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

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OBJECTIVES Objective 1: Objective 2: Objective 3: a) Utilize literature data and computer modeling to identify an SRD that specifically targets mopB (Elastase) b) Utilize literature data and computer modeling to identify a useful Cecropin (i.e., Cecropin B) c) In vitro testing of anti-<strong>Xylella</strong> activity of the mopB-specific SRD (Elastase) and Xyllela-specific Cecropin B and demonstration of synergistic killing effect due to the combined use of Elastase and Cecropin B. a) Design and construction of synthetic gene encoding Elastase-Linker-Cecropin B Chimeric protein b) Expression Elastase-Linker-Cecropin B in insect and plant cells and testing activity in vitro. a) Expression in transgenic plants b) Testing for anti-<strong>Xylella</strong> activity in planta and testing for graft transmissibility. RESULTS AND CONCLUSION Human Neutrophil Elastase (HNE) (6) was chosen as our first SRD. Neutrophils contain a variety of proteins that enable the cells to migrate toward and eliminate microbial pathogens (7). Until 1991, no specific antibacterial activity had been ascribed to HNE (8). However recent research has established that HNE is the only human neutrophil protein, which is capable of individually killing Borrelia burgdorferi, the causative agent of Lyme disease (9, 10). Furthermore, it is known that HNE can augment the cidal properties of other active proteins (11). Sequence-structure analysis of mopB revealed that it contained an specific cleavage site for HNE that is exposed on the surface. We have studied the efficacy of HNE in combination with the antibacterial peptide Cecropin B, that inserts preferentially into the lipid bilayer of gram-negative bacteria, in killing Xf. Measuring the number of colony forming units remaining after the bacterium was exposed to HNE, Cecropin B and the combination of both, we found that HNE greatly stimulates the lysis induced by Cecropin B. In addition, we found that Mop B was partially digested by HNE after incubating either purified Mop B or Xf cells with HNE for an hour. Based on these preliminary results, we have designed a chimeric protein of Cecropin B and HNE; in order to stabilize the Cecropin B peptide and enhance the overall affinity of the ligands for the bacterial surface. The covalent attachment of Cecropin B to HNE is proposed to increase the stability of the peptide by lowering the conformational entropy of its unfolded state and to increase the overall affinity for the bacterial surface by minimizing the degrees of motion at the binding site, thereby increasing binding between the ligands and the surface. Our strategy began with the generation of a 3-D model of the chimera. The modeling was based on published protein data bank (PDB) structures of HNE and nuclear magnetic resonance structures of peptides homologous to Cecropin B. A short G- S-T-A peptide linker was inserted between the C-terminus of HNE and the N-terminus of Cecropin B to allow both functional domains to make contact with the bacterial surface simultaneously without steric interference. Energy minimization and molecular dynamics analysis using the AMBER 7.0 force field indicated that the chimera forms a stable structure. The HNE-GSTA-Cecropin B chimera gene was synthesized and is currently being cloned into a baculovirus vector for overexpression in insect cells. The chimera will be purified from insect cells and tested for its activity against Xf in vitro. The chimera will be also cloned into a plant vector for transformation of grape embryogenic callus growing in a CELLline 350 bioreactor where they will be analyzed for the production and anti-Xf activity of the secreted protein. We will choose the most promising embryogenic lines for plant regeneration. The plant expression vector will have necessary regulatory sequences to facilitate transcription and extracellular delivery of the protein product. Currently we are investigating grapevine embryogenic callus for the extracellular production of the pear polygalacturonase inhibiting protein (pPGIP). This protein has been found in the xylem exudate of transgenic grapes expressing the pPGIP gene and will be used to modify delivery of the chimeric protein to grapevine xylem tissues. IVGGRRARPHAWPFMVSLQLRGGHFCGATLIAPNFVMSAAHCVANVNVRAVRVVLGAHNLSRREPTR QVFAVQRIFEDGYDPVNLLNDIVILQLNGSATINANVQVAQLPAQGRRLGNGVQCLAMGWGLLGRNRG IASVLQELNVTVVTSLCRRSNVCTLVRGRQAGVCFGDSGSPLVCNGLIHGIASFVRGGCASGLYPDAFAP VAQFVNWIDSIIQGSTAKWKVFKKIEKMGRNIRNGIVKAGPAIAVLGEAKAL Figure 1. HNE-cecropin B chimeric amino acid sequence. HNE is attached to cecropin B (shown in bold) by the GSTA linker, which is underlined. - 307 -
Neutrophil elastase Cecropin B Chimera mopB Figure 2. Design and mechanism of chimeric protein targeted to X. fastidiosa. The top panel shows the two domains of the chimera in separate planes: neutrophil elastase (1HNE from PDB) is on the left. A homology model of ceropin B is shown in the middle. The right plane shows the energy minimized model of the elastase-cecropin B chimera. The bottom panel is a schematic of the hypothetic mechanism of the chimeric protein. Elastase binds to and cleaves a specific loop on the X. fastidiosa outer membrane protein mopB. This action brings cecropin B in close contact with the membrane, where is associates with other cecropin molecules and disrupts the membrane by forming a pore, thereby disabling the bacterium. REFERENCES 1. Cohn, J., Sessa, G., and Martin, G.B. (2001). Innate immunity in plants. Curr. Opin. Immunol. 13:55-62. 2. Magor, B.G. and Magor, K.E. (2001). Evolution of effectors and receptor of innate immunity. Dev. Comp. Immunol. 25:651-682. 3. Pieters, J. (2001). Evasion of host cell defense by pathogenic bacteria. Curr. Opin. Immunol. 13:37-44. 4. Baquero, F. and Blazquez, J. (1997). Evolution of antibiotic resistance. Trends Ecol. Evol.12:482-487. 5. Bruening, G., Civerelo, E., Kirkpatrick, B., and Gilchrist, D. (2002). Virulence analysis of The Pierces Disease Agent X. fastidiosa. PD research symposium Dec 15-18 2002, San Diego, CA. 6. Sinha, S., W. Watorek, et al. (1987). Primary structure of human neutrophil Elastase. Proc. Natl. Acad. Sci.USA 84: 2228-2232. 7. Elsbach, P., and Weiss, J. (1988). Phagocytic cells: oxygen-independent anti-microbal systems. In Inflammation: basic principles and clinical correlates. Gallin, J.L., Goldstein, I.M. and Snuderman, R. (Ed.). Raven Press, New York, USA, pp 445-470. 8. Wasiluk, K. R., K. M. Skubitz, et al. (1991). Comparison of granule proteins from human polymorphonuclear leukocytes which are bactericidal toward Pseudomonas aeruginosa. Infection and Immunity 59: 4193-4200. 9. Garcia, R., L. Gusmani, et al. (1998). Elastase is the only human neutrophil granule protein that alone is responsible for in vitro killing of Borrelia burgdorferi. Infection and Immunity 66(4): 1408-1412. 10. Lusitani, D., S. E. Malawista, et al. (2002).Borrelia burgdorferi are susceptible to killing by a variety of human polymorphonuclear leukocyte components. The Journal of Infectious Diseases 185: 797-804. 11. Miyasaki, K. T. and Bodeau, A.L. (1991). In vitro killing of Actinobacillus actinomycetemcomitans and Capnocytophaga spp. by human neutrophil cathepsin G and Elastase. Infection and Immunity 59: 3015-3020. FUNDING AGENCIES Funding for this project was provided by the CDFA Pierce’s Disease and Glassy-winged Sharpshooter Board. - 308 -
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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
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 and 226: cases, positive phloem samples were
Page 227 and 228: EXPLOITING XYLELLA FASTIDIOSA PROTE
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: DESIGN OF CHIMERIC ANTI-MICROBIAL P
Page 251 and 252: and WTXb is very low (0.00-0.40%) a
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Page 257 and 258: Table 1. Single-populations descrip
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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
Page 273 and 274: We have dissected and identified al
Page 275 and 276: REALIZED LIFETIME PARASITISM AND TH
Page 277 and 278: REPRODUCTIVE AND DEVELOPMENTAL BIOL
Page 279 and 280: Mean adult longevity (days) 25 20 1
Page 281 and 282: the proposed exploratory work; thes
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
Page 289 and 290: 7. Hultmark, D., et al., Insect Imm
Page 291 and 292: Isolation of Fungal Pathogens Soil
Page 293 and 294: IDENTIFICATION OF MECHANISMS MEDIAT
Page 295 and 296: concentrations of 1.5 x 10 7 bacter
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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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