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

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and grape may differentially affect growth of Xf. Redox status also likely affects the tendency for Xf aggregation and biofilm formation. Adding reducing agents such as glutathione to artificial medium promotes Xf aggregation and biofilm formation (Leite et al., 2004). It was reported that thiols mediate the aggregation and adhesion of Xf (Leite et al., 2002). Thiolcontaining compounds in xylem fluid include cysteine, methionine and glutathione. The redox status in citrus and grape xylem fluid and its role in Xf aggregation and biofilm formation, and host plant resistance/susceptibility to Xf need to be further investigated. OBJECTIVES 1. Investigate the effect of host plant xylem fluid on Xf multiplication, aggregation and attachment. 2. Determine the biochemical mechanisms of host xylem fluid influence on Xf multiplication, aggregation and attachment. RESULTS Commercial citrus (lemon, orange and grapefruit) groves in proximity to vineyards were selected in the Temecula Valley, California. Three blocks of 30 citrus and 30 grape vines were used. A minimum of 15 citrus trees and 15 vines were randomly selected from each block (making a total of 15 trees or vines from each plant species) to extract xylem fluid. Terminal shoots from each plant were used for xylem extraction with a pressure bomb apparatus (Anderson et al., 1989). Upon collection, the xylem fluid was immediately placed on dry ice before final storage in a -80 o C freezer. The samples were used to test the impact of these xylem fluid on Xf resistance and chemical analyses of soluble carbohydrates, free amino acids, and redox status. Effects of xylem fluid of each plant species on Xf attachment were evaluated on the biofilm formation. Formation of biofilm on the abiotic surfaces was assessed as described by Espinosa-Urgel et al. (2000). The analyses of Xf multiplication and aggregation were based on the fact that optical density (540 nm) is correlated with bacterial cell numbers and aggregation state as described by Burdman et al. (2000). Our data indicated that, when the xylem fluid of grapefruit, orange and lemon was added to the PD Temecula strain of Xf in PD3 medium in glass culture tubes, there were heavy Xf cell aggregations to form large white clumps in suspension of the culture and the culture fluid was clear with no significant turbidity; in contrast, grape xylem fluid added to the same Xf culture did not cause visible clumping, but rather a visible thick biofilm was formed on the surface of glass tube and the culture was turbid (Figure 1). After homogenization of the culture, we found that the numbers of Xf cells in the grapefruit xylem fluid treatment were significantly higher at 6, 8 and 9 days after culture compared with those in the grape xylem fluid treatment (Figure 2). The numbers of Xf cells in orange or lemon xylem fluid treatments were generally lower than those in grape xylem fluid treatment (Figure 3). These data suggest that the citrus species, especially grapefruit, are suitable hosts for Xf growth and may serve as a great reservoir of the pathogen for GWSS acquisition. Our assay results revealed that xylem fluid of the citrus species significantly inhibited Xf biofilm formation compared to that of grape (Figure 4). Our attempt to investigate the biochemical mechanisms likely to be involved indicated that 96% of amino acids in grape xylem fluid was comprised of glutamine, while 47% of amino acids in grape fruit xylem fluid was proline (Figure 5). The content of total amino acids in grape xylem fluid was near 9-fold higher than that in grapefruit xylem fluid (Figure 5). Sugar contents were 1.4- to 5.5-fold higher in grape xylem fluid than those in grapefruit xylem fluid (Figure 6). Peroxidase and total thiol levels were also higher in grape xylem fluid than in citrus xylem fluid (Figures 7 and 8). CONCLUSIONS <strong>Xylem</strong> fluid of grapefruit, orange and lemon caused PD Temecula strain of Xf cells to aggregate and form large white clumps but inhibited the attachment. In contrast, grape xylem fluid did not cause visible clumping but led to heavy attachment. Grapefruit xylem fluid significantly increased multiplication of Xf cells compared with grape xylem fluid. Citrus species, especially grapefruit, appear to be suitable hosts for Xf growth and may serve as a reservoir of the pathogen for GWSS acquisition and transmission to grape vines. Further research is underway to elucidate the biochemical mechanisms. Figure 1. Effect of host plant xylem fluid on Xf aggregation. A, treatment with grape xylem fluid. B, treatment with grapefruit xylem fluid. C, treatment with orange xylem fluid. D, treatment with lemon xylem fluid. Note that white clumps of Xf aggregates are formed in the grapefruit, orange and lemon xylem fluid treatments. - 61 -
0.18 600 Optical density 0.16 0.14 Grape 0.12 Grape fruit 0.1 Control 0.08 0.06 0.04 4 5 6 7 8 9 10 Days of culture Figure 2. Effect of host plant xylem fluid on Xf growth. nM / ml xylem fluid 500 400 300 200 100 0 Glu Gln Pro Total Grape Grape Fruit Optical Density 0.18 0.16 0.14 0.12 0.1 0.08 0.06 0.04 4 5 6 7 8 9 10 Days of culture Figure 3. Effect of host plant xylem fluid on Xf growth. Optical density 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 2 4 6 Days of culture Figure 4. Effect of host plant xylem fluid on Xf biofilm formation. Grape Orange Lemon Grape Grape Fruit Orange Lemon Figure 5. Some amino acid contents in grape and grape fruit xylem fluid. nM / ml xylem fluid 4000 3500 3000 2500 2000 1500 1000 500 0 Glucose Fructose Sucrose Grape Grape Fruit Figure 6. Sugar contents in grape and grape fruit xylem fluid. Change of OD/ml xylem fluid 3 2.5 2 1.5 1 0.5 0 Grape Grape Fruit Orange Lemon OD412 / ml xylem fluid 2 1.5 1 0.5 0 Grape Grape Fruit Orange Lemon Figure 7. Peroxidase levels in host xylem fluid. Figure 8. Total thiol contents in host xylem fluid. - 62 -
Page 1: IMPACT OF HOST PLANT XYLEM FLUID ON
Page 5 and 6: OPTIMIZING MARKER-ASSISTED SELECTIO
Page 7 and 8: esistant and susceptible genotypes
Page 9 and 10: MAP BASED IDENTIFICATION AND POSITI
Page 11 and 12: esistant genotypes are in process a
Page 13 and 14: BREEDING PIERCE’S DISEASE RESISTA
Page 15 and 16: Progeny from crosses of field resis
Page 17 and 18: a = (1=low, 4= high); b = (1=green,
Page 19 and 20: - 78 -
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Page 23 and 24: v.8) for differences due to the pla
Page 25 and 26: SHARPSHOOTER FEEDING BEHAVIOR IN RE
Page 27 and 28: correlated with all other findings
Page 29 and 30: EFFECTS OF FEEDING SUBSTRATE ON RET
Page 31 and 32: REFERENCES Bextine, B. and T. Mille
Page 33 and 34: will also provide insights into the
Page 35 and 36: OBJECTIVES 1. Determine glassy-wing
Page 37 and 38: GWSS per 30 s sweep (seasonal avera
Page 39 and 40: ELISA for the presence of GWSS egg
Page 41 and 42: Project Leader: Thomas P. Freeman E
Page 43 and 44: Freeman, T. P., R. A. Leopold, D. R
Page 45 and 46: pathogen, when they move into viney
Page 47 and 48: A NOVEL IMMUNOLOGICAL APPROACH FOR
Page 49 and 50: 1.6 GWSS Adults That Fed on Protein
Page 51 and 52: 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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- 164 -
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- 166 -
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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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- 246 -
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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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Neutrophil elastase Cecropin B Chim
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and WTXb is very low (0.00-0.40%) a
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100 100 0.056 North America 100 0.0
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GENETIC DIFFERENTIATION AMONG GEOGR
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Table 1. Single-populations descrip
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MOLECULAR DISTINCTION BETWEEN POPUL
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These novel observations strongly s
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SEQUENCE DIVERGENCE IN TWO MITOCHON
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Table 3. Pairwise sequence distance
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DEVELOPMENT OF MOLECULAR DIAGNOSTIC
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A. B. Relative Density of SCAR Band
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THE ALIMENTARY TRACK OF GLASSY-WING
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We have dissected and identified al
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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
Page 299 and 300:
SYMBIOTIC CONTROL OF PIERCE’S DIS
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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
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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
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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