1a. Objectives (from AD-416)
The overall goals of this project are to reduce losses due to Xf-caused diseases during crop production and to develop effective, sustainable disease and insect vector management strategies by characterizing the host-pathogen-vector-environment interactions of these complex pathosystems. Specific objectives for the project are outlined below: Objective 1: Determine the epidemiology of exotic, emerging, re-emerging, and invasive diseases in California, including (but not necessarily limited to) Xf-caused diseases of horticultural, agronomic, and ornamental crops. Objective 2: Determine the nature and mechanism(s) of susceptibility/resistance to Xf infection and subsequent disease development in horticultural and agronomic crops, including (but not necessarily limited to) Vitis species and Prunus species. Objective 3: Develop effective, economical, environmentally sound strategies to manage exotic, emerging, re-emerging, and invasive diseases, including (but not necessarily limited to) xylella diseases. Objective 4: Improve sensitivity and specificity of diagnostics utilizing new biomarkers based on the DNA sequence of the Liberibacter bacterium associated with Huanglongbing (citrus greening) and Zebra chip diseases. (NP 303; Component 1; Problem Statement 1A)
1b. Approach (from AD-416)
Determine the epidemiology of Xylellae diseases in California. Characterize the complex host-pathogen-insect vector-environment interactions of these pathosystems. Determine the biochemical, physiological, genetic and mechanistic bases of resistance to XF infection and subsequent disease development in Vitis and selected Prunus species. Utilize genomic sequences of Liberibacter bacteria associated with Huanglongbing and Zebra Chip diseases to develop new PCR-based assays for pathogen detection. Formerly 5302-22000-007-00D.
3. Progress Report
Research progress for FY11 included breeding for resistance to Xylella fastidiosa (Xf), characterization of Xf-host interactions, ecological and biological characterization of insect vectors of Xf, and genetic analysis of ‘Candidatus Liberibacter’ species associated with citrus Huanglongbing (HLB) and potato Zebra Chip diseases. Crop improvement efforts through traditional breeding included creation and screening of fifth backcross generation table and raisin grape progeny resistant to Pierce’s disease (PD). Four table and ten raisin PD-resistant selections from earlier generations have been selected and planted in small production trials. Second generation hybrids from a peach (Xf-resistant) x almond (Xf-susceptible) cross were established in a research orchard to be evaluated for Xf resistance in subsequent years. Additional molecular markers were developed to track inheritance of 22 resistance gene analogs in grape. Molecular genetic analysis of Xf pathogenicity/virulence identified eight novel genes that are predicted to be secreted by Xf; construction of knockout mutants of these genes is in progress. The molecular mechanism of Xf PemK toxicity was investigated and shown to be due to ribonuclease activity. Candidate small RNAs potentially produced by Xf were identified via in silico analysis of the Xf genome. Three Xf knockout mutants were generated to target virulence genes associated with putative functions in biofilm formation, cell wall digestion and cell division. Changes in xylem sap chemistry associated with Xf infection were documented and shown to be consistent among three cultivars. Expression profile analysis identified differentially expressed proteins in response to Xf infection. Two host defense response genes (jasmonic acid methyltransferase and jasmonic acid methyl esterase) were identified and cloned. Eleven new weed hosts of Xf were identified that may serve as sources of the bacterium. Xf was frequently found in ornamental olive trees in urban areas, but has not spread to olive production regions. Genetic typing and pathogenicity assays of Xf strains from olive have been initiated. Research on insects that transmit Xf continued. Movement of green sharpshooters into vineyards, almond orchards, and olive groves from source habitats was monitored. Green sharpshooters were shown to enter vineyards but not establish resident populations. To better understand response of glassy-winged sharpshooter females to PD affected grapevines, egg maturation and feeding rates of females were compared on unaffected and PD affected grapevines. X-ray videos of glassy-winged sharpshooter feeding on grapevines indicated that feeding alone does not disrupt water flow in grapevine stems. Genetic diversity of ‘Candidatus Liberibacter’ species associated with HLB and ZC was evaluated via sequence polymorphism in a prophage gene, and by multilocus sequence typing. Comparative genomics was used to identify common and distinct genes of the two presumptive pathogens.
1. Fifth backcross generation grapevines were created for the development of Pierce’s disease resistant raisin and table grapes. All table and raisin grapes grown in California are susceptible to Pierce’s disease caused by Xyllela fastidiosa. ARS scientists at Parlier, CA have generated fifth backcross generations of table and raisin grapes combining Pierce’s disease resistance from Vitus arizonica with fruit quality from advanced table and raisin selections. The fifth generation progeny have an average of 98.4% V. vinifera background for high fruit quality, yet retain the Pierce’s disease resistance gene PdR1 from V. arizonica. This germplasm may be developed into table and raisin grape varieties that can be grown in areas with high incidence of Pierce’s disease.
2. Timing of glassy-winged sharpshooter invasion of California estimated based on genomic variation of an insect virus used as a surrogate population genetics data set. Most invasive insect pests are discovered only after establishment with the actual date of introduction unknown. ARS scientists at Parlier, CA developed a novel method for distinguishing among recently separated populations of an invasive insect pest. As RNA viruses evolve at rates many times greater than that of their insect hosts, nucleotide sequence polymorphism of Homalodisca vitripennis reovirus was used as a surrogate data set to determine timing of glassy-winged sharpshooter introduction to California. The data indicated that glassy-winged sharpshooter was introduced to California in 1988, two years prior to the earliest documented specimen collected from the established invasive population. This novel method of dating an invasive species population provided the first credible estimate of time elapsed between introduction and discovery of an invasive insect pest and explains why belated efforts often fail to eliminate an invasive species population.
3. Complete genomes of Xylella fastidiosa strains M12 and M23 were determined, annotated, and released. Distinct strains of X. fastidiosa differ in host range and virulence on high value horticultural crops but genetic differences among such strains remain incompletely characterized. ARS scientists at Parlier, CA completed the genome sequences of two X. fastidiosa strains that cause disease of grape and almond (M23) or almond but not grape (M12). These publically released genome sequences will facilitate ongoing molecular studies aimed at characterizing pathogen genes responsible for host range of the bacterium causing Pierce’s disease of grapevine and almond leaf scorch.
4. Comparative genome analysis of ‘Candidatus Liberibacter’ species causing citrus Huanglongbing (HLB) and potato Zebra Chip (ZC) diseases. The genomes of the presumptive pathogens causing HLB (‘Candidatus Liberibacter asiaticus’) and ZC (Candidatus Liberibacter solanacearum’) diseases are incompletely characterized. ARS scientists at Parlier, CA conducted a comparative genome analysis of these two unculturable, fastidious bacteria. Genomic and metabolic pathway analyses revealed that the two bacteria encode 884 common genes representing 65% of the genome. The remaining 35% of the two genomes were divergent, with a significant number of genes coding for membrane and transport functions that reflect the corresponding pathogenic lifestyle of these bacteria. This genomic analysis provided basic knowledge needed for functional analyses of 'Ca. Liberibacter' genes involved in pathogenicity and will facilitate identification of new targets for development of disease resistance.
5. Molecular mechanism of Xylella fastidiosa PemK toxin determined. The PemK/PemI toxin/antitoxin confers stability of inheritance of plasmid DNA in Xylella fastidiosa. ARS scientists at Parlier, CA determined that PemK toxin is an enzyme that degrades cellular RNA, thereby killing bacterial cells that do not inherent plasmid expressing the PemI/PemK system. Improved understanding of the PemK toxin mechanism has facilitated development of a gene shuttle vector for stable delivery of foreign DNA to X. fastidiosa for use in genetic analysis of pathogenicity.
6. Movement of green sharpshooters into vineyards is transient. The green sharpshooter is the most widely distributed vector of Xyllela fastidiosa in the Central Valley of California. ARS scientists at Parlier, CA, in collaboration with researchers from UC Berkeley, monitored movement of green sharpshooters into vineyards and assessed population development in vineyards. Although green sharpshooters commonly moved into vineyards, resident populations did not become established. These results indicate that distancing vineyards from known green sharpshooter habitats may reduce movement of X. fastidiosa vectors into vineyards, potentially reducing incidence of Pierce’s disease.
7. Ecological aspects affecting Xylella fastidiosa infection in almond nurseries. Distribution of X. fastidiosa infected plants in almond orchards indicate low rates of primary pathogen spread and lack of secondary spread, implicating infected nursery stock as a source of diseased plants. ARS scientists in Parlier, CA described spatiotemporal distribution of X. fastidiosa and insect vectors in weed hosts associated with commercial almond nurseries. The research revealed “hot spots” of X. fastidiosa infection and vector populations in vegetation surrounding commercial nurseries. These results indicate that suppression of vector populations and weed hosts of X. fastidiosa adjacent to almond nurseries may reduce incidence of almond leaf scorch in commercial almond orchards.
8. Identification of potato tuber chemistry involved in Zebra Chip (ZC) disease symptom development. Zebra Chip disease is a growing threat to potato growers in North America, but how ZC symptoms develop in response to infection is unknown. ARS scientists at Parlier, CA conducted research to determine relationships between potato tuber chemistry and ZC symptoms. Symptom severity was positively associated with levels of phenolic compounds and amino acids. Tubers with ZC symptoms tested positive for ‘Candidatus Liberibacter solancearum’, indicating that altered tuber chemistry may be a plant response to infection. Basic knowledge gained from this research may be used to identify specific host responses that, if mitigated, could reduce or eliminate ZC disease symptoms produced by infected plants.
9. Xylella fastidiosa transmission to grapevines by glassy-winged sharpshooter. Pierce’s disease incidence varies among California grape growing regions infested by glassy-winged sharpshooter, but no information was available on vector characteristics that potentially could lead to differential pathogen spread. ARS researchers in Parlier, CA conducted laboratory studies to determine the effects of glassy-winged sharpshooter population origin, age, and gender on X. fastidiosa transmission. The research demonstrated no effects of these vector characteristics on pathogen transmission, indicating that rates of X. fastidiosa spread in the field are determined by local ecological aspects affecting glassy-winged sharpshooter population density and movement patterns. Therefore, approaches for controlling Pierce’s disease in different grape growing regions of California should be adjusted locally to maximize efficiency of control efforts.
10. Glassy-winged sharpshooters do not cause disruption of xylem flow during feeding. Air introduced into xylem during feeding by the glassy-winged sharpshooter potentially could affect grapevine health by disruption of water flow. ARS scientists at Parlier, CA used the Argonne National Laboratory’s Advanced Photon Source to capture X-ray videos of glassy-winged sharpshooters feeding on grapevines. The images revealed that glassy-winged sharpshooters did not cause air to enter grapevine xylem during or after feeding. Knowledge gained from these studies will facilitate efforts to develop novel control strategies for Pierce’s disease aimed at preventing inoculation of bacteria during feeding by glassy-winged sharpshooters.
Chen, J., Civerolo, E.L., Lee, R.F., Jones, J., Deng, X., Hartung, J.S., Keremane, M.L., Brlansky, R. 2011. “Candidatus liberibacter sp.”, without koch's postulates completed, can the bacterium be considered as the causal agent of citrus Huanglongbing (yellow shoot disease)?. Acta Phytopathologica Sinica. 41(2):113-117.