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

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(a) Plasmid Wt R1 R2 (d) Wt R1 R2 (e) N-MopB C-OmpA (b) Wt OmpA Prom OmpA RB (c) R FA OmpA Prom N-MopB C-OmpA Figure 3. E. coli strains with the E. coli OmpA gene replaced by a chimeric MopB-OmpA gene. (a)A low copy number plasmid was prepared with an insert composed of the 5’UTR and leader peptide (small rectangle) of OmpA fused to codons 1-171 of MopB (N-MopB), which in turn is fused to codons 172-325 of OmpA (C-OmpA). (b)Representation of the wildtype chromosomal OmpA gene (Wt). (c)Desired recombinant between the plasmid and the chromosomal OmpA gene to give a chromosomal, chimeric MopB-OmpA gene in place of OmpA. (d)Analysis of a polymerase chain reaction (PCR) 916bp product expected to be amplified, by forward (FA) and reverse (RB) primers designed as indicated in part (c), only from the recombinant sequence. Lanes received PCR incubation mixtures from Wt E. coli and two candidate recombinant strains, R1 and R2. (e)Gel electro-phoresis (SDS-PAGE) of protein extracts from E. coli lines Wt, R1 and R2. Unfortunately, the loading for the Wt lane is substantially greater than the loading for lane R1, which is more heavily loaded than lane R2. Dot indicates a band that is lost in R1 and R2 compared to Wt. The star marks a band of enhanced intensity, relative to other bands in the same lane, in R1 and R2 compared to Wt. E. coli transformants displaying MopB sequences were selected using magnetic beads covalently coupled to anti-MopB IgG. Beads were plated on agar medium to recover colonies growing up from bead-selected cells. Pooled colonies were cultured, and the cells were exposed to the OmpA-specific bacteriophage K3 at a multiplicity of infection of 15 to deplete the population in cells still bearing OmpA. Fig. 3 provides evidence for the occurrence of the expected recombination events and for the production of the chimeric MopB-OmpA protein in amounts visible on a coomassie brilliant blue-stained gel [Fig. 3(d) and (e)]. The cells derived by these approaches agglutinate beads displaying anti-MopB IgG, providing evidence that some part of the MopB portion of the chimera, presumably the MopB outer loops, is displayed on the exterior of the E. coli cell. CONCLUSIONS Based on results reported here and in previous progress reports, MopB is a highly suitable target for strategies designed to interfere with the ability of Xf to initiate infections leading to development of Pierce’s disease. Our overall strategy for creating grape plants resistant to Xf is revealed by the four new objectives stated above in the Objectives section. Experimental steps (i), (ii) and (iii) outlined at the end of the Introduction reveal how we intend to satisfy new Objective 1. Results in Fig. 3 suggest that we have completed experimental step (i) and that we are ready to proceed to the selection of variant gp38 proteins capable of high affinity binding to MopB on the surface of Xf cells, i.e., experimental steps (ii) and (iii). REFERENCES Drexler, K., Dannull, J., Hindennach, I., Mutschler, B. & Henning, U. (1991). Single Mutations in a Gene for a Tail Fiber Component of an Escherichia-Coli Phage Can Cause an Extension from a Protein to a Carbohydrate as a Receptor. Journal of Molecular Biology 219, 655-664. Pautsch, A. & Schulz, G. E. (1998). Structure of the outer membrane protein A transmembrane domain. Nature Structural Biology 5, 1013-1017. Singh, S. P., Williams, Y. U., Miller, S. & Nikaido, H. (2003). The C-terminal domain of Salmonella enterica serovar Typhimurium OmpA is an immunodominant antigen in mice but appears to be only partially exposed on the bacterial cell surface. Infection & Immunity 71, 3937-3946. Funding Funding for this project was provided by the CDFA Pierce’s Disease and Glassy-winged Sharpshooter Board and the USDA Agricultural Research Service. - 289 -
CHARACTERIZATION OF NEONICOTINOIDS AND THEIR PLANT METABOLITES IN CITRUS TREES AND GRAPEVINES, AND EVALUATION OF THEIR EFFICACY AGAINST THE GLASSY-WINGED SHARPSHOOTER Project Leaders: Frank Byrne Dept. of Entomology University of California Riverside, CA 92521 Nick Toscano Dept. of Entomology University of California Riverside, CA 92521 Reporting Period: The results reported here are from work conducted from July 2004 to October 2004. ABSTRACT The toxicities of established and new members of the neonicotinoid insecticide class were assessed against the glassy-winged sharpshooter in topical application bioassays. All compounds were highly toxic to the insect. Clothianidin elicited its toxic response more rapidly than thiamethoxam and was 3-fold more toxic overall at the LD50 level. Clothianidin has been proposed as an active derivative of thiamethoxam, so it is important to establish the fate of these chemicals within plant systems that are likely to be treated for GWSS control. INTRODUCTION The primary means of controlling the spread of Pierce’s disease (PD) in California vineyards is through the elimination of its vector using insecticides. The glassy-winged sharpshooter (GWSS) Homalodisca coagulata feeds directly from the plant xylem system and, therefore, systemic insecticides are currently being evaluated on both citrus and grapes. <strong>Of</strong> the various classes of insecticide under consideration, the neonicotinoids, especially imidacloprid, have proven to be the most effective at suppressing GWSS populations. Imidacloprid (1-[(6-chloro-3-pyridinyl)methyl]-4,5-dihydro-N-nitro-1H-imidazol-2-amine) is a nicotinic acetylcholine receptor agonist that combines high potency with low mammalian toxicity and favorable persistence. As a systemic, seed, soil or foliar treatment, it has proved to be especially effective against a wide range of homopterous insect pests, including the GWSS. The success of imidacloprid in controlling GWSS is due largely to its excellent systemic properties. Systemic applications exploit the xylophagous feeding behavior of the insect, and thereby disrupt the transmission of PD and other X. fastidiosa-related diseases. This project is an extension of a one-year project that was funded by the UC Pierce’s Disease Research Grant Program. It will focus on the fate of imidacloprid and other neonicotinoid insecticides in citrus and grapevines, and the impact of these chemicals on GWSS. In a previous study, imidacloprid and two of its derivatives were shown to be highly toxic to GWSS adults (Byrne and Toscano, 2003). The aims of this study are to determine the extent to which metabolites of neonicotinoids are formed in citrus trees and grapevines, and to determine their toxicological significance towards GWSS. The presence of insecticidal metabolites in xylem sap could contribute to the excellent persistence of imidacloprid treatments against sharpshooters. As well as maintaining the toxic pressure of the initial application, the metabolism of neonicotinoids to yield equally or more toxic metabolites may also account for the stability of this chemical class to resistance. <strong>Of</strong> particular interest to us are thiamethoxam and clothianidin, which are being evaluated for use against citrus and grape pests. During the past year, it has been established that thiamethoxam is converted into clothianidin by insects and cotton plants (Nauen et al., 2003). This is an important finding, as it could have ramifications for the use of these products on grapes and citrus. When several products from the same class become available for pest management, it is important that their use be carefully monitored in order to circumvent potential resistance problems. The possibility that thiamethoxam is converted into clothianidin is, therefore, of concern when formulating management strategies based around the neonicotinoids. Receptor binding studies have suggested that thiamethoxam does not bind to the same receptor site as imidacloprid and so it has been proposed as a suitable product for alternation with imidacloprid because of the reduced resistance risk (Weisner and Kayser, 2000). Now that thiamethoxam has been shown to be a potential pro-insecticide, and clothianidin has been shown to bind to the same receptors as imidacloprid, new issues are raised about its suitability as a product for rotation with other neonicotinoids. This is an important reason for determining the fate of thiamethoxam in citrus and grapes. OBJECTIVES 1. Determine the metabolic fate of neonicotinoids within citrus trees and grapevines. 2. Determine the relative toxicities of neonicotinoids and their metabolites to the adult and egg stages of the GWSS. RESULTS The toxicity of four neonicotinoid insecticides has been assessed for GWSS adults using a topical application bioassay (Table 1). Thiamethoxam, clothianidin and acetamiprid were all more toxic than imidacloprid. Clothianidin was - 290 -
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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
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
Page 185 and 186: GENETIC VARIABILITY OF XYLELLA FAST
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Page 189 and 190: probing activities). In preliminary
Page 191 and 192: the slow release valve was opened a
Page 193 and 194: 12. Hopkins DL (1977) Diseases caus
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
Page 203 and 204: multiply to relatively high (easily
Page 205 and 206: SPATIAL DATABASE CREATION AND MAINT
Page 207 and 208: Project Leader: Thomas M. Perring D
Page 209 and 210: Figure 2. Individual leaves from a
Page 211 and 212: The state variables, process functi
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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: Figure 2. Polymer disk accumulation
Page 233 and 234: EVALUATION OF RESISTANCE POTENTIAL
Page 235 and 236: Project Leader: Doug Cook Dept. of
Page 237 and 238: Based on the in silico analysis, de
Page 239 and 240: CONTROL OF PIERCE’S DISEASE THROU
Page 241 and 242: Figure 1. Oleander ‘White’ afte
Page 243 and 244: RESULTS During the reporting period
Page 245 and 246: DEVELOPMENT OF AN ARTIFICIAL DIET A
Page 247 and 248: DESIGN OF CHIMERIC ANTI-MICROBIAL P
Page 249 and 250: Neutrophil elastase Cecropin B Chim
Page 251 and 252: and WTXb is very low (0.00-0.40%) a
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Page 255 and 256: GENETIC DIFFERENTIATION AMONG GEOGR
Page 257 and 258: Table 1. Single-populations descrip
Page 259 and 260: MOLECULAR DISTINCTION BETWEEN POPUL
Page 261 and 262: These novel observations strongly s
Page 263 and 264: SEQUENCE DIVERGENCE IN TWO MITOCHON
Page 265 and 266: Table 3. Pairwise sequence distance
Page 267 and 268: DEVELOPMENT OF MOLECULAR DIAGNOSTIC
Page 269 and 270: A. B. Relative Density of SCAR Band
Page 271 and 272: THE ALIMENTARY TRACK OF GLASSY-WING
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Page 275 and 276: REALIZED LIFETIME PARASITISM AND TH
Page 277 and 278: REPRODUCTIVE AND DEVELOPMENTAL BIOL
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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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