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

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Objective 3 Six months after inoculation (see objective 1), we also noticed an unexpectedly high percentage (35%) of inoculated vines that did not develop typical PD symptoms. One might have expected no more than 5% or so of the mutants to be non pathogenic. We sequenced the Xf DNA, flanking the Tn5 element in order to determine the specific location of the Tn5 insertion in each putatively “avirulent” mutant. Table 1 summarizes the categories of the genes that were knocked out in the avirulent Xf mutants. We then chose to further characterize insertions in open reading frames (ORFs) that code for proteins that have possible roles in Xf virulence/colonization or ORFs with no known function. Tn5 insertions in known “housekeeping” genes were not screened further. Three new Chardonnay grapevines growing in pots in the greenhouse were inoculated with each Xf mutant of interest as well as the appropriate controls. The experiment was done in duplicate. The rate of symptom development or lack there of, is being monitored as we described in objective 1. After 14 weeks, petiole samples at the point of inoculation (poi) and 12 inches above the poi will be taken from each mutant and control vines. Xf cells will be cultured from those samples in order to assess bacterial population and colonization. The insertion sites will be further confirmed by PCR. Objective 4: Develop a Xf/E. coli Shuttle that is Stable in planta. A plasmid DNA fraction was isolated from the UCLA strain of Xf and subjected to in vitro mutagenesis using the transposome technology that was previously used to create our Tn5 Xf library. This DNA was electroporated in the UCLA strain and 4 kan R colonies were obtained. These were sequenced and found to be insertions in the small 1.3kb plasmid that we previously attempted to develop as a Xf/E. coli shuttle vector. These Tn5 insertions were in different areas of the native plasmid so we tested the relative stability of these plasmids by culturing the transformants on PD3 medium with and without kanamycin. After 3 passages on non-selective media the colonies were transferred to PD3 media containing kanamycin and no colonies were observed on the plates. This indicates that the plasmids containing the Tn5 insertions were lost upon culture in non-selective medium, results that were the same as our previous attempts to engineer these small native plasmids as shuttle vectors. Future work will focus on a similar strategy to construct a shuttle vector from the 5.8kb plasmid in the UCLA strain, with the hope that this construct might be stably maintained in Xf without antibiotic selection. REFERENCES Guilhabert, M. R and Kirkpatrick, B. C. 2003. Transformation of Xylella fastidiosa with broad host range RSF1010 derivative plasmids. Mol. Plant Path. 4, 279-285. Guilhabert, M. R., Hoffman, L. M., Mills, D. A. and Kirkpatrick, B. C. 2001.Transposon mutagenesis of Xylella fastidiosa by electroporation of Tn5 synaptic complexes. Mol. Plant-Microbe Interact. 14:701-706. Hill, B. L. and Purcell, A. H. 1995. Multiplication and movement of Xylella fastidiosa within grapevine and four other plants. Phytopath. 85, 1368-1372. Purcell, A. H. and Saunders, S. R. 1999. Fate of Pierce's disease strains of Xylella fastidiosa in common riparian plants in California. Plant Dis. 83, 825-830. Simpson, A. J. G. et al. 2000. The genome sequence of the plant pathogen Xylella fastidiosa. Nature 406: 151-157. Van Sluys et al., 2003. Comparative analyses of the complete genome sequences of Pierce’s disease and citrus variegated cholorosis strains of Xylella fastidiosa. J. Bacterial. 185, 1018-1026. FUNDING AGENCIES Funding of this project was provided by the CDFA Pierce’s Disease and Glassy-winged Sharpshooter Board and the University of California Pierce’s Disease Grant program. - 205 -
DEVELOPMENT OF SSR MARKERS FOR GENOTYPING AND ASSESSING THE GENETIC DIVERSITY OF XYLELLA FASTIDIOSA IN CALIFORNIA Project Leaders: Hong Lin Crop Diseases, Pests & Genetics SJVAC, USDA-ARS Parlier, CA 93648 Andrew Walker Dept. of Viticulture & Enology University of California Davis, CA 95616 Collaborator: Edwin Civerolo SJVAC, USDA-ARS Parlier, CA 93648 Reporting period: The results reported here are from work conducted from March 2004 to September 2004. ABSTRACT Recently available genomic sequences of four Xylella fastidiosa strains (PD, CVCD, ALSD and OLSD) facilitate genome wide searches for identifying Simple Sequence Repeat (SSR) loci. Sixty SSR loci were selected for SSR marker development. We designed and validated 34 SSR primers with good reliability and specificity. These SSR primers showed various levels of polymorphism with average 11.3 alleles per locus among 43 Xylella fastidiosa isolates. These multi-locus SSR markers, distributed across the entire genome, are a useful tool for pathogen genotyping, population genetics and molecular epidemiology studies. INTRODUCTION Xylella fastidiosa (Xf) causes economically important diseases that results in significant losses in several agricultural, horticultural and landscape crops, including grape Pierce’s disease (PD), almond leaf scorch disease (ALSD), citrus variegated chlorosis disease (CVCD) and oleander leaf scorch disease (OLSD). Recent introduction and establishment of the invasive and more effective vector, the Glassy-winged Sharpshooter (Homalodisca coagulata, GWSS) has had a great impact on the California grape industry. Host plant resistance is a critical component of integrated crop management. If this insect becomes widely established, the use of resistant varieties may become the most reliable and effective way to control PD. However, the durability of resistant grape plants depends upon the variability and adaptability of the pathogen population and its interaction with the resistance genes of plants. Most resistance studies are performed by screening against a subpopulation of a given pathogen, and neglect that fact that changes in pathogen population structure that may lead to resistance breakdown. It is clear that pathogen populations with a high evolutionary potential are more likely to overcome host genetic resistance than pathogen populations with a low evolutionary potential (MacDonald and Linde, 2002). The risk becomes even greater with the recent establishment of a more effective vector, the GWSS, which dramatically increases the dispersal of Xf genes/genotypes. In California, information regarding the population structure and genetic diversity, as well as the genetic evolutionary and epidemiological relationships, among Xf strains in agricultural populations is not clear. In order to develop effective management strategies, it is critical to understand pathogen population structure and genetic diversity in the agricultural ecosystem. A tool is needed that is capable of precisely, powerfully, easily analyzing Xf diversity and genotyping strains. We developed multi-locus DNA markers to fill this need. OBJECTIVES 1. Perform genome-wide sequence analysis to identify Simple Sequence Repeat (SSR) loci from four Xf genomic sequencing databases (PD, CVCD, ALSD and OLSD). Design and develop multi-locus SSR markers. 2. Analyze genetic diversity and population structures of PD Xf statewide. Compile a large Xf allele frequency database for strain identification. 3. Use the SSR Marker system to examine interactions between hosts and Xf including adaptation, host selection and pathogenecity of Xf strains RESULTS SSR Locus Identification and Primer Design 1. A genome wide search was performed to identify SSR loci among all four Xf strains (CVC 9a5c 2.68Mbp, PD Temecula 2.52Mbp, ALS Dixon 2.67Mbp, and OLS Ann-1 2.63Mbp). Figure 1 shows the distributions of SSR loci among four strains of Xf. 2. We used the following criteria to select SSR loci for primer design; a) each locus has single hit per genome and b) each selected locus contains at least 5 or more of repeat unit lengths. - 206 -
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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
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 and 118: Inhibition of biofilm is BSA concen
Page 119 and 120: CULTURE-INDEPENDENT ANALYSIS OF END
Page 121 and 122: Borneman, J., M. Chrobak, G. Della
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
Page 131 and 132: III. Production of transgenic plant
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-
Page 145: 1995; Purcell and Saunders, 1999).
Page 149 and 150: Strain Name Host of Origin County o
Page 151 and 152: ROLE OF ATTACHMENT OF XYLELLA FASTI
Page 153 and 154: wild-type cells was not conclusive
Page 155 and 156: DETERMINATION OF GENES CONFERRING H
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Page 159 and 160: MULTILOCUS SEQUENCE TYPING TO IDENT
Page 161 and 162: GENOME-WIDE IDENTIFICATION OF RAPID
Page 163 and 164: Americas and since most of the plan
Page 165 and 166: EFFECTS OF CHEMICAL MILIEU ON ATTAC
Page 167 and 168: CONCLUSIONS Our overall objective i
Page 169 and 170: RESULTS Objective 1. We conducted t
Page 171 and 172: Redak, R. A., Purcell, A. H., Lopes
Page 173 and 174: In parallel, an “activating trans
Page 175 and 176: PATTERNS OF XYLELLA FASTIDIOSA INFE
Page 177 and 178: % of vessels 100 80 60 40 20 Mugwor
Page 179 and 180: DOCUMENTATION AND CHARACTERIZATION
Page 181 and 182: Magnolia002 showed more identity (9
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Page 189 and 190: probing activities). In preliminary
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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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