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

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OBJECTIVES 1. Study the behavior of strains of A. xylosoxidans subsp. denitrificans (Axd and Raxd) when grown under various biotic influences and, 2. Investigate and compare the growth of A. xylosoxidans subsp. denitrificans (wild type) and Raxd to that of Axd modified to express a short chain antibody against X. fastidiosa (Axd 7.7) that infects grape under different physiological conditions, such as in response to nutrient availability and energetic demands. RESULTS We have found that Axd and Raxd do not establish when introduced into soil, but can be recovered from soil that was sterilized before inoculation with Axd and Raxd. Axd and Raxd, when applied to leaf surfaces, can be recovered from established phylloplane communities of basil, strawberry, and sage, although recovery is scant to low. Co-culture experiments showed that Axd and Raxd growth are negatively affected by the presence of E. coli and P. aeruginosa. The growth of Axd modified to express an S1 scFv (single chain antibody variable region fragments) antibody (Axd 7.7) that binds specifically to a strain of X. fastidiosa that infects grape was compared to that of the wild type Axd and Raxd. Axd, Raxd, and Axd 7.7 exhibited similar growth patterns in tryptic soy broth (TSB). Axd, Raxd, and Axd 7.7 also demonstrated longer lag phases in Luria Bertani medium (LB) than for TSB. Cell numbers remained fairly constant for each strain at each growth phase. Growth studies are underway that monitor the growth of Axd, Raxd, and Axd 7.7 in dilute, R2A medium. Current studies also underway include using enzyme linked immunosorbent assays to monitor the expression of S1 scFv from Axd 7.7 under environmental challenges, such as poor nutrient availability and energetic demands. CONCLUSIONS From earlier work we have found that Raxd establishes within the mouthparts of H. coagulata (Bextine et al. 2004a) and within the xylem of several of this sharpshooter’s host plants (Bextine et al. 2004b). The bacterium, however, does not establish within soil if soil communities are in place. If the soil is sterilized and biotic competition is eliminated, then Axd and Raxd grow relatively well. Conversely, Axd and Raxd can survive and be retrieved from the leaf surfaces of plants other than citrus, such as basil, sage, and strawberry plants for up to two weeks. These data suggest that Axd and Raxd are more suited to the plant environment than to a soil environment. We conclude that Axd and Raxd will remain in the plant environment long enough to exert is anti-Xylella effect with little to no disruption of any relevant ecosystem. Raxd did not grow well in the presence of E. coli and P. aeruginosa compared to Raxd grown in pure culture. Thus, compared to a ubiquitous bacterium and a pathogen, respectively, Raxd is not as fit under standard growth conditions. Axd 7.7 growth compared to Axd and Raxd differed little under our experimental conditions. All data collectively suggest that Axd 7.7 shows potential for delivery of an anti-Xylella product with little impact on nontarget bacterial ecosystems. This statement is qualified by the fact that field tests must be implemented to assess the true behavior of strains of Axd in the environment. Laboratory studies are not suitable for a genuine assessment of risk assessment and environmental impact; nevertheless, they provide important insight. REFERENCES Bextine, B.R., C.R. Lauzon, D. Lampe, and T.A. Miller. 2004a. Delivery of a genetically marked Alcaligenes sp. to the glassy-winged sharpshooter for use in a paratransgenic control strategy. Curr. Microbiol. 48: 327-331. Bextine, B.R., C.R. Lauzon, D. Lampe and T.A. Miller. 2004b. Establishment of a genetically marked insect-derived symbiont in multiple host plants. Curr. Microbiol. In Press. Ingvorsen, K., B. Højer-Pedersen, and S. E. Godtfredsen. 1991. Novel cyanide-hydrolyzing enzyme from Alcaligenes xylosoxidans subsp. denitrificans. Appl. Environ. Microbiol. 57: 1783-1789. Velicer, G.J. 1999. Pleiotropic effects of adaptation to a single carbon source for growth on alternative substrates. Appl. Environ. Microbiol. 65(1): 264-269. FUNDING AGENCIES Funding for this project was provided by the USDA Animal and Plant Health Inspection Service. - 359 -
MANAGEMENT OF PIERCE’S DISEASE OF GRAPE BY INTERFERING WITH CELL-CELL COMMUNICATION IN XYLELLA FASTIDIOSA Project Leader: Steven E. Lindow Dept. of Plant and Microbial Biology University of California Berkeley, CA 94720 Cooperators: Karyn Lynn Newman Dept. of Plant and Microbial Biology University of California Berkeley, CA 94720 Alexander Purcell Dept. of Environmental Science Policy & Management University of California Berkeley, CA 94720 Reporting Period: The results reported here are from work conducted from December 15, 2003 to October 15, 2004. ABSTRACT Xylella fastidiosa (Xf) is an endophyte that is restricted to the xylem, a network of vessels for water transport, in which it forms an aggregated biofilm. It is transmitted from plant to plant by xylem sap-feeding insects, and forms a polar biofilm in these insects’ foreguts. In other systems, biofilms are characterized by community behavior under the control of cell densitydependent gene expression, which requires cell-cell signaling. Xf has homologs of the cell-cell signaling genes found in the important plant pathogen Xanthomonas campestris pathovar campestris (Xcc) and produces a similar alpha,beta unsaturated fatty acid signal molecule called DSF that coordinates gene expression in a community (2, 7). We have investigated DSFmediated cell-cell signaling in Xf with the aim of developing cell-cell signaling disruption as a means of controlling Pierce’s disease. We have determined that the rpfF gene is necessary and sufficient for DSF signal synthesis and that rpfF mutants of Xf are hypervirulent and non-transmissible. Lack of transmissibility was linked to an inability of the rpfF mutant to form a biofilm in the insect foregut; while taken up by insects, the mutant strain is not retained. Xf strains that overproduce DSF produce disease symptoms in grape, but only at the site of inoculation and the cells do not move within the plant as do wildtype strains. Thus elevating DSF levels in plants should reduce movement of Xf in the plant and also reduce the likelihood of transmission by sharpshooters. We screened several collections of bacterial strains isolated from plants and identified bacterial strains that can interfere with Xf signaling both by producing large amounts of DSF, by degrading DSF, or by in some way interfering with recognition of DSF. When co-inoculated into grape with Xf, both DSF-producing strains and DFS degrading strains greatly reduced the indicidence of disease in grape; DSF-producing strains consistently were the most effective in reducing disease. Given that DSF appears to mediate an attenuation of virulence in Xf we are in the process of transforming grape with the rpfF gene to enable DSF production in planta. Preliminary results indicate that transient expression of rpfF in Nicotiana benthamiana following infiltration with appropriate Agrobacterium tumefaciens strains resulted in high levels of DSF production, suggesting that it is likely that grape cells will produce DSF when transformed with the bacterial rpfF gene. Non-endophytic bacterial species were also established in high numbers inside grape leaves and petioles following spray application to plants with a high concentration of a silicon-based surfactant with a low surface tension, suggesting that it may be possible to produce protective compounds such as DSF in plants by a variety of bacteria. INTRODUCTION Endophytic bacteria such as Xylella fastidiosa (Xf) colonize the internal tissues of the host, forming a biofilm inside the plant. A key determinant of success for an endophyte is the ability to move within the plant. We expect activities required for movement to be most successful when carried out by a community of cells since individual cells may be incapable of completing the feat on their own. Cells assess the size of their local population via cell-cell communication and coordinately regulate the expression of genes required for such processes. Our study aims to determine the role of cell-cell communication in Xf in colonization and pathogenicity in grapevines and transmission by the insect vector. Xf shares sequence similarity with the plant pathogen Xanthomonas campestris pathovar campestris (Xcc) (7). In Xcc, expression of pathogenicity genes is controlled by the Rpf system of cell-cell communication, enabling a population of cells to launch a pathogenic attack in a coordinated manner (1). Two of the Rpf proteins, RpfB and RpfF, work to produce a diffusible signal factor (DSF) (1) which has recently been described as an alpha,beta unsaturated fatty acid ( 9): As the population grows, the local concentration of DSF increases. Other Rpf proteins are thought to sense the increase in DSF concentration and transduce a signal, resulting in expression of pathogenicity factors (8). The Xf genome not only contains homologs of the rpf genes most essential for cell-cell signaling in Xcc, but also exhibits striking colinearity in the arrangement of these genes on the chromosome (2). We now have shown that Xf makes a molecule that is recognized by Xcc - 360 -
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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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DEVELOPMENT OF AN ARTIFICIAL DIET A
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DESIGN OF CHIMERIC ANTI-MICROBIAL P
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Page 285 and 286: REFERENCES Hoddle, M. S. and S. V.
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Page 309 and 310: patterns differed among the lines,
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Page 315 and 316: REFERENCES Toscano, N., Byrne, F.,
Page 317 and 318: RESULTS AND CONCLUSIONS The program
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