Location: Crop Improvement and Protection Research2008 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
We prepared three V. dahliae EST libraries, one grown in complete medium, one with a limited nitrogen source, and one in the presence of lettuce root extract. These libraries were shipped to the Broad Institute of MIT and Harvard, and used in the annotation of the V. dahliae genome. In addition, the EST libraries are currently being analyzed to assess overall V. dahliae gene expression under the three different growth conditions. Because the initial screens of lettuce cDNAs for differential expression were unsatisfactory, an approach was employed to examine the expression of candidate lettuce antifungal genes. The expression of antifungal pathogenesis-related genes, such as a class IV endochitinase gene and a thaumatin-like gene were examined in the lettuce-V. dahliae interaction. The experiments revealed tissue-specific expression patterns of both genes in the susceptible interaction with V. dahliae, with higher levels of the thaumatin-like gene expressed in leaf tissues and reduced expression of the class IV endochitinase in root tissues infected with V. dahliae. A PCR method developed in collaboration with the UC Davis to distinguish between two variants, or races, of V. dahliae infecting lettuce was further tested for validity and applicability. This method was invalidated by testing isolates using the PCR method, coupled with virulence assays in greenhouses. Other methods are being pursued to distinguish races of V. dahliae. An assay was developed for use in growth chambers that enables a more rapid assessment of pathogenicity of V. dahliae on lettuce. This technique employs the use of lettuce seedlings contained in tubes. This technique offers advantages over conventional assays in greenhouses for Verticillium wilt in that it is contained, rapid, and the environment is controlled. The assay also reduces labor costs associated with lengthy greenhouse assays. Insertional mutants of V. dahliae were analyzed and preliminary analyses revealed a coalescence of filaments and reduced production of microsclerotia in one of these mutants. Currently, analyses of the DNA from this strain by thermal asymmetric interlaced (TAIL) PCR are being performed to identify the gene associated with this phenotype. We tentatively identified a novel pathogen of strawberry flowers as Pseudomonas marginalis. We conducted laboratory and field evaluations to determine if there is resistance to the pathogen in currently used commercial varieties. Indications from the first field experiment indicate that there is some level of resistance available. This work is supporting NP308 Problem Statement 1C: Identification and Mitigation of Emerging Problems.
1. Provision of Expressed Sequence Tags(EST) libraries and public release of the V. dahliae and V. albo-atrum genomic sequences. V. dahliae and V. albo-atrum cause disease worldwide and threaten the sustainability of lettuce production and other crops in the US. To facilitate genome annotation for the study of these pathogens, three normalized EST libraries from the V. dahliae lettuce isolate VdLs.17 were prepared at the by ARS scientists in the Crop Improvement and Protection Research Unit in Salinas, CA and shipped to the Broad Institute of Massachusetts Institute of Technology and Harvard, where approximately 37,000 sequence reads were obtained. Working collaboratively with laboratories at Pennsylvania State University, University of Georgia, North Carolina State University, University of California, Davis, and Agriculture and Agri-Food Canada, the assembled genomic sequences and annotation of V. dahliae and V. albo-atrum were approved by the project collaborators and publicly released in 2008. Preliminary analyses of the whole-genome comparison between the eight chromosomes of V. dahliae and DNA sequences of V. albo-atrum reveal the presence of four major regions of non-synteny and V. dahliae-specific genes in these regions. DNA sequences, tools, and knowledge developed in this project will have an immediate worldwide impact in providing insight into the genetic components important for virulence and host range, pathogen detection, and may reveal control targets alternative to methyl bromide. This work supported National Program 308, Problem Statement 1A.
2. Rapid assessment of pathogenicity of V. dahliae on lettuce. Current methods to assess Verticillium wilt of lettuce are time-consuming and labor-intensive, especially for routine assays to examine virulence of Verticillium spp. isolates. A technique that takes advantage of an early flowering plant introduction line was thoroughly tested and validated by the scientists in the Crop Improvement and Protection Research Unit at the Agricultural Research Service Station in Salinas, CA. The experiments revealed that the technique is suitable for the rapid analysis of virulence of V. dahliae isolates and thus has an impact in reducing the time required for analyses of the V. dahliae-lettuce interaction. Because resistant lettuce provides the most cost-effective control strategy alternative to the use of methyl bromide and soil fumigation in general, comparative tests also were conducted with additional lettuce cultivars. These comparative experiments revealed that the method is not suitable to screen lettuce for resistance to V. dahliae. This work supported National Program 308, Problem Statement 1C: Identification and Mitigation of Emerging Problems.
3. Bacterial Blight in Arugula. Bacterial diseases of vegetables can significantly limit yield as well as their usefulness in crop rotations. In collaboration with Washington State University, ARS scientists in the Crop Improvement and Protection Research Unit in Salinas, CA examined the etiology of bacterial pathogens. It was determined that the pathogen causing Bacterial Blight, which has recently been found in Arugula in Nevada, is from organic and conventional production fields as Pseudomonas syringae pv. alisalensis. This was the first report of bacterial blight on Arugula in Nevada; thus, eliminating them as potential rotational crops for previously diseased fields. This work supported National Program 308, Problem Statement 1A and 1C.
4. Bacterial Leaf Spot. Bacterial leaf spot of lettuce, caused by Xanthomonas campestris pv. Vitians, is an important sporadic disease of lettuce in California. In collaboration with the USDA/ARS breeding program in Salinas, ARS scientists in the Crop Improvement and Protection Research Unit are evaluating the genetic basis for resistance and developing germplasm for resistance to bacterial leaf spot. We evaluated material for resistance and wrote a germplasm release based on this and previous years data. The information learned in this project can lead to a germplasm that is resistant to Bacterial leaf spot, which means fewer crops being diseased and higher income for farmers. This work supported National Program 308, Problem Statement 1A and 1C.
5. Molecular diagnostics for Phytophthora ramorum. There are several different molecular methods that have been used to identify Phytophthora ramorum, the pathogen responsible for Sudden Oak Death, three of these are currently used by USDA-APHIS to identify the pathogen in plant samples; but, wide spread testing of this method using a broad range of isolates and species of the genus has not been done. Likewise, similar testing has not been done for the other detection methods. In an effort to assist USDA-APHIS in evaluating these molecular diagnostic techniques an ARS scientist in the Crop Improvement and Protection Research Unit in Salinas, CA has initiated and is coordinating with a researcher at the University of California Riverside a blind test of 11 of the diagnostic methods by the 7 labs that have developed them (ARS, Ft. Detrick and Salinas; USDA APHIS PPQ-CPHST, Raleigh, NC; USDA-FS, Saucier, MS; University of California, Berkeley, CA; CSL, York, UK; NRC Canada Forest Service, Ste Foy, QC, Canada) using a library of 400 isolates as well as 60 field samples. The results have been provided to APHIS-PPQ-CPHST to assist them in deciding on additional assays to include in their analysis. The results of this work will help regulatory agencies make decisions on additional diagnostic markers they can use to identify this important quarantine pathogen. This work supported National Program 308, Problem Statement 1C: Identification and Mitigation of Emerging Problems.
6. Mitochondrial haplotype determination for Phytophthora ramorum. Phytophthora ramorum causes sudden Oak death and is a pathogen under strict quarantine restrictions. The mitochondrial genomes for 2 isolates of P. ramorum were sequenced by ARS scientists in the Crops Improvement and Protection Research Unit in Salinas, CA and used to recognize polymorphic sites for identification of mitochondrial haplotypes, which, since the pathogen clonally reproduces, will be useful for monitoring spread of specific pathogen genotypes. A total of 4 haplotypes were identified in a collection of 40 isolates representing the geographic range of pathogen recovery. PCR primers for haplotype determinations were developed and published with additional details of the work forwarded to USDA-APHIS with an offer to assist them in evaluating the utility of the technique with field samples. The results of this work will help regulatory agencies make decisions on additional diagnostic markers they can use to identify this important quarantine pathogen. This work supported National Program 308, Problem Statement 1C: Identification and Mitigation of Emerging Problems.
7. Web site to support Phytophthora research. The genus Phytophthora has approximately 80 species and is responsible for a wide range of diseases on a worldwide basis; but, due to similarities in morphological features identification to a species level can be a challenge. In an effort to enhance an understanding of the genus, simplify identification, and stimulate research a collaborative project was initiated by ARS scientists in the Crop Improvement and Protection Research Unit with researchers at Penn State University, University of California at Riverside, North Carolina State University and an ARS researcher at Peoria, IL to develop a web based database for the genus. The database includes complete morphological descriptions, information on host range and geographical distribution, and a comprehensive molecular phylogeny using at least 5 nuclear genes (mitochondrial genes will be added when the sequence analysis described above is completed). The molecular database is fully searchable and designed in a way to facilitate molecular identification of unknown isolates with a section on molecular markers added in the near future. It is expected that this database will serve as a resource for researchers working on the genus as well as a repository for future work to keep the information presented current. This work supported National Program 308, Problem Statement 1C: Identification and Mitigation of Emerging Problems.
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 and university researchers at the University of California Davis, an ARS scientist in the Crop Improvement and Protection Research Unit in Salinas, CA participated on larger scale field evaluations with alternative fumigants to determine their efficacy in controlling soilborne pests (weeds, pathogens and nematodes) and yield of ornamental crops in field evaluations. The results of this work were communicated to growers by two one-day fumigation workshops and should help the grower decide on which alternative fumigation practice to utilize. This work supported National Program 308, Problem Statement 1A and 1D.
5. Significant Activities that Support Special Target Populations
• One of the SYs organized a field trip of University of California, Santa Cruz students, including minorities and women, to meet and discuss research and careers with researchers at the USDA-ARS station in Salinas, CA. • One of the SYs has trained student interns from Hartnell College and CSUMB as part of a USDA funded program to increase the exposure of minority (specifically Hispanic) students to science. • Dr. Bull works with Hartnell College on the development of a Science Institute designed to develop minority student interest in Science Careers and to increase internship possibilities for underrepresented populations. This resulted in a Science Institute Visioning Workshop held April 22, 2008 where she served as a Workshop Organizer, MC and breakout session leader. • March 5, 2007; Dr. Bull provided a pre-print of the Ruth Allen Chapter to Charmaine Scardina for Women’s history month. She provided it to all locations in the PWA. • August 2008: Dr. Bull organized and Moderated a session on Diversity for the American Phytopathological Society Centennial meeting. To prepare for this session Dr. Bull completed and began to analyze a survey and special session on the status of women and minority plant pathologists. • July 2008: Dr. Bull gave a talk about research opportunities and careers a high school bridging program at Hartnell community College designed to increase math and science skills of incoming Freshmen. • Dr. Bull serves on the Joint Committee for Women in Plant Pathology and Cultural Diversity for APS