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United States Department of Agriculture

Agricultural Research Service


Location: Crop Diseases, Pests and Genetics

2010 Annual Report

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. FY10 Program Increase.

3.Progress Report
Research progress for FY10 included.
1)breeding for resistance to Xylella fastidiosa [Xf],.
2)characterization of molecular interplay among Xf and plants,.
3)molecular typing of Xf strains & plasmids, and.
4)ecological & biological characterization of insect vectors of Xf. To address a new objective, research was conducted on DNA-based diagnostic and fingerprinting techniques for detection of Candidatus Liberibacter asiaticus and Ca. L. solanacerarum, the presumptive causal agents of citrus Huanglongbing and potato zebra chip diseases.

Crop improvement efforts through breeding included evaluation of 3rd-backcross progeny of table grapes & raisins with PD resistance from Vitis arizonica. Progeny were 97% V. vinifera and exhibited good fruit quality. Hybrids for additional backcross generations were made & screened with molecular markers for PD resistance. Breeding for Xf resistance in almonds focused on peach-almond hybrids intended for use as rootstocks. Almond-peach hybrids do not support Xf infection.

Molecular studies of Xf–plant interactions focused on genes differentially expressed in resistant vs. susceptible grapes. Genes that differed in response to Xf infection were identified & selected for further research. The model plant Arabidopsis thaliana was developed into a surrogate system to elucidate plant genes that effect response to Xf. A. thaliana ecotypes supporting high or low Xf populations were identified & are being used for gene mapping assays. Research on secondary metabolites associated with Xf infection of grape & almonds was initiated.

Xf strains causing diseases of mulberry & olive were characterized by multilocus sequence typing. Pathogenicity assays of Xf on olive are ongoing. Plasmids associated with mulberry strains were completely sequenced, annotated, & used to construct a stable shuttle vector for cloning DNA into Xf. Xf strains from almond & grape were typed by DNA fingerprinting.

Research conducted on insect vectors of Xf included assays to determine effects of egg load on glassy-winged sharpshooter (GWSS) egg laying behavior. Mechanistic studies of Xf transmission to grape plants by GWSS determined that Xf populations fluctuated in insects and that inoculation was associated with precibarium (mouthpart) flushing and deposition of Xf into saliva sheaths during GWSS feeding.

Xf infection in almond orchards indicated lack of secondary spread. Almond nursery stock infection of Xf was assessed & implicated as a source of primary infection. Weeds located in proximity to almond nurseries were evaluated as sources of Xf & vectors. Monitoring movement of green sharpshooter vectors into vineyards indicated that proximity of vectors to vineyards was required. A simulation model of Xf spread indicated that vector population suppression was more effective than removing infected plants.

Improved detection assays for Ca. Liberibacter spp. associated with Huanglongbing & zebra chip were developed. The complete genome sequence of Ca. L. solanaceraum (zebra chip) was determined & annotated. Fingerprinting assays able to distinguish strains of Ca.L. asiaticus were developed & used to differentiate pathogen populations.

1. Table grapes and raisins with high fruit quality and Pierce’s disease resistance. Introduction of Pierce’s disease resistance from wild grape species into table grapes and raisins results in small berries and poor fruit quality. ARS scientists at Parlier, CA have generated, via repeated back-crossing, table grapes and raisins that retain Pierce’s disease resistance from Vitis arizonica but have fruit of high quality. The current advanced selections have the potential to developed into new table grape and raisin cultivars with Pierce's disease resistance that will provide growers with an alternative to high quality cultivars that are susceptible to Pierce’s disease.

2. Peach-almond hybrids are non-hosts of the bacteria causing Almond Leaf Scorch disease. California’s 700,000+ acres of almonds are susceptible to Almond Leaf Scorch, a disease caused by Xylella fastidiosa, a bacterium transmitted from plant to plant by insects. ARS researchers in Parlier, CA conducted studies to determine susceptibility of peach-almond hybrids, a rootstock for almonds that boosts the tree vigor. The research demonstrated that X. fastidiosa could not survive within the peach-almond hybrid. Use of the peach-almond hybrid rootstock in commercial orchards may reduce incidence of Almond Leaf Scorch disease by eliminating one possible source of the bacterium.

3. Identification of candidate genes involved in host plant resistance to Pierce’s disease. Cultivated table and wine grapes derived from the European grape (Vitis vinifera) are susceptible to infection by Xylella fastidiosa, the causal agent of Pierce’s disease. ARS scientists at Parlier, CA used functional genomic and proteomic approaches to identify genes involved in resistance to Pierce’s disease. Among genes that were differentially expressed by susceptible and resistant progeny of crosses between V. vinifera and a wild grape, four candidate resistance genes were selected for further study. Information derived from these studies facilitates efficient selection of promising genetic backgrounds capable of limiting infection of X. fastidiosa in grapevines, a critical trait in the breeding of Pierce’s disease resistant grapevines.

4. Arabidopsis thaliana as a model host for Xylella fastidiosa. Novel control strategies for Pierce’s disease of grapes may be derived from understanding the complex genetic interplay between the pathogen (Xylella fastidiosa) and plant host. Research progress is hampered due to slow growth of grapes, long incubation periods between inoculation and expression of disease, and limited understanding of grape genes involved in susceptibility/resistance response to infection. ARS scientists at Parlier, CA have successfully developed Arabidopsis thaliana, a fast-growing plant with a completely sequenced genome and a wide variety of defined ecotypes, as a model host for X. fastidiosa. Two ecotypes of A. thaliana that differ in response to X. fastidiosa infection have been identified and are being screened for genes determining disease susceptibility/resistance. Knowledge of key host genes identified in the Arabidopsis-X. fastidiosa model system may then be transferred to grapes and other plants of economic importance to define host-pathogen interactions and develop improved resistance to X. fastidiosa infection.

5. Development of a stable shuttle vector for delivery of genes to Xylella fastidiosa. Novel targets for disease control strategies aimed at disrupting the ability of Xylella fastidiosa to induce Pierce’s disease of grapes may be gleaned from molecular genetic studies in which specific X. fastidiosa genes are rendered non-functional by targeted mutation. To confirm that loss of ability to induce disease is due to mutation of the targeted gene, it is necessary to test if the functional version of the gene restores function. USDA-ARS scientists at Parlier, CA have developed a stable plasmid shuttle vector for delivery of genes to X. fastidiosa. Use of the shuttle vector greatly simplifies the process of analyzing mutants such that basic scientific research aimed at understanding the ability of X. fastidiosa to cause disease is enhanced and expedited.

6. Ecological differences among glassy-winged sharpshooter populations requires region-specific optimization of control practices. Environmentally friendly alternatives to chemical control of the glassy-winged sharpshooter, a vector of Xylella fastidiosa, requires an understanding of the insect’s biology and ecology. Populations of glassy-winged sharpshooter in Southern and Central California are currently managed using essentially identical practices. ARS researchers at Parlier, CA have examined biological and behavioral characteristics of glassy-winged sharpshooters derived from separate populations in Southern and Central California. Results indicate key differences among the two populations with respect to timing of reproduction and longevity. Based on these findings, management practices should be optimized specifically for each region such that control measures employed are more efficient.

7. Egg load and time since last egg laying affects behavior of the glassy-winged sharpshooter. Spread of insect-transmitted pathogens is largely dependent on population dynamics of the insect vector. To better understand oviposition (egg laying) patterns of the glassy-winged sharpshooter, an insect vector of Xylella fastidiosa, ARS scientists at Parlier, CA examined the effects of egg load (number of mature eggs carried by a female) on oviposition behavior. Results indicate that glassy-winged sharpshooter females were more likely to deposit eggs and accept a less preferable plant host, when egg load and time since last oviposition increased. New knowledge derived from the research will improve understanding of plant host utilization by glassy-winged sharpshooter and facilitate development of environmentally friendly control measures to reduce glassy-winged sharpshooter populations and, consequently, transmission of X. fastidiosa.

8. Elucidation of critical events in inoculation of Xylella fastidiosa to grapes by the glassy-winged sharpshooter. Xylella fastidiosa, the bacterium causing Pierce’s disease, is transmitted to grapes by the glassy-winged sharpshooter. ARS scientists at Parlier, CA have investigated the mechanism of insect inoculation of plants. Results indicate that X. fastidiosa undergoes cyclic changes in population density within the glassy-winged sharpshooter and that inoculation of grapes involves flushing of bacteria from the insect foregut. Subsequent deposition of bacteria into insect salivary sheaths associated with plant tissues appears to be the route by which X. fastidiosa gains access to grapevine xylem to establish infection. Knowledge gained from these studies will facilitate efforts to develop novel control strategies for Pierce’s disease aimed at disrupting the inoculation process.

9. Development of a diagnostic assay for distinguishing strains of Candidatus Liberibacter asiaticus strains associated with citrus huanglongbing disease. Huanglongbing is the most damaging disease threatening citrus production worldwide. ARS scientists in Parlier, CA have developed a DNA-fingerprinting technique able to distinguish strains of the presumptive pathogen, Ca. Liberibacter asiaticus. As populations in Florida and Guangdong (China) were readily distinguished from one another, application of the diagnostic technology will facilitate monitoring potential spread of the pathogen to citrus production areas where huanglongbing disease is not currently known to occur.

10. Complete genome sequence determined for the unculturable bacterium associated with zebra chip disease of potato. Zebra chip is an emergent disease of potato that renders infected tubers unsuitable for processing. ARS scientists at Parlier, CA have determined the complete genome sequence of the unculturable bacterium (Candidatus Liberibacter solanacearum) associated with zebra chip disease. Knowledge of the genome sequence of Ca. L. solanacearum will facilitate development of new DNA-based diagnostic assays for the presumptive pathogen and potentially lead to identification of new targets for development of disease resistance.

5.Significant Activities that Support Special Target Populations
1. Project scientists represented USDA-ARS at the Career Pathways Fair hosted at Fresno State University and sponsored by the USDA-Hispanic Serving Institutions National Program (Feb. 2, 2010).

2. Project scientists represented USDA-ARS by participation in Future Scientist Student Outreach Initiative held by the USDA- Hispanic Serving Institution National Program (May 21, 2010).

Review Publications
Krugner, R., Johnson, M.W., Morgan, D.J., Morse, J.G. 2009. Production of Anagrus epos Girault (Hymenoptera: Mymaridae) on Homalodisca vitripennis (Germar) (Hemiptera: Cicadellidae) eggs. Biological Control. 51(1):122-129.

Livingston, S., Chen, J., Civerolo, E.L. 2009. Seasonal Behavior of Xylella fastidiosa Causing Almond Leaf Scorch Disease Under Field Conditions and Improved Detection of the Bacteria by Means of Array-PCR. Journal of Phytopathology. 158:40-45.

Wen, A., Mallik, I., Alvarado, V.Y., Pasche, J.S., Wang, X., Li, W., Levy, L., Lin, H., Scholthof, H., Mirkov, E., Rush, C.M., Gudmestad, N.C. 2009. Detection, Distribution and Genetic Variability of 'Candidatus Liberibacter’ Species Associated with Zebra Complex Disease of Potato in North America. Plant Disease. 93:1102-1115.

Ledbetter, C.A., Rogers, E.E. 2009. Differential Susceptibility of Prunus Germplasm (Subgenus Amygdalus) to a California Strain of Xylella fastidiosa. HortScience. 44(7):1928-1931.

Yamaguchi, M., Valliyodan, B., Zhang, J., Lenoble, M.E., Yu, O., Rogers, E.E., Nguyen, H.T., Sharp, R.E. 2010. Regulation of Growth Response to Water Stress in the Soybean Primary Root. I. Proteomic Analysis Reveals Region-Specific Regulation of Phenylpropanoid Metabolism and Control of Free Iron in the Elongation Zone. Plant Cell and Environment. 33:223-243.

Chen, J., Deng, X., Sun, X., Jones, D., Irey, M., Civerolo, E.L. 2010. Guangdong and Florida Populations of “Candidatus Liberibacter asiaticus” Distinguished by a Genomic Locus With Short Tandem Repeats. Phytopathology. 100:567-572.

Lin, H., Chen, C., Doddapaneni, H., Duan, Y., Civerolo, E.L., Bai, X., Zhao, X. 2010. A New Diagnostic system for Ultra Sensitive and Specific Detection and Quantitation of “Candidatus Liberibacter asiaticus”, the Bacterium Associated with Citrus Huanglongbing. Journal of Microbiological Methods. 81(1):17-25.

Gassman, A.J., Fabrick, J.A., Sisterson, M.S., Hannon, E.R., Stock, P.S., Carriere, Y., Tabashnik, B.E. 2009. Effects of pink bollworm resistance to Bt on phenoloxidase activity and susceptibility to entomopathogenic nematodes.. Journal of Economic Entomology. 102 (3): 1224-1232

Stenger, D.C., Lee, M.W., Rogers, E.E., Chen, J. 2010. Plasmids of Xylella fastidiosa Mulberry-Infecting Strains Share Extensive Sequence Identity and Gene Complement with pVEIS01 From the Earthworm Symbiont Verminephrobacter Eiseniae. Physiological and Molecular Plant Pathology. 74:238-245.

Sisterson, M.S., Thimmiraju, S., Daane, K., Lynn-Patterson, K., Groves, R. 2010. Epidemiology of Diseases Caused by Xylella fastidiosa in California: Evaluation of Alfalfa As A Source of Vectors and Inocula. Plant Disease. 94:827-834.

Cheng, D.W., Lin, H., Civerolo, E.L. 2010. Extracellular Genomic DNA Mediates Enhancement of Xylella fastidiosa Biofilm Formation in Vitro. Journal of Plant Pathology. 92(2):405-410.

Cheng, D.W., Lin, H., Takahashi, Y., Walker, A.M., Civerolo, E.L., Stenger, D.C. 2010. Transcriptional Regulation of the Grape Cytochrome P450 Monooxygenase Gene CYP736B Expression in Response to Xylella fastidiosa Infection. Biomed Central (BMC) Plant Biology. 10:135.

Last Modified: 4/23/2014
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