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

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REFERENCES Chen, J., and Civerolo, E. 2003. Developing a microarray-PCR-based identification and detection system for Xylella fastidiosa strains important to California. Proceedings of Pierce’s disease research symposium. California Department of Food and Agriculture. Page 49-50. December 8-11, 2003. Coronado, California. Chen, J., Groves, R., Civerolo, E. L., Viveros, M., Freeman, M., and Y. Zheng. 2004a. Simultaneous association of more than one Xylella fastidiosa genotype with almond leaf scorch disease in California. Phytopath. 94:S17. Chen, J., Groves, R., Civerolo, E. L., Viveros, M., Freeman, M., and Y. Zheng. 2004b. Simultaneous association of more than one Xylella fastidiosa genotype with almond leaf scorch disease in California. (submitted) Table 1. List of four single nucleotide polymorphisms from 38 rDNA sequences of Xylella fastidosa and the related information. Strain Name Host Geographic Origin 150 454 1261 1338 R116v11 Grape Georgia C G A C PCE-FG Grape Florida C G A C PD28-5 Grape Florida C G A C PCE-FF Grape Florida C G A C Temecula Grape California C G A C GR.8935 Grape Florida C G A C Temecula Grape California C G A C Temecula Grape California C G A C Mul-2 Mulberry Nebraska C G A C Dixon1 Almond California T A G C Dixon2 Almond California T A G C Ann-1 1 Oleander California T A G C Ann-1 2 Oleander California T A G C PWT-22 Periwinkle Florida T A G C PWT-100 Periwinkle Florida T A G C Mul1 Mulberry Massachusetts T A G C Elm Elm Washington DC T A G C OAK OAK Washington DC T A G C PLS2-9 Plum Georgia T A G C PLM G83 Plum Georgia T A G C PP4-5 Peach Georgia T A G C RGW-R Ragweed Florida T A G C ELM-1 Elm Washington DC T A G C ALS-BC Almond California T A A T MUL-3 Mulberry Massachusetts T A A T P3 Coffee Brazil T A A T B14 Citrus Brazil T A A T SL1 Citrus Brazil T A A T CRS2 Coffee Brazil T A A T CM1 Coffee Brazil T A A T CI.52 Citrus Brazil T A A T CO.01 Coffee Brazil T A A T CVC93-2 Citrus Brazil T A A T 9a5c Citrus Brazil T A A T 9a5c Citrus Brazil T A A T PE.PLS Pear Taiwan T A A C PL.788 Plum Georgia T A A C OSL92-3 Oak Florida T A A C FUNDING AGENCIES Funding for this project was provided by the University of California Pierce's Disease Grant Program. - 173 -
DNA MICROARRAY AND MUTATIONAL ANALYSIS TO IDENTIFY VIRULENCE GENES IN XYLELLA FASTIDIOSA Project Leader: Donald A. Cooksey Dept. of Plant Pathology University of California Riverside, CA 92521 Collaborators: Korsi Dumenyo Dept. of Plant Pathology University of California Riverside, CA 92521 Rufina Hernandez-Martinez Dept. of Plant Pathology University of California Riverside, CA 92521 Reporting Period: The results reported here are from work conducted from October 2003 to September 2004. ABSTRACT The development of successful management and control strategies of Pierces disease of grape requires the identification of virulence and pathogenicity genes and determining how they functions to control the disease development process. Based on the presumption that biofilm formation is a major pathogenicity factor of Xylella and that it may play a major role in the disease causing process, we have been studying the factors – genetic and environmental that affect biofilm formation by Xylella fastidiosa. We have identified that, Bovine serum albumen, a component of PW medium specifically inhibits biofilm formation in X. fastidiosa and that this inhibition is BSA concentration dependent. Because of its effect on the biofilm formation in vitro, we are studying the expression profiles of X. fastidiosa genes in the presence and absence of BSA in the media. We have also identified a global regulatory gene, rsmA (rsm = regulator of secondary metabolism) that control biofilm. An rsmA-deficient strain of X. fastidiosa forms more biofilm in vitro than the wild type. In a preliminary nylon membrane DNA macroarray experiment using about a 100 select candidate pathogenicity genes, we have determined an increased expression of 15 genes in the mutant when compared to the wild type parent. We are now using full genome microarrays of Xylella fastidiosa to catalogue the genes whose expressions are controlled by either rsmA or BSA. The results from these ongoing analyses using both approaches should help us catalogue X. fastidiosa genes which may be involved in pathogenicity and biofilm formation. Subsequent genetic analysis of the genes to be identified should give us some understanding of not only how pathogenicity is regulated in this bacterium but also how to tackle the problems posed by Pierces disease. INTRODUCTION Although the exact mechanism of Piece’s disease is not completely understood, infected grape plants show symptoms resembling those of water-stress. Moreover, the xylem-limited Xylella fastidiosa bacterium produces biofilm in vitro and in planta (4, 9, 10, 12). Putting these two observations together, it has been suggested that this biofilm clogs up the vascular tissues of the plant and occlude water and nutrient transport. Because of this assumed importance of biofilm formation in the disease mechanism of Xylella fastidiosa, we have been studying signals and factors affecting biofilm formation in a bit to identify the regulators of pathogenicity in Xylella fastidiosa. rsmA is a post-transcriptional regulatory gene that controls pathogenicity and secondary metabolism in a wide group of bacteria including Gram positive and negative organisms (1, 3, 11, 15). In Erwinia spp. and other related plant-associated bacteria, rsmA together with its regulatory noncoding RNA pair, rsmB control many phenotypes including pathogenicity, extracellular polysaccharide and enzyme production, and elicitation of hypersensitive response, pigment formation, motility and antibiotic biosynthesis). And in E. coli and related enterobacterial human pathogens, csrA and csrB, the homologues of rsmA and rsmB regulate, among others, glycogen biosynthesis and biofilm formation (6, 8, 17, 19). Because of the role of biofilm formation on the pathogenicity of many bacterial pathogens (5, 14), and the fact that rsmA or its homologs control both pathogenicity and biofilm formation in different bacteria, we wanted to determine the possible role of rsmA on biofilm formation in Xylella. We found that Xylella fastidiosa strains vary widely in their biofilm forming abilities and this is influenced by the culture medium in which the assay is carried out. We report that BSA is the specific inhibitor of biofilm formation in PW medium and that the amount of biofilm the bacterium forms if inversely proportional to the concentration of BSA in the medium. Further, we show that biofilm formation is regulated by rsmA gene as rsmA - mutants form higher levels of biofilm than the wild type parent. We confirm this observation by showing that the heterogonous expression of Xylella fastidiosa rsmA in E. coli reduces biofilm formation in this bacterium. Put together, these suggest that rsmA may regulate pathogenicity in Xylella fastidiosa through its effects on factors such as biofilm formation in the plant. - 174 -
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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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Page 67 and 68: REFERENCES Gautam, R. D. 1986. Effe
Page 69 and 70: PARASITISM OF THE GLASSY-WINGED SHA
Page 71 and 72: Table 1. Parasitism by G.ashmeadi o
Page 73 and 74: Project Leader: Robert F. Luck Dept
Page 75 and 76: A more interesting analysis using t
Page 77 and 78: MYCOPATHOGENS AND THEIR EXOTOXINS I
Page 79 and 80: POPULATION DYNAMICS AND INTERACTION
Page 81 and 82: No egg masses were recorded on olea
Page 83 and 84: EXPLORATION FOR FACULTATIVE ENDOSYM
Page 85 and 86: 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: 16S rDNA is by far the most sequenc
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
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
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EFFECTS OF CHEMICAL MILIEU ON ATTAC
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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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Impact Of Host Plant Xylem Fluid On Xylella Fastidiosa Multiplication ...

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