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.
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. Formerly 5302-22000-007-00D.
3. Progress Report
This project was initiated March 15, 2007 and replaces project 5302-22000-007-00D. Focus of the research is on diseases of horticultural and landscape crops caused by Xylella fastidiosa with emphasis on Pierce’s Disease (PD) of grapes, almond leaf scorch disease (ALSD), and sharpshooter insects that vector the pathogen. Progress has been made in the areas of X. fastidiosa-plant interactions, development of PD resistance in grape, pathogen genetic diversity/genomics, mechanisms of vector transmission, and modeling of disease and vector populations. Wide crosses among species of Vitis has resulted in seedling families that segregate for resistance and susceptibility to PD. Wide-cross family progeny provided material for use in a breeding program aimed at development of high quality raisin and tables grapes with resistance to PD. The extensive X. fastidiosa strain collection maintained at Parlier has been the source of material to examine the genetic and biological basis for strain diversity in X. fastidiosa. Basic studies designed to determine the mechanism of vector transmission have resulted in the generation of a model in which transmission is associated with salivation and flushing of sharpshooter mouthparts during feeding.
Differential Gene Expression of Pierce’s Disease Susceptible and Resistant Grapes. Information regarding genetic control of resistance and the molecular resistance mechanisms to Pierce’s disease of grape is limited. Using microarray gene expression analysis along with appropriate experimental design, ARS researcher in Crop Diseases, Pests and Genetics Research Unit, Parlier, CA indetified 9 candidate genes involved in signal transduction and defense response from PD resistant grapes. Molecular information derived from these genes led to design of new molecular markers for marker-assisted PD resistance breeding projects. Utilization of grape resistant genes for durable resistance to PD is likely the ultimate solution since other means of control are ineffective or have undesirable environmental consequences. This research supports NP 303(Plant Diseases), Component 3(Plant Disease Resistance), Problem Statement 3A (Mechanisms of Plant Disease Resistance). Complete genome sequence of two X. fastidiosa almond leaf scorch strains. Genomic variation among strains of X. fastidiosa with distinct pathogenic properties is poorly described. ARS researcher in Crop Diseases, Pests and Genetics Research Unit, Parlier, CA with the collaboration from Los Alamos National Lab, sequenced the genomes of two X. fastidiosa almond leaf scorch strains annotated, and compared to four other XF strains for which complete genome sequences are available. Significant new information on genome rearrangement, gene complement, and prophage integration was found. The information will facilitate a better understanding of X. fastidiosa evolution and host range. These results support NP303(Plant Diseases), Component 1(Disease Diagnosis: Detection, Identification and Characterization of Plant Pathogens), Problem Statement 1B (Detection, Identification, Characterization, and Classification of Plant Pathogens). Management of almond leaf scorch disease. Currently there are no effective management techniques that prevent almond trees from becoming infected by X. fastidiosa and growers must decide to replace or keep almond leaf scorch (ALS) diseased trees. As the risk of tree-to-tree spread appears to be low, the decision to replace infected trees should focus on the loss of productivity due to infection. ARS scientists in Parlier, CA compared the yield and vitality of infected and uninfected almond trees for two almond cultivars. Yields of ALS-affected trees were significantly lower for both cultivars. To aid growers, a simple economic model was developed using field data to determine conditions under which replanting infected trees would increase returns. These results support NP303(Plant Diseases), Component 4 (Biological and Cultural Strategies for Sustainable Disease Management), Problem statement 4A (Biological and Cultural Control Technologies). Genomic characterization of Candidatus Liberibacter, the bacterium associated with citrus Huanglongbing disease. Genomic information for the bacterium associated with citrus Huanglongbing disease is limited, due to its recent emergence in the US and the inability to culture Ca. Liberibacter in vitro. An innovative PCR-based genomic walking approach was developed by scientists in Parlier, CA to identify genomic DNA sequences of this pathogen. Using this technique, 19,816 bp of new DNA sequences representing nine accessions were submitted to GenBank and are now publicly available. The sequences obtained from this study provide new genome information for Ca. Liberibacter that improve understanding of the pathogen and facilitate development of new genome-based detection tools. These results support NP303(Plant Diseases), Component 1 (Disease Diagnosis: Detection, Identification and Characterization of Plant Pathogens), Problem Statement 1B (Detection, Identification, Characterization, and Classification of Plant Pathogens). Correlation of EPG X waveform with xylem penetration by sharpshooters. The mechanism by which X. fastidiosa is inoculated to plants by sharpshooters is poorly understood. Sharpshooter feeding behavior during pathogen inoculation to plants was studied via electrical penetration graph (EPG) waveform analysis. The X waveform was identified as an indicator of shrapshooter mouth part (stylet) penetration into xylem, and is likely associated with inoculation of X. fastidiosa into the plant vascular system. Information on the inoculation mechanism will be used for predictive modeling and to develop varieties of grapes resistant to feeding by the glassy-winged sharpshooter. These results support NP303 (Plant Diseases), Component 2 (Biology, Ecology, Epidemiology, and Spread of Plant Pathogens and their Relationships with Hosts and Vectors), Problem Statement 2B (Plant-Microbe-Vector Interactions). A genomic approach to classify subgroups of Xf strains. There is currently no standardized procedure to classify X. fastidiosa strains below the species level. Over 100 16S rDNA sequences were analyzed by ARS researchers in Parlier, CA. A combination of four single nucleotide polymorphisms (SNPs) sub-grouped all X. fastidiosa strains into four clusters, in agreement with the current knowledge of pathotypes and the proposed sub-species. SNPs from eight other conserve genomic regions supported this classification system. This research provides a simple method with un-ambiguous information to study X. fastidiosa taxonomy. These results support NP303 (Plant Diseases), Component 1 (Disease Diagnosis: Detection, Identification and Characterization of Plant Pathogens), Problem Statement 1B (Detection, Identification, Characterization, and Classification of Plant Pathogens). Development of an improved nested-PCR assay for citrus Huanglongbing (HLB) pathogen detection. Rapid and sensitive diagnostics for the HLB pathogen are essential for implementation of control strategies to mitigate losses in citrus associated with introduction of HLB into the US. Current PCR assays may result in false-negative diagnoses when the HLB pathogen is present in low abundance in samples. Nested-PCR for the HLB pathogen was developed by ARS scientists in Parlier, CA, which was more sensitive than standard PCR for routine detection. The new procedure will facilitate epidemiological studies designed to monitor spread of HLB within established zones and provide early detection of HLB spread to citrus production areas that currently are not affected by HLB. These results support NP303 (Plant Diseases), Component 1 (Disease Diagnosis: Detection, Identification and Characterization of Plant Pathogens), Problem Statement 1A (New Diagnostic Methods and Tools. Discovery and characterization of a novel X. fastidiosa plasmid. Extrachromosomal DNA elements associated with X. fastidiosa vary among strains and are poorly described. A novel ~30 kbp plasmid DNA was identified from strains of X. fastidiosa associated with mulberry leaf scorch disease in Southern California. Partial DNA sequence obtained for the 30kbp plasmid indicate the presence of a gene cluster (Type IV secretion system) involved in horizontal DNA transfer among bacterial cells and indicate a surprising close evolutionary relationship with a plasmid DNA derived from a bacterial symbiont of earthworms. The novel plasmid may be useful, upon modification, as a stable gene vector to introduce genes of interest into X. fastidiosa. The unexpected relationship of the X. fastidiosa 30kb plasmid with a plasmid from an unrelated bacterium inhabiting a completely different ecological niche suggests that X. fastidiosa may exchange DNA with a much wider array of microorganisms than previously realized. These results support NP303 (Plant Diseases), Component 2 (Biology, Ecology, Epidemiology, and Spread of Plant Pathogens and their Relationships with Hosts and Vectors), Problem Statement 2A (Pathogen Biology, Virulence Determinants, and Genetics of the Pathogen).
5. Significant Activities that Support Special Target Populations
Deng, X., Chen, J., Feng, Z., Li, H., Civerolo, E.L. 2007. Nested-PCR Detection and Sequence Confirmation of Candidatus Liberibacter asiaticus from Murraya paniculata in Guangdong, China. Plant Disease 91:1051.