Location: Crop Improvement and Protection Research2009 Annual Report
1a. Objectives (from AD-416)
Objective 1: Optimize delivery and evaluate performance of chemical, cultural, biological, and genetic alternatives to methyl bromide for crops/pathogen combinations currently benefiting from the use of methyl bromide. a. Evaluate alternative fumigants. b. Identify genes associated with pathogenicity of Verticillium dahliae based on a Verticillium comparative genomics study to support the development of alternative control procedures. c. Identify emerging diseases and their etiology and evaluate resistant germplasm for diseases of strawberry, lettuce, and vegetables. d. Evaluate the influence of crop rotation on pathogen populations and beneficial microbial community dynamics in the soil and severity of Verticillium wilt and other diseases. Develop tools to evaluate and understand the role of myxobacteria in agricultural and natural ecosystems in an effort to elucidate their potential for pathogen control. Objective 2: Develop molecular diagnostic tools for the identification of emerging diseases of vegetables and strawberries, and use these tools in the development of management strategies as alternatives to methyl bromide.
1b. Approach (from AD-416)
Develop integrated management approaches including crop rotation, biological control, selection of disease resistant varities, organic production, other biological practices, and combinations of biological practices with reduced concentrations of chemical fumigants to control diseases. Identifying useful commercial biological agents as well as new agents from the strawberry rhizosphere that will help to improve plant growth and disease management. Molecular tools will be developed for evaluation of the fficacy of pathogen management and modeling population dynamics of beneficial microbes. Replaces 5305-22000-009-00D(11/07).
3. Progress Report
Continued analyses of the genomes of V. dahliae and V. albo-atrum and comparison to other genomes have revealed sets of genes that are unique to V. dahliae. For additional in-depth analyses, a research proposal was written with 7 other researchers from the US and abroad and submitted to the competitive grants program of the US Agriculture and Food Research Initiative for functional genomics analyses of V. dahliae. Preliminary screens of insertional mutants of V. dahliae have identified two candidate mutant strains of V. dahliae that are potentially nonpathogenic on lettuce. We examined the expression of two defense-related genes of lettuce in response to fungal infection in an additional lettuce cultivar. The experiments revealed that both putative antifungal genes are upregulated in the susceptible interaction with V. dahliae, in leaf tissues. Results of independent differential screening also reveal that these same genes are expressed at higher levels in lettuce leaves infected with V. dahliae versus the non-inoculated control plants. The development of a PCR method in collaboration with the UC Davis to distinguish between two races of V. dahliae is ongoing, using published primer sequences. Our results indicate that this marker system may be useful for rapidly distinguishing between races of V. dahliae pathogenic on lettuce and tomato grown in California. The development of an assay for use in growth chambers that enables a rapid assessment of pathogenicity of V. dahliae on lettuce was completed. This technique employs the use of lettuce seedlings contained in tubes. This technique offers advantages over conventional assays in greenhouses for Verticillium wilt since it is contained, rapid, the environment is controlled, and reduces labor costs associated with lengthy greenhouse assays. We described six novel diseases from the Western US and made tentative identifications of the pathogens from four of the diseases. We also determined that epidemics of bacterial blight on parsley in California and Arizona since 2003 have been caused by two and possibly three different pathogens and are currently evaluating the breadth of the host range of these pathogens. We continue to monitor these emerging diseases. Rifampicin resistant variants of nine bacterial plant pathogens and environmental organisms (needed for myxobacterial enrichment studies) were generated. We are currently comparing these variants to the wild type strains for growth characteristics and virulence on plants. We concluded field trials that identified commercially available strawberry cultivars that are resistant to the pathogen causing Strawberry Blossom Blight. We have shown that the pathogen is similar but substantially different from a know plant pathogen, Pseudomonas marginalis and are concluding the identification. We have completed FAME analysis to measure changes in microbial communities in an experiment measuring the impact of 1 – 5 years vegetable rotation on organic strawberry production in collaboration with a scientist. Data analysis is in progress. We collaborated with ARS Scientists at two other locations in California and university researchers on
1. Analyses of Verticillium genomic sequences. Verticillium dahliae is a soilborne fungus that causes economic losses of high-value crops and ornamental plants throughout the U.S. In an analysis of the genomic sequence of V. dahliae, and the sequence of a related species V. albo-atrum, an ARS researcher at Salinas, California, found sets of genes that are unique to V. dahliae. These genes may underpin virulence and host range differences of V. dahliae. This knowledge may lead to development of novel, efficacious control alternatives to methyl bromide and other fumigants, thereby reducing losses to Verticillium and reducing production costs.
2. Rapid disease assay of Verticillium on lettuce. Conventional greenhouse assays for examining the pathogenicity of the soilborne fungus V. dahliae require lengthy testing periods. ARS researchers at Salinas, California developed a growth chamber technique that enables a more rapid assessment of pathogenicity of V. dahliae on lettuce. The technique takes advantage of an early flowering lettuce accession line that develops symptoms quickly and speeds analyses of the V. dahliae-lettuce interaction. The reduction in time to assess pathogenicity of V. dahliae from 100 days to 42 days represents a significant savings in time and cost.
3. Emended taxonomy of Pseudomonas cannabina. Management of bacterial blight is difficult because two ecologically distinct pathogens (P. syringae pv. alisalensis and P. syringae pv. maculicola) from the same species cause indistinguishable symptoms. An ARS researcher at Salinas, California, evaluated the relationship of these organisms and demonstrated that one of the organisms belongs to a distinct species and proposed to emend the description of the species Pseudomonas cannabina to include P. cannabina pv. alisalensis comb. nov and the creation of P. cannabina pv. cannabina pv. nov. Disease control methods can now be targeted based on the specific pathogen causing the disease in a given field.
4. Disease resistant lettuce germplasm released. Bacterial leaf spot of lettuce caused by Xanthomonas campestris pv. vitians is an important but sporadic disease of lettuce in California. ARS researchers at Salinas, California, released six breeding lines with good head quality and resistance to Bacterial leaf spot. The material is currently being used by private and public breeders for development of proprietary varieties. This will result in better yields of good quality lettuce and reduced losses for growers.
5. Improved diagnostics for the sudden oak death pathogen, Phytophthora ramorum. The several molecular methods, including three used by USDA-APHIS, for identifying Phytophthora ramorum have not been rigorously compared and validated for accuracy. ARS researchers at Salinas, California, initiated and coordinated with other ARS and university researchers blind testing of 11 of the diagnostic methods using a library of 400 isolates and 60 field samples. The results have been provided to APHIS-PPQ-CPHST to assist them in their regulatory responsibility for this pathogen, and a manuscript reporting the results has been recently published. The results of this work will help regulatory agencies make decisions on diagnostic markers to identify this important quarantine pathogen.
6. Mitochondrial haplotype determination for Phytophthora ramorum. Phytophthora ramorum is a pathogen under strict quarantine restrictions due to its causing Sudden Oak Death in central coastal California and movement around the country in the nursery industry. The mitochondrial genomes for 2 isolates of P. ramorum were sequenced and used to identify variation that would be usefulfor identification of individual isolates, which, since the pathogen reproduces by asexual means only, will be useful for monitoring spread of specific pathogen genotypes. A total of 4 differences in mitochondrial sequences were identified in a collection of 40 isolates representing the geographic range of pathogen recovery. Rather than relying on DNA sequence analysis for determining haplotypes, PCR amplification primers were developed so melt curve analysis could be used to identify the differences in mitochondrial sequences (this process is quicker and less expensive). The results of this work will provide regulatory agencies and researchers with additional tools for identification of this important quarantine pathogen.
7. Web site supports Phytophthora research. The genus Phytophthora has approximately 90 species and is responsible for a wide range of crop plant diseases on a worldwide basis, but due to similarities in morphological features their identification to a species level can be a challenge and are often incorrect. An ARS researcher at Salinas, California, initiated a collaborative project with researchers at Penn State University, University of California at Riverside, NC State University, and an ARS researcher at Peoria, Illinois, to enhance understanding of the genus, simplify identification, stimulate research, and develop a web-based database for the genus. The database includes complete morphological descriptions, information on host range and geographical distribution, a comprehensive molecular phylogeny using seven nuclear genes (four mitochondrial genes will be added shortly), and a section on molecular detection and identification. The Phytophthora database will serve as a resource for researchers working on the genus as well as a repository of future relevant research progress and information.
8. Efficacy of alternative fumigants. Data on the efficacy of alternative fumigants is needed for growers to be able to make informed decisions for pest management after the phase out of methyl bromide. In collaboration with ARS scientists at UC Davis and Parlier, California, and university researchers from UC Davis one of the SYs participated on larger scale field evaluations with alternative fumigants. They determined the efficacy of the fumigants in controlling soilborne pests (weeds, pathogens and nematodes) and yield of ornamental crops in field evaluations. The results of this work will help growers and fumigant applicators make decisions on which alternative fumigation practices to use.
5. Significant Activities that Support Special Target Populations
• An ARS scientist mentored three students (2 minority and one woman) from Hartnell College and CSUMB (both minority serving institutions) through a grant funded internship program. • An ARS scientist mentored a former minority student intern in preparation graduate school application, a USDA graduate student proposal, and personally provided support and travel for this student to the American Phytopathological Society Meeting in Portland, OR. This student was accepted to the Plant Pathology Department at the University of Wisconsin and will begin a Ph.D. program this fall. • An ARS scientist organized and participated in 5 field trips or outreach activities by the USDA for Hartnell College or CSUMB classes or students. • An ARS scientist initiated a project in which our research station is “adopting” with four 5th grade classes at a local minority serving elementary school to provide science programming and field trips. • An ARS scientist organized USDA participation and participated in a Teacher Externship Day Thirty teachers from the Salinas valley attended. • An ARS scientist participated in WISE Women In Science Exploration of Hartnell Community College as a mentor and sponsor. • An ARS scientist provided additional outreach activities to classes or administrators from 2 additional elementary schools in Salinas. • An ARS scientist provided a field trip to the station for Girl Scout Troup 3197 and their parents. • An ARS scientist serves on the Joint Committee for Women in Plant Pathology and Cultural Diversity for APS • An ARS scientist organized and delivered one summer workshop for Salinas High School teachers at the USDA-ARS station in Salinas, CA. • An ARS scientist has been working with administrators and teachers at Spreckels Elementary School on their science curriculum.
Bull, C.T., De Boer, S.H., Denny, T.P., Firrao, G., Fischer-Le Saux, M., Saddler, G.S., Scortichini, M., Stead, D.E., Takikawa, Y. 2008. Demystifying the Nomenclature of Bacterial Plant Pathogens. Journal of Plant Pathology. 90:403-417.