Impact Of Host Plant Xylem Fluid On Xylella Fastidiosa Multiplication ...

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DEVELOPMENT OF AN ARTIFICIAL DIET FOR THE GLASSY-WINGED SHARPSHOOTER Project Leader: Thomas A. Coudron USDA, ARS, BCIRL Columbia, MO 65203 Collaborators: Walker A. Jones USDA, ARS, KDLG Subtropical Agric. Res. Center Beneficial Insects Research Unit Weslaco, TX 78596 Researcher: Cynthia L. Goodman USDA, ARS, BCIRL Columbia, MO 65203 Elaine Backus USDA, ARS, PWA SJV Agricultural Sciences Center Parlier, CA 93648 Wayne Hunter USDA, ARS U.S. Horticultural Research Laboratory Subtropical Insects Research Ft. Pierce, FL 34945 Reporting Period: Funding for the study was initiated in October, 2004 and the project is in the start-up phase at the time of this reporting. ABSTRACT The intent of this project is to develop an artificial rearing system for the glassy-winged sharpshooter (Homalodisca coagulata) (GWSS), the primary vector of Pierce’s Disease (Xylella fastidiosa) (PD). In order to accomplish this, a diet delivery system will first be developed and then used to test artificial diets. Diet formulations will be based, in part, on previous studies performed by Cohen (2002) using GWSS, as well as on artificial diets developed for other Hemiptera (Mitsuhashi, 1979; Coudron et al., 2002) and on the xylem chemistry of GWSS host plants (Andersen, et al., 1992). Diets will be evaluated based on their effects on life history analyses, reproductive rate and intrinsic rate of increase of GWSS. Another aspect of our project involves investigating nitrogen source(s) for GWSS, as that may represent a nutrient limitation for xylem feeders. Two potential sources for nitrogen, i.e. proteins or peptides, will be studied by determining the fate of dietary proteins/peptides (Brandt, et al., 2004) and the ability of salivary and midgut proteolytic enzymes to digest proteins/peptides (Wright, et al., 2004). In this way, we will identify the role(s) proteins and peptides play in GWSS nutrition and their potential uses in artificial diet formulations. INTRODUCTION The formulation of an artificial diet for GWSS will greatly enhance the ability of researchers to rear this insect. Presently, the rearing of GWSS is labor-intensive and costly because of its dependence on the propagation of appropriate host plants, with researchers often needing to propagate several species of plants to enable them to rear GWSS under optimal conditions. The development of an artificial diet would likely be more cost effective and portable, increasing the availability of high quality insects for Pierce’s disease researchers and decreasing the costs and time-constraints associated with maintaining the insect in culture. The increased accessibility of GWSS to researchers can lead to more rapid developments in novel control measures for this major vector of PD, with these new measures being directly applied by growers. Furthermore, the coupling of an artificial diet with a suitable delivery system can lead to an improved understanding of the relationship between GWSS nutrition and other PD-related issues (including GWSS’ varying abilities to acquire/maintain/transmit infectious Xf under different circumstances, e.g., via artificial membranes vs. plants, Redak et al., 2004). In addition, the diet delivery system alone would have other potential uses such as in studying the interactions between GWSS, Xf, and the host plant, as well as in testing potential anti-GWSS and anti-Xf control agents. This could be accomplished by incorporating into the feeding system: 1) selected host plant-associated compounds; 2) media containing the causative agent of PD (Xylella fastidiosa, Xf) (although some studies have suggested that Xf acquired via an artificial membrane by GWSS may not be infectious, Redak et al., 2004); 3) control agents including anti-GWSS or -Xf compounds (such as proteins to be engineered into host plants to control either GWSS or Xf; Dandekar et al., 2003; Lin, 2003; Meredith and Dandekar, 2003; Reisch et al., 2003) or anti- GWSS microbials (Kaya, 2003; Mizell & Boucias, 2003). In summary, the development of an artificial diet and a corresponding delivery system for GWSS could lead to insights that can be used to generate improved methods for controlling GWSS and, therefore, Pierce’s disease. An important part of our project also involves gaining a better understanding of the digestive physiology of GWSS. This will be investigated by focusing on the role proteins and peptides play in GWSS nutrition, as these or similar compounds have been isolated from some xylem fluids (Cohen, 2002; Jain and Basha, 2003; Rep et al., 2003). We will accomplish this by determining the extent to which GWSS can digest proteins and peptides, as well as elucidating the fate of specific ingested proteins in GWSS. This information will be directly used in the generation of an optimal artificial diet for GWSS. Furthermore, GWSS’ ability to degrade proteins/peptides will also shed light on the degree to which GWSS can disable defensive proteins/peptides in plants, which is important when dealing with salivary enzymes that are secreted into plant tissues and could alter anti-Xf defense components (e.g., either naturally occurring or genetically engineered proteins/peptides; Lin, 2003; Meredith and Dandekar, 2003; Reisch et al., 2003). This knowledge could be used when modifying target plants such as grapevines to improve their resistance against Pierce’s disease (PD). Therefore, our investigation into nutritional requirements will not only aid us in the development of a suitable artificial diet for GWSS, but - 91 -
will also provide insights into the potential efficacies of anti-PD plant modifications. OBJECTIVES 1. Develop an artificial diet delivery system for rearing the glassy-winged sharpshooter (GWSS), Homalodisca coagulata. 2. Formulate and evaluate an artificial diet for the development and reproduction of GWSS. 3. Investigate the utilization of proteinaceous components in the food stream of GWSS in order to refine and improve the artificial diet using physiological and proteomic/genomic approaches. RESULTS AND CONCLUSIONS This project has just been funded. Preparation of quarantine facilities is complete and the identification of insect cultures to be used in our studies is underway. The process to hire an additional researcher has been initiated. Preliminary experiments, in collaboration with Jones and Setamou at ARS in Weslaco, have demonstrated continuous feeding by adult GWSS for over 30 days on artificial diets presented through a specialized feeding tube. Additionally, differences in survival have been noted as a result of changes in amino acid concentration and composition within the diet. REFERENCES Anderson PC, Brodbeck BV, Mizell RF, 1992. J. Insect Physiol. 38:611-612. Brandt SL, Coudron TA, Habibi J, Brown GR, Ilagan OM, Wright MK, Wagner RM, Backus EA, Huesing JE, 2004. Curr. Microbiol. 48:1-9. Cohen AC, 2002. Proc. Pierce’s Disease Research Symp., pp. 83-85. Coudron TC, Wittmeyer JL, Kim Y, 2002. J. Econ. Entomol. 95(6), 1159-1168. Dandekar A, Gupta G, McDonald K, Hong-Geller E., 2003. Proc. Pierce’s Disease Research Symp., pp. 87-88. Jain AK, Basha SM, 2003. African J. Biotechnol. 2:66-70. Kaya H., 2003. Proc. Pierce’s Disease Research Symp., pp. 263-264. Lin H, 2003. Proc. Pierce’s Disease Research Symp., 158-159. Meredith C, Dandekar A, 2003. Proc. Pierce’s Disease Research Symp., pp. 23-25. Mitsuhashi J, 1979. In: Leafhopper Vectors and Plant Disease Agents (Maramarosch, K, Harris, KF, eds). Academic Press: NY; pp. 369-412. Mizell RF, Boucias DG, 2003. Proc. Pierce’s Disease Research Symp., pp. 274-275. Reisch B, Walker A, Kikkert JR, 2003. Proc. Pierce’s Disease Research Symp., pp. 30-32. Rep M, Dekker HL, Vossen JH, de Boer AD, Houterman PM, de Koster CG, Cornelissen BJC, 2003. FEBS Letters 534, 82- 86. Wright, M.K., Wagner, R.M., Huesing, J.E., Brandt, S.L., Habibi, J., Coudron, T.A., Thoma, R., Backus, E.A. 2004. J. Insect Physiol. (submitted). FUNDING AGENCIES Funding for this project was provided by the CDFA Pierce’s Disease and Glassy-winged Sharpshooter Board. - 92 -
Page 1 and 2: IMPACT OF HOST PLANT XYLEM FLUID ON
Page 3 and 4: 0.18 600 Optical density 0.16 0.14
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Page 23 and 24: v.8) for differences due to the pla
Page 25 and 26: SHARPSHOOTER FEEDING BEHAVIOR IN RE
Page 27 and 28: correlated with all other findings
Page 29 and 30: EFFECTS OF FEEDING SUBSTRATE ON RET
Page 31: REFERENCES Bextine, B. and T. Mille
Page 35 and 36: OBJECTIVES 1. Determine glassy-wing
Page 37 and 38: GWSS per 30 s sweep (seasonal avera
Page 39 and 40: ELISA for the presence of GWSS egg
Page 41 and 42: Project Leader: Thomas P. Freeman E
Page 43 and 44: Freeman, T. P., R. A. Leopold, D. R
Page 45 and 46: pathogen, when they move into viney
Page 47 and 48: A NOVEL IMMUNOLOGICAL APPROACH FOR
Page 49 and 50: 1.6 GWSS Adults That Fed on Protein
Page 51 and 52: Hagler, J.R. & S.E. Naranjo. 2004.
Page 53 and 54: RESULTS: Oviposition Survey Wild gr
Page 55 and 56: Host specificity testing: No-choice
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Page 61 and 62: BIOLOGY AND MORPHOMETRIC ANALYSIS O
Page 63 and 64: Table 1. Mean a developmental durat
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Page 67 and 68: REFERENCES Gautam, R. D. 1986. Effe
Page 69 and 70: PARASITISM OF THE GLASSY-WINGED SHA
Page 71 and 72: Table 1. Parasitism by G.ashmeadi o
Page 73 and 74: Project Leader: Robert F. Luck Dept
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Page 79 and 80: POPULATION DYNAMICS AND INTERACTION
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EXPLORATION FOR FACULTATIVE ENDOSYM
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Although Baumannia and Wolbachia we
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EFFECTS OF SUBLETHAL DOSES OF IMIDA
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Table 2. Mortality and flight perfo
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A NOVEL METHOD TO INDUCE OVIPOSITIO
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REFERENCES Brodbeck, B. V., P. C. A
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Cohorts H. coagulata neonates were
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REFERENCES Blua, M. J. and D. J. W.
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Figure 2 shows the average numbers
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REPRODUCTIVE BIOLOGY AND PHYSIOLOGY
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REFERENCES Blua, M. J., Phillips, P
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RESULTS Some plant families had no
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Table 5. Winter, spring and summer
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16S rDNA is by far the most sequenc
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DNA MICROARRAY AND MUTATIONAL ANALY
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Inhibition of biofilm is BSA concen
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CULTURE-INDEPENDENT ANALYSIS OF END
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Borneman, J., M. Chrobak, G. Della
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P (CFU P OBJECTIVE 1. Determine the
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Purcell, AH and SR Saunders. 1999a.
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Figure 1: Southern blot of tolC::ap
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Project Leader: David Gilchrist Dep
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III. Production of transgenic plant
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RESULTS Construction of cDNA Librar
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CONCLUSIONS Genetic resistance and
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Mutagenesis of Xylella The EZ::TN T
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ISOLATION OF BACTERIOPHAGES SPECIFI
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Project Leader: Michele M. Igo Sect
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outer membrane fractions using two-
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1995; Purcell and Saunders, 1999).
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DEVELOPMENT OF SSR MARKERS FOR GENO
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Strain Name Host of Origin County o
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ROLE OF ATTACHMENT OF XYLELLA FASTI
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wild-type cells was not conclusive
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DETERMINATION OF GENES CONFERRING H
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S1 S2 S3 S4 Nb123456789bb123456789b
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MULTILOCUS SEQUENCE TYPING TO IDENT
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GENOME-WIDE IDENTIFICATION OF RAPID
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Americas and since most of the plan
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EFFECTS OF CHEMICAL MILIEU ON ATTAC
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CONCLUSIONS Our overall objective i
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RESULTS Objective 1. We conducted t
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Redak, R. A., Purcell, A. H., Lopes
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In parallel, an “activating trans
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PATTERNS OF XYLELLA FASTIDIOSA INFE
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% of vessels 100 80 60 40 20 Mugwor
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DOCUMENTATION AND CHARACTERIZATION
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Magnolia002 showed more identity (9
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PLASMID ADDICTION AS A NOVEL APPROA
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GENETIC VARIABILITY OF XYLELLA FAST
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probing activities). In preliminary
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the slow release valve was opened a
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12. Hopkins DL (1977) Diseases caus
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The almost complete absence of info
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QUANTIFYING LANDSCAPE-SCALE MOVEMEN
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Table 1. The mean (±SD) ELISA read
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EPIDEMIOLOGICAL ASSESSMENTS OF PIER
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multiply to relatively high (easily
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SPATIAL DATABASE CREATION AND MAINT
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Project Leader: Thomas M. Perring D
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Figure 2. Individual leaves from a
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The state variables, process functi
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DEVELOPMENT OF A FIELD SAMPLING PLA
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Figure 1. Three main dispersion pat
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OBJECTIVES 1. Track the movement of
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REFERENCES 1. Beard, C.B., Cordon-R
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cases, positive phloem samples were
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EXPLOITING XYLELLA FASTIDIOSA PROTE
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Figure 2. Polymer disk accumulation
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CHARACTERIZATION OF NEONICOTINOIDS
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EVALUATION OF RESISTANCE POTENTIAL
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Project Leader: Doug Cook Dept. of
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Based on the in silico analysis, de
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CONTROL OF PIERCE’S DISEASE THROU
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Figure 1. Oleander ‘White’ afte
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RESULTS During the reporting period
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DEVELOPMENT OF AN ARTIFICIAL DIET A
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DESIGN OF CHIMERIC ANTI-MICROBIAL P
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Neutrophil elastase Cecropin B Chim
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and WTXb is very low (0.00-0.40%) a
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100 100 0.056 North America 100 0.0
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GENETIC DIFFERENTIATION AMONG GEOGR
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Table 1. Single-populations descrip
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MOLECULAR DISTINCTION BETWEEN POPUL
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These novel observations strongly s
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SEQUENCE DIVERGENCE IN TWO MITOCHON
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Table 3. Pairwise sequence distance
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DEVELOPMENT OF MOLECULAR DIAGNOSTIC
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A. B. Relative Density of SCAR Band
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THE ALIMENTARY TRACK OF GLASSY-WING
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We have dissected and identified al
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REALIZED LIFETIME PARASITISM AND TH
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REPRODUCTIVE AND DEVELOPMENTAL BIOL
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Mean adult longevity (days) 25 20 1
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the proposed exploratory work; thes
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SEARCHING FOR AND COLLECTING EGG PA
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REFERENCES Hoddle, M. S. and S. V.
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some Gram(+) bacteria, but are inac
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7. Hultmark, D., et al., Insect Imm
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Isolation of Fungal Pathogens Soil
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IDENTIFICATION OF MECHANISMS MEDIAT
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concentrations of 1.5 x 10 7 bacter
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anchor it in the outer membrane (sh
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SYMBIOTIC CONTROL OF PIERCE’S DIS
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MANAGEMENT OF PIERCE’S DISEASE OF
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pfF mutants are taken up by insects
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Simpson, A. J. G., F. C. Reinach, P
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Al-Wahaibi, A.K., Owen, and J. G. M
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patterns differed among the lines,
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Punja, Z.K. 2001. Genetic engineeri
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mind, we conducted an additional st
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REFERENCES Toscano, N., Byrne, F.,
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RESULTS AND CONCLUSIONS The program
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COMPATIBILITY OF INSECTICIDES WITH
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More tests are in progress to addre
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Impact Of Host Plant Xylem Fluid On Xylella Fastidiosa Multiplication ...

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