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

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To determine what genes were affected that resulted in restored transmission, we will clone and sequence the DNA flanking the transposon using standard protocols for determining genomic DNA sequence flanking insertion DNA. The identity of these genes may enable us to grasp key features of the bacterial mechanism driving transmission. For example, we may find that certain adhesins are required for attachment to the foregut if activating transposons near adhesin genes restore transmissibility. REFERENCES Dow, J. M. and M.J. Daniels. 2000; “Xylella genomics and bacterial pathogenicity to plants”; Yeast; 17: 263-271. Gullharbert, M. R.,Hoffman, L.M., Mills, D. A. and Kirkpatrick, B. C. 2001. Transposon mutagenesis of Xylella. MPMI Vol. 14, N 6 pp 701-706. Newman K.L, Almeida R. P.P, Purcell A.H. and Lindow S.E. 2004. Cell-cell signaling control Xylella fastidiosa interaction with both insects and plants. Proc. Nat. Acad. Sciences. 101:1737-1742. Newman K.L, Almeida R. P.P, Purcell A.H. and Lindow S.E 2003. Analysis of colonization of Vitis vinifera by Xylella fastidiosa by using a green fluorescent strain. Applied and Envir. Microbiology. 69: 7319-7327. FUNDING AGENCIES Funding for this project was provided by the CDFA Pierce’s Disease and Glassy-winged Sharpshooter Board. - 233 -
PATTERNS OF XYLELLA FASTIDIOSA INFECTION IN PLANTS AND EFFECTS ON ACQUISITION BY INSECT VECTORS Project Leaders: Alexander Purcell ESPM-Division of Insect Biology University of California Berkeley, CA 94720 Steve Lindow Plant and Molecular Biology University of California Berkeley, CA 94720 Researcher: Christina Wistrom ESPM – Division of Insect Biology University of California Berkeley, CA 94720-3112 Reporting Period: The results reported here are from work conducted from July 2003 to September 2004. ABSTRACT We are studying the effect of host plant tolerance on insect vector acquisition of Xylella fastidiosa (Xf) from plants tolerant, moderately susceptible, and highly susceptible to Xf infection. We are observing Xf population and distribution in tolerant and susceptible plants, and its relationship to xylem anatomy, symptom development, and bacterial acquisition by sharpshooters. Since host plant resistance is an important component in the long-term goal of curing PD, it is important to know how resistant plants affect PD spread in areas permanently infested with sharpshooter vectors. We also address the short-term goal of controlling PD spread by comparing grape cultivars in their ability to provide inoculum for vine-to-vine spread of Pierce’s disease. Anatomical comparisons of three cultivars, Sylvaner’, ‘Cabernet Sauvignon’ and ‘Pinot Noir’ showed that all three varieties had similar numbers, lengths and distributions of vessels. The only significant difference was that tolerant ‘Sylvaner’ had ~ 20 % more rays than the more susceptible ‘Cabernet Sauvignon’ or ‘Pinot Noir’ (n = 25, P = 0.01) in canes of similar age, length and diameter. In all four alternate hosts, morning glory (Ipomoea purpurea), mugwort (Artemisia douglasiana), sunflower (Helianthus annuus) and annual bur-sage (Ambrosia acanthicarpa), the longest vessels measured were less than 13 cm long, while in grapes the longest vessels averaged 62 cm. Though alternate hosts had various vascular morphologies and stem lengths, all had shorter vessels than grapes. Blue-green sharpshooters failed to efficiently inoculate wild-type Xf and green fluorescent protein-expressing (GFP) Xf into both grapes and alternate hosts; only one of 44 grapes inoculated with BGSS became infected. In order to generate GFP-Xf infected plants for microscopy, we are mechanically inoculating alternate hosts and grapes. Ongoing work focuses on refining microscopic techniques to visualize small numbers of Xf in plant stems, and generating large numbers of Xf infected grapevines to serve as new sources for sharpshooter bacterial acquisition. INTRODUCTION Alternate hosts of Xf were selected for their different patterns of Xf colonization after vector inoculation, lack of stem lignification, varying morphology, and absence of green autofluorescence under blue light. In previous experiments, Xfcarrying sharpshooters infected morning glory and sunflower more than 80% of the time. Xf spread systemically throughout both plants and reached populations over 10 5 colony-forming units (CFU)/gram. Quinoa and mugwort were less-frequently infected (32% and 16%, respectively) by Xf and supported lower bacterial populations (10 3 CFU/g for quinoa, 10 6 CFU/g for mugwort). Xf moved systemically to a limited extent in quinoa, but not in mugwort (8, 16). Grape cultivars with varying tolerance to PD selected for evaluation are tolerant ‘Sylvaner’, moderately susceptible ‘Cabernet Sauvignon’ and highly susceptible ‘Pinot Noir’ cultivars of Vitis vinifera (12, 13). Both blue-green sharpshooters (BGSS) and glassy-winged sharpshooters (GWSS) will be used to infect plants and assess the efficiency of insect acquisition of Xf (1, 7, 11). We are using wild type and transformed isolates of Temecula Xf in our experiment. The transformed isolate continually expresses green fluorescent protein (GFP) when illuminated with blue light. GFP-Xf was transmitted by blue-green sharpshooters, retained typical virulence in grape, and allowed examination of plant tissues without the extensive fixation required with electron microscopy. With confocal microscopy, GFP-expressing Xf can be observed in small and large colonies in vessels, and passing through bordered pits between vessels in symptomatic ‘Cabernet Sauvignon’ petioles (10). Anatomical comparisons between alternate hosts and grape cultivars included measurements of vessel length and number, and vascular bundle number and distribution based on the techniques of Tyson et al. (15), and Ewers and Fischer, modified to infuse the pigment via 100kPa pressure applied to the proximal end of the cutting (5). We evaluated primary vegetative growth rather than secondary xylem due to the difficulties in sectioning, culturing from, and feeding BGSS on partially lignified stems. GFP-Xf inoculation and colonization of all plants will be measured similarly in all plants: groups of four GFP-Xf carrying sharpshooters inoculated a 3-cm stem section, and the plants were evaluated for the presence of GFP-Xf approximately 8 weeks after inoculation. Colonized vessels will be counted, and populations estimated by culture on PWG media (2, 8). - 234 -
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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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Page 125 and 126: Purcell, AH and SR Saunders. 1999a.
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Page 133 and 134: RESULTS Construction of cDNA Librar
Page 135 and 136: CONCLUSIONS Genetic resistance and
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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 159 and 160: MULTILOCUS SEQUENCE TYPING TO IDENT
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Page 167 and 168: CONCLUSIONS Our overall objective i
Page 169 and 170: RESULTS Objective 1. We conducted t
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Page 189 and 190: probing activities). In preliminary
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Page 197 and 198: QUANTIFYING LANDSCAPE-SCALE MOVEMEN
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Page 205 and 206: SPATIAL DATABASE CREATION AND MAINT
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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
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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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