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

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several species; however, additional studies must be completed to further elucidate the role of this pathogen in causing widespread disease in the urban setting as well on crops of agronomic importance in California. PE.PLS-PEAR 93 99 62 53 Xanthomona 10 NEGRITO005 LAGESTRO021 PCERASI076 DIXON-ALS PCERASI057 PCERASI086 187-ALS LIQUIDAUI12 PCERASI045 LIQUIDA020 PCERASI053 PCERASIACl12 GINGKO ALS2ALMOND PCERASI047 ALS6ALMOND PCERASI052 OLIVE ** * * ** ** * ** ** ** * * ** ** * GLENNALMON CO01COFFEE 91 CI11067CIT 89 75 5R1Peac h-PELS 2.4Pl um-PLS Peach Leaf Scorch (PELS) 92. 10O ak-OkLS Oak Leaf Scorch (OkLS) ContraCos ta-ALS Almond Leaf Scorch (ALS) “Dixon-type” MAGNOLI038 JAC AR AN028 HEMEROCALL MULBERR024 MULBERR063 MULBERR012 MULBERRYVA NANDINA ** ** ** ** * ** * * ** ** ** ** MULBERR059 MAGNOLI002 ALMONDS036 PEACH 018 STANISLAUS CERCISORNA PEACHES019 A05PIERCES Bacterial Leaf Scorch of Coffee (BLSCo) Citrus Varigated Chlorosis (CVC) PF1OLSOLEA ANN1OLEAND Oleander Leaf Scorch (OLS) Mulberry Leaf Scorch (MLS) Pierce’s Disease (PD) Figure 1. Preliminary phylogenetic tree constructed using the neighbor joining method, based on 16S rDNA sequence data for Xylella fastidiosa with the sequence of Xanthomonas vesicatoria (AF203392) as the outgroup. The numbers above the branches represent bootstrap values obtained for 100 replications. * Indicates isolates collected in 2003, ** indicates isolates collected in 2004. REFERENCES Felsenstein, J. (1995). PHYLIP version 3.57 manual. U.Washington, Seattle. Hendson, M., Purcell, A. H., Chen, D.,Smart, C., Guilhabert, M., and Kirkpatrick, B. (2001) App. and Env. Microbio. 67:895-903. Hill, B. L. and Purcell, A. H. (1995) Phytopath. 85:1368-1372. Huang,Q. and Sherald,J.L. (2004) Curr. Microbiol. 48 (1), 73-76. Minsavage, G. V., Thompson, C. M., Hopkins, D. L., Leite, R.M.M.V.B.C., and Stall, R. E. (1994) Phytopath. 84: 456-461. Thompson, J.D., Higgins, D.G. and Gibson, T.J. (1994) Nucleic Acids Research, 22:4673-4680 FUNDING AGENCIES Funding for this project was provided by the CDFA Pierce’s Disease and Glassy-winged Sharpshooter Board, with additional contributions made by the California Association of Nurseries and Garden Centers' CANERS Foundation. - 241 -
PLASMID ADDICTION AS A NOVEL APPROACH TO DEVELOPING A STABLE PLASMID VECTOR FOR XYLELLA FASTIDIOSA Project Leaders: Glenn M. Young Dept. of Food Science and Technology University of California Davis, CA 95616 Michele Igo Section of Microbiology Division of Biological Sciences University of California Davis, CA 95616 Cooperator: Bruce Kirkpatrick Dept. of Plant Pathology University of California Davis, CA 95616 Reporting Period: This report summarizes the goals of this project. Funding for this project initiated September 1, 2004. INTRODUCTION Current approaches to understanding the progression of Pierce’s disease are limited by the lack of genetic techniques that can be used to study the biology of Xylella fastidiosa (Xf). In particular, extrachromosomal elements, such as plasmids, having long-term stability in Xf when grown in lab cultures or en planta, have not yet been satisfactorily developed. We will develop vectors that exhibit stable maintenance by Xf by adapting previously described genetic and microbiological techniques. Our particular research efforts will focus on taking advantage of a well-studied bacteriological phenomenon called plasmid addiction (2, 4, 10). The major mechanistic principle of plasmid addiction is that the plasmid carries a genetic trait that the host bacterium requires for viability. The trait does not affect the metabolic properties of the bacterium nor does it affect reproduction. However, loss of the plasmid-encoded trait is a lethal event, so by definition plasmid addiction ensures vector stability. In addition, we will systematically evaluate other genetic mechanisms for increasing plasmid stability including multimer resolution and active partitioning systems. Finally, we will examine the stability of each of the newly developed vectors for Xf in vitro and en planta. The results of this analysis will allow us to construct one or more stable plasmid vectors that can be used by all researchers using genetic approaches to develop methods that limit Xf-related diseases. Xylella fastidiosa is a Gram-negative, endophytic bacterium, which is responsible for a number of economically important plant diseases (for recent reviews, see (5, 7, 8)). Diseases that are important to the California agricultural economy include Pierce’s disease of grapevine, almond leaf scorch, alfalfa dwarf, and oleander leaf scorch. Some strains of Xf, such as the Pierce’s disease strains, have very wide host ranges and are capable of colonizing the xylem of widely divergent plant species. In many plant species, infection by Xf does not provoke symptoms or noticeable distress. However, the colonization of certain plants, such as grapevines, leads to the development of disease symptoms and of plant decline. Although the specific details of the disease process are not fully understood, it is known that Xf forms a biofilm within xylem vessels that has a major impact on the movement of sap within the xylem tissue. Disease symptoms seem to be dependent on the rate and extent of colonization of the xylem tissue by Xf. Some of the symptoms observed in infected grapevines include leaf marginal necrosis, severe leaf scorch, and dieback. Another important aspect of the disease cycle involves the insect vector. Xf is transmitted from plant to plant by xylemfeeding insects including the glassy-winged sharpshooter (5, 7, 8). The insect vectors acquire the bacterium by feeding on infected plants. Since the Pierce’s disease strain can colonize numerous plant species, the source of inoculum can be infected grapevines or symptomless plants present in the riparian habitats surrounding the vineyard. In vectors showing the highest transmission efficiencies, Xf is present as a polar biofilm in the insect foregut and is transmitted to uninfected plants during subsequent feeding events. In susceptible plants, efficient transmission of Xf occurs at low bacterial cell numbers (
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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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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
Page 139 and 140: ISOLATION OF BACTERIOPHAGES SPECIFI
Page 141 and 142: Project Leader: Michele M. Igo Sect
Page 143 and 144: outer membrane fractions using two-
Page 145 and 146: 1995; Purcell and Saunders, 1999).
Page 147 and 148: DEVELOPMENT OF SSR MARKERS FOR GENO
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: Magnolia002 showed more identity (9
Page 185 and 186: GENETIC VARIABILITY OF XYLELLA FAST
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Page 189 and 190: probing activities). In preliminary
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Page 195 and 196: The almost complete absence of info
Page 197 and 198: QUANTIFYING LANDSCAPE-SCALE MOVEMEN
Page 199 and 200: Table 1. The mean (±SD) ELISA read
Page 201 and 202: EPIDEMIOLOGICAL ASSESSMENTS OF PIER
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Page 205 and 206: SPATIAL DATABASE CREATION AND MAINT
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Page 209 and 210: Figure 2. Individual leaves from a
Page 211 and 212: The state variables, process functi
Page 213 and 214: DEVELOPMENT OF A FIELD SAMPLING PLA
Page 215 and 216: Figure 1. Three main dispersion pat
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Page 221 and 222: OBJECTIVES 1. Track the movement of
Page 223 and 224: REFERENCES 1. Beard, C.B., Cordon-R
Page 225 and 226: cases, positive phloem samples were
Page 227 and 228: EXPLOITING XYLELLA FASTIDIOSA PROTE
Page 229 and 230: Figure 2. Polymer disk accumulation
Page 231 and 232: 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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