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

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1. Natural populations of GWSS are commonly found thriving on several citrus varieties. 2. Axd colonized and grew best in the citrus varieties tested. 3. Axd colonized the xylem vessels of test plants, the same tissue from which GWSS feed. 4. Axd passively moved through populations of GWSS. 5. Axd did not negatively affect GWSS. Interestingly, Axd appears to mirror the host range of GWSS. Genetically marked Axd colonizes several host plants. This suggests that genetic modification does not interfere with the biology of Axd, which should enter into the insect-plant cycle and be transmitted along with the pathogenic bacteria target. While GWSS is the vector of greatest interest in California, two other native sharpshooters also transmit the vehicle bacterium, Axd, and several plants can serve as hosts. In the laboratory, inhibition of Xf-transmission by GWSS was demonstrated using two different categories of reagents, a surface antibody fragment and an antibiotic peptide (Indolicidin). The antibody fragment was specific to Xf. In our trials the antibody fragment was being expressed in the coat of a phage, so the effects on transmission might be greater when the antibody fragment is expressed on the surface of Axd. Indolicidin inhibited Xf growth in vitro, but did not affect growth of Axd. Transformation of Axd to produce each/or both of these reagents is currently under way. We concluded that Axd will be an effective delivery agent of a symbiont control strategy for combating Xf. GWSS readily acquired Axd from a plant source and this bacterium translocated and colonized a variety of plants tested. We have yet to determine the effect of the reagents on Xf in infected grapevines. Previously, plant symptoms confirmed by ELISA or PCR detection were used to determine if transmission had occurred. Unfortunately, these systems require the bacterium to colonize and infect the host plant to determine transmission. If an infected plant is asymptomatic, important but less obvious transmission events may be missed. Our system removes the plant “unknowns” from the equation. However, we recognize the importance of actual plant infection as a measure of transmission importance, but suggest using the artificial disease cycle as an initial rapid measure of vector competence. REFERENCES 1. Almeida RPP, Purcell AH (2003) Homalodisca coagulata (Hemiptera, Cicadellidae) transmission of Xylella fastidiosa to almond. Plant Dis. 87:1255-1259 2. Almeida RPP, Purcell AH (2003) Transmission of Xylella fastidiosa to grapevines by Homalodisca coagulata (Hemiptera: Cicadellidae). J. Econ. Entomol. 96:264-271 3. Beard CB, Cordon-Rosales C, Durvasula RV (2002) Bacterial symbionts of the Triatominae and their potential use in control of Chagas disease transmission. Annu. Rev. Entomol. 47:123-141 4. Beninati C (2000) Therapy of mucosal candidiasis by expression of an anti-idiotype in human commensal bacteria. Nat. Biotechnol. 18:1060-1064 5. Bextine B, Lampe D, Lauzon C, Jackson B, Miller TA (2004) Establishment of a genetically marked insect-derived symbiont in multiple host plants. Curr. Microbiol. In Press. 6. Bextine B, Lauzon C, Potter S, Lampe D, Miller TA (2004) Delivery of a genetically marked Alcaligenes sp. to the glassy-winged sharpshooter for use in a paratransgenic control strategy. Curr. Microbiol. 48:327-331 7. Bextine B, Miller TA (2004) Comparison of whole-tissue and xylem fluid collection techniques to detect Xylella fastidiosa in grapevine and oleander. Plant Dis. 88:600-604 8. Chang TL-Y, Chang CH, Simpson DA, Xu Q, Martin PK, Lagenaur LA, Schoolnik GK, Ho DD, Hillier SL, Holodniy M, Lewicki JA, Lee PP (2003) Inhibition of HIV infectivity by a natural human isolate of Lactobacillus jensenii engineered to express functional two-domain CD4. Proc. Nat. Acad. Sci. 100:11672-11677 9. Costa HS, Blua MS, Bethke JA, Redak RA (2000) Transmission of Xylella fastidiosa to oleander by the glassy-winged sharpshooter, Homalodisca coagulata. Hortsci. 35:1265-1267 10. Meade MJ, Waddell RL, Callahan TM (2001) Soil bacteria Pseudomonas putida and Alcaligenes xylosoxidans subsp. denitrificans inactivate triclosan in liquid and solid substrates. Microbiol. Let. 204:45-48 11. Steidler L, Hans W, Schotte L, Neirynck S, Obermeier F, Falk W, Fiers W, Remaut E (2000) Treatment of Murine Colitis by Lactococcus lactis Secreting Interleukin-10. Science 289:1352-1354 12. Yang HL, Sun XL, Song W, Wang YS, Cai MY (1999) Screening, identification and distribution of endophytic associative diazotrophs isolated from rice plants. Acta Bot. Sinica. 41:927-931 FUNDING AGENCIES Funding for this project was provided by the USDA Animal and Plant Health Inspection Service and the CDFA Pierce’s Disease and Glassy-winged Sharpshooter Board. - 285 -
EXPLOITING XYLELLA FASTIDIOSA PROTEINS FOR PIERCE’S DISEASE CONTROL Project Leaders: George Bruening Dept. of Plant Pathology University of California Davis, CA 95616 Cooperators: Abhaya M. Dandekar Department of Pomology University of California Davis, CA 95616 Edwin Civerolo USDA, ARS, PWA San Joaquin Valley Agricultural Sciences Center Parlier, CA 93648 Goutam Gupta Biology Division Los Alamos National Laboratory Los Alamos, New Mexico Reporting Period: The results reported here are from work conducted from October 15, 2003 to October 11, 2004. ABSTRACT The Xylella fastidiosa (Xf) is the causal agent of Pierce’s disease of grape. In previous work, we discovered, partially purified, and investigated the processing of the Xf protein MopB, which previously had been known only from the nucleotide sequence of its gene. The amino acid sequence of MopB, the uniform staining of Xf cells with fluorescent anti-MopB antibody and the abundance of MopB in total protein extracts of Xf cells suggest that MopB is the major outer membrane protein of Xf. As such, MopB is expected to participate in Xf colonization of grape xylem elements. We previously demonstrated that partially purified MopB binds to (xylem-rich) balsa wood or cellulose (filter paper) disks under conditions in which other proteins do not adhere. Here we report improvements in our MopB purification procedure and observations on adherence of MopB in Xf cells to cellulose disks under conditions that eluted other Xf proteins. A high (0.25mM) concentration of the cellulose fragment cellotetraose did not interfere with the binding of MopB to cellulose, suggesting that the binding reaction of MopB is not specific for cellulose. We exposed Xf cells or MopB to each of three fibrous polymer disks and to cellulose disks and observed similar adherence of MopB from both sources to all four polymer disk types. Thus, MopB appears to associate with porous materials generally when it is exposed to such materials in purified form or as Xf cells. The abundance and exterior exposure of MopB makes MopB an ideal target for Pierce’s disease control strategies. We seek to develop soluble proteins with high affinity for MopB. We will apply, as an anti-Xf agent, a selected MopB-binding protein alone or as a chimera with a bacterial cell-inactivating peptide or protein. Our expectation is that expression of the anti-Xf protein, targeted to the xylem in grape rootstock, may result in the anti-Xf protein moving into and protecting the grafted scion. In this reporting period, experiments were initiated with the objective of creating a protein having high affinity for MopB. As a first step towards this objective, Project Scientist Paul Feldstein developed E. coli strains expressing surface elements of MopB protein, so that the experimentally compliant E. coli can be used to select proteins with high affinity for Xf MopB. INTRODUCTION We have been investigating an abundant protein of Xf, MopB. We showed that MopB is the major outer membrane protein of Xf and is partly exposed on the outside of the bacterial cell. We purified MopB, prepared antibodies against it, and demonstrated an apparent affinity of MopB for cellulose. This last observation and the abundance of MopB suggested that MopB may participate in the initial attachment of Xf to the inner surface of the xylem vascular elements or in some other critical event in the initiation of infection leading to the development of Pierce’s disease. Regardless of whether MopB is critical in this process, its location and prevalence support our contention that MopB is an ideal target for a Xf-specific bactericide or for a reagent that would coat and thereby inactivate Xf cells. Our strategy for creating a high-affinity MopBbinding protein is to begin with a protein that has evolved to bind tightly to the major outer membrane protein of E. coli, OmpA, and to convert the specificity of that protein from OmpA-binding to MopB-binding. The T2-like E. coli bacteriophage K3 has OmpA as its receptor. The K3 tail fiber adhesion gp38 is responsible for binding of bacteriophage K3 to OmpA in a reaction whose rate and irreversibility suggest a high-affinity association. Mutational conversion of gp38 from its natural receptor OmpA to other E. coli surface proteins has been demonstrated in several publications (Drexler et al., 1991, and references cited therein). In outline, our planned experimental steps for creating an anti-Xf protein are (i) replace the OmpA protein of E. coli with a protein that has MopB sequences displayed on the cell exterior, (ii) select variants of bacteriophage K3 that can infect the modified E. coli and also can bind to Xf cells, (iii) isolate the variant bacteriophage K3 gene gp38 (expected to encode a MopB-binding gp38 protein), and (iv) genetically modify the MopB-binding gp38 to confer solubility and (in collaboration with the Gupta laboratory) possibly fuse the gp38 to a bactericidal peptide-encoding sequence. Step (v) will be the expression of a xylem-targeted version of the gp38 or gp38 fusion protein in rootstock and will be performed in collaboration with the Dandekar laboratory. - 286 -
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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
Page 175 and 176: PATTERNS OF XYLELLA FASTIDIOSA INFE
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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
Page 213 and 214: DEVELOPMENT OF A FIELD SAMPLING PLA
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: cases, positive phloem samples were
Page 229 and 230: Figure 2. Polymer disk accumulation
Page 231 and 232: CHARACTERIZATION OF NEONICOTINOIDS
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
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Page 271 and 272: THE ALIMENTARY TRACK OF GLASSY-WING
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Page 275 and 276: 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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