage were inoculated by two different methods. By grafting inoculation, segments 2 to 4 cm 

long of Ca. L. asiaticus-infected citrus plants were grafted in the test plants. By insect 

inoculation, 20 psyllids (Diaphorina citri) were transferred from donor plants to each test plant. 

The insects were kept in a net cage on the plant for 2 weeks. After killing the psyllids by 

insecticide spray, the plants were maintained under greenhouse conditions for further observation 

of symptom development. Non-transformed plants of the same cultivar of sweet orange were 

used as control. The plants were examined regularly for HLB symptom development and 

evaluated by PCR for HLB bacterium presence 6 and 13 months after inoculation. Leaf DNA 

was extracted from 500 mg of central leaf veins according to the CTAB protocol of Murray and 

Thompson (1980). PCR was performed by using rplA2/rplJ5 primers to identify Ca. L. asiaticus 

(Teixeira et al., 2005). In both methods of inoculation, the plants showed symptoms of the 

disease approximately 10 months after inoculation. Disease incidence ranged from 20% to 100% 

among the transgenic and control plants (Fig. 1). The results show an improvement in resistance 

to HLB of three transgenic events, STX-3, STX-5, and STX-11 (Fig. 1). However, the 

concentration of the HLB bacterium was not determined in the different tested plants. 

Nevertheless, the variation in symptom expression indicates increased HLB resistance in some 

transgenic events. 


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This work has been supported by the Fundo de Defesa da Citricultura (FUNDECITRUS), 

Araraquara, SP, Brazil. 

References 

Bespalhok Filho, J.C., Kobayashi, A.K., Pereira, L.F.P., Vieira, L.G.E. 2001. Laranja 

transgênica. Biotecnologia, Ciência e Desenvolvimento 23:62-66. 



da Graça, J.V., Korsten, L. 2004. Citrus huanglongbing: review, present status and future 

strategies. In: S.A.M.H. Naqui (ed.). Diseases of Fruits and Vegetables. Kluwer Academic, 

The Netherlands, p. 229-245. 

Okada, M., Natori, S. 1983. Purification and characterization of an antibacterial protein from 

hemolinph of Sarcophaga peregrine (flesh-fly) larvae. Biochemical Journal 211:727-734. 

Teixeira, D.C., Danet, J.L., Eveillard, S., Martins, E.C., Jesus, W.C., Jr., Yamamoto, P.T., Lopes, 

S.A., Bassanezi, R.B., Ayres, A.J., Saillard, C., Bové, J.M. 2005. Citrus huanglongbing in 

São Paulo state, Brazil: PCR detection of the Candidatus Liberibacter species associated 

with the disease. Molecular and Cellular Probes 19:173-179. 

Fig. 1. Frequency of HLB-diseased plants of transgenic events and non-transformed control 

plants 6 months after inoculation with Ca. Liberibacter asiaticus. Based on PCR results by using 

the rplA2/rplJ5 primers 6 months after inoculation. 


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11.8 Rootstocks and Pruning Effects on Huanglongbing Incidence on Tahiti Limes in 

Bebedouro, Northern São Paulo State, Brazil 

Stuchi, E.S. 1,2 , Reiff, E.T. 2 , Sempionato, O.R. 2 , Cantuarias-Avilés, T. 2 , Girardi, E,A. 1 , Parolin, 

L.G. 2 , Toledo, D.A. 2 



In 2004, huanglongbing (HLB), caused by Candidatus liberibacter asiaticus and 

Ca. L. americanus and vectored by Diaphorina citri Kuwayama, was first reported in São Paulo 

State, Brazil. Tahiti (Persian) lime is an important regional fruit crop, but HLB symptoms are not 

so clearly expressed as in sweet oranges. In 2001, an experiment was planted in Bebedouro, 

Northern São Paulo State, aimed to characterize the following 12 rootstocks (treatments) for 

Tahiti lime: two Rangpur limes (FCAV and Limeira), three trifoliate oranges (Rubidoux, FCAV, 

and Flying Dragon (FD)), Swingle citrumelo, Sunki, and Sun Chu Sha Kat mandarins, Orlando 

tangelo, Carrizo citrange, and the hybrids Changsha × English Small (HRS 801) and Rangpur 

lime × Swingle citrumelo (HRS 827). Except on FD, all the other rootstocks induced high vigor 

to Tahiti lime canopies. In May 2009, after a first 7-year evaluation cycle (Cantuarias-Avilés, 

2009), trees were pruned to a 3 m height. Most of the trees showed HLB-like symptoms in 

October 2009, and on December 2009, they were sampled for Ca. Liberibacter spp. detection by 

PCR. All the suspected trees were positive. Until December 2009, the average cumulative 

incidence (CI) of HLB in the plots was 72.1%, with CI values varying from 10 to 100%. For 

most of the rootstocks, CI values were equal or higher than 60%, while the trees budded onto the 

FD rootstock showed the lower HLB incidence (10%). Pruning may be an extremely hazardous 

practice in areas under high HLB pressure if a careful vector control program is not applied. 


Financial Support: FAPESP. 

Reference 

Cantuarias-Aviles, T. 2009. Avaliação horticultural da laranjeira ‘Folha Murcha’, tangerineira 

‘Satsuma’ e limeira ácida ‘Tahiti’ sobre doze porta-enxertos. Universidade de São Paulo 

Escola Superior de Agricultura “Luiz de Queiroz”, Piracicaba/SP, Brazil, 129 p. 


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11.9 Candidatus Liberibacter asiaticus (CLas) Titer in Field HLB-Exposed Commercial 

Citrus Cultivars 

Stover, E., McCollum, G. USDA-ARS-USHRL, Fort Pierce, FL, USA 

There are reports that some citrus scion cultivars are slower to develop huanglongbing (HLB; 

e.g., Lange et al., 1985; Shokrollah et al., 2009). To evaluate this in Florida, eight groves with 

four or more diverse cultivars planted in close proximity were surveyed. Twenty trees of each 

cultivar in each grove were randomly selected to avoid bias and edge effects, and an HLB 

diagnostic leaf sample was collected from each. CLas 16S rDNA primers (Li et al., 2006) were 

used in qPCR, all standardized to 3 ng nucleic acid/reaction. No cultivar was low in CLas titer in 

all groves. Across all groves, 22% of the 760 trees tested had a Ct value 5.8 CLas 

genomes/sample). In two groves, there was little HLB. Data on the six groves with high 

incidence of HLB were subjected to ANOVA, using CT values. Minneola, Sweet Orange, and 

Murcott displayed the greatest CLas titer, averaging 304, 236, and 168 CLas/sample. In the 

lowest titer group, Temple, Fallglo, grapefruit, and Sunburst displayed the lowest titers, at 9, 13, 

40, and 107, respectively. Minneola and Temple differed 30-fold in CLas titer and were 

compared adjoining each other in all the same groves. Assessment of Las/HLB in replicated 

cultivar trials and breeding populations exposed to high disease pressure and controlled psyllid 

challenges have all been initiated. Since Temple, Sunburst, Fallglo, and their parents are widely 

used in our breeding program, we hope to identify diverse scion types with greater HLB 

resistance for near-term deployment. 

References 

Lange, J.H. de, Vincent, A.P., Nel, M. 1985. Breeding for resistance to greening disease in 

citrus. Citrus and Subtropical Fruit Journal 614:6-9. 

Li, W., Hartung, J.S., Levy, L. 2006. Quantitative real-time PCR for detection and identification 



Shokrollah, H., Abdullah, T.L., Sijam, K., Abdullah, S.N.A., Abdullah, N.A.P. 2009. Differential 

reaction of citrus species in Malaysia to huanglongbing (HLB) disease using grafting 

method. American Journal of Agricultural and Biological Sciences 4:32-38. 


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11.10 Host Response of Different Citrus Genotypes and Relatives to Candidatus 

Liberibacter asiaticus Infection 

Boscariol-Camargo, R.L., Cristofani-Yaly, M., Malosso, A., Coletta-Filho, H.D., Machado, 

M.A. Centro APTA Citros Sylvio Moreira – IAC, Cordeirópolis/SP, Brazil 

Citrus huanglongbing (HLB) or greening, reported in São Paulo State in 2004, is considered the 

main disease of the crop worldwide and has caused significant damage. In Brazil, the disease is 

caused by two species of the bacteria Candidatus Liberibacter, and disease control is 

accomplished by eradicating infected plants and insecticidal spraying, but there is a demand for 

alternative strategies with lower economic and environmental costs. Considering that much of 

the germplasm of citrus in Brazil had not yet been evaluated for susceptibility or resistance to the 

HLB bacterium, this study aimed to evaluate the multiplication of bacteria and the development 

of symptoms in species and varieties of citrus and related genera. Five replicates of each plant 

were inoculated with infected Ca. Liberibacter asiaticus buds and evaluated for disease 

symptoms and presence of bacteria during 12 months. Grafted plants with healthy budwood were 

used as negative control. Evaluation was carried out every 2 months using Real Time 

Quantitative PCR with TaqMan probes. After infection with infected buds, the bacteria could be 

detected in all genotypes, although with variation in the amount of bacteria over time. Genotypes 

such as Atalantia, Poncirus trifoliata, and Citrus limettioides Tanaka (Persian lime) showed a 

reduction in the bacteria multiplication rate, indicating some degree of tolerance. Actually, there 

was no relationship between the titer of the bacteria in the tissues, presence, and intensity of 

symptoms. 


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11.11 Candidatus Liberibacter asiaticus (CLas) Titer in Poncirus trifoliata and P. trifoliata 

Hybrids: Inferences on Components of HLB Resistance 

Stover, E., Shatters, R.G., Jr., McCollum, G., Hall, D.G., Duan, Y.-P. USDA-ARS-USHRL, 

Fort Pierce, FL, USA 

Poncirus trifoliata hybrids field grown at USHRL on Sun Chu Sha rootstock were tested for 

CLas 16S rDNA and Citrus dehydrin by qPCR, assessing random quadrant samples, a diagnostic 

“worst” sample, and rootstock suckers (November 2009). Resulting data were expressed as 

abundance of CLas relative to Citrus dehydrin. The two P. trifoliata had non-detectable or low 

CLas abundance, as did two citranges, except that citrange diagnostic samples and rootstock 

samples had very high CLas (20-24 CLas rDNA/Citrus dehydrin). Variability was observed in 

relative CLas abundance among the 10 citranges tested with most showing high abundance in 

quadrants (20 CLas/citrus gene), and all showed high CLas in rootstock suckers. The data 

suggest that Poncirus and some Poncirus hybrids suppress CLas even when grafted onto a 

high-titer source. Data suggest that in some citranges, CLas increases in small populations of 

leaves with the possibility that leaves undergoing senescence may permit proliferation as host 

defenses decline. Using only most-symptomatic diagnostic samples may obscure important 

differences in CLas proliferation. Theoretically, slower development of HLB/CLas could be due 

to alteration in several components: attractiveness of trees to ACP, CLas establishment at ACP 

feeding, CLas proliferation following ACP inoculation, systemic movement of CLas with 

subsequent further proliferation, and development of plant responses observed as HLB 

symptoms. Reduction or slowing of any of these steps may slow disease development and spread 

but with different implications in management and commercial significance. Careful 

consideration needs to be given to the value and implications of such resistance or tolerance. 

References 

Folimonova, S.Y., Robertson, C.J., Garnsey, S.M., Gowda, S., Dawson, W.O. 2009. 

Examination of the responses of different genotypes of citrus to huanglongbing (citrus 

greening) under different conditions. Phytopathology 99:1346-1354. 

Li, W., Hartung, J. S., Levy, L. 2006. Quantitative real-time PCR for detection and identification 




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11.12 The Role of Salicylic Acid and Systemic Acquired Resistance in the Response of 

Citrus to HLB 

Khalaf, A. 1 , Febres, V.J. 1 , Brlansky, R.H. 2 , Gmitter, F.G., Jr. 2 , Moore, G.A. 1 

1 UF-IFAS Horticultural Sciences, Gainesville, FL, USA 


To study the mechanisms of induced resistance in citrus, we designed microarray experiments to 

compare the response of citrus types to exogenous application of salicylic acid (SA) and 

infection with Candidatus Liberibacter asiaticus, the etiological agent of huanglongbing (HLB). 

SA has been shown to induce the expression of genes associated with plant defense and the 

establishment of systemic acquired resistance (SAR). Our custom Agilent Citrus GeneChip 

(4x44K format) was used in these experiments to compare treated and untreated grapefruit 

(Citrus paradisi) plants. Functional expression analysis of the microarray data confirmed 

up-regulation of some genes previously associated with SA/SAR (pathogenesis-related proteins, 

LRR, lipid metabolic processes, etc.) in treated but not untreated plants. We also evaluated gene 

expression responses of sweet orange (C. sinensis) and rough lemon (C. jambhiri) to HLB. In our 

greenhouse observations, sweet orange developed yellow blotchy mottle symptoms earlier than 

rough lemon. Plants were also tested by PCR for the presence of HLB and only positive plants 

were further used in the functional gene expression analyses. More than half of the genes 

significantly induced by SA were also induced in rough lemon but not sweet orange 27 weeks 

after inoculation with HLB. These results indicate that HLB induced a response that overlapped 

in that part of SA. This information could be exploited to improve disease management strategies 

in the field. 


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11.13 Observations of Citrus × Poncirus Hybrid Tolerance to Infection with Candidatus 


Bowman, K.D., Albrecht, U. USDA-ARS, Fort Pierce, FL, USA 

This study used greenhouse inoculation tests and field trials to investigate the apparent tolerance 

of some Citrus × Poncirus hybrids to infection with Candidatus Liberibacter asiaticus (Las). The 

citrus rootstock US-897 (Citrus reticulata Blanco × Poncirus trifoliata L. Raf.) was observed to 

be tolerant to HLB in field plantings. Where US-897 trees were naturally exposed to high 

populations of Asian citrus pysllid, Diaphorina citri (ACP), infected with Las, they did not 

exhibit distinct symptoms typical for HLB disease. Greenhouse tests were completed to compare 

the response of US-897 seedlings inoculated with Las to those of susceptible genotypes. 

Although ACP-exposed field trees and Las-inoculated greenhouse trees of US-897 became 

PCR-positive for the pathogen, they did not develop leaf symptoms nor develop faint leaf 

symptoms only much longer after inoculation. In addition, growth of US-897 plants was 

unaffected or only slightly affected by Las infection. Other hybrids of P. trifoliata with Citrus 

spp. were also observed to exhibit some tolerance to Las infection, including US-802 (Citrus 

grandis × P. trifoliata). Some variability was observed in the tolerance reaction of different 

Poncirus hybrids to Las infection. Preliminary greenhouse and field studies with susceptible 

scions grafted on US-897 and some other Poncirus hybrids suggest that these rootstocks may 

provide some delay in HLB disease progression or moderation of symptom expression, but that 

all trees, regardless of rootstock, eventually decline from the disease. Studies are underway now 

to examine the effect of the Poncirus-type tolerance in a scion and to determine its physiological 

basis. 


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11.14 Performance of a Phage Gene in Transgenic Citrus Resistant to Citrus Greening 

Jiang, Y. 1 , Perazzo, G. 1 , Septer, A. 1 , Kress, R. 2 , Gabriel, D.W. 1,3 

1 Integrated Plant Genetics, Inc, Alachua, FL, USA 

2 Southern Gardens Citrus, Clewiston, FL, USA 

3 UF-IFAS Plant Pathology Department, Gainesville, FL, USA 

Huanglongbing (HLB), also known as citrus greening, is a lethal disease of citrus caused by 

several species of Candidatus Liberibacter, a psyllid transmitted, phloem-limited, 

α-proteobacteria. One of these species, Ca. Liberibacter asiaticus (Las), is widespread in Florida 

citrus. Additional species of Liberibacter have emerged as new infectious agents of tomato and 

potato. There are no practical chemical controls for Liberibacter. A gene was isolated from phage 

Xp15 of Xanthomonas campestris pv. pelargonii that encodes a protein, BC, that is not directly 

lethal to bacteria but affects ability to resist natural plant anti-microbial compounds, including 

phytoalexins. More than 40 transgenic citrus lines (cv. Carrizo, Pineapple sweet orange, and 

Duncan grapefruit) expressing BC were generated and half were clonally propagated. Expression 

of BC did not obviously affect the morphology or development of transgenic citrus trees, many 

of which are over 3 years old. Western blot analyses showed that BC can be stably expressed in 

citrus. For challenge inoculations, Las was introduced by grafting Las-infected grapefruit as 

scions onto BC expressing transgenic and control lines as rootstock. Las-infected, grafted scions 

transmitted Las to previously uninfected rootstock, including some transgenic BC lines, based on 

positive nested PCR tests of rootstock shoots. However, after removal of the infected scions, all 

transgenic lines expressing BC became Las negative within 1-2 months, and remained so after 

9 months of testing to date, i.e., the BC expressing lines appeared to be self-curing. 

Non-transgenic and/or silenced citrus lines that became positive remained positive. Las was also 

introduced into tobacco by use of infected dodder; all transgenic tobacco plants tested that 

expressed BC remained Las-negative after dodder challenge. Transgenic citrus plants expressing 

BC are in field trials conducted by Southern Gardens Citrus. 


Financial Support: Southern Gardens Citrus and USDA-APHIS. 


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11.15 Genome Sequences of Haploid Clementine Mandarin and Diploid Sweet Orange 

Gmitter, F.G., Jr. UF-IFAS Citrus Research and Education Center, Lake Alfred, FL, USA 

The International Citrus Genome Consortium (ICGC) is releasing a preliminary annotated 

genome assembly from a Clementine mandarin haploid plant, sequenced using Sanger 

technology. Sequencing, assembly, and annotation were performed by Genoscope (France), 

USDOE-JGI and HudsonAlpha (USA), and IGA (Italy) with contributions from Lifesequencing 

(Spain). Brazil, France, Italy, Spain, and the USA (i.e., the Florida citrus industry) provided 

financial support for the project. The assembly represents 6.3x coverage. Preliminary annotation 

is underway (November 2010) to be completed before January 2011. Additional steps to refine 

and improve the sequence, including haploid BAC end sequencing, a high-density linkage map, 

and additional sequencing, will be described; a chromosome-based assembly should be complete 

by late summer 2011. Additionally, an annotated assembly of the heterozygous diploid sweet 

orange is being released by a collaboration of UF, Roche/454 Life Sciences, USDOE-JGI, and 

Georgia Tech University. This genome was assembled from shotgun reads using 454 FLX and 

FLX Titanium platforms (14x coverage), 3 kb and 8 kb insert paired end library FLX reads 

(9 and 6.7x coverage, respectively), plus an additional 1.2x Sanger coverage produced previously 

at JGI. The genome currently represents ~320Mb, anchored to more than 12,500 scaffolds; 

however, more than 50% of the sequence is within only 256 scaffolds. Annotation integrated 

EST information with ab initio GeneMark models, predicting 25,376 protein coding loci and 

more than 46,000 transcripts. Future perspectives on citrus genome sequencing will be discussed, 

as well as the potential applications of these genome sequences to the efforts to provide 

long-term genetic solutions to HLB. 


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11.16 Exploring Metabolic Profiles of Plant Tissue with Increased or Decreased 

Susceptibility 

Malik, N.S.A., Pérez, J.L., Brockington, J., Mangan, R.L. USDA-ARS, Kika de la Garza 

Subtropical Agricultural Research Center, Weslaco, TX, USA 

Studies were initiated to investigate any links between metabolic profiles of the citrus leaves and 

the extent of ACP infestation. Exploratory studies were therefore conducted to investigate 

differences in metabolic profiles between young and mature leaves. Initially, profile of 

polyphenols in young and susceptible leaves versus mature and not susceptible leaves showed 

that with the age of grapefruit (Rio Red) leaves there occurs a drastic decline in flavonoids. For 

example, the levels of flavonoid naringin declined by 90%, while there were substantial losses in 

apigenin and Flavonid-1 (identity yet to be determined), when young leaves matured. In addition, 

our initial studies indicated that sour orange plants first subjected to low temperature (5 ± 1°C) 

for 5-6 days attracted more psyllids than the control plants, i.e., plants that are continuously kept 

under warm temperature (26 ± 1°C). Polyphenol profiles of leaves exposed to low temperature 

showed specific difference from the polyphenol profiles of control plants. In addition, we 

compared the changes in leaf polyphenols resulting from psyllid infestation (psyllid stress) to 

changes resulting from water stress. These studies provided interesting insight to metabolic 

changes in satsuma leaves in response to different stresses. For example, two flavonoids appear 

only in leaves infected with ACP, i.e., they are absent in normal and water stressed leaves. In 

addition, caffeic acid levels increased nearly 100% in water stressed leaves but declined nearly 

80% in leaves infested with psyllids. The samples will now be analyzed for free amino acids, 

polyamine, sugars, and pigments. 


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Take-Home Messages: 

What Can Be Implemented 

Now or in the Near Future? 

2011 International HLB Meeting 

Orlando, Florida January 2011 


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HLB Pathology 

Take-Home Messages 

Megan Dewdney 

and 

Timothy Schubert 

Grower Day Summary: 

HLB Pathology Lessons 


Orlando, FL 


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What Can Be Implemented Shortly? 

Ca. Liberibacter spp. Lessons 

Considerations for Sampling 

• Uneven distribution of Ca. Liberibacter spp. makes diagnosis very challenging 

‣ Because you can’t find it, doesn’t mean it’s not there 

‣ Insufficient sampling or wrong tissue 

• Need to think about HLB as affecting a population rather than individual plants for 

management 

‣ Population examples: grove, block, etc. 

Fruit of New Technologies 

• New technologies allowed advancements not possible 10 years ago 

• Indications of at least 2 Florida Las strains 

‣ Separate introductions? Present in Florida potentially for many years based on genetic 

diversity 

‣ Disease appearance late 2005 due to psyllid arrival 1998 and long latent period 

• 4 main strains worldwide 

‣ Brazilian, USA, China, and India (most different) 

Distinguishing Strains with Sequencing 

• Should more than a small portion of the ribosome be used to 

differentiate between strains? 

‣ Probably yes but which ones ─ still being debated 

‣ Is there a biological/phenological/epidemiological significance? 

Culturing the Liberibacter spp. 

• Progress with insect cell lines 

Michael Rogers 

• Used several insect cell lines in a co-culture 

‣ D. citri cell lines recently received 

‣ In mosquito and fruit fly cell lines able to transfer between 18-20 times 


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New Detection Methods 

• Antibodies specific for Las to develop cheaper and faster methods of detection in the 

field 

‣ Dot blots 

‣ ELISA perhaps to be used in a dip stick test 

• Could be very useful for new experimental protocols 

‣ Immunocapture 

‣ Gold labeling ─ Is that really Las? 

• Spectroscopic techniques 

‣ Needs more work because can’t tell diseases apart in the field 

PCR – New and Improved?!? 

• A new way to test for 4 Ca. Liberibacter spp. 

‣ A better tool for regulatory agencies? PPQ? DPI? 

• A more sensitive/reliable technique for diagnostic qPCR 

‣ Based on a prophage (bacterial virus) which may not always be there 

‣ Not quantitative ─ only a yes/no answer 

• Developing strips for PCR 

‣ Isothermal DNA detection 

‣ Constant temperatures for 20 minutes 

Are Liberibacters the microscopic equivalent of a 1960’s Rock and Roll Band? 

Trash the hotel room and leave quickly 


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• Asymptomatic new flush has the highest Las titers in the plant under controlled 

conditions 

‣ First 3-6 months 

• Symptoms then commence but the bacterial titer drops 

‣ Under electron microscopy, bacteria are degrading or absent 

• Under natural inoculation of young trees, titer found in symptomatic tissue at higher 

levels than asymptomatic tissues 

‣ Recently symptomatic trees ─ is the syndrome from a natural inoculation process 

different from artificial? 

Early Physiological Changes 

• First microscopic symptom is swelling between cells 

• Callose deposits in phloem 

‣ Phloem collapse complete by 9 months with surrounding cells full of starch 

• At 6-9 months, first symptoms are observed 

Seasonality of Liberibacter 

• U.S. Sugar (Southern Gardens) Diagnostic Lab samples 

‣ Best diagnostic accuracy is from August to October 

‣ August to January/February, all typical symptoms are suitable to identify a positive 

‣ Worst time to send in samples is in June and July 

‣ Only blotchy mottle will come back positive year-round 

• Experimental evidence confirms these trends in the field 

Las Shuts Down Part of Defense 

• Common plant defense pathway is short-circuited by Las 

‣ The bacteria has an enzyme that is capable of degrading salicylic acid (SA) 

‣ Looking for an inhibitor of this enzyme 

• Is this why applying salicylic acid-containing products has had mixed results? 

Revisiting Seed Transmission 

• Some debate over seed transmission (still!) 

• Bacteria can be found in seed coat 

‣ Especially the phloem of developing seed 

‣ Embryos all tested negative 

• Numbers diminish over time 

‣ Could detect sometimes but did not distinguish between live and dead bacteria 

‣ Seedlings all tested negative after 1 year 

• Conclusion: no seed transmission 


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Interesting Facts About Ca. Liberibacter spp. Some day useful? 

Garnier and Bové, 1984 

‘Omics ─ What on Earth Are They? 

• Genomics ─ What genetic information is present in an organism? 

‣ Sequencing starts the process 

• Transcriptomics ─ What genes are being used? 

‣ Up regulated ─ identifies what genes are in use more 

frequently than normal (as defined by research) 

‣ Down regulated ─ identifies what genes are less active 

than normal 

• Proteomics ─ What proteins (structural and enzymes) are 

being made? 

• Metabolomics ─ What are the final gene products? 

So What? 

• These are excellent techniques for developing ideas for new research projects 

• Need to be followed up by experiments that prove the hypotheses 

• Sorting through the data 

• Allowed for great strides in host pathogen relations in Xylella fastidiosa 

Ca. Liberibacter Genomes Small 

• Smaller than other closely related bacteria 

• Some genes for flagellum present but never seen in plant or insect ─ Does it work? 

• Carries genes that cause disease in other organisms, especially animals like insects 

• Contains virus sequences that are harmful when bacteria are stressed 

‣ Can we use this to kill Liberibacter in the plant? 

• Bacteria take form of tangled knots within cells 

Ca. Liberibacter americanus 

• Ca. L. americanus genome is close to complete allowing 

for the discovery of common disease causing elements 

‣ Now almost out-competed by Ca. L. asiaticus in Brazil 

‣ L. americanus does not contain the prophage 


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Entomology 


Lukasz Stelinski (FL) and 

Mamoudou Setamou (TX) 


Entomology Lessons 


Orlando, FL 


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Session 2: Asian Citrus Psyllid 

Biology and Genomics 

2.1: de León et al. 

• Reported evidence for separate ACP invasions in North and South America apparently 

resulting from ACP that came from different countries in Asia 

• Little gene flow was evident between these two groups ─ so they have remained separate 

• Suggested that any biological control agent would likely need to originate from the 

source country of the founding ACP population 

2.2: Bextine et al. 

• Compared the microbial diversity in potato psyllid and ACP. The greatest bacterial 

diversity occurred in eggs 

• When both psyllid species were infected with Liberibacter, there was another bacterial 

species present which is not there when Liberibacter was absent 

• The species of bacteria associated with Liberibacter infections was unique for each 

psyllid and are potential targets for psyllid control 

2.3: Borovsky et al. 

• Development of RNAi for ACP control 

• Demonstrated the efficacy of an artificial feeding system 

• Used three double stranded RNAs to target three specific ACP genes and showed 

increased ACP mortality 

• By using all three types simultaneously, mortality was greatest 

2.5: Ammar et al. 

• Using a detached citrus leaf placed in a polypropylene tube allowed more rapid 

assessments (2-3 weeks) of Las inoculation by infected ACP compared to whole plant 

assays (3-12 months) 

2 .6: Bextine et al. 

• Antennae of ACP and potato psyllid are slightly different. Suggested ACP might be 

better at finding host plants, while potato psyllid might be better at finding mates 


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Biology and Genomics (Posters) 

2.7: Hunter et al. 

• Production and mining of ACP genetic libraries identified several genes which function 

in psyllid responses to stress such as temperature, insecticides, and disease 

• Knowledge of ACP functional genomics will be used to develop RNAi gene disruption 

methods for ACP suppression 


Ecology and Transmission 

3.1: Robbins et al. 

• Made a recent research advancement in recording ACP antennal signals with a device 

that is coupled to a gas chromatograph. These recordings allow us to precisely narrow 

down which chemicals psyllids are able to smell for mixtures of chemicals that are 

injected onto the chromatograph 

• Establishing this method should speed up the process of identifying attractants for ACP ─ 

both host plant volatile and potential pheromone components 

• Seven compounds of interest identified that are being pursued 

3.2: Lopez-Arroyo et al. 

• Discussed ACP distribution in Northern Sinaloa, Mexico 

• Populations of ACP greatest on Mexican Lime > Orange > Grapefruit 

• Seasonal population abundance greatest September to February 

• 18 generations of ACP/year 

3.3: Ammar et al. 

• ACP salivary glands had the fewest Las bacteria compared with the alimentary canal and 

other body parts 

• Authors suggest that salivary glands are the major barrier to Las infection and 

transmission ─ perhaps a target site to exploit 

3.4: Pelz-Stelinski et al. 

• Acquisition greatest during nymphal development and at 25°C 

• Low efficiency of Las inoculation by Florida ACP (5%) 

• Las decreases over lifespan of ACP 

• Las alters ACP fitness: increased egg laying, faster development time, shorter lifespan 

• Increased egg laying by infected ACP suggest that Las is well-adapted to psyllids and 

may contribute to disease spread 


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3.5: Ebert et al. 

• Seasonal changes in numbers of ACP carrying the HLB-causing pathogen 

• The more aggressive the management, the lower the incidence of HLB infection 

• Infection can vary from 2-3% to 40% or more and appears to be correlated with how 

aggressively psyllids are managed and whether management is done in coordinated 

fashion 

• October/November/December ─ period of greatest threat from infected ACP in Florida 

3.6: Mann et al. 

• ACP initially more attracted to HLB-infected plants, but subsequently they choose 

healthy plants as their final location for long-term setting 

• This may be a mechanism for rapid disease spread 

• Chemical and nutritional factors may underlie this behavior, which are being identified to 

prevent this movement from diseased to healthy plants 

• Candidatus Liberibacter asiaticus is transferred at a low rate 3-5% from males to females 

during mating 

3.7: Serikawa et al: 

• Measured feeding behavior of ACP on plants treated with systemic and foliar insecticides 

• Chlorpyrifos, fenpropathrin, imidacloprid, and spinetoram were best in disturbing ACP 

feeding and thus may be best in preventing acquisition/inoculation 

• Aldicarb and spirotetramat did not disrupt ACP feeding, which may possibly allow 

pathogen inoculation prior to death 

3.8: Miranda et al. 

• Also measured effect of insecticides on ACP feeding 

• Systemically applied imidacloprid and thiamethoxam reduced psyllid probing and killed 

ACP fast 

• Effect can be very long lasting; upwards of 95 days 

• Contact insecticides (Dimethoate, λ-cyhalothrin) can initially prevent feeding on older 

leaves, but protection of young shoots was much worse. Also effect was not as long 

lasting as with systemic insecticides. In some cases, ACP could probe on newly treated 

shoots or within a few days 


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Ecology and Transmission (Posters) 

3.10: Barbosa et al. 

• Las acquired more efficiently than Lam by ACP 

3.11: Halbert et al. 

• Do ACP use other host plants? 

• ACP produced offspring on citrus, but not on Zanthoxylum species (in the citrus or rue 

family) 

• ACP adults lived longer on citrus and Z. clava-herculis than on other Zanthoxylum 

species 

• ACP might be able to transmit Las to Zanthoxylum species, although development of 

HLB may only be possible in Z. fagara 

3.12: Jasso-Arumedo et al. 

• In Mexico, infestations of ACP were greater on orange jasmine than in backyard citrus 

• Abundance of natural enemies was approximately 3:1 higher in natural jasmine than in 

backyard citrus 

• Cycloneda sanguinea was the most abundant; beneficial in both plants 

• T. radiata was more abundant in orange jasmine than in backyard citrus 

3.14: Jasso-Argumedo et al. 

• Infestation of young trees in Mexican orange groves was constant throughout the year 

(10-30%); lower infestations occurred in mature orange trees 

• The average annual infestation of Persian lime trees was 21% 

3.15: Thomas et al. 

• In Texas, ACP adults have been found on hackberry, mesquite, potato, acacia, and 

torchwood 

• ACP adults and nymphs have been found on common fig 

• Alternate ACP hosts may serve as reservoirs for Las 

• Other psyllid species utilizing these hosts may by potential vectors of Las 


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Session 9: Asian Citrus Psyllid Management 

Moderator: Michael Rogers 

7 Oral presentations 

19 posters 

Geographic areas of studies presented: 

• U.S. (CA, FL, TX, and AL) 

• Mexico 

• Brazil 

Topics covered in Oral Presentations: 

1. Database for ACP population in commercial groves 

2. Efficacy evaluation of dormant sprays for ACP for psyllid control and conservation of 

natural enemies 

3. Testing of insecticides for ACP control (type, rates, application methods) 

4. New strategies for ACP control (RNA interference) 

5. Studies on imidacloprid for ACP management 

6. Insecticide resistance and susceptibility of uninfected and CLas infected ACP 

9.1: Gast et al. (FL) 

• U.S. Sugar has a “monster” plant and has strong interest in continuing the supply of fruit 

to the plant 

• Second location where HLB was detected in FL (Oct 2005). Initially, no ACP control on 

mature groves, but currently aggressive ACP control and grove scouting for both ACP 

and HLB 

• Monitoring program is expensive (4 scouts for ACP and 15 HLB inspectors), but required 

• Two ACP scouting methods (tap sampling for adult and visual inspections of flush shoots 

for all life stages) 

• Comprehensive database developed: good correlation between HLB infection rate and 

% sampling sites with psyllids and flush shoots with ACP nymphs 

• Factors that affect HLB infection: young trees, neighbor not doing anything, and strong 

border effects 

• To keep HLB infection

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• Technology, environmentally friendly. Working w/APHIS and EPA for regulatory issues, 

and w/companies for commercialization 

• Psyllid genome mapped and ready for release (in 3-4 months) 

9.3: Qureshi and Stansly (FL) 

• Demonstrated the importance of winter dormant sprays for ACP control 

• 1 or 2 winter dormant sprays effective for providing excellent ACP control for up to 

4-5 months 

• Winter dormant sprays (Jan 15) also allow judicial use of beneficials (minimal to nil 

effects on natural enemies) 

• For effective psyllid control, make at least one winter spray, then during the season spray 

before each flush cycle based on psyllid counts (similar recommendation made by Bartels 

et al. from TX, poster 9.8) 

• Tamarixia main parasitoid in FL, but parasitism rate is moderate (20%). Inoculative and 

augmentative releases improve this parasitism rate, up to 50% in some locations 

9.4: Byrne et al. (CA) 

• Evaluate the efficacy of imidacloprid in three settings (backyard citrus, nursery and 

commercial groves) 

• CA is in ACP eradication mode, once a find made eradication efforts are immediately 

implemented at the sites and vicinity 

• Treatment consists of foliar application of a pyrethroid (e.g., cyfluthrin) and a soil 

application of neonicotinoid (imidacloprid) 

• Negative correlation between leaf tissue content of imidacloprid and trunk diameter. As 

the tree ages, lower imidacloprid titer in leaf tissue; thus needs for higher application 

rates! 

• Nursery stock regulation: requirement to treat nursery plants with both a systemic and a 

foliar insecticide in order to move plants. This treatment is performed under supervision 

• In commercial citrus both foliar sprays and imidacloprid applied; takes time to reach 

lethal dose in plant (200-250 ppb) 

9.5: Lopez-Arroyo et al. (Mexico) 

• ACP detected in 2002 and by 2008 has spread almost everywhere (even in states with no 

commercial citrus) 

• Infective ACP (i.e., carrying CLas) found in July 2009. ACP did not catch stakeholder 

attention until this detection 

• 40 insecticides from various classes tested for their efficacy in ACP control and 

non-target impacts on indicator beneficials 

• Based on their test results, 12 insecticides (each from a different class) were selected and 

recommended for use (once per year to avoid resistance buildup) 

• Augmentative releases of Tamarixia is also highly recommended because of the excellent 

host finding ability of Tamarixia that is found where its host is present 

• A pilot program on AWM of ACP will start soon (or under way) 


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9.6: Yamamoto et al. (Brazil) 

• Introduced the 3 strategies that are important for HLB management (elimination of 

sources of inoculum, ACP control, and healthy nursery trees), but stressed the importance 

of ACP control as a key element 

• Both foliar and soil applied insecticides can be used for ACP control, but because soil 

systemic insecticides are compatible with BC, emphasis is placed on them 

• Their studies on imidacloprid showed excellent control on non-bearing trees, but 

inconsistent results with bearing trees 

• To elucidate the inconsistency observed, field trials were performed to evaluate the 

impact of application rate and volume, phosphate, soil type, and rootstock on 

imidacloprid efficacy 

• Good control obtained in Sept in contrast to Nov application 

• Poor control when neonic titer 400 ppb 

9.7: Tiwari et al. (FL) 

• Studied insecticide resistance in field-collected ACP populations in FL using different 

methods (diagnosis dose method, leaf disc) 

• 12 insecticides were tested, and resistance has been documented in 4 of them 

(chlorpyrifos, imidacloprid, thimaethoxam, and fenpropathrin) at various levels 

(resistance ratio for imidacloprid is up to 35 in some locations), thus requiring that a 

resistance management program must be implemented 

• This resistance seems to be regulated by enzymatic activity, with general esterase playing 

a major role 

• They also observed that CLas infected ACP (w/lower general esterase activity) were 

more susceptible to insecticides than their non-infective counterparts, which is somehow 

good news 

• Various resistance levels across the state: resistance monitoring, pesticide rotation 

Session 9: Asian Citrus Psyllid Management (Posters) 

Topics covered in Posters: 

1. Area-wide management of ACP (TX, CA, and FL: 9.8, 9.9, and 9.10) 

• ACP better managed on AW basis than local levels 

• Importance of winter dormant spray application is demonstrated and two dormant (Nov 

and Jan) are recommended in TX, one in FL 

• Spray prior to onset of new flush 

• Aerial application of broad spectrum insecticide as effective as ground airblast sprays 

• In CA, eradication mode and any finds immediately trigger spray application of find sites 

and neighboring sites 


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2. Low Volume Spray Technology (FL: 9.11) 

• LV is effective technology for ACP control 

• Minimum droplet size (DV0.5) of 90 microns is required to meet requirement 

• Engine speed reduction for both airblast type sprayers and air-assisted sprayers, and 

increased flow rate for air-assisted sprayers can increase droplet size to meet the 

requirement 

• LV8-V2, LV8, London Fog 18-20 Citrus sprayer are all examples of machines in which 

droplet size increase is obtained by adjusting engine from full throttle to lower rpm 

3. Molecular analysis of Tamarixia and Development of Diaphorina specific marker 

(TX, Mexico: 9.12, 9.16, 9.17, 9.20) 

• Morphological and genetic variations observed within Tamarixia. 4 groups of Tamarixia 

observed in N and S America. It is possible that cryptic sp. exists 

• Highly specific markers developed to determine whether predators have consumed ACP 

• This tool will assist in understanding which natural enemies consume ACP and play a 

role in its control 

4. Host specificity testing of Tamarixia (CA, 9.13) 

• Before releasing Tamarixia, host range testing must be performed 

• Three indigenous psyllids to CA (acacia psyllid, prosopis psyllid, Rhus psyllid) were not 

accepted as hosts by Tamarixia 

• Completing such tests with additional psyllids can facilitate the release of Tamarixia into 

new areas such as CA and TX 

5. Evaluation of ACP predators (FL, TX: 9.14 and 9.15) 

• Spiders were observed as the most abundant predators in non-commercial citrus in TX; 

however, most of these spiders first attack and then reject ACP preys 

• Convergent ladybeetle is an important predator for soft-bodied insect such as ACP, 

brown citrus aphid and green aphid. Its larvae prefer psyllid nymph over BCA 

• Because these 3 preys are equally suitable for convergent LB, they offer avenue for the 

persistence and better efficacy of this natural enemy 

6. Novel approaches for ACP management (9.17, 9.18, 9.19, 9.21, 9.24, 9.25, and 9.26) 

• New fungus sp (Isaria poprawski) w/potential of ACP control has been genetically 

characterized 

• RNAi technology (previously described) 

• Photonic fence technology can identify and zapped ACP by delivering levels of photonic 

energy. PFT used in mosquito control. Preliminary test done in TX and could be useful in 

small areas citrus production 

• An auto-dissemination system developed that will permit ACP to naturally disperse 

spores of pathogenic fungi. System can be used in urban area 

• Use of DMDS (for ACP control): studies conducted on release profile and release method 

of this compound 


P a g e | 228 

7. Other topics (9.22, 9.23) 

• Plant growing environment has been modified (change of day and night T, length of 

induction period) to allow continuous new flush production in citrus plants. Can be used 

to support BC programs for n.e. rearing and also in nurseries to induce branching 

• Field trial under way to determine the best ACP control timing. Calendar sprays are 

compared to spray programs triggered by threshold. All spray timing significantly 

reduces ACP pop within 6 months, but impact on yield not yet quantified. This study will 

assist grower in knowing when to spray 

Session 9: Asian Citrus Psyllid Management 

Key Points 

• ACP control is of paramount importance 

• Better achieved when done in an AW basis 

• Winter dormant sprays are a MUST! 

• Aerial, ground airblast, and LV spray are effective tools 

• Important to preserve natural enemies as they contribute to ACP control 

• Resistance development is a real problem; monitor and manage resistance 

• New control methods are being developed 


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Horticulture 


Ed Stover 


Horticulture Lessons 


Orlando, FL 


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3 full days 

400 participants from 20 countries 

75 oral presentations 

96 posters 

(20 pages of notes) 

Urgency of HLB as a threat to citrus production and the engine of substantial grower 

investment has fully engaged numerous researchers to find solutions 

• In 2 years of this effort, there are SOME new ideas or mature recommendations ready for 

implementation 

• Tools and knowledge are being assembled for much greater future progress and I assume 

growers want a glimpse 

• In this meeting, researchers shared ideas and data and many came away with a piece of 

the puzzle that will plug in to advance their efforts or established collaborations that will 

accelerate progress 

• MANY of these “aha” moments occur over dinner or during breaks, and so these 

summaries can only tell a part of the story 

Urgency of need to save existing trees has compelled strong interest in developing 

therapeutics to lessen effect of HLB 

• Nutritional treatments are controversial, seem to provide a benefit in some situations ─ 

great talk by Irey to suggest threshold of 4-5% tree loss per year as transition point from 

Scouting/Roguing to Nutritional Life Support for infected trees 

• Antibiotic treatments Zhang et al. (UF-IRREC and ARS) have provided marked benefits 

in pilot studies in the greenhouse and initial field studies....may face tough sledding for 

deregulation and likely require cheaper delivery (sprays rather than injections) for 

commercial delivery 

• Jim Graham, Irey, and Miele ─ Injection of Cu compounds (Magna-Bon) reported to 

reduce CLas 

More therapeutics to save existing trees: 

• Ping Duan (ARS ─ Ft. Pierce) reported that in initial field trials, a form of therapy, 

appears to have eliminated HLB symptoms .... Stay tuned 

• RNAi for psyllid control (Falk/Hunter/Borovsky et al.) ─ Biopesticide today/transgenic 

tomorrow? 

• Bill Dawson ─ CREC ─ provided update on the CTV expression vector. Best greatly 

suppress HLB and have developed variants that can infect CTV+ trees and likely will 

protect 80% of trees for 10+ years. BIG regulatory hurdles, but in field trials 


P a g e | 231 

• Dean Gabriel UF ─ Gainesville Plant Path, reported discovery that a virus embedded in 

the CLas DNA may provide seeds of CLas destruction. Signal that causes the virus to 

replicate would kill CLas cells. Possible therapeutic, transgenic? 

New Understanding of HLB/citrus biology with relevance to citrus industry? 

• The question as to whether HLB can be seed transmitted continues to compel new work 

• Two studies reported that seed from HLB infected highly symptomatic fruit did not result 

in plants with HLB. One study reported CLas transmitted often, but likely not causing 

HLB 

• In a study reported by Mark Hilf, USDA/ARS, Ft. Pierce, he showed that CLas is often 

present in vascular bundles of seed coats which may explain the reports of early PCR+ 

seedlings 

In our breeding program, we remove seed coats before planting seeds from infected fruit ─ 

Citrus Breeding Programs throughout U.S. and the world are squarely focused on HLB 

• Developing tools that will open up cutting edge technologies that make this “a golden age 

for biology.” Steve Lindow noted that “HLB is a tough nut to crack, and you are lucky 

you didn’t get it even a few years ago” 

• Exploring genes related to resistance/susceptibility 

• Assessing resistance in conventional breeding/ scions/rootstocks 

• Developing transgenics 

Loading the tool box! 

• Tremendous progress in genomics of host, pathogen, and vector ─ FINALLY we will 

have the full wiring diagram to fix “what’s broken” 

• Citrus: Gmitter (CREC ─ leading international consortium) Full genome (DNA 

sequence) of Haploid Clementine and Ridge Pineapple Orange genome will provide 

RNAi targets, etc. 

• Liberibacter: Gabriel and Wulff (Brazil) CLam and CLas comparison; Hong Lin (ARS ─ 

Parlier) strains of CLas; Duan; Hartung.... Understanding differences reveals weaknesses 

to exploit 

• Antibodies to CLas!!!! Like shining a spotlight at night 

Exploring resistance and susceptibility genes 

• Identifying genes that may confer resistance, better understanding of HLB biology and 

gene expression in resistant and susceptible Citrus. Targets for action! 

• Understanding of Citrus defense systems and HLB, e.g., some SA response to HLB 

(Moore ─ UF Gainesville); but shuts down some SAR systems (Dandekar ─ UCD); turns 

up JA (Machado ─ Brazil); produces antimicrobials (Albrecht and Bowman) 

• Identified CLas “virulence” genes that cause plant disease response (Wang ─ CREC) 

• Better understanding of HLB source/sink metabolism, etc., for therapeutics or transgenics 

(Chen et al. ─ CREC; He et al. ─ IRREC) 

• Understanding of phloem plugging (Albrigo ─ CREC) 


P a g e | 232 

Existing Resistance? Not ready for prime time.... 

• No strong HLB resistance has been identified in cultivated Citrus scion varieties 

• But evidence that there are differences in susceptibility, seemingly even between sweet 

oranges, which may have economic value (Stuchi et al. ─ Brazil) 

• Some specialty types (e.g., ‘Temple’) with much lower CLas than sweet orange (Stover 

et al.) 

• Evidence that some rootstocks may enhance HLB tolerance of scions (Grosser; Stuchi) 

Transgenics 

• Exploring antimicrobial peptides and other transgenes that will enhance plant resistance 

(many labs reported) 

• Comparing promoters that direct where genes are “turned on” (ARS ─ Ft Pierce; CREC) 

• Tomato and LSol as a model system for testing transgenics (Manjunath and Lee ─ ARS 

─ Riverside) 

• Improved methods for resistance screening of transgenics (Grosser ─ CREC) 

• Many transgenics being tested by many labs 

• Most advanced is GE citrus with virus gene that kills bacteria, looks excellent in GH, and 

now in field trials (Jiang et al., Gabriel lab ─ UF) 

Take home? 

• If knowledge is power........ 

• Our knowledge of HLB, Liberibacter, ACP, and their interaction with Citrus has 

expanded many-fold over the last 2 years 

• The assembled understanding is reaching a critical mass that will soon reveal outstanding 

tools for living with HLB 

• And a series of ever better solutions will emerge over the coming years 


P a g e | 233 

Chris Oswalt - UF/IFAS - Polk County 

Bartow, FL 

Specific Topics 

• Statewide Disease Levels 

• Tree Removal 

• Fruit Quality 

• Novel Alternative Treatments 

• Regional HLB Management 

• Foliar Nutrition 

Statewide Disease Levels 

• In 2008 HLB disease level 1.6 to 2.3% 

• In 2009 low response combined number at 6.4% for oranges only 

• Survey (2009) adjusted to HLB infection at 8% statewide for all varieties (2X increase 

per year) 

• It is estimated today that HLB disease level is approximately18% 

• Highest levels in south and east 

• Central, northern, and western 1% (2009) 

Tree Removal 

• Factors include: tree age, infection levels, estimated future infection rates and production, 

time horizon, and costs of treatments (nutritional) 

• Average annual infection rate exceeds 4-5% 

• Dependent on production costs and fruit prices (along with a number of other 

assumptions) 

• Will be implementing a foliar nutritional program 

• At $1.50/pound annual HLB infection rates >3.9% 

• At $1.25/pound annual HLB infection rates >4.4% 

HLB Symptomatic Fruit Quality 

• Dependent on harvest date 

• Brix ─ lower to no change 

• Generally higher in acid 

• Generally lower ratios 

• Higher levels (below thresholds) of Limonin and Nomilin 

• A 25% blend is detectable by panelists 


P a g e | 234 

Novel Alternative Treatments 

• Trunk injection of Magna-Bon (copper sulfate pentahydrate) 

• Positive response in appearance 

• Resulted in higher ct values with treatment (lower HLB) 

• Magna-Bon systemic in tree 

• Penicillin and streptomycin trunk injection 

• Reduced HLB levels in treated citrus 

Regional HLB Management 

• Brazilian study (regional vs. local) on inoculum reduction and vector control 

• Regional management resulted in delayed epidemic of HLB by 10 months 

• Reduced HLB incidence by 90% 

• Reduced HLB progression by 75% 

• Attributed to smaller ACP populations and lower frequency of HLB infected ACP 

Foliar Nutrition Programs 

• HLB infected trees are deficient in Ca, Mg, Mn, Zn, and B compared to healthy citrus 

trees 

• These mineral nutrients are important for many plant functions 

• Program started early at first HLB detection 

• Tree health, fruit quality, and yields (above area average) maintained for 5 years in grove 

• Maintained aggressive psyllid control throughout 

• Today grove close to 100% HLB infected 

• Tree death attributed to blight not HLB 

• This is not a replicated statistically valid experiment ─ a real world observation 

• Has been repeated with similar success 

• One statistical study 

• Trees with mild symptoms HLB + 

• Study compared to an ACP control program 

• Multiple combinations of nutritional materials including reportedly successful 

combinations 

• No treatment was statistically better than control 

• Treatments had no effect on tree HLB levels 

• Provides for the continued buildup of HLB inoculum 

• Enhanced nutritional programs along with good grove management has been shown to 

increase productivity 


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A1. Author Index 

Author’s Name 

Page number(s) 

Acevedo, G. 97 

Achor, D.S. 117, 128 

Acosta, I. 109 

Adamczyk, J. 167 

Afunian, M. 70 

Akula, N. 182 

Alanís-Martínez, E.I. 81 

Albano, J.P. 71 

Albrecht, U. 120, 124, 209 

Albrigo, L.G. 128 

Aldama-Aguilera, C. 115 

Alessandro, R.T. 49 

Alonso, E. 109 

Ammar, E.-D. 44, 51, 57 

Amorim, L. 127, 176 

Arevalo, H.A. 156, 169, 188 

Aritua, V. 132 

Arras, J. 45 

Atwood, R. 157 

Aubert, B. 192 

Ayres, A.J. 20, 88, 107 

Backus, E.A. 55 

Bagio, T.Z. 202 

Bai, J. 91, 93, 96 

Bai, Y. 21, 77 

Baldwin, E. 91, 92, 93, 96 

Barbosa, J.C. 59, 107 

Bar-Joseph, M. 138 

Barreto, T.P. 24 

Barroso-Aké, H. 63 

Bartels, D.W. 154 

Barthe, G.A. 198 

Bassanezi, R.B. 88, 127, 176 

Batista, L. 109 

Batkin, T. 112 

Beattie, G.A.C. 88 

Belasque, J., Jr. 59, 107 

Benyon, L.S. 18, 199 

Bergamin Filho, A. 59, 127, 176 

Bespalhok, J. 202 


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Bethke, J. 150 

Bextine, B.R. 41,45, 46, 148, 164 

Bohannon, R. 83 

Borbón, J. 108 

Borovsky, D. 42 

Borroto, A. 109 

Boscariol-Camargo, R.L. 206 

Bové, J.M. 20, 107 

Bowman, K.D. 120, 124, 199, 209 

Brady, B. 70 

Briefman, L. 72 

Bright, D.B. 139, 187 

Brlansky, R.H. 53, 68, 125, 208 

Brockington, J. 168, 212 

Brown, L.G. 191 

Brown, S.E. 114 

Bryan, C. 129 

Buenahora, J. 40, 162 

Burns, J.K. 130 

Byrne, F. 150 

Cabanillas, H.E. 163 

Cabrera-Mireles, H. 151 

Cáceres, S. 40, 162 

Cai, Z.J. 134, 137 

Camargo, L.E.A. 88 

Cancalon, P.F. 129 

Cantuarias-Avilés, T. 204 

Carrillo Medrano, S.H. 110 

Casín, J.C. 109 

Cayetano, X. 108 

Chen, C. (China) 21 

Chen, C. (CREC) 125 

Chen, J. 34, 38 

Chiyaka, C. 104 

Ciomperlik, M.A. 154 

Civerolo, E.L. 21, 70, 77 

Coletta-Filho, H.D. 67, 206 

Collazo, C. 109 

Cortez-Mondaca, E. 50, 151 

Cote, J. 72 


P a g e | 237 




Cristofani-Yaly, M. 206 

Cruz-García, R. 158 

Curtí-Díaz, S.A. 81, 151 

D’Souza, R. 124 

da Graça, J.V. 76, 154, 197 

Dandekar, A.M. 124 

Davis, M.J. 19 

Davis, R. 22 

Dawson, W.O. 138 

de León, J.H. 40, 162, 163, 166 

de Miranda, M.P. 56, 152 

De Souza, A.A. 67 

Dea, S. 91, 92, 93, 96 

Deng, X. 34, 38 

Dewdney, M.M. 103, 214 

Díaz-Zorrilla, U. 151 

Dickstein, E.R. 105 

Dima, C.S. 82 

Dollet, M. 32, 192 

Duan, Y.-P. 18, 19, 21, 31, 29, 87, 134, 136, 137, 175, 181, 186, 207 

Dunlap, C. 167 

Dutt, M. 198 

Ebert, T.A. 53, 103 

Eckstein, B. 59 

Ehsani, R. 82 

Estes, M. 73 

Fabre, S. 32 

Fan, G.-C. 31, 134, 137 

Fan, J. 125 

Febres, V.J. 208 

Felipe, R.T.A. 200 

Felippe, M.R. 56, 152 

Feliz, A. 108 

Fiehn, O. 120 

Fleites, L.A. 19 

Flores Virgen, R. 110 

Flores-Cruz, Z. 19 

Folimonova, S.Y. 108, 117 

Fontaine-Bodin, L. 32 

Francisco, C.S. 118 


P a g e | 238 




Frare, G.F. 75, 88 

Freitas-Astúa, J. 118 

Fritz, B. 157 

Gabriel, D.W. 19, 20, 210 

Gadea, P. 72, 180 

Galindo, T. 111 

Galindo-Mendoza, M.G. 115 

Garcia, R.B. 56 

Garnsey, S.M. 141 

Gasparoto, M.C.G. 127 

Gast, T. 72, 143, 187 

Gastaminza, G.A. 40, 162 

Gatineau, F. 32, 192 

Ghosh, D.K. 141 

Girardi, E.A. 189, 196, 204 

Glick, E. 148, 164 

Glynn, J.M. 21 

Gmitter, F.G., Jr. 125, 208, 211 

Gomez, H. 89 

Gómez, R. 97 

González, V.M. 50 

González, C. 109 

González-Hernández, A. 61, 158 

Gonzalez-Mora, J. 82 

Gooch, M.D. 19 

Gottwald, T.R. 73, 74, 100, 176, 178, 185 

Gowda, S. 136, 141 

Grafton-Cardwell, E.E. 155 

Graham, J.H. 72, 139, 174, 178, 185, 187 

Grange, L. 202 

Grosser, J.W. 198 

Hagler, J.R. 166 

Hail, D. 41 

Hajeri, S. 136 

Halbert, S.E. 60, 101, 102, 104, 193 

Hall, D.G. 44, 46, 51, 57, 74, 80, 207 

Hamel, R. 156 

Hartung, J.S. 17, 68 

Hartzog, H.M. 191 

Haun, C. 129 


P a g e | 239 




Hawkins, S.A. 71 

He, Z. 175, 186 

Heitschmidt, J. 71 

Hernández-Fuentes, L.M. 151 

Hilf, M.E. 108, 117, 126, 138, 140 

Hoddle, M.S. 159 

Hoffman, M. 31, 87 

Hoffmann, C. 157 

Hou, H. 143 

Humber, R.A. 163 

Hunter, W.B. 41, 45, 46, 148, 164 

Hurner, T. 157 

Imbert, E. 192 

Irey, M.S. 70, 72, 73, 74, 78, 80, 82, 91, 92, 93, 95, 96, 139, 141, 

Iwanami, T. 22 

Jackson, J.L. 157 

Jackson, M. 167 

Jantasorn, A. 87 

Jasso-Argumedo, J. 61, 63, 151, 158 

Jasso-Laucirica, T. 61 

Jaynes, J. 201 

Jiang, Y. 210 

Johanson, E. 165 

Johnson, E.G. 139, 187 

Jones, E.M. 191 

Jones, W.A. 163 

Jordan, R. 68 

Kang, B.-K. 19 

Kanga, L.H.B. 162 

Kato, H. 22 

Ke, C. 137 

Khalaf, A. 208 

Kishi, L.T. 118 

Kobayashi, A.K. 202 

Kress, R. 210 

Kund, G. 194 

Kunta, M. 76 

Kuykendall, L.D. 17 

Kwok, K. 78 

Lapointe, S.L. 49 


P a g e | 240 




Lawrence, K.C. 71 

Ledebuhr, M. 157 

Lee, R.F. 70, 99, 102, 193, 194 

Leicht, E. 124 

Leite, R.P., Jr. 24, 202 

Lemos, M.V.F. 75 

Levesque, C.S. 70, 78 

Li, J. 30, 33 

Li, W.B. 31 

Li, Y. 30, 33 

Li, Z.-G. 125 

Liao, H.-L. 131 

Lin, H. 21, 70, 77 

Lin, Y.-Z. 134 

Lindeberg, M. 23 

Liptay, A. 186 

Liu, B. 31, 134, 137 

Liu, R. 38 

Liu, T. 47 

Llauger, R. 109 

Locali-Fabris, E.C. 118 

Loera-Gallardo, J. 181 

Logarzo, G.A. 40, 162 

Lopes, J.R.S. 56 

Lopes, S.A. 75, 88, 200 

López, D. 109 

López-Arroyo, J.I. 50, 61, 63, 81, 97, 151, 156 

Loredo-Salazar, R.X. 81 

Lourenço, S.A. 127 

Lozano-Contreras, M. 61, 64, 158 

Luis, M. 109 

Machado, M.A. 118, 206 

Mafra, V.S. 118 

Malik, N.S.A. 168, 212 

Malosso, A. 206 

Mangan, R.L. 168, 212 

Manjunath, K.L. 69, 70, 78, 99, 102, 133, 193, 194 

Mann, R.S. 54 

Manthey, J. 91, 92, 93 

Manzanilla Ramirez, M.A. 110 


P a g e | 241 




Marques, V.V. 24, 202 

Martin, D. 157 

Martinelli, F. 124 

Martins, P.K. 118 

Massari, C.A. 107 

Matos, L. 108 

McCollum, G. 180, 199, 201, 205, 207 

McGowen, N. 83 

McLaughlin, W.A. 114 

Mears, P. 102 

Medina Urrutia, V.M. 110 

Meikle, W. 167 

Mendes, B.M.J. 200 

Méndez, F. 108 

Meneguim, L. 24, 202 

Merlin, T. 186 

Miele, F. 174 

Mills, D.R. 170, 281 

Minenkova, O. 68 

Miyata, S. 22, 141 

Montesino, L.H. 127, 176 

Monzó, C. 169 

Moore, G.A. 208 

Mora-Aguilera, G. 97 

Morales-Koyoc, D. 61 

Moran, P. 163 

Morgan, J.K. 69 

Morris, R.A. 73, 94 

Morse, J.G. 150, 155 

Mourão-Filho, F.A. 200 

Mullen, E. 165 

Muraro, R.P. 94 

Murata, M.M. 24 

Murray, K.D. 163 

Myers, M.E. 185 

Nageswara Rao, N. 141 

Narciso, J. 93 

Neuman, R.D. 170, 171, 172, 281, 287 

Niedz, R. 199 

Nunes da Rocha, U. 105 


P a g e | 242 




Oberheim, A.P. 114 

Okuma, D.M. 55, 62 

Olvera-Vargas, L.A. 115 

Orozco Santos, M. 110 

Paccola-Meirelles, L.D. 24 

Paldi, N. 148, 164 

Pandey, R.R. 159 

Park, B. 71 

Parkunan, V. 103 

Parnell, S.R. 74, 100 

Parolin, L.G. 196, 204 

Parra, J.R. 162 

Patne, S. 133 

Patt, J. 165, 167 

Pegram, N. 165 

Pelz-Stelinski, K.S. 52, 54 

Peña, I. 109 

Perazzo, G. 210 

Pereira, E.V., Jr. 200 

Pereira, L.F.P. 202 

Pérez, D. 109 

Pérez, J.L. 109, 168, 212 

Pérez-Márquez, J. 50 

Peroni, L.A. 67 

Pfannenstiel, R.S. 160 

Phahladira, M.N.B. 101 

Pietersen, G. 101 

Plotto, A. 91, 92, 93, 96 

Polek, M.L. 70, 78, 112, 113, 157 

Poole, G.H. 71 

Powell, C.A. 31, 42, 175, 181, 186 

Puello, H. 108 

Puttamuk, T. 87 

Qureshi, J.A. 149, 161 

Ramadugu, C. 70, 99, 102, 193, 194 

Reagan, R.L. 124 

Reese, J. 46 

Reiff, E.T. 196, 204 

Ribeiro-Alves, M. 118 

Riley, T.D. 74, 89 


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Robbins, P.S. 49 

Roberts, P.D. 60 

Robertson, C. 138 

Robl, D.J. 89 

Robles-González, M.M. 97, 110, 151 

Rodríguez-Quibrera, C.G. 81 

Rogers, M.E. 52, 53, 55, 62, 103, 141, 153 

Roose, M.L. 133 

Rouse, R.E. 177, 188 

Rouseff, R.L. 54, 91, 93 

Ruan, C.-Q. 134 

Russell, D.N. 60 

Russell, P.F. 83 

Rutschman, P. 165 

Saha, S. 23 

Salcedo, D. 97 

Sandoval, J.L., II 197 

Santiago, O. 72 

Santos, M.A. 75 

Schumann, A.W. 177 

Sempionato, O.R. 196, 204 

Septer, A. 210 

Sequeira, R. 179 

Serikawa, R.H. 55, 62 

Sétamou, M. 40, 76, 154, 162, 166, 197 

Shao, J. 17 

Shatters, R.G., Jr. 42, 43, 46, 51, 69, 207 

Shelby, K.S. 46 

Shelton, A.B. 170, 171, 172, 281, 287 

Shohael, A. 198 

Singer, B. 104 

Skaria, M. 76 

Skogerson, K. 120 

Smith, M.W. 22 

Sousa, M.C. 75, 200 

Spann, T.M. 177 

Sreedharan, A. 119 

Stach-Machado, D.R. 67 

Stange, R. 201 

Stañgret, C.R.W. 40, 162 


P a g e | 244 




Stansly, P.A. 149, 156, 161, 169, 188 

Stelinski, L.L. 54, 153 

Stoffella, P.J. 186 

Stover, E. 164, 175, 180, 199, 201, 205, 207 

Stuchi, E.S. 189, 196, 204 

Su, H.N. 34 

Sugahara, V.H. 202 

Takita, M.A. 67 

Tandy, M. 157 

Taylor, B.J. 112, 155 

Teixeira, D.C. 88 

Thaveechai, N. 87 

Thomas, D.B. 64, 166 

Tian, S. 47 

Tiwari, S. 153 

Toledo, D.A. 196, 204 

Tomas, J. 184 

Tomimura, K. 22 

Trivedi, P. 135 

Trumble, J. 194 

Unruh, T.R. 160 

Uratsu, S.L. 124 

Uribe-Bustamante, A. 81 

Vahling, C.M. 18 

Valim, F. 93 

van Bruggen, A.H.C. 104, 105 

van den Bosch, F. 100 

Vasquez-Souza, G.V. 24, 202 

Velázquez-Monreal, J.J. 81, 97, 110 

Viana, E. 186 

Vidalakis, G. 70 

Vieira, L.G.E. 202 

Viljoen, R. 101 

Villanueva-Jiménez, J.A. 151 

Villanueva-Segura, O.K. 158 

Villas-Boas, L.A. 24 

Walter, A. 57 

Wang, A. 30 

Wang, N.-Y. 103, 119, 132, 135, 182 

Wang, X.F. 34 


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Wang, Z. 30, 33, 47 

Wei, S. 119 

Weinert, M. 22 

Weng, Q.Y. 127 

Windham, W.R. 71 

Wisler, G. 113, 157 

Wood, B. 178 

Wulff, N.A. 19, 20, 88, 127 

Xia, Y. 179 

Xian, J. 30 

Xiao, R.-F. 134 

Xie, P. 33 

Yamamoto, P.T. 40, 56, 152, 176 

Yin, Y. 30, 33, 47 

Yu, Q. 125 

Yuan, Q. 68 

Zamora, V. 109 

Zee, R.H. 172 

Zekri, M. 156 

Zhang, J. 129 

Zhang, M.-Q. 175, 181, 186 

Zhang, P. 38 

Zhang, S. 87 

Zhang, S.J. 19, 20 

Zheng X.-F. 134 

Zhou, C.Y. 34 

Zhou, L.J. 19, 31, 69, 137, 175 

Zhu, Y.-J. 134 


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A2. List of Registered Participants (those registered at the time of the meeting are included) 

Full Name Affiliation Address City State ZIP Country Email 

Ms. Diann Achor UF/IFAS CREC 700 Experiment Station Rd Lake Alfred FL 33850 USA dsar@ufl.edu 

Mr. Bob Adair Florida Rsrch Ctr for Ag 

7055 33rd Street Vero Beach FL 32966 USA bob@flaresearch.com 

Sustainability 

Dr. Ute Albrecht USDA ARS 2001 S Rock Rd Fort Pierce FL 34945-3030 USA ute.albrecht@ars.usda.gov 

Dr. Gene Albrigo UF/IFAS CREC 700 Experiment Station Rd Lake Alfred FL 33850 USA albrigo@ufl.edu; 

albrigo@crec.ifas.ufl.edu 

Dr. Cristobal Aldama Universidad Autonoma de San Luis Sierra Leona 550, Col San Luis, 

78250 Mexico cristobal.aldama@uaslp.mx 

Potosi - SINAVEF 

Lomas II Seccion 

Potosi 

Mr. Eduardo Almada USDA APHIS IS Carr Reynosa-Matamoros Reynosa, Tam 88501 Mexico eduardo.almada@aphis.usda.gov 

KM79 Brecha 102 

Mr. Allen Altman Altman*Allen 12445 US Highway 301 Dade City FL 33525 USA allenpcfb@aol.com; 

allen.altman@ffbic.com 

Mr. Fernando Agricultural Integrated Solutions USA 6505 SW 113th Ave Miami FL 33173 USA fadelavega@gmail.com 

Alvarez 

Mr. Ricardo Alvarez Comite Estatal de Sanidad Vegetal de Carr. Victoria Monterrey Guemez, 

87230 Mexico rialra@yahoo.com 

Ramos 

Tamaulipas 

Km 22 

Tamaulipas 

Dr. El-Desouky USDA ARS USHRL 2001 S Rock Rd Fort Pierce FL 34945 USA Eldesouky.Ammar@ars.usda.gov 

Ammar 

Dr. Lilian Amorim University of Sao Paulo - ESALQ Av. Pádua Dias 11 Piracicaba, 

13418-900 Brazil liamorim@esalq.usp.br 

Sao Paulo 

Ms. Vickie Anthony Citrus Solutions, LLC PO Box 1341 Zolfo Springs FL 33890-1341 USA 

Dr. Doug Archer University of Florida/IFAS PO Box 110200 Gainesville FL 32611-0200 USA dlarcher@ufl.edu 

Dr. Calvin Arnold USDA ARS USHRL 2001 S Rock Rd Fort Pierce FL 34945 USA Calvin.Arnold@ars.usda.gov 

Ms. Marina Arouca UF/IFAS CREC 700 Experiment Station Rd Lake Alfred FL 33850 USA marinarouc@yahoo.com.br 

Mr. Hugo Arredondo COMITE ESTATAL DE SANIDAD EMILIANO ZAPATA 482, Tecoman, 

28110 Mexico hugo.arredondo@senasica.gob.mx 

VEGETAL DE COLIMA 

COL. TEPEYAC 

Colima 

Mr. Ryan Atwood University of Florida Extension 1951 Woodlea Rd Tavares FL 32778 USA ryan@keyplex.com 

Mr. Juliano Ayres Fundecitrus Av. Dr. Adhemar Pereira de Arraraquara, 

14807-040 Brazil ayres@fundecitrus.com.br 

Barros 201 

Sao Paulo 

Dr. Murat Azik Florida Department of Citrus 700 Experiment Station Rd Lake Alfred FL 33850 USA mazik@citrus.state.fl.us 

Dr. Jinhe Bai USDA ARS Citrus & Subtropical 600 Avenue S, NW Winter Haven FL 33881 USA jinhe.bai@ars.usda.gov 

Products Lab 

Dr. Elizabeth USDA ARS Citrus & Subtropical 600 Avenue S, NW Winter Haven FL 33881 USA liz.baldwin@ars.usda.gov 

Baldwin 

Products Lab 

Mr. Botond Balogh Nichino America, Inc. 6913 Monarch Park Dr Apollo Beach FL 33572 USA bbalogh@nichino.net 

Mr. Bobby Barben Barben Fruit Company, Inc. 21 E Pine St Avon Park FL 33825-3946 USA rhb@barbenfruit.com 

Mr. John Barben Barben Fruit Company, Inc. 21 E Pine St Avon Park FL 33825-3946 USA jpb@barbenfruit.com 

Mr. Bill Barber Lykes Citrus Management PO Box 1690 Tampa FL 33601-1690 USA bill.barber@lykes.com 

Mr. Júlio Barbosa University of Sao Paulo - ESALQ Av. Pádua Dias 11 Piracicaba, 

Sao Paulo 

Mr. Juan Barcelo Consorcio Citricola del Este Carratera Hato Mayor 

Sabana de la Mar Km 1 

Hato Mayor 25000 Dominican 

Republic 

13418-900 Brazil juliobarbosao@gmail.com 

jbarcelos@citricola.com; 

citricola@citricola.com 


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Mr. Joe Barcinas Foothill Agricultural Research, Inc 550 W Foothill Parkway Corona CA 92882 USA farinc@att.net 

Mr. Shahar Bar- ANI 3006 Longhorn Ave Austin TX 78758 USA sbar-cohav@appliednanotech.net 

Cohav 

Ms. Michele Barefoot New Zealand Institute for Plant & 430 F St Ste F Davis CA 95616 USA michele.barefoot@plantandfood.co 

Food Research 

.nz 

Dr. Moshe Bar- S. Tolkowsky Laboratory ARO, The Volcani Center Rehovot 76211 Israel mbjoseph@gmail.com 

Joseph 

Bet Dagan 

Mr. Alfred Barrett Jamaica Citrus Protection Agency P.O. 108 Bog Walk Saint 

55555 Jamaica jcpa@anbell.net; 

Catherine 

barrettalfred@hotmail.com 

Mr. Giovane Barroti Citrus Consultants Group - GCONCI Rua Carlos Gomes, 144 - Cordeiropolis, 

13490000 Brazil gbarroti@uol.com.br 

Centro - CP 39 

Sao Paulo 

Dr. David Bartels USDA APHIS PPQ CPHST Moore Air Base #6414 22675 Edinburg TX 78541 USA david.w.bartels@aphis.usda.gov 

N Moorfield Rd 

Dr. Renato Bassanezi Fundecitrus Av. Dr. Adhemar Pereira de Arraraquara, 

14807-040 Brazil rbbassanezi@fundecitrus.com.br 

Barros 201 

Sao Paulo 

Ms. Shirley 

California Citrus Mutual 512 N Kaweah Ave Exeter CA 93221-1200 USA shirleyb@cacitrusmutual.com 

Batchman 

Mr. Ted Batkin California Citrus Research Board PO Box 230 Visalia CA 93279-0230 USA ted@citrusresearch.org 

Mr. Clark Baxley Florida Citrus Mutual 25942 Risen Star Dr Wesley FL 33544 USA clarkbaxley@juno.com 

Chapel 

Dr. Jose Belasque Fundecitrus 

Av. Dr. Adhemar Pereira de Arraraquara, 

14807-040 Brazil belasque@fundecitrus.com.br 

Junior 

Barros 201 

Sao Paulo 

Mr. John Bell Bayer CropScience LP PO Box 12014 Research NC 27709-2014 USA john.bell@bayercropscience.com 

Triangle Park 

Mr. Jose Bellotte Louis Dreyfuss Commodities Rod.Armando Salles Bebedouro, 

14707900 Brazil jose.bellotte@ldcom.com 

Oliveira, Km 396 

Sao Paulo 

Dr. Lesley Benyon USDA ARS USHRL 2001 S Rock Rd Fort Pierce FL 34945 USA lesley.benyon@ars.usda.gov 

Dr. Armando University of Sao Paulo - ESALQ Av. Pádua Dias 11 Piracicaba, 

13418-900 Brazil abergami@esalq.usp.br 

Bergamin Filho 

Sao Paulo 

Dr. Philip Berger USDA APHIS PPQ CPHST 1730 Varsity Drive Ste 400 Raleigh NC 27606 USA philip.h.berger@aphis.usda.gov 

Dr. Blake Bextine University of Texas at Tyler 3900 University Blvd Tyler TX 75799 USA bbextine@uttyler.edu 

Ms. Kathie Blyskal Sunkist Growers, Inc. 10509 Business Dr Unit B Fontana CA 92337 USA kblyskal@sunkistgrowers.com 

Dr. Robert Bohannon Agdia, Inc. 30380 County Rd 6 Elkhart IN 46514 USA bob@agdia.com 

Dr. Dov Borovsky University of Florida FMEL 200 9th St SE Vero Beach FL 32962 USA dobo@ufl.edu 

Mr. Daniel Botts Florida Fruit & Vegetable Association PO Box 948153 Maitland FL 32794-8153 USA daniel.botts@ffva.com 

Mr. Doug Bournique Indian River Citrus League PO Box 690007 Vero Beach FL 32969-0007 USA info@ircitrusleague.org 

Mr. Lawrence Bowie Trade Winds Citrus Limited Bog Walk Saint 

55555 Jamaica leon.stewart@tradewindscitrus.co 

Catherine 

m 

Dr. Kim Bowman USDA ARS 2001 S Rock Rd Fort Pierce FL 34945 USA kim.bowman@ars.usda.gov 

Mr. Maury Boyd Boyd*Maurice PO Box 979 Oakland FL 34760-0979 USA mboyd.mcorp@mpinet.net 

Mr. Dennis 

Haines City Citrus Growers 

PO Box 337 Haines City FL 33845-0337 USA dennisb@gate.net 

Broadaway 

Association 


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Mr. Jim Brockington USDA ARS 2413 E Highway 83 Weslaco TX 78596 USA jim.brockington@ars.usda.gov 

Mr. Andrew Brown California Citrus Mutual 512 N Kaweah Ave Exeter CA 93221-1200 USA 

Dr. H. Donovan TicoFrut S.A. Cerro Cortez, San Carlos Aguas Zarcas, 

3535456 Costa Rica dbrown@ticofrut.com 

Brown 

Alajuela 

Dr. Lawrence Brown USDA APHIS PPQ CPHST PERAL 1730 Varsity Dr Ste 300 Raleigh NC 27606 USA lawrence.g.brown@aphis.usda.gov 

Dr. Sherline Brown University of the West Indies Mona Kingston 7 Kingston Jamaica sherline.brown02@uwimona.edu.j 

m 

Dr. Harold Browning UF/IFAS CREC 700 Experiment Station Rd Lake Alfred FL 33850 USA hwbr@crec.ifas.ufl.edu 

Mr. Dan Brunetti KeyPlex 1155 Louisiana Ave Ste 207 Winter Park FL 32789 USA dbrunetti@keyplex.com 

Dr. Marco T. 

Texas AgriLife Research 2415 E Highway 83 Weslaco TX 78596 USA marcobn@me.com 

Buenrostro-Nava 

Dr. Richard Buker Helena Chemical Company 2405 N 71st St Tampa FL 33619-2952 USA bukerr@helenachemical.com 

Mr. Leon Bunce USDA APHIS PPQ 920 Main Campus Dr Ste Raleigh NC 27606 USA leon.k.bunce@aphis.usda.gov 

200 

Mr. Bill Burchenal Cee Bee's Grove, Inc. 16907 Boy Scout Rd Odessa FL 33556-2101 USA bill@ceebeescitrus.com 

Dr. Jackie Burns UF/IFAS CREC 700 Experiment Station Rd Lake Alfred FL 33850 USA jkbu@ufl.edu 

Dr. Frank Byrne University of California, Riverside Department of Entomology Riverside CA 92521 USA frank.byrne@ucr.edu 

Mr. Zijian Cai 

Fujian Academy of Agricultural 

Sciences 

Research Center for High 

Technology of Ag Sciences 

Wusi Road 251, Room 1309 

Fuzhou, 

Fujian 

350003 PR China 

Mr. Justin Cain Chemical Dynamics, Inc. PO Box 486 Plant City FL 33564-0486 USA JCain03@msn.com 

Dr. Hernán Camacho TicoFrut, S.A. Muelle de San Carlos Ciudad, 

3000 Costa Rica hcamachov@hotmail.com 

Quesada 

Dr. Paul Cancalon Florida Department of Citrus 700 Experiment Station Rd Lake Alfred FL 33850 USA pcancalo@citrus.state.fl.us 

Dr. Kitty Cardwell USDA/NIFA 800 9th St SW Washington DC 20024 USA kcardwell@nifa.usda.gov 

Mr. Eduardo Carlos IAPAR - Instituto Agronomico do Rod. Celso Garcia Cid, km Londrina, PR 86001-970 Brazil efcarlos@iapar.br 

Parana 

375 

Mr. Doug Carman California Citrus Mutual 512 N Kaweah Ave Exeter CA 93221-1200 USA 

Mr. Mike Carrere Lykes Citrus Management PO Box 1690 Tampa FL 33601-1690 USA mike.carrere@lykes.com 

Mr. Stan Carter McArthur Farms, Inc. 1550 NE 208th St Okeechobee FL 34972-7295 USA stanjcfl@aol.com 

Mr. Steve Caruso Florida's Natural Growers/Citrus PO Box 1111 Lake Wales FL 33859-1111 USA steve.caruso@citrusworld.com 

World 

Dr. Bill Castle UF/IFAS CREC 700 Experiment Station Rd Lake Alfred FL 33850 USA bcastle@ufl.edu 

Mr. Wiley Cauthen McKinnon Corporation PO Box 979 Oakland FL 34760-0979 USA 

Mr. Peter Chaires New Varieties Development & PO Box 1113 Lakeland FL 33802 USA pchaires@flcitruspackers.org 

Management Corporation 

Ms. Holly 

Pest & Disease Management, LLC PO Box 1208 Avon Park FL 33826-1208 USA HLChamberlain@embarqmail.com 

Chamberlain 

Dr. Chunxian Chen UF/IFAS CREC 700 Experiment Station Rd Lake Alfred FL 33850 USA cxchen@ufl.edu 

Dr. Jianchi Chen USDA ARS 9611 S Riverbend Ave Parlier CA 93648 USA Jianchi.chen@ars.usda.gov 

Mr. Wilson Roberto Citrus Consultants Group - GCONCI Rua Carlos Gomes, 144 - Cordeiropolis, 

13490000 Brazil wrchignolli@linkway.com.br 

Chignolli 


Sao Paulo 


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Dr. Matthew 

USDA APHIS PPQ CPHST Moore Air Base #6414 22675 Edinburg TX 78541 USA matt.a.ciomperlik@aphis.usda.gov 

Ciomperlik 

N Moorfield Rd 

Dr. Edwin Civerolo USDA ARS San Joaquin Valley Ag 9611 S Riverbend Ave Parlier CA 93648 USA edwin.civerolo@ars.usda.gov 

Sciences 

Dr. Paul Clayson UF/IFAS CREC 700 Experiment Station Rd Lake Alfred FL 33850 USA drpaulsmc@gmail.com 

Mr. James F. A Elizabeth Alexander PO Box 524 Bartow FL 33831-0524 USA jfclements@gmail.com 

Clements 

Mr. Rex Clonts Clonts Groves, Inc. PO Box 622916 Oviedo FL 32762-2916 USA wrclonts@yahoo.com 

Mr. Andy Coker California Citrus Mutual 512 N Kaweah Ave Exeter CA 93221-1200 USA 

Dr. Helvecio Coletta- Centro de Citricultura/IAC Rod Anhanguera, Km 158 Cordeiropolis, 

13490-970 Brazil helvecio@centrodecitricultura.br 

Filho 

Sao Paulo 

Mr. Jim Conrad DuPont Crop Protection 3680 Winding Lake Cir Orlando FL 32835 USA jim.conrad-1@usa.dupont.com 

Mr. Vic Corkins Central California Tristeza 

22847 Road 140 Tulare CA 93274-9367 USA jbarnier@cctea.org 

Eradication Agency 

Mr. Aaron Corkum Cutrale Citrus Juices USA, Inc. 602 McKean St. Auburndale FL 33823-4070 USA acorkum@cutrale.com 

Mr. Reinaldo Citrus Consultants Group - GCONCI Rua Carlos Gomes, 144 - Cordeiropolis, 

13490000 Brazil corte@gconci.com.br 

Donizeti Corte 


Sao Paulo 

Mr. James Cranney California Citrus Quality Council 210 Magnolia Ave Ste 3 Auburn CA 95603 USA jcranney@calcitrusquality.org 

Dr. André Creste Eduardo de Paula Machado e Outros Fazenda Sao Jose, Caixa Rio Clara, Sao 

13500970 Brazil acreste@uol.com.br 

Postal 31 

Paulo 

Dr. John daGraca Texas A&M University - Kingsville, 312 N International Blvd Weslaco TX 78596 USA jdagraca@ag.tamu.edu 

Citrus Center 

Mr. Tad Dallas USDA ARS USHRL 2001 S Rock Rd Fort Pierce FL 34945-3030 USA tad.dallas@ars.usda.gov 

Dr. Abhaya Dandekar University of California - Davis One Shields Ave Davis CA 95616 USA amdandekar@ucdavis.edu 

Mr. Craig Davis UF/IFAS CREC 700 Experiment Station Rd Lake Alfred FL 33850 USA 

Dr. William Dawson UF/IFAS CREC 700 Experiment Station Rd Lake Alfred FL 33850 USA wodtmv@crec.ifas.ufl.edu 

Dr. Leonardo De La Auburn University 

209 Rouse Life Sciences Auburn AL 36849 USA lzd0005@auburn.edu 

Fuente 

Building 

Dr. Jesse de Leon USDA ARS 2413 East Highway 83 Weslaco TX 78596 USA jesus.deleon@ars.usda.gov 

Mr. Arlindo de Salvo Improcrop do Brasil Ltda Curio 312 B Capela Velha Araucaria, 

83705552 Brazil arlindoconsulton@gmail.com 

Parana 

Dr. Sharon Dea USDA ARS Citrus & Subtropical 600 Avenue S, NW Winter Haven FL 33881 USA sharon.dea@ars.usda.gov 

Products Lab 

Mr. Juan Delgado TicoFrut S.A. 18-4433, Aguas Zarcas San Carlos, 

18-4433 Costa Rica jdelgado@ticofrut.com 

Alajuela 

Dr. Christian Agdia, Inc. 30380 County Rd 6 Elkhart IN 46514 USA christian@agdia.com 

Delgado Palomino 

Dr. Xiaoling Deng South China Agricultural University Wushan Rd 483 Guangzhou, 

510642 PR China xldeng@scau.edu.cn 

Guangdong 

Dr. Ziniu Deng Hunan Agricultural University Furong Qu Changsha, 

410128 PR China deng7009@163.com 

Hunan 

Mr. Mike Dennison Cee Bee's Grove, Inc. 16907 Boy Scout Rd Odessa FL 33556-2101 USA 


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Dr. Megan Dewdney UF/IFAS CREC 700 Experiment Station Rd Lake Alfred FL 33850 USA mmdewdney@crec.ifas.ufl.edu 

Mr. Fabio Di Giorgi Louis Dreyfuss Commodities Rod.Armando Salles Bebedouro, 

14707900 Brazil fabio.di.giorgi@ldcom.com 


Sao Paulo 

Mr. Todd Dicks L Dicks, Inc. PO Box 1809 Dundee FL 33838-1809 USA 

Dr. Fang Ding Huazhong Agricultural University No 1 Shizishan St Wuhan 430070 PR China dingfang2008@gmail.com 

Dr. Wayne Dixon Florida Dept. of Agriculture & 1911 SW 34th St Gainesville FL 32608 USA Wayne.Dixon@freshfromflorida.c 

Consumer Services 

om 

Dr. Michel Dollet CIRAD Campus de Baillarquet TA Montpelier 34398 France michel.dollet@cirad.fr 

A-29/F 

Dr. Hamed Doostdar KeyPlex 1155 Louisiana Ave Ste 207 Winter Park FL 32789 USA hdoostdar@keyplex.com 

Mr. Aedan Dowling Tropicana Products, Inc. PO Box 338 Bradenton FL 34206-0338 USA aedan.dowling@pepsico.com 

Mr. Randy Driggers USDA ARS USHRL 2001 S Rock Rd Fort Pierce FL 34945 USA randy.driggers@ars.usda.gov 

Mr. Yoel Drishpoun Mehadrin Tnuport Export 1 Power Center Beerot 

60905 Israel yoeld@mtex.co.il 

Yitzhak 

Dr. Yong-Ping Duan USDA ARS USHRL 2001 S Rock Rd Fort Pierce FL 34945 USA yongping.duan@ars.usda.gov 

Dr. Joan Dusky UF/IFAS Extension Administration PO Box 110210 Gainesville FL 32611 USA jadu@ufl.edu 

Dr. Timothy Ebert UF/IFAS CREC 700 Experiment Station Rd Lake Alfred FL 33850 USA tebert@ufl.edu 

Mr. Mike Edenfield Bayer CropScience LP 11097 Ledgement Ln Windermere FL 34786 USA mike.edenfield@bayercropscience. 

com 

Mr. Choaa El Mohtar UF/IFAS CREC 700 Experiment Station Rd Lake Alfred FL 33850 USA mohtarc@crec.ifas.ufl.edu 

Mr. Jim Ellis 

FDACS - Division of Fruit & 

PO Box 1072 Winter Haven FL 33882-1072 USA James.Ellis@freshfromflorida.com 

Vegetables 

Mr. Gary England University of Florida 7620 State Road 471 Ste 2 Bushnell FL 33513 USA gke@ifas.ufl.edu 

Mrs. Kate English Pavese Law Firm PO Drawer 1507 Fort Myers FL 33902 USA kre@paveselaw.com 

Mr. Bret Erickson Texas Citrus Mutual 901 Business Park Dr Ste Mission TX 78572 USA 

400 

Dr. Akif Eskalen University of California, Riverside Dept of Plant Pathology & Riverside CA 92521 USA akif.eskalen@ucr.edu 

Microbiology 900 University 

Ave, Fawcett Lab #232 

Mr. Mark Estes FDACS 3027 Lake Alfred Rd Winter Haven FL 33881-1438 USA mark.estes@freshfromflorida.com 

Miss Jennie Fagen University of Florida PO Box 110700 Gainesville FL 32611 USA jenn1eruth@ufl.edu 

Mr. Mauro Fagotti Citrus Consultants Group - GCONCI Rua Carlos Gomes, 144 - Cordeiropolis, 

13490000 Brazil fagotti@gconci.com.br 


Sao Paulo 

Dr. Bryce Falk University of California, Davis Dept. of Plant Pathology 1 Davis CA 95616-5270 USA bwfalk@ucdavis.edu 

Shields AVE 

Dr. Guocheng Fan Fujian Academy of Agricultural Research Center for High Fuzhou, 

350003 PR China 

Sciences 

Technology of Ag Sciences Fujian 


Mr. Steve Farr Ben Hill Griffin, Inc. PO Box 127 Frostproof FL 33843-0127 USA sfarr@bhgriffin.com 

Dr. Vicente Febres University of Florida 1301 Fifield Hall Gainesville FL 32611 USA vjf@ufl.edu 

Miss Rafaella Felipe University of Sao Paulo - ESALQ Avenida Padua Dias, 9 Piracicaba, 

13416-145 Brazil raffynha@yahoo.com.br 

Sao Paulo 


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Dr. Latanya Fisher University of Florida 1301 Fifield Hall Gainesville FL 32611 USA lfisher@ufl.edu 

Mrs. Lidia Flores Comite Estatal de Sanidad Vegetal de Hacienda las Toronjas #140 Montemorelos 

67500 Mexico lidiaf2004@yahoo.com.mx 

Nuevo Leon 

, Nuevo Leon 

Mr. John Floyd Floyd & Associates, Inc. 10548 Singletary Rd Dade City FL 33525-1568 USA floydcitrus@yahoo.com 

Dr. Svetlana 

UF/IFAS CREC 700 Experiment Station Rd Lake Alfred FL 33850 USA svetlana@ufl.edu; 

Folimonova 

sveta@crec.ifas.ufl.edu 

Dr. Lisa Fontaine- CIRAD 

Campus de Baillarquet TA Montpelier 34398 France lisa.fontaine_bodin@cirad.fr 

Bodin 

A-29/F 

Miss Ana Aurora COMITE ESTATAL DE SANIDAD PEDRO VILLEGAS 51 HERMOSILL 

83000 MEXICO aurorafontes@gmail.com 

Fontes Puebla VEGETAL DE SONORA 

COL. DEL RAZO 

O, SONORA 

Mr. Dick Fort Fort*Richard Jr 500 5th St NE Fort Meade FL 33841-2612 USA rafort@embarqmail.com 

Dr. Juliana Freitas- Embrapa/Centro de Citricultura Rod. Anhanguera km 158 Cordeiropolis, 

13490-970 Brazil juliana@cnpmf.embrapa.br 

Astua 

Sylvio Moreira 

Sao Paulo 

Mr. Joe Froehlich Froehlich*Joseph J. 9 Cheshire Rd Bethpage NY 11714 USA jjfro0609@msn.com 

Mr. Jack Frost Wedgworth's, Inc. PO Box 2076 Belle Glade FL 33430 USA jack@wedgworth.com 

Mr. Satoshi Fusayasu Plant Protection Station, Naze Branch 1-1 Nazenagahama-cho Amami City, 

894-0036 Japan fusayasus@pps.maff.go.jp 

Kagoshima 

Dr. Stephen Futch University of Florida Extension 700 Experiment Station Rd Lake Alfred FL 33850 USA shf@ufl.edu 

Dr. Dean Gabriel University of Florida - Plant 

PO Box 110680 Gainesville FL 32611-0680 USA dgabr@ufl.edu; 

Pathology Dept. 

gabriel@ipgenetics.com 

Ms. Paula Gadea United States Sugar Corporation 111 Ponce de Leon Ave Clewiston FL 33440 USA pgadea@ussugar.com 

Mr. Dan Galbraith California Citrus Mutual 512 N Kaweah Ave Exeter CA 93221-1200 USA dgalbraith@limoneira.com 

Mrs. Celestina California Department of Food & 13915 Saticoy St Van Nuys CA 91402 USA tgalindo@cdfa.ca.gov 

Galindo 

Agriculture 

Dr. Steve Garnsey UF/IFAS CREC 700 Experiment Station Rd Lake Alfred FL 33850 USA steve.garnsey@gmail.com 

Mr. Timothy Gast Southern Gardens Citrus 1820 County Road 833 Clewiston FL 33440 USA tgast@southerngardens.com 

Mr. Tim Gaver Saint Lucie County Extension Service 8400 Picos Rd Ste 101 Fort Pierce FL 34945-3045 USA tgaver.49@ufl.edu 

Mr. Jeff Geuder USDA Florida Ag Statistics PO Box 530105 Orlando FL 32853-0105 USA jeff_geuder@nass.usda.gov; nassfl@nass.usda.gov 

Mr. Frank Giles Florida Grower Magazine/Meister 1555 Howell Branch Rd Ste Winter Park FL 32789 USA frgiles@meistermedia.com; 

Media 

C-204 

fgiles@meistermedia.com 

Mr. James Giles ProPak Software, LLC 150 3rd St SW 2nd Floor Winter Haven FL 33880 USA jamesg@propaksoftware.com 

Mr. Luiz Girotto Fischer S/A - Comercio Industria e Sao Lourenco St #81 Matao, Sao 

15990-200 Brazil salves@terralagro.com.br 

Agricultura 

Paulo 

Dr. Fred Gmitter UF/IFAS CREC 700 Experiment Station Rd Lake Alfred FL 33850 USA fgg@crec.ifas.ufl.edu 

Mr. Pat Gomes USDA APHIS PPQ 920 Main Campus Dr Ste Raleigh NC 27606-5213 USA patrick.j.gomes@aphis.usda.gov 

200 

Mrs. Hilda Gomez USDA APHIS PPQ CHRP 1700 NW 66th Ave Ste 112 Plantation FL 33313 USA hilda.gomez@aphis.usda.gov 

Mr. Exau Vicente Comite Estatal de Sanidad Vegetal de Col. Centro. Tuxtla Gutierrez Chiapas 29000 Mexico 

Gonzalez 

Chiapas 

Calle 7 Poniente Norte #151 

Mr. Pedro Gonzalez UF/IFAS CREC 700 Experiment Station Rd Lake Alfred FL 33850 USA pcgo@crec.ifas.ufl.edu 


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Dr. Jose Gonzalez- NREC, Carnegie Mellon University 10 40th St Pittsburgh PA 15201 USA jgmora@nrec.ri.cmu.edu 

Mora 

Mr. Jim Gorden California Citrus Research Board PO Box 230 Visalia CA 93279-0230 USA jim@gordenag.com 

Dr. Tim Gottwald USDA ARS USHRL 2001 S Rock Rd Fort Pierce FL 34945 USA tim.gottwald@ars.usda.gov 

Dr. Luiz Goulart University of California Davis MMI Dept., GBSF Ste 5503 Davis CA 95616 USA 

Rm 5521 

Dr. Siddarame Gowda UF/IFAS CREC 700 Experiment Station Rd Lake Alfred FL 33850 USA gowda@ufl.edu 

Ms. Cheryl Graffam UF/IFAS CREC 700 Experiment Station Rd Lake Alfred FL 33850 USA 

Dr. Elizabeth 

University of California, Riverside Department of Entomology Riverside CA 92521 USA bethgc@uckac.edu 

Grafton-Cardwell 

Dr. James Graham UF/IFAS CREC 700 Experiment Station Rd Lake Alfred FL 33850 USA jhg@crec.ifas.ufl.edu; 

jhgraham@ufl.edu 

Dr. Dennis Gross Texas A&M University Department of Plant 

College TX 77843-2132 USA d-gross@tamu.edu 

Pathology & Microbiology Station 

2132 TAMU 

Dr. Jude Grosser UF/IFAS CREC 700 Experiment Station Rd Lake Alfred FL 33850 USA jgrosser@ufl.edu 

Mr. Gus Gunderson California Citrus Mutual 512 N Kaweah Ave Exeter CA 93221-1200 USA 

Dr. Dan Gunter Citrus Research and Development 700 Experiment Station Rd Lake Alfred FL 33850-2299 USA dgunter@sunorchard.com 

Foundation, Inc. 

Dr. Goutam Gupta Los Alamos National Labs Mail Stop M888, TA-43, Los Alamos NM 87545 USA gxg@lanl.gov 

HRL 

Mr. Francisco Belize Agricultural Health Authority Belmopan 35467 Belize frankpest@yahoo.com 

Gutierrez 

Dr. Kevin Hackett USDA ARS 5601 Sunnyside Ave Beltsville MD 20705 USA Kevin.Hackett@ars.usda.gov 

Dr. Subhas Hajeri UF/IFAS CREC 700 Experiment Station Rd Lake Alfred FL 33850 USA hajeriss@ufl.edu 

Dr. Susan Halbert FDACS - Division of Plant Industry PO Box 147100 Gainesville FL 32614 USA Susan.Halbert@freshfromflorida.c 

om 

Dr. David Hall USDA ARS 2001 S Rock Rd Fort Pierce FL 34945 USA david.hall@ars.usda.gov 

Mr. Ron Hamel Gulf Citrus Growers Association, Inc. PO Box 1319 LaBelle FL 33975-1319 USA gulfcitruscapron@embarqmail.com 

Mr. Tom Hammond Hammond Groves PO Box 643278 Vero Beach FL 32964 USA tomshammond@aol.com 

Mr. Craig Hampton Tropicana Products, Inc. 1001 13th Ave E Bradenton FL 34208 USA craig.hampton@pepsico.com 

Mr. Michael Harowitz ProSource One 4094 Paul Buchman Hwy Plant City FL 33565 USA maharowitz@landolakes.com 

Dr. Scott Harper UF/IFAS CREC 700 Experiment Station Rd Lake Alfred FL 33850 USA sjharper@ufl.edu 

Mr. Bert J. Harris B J Harris & Son, Inc. 514 Lake Mirror Dr Lake Placid FL 33852-5966 USA bande@htn.net 

Mr. Gregg Hartt S Y Hartt & Son, Inc. PO Box 1429 Avon Park FL 33826-1429 USA theorangedoctor@htn.net 

Dr. John Hartung USDA ARS Molecular Plant 

10300 Baltimore Ave Beltsville MD 20705-2350 USA john.hartung@ars.usda.gov 

Pathology Lab 

Mr. Louis Haverlock Lou Ross Citrus, Inc. PO Box 567 Balm FL 33503-0567 USA rosslou2@aol.com 

Dr. Zhenli He University of Florida 2199 S Rock Rd Fort Pierce FL 34945-3138 USA zhe@ufl.edu 

Mr. Paul Heller Texas Citrus Mutual 901 Business Park Dr Ste Mission TX 78572 USA martha@valleyag.org 

400 


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Mrs. Megan 

USDA APHIS PPQ CPHST 1730 Varsity Drive Ste 400 Raleigh NC 27606 USA megan.w.henderson@aphis.usda.g 

Henderson 

ov 

Dr. Katherine University of Florida - SFREC 2685 State Road 29 N Immokalee FL 34142 USA jinxkat@ufl.edu 

Hendricks 

Dr. Jorge Hernandez SAGARPA Karina 116 Celaya, Gto. 38050 Mexico j.hernandezbaeza@yahoo.com.mx 

Dr. Mark Hilf USDA ARS USHRL 2001 S Rock Rd Fort Pierce FL 34945 USA mark.hilf@ars.usda.gov 

Mr. Nick Hill California Citrus Mutual 512 N Kaweah Ave Exeter CA 93221-1200 USA ccm@cacitrusmutual.com 

Dr. Nguyen Van Hoa Southern Horticultural Research 203, My Tho Tien Giang 860000 Viet Nam hoavn2003@gmail.com 

Institute 

Ms. Joanne Hodge USDA ARS USHRL 2001 S Rock Rd Fort Pierce FL 34945-3030 USA 

Dr. Greg Hodges FDACS - Division of Plant Industry 1911 SW 34th St Gainesville FL 32608 USA Greg.Hodges@freshfromflorida.co 

m 

Dr. Michele Hoffman USDA ARS USHRL 2991 S Rock Rd Fort Pierce FL 34945 USA michele.hoffman@ars.usda.gov 

Dr. Charla 

USDA APHIS PPQ CPHST 1730 Varsity Drive Ste 400 Raleigh NC 27606 USA Charla.Hollingsworth@aphis.usda. 

Hollingsworth 

gov 

Mr. Kieth 

Chemical Containers, Inc. PO Box 1307 Lake Wales FL 33859-1307 USA kiethh@embarqmail.com 

Hollingsworth 

Mr. Curt Holmes California Citrus Mutual 512 N Kaweah Ave Exeter CA 93221-1200 USA 

Mr. David Freeman Graves Brothers Company 

2770 Indian River Blvd Ste Vero Beach FL 32960-4230 USA 

Howard 

201 

Dr. Jiahuai Hu UF/IFAS CREC 700 Experiment Station Rd Lake Alfred FL 33850 USA hjh@ufl.edu 

Dr. John Hu University of Hawaii 3190 Maile Way Honolulu HI 96822 USA johnhu@hawaii.edu 

Dr. Wayne Hunter USDA ARS 2001 S Rock Rd Fort Pierce FL 34945 USA wayne.hunter@ars.usda.gov 

Mrs. Sandy Hurner Hurner*G Tim 3604 Golfview Rd Sebring FL 35875-5028 USA 

Mr. Tim Hurner University of Florida Extension 4509 George Blvd Sebring FL 33875 USA plowboy@ufl.edu 

Mr. Thiago Iost Promotora Citricola del Golfo SA de Constitucion 405 Pte Monterrey, 

64000 Mexico thiago.iost@citrofrut.com.mx 

CV 

Colonia Centro 

NL 

Mr. Michael Irey United States Sugar Corporation 111 Ponce de Leon Ave Clewiston FL 33440 USA msirey@ussugar.com; 

mirey@ussugar.com 

Mr. Ed Ishida Bayer CropScience LP 1773 Powell Dr. Ventura CA 93004 USA Ed.Ishida@bayercropscience.com 

Mr. John Jackson FCIRCC 36545 E Eldorado Lake Dr Eustis FL 32736 USA jackson7@ufl.edu 

Mr. Arom Jantasorn University of Florida 2199 S Rock Rd Fort Pierce FL 34945 USA am_pam@hotmail.com 

Dr. Juan Jasso INIFAP Calle 6, No. 398 Av Correra Merida, 

97130 Mexico jasso.juan@inifap.gob.mx 

Racho 

Yucatan 

Mr. Tom Jerkins Blue Goose Growers, LLC 16050 W Orange Ave Fort Pierce FL 34945 USA tjerkins@bluegoosegrowers.com 

Dr. Yingnan Jiang Integrated Plant Genetics, Inc. 13420 Progress Blvd Ste 100 Alachua FL 32615 USA jiang@ipgenetics.com 

Mr. 3ric Johanson Intellectual Ventures Laboratory 1600 132nd Ave NE Ste 100 Bellevue WA 98134 USA 3ricj@intven.com 

Ms. Christen Johnson UF/IFAS CREC 700 Experiment Station Rd Lake Alfred FL 33850 USA cajohnson295@gmail.com 

Dr. Evan Johnson UF/IFAS CREC 700 Experiment Station Rd Lake Alfred FL 33850 USA egjohnson@ufl.edu 

Mr. Jason Johnson FDACS - Citrus Nursery Inspection 3397 US 27 S Avon Park FL 33825 USA Jason.Johnson@freshfromflorida.c 

om 

Dr. Moneen Jones University of Florida - SFREC 2685 State Road 29 N Immokalee FL 34142 USA mmjones2@ufl.edu 


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Dr. Joseph Joyce University of Florida/IFAS PO Box 110180 Gainesville FL 32611-0180 USA jcj@ifas.ufl.edu 

Mr. Marvin Kahn Kahn Grove Service Company PO Box 3346 Sebring FL 33871-3346 USA mkahn@kahngrove.com 

Mr. David Karp University of California, Riverside 9050 Lloyd Pl Los Angeles CA 90069 USA dkarp@ucr.edu 

Dr. Manjunath USDA ARS Citrus Germplasm 1060 Martin Luther King Riverside CA 92507 USA manjunath.keremane@ars.usda.go 

Keremane 

Repository 

Blvd 

v 

Dr. Abeer Khalaf University of Florida 1301 Fifield Hall Gainesville FL 32611 USA abeera@ufl.edu 

Dr. Nabil Killiny UF/IFAS CREC 700 Experiment Station Rd Lake Alfred FL 33850 USA nabilkilliny@ufl.edu 

Dr. Dan King Florida Department of Citrus 700 Experiment Station Rd Lake Alfred FL 33850 USA dking@citrus.state.fl.us 

Mr. Richard Kinney Florida Citrus Packers PO Box 1113 Lakeland FL 33802-1113 USA rkinney@flcitruspackers.org 

Mr. Thomas 

Cooperative Producers, Inc. PO Box 3147 Immokalee FL 34143 USA tfkirschner@yahoo.com; 

Kirschner 

kirschnertfk@yahoo.com 

Mr. Maris Klavins Bayer CropScience LP 2 T.W. Alexander Dr Research NC 27709 USA maris.klavins@bayer.com 

Triangle Park 

Mr. Ben Krupski Lennon Grove Service, Inc. 2701 Dean Ridge Rd Orlando FL 32825-8724 USA lgscitrus@aol.com 

Dr. Stephen Lapointe USDA ARS 2001 S Rock Rd Fort Pierce FL 34945 USA stephen.lapointe@ars.usda.gov 

Mr. Bruno Laureano Comite Estatal de Sanidad Vegetal Calle Nogal Lote 11, Mz2 Chilpancingo, 

39090 Mexico bruno_laureano@yahoo.com.mx 

Ahuelican 

del Guerrero 

Jacarandas 2a Seccion Guerrero 

Dr. Richard Lee USDA ARS Citrus Germplasm 1060 Martin Luther King Riverside CA 92507 USA richard.lee@ars.usda.gov 

Repository 

Blvd 

Dr. Rui Leite 

IAPAR - Instituto Agronomico do Rod. Celso Garcia Cid, km Londrina, PR 86001-970 Brazil ruileite@iapar.br 

Parana 

375 

Mr. Bill Lennon Lennon Grove Service, Inc. 2701 Dean Ridge Rd Orlando FL 32825-8724 USA lgscitrus@aol.com 

Mrs. Jewel 

Florida Citrus Mutual PO Box 89 Lakeland FL 33802 USA jewell@flcitrusmutual.com 

Letchworth 

Dr. Cynthia LeVesque California Citrus Research Board PO Box 230 Visalia CA 93279-0230 USA cynthia@citrusresearch.org 

Dr. Laurene Levy USDA APHIS PPQ CPHST BARC-East, Bldg-580 Beltsville MD 20705 USA laurene.levy@aphis.usda.gov 

Mr. Wayne Lewis Lewis & Associates, Inc. PO Box 16518 Tampa FL 33687 USA wlewis@la-fl.com 

Dr. Wenbin Li USDA APHIS PPQ Building 580, Powder Mill Beltsville MD 20708 USA wenbin.li@aphis.usda.gov 

Rd 

Dr. Hui-Ling Liao University of Florida/IFAS 700 Experiment Station Rd Lake Alfred FL 33850 USA bigface@ufl.edu 

Dr. Lia Liefting MAF Biosecurity New Zealand PO Box 2095 Auckland 1140 New lia.liefting@maf.govt.nz 

Zealand 

Mr. Rene Lima Citrovita Agropecuaria Ltda Estrada Municipal, s/n, km Itapetininga, 

18203-000 Brazil rene.lima@citrovita.com.br 

01 ditrito Recha 

Sao Paulo 

Dr. Hong Lin USDA ARS 9611 S Riverbend Ave Parlier CA 93648 USA hong.lin@ars.usda.gov 

Dr. Magdalen Cornell University 

Department of Plant Ithaca NY 14853 USA ML16@cornell.edu 

Lindeberg 

Pathology 

Dr. Steven Lindow University of California, Berkeley Plant & Microbial Biology Berkeley CA 94720 USA icelab@berkeley.edu 

Department 111 Koshland 

Hall 

Mr. Horacio Lomba Consorcio Citricos Dominicanos S.A. Carretera Duarte Km 46 Villa 

Altagracia 

10112 Dominican 

Republic 

hlomba@gruporica.com 


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Mr. Martin Loosli Louis Dreyfuss Commodities Rod.Armando Salles Bebedouro, 

14707900 Brazil martin.loosli@ldcom.com 


Sao Paulo 

Dr. Silvio Lopes Fundecitrus Av. Joao Luis Gentil Araraquara, 

14805-289 Brazil slopes@fundecitrus.com.br 

Fernandes 243 

San Paulo 

Mr. Abel Lopez SENASICA SAGARPA INSURGENTES SUR 489 CUAUHTEM 

06100 MEXICO abel.lopez@senasica.gob.mx 

Buenfil 

PISO 16 

OC, MEXICO 

Dr. Jose Lopez- INIFAP Campo Experimental General 

64700 Mexico jila64@yahoo.com; 

Arroyo 

Teran,Nuevo 

lopez.jose@inifap.gob.mx 

Leon 

Dr. Eliezer Louzada Texas A&M University - Kingsville, 312 N International Blvd Weslaco TX 78596 USA elouzada@ag.tamu.edu 

Citrus Center 

Mr. David Lowe USDA APHIS PPQ CHRP 1700 NW 66 Ave Ste 112 Plantation FL 33313 USA david.lowe@aphis.usda.gov 

Dr. Monica Lozano INIFAP Calle 6, No. 398 Av Correra Merida, 

97130 Mexico lozano.monica@inifap.gob.mx 

Racho 

Yucatan 

Mr. Fred Lozo Florida Phosphorus, Inc. 6 Abaco Rd Key Largo FL 33470 USA flaphosphorus@comcast.net 

Mr. Charlie Lucas Consolidated Citrus LP 63 Barn Rd Venus FL 33960 USA clucas@cclpcitrus.com 

Mr. Eduardo Antonio Citrus Consultants Group - GCONCI Rua Carlos Gomes, 144 - Cordeiropolis, 

13490000 Brazil ealucato@uol.com.br 

Lucato 


Sao Paulo 

Ms. Magally Luque- CDFA 3180 Cridge St Riverside CA 92507 USA mlwilliams@cdfa.ca.gov 

Williams 

Dr. Marcos Machado Centro de Citricultura Sylvio Moreira Caixa Postal 04 

Cordeiropolis, 

13493970 Brazil marcos@centrodecitricultura.br 

- IA 

SP 

Mr. David Machlitt Consulting Entomology Services 12142 River Grove St Moorpark CA 93021 USA machlitt@gmail.com 

Dr. Agenor Mafra- ISCA Technologies, Inc. 1230 W Spring St Riverside CA 92507 USA president@iscatech.com 

Neto 

Dr. Robert Mangan USDA ARS 2413 E Highway 83 Weslaco TX 78596 USA robert.mangan@ars.usda.gov 

Dr. Rajinder Mann UF/IFAS CREC 700 Experiment Station Rd Lake Alfred FL 33850 USA mannrs@crec.ifas.ufl.edu 

Dr. John Manthey USDA ARS CPRL 600 Avenue S. NW Winter Haven FL 33881 USA John.Manthey@ars.usda.gov 

Mrs. Veronica Coordinator, Belize MIle 9 SCK Valley SCK District Belize vema600@yahoo.com 

Manzanero Majil 

Dr. Viviani Marques Agronomic Institute of Parana Celso Garcia Cid km 375 Londrina, 

86001-970 Brazil viviani@iapar.br 

Parana 

Mr. Jesus Marquez CESAVENY Primero de Diciembre No 36 San Leonel, 

63830 Mexico coordinacioncitricos@cesavenay.o 

Norte 

Nayarit 

rg.mx 

Mr. Larry Marsh Florida Citrus Management, Inc. PO Box 1347 LaBelle FL 33975 USA larrym3041@aol.com 

Mr. Julio Martich Consorcio Citricos Dominicanos S.A. Carretera Duarte Km 46 Villa 

10112 Dominican juliomartich@codetel.net.do 

Altagracia 

Republic 

Dr. Jose Luis 

Citricola Del Yaqui, S.A. de C.V. Allende 615 PTE Local 3 CD. Obregon, 

85000 Mexico jlmarca@hotmail.com 

Martinez 

Altos 

Son 

Mr. Eloy Martinez COMITE ESTATAL DE SANIDAD PROL. MIGUEL HIDALGO SAN PEDRO 

72160 MEXICO eema_59@hotmail.com 

Aguilar 

VEGETAL DE PUEBLA 

No. 2107 

CHOLULA,P 

UEBLA 


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Mr. Perry Mason Mason Groves PO Box 286 Lake Placid FL 33862-0286 USA masongroves@embarqmail.com 

Dr. Philip Mason USDA APHIS PPQ 2150 Centre Ave Bldg B Fort Collins CO 80526 USA phillip.a.mason@aphis.usda.gov 

Mr. Luis Matos UF/IFAS CREC 700 Experiment Station Rd Lake Alfred FL 33850 USA luismatos@crec.ifas.ufl.edu 

Dr. Takayuki 

Yokohama Plant Protection Station 1-16-10, Shinyamashita, Yokohama, 

231-0801 Japan matsuurat@pps.maff.go.jp 

Matsuura 

Naka-ku 

Kanagawa 

Dr. Richard Mayer KeyPlex 1155 Louisiana Ave Ste 207 Winter Park FL 32789 USA rtmayer@Keyplex.com 

Mr. Steve Mayo Mayo Grove Management 6601 North Blvd Fort Pierce FL 34951-5202 USA Stephen.Mayo@ars.usda.gov 

Mr. Peter McClure Central Florida Grove Service, Inc. 5325 Summerlin Rd Port Saint FL 34987 USA pmcclure@evansprop.com 

Lucie 

Dr. Greg McCollum USDA ARS USHRL 2001 S Rock Rd Fort Pierce FL 34945 USA greg.mccollum@ars.usda.gov 

Mr. Donald 

Dixie Farms, LLC 5 Hill Ave Orlando FL 32801 USA 

McCosham 

Mr. George McEwan California Citrus Mutual 512 N Kaweah Ave Exeter CA 93221-1200 USA 

Dr. Alistair McKay Sunkist Growers, Inc. 1756 Eastman Ave #112 Ventura CA 93003 USA amckay@sunkistgrowers.com 

Mr. Marty McKenna McKenna & Associates Citrus, Inc. 2551 Lakeview Dr Sebring FL 33870-2759 USA mck80@earthlink.net; 

donna@mckennabros.com 

Mr. Pat McKenna McKenna Brothers, Inc. 70 Mammoth Grove Road Lake Wales FL 33898 USA pat@mckennabros.com 

Mr. Ben McLean McLean Ag Chemical, Inc. PO Box 1044 Minneola FL 34755 USA benmclean@aol.com 

Mr. Mark McLean Citrus Marketing Services 3335 US Highway 27 S Sebring FL 33870-5441 USA markvmclean@gmail.com 

Mr. Ryan McLean Citrus Marketing Services 3335 US Highway 27 S Sebring FL 33870-5441 USA 

Dr. Camilo Lazaro Citrus Consultants Group - GCONCI Rua Carlos Gomes, 144 - Cordeiropolis, 

13490000 Brazil clmedina@conplantferti.com.br 

Medina 


Sao Paulo 

Mr. Mauricio Lemos Citrus Consultants Group - GCONCI Rua Carlos Gomes, 144 - Cordeiropolis, 

13490000 Brazil mauricio.mendes@agrafnp.com.br 

Mendes da Silva 


Sao Paulo 

Dr. Ana Maria IAPAR - Instituto Agronomico do Rod. Celso Garcia Cid, km Londrina, PR 86001-970 Brazil meneguim@iapar.br 

Meneguim 

Parana 

375 

Ms. Tatiana Merlin Sao Paulo State University 1780 Dr Jose Barbosa de Botucatu, Sao 

18610307 Brazil trpdalmeida@hotmail.com 

Barros 

Paulo 

Mr. John Merritt Consolidated Citrus LP 63 Barn Rd Venus FL 33960 USA jmerritt@cclpcitrus.com 

Mr. Paul Metheney Central California Tristeza 

22847 Road 140 Tulare CA 93274-9367 USA pmetheney@cctea.org 

Eradication Agency 

Mr. Kevin Metheny Florida Citrus Mutual PO Box 89 Lakeland FL 33802 USA kevinm@flcitrusmutual.com 

Mr. Manuel Meza USDA APHIS IS Carr Reynosa-Matamoros Reynosa, Tam 88501 Mexico manuel.meza@aphis.usda.gov 


Mr. Frank Miele Magna - Bon 2421 SW 127 Ave Davie FL 33325 USA mielemgt@yahoo.com 

Dr. Marcelo Miranda Fundecitrus Av. Dr. Adhemar Pereira de Araraquara, 

14807040 Brazil mpmiranda@fundecitrus.com.br 

Barros 201 

Sao Paulo 

Dr. Shin-ichi Miyata National Institute of Fruit Tree Fujimoto 2-1 

Tsukuba, 

305-8605 Japan smiyata@ufl.edu; 

Science 

Ibaraki 

smiyata@affrc.go.jp 

Mr. Michael Monroe Sun Ag, Inc. 7735 County Road 512 Fellsmere FL 32948-7802 USA mmonroe@sunaginc.com 

Mr. Rick Montney ProPak Software, LLC 150 3rd St SW 2nd Floor Winter Haven FL 33880 USA rickm@propaksoftware.com 

Dr. Cesar Monzo University of Florida - SFREC 2685 State Road 29 N Immokalee FL 34142 USA cmonzo@ufl.edu 


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Mr. Gene Mooney Ben Hill Griffin, Inc. PO Box 127 Frostproof FL 33843-0127 USA gmooney@bhgriffin.com 

Dr. Gloria Moore University of Florida 1301 Fifield Hall Gainesville FL 32611 USA gamoore@ufl.edu 

Ms. Pamela Moreno USDA APHIS IS Carr Reynosa-Matamoros Reynosa, Tam 88501 Mexico pamelamoreno0207@hotmail.com 


Dr. Kent Morgan USDA ARS USHRL 2001 S Rock Rd Fort Pierce FL 34945 USA kent.morgan@ars.usda.gov 

Mr. Robert A. Morris UF/IFAS CREC 700 Experiment Station Rd Lake Alfred FL 33850 USA ramorris@ufl.edu 

Dr. Joseph Morse University of California, Riverside Department of Entomology Riverside CA 92521 USA joseph.morse@ucr.edu 

3401 Watkins Dr 

Mr. Matt Moye Citrus Solutions, LLC PO Box 1341 Zolfo Springs FL 33890-1341 USA moyeboy2@hotmail.com 

Ms. Emma Mullen Intellectual Ventures Laboratory 1600 132nd Ave NE Ste 100 Bellevue WA 98134 USA emullen@intven.com 

Mr. Ronald P. UF/IFAS CREC 700 Experiment Station Rd Lake Alfred FL 33850 USA rpm@crec.ifas.ufl.edu 

Muraro 

Mr. Michael Murphy Cooperative Producers, Inc. PO Box 3147 Immokalee FL 34143 USA msmurf1fl@yahoo.com 

Mr. Monty Myers University of Florida - IRREC 2199 S Rock Rd Fort Pierce FL 34945 USA montyemyers@crec.ifas.ufl.edu 

Mr. Dan Myhaver EnviroLogix, Inc. 530 Riverside Industrial Portland ME 04039 USA dan.myhaver@envirologix.com 

Pkwy 

Ms. Cheryl Nagle Tropicana 1001 13th Ave E Bradenton FL 34208 USA cheryl.nagle@pepsico.com 

Dr. Jan Narciso USDA ARS Citrus & Subtropical 600 Avenue S, NW Winter Haven FL 33881 USA jan.narciso@ars.usda.gov 

Products Lab 

Mr. Ernie Neff Southeast AgNet/Citrus Industry 302 S Massachusetts Ave Lakeland FL 33801 USA ernie@southeastagnet.com 

Mag. 

Ste 114 

Dr. Ronald Neuman Auburn University Department of Chemical Auburn AL 36849 USA neumard@auburn.edu 

Engineering 

Mr. Jerry Newlin Orange - Co, LP 12010 NE Hwy 70 Arcadia FL 34266-4267 USA jnewlin@orangecofla.com 

Dr. Ru Nguyen FDACS - Division of Plant Industry PO Box 147100 Gainesville FL 32614-7100 USA ru.nguyen@freshfromflorida.com 

Mr. James Nickel California Citrus Mutual 512 N Kaweah Ave Exeter CA 93221-1200 USA 

Mrs. Cheryl Noble Asset Protection Partners of Central PO Box 160398 

Altamonte FL 32716 USA cnoble@sihle.com 

Fla 

Springs 

Mr. Antonio Novelo Comite Estatal de Sanidad Vegetal de Col. Cd. Industrial Calle 19 Merida, 

Mexico nnovelo4@hotmail.com; 

Cocom 

Yucatan 

No. 443 X 26 Y 28 

Yucatan 

psv@yct.sagarpa.gob.mx 

Dr. Ulisses Nunes da University of Florida - Plant 

1453 Fifield Hall Gainesville FL 32611 USA u.nunesdarocha@ufl.edu 

Rocha 

Pathology 

Mr. Gerald O'Connor KeyPlex 1155 Louisiana Ave Ste 207 Winter Park FL 32789 USA goc@keyplex.com 

Miss Daniela Okuma University of Florida 700 Experiment Station Rd Lake Alfred FL 33850 USA dmokuma@ufl.edu 

Mr. Donny Oleniczak Bayer CropScience LP 203 Winghurst Blvd Orlando FL 32828 USA 

Mr. Chris Oswalt University of Florida Extension 1702 Highway 17-98 S Bartow FL 33830 USA wcoswalt@ufl.edu 

Mr. Paulo Paiva Louis Dreyfuss Commodities Rod.Armando Salles Bebedouro, 

14707900 Brazil paulo.paiva@ldcom.com 


Sao Paulo 

Dr. Raju Pandey University of California, Riverside Department of Entomology Riverside CA 92521 USA pandeyr@ucr.edu 

900 University Ave 

Dr. Venkatesan UF/IFAS CREC 700 Experiment Station Rd Lake Alfred FL 33850 USA venki@ufl.edu 

Parkunan 


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Dr. Stephen Parnell Rothamsted Research West Common Harpenden, 

AL5 2JQ United stephen.parnell@bbsrc.ac.uk 

Hertfordshire 

Kingdom 

Dr. Larry Parsons UF/IFAS CREC 700 Experiment Station Rd Lake Alfred FL 33850 USA lparsons@ufl.edu 

Mr. Minesh Patel Dixie Farms, LLC 5 Hill Ave Orlando FL 32801 USA min@beaconlandscaping.com 

Mrs. Sai Patne University of California, Riverside 900 University Ave Riverside CA 92521 USA spatn001@ucr.edu 

Dr. Joseph Patt USDA ARS 2413 E Highway 83 Weslaco TX 78596 USA Joseph.Patt@ars.usda.gov 

Ms. Cristina Paul USDA ARS MPPL Citrus Quarantine Unit Bldg Beltsville MD 20705 USA cristina.paul@ars.usda.gov 

004 Rm 118 

Mr. James Paul Gulf Coast Citrus PO Box 7369 Naples FL 34101-7369 USA 

Mr. Jay Pearson Sinensis, Inc. PO Box 2275 Winter Haven FL 33883-2275 USA jay@ejpearsonjr.com 

Mr. Nathan Pegram Intellectual Ventures Laboratory 1600 132nd Ave NE Ste 100 Bellevue WA 98005 USA npegram@intven.com 

Mr. Doug Peltzer California Citrus Mutual 512 N Kaweah Ave Exeter CA 93221-1200 USA 

Mr. Tim Peltzer California Citrus Mutual 512 N Kaweah Ave Exeter CA 93221-1200 USA 

Dr. Kirsten Pelz- UF/IFAS CREC 700 Experiment Station Rd Lake Alfred FL 33850 USA pelzstelinski@ufl.edu 

Stelinski 

Mr. Ezequiel Perez Consorcio Citricola del Este Carratera Hato Mayor Hato Mayor 25000 Dominican eperez@citricola.com 


Republic 

Mr. Federico Perez Comite Estatal de Sanidad Vegetal de Circunvalacion Agustin Guadalajara, 

44150 Mexico psv@jal.sagarpa.gob.mx 

Mejia 

Jalisco, A.C. 

Yanez No. 2175, Col Barrera Jalisco 

Ms. Edith Pettis Pettis Groves PO Box 2539 Wauchula FL 33873 USA epettis06@hotmail.com 

Dr. Robert 

USDA ARS 2413 E Highway 83 Weslaco TX 78596 USA Bob.Pfannenstiel@ars.usda.gov 

Pfannenstiel 

Mr. Steve Pfleger Tropicana Products, Inc. PO Box 338 Bradenton FL 34206-0338 USA 

Mr. J. Don Phillips Certi-Fine Fruit Company, Inc. 125 Terra Mango Loop Ste Orlando FL 32835 USA cff@cfl.rr.com 

A 

Mr. Floyd Philmon Philmon*Floyd 13456 Happy Hill Rd Dade City FL 33525-8125 USA 

Mr. Mark Philmon Philmon*Mark A 18034 Clay Hill Rd Dade City FL 33523-6441 USA 

Mr. Francisco Pierri Citrus Consultants Group - GCONCI Rua Carlos Gomes, 144 - Cordeiropolis, 

13490000 Brazil fpn14@terra.com.br 

Neto 


Sao Paulo 

Dr. Gerhard Pietersen ARC - Plant Protection Research Private Bag X134 Pretoria 0121 South Africa gerhard.pietersen@up.ac.za 

Institute 

Mr. Andy Pike Sun Ray Farms 2400 Old State Road 8 Lake Placid FL 33852-9591 USA 

Mr. Ernesto Luiz Citrus Consultants Group - GCONCI Rua Carlos Gomes, 144 - Cordeiropolis, 

13490000 Brazil elpa@btu.flash.tv.br 

Pires de Almeida 


Sao Paulo 

Mr. Shane Platt Farm Credit PO Box 8009 Lakeland FL 33802-8009 USA splatt@farmcreditcfl.com 

Dr. Anne Plotto USDA ARS Citrus & Subtropical 600 Avenue S, NW Winter Haven FL 33881 USA Anne.Plotto@ars.usda.gov 

Products Lab 

Dr. MaryLou Polek California Citrus Research Board PO Box 230 Visalia CA 93279-0230 USA marylou@citrusresearch.org 

Dr. Gavin Poole USDA ARS USHRL 2001 S Rock Rd Fort Pierce FL 34945 USA gavin.poole@ars.usda.gov 

Dr. Elena Postnikova USDA 1301 Ditto Ave Fort Detrick MD 21702 USA elena.postnikova@ars.usda.gov 

Dr. Charles Powell University of Florida 2199 S Rock Rd Fort Pierce FL 34945-3138 USA CApowell@ufl.edu 


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Mr. Ray Prewett Texas Citrus Mutual 901 Business Park Dr Ste Mission TX 78572 USA ray@valleyag.org 

400 

Mr. Pickens Price Price Groves PO Box 1165 Dade City FL 33526-1165 USA pricedevco@embarqmail.com 

Dr. Humberto Puello Consorcio Citricola del Este Carratera Hato Mayor Hato Mayor 25000 Dominican chpuello@yahoo.com; 


Republic hpuello@citricola.com 

Dr. Jawwad Qureshi University of Florida/IFAS SWFREC 2685 State Road 29 N Immokalee FL 34142 USA jawwadq@ufl.edu 

Mrs. Jacqueline SA San Miguel Lavalle 4001 San Miguel, 

4000 Argentina jramallo@sa-sanmiguel.com 

Ramallo 

Tucuman 

Mr. Brian Randolph IMG Citrus, Inc. 2600 45th St Vero Beach FL 32967-1384 USA brianr@imgcitrus.com; 

brian.randolph@imgcitrus.com 

Mr. Enrique Rangel USDA APHIS IS Carr Reynosa-Matamoros Reynosa, Tam 88501 Mexico enrique.rangel@aphis.usda.gov 


Dr. Jose Reyes UF/IFAS CREC 700 Experiment Station Rd Lake Alfred FL 33850 USA jireyes@ufl.edu 

Mr. Taw Richardson AgroSource, Inc. PO Box 1341 Mountainside NJ 07092-0341 USA taw.richardson@agrosource.net 

Mr. Steve Riffle Tropicana Products, Inc. PO Box 338 Bradenton FL 34206-0338 USA 

Mr. Timothy Riley USDA APHIS PPQ 10806 Palmbay Dr. Orlando FL 32824 USA timothy.riley@aphis.usda.gov 

Dr. Mark Ritenour UF - Indian River REC 2199 S Rock Rd Fort Pierce FL 34945 USA ritenour@ufl.edu 

Dr. Paul Robbins USDA ARS 2001 S Rock Rd Fort Pierce FL 34945-3030 USA paul.robbins@ars.usda.gov 

Mr. David Roberts California Citrus Mutual 512 N Kaweah Ave Exeter CA 93221-1200 USA ccm@cacitrusmutual.com 

Dr. Pamela Roberts UF/IFAS Southwest Fla. Research & 2686 SR 29 N Immokalee FL 34142 USA pdr@ufl.edu 

Education Center 

Mr. Thomas Roberts Integrated Consulting Entomology 120 Stanislaus Ave Ventura CA 93004 USA troberts@dock.net 

Ms. Cecile Robertson UF/IFAS CREC 700 Experiment Station Rd Lake Alfred FL 33850 USA 

Mr. Daniel Robl USDA 1833 57th St Sarasota FL 34243 USA daniel.robl@aphis.usda.gov 

Dr. Pedro Robles SENASICA 

Guillermo Perez Valenzuela Col. Del 

04100 Mexico pedro.robles@senasica.gob.mx 

Garcia 

127 

Carmen,Coyo 

acan 

Mr. Hamilton Rocha Citrus Consultants Group - GCONCI Rua Carlos Gomes, 144 - Cordeiropolis, 

13490000 Brazil c.dalfre@uol.com.br 


Sao Paulo 

Dr. Mario Rocha- Universidad Autonoma de Nuevo Cd. Universitaria 

Monterrey, 

66450 Mexico mrocha@fcb.uanl.mx; 

Peña 

Leon 

Nuevo Leon 

mario.rochape@uanl.edu.mx 

Mr. Jason Rogers J. B. Rogers Citrus, Inc. 9149 Lake Lynn Dr. Sebring FL 33876 USA agrogers5@gmail.com 

Dr. Michael Rogers UF/IFAS CREC 700 Experiment Station Rd Lake Alfred FL 33850 USA mrgrs@ufl.edu 

Dr. Steve Rogers Citrus Research and Development 700 Experiment Station Rd Lake Alfred FL 33850-2299 USA steve@stever.com 


Dr. Mikeal Roose University of California, Riverside Department of Botany & Riverside CA 92521-0001 USA mikeal.roose@ucr.edu 

Plant Sciences 

Mr. Sidney Marcos Citrus Consultants Group - GCONCI Rua Carlos Gomes, 144 - Cordeiropolis, 

13490000 Brazil s.mrosa@uol.com.br 

Rosa 


Sao Paulo 

Mr. Richard Rosario University of Florida PO Box 110700 Gainesville FL 32611 USA richardrosariopa@ufl.edu 

Mr. David Roth California Citrus Mutual 512 N Kaweah Ave Exeter CA 93221-1200 USA 

Dr. Robert Rouse University of Florida 2686 State Rd 29 N Immokalee FL 34142-9514 USA rrouse@ufl.edu 


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Dr. Chuanqing Ruan Fujian Academy of Agricultural Research Center for High Fuzhou, 

350003 PR China ruanchuanqing@163.com 

Sciences 

Technology of Ag Sciences Fujian 


Mr. Phillip Rucks Phillip Rucks Citrus Nursery, Inc. PO Box 1318 Frostproof FL 33843 USA rucksnursery@aol.com 

Mr. Nick Russakis Russakis Groves 8801 Indrio Rd Fort Pierce FL 34951-1615 USA jimgrussakis@aol.com 

Mr. Paul Russell Agdia, Inc. 30380 County Rd 6 Elkhart IN 46514 USA prussell@agdia.com 

Mr. Phillip 

Intellectual Ventures Laboratory 1600 132nd Ave NE Ste 100 Bellevue WA 98005 USA prutschman@intven.com 

Rutschman 

Mr. Earl Rutz California Citrus Research Board PO Box 230 Visalia CA 93279-0230 USA earlrutz1@mac.com 

Dr. Surya Saha Cornell University 334 Plant Science Ithaca NY 14850 USA ss2489@cornell.edu 

Dr. Bacilio Salas USDA APHIS Moore Air base Bldg-6414 Edinburg TX 78541 USA bacilio.salas@aphis.usda.gov 

22675 N Moorefield Rd 

Mr. Antonio Celso Citrus Consultants Group - GCONCI Rua Carlos Gomes, 144 - Cordeiropolis, 

13490000 Brazil acsanches@terra.com.br 

Sanches 


Sao Paulo 

Mr. Sergio Sanchez USDA APHIS IS Carr Reynosa-Matamoros Reynosa, Tam 88501 Mexico sergio.sanchez@aphis.usda.gov 

Mr. Hector Sanchez 

Anguiano 

SENASICA 


Guillermo Perez Valenzuela 

127 

Col. Del 

Carmen,Coyo 

acan 

04100 Mexico hector.sanchez@senasica.gob.mx 

Bebedouro, 

14711-114 Brazil mrsantos@jfcitrus.com.br 

Sao Paulo 

Orlando FL 32826 USA ssantra@mail.ucf.edu 

Mr. Francisco Santos JF Citrus Agropecuaria Ltda Rua Cel. Candido Procopio 

De Oliveira, 353 

Dr. Swadeshmukul NanoScience Technology Center, 12424 Research Parkway Ste 

Santra 

UCF 

400 

Mr. Greg Sapp Sapp*Greg PO Box 1783 Lake Placid FL 33862-1783 USA sappbros@embarqmail.com 

Mr. Dyson Schneider California Citrus Mutual 512 N Kaweah Ave Exeter CA 93221-1200 USA 

Mr. Keith Schorsch Intellectual Ventures Laboratory 1600 132nd Ave NE Ste 100 Bellevue WA 98005 USA nsmith@intven.com 

Dr. Tim Schubert Florida Department of Ag & 

1911 SW 34th St Gainesville FL 32614 USA Tim.Schubert@freshfromflorida.co 

Consumer Services 

m 

Ms. Rhonda 

UF/IFAS CREC 700 Experiment Station Rd Lake Alfred FL 33850 USA rschuman@ufl.edu 

Schumann 

Mr. Don Seaver USDA APHIS PPQ CPHST 1730 Varisty Dr Raleigh NC 27606 USA donald.m.seaver@aphis.usda.gov 

Miss Rosana 

University of Florida 700 Experiment Station Rd Lake Alfred FL 33850 USA rserikawa@ufl.edu 

Serikawa 

Dr. Mamoudou Texas A&M University - Kingsville, 312 N International Blvd Weslaco TX 78596 USA msetamou@ag.tamu.edu 

Setamou 

Citrus Center 

Mr. Kevin Severns California Citrus Mutual 512 N Kaweah Ave Exeter CA 93221-1200 USA 

Mr. Randy Sexton Sexton Grove Service 4325 17th St SW Vero Beach FL 32968-5912 USA sextongrov@aol.com 

Mr. Jonathan Shao USDA ARS Molecular Plant 

10300 Baltimore Ave Beltsville MD 20705-2350 USA jonathan.shao@ars.usda.gov 

Pathology Lab 

Dr. Robert Shatters USDA ARS USHRL 2001 S Rock Rd Fort Pierce FL 34945 USA robert.shatters@ars.usda.gov 

Mrs. Shannon Shepp FDACS - Division of Fruit & 

PO Box 1072 Winter Haven FL 33882-1072 USA Shannon.Shepp@freshfromflorida. 

Vegetables 

com 


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Mr. Joby Sherrod Duda PO Box 788 LaBelle FL 33975 USA joby.sherrod@duda.com 

Mr. Turksen Shilts UF/IFAS CREC 700 Experiment Station Rd Lake Alfred FL 33850 USA 

Mr. Jose Javier Agricultural Integrated Solutions 6505 SW 113th Ct Miami FL 33173 USA jtomas@aiscosolutions.com 

Siguenza 

USA 

Mr. Flavio Silva JF Citrus Agropecuaria Ltda Rua Cel. Candido Procopio Bebedouro, 

14711-114 Brazil jugsalvo@gmail.com 

De Oliveira, 353 

Sao Paulo 

Mr. Wayne Simmons Simmons*Wayne H 1600 Hwy 29 S LaBelle FL 33935 USA whs58@yahoo.com 

Mr. Rocco Simonetta PepsiCo 617 W Main St Barrington IL 60010 USA rocco.simonetta@pepsico.com 

Mr. Oscar Augusto Citrus Consultants Group - GCONCI Rua Carlos Gomes, 144 - Cordeiropolis, 

13490000 Brazil oscar.simonetti@yahoo.com.br 

Simonetti 


Sao Paulo 

Dr. Trevor Smith FDACS - Division of Plant Industry PO Box 147100 Gainesville FL 32614-7100 USA trevor.smith@freshfromflorida.co 

m 

Mr. James Snively Southern Gardens Citrus 1820 County Road 833 Clewiston FL 33440 USA jsnively@southerngardens.com 

Mr. Armando Solorio Comite Estatal de Sanidad Vegetal de Circunvalacion Agustin Guadalajara, 

44150 Mexico sagarpa_jalisco@yahoo.com.mx 

Hernandez 

Jalisco, A.C. 

Yanez No. 2175, Col Barrera Jalisco 

Mr. Carl Sons Sons*Carl 32 Bass St Haines City FL 33844-9612 USA 

Dr. Timothy Spann UF/IFAS CREC 700 Experiment Station Rd Lake Alfred FL 33850 USA spann@ufl.edu 

Mr. Mike Sparks Florida Citrus Mutual PO Box 89 Lakeland FL 33802 USA mikes@flcitrusmutual.com 

Mr. Bob Stambaugh Cooperative Ventures LLC PO Box 9498 Winter Haven FL 33883-9498 USA bobstambaugh@winterhavenlaw.c 

om 

Dr. Richard Stange USDA ARS USHRL 2001 S Rock Rd Fort Pierce FL 34945 USA 

Dr. Philip Stansly University of Florida 2685 State Road 29 N Immokalee FL 34142 USA pstansly@ufl.edu 

Mrs. Beatriz Stein Estacion Experimental Agroindustrial Avda William Cross 3150 Las Talitas, 

4101 Argentina bstein@sinectis.com.br 

Obispo Col. 

Tucuman 

Dr. Lukasz Stelinski UF/IFAS CREC 700 Experiment Station Rd Lake Alfred FL 33850 USA stelinski@ufl.edu 

Ms. Denise 

Rabo AgriFinance, Inc. 

6704 Professional Parkway Sarasota FL 34240 USA denise.stembridge@raboag.com 

Stembridge 

W 

Dr. Neusa Stenzel IAPAR - Instituto Agronomico do Rod. Celso Garcia Cid, km Londrina, PR 86001-970 Brazil nstenzel@iapar.br 

Parana 

375 

Mr. Mike Stewart Consolidated Citrus LP 63 Barn Rd Venus FL 33960 USA mstewart@cclpcitrus.com 

Mr. Thomas Stopyra The Packers of Indian River, Ltd. PO Box 12969 Fort Pierce FL 34979-2969 USA tstopyra@packerscitrus.com 

Mrs. Vera Stopyra Gresham Enterprises LLC 4 Bogey Dr Winter Haven FL 33881 USA 

Mr. Matt Story Story*Mathew PO Box 1221 Lake Wales FL 33859-1221 USA matt@storycompanies.com 

Mr. Paul Story California Citrus Mutual 512 N Kaweah Ave Exeter CA 93221-1200 USA 

Mr. Vic Story Story Groves, Inc. PO Box 857 Babson Park FL 33827-0857 USA vic@storycompanies.com; 

storycitrus@aol.com 

Dr. Ed Stover USDA ARS USHRL 2001 S Rock Rd Fort Pierce FL 34945 USA ed.stover@ars.usda.gov 

Mr. John Strang Gapway Grove Corporation PO Box 1364 Auburndale FL 33823-1364 USA john@gapwaygroves.com 

Mr. Frank Strazzulla Citrosuco North America, Inc. 5937 Highway 60 E Lake Wales FL 33898 USA fstrazzulla@citrosuco.com 

Dr. Eduardo Stuchi Embrapa Cassava & Tropical Fruits PO Box 74 Bebedouro, 

14700-970 Brazil stuchi@estacaoexperimental.com.b 

SP 

r 

Mr. Keith Sullivan Consolidated Citrus LP 63 Barn Rd Venus FL 33960 USA ksullivan@cclpcitrus.com 


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Dr. Xiaoan Sun FDACS - Division of Plant Industry PO Box 147100 Gainesville FL 32614-7100 USA Xiaoan.Sun@freshfromflorida.com 

Dr. Jim Syvertsen UF/IFAS CREC 700 Experiment Station Rd Lake Alfred FL 33850 USA jmsn@ufl.edu 

Dr. Alexandre Fischer S/A - Comercio Industria e Sao Lourenco St #81 Matao, Sao 

15990-200 Brazil atachibana@citrosuco.com.br 

Tachibana 

Agricultura 

Paulo 

Mr. Brian Taylor California Citrus Research Board PO Box 230 Visalia CA 93279-0230 USA brian@citrusresearch.org 

Mr. David Taylor Trade Winds Citrus Limited Bog Walk Saint 

5555 Jamaica leon.stewart@tradewindscitrus.com 

Catherine 

Dr. Earl Taylor USDA ARS USHRL 2001 S Rock Rd Fort Pierce FL 34945-3030 USA earl.taylor@ars.usda.gov 

Mr. John Taylor Syngenta Crop Protection 110 Dory Rd N North Palm FL 33408 USA john.taylor@syngenta.com 

Beach 

Miss Stephanie Shea University of Florida - SFREC 2685 State Road 29 N Immokalee FL 34142 USA ssteems@ufl.edu 

Teems 

Mr. Bruce Templeton Templeton*Bruce 3948 Templeton Rd Lake Wales FL 33853-8891 USA arcoco@yahoo.com 

Mr. Jose Eduardo Citrus Consultants Group - GCONCI Rua Carlos Gomes, 144 - Cordeiropolis, 

13490000 Brazil jeteofilo@vivax.com.br 

Teofilo 


Sao Paulo 

Mr. Len Therrien USDA ARS USHRL 2001 S Rock Rd Fort Pierce FL 34945-3030 USA leonard.therrien@ars.usda.gov 

Dr. Donald Thomas USDA ARS 2413 East Highway 83 Weslaco TX 78596 USA donald.thomas@ars.usda.gov 

Dr. Lavern Timmer University of Florida 700 Experiment Station Rd Lake Alfred FL 33850 USA lwtimmer@ufl.edu; 

lwt@crec.ifas.ufl.edu 

Dr. Siddharth Tiwari University of Florida 700 Experiment Station Rd Lake Alfred FL 33850 USA stiwari@ufl.edu 

Mr. Jose Tomas Agricultural Integrated Solutions 6505 SW 113th Ct Miami FL 33173 USA jtomas@aiscosolutions.com 

USA 

Mr. Gilberto Tozatti Citrus Consultants Group - GCONCI Rua Carlos Gomes, 144 - Cordeiropolis, 

13490000 Brazil tozatti@gconci.com.br; 


Sao Paulo 

gilberto.tozatti@hotmail.com 

Mr. Chris Troesch Simpson Fruit Company 445 Limit Ave Mount Dora FL 32757-2999 USA 

Mr. Alex Truszkowski DuPont Crop Protection 3680 Winding Lake Cir Orlando FL 32835 USA alex.t.truszkowski@usa.dupont.co 

m 

Dr. Thomas Turpen Citrus Research and Development 700 Experiment Station Rd Lake Alfred FL 33850-2299 USA tom.turpen@innovationmatters.co 


m 

Mr. John Updike Updike Jr.*John PO Box 231 Lake Wales FL 33859-0231 USA jupdike@alcoma.net 

Dr. Filomena Valim Florida Department of Citrus 700 Experiment Station Rd Lake Alfred FL 33850 USA fvalim@citrus.state.fl.us 

Dr. Ariena H.C. van Emerging Pathogens Institute and 1453 Fifield Hall Gainesville FL 32611 USA ahcvanbruggen@ufl.edu 

Bruggen 

Plant Pathology 

Dr. Frank van den Rothamsted Research West Common Harpenden, 

AL5 2JQ United frank.vandenbosch@bbsrc.ac.uk 

Bosch 

Hertfordshire 

Kingdom 

Ms. April Vega UF/IFAS CREC 700 Experiment Station Rd Lake Alfred FL 33850 USA aprilv26@ufl.edu 

Dr. Jose Velazquez INIFAP Km. 35 Carretera Colima Tecoman, 

28100 Mexico jvelazquezmon@yahoo.com.mx 

Manzanillo 

Colima 

Dr. Georgios 

University of California, Riverside Department of Plant Riverside CA 92521 USA vidalg@ucr.edu 

Vidalakis 

Pathology & Microbiology 

Mr. Ricardo Violante Cutrale Citrus Juices 602 McKean St Auburndale FL 33823 USA violante@cutrale.com.br 


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Ms. Ashley Voss USDA ARS USHRL 2001 S Rock Rd Fort Pierce FL 34945-3030 USA ford.ashley@gmail.com; 

Ashley.Voss@ars.usda.gov 

Ms. Callie Walker Pavese Law Firm PO Drawer 1507 Fort Myers FL 33902 USA cwalker@oldecypressbank.com 

Dr. Abigail Walter USDA ARS USHRL 2001 S Rock Rd Fort Pierce FL 34945-3030 USA abigail.walter@ars.usda.gov 

Mr. Nan-Yi Wang UF/IFAS CREC 700 Experiment Station Rd Lake Alfred FL 33850 USA nywang@ufl.edu 

Dr. Nian Wang UF/IFAS CREC 700 Experiment Station Rd Lake Alfred FL 33850 USA nianwang@ufl.edu 

Mr. Xuefeng Wang USDA ARS 9611 S Riverbend Ave Parlier CA 93648 USA 

Mr. Malcolm C Watters*Malcolm C 2220 County Road 17 N Lake Placid FL 33852 USA malwatters@htn.net 

Watters 

Mr. Tripp Watters Watters*Malcolm C 2220 County Road 17 N Lake Placid FL 33852 USA 

Mr. Jed Weeks Crop Production Services, Inc. 2100 Moores Ln Mulberry FL 33860 USA jed.weeks@cpsagu.com 

Dr. Shawron 

Orange - Co, LP 12010 NE Hwy 70 Arcadia FL 34266-4267 USA sweingarten@orangecofla.com 

Weingarten 

Mr. David Wheeler Wheeler Farms, Inc. PO Box 2715 Lake Placid FL 33862-2715 USA wheelerfarms@earthlink.net 

Mr. Mitch Willis PepsiCo 1001 13th Ave E Bradenton FL 34208 USA mitch.willis@pepsico.com 

Dr. Gail Wisler USDA ARS 5601 Sunnyside Ave Beltsville MD 20705 USA Gail.Wisler@ARS.USDA.GOV 

Dr. Glenn Wright University of Arizona - Yuma 6425 W 8th St Yuma AZ 85364 USA gwright@ag.arizona.edu 

Agriculture Center 

Dr. Nelson A. Wulff Fundecitrus Av. Dr. Adhemar Pereira de Arraraquara, 

14807-040 Brazil nelsonwulff@fundecitrus.com.br 

Barros 201 

Sao Paulo 

Dr. Yulu Xia North Carolina State University 1730 Varsity Dr Ste 110 Raleigh NC 27606 USA yulu_xia@ncsu.edu 

Miss Pan Xie 

Chongqing University, 

Genetic Engineering 

Chongqing 400044 PR China xpdd840605@163.com 

Bioengineering College 

Research Center No. 174 

Shazhengjie Dist. 

Dr. Pedro Yamamoto University of Sao Paulo - ESALQ Av. Padua Dias, 11 Piracicaba, 

13418900 Mexico pedro.yamamoto@usp.br 

Sao Paulo 

Dr. Gal Yarden Beeologics 11800 SW 77th Ave Miami FL 33156 USA gal@beeologics.com 

Miss Jamie Yates UF/IFAS CREC 700 Experiment Station Rd Lake Alfred FL 33850 USA jdyates@ufl.edu 

Dr. Raymond Yokomi USDA ARS 9611 S Riverbend Ave Parlier CA 93648 USA ray.yokomi@ars.usda.gov 

Ms. Cassie Young UF/IFAS IRREC 2199 S Rock Rd Fort Pierce FL 34945 USA 

Dr. Qibin Yu UF/IFAS CREC 700 Experiment Station Rd Lake Alfred FL 33850 USA qibin@crec.ifas.ufl.edu; 

qibin@ufl.edu 

Dr. Qing Yuan USDA ARS Molecular Plant 

10300 Baltimore Ave Beltsville MD 20705-2350 USA qing.yuan@ars.usda.gov 

Pathology Lab 

Mr. Kazuyoshi Yuasa Japan International Cooperation 16th Floor, Daeha Business Hanoi 100000 Viet Nam Kazuyoshi.Yuasa@gmail.com 

Agency 

Center, 360 Kim Ma 

Dr. Cecilia Zapata UF/IFAS CREC 700 Experiment Station Rd Lake Alfred FL 33850 USA ceciliaz@ufl.edu 

Mr. Jorge Zatarain COMITE ESTATAL VEGETAL DE PROL. MIGUEL HIDALGO SAN PEDRO 

72160 MEXICO zatarainestolano@hotmail.com 

Estalano 

SANIDAD DE PUEBLA 

No. 2107 

CHOLULA,P 

UEBLA 

Dr. Mongi Zekri University of Florida Extension PO Box 68 LaBelle FL 33975 USA maz@ufl.edu 

Dr. Jiuxu Zhang Florida Department of Citrus 700 Experiment Station Rd Lake Alfred FL 33850 USA jzhang@citrus.state.fl.us 


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Dr. Muqing Zhang UF/IFAS IRREC 2199 S Rock Rd Fort Pierce FL 34945 USA mqzhang@ufl.edu 

Dr. Shujian Zhang University of Florida Plant Pathology Department Gainesville FL 32611 USA sjzhang@ufl.edu 

1453 Fifield Hall 

Dr. Lijuan Zhou USDA ARS USHRL 2001 S Rock Rd Fort Pierce FL 34945 USA lijuan.zhou@ars.usda.gov 


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A3. Meeting Agenda 

2 nd International Research Conference on 

Huanglongbing 

January 10-14, 2011 

Agenda 

Day 1: Monday, 10 January 2011 

7:00 am – 9:00 pm Registration (Foyer) 

7:00 – 8:00 am Continental Breakfast (tour attendees only) (Foyer) 

8:15 am – 4:30 pm Preconference Tour (by reservation only, tour sold out) (Departs from Foyer) 

7:00 – 9:00 pm Welcome Reception (Boca Foyer and Boca Patio) 

Day 2: Tuesday, 11 January 2011 


7:30 – 8:30 am Continental Breakfast (Foyer) 

8:30 – 9:15 am Welcome (Caribbean Ballroom IV and V) 

Introduction – Mike Sparks, Jim Graham 

Mission, Goals, and Objectives – Wayne Dixon 

Rules of the House – Jackie Burns 

9:15 – 10:15 am Opening Keynote Address (Caribbean Ballroom IV and V) 

HLB – A Clear and Present Danger: An Epidemiological Perspective – Tim 

Gottwald 

10:15 – 10:30 am Break (Foyer) 


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10:30 am – 12:00 pm Session 1 (Caribbean Ballroom IV and V) 

Pathogen Genomics, Bioinformatics, Phylogenetics, and Culturing – John 

Hartung, Moderator 

Oral Presentations 

10:30 1.1 Genomic Comparisons of the Ca. Liberibacter asiaticus Chromosome with 

Other Members of the Rhizobiales – Hartung, J.S., Shao, J., Kuykendall, L.D. 

10:45 1.2 Genetic and Functional Characterization of the znu Operon in the 

Intracellular Citrus Pathogen, Candidatus Liberibacter asiaticus – Vahling, 

C.M., Benyon, L.S., Duan, Y.-P. 

11:00 1.3 Comparison of the Ca. Liberibacter asiaticus Genome with a Draft Ca. L. 

americanus Genome Reveals Similar Prophage with Likely Pathogenicity 

Factors – Zhang, S.J., Wulff, N.A., Flores-Cruz, Z., Zhou, L.J., Kang, B.-K., 

Fleites, L.A., Gooch, M.D., Davis, M.J., Duan, Y.-P., Gabriel, D.W. 

11:15 1.4 Analysis of Candidatus Liberibacter americanus Genome – Wulff, N.A., Zhang, 

S., Ayres, A.J., Bové, J.M., Gabriel, D.W. 

11:30 1.5 Population Genetics Analysis of Candidatus Liberibacter asiaticus from 

Multiple Continents – Glynn, J.M., Bai, Y., Chen, C., Duan, Y.-P., Civerolo, E.L., 

Lin, H. 

11:45 1.6 Phylogenetic Analysis of Asian Candidatus Liberibacter asiaticus; Asian 

Common Strains Are Distributed in Northeast India, Papua New Guinea, and 

Timor-Leste – Miyata, S., Kato, H., Tomimura, K., Davis, R., Smith, M.W., 

Weinert, M., Iwanami, T. 

Posters 

1.7 Bioinformatic Analysis of Genome Sequence Data for Ca. Liberibacter 

asiaticus – Lindeberg, M., Saha, S. 

1.8 Genetic Diversity of Candidatus Liberibacter asiaticus Isolates from Paraná 

State, Brazil – Meneguim, L., Marques, V.V., Murata, M.M., Barreto, T.P., 

Vasquez-Souza, G., Villas-Boas, L.A., Paccola-Meirelles, L.D., Leite, R.P., Jr. 

1.9 Analysis of Endophytic Bacterial Diversity from Huanglongbing Pathogen- 

Infected Citrus Tissues – Wang, A., Yin, Y., Li, Y., Li, J., Xian, J., Wang, Z. 

1.10 Evolving Diversity of Candidatus Liberibacter asiaticus Revealed by 

Comparative Analysis of Two Intragenic Tandem Repeat Genes – Zhou, L.J., 

Powell, C.A., Hoffman, M., Li, W.B., Fan, G.-C., Liu, B., Duan, Y.-P. 

1.11 In vitro Culture of the Fastidious Bacteria Candidatus Liberibacter asiaticus 

in Association with Insect Feeder Cells – Fontaine-Bodin, L., Fabre, S., 

Gatineau, F., Dollet, M. 

1.12 Preliminary Report of Cultivation of Candidatus Liberibacter asiaticus from 

Citrus Tissue with Huanglongbing – Xie, P., Yin, Y., Li, Y., Li, J., Wang, Z. 

1.13 Characterization of Highly Mosaic Genomic Loci of Candidatus Liberibacter 

asiaticus in Southern China and Florida – Wang, X.F., Zhou, C.Y., Deng, X., 

Su, H.N., Chen, J. 

1.14 Further Evidence That U.S. and Chinese Populations of Candidatus 

Liberibacter asiaticus are Different – Deng, X., Liu, R., Zhang, P., Chen, J. 


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12:00 – 1:30 pm Lunch and Keynote Lecture 1 (Caribbean Ballroom VI and VII) 

RNAi Strategies for Insect Vectors of Plant Pathogens – Bryce Falk 

1:30 – 2:30 pm Session 2 (Caribbean Ballroom IV and V) 

Asian Citrus Psyllid Biology and Genomics – David Hall, Moderator 


1:30 2.1 Phylogeographic and Population Genetic Studies Uncover Two Founding 

Events in Asian Citrus Psyllid Populations Collected in the Americas – de 

León, J.H., Sétamou, M., Gastaminza, G.A., Buenahora, J., Cáceres, S., 

Yamamoto, P.T., Logarzo, G.A., Stañgret, C.R.W. 

1:45 2.2 Alteration of Microbiome of Bactericera cockerelli and Diaphorina citri Based 

on Candidatus Liberibacter sp. Infection – Hail, D., Hunter, W., Bextine, B. 

2:00 2.3 Oral Uptake of dsRNA Increases Mortality in Diet Fed Psyllids – Shatters, 

R.G., Jr., Powell, C.A., Borovsky, D. 

2:15 2.4 The Psyllid Feeding Process: Composition and Biosynthetic Inhibition of the 

Salivary Sheath – Shatters, R.G., Jr. 

Posters 

2.5 A New Method for Short-Term Rearing of Psyllid Adults and Nymphs on 

Detached Citrus Leaves and Young Terminal Shoots – Ammar, E.-D., Hall, 

D.G. 

2.6 Comparative Analysis of Asian Citrus Psyllid and Potato Psyllid Antennae 

– Arras, J., Hunter, W., Bextine, B. 

2.7 The Emerging Psyllid Genome: RNA-Interference and Insect Biology – 

Hunter, W.B., Bextine, B.R., Shatters, R.G., Reese, J., Shelby, K.S., Hall, D.G. 

2.8 Bacterial Population Diversity in Diaphorina citri: Analysis by PCR-DGGE 

and RFLP Methodology – Wang, Z., Tian, S., Liu, T., Yin, Y. 

2:30 – 2:45 pm Break (Foyer) 


Asian Citrus Psyllid Ecology and Transmission – Lukasz Stelinski, 

Moderator 


2:45 3.1 Antennal Responses of Diaphorina citri to Host Plant Volatiles Recorded 

Using a Coupled Gas Chromatograph Electroantennogram Detector 

System – Robbins, P.S., Alessandro, R.T., Lapointe, S.L. 

3:00 3.2 Population Dynamics of the Asian Citrus Psyllid and Potential 

Generations in Northern Sinaloa, Mexico – Cortez-Mondaca, E., López- 

Arroyo, J.I., Pérez-Márquez, J., González, V.M. 

3:15 3.3 Localization of Candidatus Liberibacter asiaticus in Dissected Organs of 

Its Psyllid Vector Diaphorina citri Using Fluorescent in situ Hybridization 

and Quantitative PCR – Ammar, E.-D., Shatters, R.G., Hall, D.G. 

3:30 3.4 Interactions of the Asian Citrus Psyllid, Diaphorina citri, with Candidatus 

Liberibacter asiaticus – Pelz-Stelinski, K.S., Rogers, M.E. 


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3:45 3.5 Seasonal Changes in Numbers of Asian Citrus Psyllids Carrying 

Candidatus Liberibacter asiaticus – Ebert, T.A., Brlansky, R.H., Rogers, 

M.E. 

4:00 3.6 standing Diaphorina citri-Candidatus Liberibacter asiaticus Interactions 

and D. citri Behavior for Managing Huanglongbing (HLB) in Florida – 

Mann, R.S., Pelz-Stelinski, K.S., Rouseff, R.L., Stelinski, L.L. 

4:15 3.7 Effects of Soil-Applied and Foliar-Applied Insecticides on Asian Citrus 

Psyllid (Diaphorina citri) Feeding Behavior and Their Possible Implication 

for HLB Transmission – Serikawa, R.H., Okuma, D.M., Backus, E.A., Rogers, 

M.E. 

4:30 3.8 Effect of Insecticides and Mineral Oil on Probing Behavior of Diaphorina 

citri Kuwayama (Hemiptera: Psyllidae) in Citrus – de Miranda, M.P., 

Felippe, M.R., Garcia, R.B., Yamamoto, P.T., Lopes, J.R.S. 

Posters 

3.9 A New Detached-Leaf Assay Method to Test the Inoculativity of Psyllids 

with Candidatus Liberibacter asiaticus Associated with Huanglongbing 

Disease – Ammar, E.-D., Walter, A., Hall, D.G. 

3.10 Preliminary Study of Comparative Acquisition of Candidatus Liberibacter 

asiaticus and Ca. L. americanus by Diaphorina citri Under Different 

Temperatures – Barbosa, J.C., Eckstein, B., Belasque, J., Jr., Bergamin 

Filho, A. 

3.11 Host Range of Diaphorina citri Kuwayama and Leuronota fagarae on 

Citrus and Zanthoxylum spp. – Russell, D.N., Halbert, S.E., Roberts, P.D. 

3.12 Abundance of Diaphorina citri (Hemiptera: Psyllidae) in Orange Jasmine 

and Backyard Citrus of Yucatán, Mexico – Lozano-Contreras, M., Jasso- 

Argumedo, J., Morales-Koyoc, D., Jasso-Laucirica, T., González-Hernández, 

A., López-Arroyo, J.I. 

3.13 Difference of Gender and Effect of Photoperiod on Asian Citrus Psyllid 

Feeding Behavior – Okuma, D.M., Serikawa, R.H., Rogers, M.E. 

3.14 Seasonal Abundance of Diaphorina citri (Hemiptera: Psyllidae) and 

Natural Enemies in Citrus Groves of Yucatán, Mexico – Jasso-Argumedo, 

J., Lozano-Contreras, M., Barroso-Aké, H., López-Arroyo, J.I. 

3.15 Host Plants of Psyllids in South Texas – Thomas, D.B. 

4:45 – 6:00 pm Poster Session 1 (Caribbean Ballroom III) 

7:00 – 8:15 pm Conference Dinner (Caribbean Ballroom VI and VII) 

Day 3: Wednesday, 12 January 2011 




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8:00 – 10:15 am Session 4 (Caribbean Ballroom IV and V) 

Survey, Detection and Diagnosis – John da Graça, Moderator 


8:00 4.1 Development and Reactivity of Polyclonal Antibodies Based on OMP 

Sequences of Candidatus Liberibacter asiaticus – Coletta-Filho, H.D., 

Peroni, L.A., De Souza, A.A., Takita, M.A., Stach-Machado, D.R. 

8:15 4.2 Development of Single-Chain Antibody Fragments (scFVs) Against 

Candidatus Liberibacter asiaticus by Phage Display – Yuan, Q., Jordan, R., 

Brlansky, R., Minenkova, O., Hartung, J. 

8:30 4.3 Highly Sensitive Detection by Real-Time PCR Targeting the Multiple 

Tandem Repeats of Two Prophage Region Genes of the Citrus 

Huanglongbing Disease Bacterium, Candidatus Liberibacter asiaticus – 

Morgan, J.K., Zhou, L., Shatters, R.G., Jr., Manjunath, K., Duan, Y.-P. 

8:45 4.4 Comparison of Different Extraction and Assay Protocols in Different 

Laboratories to Develop a Standardized Assay for Detection of 

Huanglongbing-Associated Bacteria from Psyllids – Manjunath, K., Irey, 

M., Ramadugu, C., Lee, R.F., Levesque, C., Brady, B., Polek, M., Lin, H., 

Civerolo, E., Afunian, M., Vidalakis, G. 

9:00 4.5 Assessment of Various Spectroscopic Techniques for Detection of HLB – 

Poole, G.H., Hawkins, S.A., Windham, W.R., Heitschmidt, J., Albano, J.P., 

Park, B., Lawrence, K.C., Gottwald, T.R. 

9:15 4.6 Seasonal Variability in HLB Testing Data in Plant and Psyllid Samples in 

Florida – Irey, M., Gast, T., Cote, J., Gadea, P., Santiago, O., Briefman, L., 

Graham, J. 

9:30 4.7 Survey to Estimate the Rate of HLB Infection in Florida Citrus Groves – 

Irey, M., Morris, R.A., Estes, M. 

9:45 4.8 Two Survey Protocols to Detect Newly Introduced HLB and Other Exotic 

Pathogens and Pests – Gottwald, T., Riley, T., Irey, M., Parnell, S., Hall, D. 

10:00 4.9 Distribution of Candidatus Liberibacter Americanus and Ca. L. asiaticus 

in Foliage of Naturally Infected Citrus Trees – Sousa, M.C., Lemos, M.V.F., 

Frare, G.F., Santos, M.A., Lopes, S.A. 

Posters 

4.10 A Perspective on the Activities of Texas HLB Diagnostic Laboratory – 

Kunta, M., da Graça, J.V., Sétamou, M., Skaria, M. 

4.11 Two New Real-Time PCR-Based Surveillance Systems for Candidatus 

Liberibacter Species Detection – Lin, H., Bai, Y., Civerolo, E.L. 

4.12 Detection of Candidatus Liberibacter solanacearum in Potato Psyllid 

Isolated from Sticky Traps – Kwok, K., Levesque, C.S., Manjunath, K., Irey, 

M., Polek, M. 

4.13 Detection of Candidatus Liberibacter asiaticus (Las) on Yellow Sticky 

Traps by Real-Time PCR – Irey, M., Gadea, P., Hall, D. 

4.14 Validation of the Starch-Iodine Reaction for Field Pre-Diagnosis of 

Huanglongbing in Citrus of México – Loredo-Salazar, R.X., Uribe- 

Bustamante, A., Rodríguez-Quibrera, C.G., Curtí-Díaz, S.A., Alanís-Martínez, 

E.I., Velázquez-Monreal, J.J., López-Arroyo, J.I. 


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4.15 Detecting HLB Using NIR Remote Sensing – Gonzalez-Mora, J., Dima, 

C.S., Irey, M., Ehsani, R. 

4.16 Isothermal Detection of Huanglongbing in Psyllids and Citrus Tree 

Samples – Russell, P.F., McGowen, N., Bohannon, R. 

4.17 Assessment of Candidatus Liberibacter asiaticus in the Psyllids, 

Diaphorina citri Collected from Murraya paniculata in Thailand – 

Jantasorn, A., Duan, Y.-P., Hoffman, M., Zhang, S., Puttamuk, T., Thaveechai, 

N. 

4.18 Liberibacter Reservoirs in Cities and Villages in the State of São Paulo, 

Brazil – Lopes, S.A., Frare, G.F., Camargo, L.E.A., Wulff, N.A., Teixeira, D.C., 

Bassanezi, R.B., Beattie, G.A.C., Ayres, A.J. 

4.19 Pictorial Gallery of Foliar HLB Symptoms on Various Citrus Varieties and 

Citrus Relatives – Robl, D.J., Riley, T.D., Gomez, H. 

10:15 – 10:30 am Break (Foyer) 


Economics, Fruit Quality, and Crop Loss – Mike Irey, Moderator 


10:30 5.1 Evaluation of Chemical Flavor Compounds in Orange Juice from Multiple 

Harvests of Hamlin and Valencia Fruit from HLB-Symptomatic Versus 

Healthy Trees – Baldwin, E., Bai, J., Dea, S., Plotto, A., Manthey, J., Rouseff, 

R., Irey, M. 

10:45 5.2 Evaluation of Bitterness Caused by Huanglongbing Disease in Orange 

Juice – Dea, S., Plotto, A., Manthey, J., Baldwin, E., Irey, M. 

11:00 5.3 Sensory Evaluation of Juice Made with Fruit from Huanglongbing (HLB) 

Affected Trees – Plotto, A., Valim, F., Rouseff, R., Dea, S., Manthey, J., 

Narciso, J., Bai, J., Irey, M., Baldwin, E. 

11:15 5.4 Economic Considerations to Treating HLB with the Standard Protocol or 

an Enhanced Foliar Nutritional Program – Morris, R.A., Muraro, R.P. 

11:30 5.5 When Should a Grower with HLB Stop Removing Trees? – Irey, M. 

Posters 

5.6 Use of Electronic Sensor Technology to Discriminate between Juices 

from Huanglongbing Infected and Healthy Orange Trees – Bai, J., Dea, S., 

Plotto, A., Baldwin, E., Irey, M. 

5.7 A Regional Epidemiological Approach for Yield Loss Estimates Due to 

Candidatus Liberibacter Under Different Risk Scenarios – Mora-Aguilera, 

G., Acevedo, G., López-Arroyo, J.I., Velazquez, J., Gómez, R., Robles, M., 

Salcedo, D. 


Lessons from Zebra Chip: Prospects for HLB Management – 

Dennis Gross 


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Epidemiology – Tim Gottwald, Moderator 


1:30 6.1 Five Years of Experience with Huanglongbing in Florida: Current 

Assessment; How Did We Get Here? – Halbert, S.E., Manjunath, K., 

Ramadugu, C., Lee, R.F. 

1:45 6.2 Designing Sampling Schemes to Maximize the Probability of Early 

Detection of a Huanglongbing Outbreak – Parnell, S.R., Gottwald, T.R., van 

den Bosch, F. 

2:00 6.3 Candidatus Liberibacter africanus Subspecies capensis on Calodendrum 

capense in South Africa – Phahladira, M.N.B., Viljoen, R., Pietersen, G. 

Posters 

6.4 Distribution of Psyllids Positive for Candidatus Liberibacter asiaticus in 

Citrus Groves in Southwest Florida – Halbert, S.E., Manjunath, K., 

Ramadugu, C., Mears, P., Lee, R.F. 

6.5 Seasonal Prevalence of Citrus Huanglongbing (Candidatus Liberibacter 

asiaticus) in a Central Florida Sweet Orange Grove – Parkunan, V., Wang, 

N.-Y., Ebert, T.A., Rogers, M.E., Dewdney, M.M. 

6.6 A Mathematical Model for Transmission of HLB by Psyllids – Chiyaka, C., 

Singer, B., Halbert, S., van Bruggen, A.H.C. 

6.7 Potential Spread of Huanglongbing Through Soil – Nunes da Rocha, U., 

Dickstein, E.R., van Bruggen, A.H.C. 


International Citrus Industries, Regulation, and Grower Experiences – 

MaryLou Polek, Moderator 


2:15 7.1 Laws, Huanglongbing Management, and the Current Status of the Disease 

in São Paulo, Brazil – Belasque, J., Jr., Ayres, A.J., Barbosa, J.C., Massari, 

C.A., Bové, J.M. 

2:30 7.2 Distribution of Citrus Huanglongbing in the Dominican Republic – Matos, 

L., Hilf, M.E., Cayetano, X., Feliz, A., Puello, H., Méndez, F., Borbón, J., 

Folimonova, S.Y. 


3:00 7.3 Citrus Huanglongbing in Cuba: Current Situation, Management, and Main 

Research – López, D., Luis, M., Collazo, C., Batista, L., Peña, I., González, C., 

Pérez, L., Zamora, V., Borroto, A., Pérez, D., Alonso, E., Acosta, I., Llauger, R., 

Casín, J.C. 

3:15 7.4 Spreading and Symptoms of Huanglongbing in Mexican Lime Groves in 

the State of Colima, Mexico – Robles Gonzalez, M.M., Velázquez Monreal, 

J.J., Manzanilla Ramirez, M.A., Orozco Santos, M., Flores Virgen, R., Medina 

Urrutia, V.M., Carrillo Medrano, S.H. 

3:30 7.5 The Asian Citrus Psyllid/Huanglongbing Detection, Treatment, and 

Regulatory Program in California – Galindo, T. 


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3:45 7.6 Detection and Reporting of Asian Citrus Psyllid and Huanglongbing in 

Commercial Citrus Within California: An Industry Program – Taylor, B.J., 

Polek, M.L., Batkin, T. 

4:00 7.7 Citrus Health Research Forum: A National Research Effort – Polek, M., 

Wisler, G. 

Posters 

7.8 The Identification and Distribution of Citrus Greening Disease in Jamaica 

– Oberheim, A.P., Brown, S.E., McLaughlin, W.A. 

7.9 Fitting a Spatial Analysis Grid for Research on Huanglongbing in Mexico 

– Aldama-Aguilera, C., Olvera-Vargas, L.A., Galindo-Mendoza, M.G. 


6:00 pm Dinner on your own 

Day 4: Thursday, 13 January 2011 



8:00 – 10:00 am Session 8 (Caribbean Ballroom IV and V) 

Host-Pathogen Interactions – Bill Dawson, Moderator 


8:00 8.1 Examination of Stages of the HLB Disease Development in Citrus Trees – 

Folimonova, S.Y., Achor, D.S., Hilf, M.E. 

8:15 8.2 New Defense Response Insights of Sweet Orange Infected with Two 

Candidatus Liberibacter Species – Mafra, V.S., Martins, P.K., Locali-Fabris, 

E.C., Ribeiro-Alves, M., Francisco, C.S., Freitas-Astúa, J., Kishi, L.T., 

Machado, M.A. 

8:30 8.3 Differential Expression of Potential Virulence Genes of Candidatus 

Liberibacter asiaticus in Infected Plants and Psyllids – Sreedharan, A., 

Wei, S., Wang, N. 

8:45 8.4 Metabolome Analysis of Tolerant and Susceptible Citrus Varieties in 

Response to Infection with Candidatus Liberibacter asiaticus – Albrecht, 

U., Skogerson, K., Bowman, K.D., Fiehn, O. 

9:00 8.5 Deep Transcriptome Profiling of Citrus Fruit in Response to 

Huanglongbing Disease – Martinelli, F., Uratsu, S.L., Albrecht, U., Reagan, 

R.L., Leicht, E., D’Souza, R., Bowman, K.D., Dandekar, A.M. 

9:15 8.6 Carbohydrate Metabolism and Related Gene Expression Changes in 

Huanglongbing-Affected Sweet Orange – Chen, C., Fan, J., Yu, Q., 

Brlansky, R., Li, Z.-G., Gmitter, F., Jr. 

9:30 8.7 Analysis of Colonization of Citrus Seeds by Ca. Liberibacter asiaticus and 

Its Possible Role in Seed Transmission – Hilf, M.E. 


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9:45 8.8 Natural Transmission of Huanglongbing Caused by Candidatus 

Liberibacter americanus and Ca. L. asiaticus and with Two Different 

Sources of Inoculum Plants (Citrus sinensis or Murraya exotica) – 

Gasparoto, M.C.G., Bassanezi, R.B., Amorim, L., Montesino, L.H., Lourenço, 

S.A., Wulff, N.A., Bergamin Filho, A. 

Posters 

8.9 Callose Predominates over Phloem Protein 2 in Phloem Plugging of Trees 

Affected with Huanglongbing – Albrigo, L.G., Achor, D.S. 

8.10 Influence of Huanglongbing (HLB) on the Composition of Citrus Juices 

and Mature Leaves – Cancalon, P.F., Bryan, C., Haun, C., Zhang, J. 

8.11 Gene Expression in Citrus sinensis Fruit Tissues Harvested from 

Huanglongbing-Infected Trees – Liao, H.-L., Burns, J.K. 

8.12 Expression Profiling of Host Response of Citrus to Candidatus 

Liberibacter asiaticus Infection – Aritua, V., Wang, N. 

8.13 Arabidopsis Responses to the HLB-Relative Candidatus Liberibacter 

psyllaurous – Patne, S., Manjunath, K.L., Roose, M.L. 

8.14 Comparative Studies of the Endophytic Microbial Community Structures 

in Huanglongbing-Infected and Non-Infected Citrus Plants – Zheng X.-F., 

Liu, B., Ruan, C.-Q., Lin, Y.-Z., Xiao, R.-F., Zhu, Y.-J., Fan, G.-C., Cai, Z.J., 

Duan, Y.-P. 

8.15 HLB Influences the Diversity, Structure, and Function of the Bacterial 

Community Associated with Citrus – Trivedi, P., Wang, N. 

8.16 Functional Studies of Putative Effectors of Candidatus Liberibacter 

asiaticus Using Citrus Tristeza Virus Vector – Hajeri, S., Duan, Y.-P., 

Gowda, S. 

8.17 First Report of a New Host (Pithecellobium lucidum Benth) of the Citrus 

Huanglongbing Bacterium, Candidatus Liberibacter asiaticus – Fan, G.-C., 

Cai, Z.J., Weng, Q.Y., Ke, C., Liu, B., Zhou, L.J., Duan, Y.-P. 

8.18 Citrus Seed Grafting: A Simple Technology for Testing Seed 

Transmission of Citrus Greening/HLB and of Other Pathogenic Agents – 

Bar-Joseph, M., Robertson, C., Hilf, M., Dawson, W.O. 

8.19 Lack of Transmission of HLB by Citrus Seed – Graham, J.H., Johnson, 

E.G., Bright, D.B., Irey, M.S. 

8.20 Visualization of Ca. Liberibacter asiaticus in Immature Citrus Seed Coats 

by Fluorescent In Situ Hybridization (FISH) of 16S rRNA – Hilf, M.E. 

8.21 Rapid, Sensitive, and Non-Radioactive Tissue-Blot Diagnostic Method for 

the Detection of Citrus Greening Disease (HLB) – Gowda, S., Nageswara 

Rao, N., Miyata, S., Ghosh, D.K., Irey, M.S., Rogers, M.E., Garnsey, S.M. 

10:00 – 10:15 am Break (Foyer) 


Asian Citrus Psyllid Management – Michael Rogers, Moderator 


10:15 9.1 A Database for Analysis of Diaphorina citri Population Monitoring Data 

from Commercial Groves – Gast, T., Irey, M., Hou, H. 


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10:30 9.2 RNAi Strategy in Citrus Trees to Reduce Hemipteran Pests: Psyllids and 

Leafhoppers – Hunter, W., Glick, E., Bextine, B.R., Paldi, N. 

10:45 9.3 Application of Insecticidal Sprays to Citrus in Winter Provides Significant 

Reduction in Asian Citrus Psyllid Diaphorina citri Populations and 

Opportunity for Additional Suppression Through Conservative and 

Augmentative Biological Control – Qureshi, J.A., Stansly, P.A. 

11:00 9.4 Studies on Imidacloprid and Management of ACP in California – Byrne, F., 

Morse, J., Bethke, J. 

11:15 9.5 Selection and Dosage of Insecticides for the Control of the Asian Citrus 

Psyllid in the Citrus Groves of Mexico – López-Arroyo, J.I., Díaz-Zorrilla, 

U., Hernández-Fuentes, L.M., Cortez-Mondaca, E., Robles-González, M.M., 

Villanueva-Jiménez, J.A., Cabrera-Mireles, H., Loera-Gallardo, J., Jasso- 

Argumedo, J., Curti-Díaz, S. 

11:30 9.6 Asian Citrus Psyllid (ACP) Control: Potential Use of Systemic Insecticides 

in Citrus Bearing Trees – Yamamoto, P.T., Miranda, M.P., Felippe, M.R. 

11:45 9.7 Insecticide Resistance and Susceptibility of Uninfected and Candidatus 

Liberibacter asiaticus-Infected Asian Citrus Psyllid in Florida – Tiwari, S., 

Rogers, M.E., Stelinski, L.L. 

Posters 

9.8 Development of Area-Wide Asian Citrus Psyllid Management Strategies in 

Texas – Bartels, D.W., Sétamou, M., Ciomperlik, M.A., da Graça, J.V. 

9.9 Citrus Psyllid Management Strategies for California Citrus Growing 

Regions – Grafton-Cardwell, E.E., Morse J.G., Taylor, B.J. 

9.10 Area-Wide Management of Asian Citrus Psyllid in Southwest Florida – 

Stansly, P.A., Arevalo, H.A., Zekri, M., Hamel, R. 

9.11 Evaluation of Low Volume Sprayers Used in Citrus Psyllid Control 

Applications – Hoffmann, C., Fritz, B., Martin, D., Atwood, R., Hurner, T., 

Ledebuhr, M., Tandy, M., Jackson, J.L., Wisler, G., Polek, M. 

9.12 Identification of Parasitoids and Haplotypes of Tamarixia radiata 

(Waterston) (Hymenoptera: Eulophidae) from Diaphorina citri in Yucatán, 

México – González-Hernández, A., Jasso-Argumedo, J., Cruz-García, R., 

Lozano-Contreras, M., López-Arroyo, J.I., Villanueva-Segura, O.K. 

9.13 Host Specificity Testing of Tamarixia radiata for the Classical Biological 

Control of Asian Citrus Psyllid, Diaphorina citri, in California – Pandey, 

R.R., Hoddle, M.S. 

9.14 Predators in Non-Commercial Citrus and Preliminary Evaluation of Their 

Potential Against the Asian Citrus Psyllid in Texas – Pfannenstiel, R.S., 

Unruh, T.R. 

9.15 Suitability of Diaphorina citri, Toxoptera citricida, and Aphis spiraecola as 

Prey for Hippodamia convergens – Qureshi, J.A., Stansly, P.A. 

9.16 Molecular Analysis of Tamarixia radiata from America Uncovers Extensive 

Haplotype Variation: Multiple Groups? – de León, J.H., Gastaminza, G.A., 

Sétamou, M., Cáceres, S., Kanga, L.H.B., Buenahora, J., Parra, J.R., Logarzo, 

G.A., Stañgret, C.R.W. 


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9.17 Molecular Characterization of a New Entomopathogenic Fungus Isaria 

poprawskii: A Potential Biocontrol Agent for Diaphorina citri. 

Development of Isaria-Specific Molecular Markers – de León, J.H., 

Cabanillas, H.E., Humber, R.A., Murray, K.D., Moran, P., Jones, W.A. 

9.18 RNAi – Evaluating Injection into Citrus Trees and Grapevine to Target 

Psyllids and Leafhoppers – Hunter, W., Stover, E., Glick, E., Bextine, B.R., 

Paldi, N. 

9.19 Using Novel Photonic Fence Technology to Protect Foundation Block and 

Nursery Stock from Asian Citrus Psyllid – Johanson, E., Patt, J., Mullen, E., 

Rutschman, P., Pegram, N. 

9.20 Development of a Diaphorina citri-Specific Molecular Diagnostic Marker 

for Gut Content Examinations – de León, J.H., Thomas, D., Sétamou, M., 

Hagler, J.R. 

9.21 Development of a Pathogen Dispenser to Control Asian Citrus Psyllid 

(ACP) in Residential Citrus – Patt, J., Jackson, M., Dunlap, C., Meikle, W., 

Adamczyk, J. 

9.22 Producing New Flush at Will in Citrus to Study ACP-Plant Interactions – 

Malik, N.S.A., Brockington, J., Perez, J.L., Mangan, R.L. 

9.23 Thresholds for Vector Control in Young Citrus Treated for Symptoms of 

HLB with a Nutrient/SAR Package – Monzó, C., Arevalo, H.A., Stansly, P.A. 

9.24 Experimental Release Rate Analysis of Volatile Compounds from Wax- 

Based Dispensers – Neuman, R.D., Shelton, A.B., Mills, D.R. 

9.25 Vegetation Canopy Airflow Modeling for Airborne Dispersion of DMDS – 

Shelton, A.B., Neuman, R.D. 

9.26 Methods and Systems to Deliver Volatile Compounds for Biological 

Control Strategies – Neuman, R.D., Shelton, A.B., Zee, R.H. 


Viral Vectors and Prospects for HLB Control – Bill Dawson 


HLB Management – Tim Spann, Moderator 


1:30 10.1 Trunk Injection of Copper Sulfate Pentahydrate (Magna-Bon) Affects 

Expression of HLB – Graham, J.H., Irey, M.S., Miele, F. 

1:45 10.2 Chemical Compounds Effective Against the Citrus Huanglongbing 

Bacterium, Candidatus Liberibacter asiaticus In Planta – Zhang, M., Powell, 

C.A., Zhou, L., He, Z., Stover, E., Duan, Y.-P. 

2:00 10.3 Regional HLB Management on the Effectiveness of Local Strategies of 

Inoculum Reduction and Vector Control – Bassanezi, R.B., Yamamoto, 

P.T., Montesino, L.H., Gottwald, T.R., Amorim, L., Bergamin Filho, A. 

2:15 10.4 The Theory of Managing Huanglongbing with Plant Nutrition and Real 

World Success in Florida – Spann, T.M., Rouse, R.E., Schumann, A.W. 

2:30 10.5 Nutritional Treatments: Inconsequential Effect on HLB Control and 

Promote Area-Wide Titer Increase and Disease Spread – Gottwald, T., Irey, 

M., Graham, J., Wood, B. 


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2:45 10.6 Nutritional Approaches for Management of Huanglongbing (Citrus 

Greening) in China – Xia, Y., Sequeira, R. 

Posters 

10.7 First Steps Towards Rescuing Las-Infected Citrus Germplasm – 

McCollum, G., Stover, E. 

10.8 Screening Chemical Compounds Against Citrus Huanglongbing Using an 

Optimized Grafting System from Candidatus Liberibacter asiaticus- 

Infected Citrus Scions – Zhang, M.-Q., Duan, Y.-P., Powell, C.A. 

10.9 Discovery of Antimicrobial Small Molecules Against Candidatus 

Liberibacter asiaticus by Screening Novel SecA Inhibitors Using Structure 

Based Design – Akula, N., Wang, N. 

10.10 The Low Pressure Trunk Injection System: A Technology to Fight Against 

HLB – Tomas, J. 

10.11 Does Systemic Acquired Resistance (SAR) Control HLB Disease 

Development? – Graham, J.H., Myers, M.E., Irey, M.S., Gottwald, T.R. 

10.12 Use of Growth-Priming Agents to Extend the Growth of HLB-Affected 

Citrus – He, Z., Zhang, M.-Q., Viana, E., Merlin, T., Duan, Y.-P., Stoffella, P.J., 

Liptay, A., Powell, C.A. 

10.13 Evaluation of Foliar, Zinc, and Manganese for Control of HLB or 

Associated Symptom Development – Johnson, E.G., Irey, M.S., Gast, T., 

Bright, D.B., Graham, J.H. 

10.14 Role of Nutritional and Insecticidal Treatments in Mitigation of HLB: Main 

Effects and Interactions – Stansly, P.A., Arevalo, H.A., Rouse, R.E. 

10.15 Use of Horticultural Practices in Citriculture to Survive Huanglongbing – 

Stuchi, E.S., Girardi, E.A. 

10.16 Critical Control Point (CCP) Analysis to Build a Model System for 

Measuring Citrus Propagation Risk Mitigations. II. Sampling and 

Monitoring – Brown, L.G., Jones, E.M., Hartzog, H.M. 

10.17 The Need of an Epidemio-Surveillance Network to Prevent Huanglongbing 

Arrival in the South Mediterranean Basin – Dollet, M., Aubert, B., Imbert, E., 

Gatineau, F. 

10.18 Presence of Candidatus Liberibacter asiaticus in Diaphorina citri 

Kuwayama Collected from Plants for Sale in Florida – Halbert, S.E., 

Manjunath, K., Ramadugu, C., Lee, R.F. 

10.19 A Model System for Studying Huanglongbing – Manjunath, K., Ramadugu, 

C., Kund, G., Trumble, J., Lee, R.F. 



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Host Tolerance and Resistance – Fred Gmitter, Moderator 


3:15 11.1 Incidence of Huanglongbing on Several Sweet Orange Cultivars Budded 

onto Different Rootstocks at the Citrus Experimental Station (EECB), 

Bebedouro, São Paulo, Brazil – Stuchi, E.S., Reiff, E.T., Sempionato, O.R., 

Girardi, E.A., Parolin, L.G., Toledo, D.A. 

3:30 11.2 Host Preference and Suitability of Native North American Rutaceae for the 

Development of the Asian Citrus Psyllid, Diaphorina citri Kuwayama – 

Sandoval, J.L., II, Sétamou, M., da Graça, J.V. 

3:45 11.3 Progress Using Transgenic Approaches and Biotechnology-Facilitated 

Conventional Breeding to Develop Genetic Resistance/Tolerance to HLB 

in Commercial Citrus – Grosser, J.W., Dutt, M., Shohael, A., Barthe, G.A. 

4:00 11.4 Promoter Regulation of the β-Glucoronidase (GUS) Gene and 

Antimicrobial Peptide D4E1 in a Citrus Rootstock – Benyon, L.S., Stover, 

E., Bowman, K., McCollum, G., Niedz, R. 

4:15 11.5 Responses of Transgenic Hamlin Sweet Orange Plants Expressing the 

attacin A Gene to Candidatus Liberibacter asiaticus Infection – Felipe, 

R.T.A., Mourão-Filho, F.A., Pereira, E.V., Jr., Lopes, S.A., Sousa, M.C., 

Mendes, B.M.J. 

4:30 11.6 Screening Antimicrobial Peptides In-Vitro for Use in Developing 

Huanglongbing and Citrus Canker Resistant Transgenic Citrus – Stover, 

E., Stange, R., McCollum, G., Jaynes, J. 

4:45 11.7 Response of Citrus Transgenic Plants Expressing STX IA Gene to 

Candidatus Liberibacter asiaticus – Marques, V.V., Bagio, T.Z., Sugahara, 

V.H., Vasquez, G.V., Grange, L., Meneguim, L., Kobayashi, A.K., Bespalhok, 

J., Pereira, L.F.P., Vieira, L.G.E., Leite, R.P., Jr. 

Posters 

11.8 Rootstocks and Pruning Effects on Huanglongbing Incidence on Tahiti 

Limes in Bebedouro, Northern São Paulo State, Brazil – Stuchi, E.S., Reiff, 

E.T., Sempionato, O.R., Cantuarias-Avilés, T., Girardi, E.A., Parolin, L.G., 

Toledo, D.A. 

11.9 Candidatus Liberibacter asiaticus (CLas) Titer in Field HLB-Exposed 

Commercial Citrus Cultivars – Stover, E., McCollum, G. 

11.10 Host Response of Different Citrus Genotypes and Relatives to Candidatus 

Liberibacter asiaticus Infection – Boscariol-Camargo, R.L., Cristofani-Yaly, 

M., Malosso, A., Coletta-Filho, H.D., Machado, M.A. 

11.11 Candidatus Liberibacter asiaticus (CLas) Titer in Poncirus trifoliata and P. 

trifoliata Hybrids: Inferences on Components of HLB Resistance – Stover, 

E., Shatters, R.G., Jr., McCollum, G., Hall, D., Duan, Y.-P. 

11.12 The Role of Salicylic Acid and Systemic Acquired Resistance in the 

Response of Citrus to HLB – Khalaf, A., Febres, V.J., Brlansky, R.H., 

Gmitter, F.G., Moore, G.A. 

11.13 Observations of Citrus × Poncirus Hybrid Tolerance to Infection with 

Candidatus Liberibacter asiaticus – Bowman, K.D., Albrecht, U. 


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11.14 Performance of a Phage Gene in Transgenic Citrus Resistant to Citrus 

Greening – Jiang, Y., Perazzo, G., Septer, A., Kress, R., Gabriel, D.W. 

11.15 Genome Sequences of Haploid Clementine Mandarin and Diploid Sweet 

Orange – Gmitter, F.G., Jr. 

11.16 Exploring Metabolic Profiles of Plant Tissue with Increased or Decreased 

Susceptibility – Malik, N.S.A., Perez, J.L., Brockington, J., Mangan, R.L. 


7:00 – 8:15 pm Conference Banquet and (Caribbean Ballroom VI and VII) 

Keynote Lecture 4 

Understanding the Lifestyle of Plant Pathogens: Towards Successful 

Management of Vectored Plant Diseases – Steven Lindow 

8:15 – 9:30 pm Entertainment (Caribbean Ballroom VI and VII) 


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Day 5: Friday, 14 January 2011 – Grower Day 

7:00 am – noon Registration (Foyer) 


8:00 am – 12:45 pm Session 12 (Caribbean Ballroom IV and V) 

Grower Day – Megan Dewdney, Moderator 

8:00 – 9:30 am Take Home Messages: What Can Be Implemented Now or in the Near Future? 

8:00 HLB Pathology Lessons – M. Dewdney / T. Schubert 

8:30 Entomology Lessons – L.L. Stelinski / M. Sétamou 

9:00 Horticulture Lessons – C. Oswalt / E. Stover 

9:30 – 11:15 am Managing HLB in Florida and Brazil 

9:30 The Florida Experience – M. Irey 

10:00 The Brazil Experience – R. Bassanezi 

10:30 – 10:45 am Break (Foyer) 

10:45 Citrus Health Management Areas – M.E. Rogers 

11:15 – 12:15 pm WORKSHOP: Nutrition and Tree Health – T.M. Spann / J.H. Graham / Y. Xia 

12:15 – 12:45 pm Grower/Industry Responses – Jerry Newlin / Bobby Barben / Others 


Sustaining Productivity in an Endemic HLB Environment – J. Ayres 

2:15 – 5:00 pm CHRP HLB Research Coordinating Group Meeting – G. Wisler, M.L. Polek 

(Boca Room II, III, and IV) 


P a g e | 280 

A4. Session 9 Addendum 


Management 

Extended Papers 

9.24 & 9.25 


P a g e | 281 

9.24 Experimental Release Rate Analysis of Volatile Compounds from Wax-Based 

Dispensers 

Neuman, R.D., 1 Mills, D.R., 1 Shelton, A.B. 2 

1 Department of Chemical Engineering, Auburn University, Auburn, AL, USA 

2 Department of Aerospace Engineering, Auburn University, Auburn, AL, USA 

While long-term efforts against the citrus disease Huanglongbing focus on the bacteria 

responsible and plant resistance, a short-term tool for mitigation of the problem appears to be 

exploitation of dimethyl disulfide as a vector repellent. An experimental apparatus is set up to 

investigate the release of volatile substances from a wax-based dispenser under well-defined 

conditions of geometry, air flow, temperature, and surface area. The release rate is modeled as a 

convective mass transfer process taking place in laminar boundary layer flow parallel to a flat 

surface. The gas-phase mass-transfer coefficient and, hence, the release rate per unit surface area 

is predicted. 

Introduction 

The goal of this research project is an engineering evaluation of deployment strategies for 

dimethyl disulfide (DMDS) as an effective repellent of the Asian citrus psyllid in Florida orange 

groves. This study, per the research project deliverables, was to focus on wax-based dispensers. 

In particular, a flowable emulsified paraffin wax (SPLAT) manufactured and developed by ISCA 

Technologies was selected as a candidate DMDS dispenser because of its prior success in the 

delivery of a variety of semiochemicals for insect pest management. In addition, the rheological 

properties of SPLAT make it suitable for mechanical delivery using spray or drip applicator 

systems. Notwithstanding, the release rate of semiochemicals from SPLAT can be studied under 

controlled conditions using a model system of well-defined physical geometry, namely, a flat 

surface that permits improved experimental measurements that will advance the fundamental 

understanding of the release mechanism of semiochemicals from wax-based dispensers. 

Apparatus and Procedure 

A laboratory facility set up to investigate the release rate of DMDS was modified for the DMDS 

release experiments. The existing drying tray was replaced by a Lucite plate machined with a 

rectangle-shape well (25.4 mm wide × 76.2 mm long × 6.35 mm deep) designed to contain the 

emulsified paraffin wax dispenser. The tray was placed on a support that extends through the 

tunnel floor to an Ohaus electronic balance. Mass data were acquired through an RS-232 serial 

connection, and the data were logged using a VBA macro in MS Excel. 

The experimental procedures were as follows: prior to starting a release rate experiment, 

the fan speed and electrical heaters were adjusted to provide a bulk air velocity of about 1.4 m/s 

(or 3 mph) above the dispensing tray and a bulk air temperature of 43°C, respectively. The 

selected air velocity is representative of the average air velocity in Central Florida over a 

one-year period. Although the selected air temperature is somewhat higher than the summer 


P a g e | 282 

temperatures in Central Florida, it represents a temperature that permits release rate 

measurements over long experimental time periods with reasonable accuracy. Twenty-four hours 

at minimum were allowed for apparatus stabilization. An experimental methodology was 

developed for spreading the SPLAT formulation with or without dimethyl disulfide in the tray 

well and smoothing it flush with the plate surface. The tray with the SPLAT-DMDS sample was 

immediately placed in the experimental apparatus, and mass data were collected as a function of 

time. 

The performance of SPLAT-DMDS formulations was initially evaluated by applying 1-g 

drip samples from a syringe onto roughened plastic strips hanging in crepe myrtle trees (to 

simulate drip applications in citrus groves for reference). The SPLAT-DMDS formulation 

consisted of ca. 39.6% solids (wax, soy oil, surfactant, antioxidant, etc.), 50.4% H 2 O, and 10.0% 

DMDS. Figure 1 shows the typical characteristics of the release rate as a function of time. Three 

regimes of mass loss are clearly evident: (a) an initial rapid loss, (b) a transition region, and (c) 

an approximate small linear decrease over time. After 6 hours of exposure to the elements, there 

was a mass loss of 33%; and after 24 hours, the mass loss was about 44%. In a little over 2 days, 

50% of the mass was lost in the simulated field experiment. It is important to recognize that the 

mass loss represents a combined loss of water (and other unknown volatiles or degradation 

products from the original proprietary SPLAT formulation) and DMDS, and as such, the actual 

loss of DMDS is difficult to resolve by gravimetric measurements alone. The lower horizontal 

line in Fig. 1 represents the best estimate of the dry solids in the proprietary SPLAT formulation. 


Figure 2 shows the typical release rate behavior for the model (flat surface) system of a 

SPLAT-DMDS (10% w/w) formulation under controlled laboratory conditions of fixed airflow 

(ca. 1.4 m/s) and temperature (43°C) in the experimental apparatus. As before, there are three 

regimes of mass loss: (a) an initial rapid loss that appears to be linear with time, (b) a transition 

region, and (c) a much smaller rate of decrease over time. It is significant that the two release 

rate profiles are very similar for the small drip sample and the relatively large flat model system. 

The release rate of DMDS, which is the negative of the slope of the mass versus time curve, 

varies by about two orders of magnitude. 

An advantage of the experimental approach employed in this study is that the release rate 

can be modeled as a convective mass transfer process taking place in laminar boundary layer 

flow parallel to a flat surface. Therefore, the gas-phase mass-transfer coefficient and, hence, the 

mass transfer flux (or release rate per unit surface area) can be predicted for DMDS as well as for 

H 2 O under controlled conditions (such as air velocity, temperature, surface area, etc.) and 

compared with the experimental release rate data for SPLAT-DMDS formulations. 

The molecular diffusivity of DMDS in air and the vapor pressure of DMDS were evaluated 

using data provided in Table C-1 and Table C-2 of USEPA (1994). The molecular diffusivity 

(D AB ) of DMDS in air corrected for temperature (D AB is proportional to T 1.75 ) is estimated to be 

0.919 × 10 -5 m 2 /s at 43°C. For comparison, a diffusivity value of 1.02 × 10 -5 m 2 /s was calculated 

using the semi-empirical method of Fuller et al. (1966). The local convective (gas-phase) 


P a g e | 283 

mass-transfer coefficient k c was evaluated for the case of laminar flow of fluid past a flat plate 

using the standard correlation equation for the Sherwood number in boundary-layer flow. The 

average value of k c over the SPLAT-DMDS surface of 3-inch length was determined to be 5.01 

× 10 -3 m/s. The mass transfer flux N A /A can be found as follows: 

N A /A = k c (C Ai − C AG ) 

where N A is the mass transfer rate, A is the surface area of the solid (SPLAT), k c is the 

gas-phase convective mass transfer coefficient, C Ai is the concentration of the semiochemical 

(DMDS) in the fluid (air phase) just adjacent to the surface of the solid, and C AG is the 

concentration in the bulk air. 

The mass transfer flux and the rate of evaporative loss from a free surface of DMDS were 

calculated to be 1.65 g/s ⋅ m 2 and 11.3 g/h for the model system employed in this study. For 

these calculations, it was assumed that DMDS exists as a free liquid surface at the solid-air 

interface, and C Ai was evaluated by assuming that the DMDS-air mixture behaves as an ideal gas 

and that local equilibrium exists at the solid-air interface. Furthermore, the vapor pressure of 

DMDS was evaluated to be 9.20 kPa at 43°C using the Antoine equation coefficients of USEPA 

(1994). In a similar manner, under the conditions of the experimental measurements (ca. 10% 

relative humidity), the mass transfer flux, and the rate of evaporative loss from a free surface of 

H 2 O were estimated to be 0.269 g/s ⋅ m 2 and 1.85 g/h. 

It is possible to estimate with certain assumptions the combined release rate of DMDS and 

H 2 O from SPLAT-DMDS formulations in the initial rapid loss regime. SPLAT is a complex 

fluid (emulsified paraffin wax) with unique microstructure and, most certainly, is not 

homogeneous in the traditional sense. To a first approximation, the SPLAT-DMDS formulation 

can be thought of as a three-component system consisting of DMDS, H 2 O, and a hypothetical 

SPLAT (water-free) molecule with a weight-averaged molecular mass (418 Da) of the major 

constituents of the blank SPLAT formulation presumed to be 30% paraffin wax, 4% soy oil, 10% 

Span 60, and 5% vitamin E. Additionally, it can be assumed that domains of DMDS and H 2 O 

coexist at the surface of emulsified paraffin wax in admixture with added DMDS in the same 

ratio as that in the bulk phase. If so, and assuming that the surface area ratio is proportional to the 

volume ratio, the combined release rate of DMDS and H 2 O can be estimated to be 1.85 g/h for 

the SPLAT-DMDS formulation, whereas the release rate of H 2 O in the blank SPLAT 

formulation is estimated to be 0.85 g/h. 

In this initial study, a well-defined physical geometry (i.e., flat surface) was utilized for a 

simple experimental model of a wax-based (SPLAT) dispenser. As noted earlier, the mass loss or 

release rate was measured as a function of time for a SPLAT-DMDS (10% w/w) formulation 

(see Fig. 2). In addition, release rate-time curves were obtained for the same SPLAT-DMDS 

formulation, but for a sample of one-half the thickness, and for the blank (or control) SPLAT 

formulation. The release rate-time curves are not shown for the latter two cases because they are 

similar in features to that of Fig. 2. However, the details of the release rate-time curves are 

analyzed and discussed herein in order to shed new insight into the mechanism of the release of 

DMDS (and related volatile substances) from flowable wax-based dispensers. 


P a g e | 284 

First, the release rate of H 2 O from the blank SPLAT formulation (whose surface is 

estimated to consist of 46% water) was examined under the same experimental conditions as 

those employed for the SPLAT-DMDS (10% w/w) formulation. Once again, three regimes of 

mass loss are evident in the release rate-time curve. In the initial rapid loss regime, the mass 

versus time curve is found to be linear, and thus the release rate is constant. The constant rate 

was statistically measured to be 0.87 g/h. Significantly, this value for the release rate is found to 

be in excellent agreement with the theoretical calculations (0.85 g/h) of the mass loss of water 

from the blank SPLAT formulation. The constant rate period lasts for about 60 min under the 

experimental conditions employed in this study. 

A simple model which is consistent with these findings can explain the release of H 2 O 

from flowable wax-based dispensers based on SPLAT technology. The surface of a blank 

SPLAT formulation is assumed to consist of water domains and (water-free) SPLAT domains. In 

the constant release-rate regime, the water evaporates from the surface of the water domains. 

This water behaves as if the hydrocarbon components of SPLAT were not present. The rate of 

evaporation (or release rate) under the given conditions is independent of the (water-free) 

SPLAT and is essentially the same as the rate of evaporation from a free liquid surface. The 

constant rate period continues as long as water is supplied to the surface of the SPLAT 

formulation as fast as it is evaporated. Then at some point the release rate is no longer constant, 

and it continues to decrease with time. Initially, it may be that the wetted surface area decreases 

until the surface is completely dry and/or the evaporation slowly recedes into the interior of the 

SPLAT formulation with diffusion likely controlling the transport of water. The actual 

mechanisms involved in the movement of water in the SPLAT formulation are likely quite 

complicated. Shrinkage of the SPLAT formulation is observed to occur, and shrinkage further 

complicates the mechanism of the release of water (as well as other volatile substances such as 

DMDS). 

Second, the effect of added DMDS on the mass loss from SPLAT formulations was 

examined. From the results illustrated in Fig. 2, the mass loss is initially linear with time 

(constant rate period), and the release rate in the constant rate period was statistically measured 

to be 0.63 g/h for SPLAT-DMDS (10% w/w) formulations. Furthermore, the constant rate 

period lasts for about 150 min. Importantly, this release rate in the initial rapid loss regime is less 

than that for the blank SPLAT formulation. Also, it is significantly lower than that of the 

theoretical calculations (1.85 g/h) for the combined mass loss of both water and DMDS from 

the SPLAT-DMDS formulation. In marked contrast, the results are in much better agreement 

with the theoretical calculations (0.86 g/h) that assume the mass loss results from only water. In 

the case of SPLAT-DMDS formulations, chemical (interaction) effects appear to strongly 

influence the mass loss in the constant rate period as opposed to the former case of the blank 

SPLAT formulation. DMDS is essentially insoluble in water, whereas it is completely soluble in 

many organic solvents. Although the release of DMDS is detected during the experimental 

measurements, it is likely that DMDS preferentially partitions into the (water-free) 

hydrocarbonaceous SPLAT domains. Therefore, the combined release rate of DMDS and water 

in SPLAT-DMDS formulations appears to be affected by both chemical and structural effects, 

whereas the release rate of water in blank SPLAT formulations appears to be only affected by 


P a g e | 285 

structural effects. Clearly, much remains to be learned about the mechanisms of the release of 

volatile substances in flowable wax-based dispensers. 

Third, the effect of sample thickness on the mass loss from SPLAT-DMDS formulations 

was also examined. A release rate-time curve was obtained for the same SPLAT-DMDS (10% 

w/w) formulation, but for a sample of one-half the thickness (0.317 mm). Again, the release rate 

initially was linear with time. Significantly, the rate of mass loss in the constant rate period was 

statistically measured to be 0.64 g/h, a value essentially the same as the release rate (0.63 g/h) 

for the thicker SPLAT-DMDS formulation. Clearly, the thickness of the SPLAT-DMDS 

formulation does not significantly influence the mass loss in the constant rate period of 

volatilization. Indeed, this behavior is what is to be expected on the basis of the simple model; 

that is, the release rate is independent of the thickness of the SPLAT formulation in the constant 

rate regime. However, the time for a specific mass loss is directly proportional to the thickness 

(or diameter of sphere) of the formulation sample. Also, the experimental measurements confirm 

that the time duration of the constant rate period is not as long in samples of smaller thickness. 

Conclusions 

An experimental apparatus was set up to investigate the release of volatile substances such as 

DMDS from a flowable wax-based dispenser under controlled and well-defined conditions of 

physical geometry, air flow, temperature, surface area, etc. A simple model that reasonably 

explains the release rate behavior observed in SPLAT formulations without and with added 

DMDS is proposed. Although high-quality mass loss measurements were obtained, it is difficult 

to resolve unambiguously the actual DMDS loss. Future studies should simultaneously measure 

the sulfur content to obtain an improved understanding of the coupled mass transfer processes 

involved in wax-based dispensers. The experimental apparatus can be improved by equipping it 

with multi-channel tray wells for chemical analysis sampling, on-line analytical sensors, 

feedback control for improved temperature stability, and redesigned support/balance for refined 

mass measurements. 

References 

Fuller, E., Schettler, P., Giddings, J. 1966. A new method for prediction of binary gas-phase 

diffusion coefficients. Journal of Industrial and Engineering Chemistry 58:19-27. 

USEPA. 1994. Air Emissions Models for Waste and Wastewater, Technical Report 

EPA-453/R-94-080A (Environmental Protection Agency). 


P a g e | 286 

Fig. 1. Release rate-time curve for simulated field test of SPLAT-DMDS formulation. 

Fig. 2. Release rate-time curve for model system of SPLAT-DMDS formulation. 


P a g e | 287 

9.25 Vegetation Canopy Airflow Modeling for Airborne Dispersion of DMDS 

Shelton, A.B., Neuman, R.D. 

1 Department of Aerospace Engineering, Auburn University, Auburn, AL, USA 

2 Department of Chemical Engineering, Auburn University, Auburn, AL, USA 

While long-term efforts against the citrus disease Huanglongbing focus on the bacteria 

responsible and plant resistance, a short-term tool for mitigation of the problem appears to be 

exploitation of dimethyl disulfide as a vector repellent. A computational fluid dynamics model of 

airflow in and around vegetation is used to examine the airborne dispersion of dimethyl disulfide 

vapor within a citrus orchard under a narrowly-scoped set of conditions. 

Introduction 

Spray, deposition, and volatilization of pesticides or repellents in agricultural fields and orchards 

are highly dependent upon the local meteorological conditions. Since both mean advection and 

turbulent diffusion have a direct effect on the overall dispersion of the active ingredients, the 

complex aerodynamic interactions between plants and atmosphere can strongly affect local air 

quality and efficacy of the application. The scope of the current work is the post-volatilization 

airborne dispersion of dimethyl disulfide (DMDS) within a citrus orchard as a potential repellent 

for the Asian citrus psyllid. The objective is to mathematically model and numerically simulate 

the aerodynamic effects induced by citrus vegetation canopy drag on the local wind and 

turbulence fields in and around a typical orchard configuration. Since the roughness layer of 

canopy flow is quite different from typical turbulent boundary layer flow, this detail requires 

special attention. The various vegetation elements (e.g., branches, stems, leaves, fruits, seeds, 

etc.) impart drag on the flow with a corresponding increase in wake turbulence at length scales 

smaller than the background shear-generated turbulence. However, instead of a simple 

superposition of the small-scale turbulent fluctuations from vegetation element wakes onto the 

standard boundary layer fluctuations, large and intermittent coherent structures dominate canopy 

turbulence (Finnigan, 2000). The result is a bypass of the classical energy dissipation cascade for 

wall-bounded flows. 

Computational fluid dynamics (CFD) is capable of describing these flow details by 

numerically solving the physical conservation laws of mass, momentum, and energy. However, 

the foremost challenge to computing resources is the wide range of spatial and temporal scales 

involved in turbulent flows. To further complicate matters, the scales of interest are much 

typically larger than the individual vegetation elements responsible for the dominant flow 

characteristics. A fully resolved, three-dimensional, unsteady solution is simply impractical for 

analysis of realistic flowfields; therefore, the mathematical model must represent the 

phenomenology described above in some average sense. The Reynolds-averaged Navier-Stokes 

(RANS) formulation involves time averaging of the governing equations, requiring modeling of 

the overall effect of the entire turbulent energy spectrum on the mean flow. Furthermore, the 

entire vegetation canopy is viewed as porous body with through flow (Finnigan et al., 2009; 

Katul et al., 2004; Shaw and Patton, 2003). Due to a combination of economy and accuracy, the 


P a g e | 288 

RANS porous body approach is playing an increasing role in a wide range of vegetation 

aerodynamic analyses including agricultural spray drift and deposition (Da Silva, 2006), forest 

windthrow (Frank and Ruck, 2008), air quality windbreak (shelterbelt) (Rosenfeld et al., 2010), 

and street canyon plantings (Gromke et al., 2008). The current work utilizes a RANS k − ω 

formulation (Sogachev, 2009; Sogachev and Panferov, 2006) that includes vegetation effects 

implemented through source terms in the momentum and turbulence model closure equations. 

Airflow Mathematical Model 

Basic Turbulence Formulation 

The standard way of coping with wide-ranging scales is to apply an averaging process to the 

flowfield and postulate a strategy for the closure problem of unresolved (small scales) effects 

upon the mean (extensive details may be found in Pope (2000)). In the approach taken here, the 

entire effect of turbulent motion on the mean flow is reduced to a contribution to an effective 

viscosity μ + μ t . On dimensional grounds, the eddy viscosity may be written μ t = ρk⁄ ω, 

where k is the turbulent kinetic energy and ω is the specific dissipation rate. The flowfield 

solution requires solving the turbulence closure model equations in addition to the classical 

conservation laws. The rate of change of mean momentum, mean volatile concentration, 

turbulent kinetic energy, and specific dissipation rate are 

ρ D Dt u j = 

∂ 

T u 

∂x ij + S u j , ρ D 

i Dt φ = ∂ 

T φ 

∂x i 

+ S φ 

i 

ρ D Dt k = ∂ 

T k 

∂x i + P − D + S k , ρ D 

i Dt ω = ∂ 

T ω ω 

∂x i + C ω1 

i k P − C ω 

ω2 D + Sω 

k 

The transport terms are 

T ij = − p̂ + 2 3 ρk δ ij + (μ + μ t ) ∂u i 

∂x j 

+ ∂u j 

∂x i 

 

T i φ = μ Sc + μ t 

σ φ 

∂φ 

∂x i 

, T i k = μ + μ t 

σ κ 

∂k 

∂x i 

, T i ω = μ + μ t 

σ ω 

∂ω 

∂x i 

where the pressure includes the hydrostatic contribution p̂ = p + ρgz. The shear production 

and viscous dissipation appearing in the turbulence closure are 

P = μ t 

∂u i 

∂x j 

∂u i 

∂x j 

+ ∂u j 

∂x i 

, D = C μ ρkω 

The purpose of the additional source terms S (·) in the transport equations is to provide modeling 

flexibility. In the present application, S φ allows definition of the release rate and release 


P a g e | 289 

points(s) of volatile material into the air environment, while S j u , S k , and S ω serve to mimic the 

important subscale effects of vegetation on the flow. 

The standard Wilcox k − ω model (Wilcox, 1998) uses the following model constants and 

turbulent Prandtl/Schmidt numbers: C μ = 9⁄ 100, C ω1 = 13⁄ 25, C ω2 = 100⁄ 125, σ k = σ ω = 2, 

and σ φ = 9⁄ 10. The molecular Schmidt number is Sc = μ/ρD φ , where the diffusivity D φ is a 

material property of the passive scaler. 

Vegetation Effects 

Since the details of individual vegetation elements are beyond the resolution requirements of a 

reasonable simulation, the vegetation crown is considered a porous body with through flow. This 

approach treats the effects of the unresolved vegetation on the flow as a local volume-averaged 

body force term due to aerodynamic drag. The conventional parameterization of the body force 

is 

f j = −ρC D Au j u j (no summation). 

Given a particular vegetation type and canopy configuration, the fundamental challenge is to 

determine an appropriate value of the drag index C D A, where C D is the form drag coefficient and 

A is an appropriate frontal area per unit volume. Traditionally, A is taken as the leaf area 

density (LAD), defined as the total one-sided surface area of leaves per unit volume of canopy. 

In the context of vegetation canopy flow, the terms P and D represent “free-air” turbulent 

kinetic energy production and dissipation, respectively. Due to interaction of the vegetation with 

the flow, the production is enhanced by the rate at which the mean flow does work against drag, 

while the dissipation is enhanced by the rate at which turbulent fluctuations do work against drag 

(Finnigan, 2000). Production and dissipation of turbulent kinetic energy due to the unresolved 

vegetation thus take the form 

P v = β p ρc d 

a(u i u i ) 3⁄ 2 , D v = β d ρc d a(u i u i ) 1⁄ 2 k 

where β p and β d are additional turbulence model constants. The baseline form of the source 

terms for vegetation canopy / boundary layer flow are 

S j u = f j , S k = P v − D v , S ω = α p 

ω 

k Pv − α d 

ω 

k Dv 

Analytically-derived model constants are summarized in Sogachev and Panferov (2006) as 

β p = 1, β d = 4, and α p = α d = 1⁄ 2. 

The implementation of vegetation contributions to the turbulence model equations as 

described above is common in practice (Da Silva, 2006; Endalew et al., 2009). However, the 

essential link between turbulent kinetic energy and total dissipation is broken since the 

conservation equation for ω is reduced to the role of an additional contribution of dissipation D 


P a g e | 290 

compared to D v . A modification of the vegetation element drag formulation (Sogachev and 

Panferov, 2006) relies on two assumptions. First, the additional dissipation observed within a 

vegetation canopy compared to open regions is due to the slower decay of dissipation rather than 

increased production of dissipation. Second, at the scale of the vegetation elements, the 

production of turbulent kinetic energy is almost instantly balanced by its dissipation into heat. 

The recommended form (Sogachev, 2009) for vegetation canopy flow is 

S j u = f j , S k = 0, S ω = −(C ω1 − C ω2 )D v ω k 

where the dissipation model constant is β d = 12C μ . 

Numerical Solution 

In the scope of the present work, the mean wind is steady and unidirectional and the flow is in 

equilibrium with the present ground conditions. The left/right and back/front boundaries of the 

simulation domain are periodic pairs. The flow is driven from the sky above by prescribed values 

of ambient quantities and from buoyancy below by differing surface temperatures corresponding 

to shade and full sun. The ceiling is placed at an altitude above ground level of 10H, where H = 

4.6 m is the height of the hedge. The hedge width (shade) and hedge gap (full sun) spacings are 

assumed as W = H and G = H/2, respectively. 

The computational mesh is comprised of Cartesian control volumes. Since the mesh 

spacing implies the averaging volume of the vegetation body force on the airflow, care must be 

taken to ensure that the mesh spacing is large enough to eliminate details of the vegetation 

elements, but remain small enough to accurately resolve the characteristic variations of the 

airflow and scalar concentration. For elevations z < 2H near the vegetation canopy, the mesh 

has a uniform spacing of Δx = Δy = Δz = 0.1 m. For elevations z > 2H, the mesh is 

stretched in the vertical direction at a geometric rate of 1.1. 

Using the commercially available software STAR-CCM+, the equations of motion are 

discretized with a density-based finite volume formulation and solved with an iterative implicit 

scheme. The various vegetation drag source terms are implemented through user-defined field 

functions. The implementation here does not consider variation in the drag force density within 

the canopy (i.e., from trunk to crown). A drag coefficient of C D = 0.3 is typical for vegetation 

and accounts for element deformation and sheltering. For citrus, the leaf area density is A = 

6.5 m −1 (Farooq and Salyani, 2004). 

While air is modeled as a perfect gas with constant transport properties, DMDS is modeled 

as a passive scalar having a constant diffusivity into air of D = 0.0834 cm 2 /s (USEPA, 1994). 

The Schmidt number is Sc = 1.92. A volumetric source prescribes the release point and rate of 

DMDS into the air environment as 

φ 

s release 

= ṁ φ release 

V 


P a g e | 291 

where V is the local mesh cell volume. The release is assumed to have a concentration of pure 

DMDS (φ release = 1 g/g) at a rate of ṁ = 0.2 g/d. Since the airflow itself is periodic across 

the simulation domain, a volumetric sink prescribes absorption of DMDS in a region located 

near the outflow boundary as 

φ 

s absorb 

= − ρφ τ 

V1 

3 

, τ = ⁄ 

(u i u i ) 1⁄ 

2 

to prevent recirculation of the passive scalar. The absorption timescale τ is simply the advection 

time across a mesh cell. 

The boundary condition for winds aloft is based on a log-layer atmospheric boundary layer 

u = u ∗ 

κ log z − d 0 

 

where the von Karman constant is κ = 0.41 and the roughness and displacement heights are 

assumed as z 0 ⁄ H = 0.01 and d 0 ⁄ H = 0.7, respectively. For equilibrium flow, dissipation of 

kinetic energy equals its production, D k = p k , resulting in 

k = u ∗ 2 

, ω = 

C μ 

z 0 

u3 

∗ 

C μ κ(z−d 0 )k 

Given a target altitude z 1 and corresponding wind speed u(z 1 ), the friction velocity u ∗ may be 

computed to yield κ(z) and ω(z). The Florida Automated Weather Network (FAWN) records a 

host of ambient field conditions, including temperature and wind data at an altitude of z 1 = 

10 m above ground level. Typical ambient values of wind speed and temperature are assumed as 

u(z 1 ) = 2.5 m/s and T a = 303 K. Furthermore, it is assumed that the full-shade ground surface 

temperature is ambient T a , while the full-sun ground surface temperature is T s = 323 K. 

Results 

Numerical solution of the flow model described above provides the steady state concentration 

field of DMDS. Figure 1 shows the plume geometry resulting from airflow oblique to the 

hedgerows by a series of isosurfaces. Selected isosurface levels are by decades, with the highest 

concentration value of 10 ppb likely to be in excess of the human olfactory threshold (MSDS, 

2011). Solutions were also computed for airflow parallel and perpendicular to the hedgerows but 

are unlikely scenarios in practice and not shown here. All cases show that with increasing 

distance from the point source, the plume spreads and the maximum concentration decreases, 

both at a rapid rate consistent with the high diffusivity of DMDS into air. Furthermore, the right 

image shows that DMDS concentration in excess of the 10 ppb barrier is confined to a relatively 

small volume as a vertical column having a diameter of ca. H/10. Coverage of 0.1 ppb is 

obtained in a region that is 1H to 2H wide for a distance of 5H downwind. Proposed future work 

includes multiple point sources with an optimization algorithm to drive the source location and 

release rate for desired coverage and concentration. 


P a g e | 292 

References 

Da Silva, A. 2006. A Lagrangian model for spray behaviour within vine canopies. Aerosol 

Science 37:658-674. 

Endalew, A., Hertog, M., Gebreslasie Gebrehiwot, M., Balemans, M., Ramon, H., Nicolaï, B., 

Verboven, P. 2009. Modelling airflow within model plant canopies using an integrated 

approach. Computers and Electronics in Agriculture 66:9-24. 

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Fig. 1. Perspective view of DMDS plume with flow from front-right to back-left, oblique to 

hedge. The light grey isosurfaces indicate the plume boundary encapsulating concentrations of at 

least 0.1 ppb (left), 1 ppb (middle), and 10 ppb (right) for a point release at a rate of 0.2 g/d.

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