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

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Work in Progress We have developed whole genome arrays of Xylella fastidiosa and are presently analyzing gene expression levels between the wild type and rsmA mutant, growth with and without BSA and in vivo versus in vitro conditions. We hope to catalogue the genes whose expressions are associated with biofilm formation, rsmA mutation and infection. Those genes which will overlap with more than one approach will be especially interesting for further analysis. Genetic analysis of these genes therefore should open a window for us into what goes on during the infection process. The rsmA mutant together with its parent is also being assayed for pathogenicity on grapes. In addition, we have constructed several mutants in a select candidate pathogenicity genes and are in the process of analysis these for the effects of the mutations and hence the roles of these genes in the bacterium. REFERENCES 1. Blumer, C., and D. Haas. 2000. FEMS Microbiol Lett 187:53-58. 2. Bonafonte, M. A., C. Solano, B. Sesma, M. Alvarez, L. Montuenga, D. Garcia-Ros, and C. Gamazo. 2000. FEMS Microbiol Lett 191:31-6. 3. Cui, Y., L. Madi, A. Mukherjee, C. K. Dumenyo, and A. K. Chatterjee. 1996. Mol Plant Microbe Interact 9:565-73. 4. de Souza, A. A., et al., 2004. FEMS Microbiol Lett 237:341-53. 5. Deziel, E., Y. Comeau, and R. Villemur. 2001. J Bacteriol 183:1195-204. 6. Gudapaty, S., P. Babitzke, and T. Romeo. 2000. Abstracts of the ASM General Meeting [print] 100:366. 7. Hernandez-Martinez, R., C. K. Dumenyo, and D. A. Cooksey. 2002. Phytopath 92:S36. 8. Jackson, D. W., K. Suzuki, L. Oakford, J. W. Simecka, M. E. Hart, and T. Romeo. 2002. J Bacteriol 184:290-301. 9. Marques, L. L. R., H. Ceri, G. P. Manfio, D. M. Reid, and M. E. Olson. 2002. Plant Dis 86:633-638. 10. Marques, L. L. R., G. P. Manfio, D. M. Reid, H. Ceri, and M. E. Olson. 2001. Phytopath 91:S58. 11. Mukherjee, A., Y. Cui, Y. Liu, C. K. Dumenyo, and A. K. Chatterjee. 1996. Microbiol 142 ( Pt 2):427-34. 12. Newman, K. L., R. P. Almeida, A. H. Purcell, and S. E. Lindow. 2004. Proc Natl Acad Sci U S A 101:1737-42. 13. Osiro, D., L. A. Colnago, A. M. Otoboni, E. G. Lemos, A. A. de Souza, H. D. Coletta Filho, and M. A. Machado. 2004. FEMS Microbiol Lett 236:313-8. 14. Parkins, M. D., H. Ceri, and D. G. Storey. 2001. Mol Microbiol 40:1215-26. 15. Romeo, T. 1998. Mole Microbiol 29:1321-1330. 16. Simpson, A. J. G., et al. 2000. Nature (London) 406:151-157. 17. Throup, J. P., K. P. Francis, T. Romeo, P. Williams, and G. S. A. B. Stewart. 1997. Abstracts of the ASM General Meeting. 18. Van Sluys, M. A., et al., 2003. J Bacteriol 185:1018-26. 19. Yang, H., Y. Liu Mu, and T. Romeo. 1996. J Bacteriol 178:1012-1017. FUNDING AGENCIES Funding for this project was provided by the CDFA Pierce’s Disease and Glassy-winged Sharpshooter Board and the University of California Agricultural Experiment Station. - 177 -
CULTURE-INDEPENDENT ANALYSIS OF ENDOPHYTIC MICROBIAL COMMUNITIES IN GRAPEVINE IN RELATION TO PIERCE’S DISEASE Project Leaders: Donald A. Cooksey Dept. of Plant Pathology University of California Riverside, CA 92521 James Borneman Dept. of Plant Pathology University of California Riverside, CA 92521 Reporting Period: The results reported here are from work conducted from October 1, 2003 to September 30, 2004. ABSTRACT Culture-independent, nucleic acid-based methods of assessing microbial diversity in natural environments have revealed far greater microbial diversity than previously known through traditional plating methods. If true for grapevines, then this has important consequences for Pierce’s disease management strategies that involve the establishment of introduced bacteria systemically in the grapevine xylem. Such establishment will likely be influenced by the presence of yet uncharacterized microorganisms, and knowledge of endophytic communities and their dynamics will therefore be important to the successful implementation of these strategies. In addition, analysis of microbial community composition in different hosts and conditions could lead to the identification of new biological control agents. We are employing a novel method, called oligonucleotide fingerprinting of rRNA genes (OFRG), that was recently developed by the Co-PI for analyzing microbial community composition in environmental samples. INTRODUCTION In recent years, culture-independent, nucleic acid-based methods of assessing microbial diversity in natural environments have revealed far greater microbial diversity than previously known through traditional plating methods (Amann et al., 1995). This is true for water, soil, the plant rhizosphere, and the plant leaf surface (Yang et al. 2001). A recent culture-independent analysis of bacterial populations inside of citrus plants in relation to Xylella fastidiosa also suggested that bacterial endophytic populations are much more diverse than previously realized (Araújo et al., 2002). If true for grapevines, then this has important consequences for Pierce’s disease management strategies. Several strategies are being investigated to biologically control Xylella fastidiosa in grapevines, including the use of antibiotic-producing endophytes (Kirkpatrick et al., 2001), endophytes that disrupt cell-to-cell signaling by the pathogen (Lindow, 2002), endophytes that degrade xanthan gum (Cooksey, 2002a), and the use of nonpathogenic strains of Xylella for competitive exclusion of pathogenic strains (Cooksey, 2002b). These strategies have in common the need to establish an introduced strain systemically in the grapevine xylem. Such establishment will likely be influenced by the presence of yet uncharacterized microorganisms, and knowledge of endophytic communities and their dynamics will therefore be important to the successful implementation of these strategies. In addition, analysis of microbial community composition in different hosts and conditions could lead to the identification of new biological control agents. We are employing a novel method that was recently developed by the Co-PI for analyzing microbial community composition in environmental samples. This method can be used to characterize both bacterial and fungal communities (Valinsky et al., 2002a; 2002b). Previous culture-independent methods, such as denaturing gradient gel electrophoresis (DGGE), generate only superficial descriptions of microbial community composition (Araújo et al., 2002). A far more complete view of total microbial community composition can be achieved by amplifying, cloning, and sequencing of conserved rRNA genes from the hundreds or thousands of microorganisms present in an environmental sample, but this is prohibitively expensive for any significant number of experiments. The new methodology, called oligonucleotide fingerprinting of rRNA genes (OFRG), represents a significant advance in providing a cost-effective means to extensively analyze microbial communities. The method involves the construction of clone libraries of rDNA molecules that are PCR amplified from environmental DNA, arraying of the rDNA clones onto nylon membranes or specially-coated glass slides, and subjecting the arrays to a series of hybridization experiments using 27 different end-labeled DNA oligonucleotide discriminating probes (Borneman et al., 2001). The process generates a hybridization fingerprint and identification for each clone that is essentially like sequencing the individual clones. The state of knowledge of the relationship between Xylella fastidiosa and the resident endophytic flora of grapevines is at a very early stage. Work to date has been limited to the culturing of endophytes from grapevines, but even this has led to the realization that grapevine xylem sap contains a complex community of microorganisms. Bell et al. (1995) cultured over 800 bacterial strains from grapevine xylem fluid in Nova Scotia. Dr. Bruce Kirkpatrick has also isolated several hundred bacterial strains from grapevine xylem fluid in two counties of California (Kirkpatrick et al., 2001). In citrus, the cultureindependent DGGE method of microbial community analysis was compared with culturing of endophytes in relation to the citrus variegated chlorosis strain of X. fastidiosa (Araújo et al., 2002). It was found that DGGE detected the major bacteria that were cultured from citrus xylem, but it also detected other bacterial species that had not been cultured. In addition, this method showed differences in microbial communities in different plant varieties, and most importantly, between citrus that was infected vs. non-infected with X. fastidiosa. This provides support to our hypothesis that there are likely to be important - 178 -
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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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Page 79 and 80: POPULATION DYNAMICS AND INTERACTION
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Page 89 and 90: Table 2. Mortality and flight perfo
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Page 93 and 94: REFERENCES Brodbeck, B. V., P. C. A
Page 95 and 96: Cohorts H. coagulata neonates were
Page 97 and 98: REFERENCES Blua, M. J. and D. J. W.
Page 99 and 100: Figure 2 shows the average numbers
Page 101 and 102: REPRODUCTIVE BIOLOGY AND PHYSIOLOGY
Page 103 and 104: REFERENCES Blua, M. J., Phillips, P
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Page 109 and 110: RESULTS Some plant families had no
Page 111 and 112: Table 5. Winter, spring and summer
Page 113 and 114: 16S rDNA is by far the most sequenc
Page 115 and 116: DNA MICROARRAY AND MUTATIONAL ANALY
Page 117: Inhibition of biofilm is BSA concen
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Page 123 and 124: P (CFU P OBJECTIVE 1. Determine the
Page 125 and 126: Purcell, AH and SR Saunders. 1999a.
Page 127 and 128: Figure 1: Southern blot of tolC::ap
Page 129 and 130: Project Leader: David Gilchrist Dep
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Page 133 and 134: RESULTS Construction of cDNA Librar
Page 135 and 136: CONCLUSIONS Genetic resistance and
Page 137 and 138: Mutagenesis of Xylella The EZ::TN T
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Page 141 and 142: Project Leader: Michele M. Igo Sect
Page 143 and 144: outer membrane fractions using two-
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Page 147 and 148: DEVELOPMENT OF SSR MARKERS FOR GENO
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Page 153 and 154: wild-type cells was not conclusive
Page 155 and 156: DETERMINATION OF GENES CONFERRING H
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Page 161 and 162: GENOME-WIDE IDENTIFICATION OF RAPID
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Page 165 and 166: EFFECTS OF CHEMICAL MILIEU ON ATTAC
Page 167 and 168: 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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