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

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Proportion of sharpshooters that tested positive 1 0.8 0.6 PD Strain 0.4 Grape 0.2 Chrysanthemum 0 0 2 4 6 8 1 0.8 0.6 0.4 0.2 OLS Strain Oleander Chrysanthemum 0 0 2 4 6 8 Days after transfer from source plant Results from retention experiments using the OLS strain acquired from oleander showed no significant difference in the mean proportion of insects testing positive when insects were subsequently fed for on either a host (oleander), or a non-host (chrysanthemum) of the OLS strain for 1, 3, or 7 days after acquisition. Similarly experiments using the PD strain acquired from grapevine also found no significant difference in the mean proportion of insects testing positive at 1, 3 or 7 d after acquisition regardless of whether the insects were subsequently fed on either a host (grapevine) or a non-host (chrysanthemum) of the PD strain. Thus, both PD and OLS strains of X. fastidiosa remained detectable in GWSS, even when the insects fed on a non-host of the strain for 7 d. Objectives 2 and 3 To test if feeding substrate can influence the ability of insects to acquire and transmit a particular strain of X. fastidiosa, we plan to use a pathogen delivery system to allow us to either maintain or manipulate the feeding substrate as desired. The method described by Bextine and Miller (2002) was originally used for an Alcaligenes sp. of bacteria. This technique was modified to provide an environment suitable to survival of Xylella fastidiosa and the test plants used. We are using sections of chrysanthemum stem about 10 cm long that are connected by tubing to a syringe with a suspension of X. fastidiosa in PBS. The distal end of the stem is also cut and left open. The syringe is depressed until liquid is extruded from the distal end of the cut stem. Then GWSS are allowed to feed on these stems. To demonstrate that live X. fastidiosa cells could survive movement through a cut stem, X. fastidiosa was suspended in a PBS buffer, and the syringe was depressed until liquid was extruded from the distal end of the cut stem. Droplets forming on the distal end were collected and analyzed using PCR to determine if X. fastidiosa cells were present. In all cases, Xylella was detected within the first 10 drops extruded. Thus, in these experiments, material was injected into stems until at least 10 drops of material was extruded from the distal cut end to ensure that the bacteria have been moved the entire distance of the stem. In transmission experiments, adult insects were fed for 24 hours on either infected plants, or media-grown bacteria delivered through the cut stem system as described above. Adults were then individually moved to uninfected test plants and allowed to feed for 4 d. When GWSS adults were fed on PD-infected grapevines, 12/26 (46%) transmitted the pathogen to healthy grapevine test plants. In contrast, when insects were fed on media-grown PD bacteria through the cut stem method, no individuals (0/48) transmitted the pathogen to test pants. Similar results were found with OLS-infected plants (9/37,or 24% of individuals transmitted) compared to media-grown OLS delivered through cut stems (0/22 transmitted). Thus, insects did not transmit PD or OLS strains when media-grown bacteria were delivered through the cut-stem system. Purcell et al. (personal communication) found similar results when leafhoppers were fed X. fastidiosa through parafilm sachets. Additional studies are being conducted to determine why insects are unable to transmit the pathogen from the cut stem delivery system. For example, a recent study demonstrated that X. fastidiosa cultures will produce different levels of “biofilm formation” when grown on different types of media (Leite et al. 2004). We will test if growing our strains on different media may help induce transmissibility by insects. In addition, we will conduct further studies to determine the pathway of the bacteria through the system. For example, by testing the honeydew of insects feeding on the cut stem system, we can determine if the bacteria are successfully passing through the insect. In the interim, work on the remaining objectives will continue using insects fed on PD and OLS infected plants. CONCLUSIONS In retention experiments (Objective 1) for both the PD and OLS strains, we found the proportion of insects retaining the pathogen was the same, regardless of whether insects subsequently fed on a host or a non-host of that strain. This indicates that retention of a particular strain of the pathogen by an individual insect is not dependant on host-specific xylem content of the plant on which it is feeding. In transmission experiments (Objectives 2 and 3) insects successfully transmitted the PD and OLS when they acquired the pathogen from infected grapevine and oleander plants respectively, but did not transmit either the PD or OLS strains when the media-grown bacteria were delivered through the cut-stem system. This could be the result of biological characteristics of media-grown bacteria that contribute to non-transmissibility by insects, or failure of the cut stem system to properly deliver bacteria to the insect. Further experiments are being conducted to determine the basis for lack of transmission of media-grown bacteria by GWSS. - 89 -
REFERENCES Bextine, B. and T. Miller. 2002. Insect-symbiotic bacteria inhibitory to Xylella fastidiosa in sharpshooters: pressure bomb extraction of xylem fluid to improve bacterial detection of Xylella in plants. Symposium Proceedings: Pierce’s Disease Research Symposium, Dec. 2002, California Department of Food and Agriculture, Sacramento. Pp. 29-30. Leite, B., P. C. Andersen, and M. L. Ishida. 2004. Colony aggregation and biofilm formation in xylem chemistry-based media for Xylella fastidiosa. FEMS Microbiol. Lett. 230: 283-290. FUNDING AGENCIES Funding for this project was provided by the CDFA Pierce’s Disease and Glassy-winged Sharpshooter Board. - 90 -
Page 1 and 2: IMPACT OF HOST PLANT XYLEM FLUID ON
Page 3 and 4: 0.18 600 Optical density 0.16 0.14
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,
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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: EFFECTS OF FEEDING SUBSTRATE ON RET
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.
Page 53 and 54: RESULTS: Oviposition Survey Wild gr
Page 55 and 56: Host specificity testing: No-choice
Page 57 and 58: currently employed morphological ch
Page 59 and 60: increase our present understanding
Page 61 and 62: BIOLOGY AND MORPHOMETRIC ANALYSIS O
Page 63 and 64: Table 1. Mean a developmental durat
Page 65 and 66: EFFECTS OF USING CONSTANT AND CYCLI
Page 67 and 68: REFERENCES Gautam, R. D. 1986. Effe
Page 69 and 70: PARASITISM OF THE GLASSY-WINGED SHA
Page 71 and 72: Table 1. Parasitism by G.ashmeadi o
Page 73 and 74: Project Leader: Robert F. Luck Dept
Page 75 and 76: A more interesting analysis using t
Page 77 and 78: MYCOPATHOGENS AND THEIR EXOTOXINS I
Page 79 and 80: 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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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
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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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