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

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Electron microscopy substantiated the presence of polar pili on the wildtype and many of the mutant strains. Negative staining of TEM preparations of the wild-type strain revealed an abundance of pili, the majority of which were 0.4-1.0 µm in length with many additional filaments 1.0-5.8 µm in length. Mutant strains 1A2 and 5A7 had only the shorter class of pili, whereas strain 6E11 had predominantly long pili. The correlation between the presence of long and short pili on the wild-type X. fastidiosa strain, the occurrence of essentially only long pili on the twitching, biofilm-deficient strain (6E11), and the absence of long pili on the twitching-minus, biofilm-enhanced mutants (1A2 and 5A7), clearly relates to distinct functional roles for two length classes of pili. SEM and TEM of wild-type cells attached to the substratum at the pili-bearing polar ends. Mutant strains 1A2 and 6E11 depicting only short pili and only longer type-IV pili, respectively. CONCLUSIONS Microfabricated fluidic chambers were created to mimic plant xylem vessels, in which we studied the non-flagellated Xylella fastidiosa bacterium. We discovered that the bacteria migrate ‘upstream’ by twitching motility, which explains, in part, how they are able to travel against the flow direction of xylem sap to invade non-colonized plant regions. REFERENCES Alm, R. A. and Mattick, J. S. 1997. Genes involved in the biogenesis and function of type-4 fimbriae in Pseudomonas aeruginosa. Gene 192:89-98. Braun, P. and Schmid, J. 1999a. Sap flow measurements in grapevines (Vitis vinifera L.) - 1. Stem morphology and use of the heat balance method. Plant Soil 215:39-45. Braun, P. and Schmid, J. 1999b. Sap flow measurements in grapevines (Vitis vinifera L.) - 2. Granier measurements. Plant Soil 215:47-55. Davis, M. J., French, W. J. and Schaad, N. W. 1981. Axenic Culture of the Bacteria Associated with Phony Disease of Peach and Plum Leaf Scald. Curr. Microbiol. 6:309-314. Feil, H., et al. 2003. Site-directed disruption of the fimA and fimF genes of Xylella fastidiosa. Phytopathology 93:675-682. Guilhabert, M. R., Hoffman, L. M., Mills, D. A. and and Kirkpatrick, B. C. 2001. Transposon mutagenesis of Xylella fastidiosa by electroporation of Tn5 synaptic complexes. Mol. Plant Microbe In. 14:701-706. Huang, B., Whitchurch, C. B. and Mattick, J. S. 2003. FimX, a multidomain protein connecting environmental signals to twitching motility in Pseudomonas aeruginosa. J. Bacteriol. 185:7068-7076. Kang, Y. W., et al. 2002. Ralstonia solanacearum requires type 4 pili to adhere to multiple surfaces and for natural transformation and virulence. Mol. Microbiol. 46:427-437. Lascano, R. J., Baumhardt, R. L. and Lipe, W. N. 1992. Measurement of water flow in young grapevines using the stem heat balance method. Am. J. Enol. Vitic. 43:159-165. Mollenhauer, H. H. and Hopkins, D. L. 1976. Xylem morphology of Pierce's disease-infected grapevines with different levels of tolerance. Physiol. Plant Pathol. 9:95-100. Newman, K. L., Almeida, R. P. P., Purcell, A. H. and Lindow, S. E. 2004. Cell-cell signaling controls Xylella fastidiosa interactions with both insects and plants. Proc. Natl. Acad. Sci. U.S.A. 101:1737-1742. Orndorff, P. E., et al. 2004. Immunoglobulin-mediated agglutination of and biofilm formation by Escherichia coli K-12 require the type 1 pilus fiber. Infect. Immun. 72:1929-1938. Peuke, A. D. 2000. The chemical composition of xylem sap in Vitis vinifera L. cv. riesling during vegetative growth on three different franconian vineyard soils and as influenced by nitrogen fertilizer. Am. J. Enol. Vitic. 51:329-339. Simpson, A. J. G., et al. 2000. The genome sequence of the plant pathogen Xylella fastidiosa. Nature 406:151-157. Strom, M. S. and Lory, S. 1993. Structure-Function and Biogenesis of the Type Iv Pili. Annu. Rev. Microbiol. 47:565-596. Wall, D. and Kaiser, D. 1999. Type IV pili and cell motility. Mol. Microbiol. 32:1-10.R. Alm, R. A. and Mattick, J. S. 1997. Genes involved in the biogenesis and function of type-4 fimbriae in Pseudomonas aeruginosa. Gene 192:89-98. FUNDING AGENCIES Funding for this project was provided by the University of California Pierce’s Disease Grant Program. - 197 -
ISOLATION OF BACTERIOPHAGES SPECIFIC FOR XYLELLA FASTIDIOSA Project Leader: Michele M. Igo Section of Microbiology Division of Biological Sciences and California Agricultural Experimental Station University of California Davis, CA 95616 Cooperators: Carol Lauzon Dept. of Biological Sciences California State University Hayward, CA 94542 Bruce Kirkpatrick Dept. of Plant Pathology University of California Davis, CA 95616 Reporting Period: Funding for this project was received in September 2004. ABSTRACT This report gives an overview of the project. The goal of this project is to isolate a collection of viruses (phages) that can infect and replicate in X. fastidiosa (Xf). This collection will then be screened to identify phage exhibiting useful biological properties. INTRODUCTION The causative agent of Pierce’s disease (PD) is the Gram-negative bacterium Xylella fastidiosa (Xf). Xf is highly specialized and is capable of multiplying in both the foregut of xylem-feeding insects, such as the glassy-winged sharpshooter and in the xylem system of the host plant (for recent reviews, see 4, 6, 7). The complex nature of the bacterial-host interactions that take place during the PD infectious cycle and the fastidious growth properties of Xf in the laboratory present a formidable challenge to researchers working with this bacterium. At present, there are only a few methods available to perform such basic operations as genetic exchange, mutant isolation, strain construction, and complementation. Further complications of working with Xf arise because of its slow generation time, its tendency to form aggregates, and its poor plating efficiency. Finally, few methods are available for disrupting the interaction between Xf and its hosts, which is a key component of the PD infectious cycle. As a result, there are currently no effective treatments to cure infected vines. In other Gram-negative bacteria, bacteriophages, phage derivatives and phage components have played a major role in overcoming these issues (1, 3, 8). For example, phages have been used to move genetic markers between strains, for complementation, and as cloning vectors. In addition, phages have been used as diagnostic reagents to detect pathogenic bacteria, and as therapeutic agents in bacterial infections. Unfortunately, since not all phages possess exploitable properties, it is usually necessary to isolate a collection of phages that infect the bacteria of interest and then to screen the individual phages for desirable properties. Based on studies of environmental samples, it has been estimated that there are >10 30 tailed phages in the biosphere and that phage typically outnumber bacterial cells 10 to 1 (2). These studies also revealed that phages could be found anywhere that their bacterial hosts are present. This observation has already proven true for Xf. Carol Lauzon and her colleagues have reported the presence of two phages associated with Xf from infected grapevines (5). The goal of this project is to isolate a collection of phages that are capable of infecting and replicating in Xf (Aim 1). These phages will then be screened individually to identify specific phages that have the potential to be used as genetic tools and for killing Xf en planta (Aim 2). Phages capable of moving genetic markers between Xf strains would give researchers in the field a powerful tool for investigating the properties of this unusual bacterium and establishing which parts of its genetic material make it such a deadly pathogen for certain varieties of grapes. Furthermore, phage or mixtures of phages capable of killing Xf would provide the tools necessary to determine the feasibility of using phage therapy to control the spread of PD. OBJECTIVES The primary goal of this project is to isolate a collection of phages as pure stocks and to screen this collection for phages that exhibit useful biological properties for studying and controlling the growth of Xf. Specific Aim 1: Generate a collection of pure phage stocks that infect Xf. 1A) Collect environmental samples that potentially contain Xf specific phages. 1B) Isolate and obtain pure stocks of phages from the samples. - 198 -
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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
Page 87 and 88: EFFECTS OF SUBLETHAL DOSES OF IMIDA
Page 89 and 90: Table 2. Mortality and flight perfo
Page 91 and 92: A NOVEL METHOD TO INDUCE OVIPOSITIO
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 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: Mutagenesis of Xylella The EZ::TN T
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 and 182: Magnolia002 showed more identity (9
Page 183 and 184: PLASMID ADDICTION AS A NOVEL APPROA
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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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Impact Of Host Plant Xylem Fluid On Xylella Fastidiosa Multiplication ...

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