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

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SYMBIOTIC CONTROL OF PIERCE’S DISEASE: CONSTRUCTION OF TRANSGENIC STRAINS OF ALCALIGENES XYLOSOXIDANS DENITRIFICANS EXPRESSING SURFACE ANTI-XYLELLA FACTORS AS MICROBIAL PESTICIDES FOR PIERCE’S DISEASE CONTROL Project Leader: David Lampe Dept. of Biological Sciences Duquesne University Pittsburgh, PA 19219 Cooperators: Carol Lauzon Dept. of Biological Sciences California State University Hayward, CA 94542 Blake Bextine Dept. of Entomology University of California Riverside, CA 92521 Project Director: Thomas A. Miller Dept. of Entomology University of California Riverside, CA 92521 Don Cooksey Deparment of Plant Pathology University of California Riverside, CA 92521 Steven Lindow Dept. of Plant and Microbial Biology University of California Berkeley, CA 94720 Consultant: Frank Richards Yale University New Haven, CT 06520 Reporting period: The results reported here are from research conducted from April 2003 to October 2004. INTRODUCTION The glassy-winged sharpshooter (GWSS) is the principle vector of the xylem-limited bacterium Xylella fastidiosa (Xf), which causes Pierce’s disease (PD) in grapes. Limiting the spread of this pathogen by rendering GWSS incapable of pathogen transmission (paratransgenesis) is a promising method of pathogen control. Paratransgenesis seeks to modify the phenotype of an organism indirectly by modifying its symbiotic bacteria to confer vector-incompetence Paratransgenic approaches to disrupt pathogen infection of humans are being developed by several groups. These include interference with the ability of triatomid bugs to transmit pathogens causing Chagas’ disease 1 , interference with HIV attachment to its target cells in the reproductive tracts of humans 2 , and the elimination of persistent Candida infections from biofilms in chronically infected patients 3 . Paratransgenesis has also been applied to deliver cytokines mammalian guts to relieve colitis 4; 5 . Thus, the method has wide applicability. Alcaligenes xylosoxidans denitrificans (Axd) is Gram negative, beta proteobacterial species that can colonize the GWSS foregut and cibarium, as well as various plant tissues, including xylem. It is non-pathogenic in insects, plants and healthy humans. Given these characteristics, Axd has become the focus of our paratransgenesis efforts to control PD in grapes. Over the past two years we developed the technology to stably modify Axd by inserting genes into its chromosome and also isolated as single chain antibody that recognized an epitope on the surface of the Pierce’s Disease strain of Xf. 6 . We report here the construction of strains of Axd that express an anti-Xylella single chain antibody (scFv) on the outer surface of Axd as fusions to three different heterologous outer membrane proteins. In each case, strains of varying fitness were recovered as measured by growth rate as compared to wild-type strains. OBJECTIVES 1. Construct anti-Xylella scFv-membrane protein fusions; 2. Construct strains of Axd that express the scFv-membrane protein fusions in the outer membrane; 3. Construct transgenic Axd strains of varying fitness. RESULTS A. Membrane Protein-scFv Gene Fusions We fused an anti-Xylella scFv gene to three different outer membrane protein genes in order to display the scFv on the outer membrane of Axd. These were a lipoprotein-outer membrane protein A (lpp-OmpA) fusion from E. coli 7 ; the ice nucleation protein Z (inaZ) from Pseudomonas syringae (a gift of Steven Lindow); and an internally-deleted form of inaZ that eliminates the internal ice nucleation repeat sequence but retains the N and C terminus of the protein necessary to export and - 355 -
anchor it in the outer membrane (short-inaZ) 8 . Each of these was placed on a Himar1 mariner transposon and random chromosomal insertions were obtained for each generating multiple strains 9 (see Table 1). B. Expression of scFv Fusions on the Surface of Axd. We determined the degree of surface expression of the scFv fusions on Axd by two methods. The first was a “spun cell ELISA” 7 . This method uses a suspension of cells that express a target epitope as the substrate for an ELISA. Detection of the scFv was accomplished before and after induction of the lac promoter by either reaction with Protein L-conjugated HRP (which detects scFv light chains) or with a HPR conjugated antibody that reacts with the haemagglutinin epitope tag on the scFv. Results of spun cell ELISAs on different strains are shown in Table 1. Strains varied considerably in their scFv surface expression levels, presumably due to the site of insertion. Most strains of short-inaZ fusions, for example were poor expressers when induced and strain AL8.2 only showed appreciable levels of surface expression when uninduced. Table 1. Characteristics of transgenic A. xylosoxidans strains expressing an anti-Xylella single chain antibody as an outer membrane protein fusion. Strain scFv fusion Surface expression 1 Relative Fitness 2 Insert location 3 AL7.2 P. syringae inaZ ++ G - major facilitator superfamily transporter AL7.5 “” +++ G - inorganic pyrophosphatase AL7.7 “” ++ S -fructose transport system repressor AL7.10 “” + G ND AL8.2 P. syringae short inaZ +++ (uninduced only) G/P -probable transporter AL8.3 “” BK P ND AL9.1 E. coli lpp-ompA + S ND AL9.4 “” +++ S/G ND AL9.5 “” + S ND 1 These values are relative to background as measured in a spun cell ELISA: BK = background levels; + noticeable expression, ++ strong expression; +++ very strong expression. 2 Fitness values are measured as growth rates in liquid culture relative to that of wild-type A. xylosoxidans. S (= strong, essentially wild type); G (= good, but slower than wild type); P (= poor) 3 Most likely identity of genes where transgenes were inserted. These were obtained using tblastx with flanking insertion sequences against the microbial nucleotide database from Genbank. ND= not determined. The second method used to determine whether expression was occurring in the outer membrane of Alcaligenes was a test for ice nucleation. Wild-type Axd cannot nucleate ice (unpublished observations). We tested whether or not AL7 and AL8 strains could nucleate ice. All of the AL7 strains could nucleate ice while neither of the AL8 strains did so. This is consistent with surface expression of the full-length P. syringae ice nucleation protein on the surface of the AL7 strains. AL8 strains express a form of inaZ that has the internal repeat region removed. This is the region that is responsible for ice nucleation in these proteins. C. Fitness of transgenic Axd strains Our strains are built via transposon insertion and so should vary in fitness depending on the site of insertion in the chromosome. We measured the fitness of each strain compared to wild type by measuring their growth rates in log phase in liquid culture. These relative fitness values are shown in table 1 along with the most likely site of insertion of the transposon used to make the strain. We determined the site of insertion by sequencing outward from the transposable element inverted terminal repeats into the flanking genomic DNA and then using tblastx against the microbial genomic database in Genbank. There are no Axd sequences in Genbank, so the matches we obtained were typically to species in the genus Pseudomonas, another basal beta proteobacterial group. Strains were highly variable in their fitnesses. Some strain fitnesses were indistinguishable from wild type (e.g., AL7.7 and AL9.5), while others were obviously affected in their growth rates (e.g., AL8.3). There was no obvious correlation between fitness and ability to surface express the scFv fusions. Indeed, one of our best expressing strains was only a modest grower (AL7.5) while other strains grew well and expressed the transgene poorly (e.g., AL9.5). The ability to isolate strains that - 356 -
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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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III. Production of transgenic plant
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RESULTS Construction of cDNA Librar
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CONCLUSIONS Genetic resistance and
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Mutagenesis of Xylella The EZ::TN T
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ISOLATION OF BACTERIOPHAGES SPECIFI
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Project Leader: Michele M. Igo Sect
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outer membrane fractions using two-
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1995; Purcell and Saunders, 1999).
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DEVELOPMENT OF SSR MARKERS FOR GENO
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Strain Name Host of Origin County o
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ROLE OF ATTACHMENT OF XYLELLA FASTI
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wild-type cells was not conclusive
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DETERMINATION OF GENES CONFERRING H
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S1 S2 S3 S4 Nb123456789bb123456789b
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MULTILOCUS SEQUENCE TYPING TO IDENT
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GENOME-WIDE IDENTIFICATION OF RAPID
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Americas and since most of the plan
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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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Page 283 and 284: SEARCHING FOR AND COLLECTING EGG PA
Page 285 and 286: REFERENCES Hoddle, M. S. and S. V.
Page 287 and 288: some Gram(+) bacteria, but are inac
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Page 291 and 292: Isolation of Fungal Pathogens Soil
Page 293 and 294: IDENTIFICATION OF MECHANISMS MEDIAT
Page 295: concentrations of 1.5 x 10 7 bacter
Page 299 and 300: SYMBIOTIC CONTROL OF PIERCE’S DIS
Page 301 and 302: MANAGEMENT OF PIERCE’S DISEASE OF
Page 303 and 304: pfF mutants are taken up by insects
Page 305 and 306: Simpson, A. J. G., F. C. Reinach, P
Page 307 and 308: Al-Wahaibi, A.K., Owen, and J. G. M
Page 309 and 310: patterns differed among the lines,
Page 311 and 312: Punja, Z.K. 2001. Genetic engineeri
Page 313 and 314: mind, we conducted an additional st
Page 315 and 316: REFERENCES Toscano, N., Byrne, F.,
Page 317 and 318: RESULTS AND CONCLUSIONS The program
Page 319 and 320: COMPATIBILITY OF INSECTICIDES WITH
Page 321 and 322: 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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