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

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MICROBIAL CONTROL OF THE GLASSY-WINGED SHARPSHOOTER WITH ENTOMOPATHOGENIC FUNGI Project Leader: Harry K. Kaya Dept. of Nematology University of California Davis, CA 95616 Researcher: Surendra K. Dara Shafter Research and Extension Center University of California-Davis Shafter, CA 93263 Cooperator: Michael R. McGuire USDA, ARS Shafter Research and Extension Center Shafter, CA 93263 Reporting Period: The results reported here are from work conducted from April 2004 to September 2004. ABSTRACT Objectives of our study were to search for fungal pathogens of the glassy-winged sharpshooter (GWSS), Homalodisca coagulata (Say) and evaluate their potential against the host. Searches within citrus orchards in Tulare and Riverside counties revealed no natural infections of entomopathogenic fungi in GWSS populations. Entomopathogenic fungi were also absent in cadavers of GWSS periodically collected from Riverside citrus orchards (courtesy CDFA) when incubated in the laboratory under ideal conditions for fungal emergence. However, about 140 isolates of Beauveria bassiana (Balsamo) Vuillemin and four isolates of Metarhizium anisopliae (Metschnikoff) Sorokin, both hyphomycetous fungi, were isolated from soil in GWSS habitats and other insect hosts. Some of these isolates along with a Weslaco isolate of B. bassiana from GWSS and a commercial B. bassiana isolate have been tested against GWSS. Preliminary results indicate that GWSS is susceptible to high concentrations of these fungi. INTRODUCTION The glassy-winged sharpshooter (GWSS), Homalodisca coagulata (Say), native to the southeastern United States, is a serious pest of the California grape industry because it vectors <strong>Xylella</strong> fastidiosa (Wells et al. 1987), a xylem-limited bacterium that causes Pierce’s disease (PD). Although PD has been in California for a long time, the introduction and rapid spread of GWSS made the situation worse. In addition to grapes, GWSS has a wide host range and spreads various diseases in those hosts caused by X. fastidiosa. Vector control or avoidance has been a key tactic in controlling PD. Widely practiced chemical control with imidacloprid and application of kaolin particles have their limitations. While kaolin particles, although non-toxic, can leave unwanted deposits on the harvested grape bunches, chemical insecticides have undesirable effects including human health, impact on non-target organisms, and environmental concerns. Moreover, use of chemical insecticides in citrus disrupts the successful, long-term control afforded by IPM of many different citrus pests (Grafton- Cardwell and Kallsen 2001). Use of microbial agents, such as entomopathogenic fungi, can be a viable alternative that is compatible with IPM practices. Entomopathogenic fungi invade the host by penetrating through the integument and are appropriate candidates for GWSS that has piercing and sucking mouthparts. Entomopathogenic fungi have been isolated from GWSS (Mizell and Boucias 2002, Jones - personal communication) and other cicadellids (Galaini-Wraight et al. 1991, Hywel-Jones et al. 1997, Magalhaes et al. 1991, Matsui et al. 1998, McGuire et al. 1987). The purpose of our study is to discover additional isolates of entomopathogenic fungi active against GWSS. OBJECTIVES 1. Conduct surveys to find fungal infections in GWSS populations or insects closely related to GWSS and isolate soilborne entomopathogens from GWSS habitats. 2. Culture and isolate the fungi and evaluate their pathogenicity against GWSS. 3. Evaluate the host range of fungi that infect GWSS. 4. Conduct small-scale field tests to evaluate selected pathogens against GWSS on citrus in fall and winter. RESULTS Natural Infections in GWSS Populations Citrus orchards in Tulare and Riverside counties were surveyed, in vain, for infected GWSS. GWSS cadavers from CDFA collections in the Riverside area were periodically obtained and incubated in the laboratory for fungal development. No entomopathogenic fungus has so far been found from these cadavers. However, cultures of Beauveria bassiana (Balsamo) Vuillemin from infected GWSS collected in Texas by Jones and Hirsutella spp collected in Florida by Mizell and Boucias were received in the past two months for testing against California GWSS. - 349 -
Isolation of Fungal Pathogens Soil samples were collected from an organic citrus orchard and a conventional pomegranate orchard in Tulare Co, CA and a citrus orchard at AgOps at UC Riverside. Fungal pathogens were isolated using larvae of the greater wax moth, Galleria mellonella L. and by soil plating on selective media. Waxworms were incubated in Petri plates with moist soil samples and fungal pathogens were isolated from cadavers. Alternatively, aliquots of soil suspensions were plated on media selective for B. bassiana and Metarhizium anisopliae (Metschnikoff) Sorokin. So far, 140 B. bassiana isolates and 4 M. anisopliae isolates have been isolated (Table 1). Additionally, B. bassiana was also isolated from the California harvester ant, Pogonomyrmex californicus Buckley, collected in Shafter, CA and the three-cornered alfalfa hopper, Spissistilus festinus (Say), collected in Parlier, California. Fungal isolates were cultured on selective and non-selective media to multiply the inoculum. Table 1. Fungal pathogens isolated from citrus and pomegranate orchards and infected insects Source Method B. bassiana M. anisopliae Organic citrus in Tulare Co Waxworm bait 37 - Pomegranate in Tulare Co Waxworm bait 3 4 Riverside citrus Waxworm bait 78 - Riverside citrus Selective media 22 - California harvester ant Selective medium 1 N/A Three-cornered alfalfa hopper Selective medium 1 N/A Pathogenicity of Entomopathogenic Fungi to GWSS Laboratory-reared or field-collected GWSS adults supplied by CDFA, Arvin were used for the bioassays. GWSS were either placed at -5 o C for 5 min or exposed to CO 2 for 15 sec to immobilize them and were inoculated by rolling them in a 10 µL drop of conidial suspension. Controls were treated with 0.01% of SilWet, an adjuvant used for preparing conidial suspensions. GWSS were individually incubated in a Petri plate with an excised citrus leaf and a moist filter paper. Petri plates were placed in a plastic box with moist paper towels and incubated at 27 o C and 16:8 L:D photophase. GWSS were observed daily for mortality. Dead GWSS were surface sterilized in 3% sodium hypochlorite solution followed by rinsing in deionized water and incubated in sealed Petri plates on water agar or moist filter paper at 27 o C in the dark. Bioassay 1 The isolate of B. bassiana from P. californicus (PcBb1) was tested against laboratory-reared GWSS at four concentrations 10 1 , 10 3 ,10 5 , and 10 7 conidia/ml in comparison with controls. Each treatment and control had 10 adult GWSS. Infections were observed only at higher concentrations with 50% infection in GWSS treated with 10 7 conidia/ml and 10% in those treated with 10 5 conidia/ml. Bioassay 2 Five B. bassiana isolates and a M. anisopliae isolate were tested against field-collected GWSS at four concentrations of 10 3 , 10 5 ,10 7 , and 10 9 (or 10 8 in case of M. anisopliae) conidia/ml along with untreated and SilWet (0.01%) treated controls. Isolates of B. bassiana included one from P. californicus (PcBb1), two from soil samples from citrus orchards in Tulare (GmBb25) and Riverside (GmBb41) counties, CA, one from H. coagulata in Weslaco, TX (TxBb) and a commercial isolate (designated GHA). The isolate of M. anisopliae (GmMa1) was from a soil sample from the pomegranate orchard in Tulare Co, CA. Each treatment and controls had 20 GWSS. Although all tested isolates were infective (Figures 1 and 2), all GWSS in this bioassay, including controls, suffered from a high mortality. Figure 1. Pathogenicity of B. bassiana and M. anisopliae to GWSS - 350 -
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IMPACT OF HOST PLANT XYLEM FLUID ON
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0.18 600 Optical density 0.16 0.14
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OPTIMIZING MARKER-ASSISTED SELECTIO
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esistant and susceptible genotypes
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MAP BASED IDENTIFICATION AND POSITI
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esistant genotypes are in process a
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BREEDING PIERCE’S DISEASE RESISTA
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Progeny from crosses of field resis
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a = (1=low, 4= high); b = (1=green,
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v.8) for differences due to the pla
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SHARPSHOOTER FEEDING BEHAVIOR IN RE
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correlated with all other findings
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EFFECTS OF FEEDING SUBSTRATE ON RET
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REFERENCES Bextine, B. and T. Mille
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will also provide insights into the
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OBJECTIVES 1. Determine glassy-wing
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GWSS per 30 s sweep (seasonal avera
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ELISA for the presence of GWSS egg
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Project Leader: Thomas P. Freeman E
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Freeman, T. P., R. A. Leopold, D. R
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pathogen, when they move into viney
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A NOVEL IMMUNOLOGICAL APPROACH FOR
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1.6 GWSS Adults That Fed on Protein
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Hagler, J.R. & S.E. Naranjo. 2004.
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RESULTS: Oviposition Survey Wild gr
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Host specificity testing: No-choice
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currently employed morphological ch
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increase our present understanding
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BIOLOGY AND MORPHOMETRIC ANALYSIS O
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Table 1. Mean a developmental durat
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EFFECTS OF USING CONSTANT AND CYCLI
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REFERENCES Gautam, R. D. 1986. Effe
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PARASITISM OF THE GLASSY-WINGED SHA
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Table 1. Parasitism by G.ashmeadi o
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Project Leader: Robert F. Luck Dept
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A more interesting analysis using t
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MYCOPATHOGENS AND THEIR EXOTOXINS I
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POPULATION DYNAMICS AND INTERACTION
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No egg masses were recorded on olea
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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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Page 285 and 286: REFERENCES Hoddle, M. S. and S. V.
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Page 307 and 308: Al-Wahaibi, A.K., Owen, and J. G. M
Page 309 and 310: patterns differed among the lines,
Page 311 and 312: Punja, Z.K. 2001. Genetic engineeri
Page 313 and 314: mind, we conducted an additional st
Page 315 and 316: REFERENCES Toscano, N., Byrne, F.,
Page 317 and 318: RESULTS AND CONCLUSIONS The program
Page 319 and 320: COMPATIBILITY OF INSECTICIDES WITH
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Impact Of Host Plant Xylem Fluid On Xylella Fastidiosa Multiplication ...

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