2011 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).
Comparative analyses of the genomes of fungal vascular wilt pathogens V. dahliae, Fusarium oxysporum, and V. albo-atrum and those of other fungi revealed candidate genes to pursue in functional analyses. Further analyses of some candidate genes identified were initiated in a collaborative effort with other researchers.
Research is ongoing to quantify V. dahliae in commercial spinach seed lots.
Research is ongoing to examine the localization of the fungus in spinach seeds, in collaboration with the University of California, Davis.
An alternative and rapid method to prepare targeted gene deletion constructs for fungi was analyzed and verified.
We continued the DNA sequence analyses of lettuce gene homologs associated with plant resistance to the fungus, V. dahliae, and initiated collaboration with the University of California, Davis to characterize the role of one these genes in disease resistance.
We completed analyses of a method to distinguish between two races of V. dahliae, in collaboration with the University of California, Davis.
Two genes important for colonization of lettuce were identified in V. dahliae, by insertional mutagenesis and pathogenicity assays.
Research was initiated for comprehensive analyses of transposable elements in the fungal pathogen, V. dahliae.
We demonstrated that P. cannabina pv. alisalensis strains from Australia were the causal agents for bacterial blight of mustard, although the pathogen was previously identified as P. syringae pv. maculicola.
We completed host range and phage sensitivity tests that supported the hypothesis that bacterial blight of radish in France was caused by P. cannabina pv. alisalensis in collaboration with INRA.
We demonstrated that lettuce cultivars influence population dynamics of Xanthomonas campestris pv. vitians on lettuce leaves.
We identified a third pathogen from parsley as a close relative of Pseudomonas viridiflava.
We identified a pathogen of fennel as Pseudomonas syringae pv. apii, thus expanding the known host range of this pathogen.
We documented genomic changes in P. syringae pv. apii populations.
We isolated bacteriophage specific to Xanthomonas campestris pv. vitians.
A comprehensive, multigene, phylogenetic analysis of four Phytophthora species and species complexes was completed in collaboration with three other labs.
A comprehensive, mitochondrial, multigene phylogeny of the genus Phytophthora was concluded.
A new diagnostic marker system for Phytophthora was developed and validated with field samples from the USDA-FS P. ramorum national survey.
A real time PCR soil quantification assay for Verticillium dahliae was developed and validated with field soils.
Molecular diagnostic technique for detection of Phytophthora genere and species. The fungal pathogen phytophthera causes disease on many crops. A new, real time PCR molecular diagnostic technique developed by ARS researchers at Salinas, CA, for detection and identification of Phytophthora spp. exploits a gene order difference between Phytophthora and other related genera and plants. The technology allows in one reaction to determine if a Phytophthora spp. has infected the plant, and if it is the species of interest (not possible with techniques currently used). Genus-specific probes were developed for 14 economically important Phytophthora spp., including several invasive and quarantine species, using sequence data from 500+ isolates representing all species in the genus. Validation is with close to 400 field samples provided by 3 labs participating in the national survey for Phytophthora ramorum. This technique will provide a new tool for diagnosticians to determine not only if a particular species is associated with plant infection, but if any member of the genus is present in the sample as well as a systematic approach for developing new species specific markers.
Rapid identification of more than 60 plant pathogens demonstrated. Identification of pathogens of specific plants, also called pathovars, within the Pseudomonas syringae group has been difficult or impossible since the inception of this taxon in the 1980s. These bacterial pathogens cause diseases on thousands of plants world-wide in a pathogen/host specific manner. An ARS researcher at Salinas, California, and colleagues demonstrated that MultiLocus Sequence Typing (MLST) can be used to rapidly identify members of the genus Pseudomonas to the genus and pathovar level. Four genes were sequenced for all 61 known pathotypes from the P. syringae group and related strains and were used to rapidly identify two pathogens from parsley. Additionally, a significant correlation between DNA/DNA hybridization values and % similarity was demonstrated. Using the methods and tools described, the time required to make identifications of plant pathogenic bacteria was reduced from several months to less than one week. The sequences are publicly available and will reduce the number of strains to be moved among laboratories for identification purposes as well as speed identification of these pathogens.
Identification of genes associated with Verticillium wilt symptom development. Verticillium wilt disease on lettuce causes severe symptoms that can render an entire field unmarketable. The objective of this research was to identify genetic responses of lettuce during wilt symptom development. ARS researchers in Salinas, California, identified sets of lettuce genes, including those associated with symptom development, which are repressed or induced in response to infection by the plant pathogenic fungus, Verticillium dahliae. Insights on the regulation of symptom development may contribute to the development of plants that are more tolerant to symptoms associated with Verticillium wilt and other fungal diseases.
Reclassification of Pseudomonas syringae pv. alisalensis as P. cannabina pv. alisalensis. Appropriate and specific management strategies could not be applied to manage bacterial blight and pepper spot diseases of crucifers because the pathogens causing these diseases had been indistinguishable. MultiLocus Sequence Typing (MLST) and phenotype based analyses of P. syringae pv. alisalensis and related strains resulted in the transfer of this pathogen to a new species and the demonstration that a well studied pathogen previously identified as P. syringae pv. maculicola is P. syringae pv. alisalensis. MLST data also provides information needed for rapid identification of the pathogens. Correct and rapid identification of these pathogens leads to more rapid appropriate disease management responses for crucifer production. This is significant for these pathogens because P. cannabina pv. alisalensis causes a severe bacterial blight on crucifers while the disease caused by P. syringae pv. maculicola is significantly milder.
A new tool for genetic studies of Verticillium dahliae and other fungi. Targeted gene deletion in fungi enables researchers to examine the function of genes of interest. Existing methods for generating DNA constructs for gene deletions in fungi are cumbersome, often involving multiple steps. The objective of this research was to develop an alternate approach for the creation of gene deletion constructs. An ARS researcher in Salinas, California, along with University colleagues, developed and verified an alternative method to quickly generate DNA constructs for targeted deletion of genes. Thus, the tool will enable rapid preparation of constructs for the deletion of genes associated with pathogenicity of Verticillium dahliae and other fungi important to US Agriculture.
Web site to support Phytophthora research. The fungal genus Phytophthora is responsible for many diseases of crop plants worldwide and consists of approximately 117 species that are difficult to identify. ARS researchers at Salinas, CA, and other ARS locations, and university researchers developed a web-based database for Phytophthora research to enhance the understanding of the genus, simplify species identification and stimulate further research on the genus. The database includes complete morphological descriptions, information on host range and geographical distribution, a comprehensive molecular phylogeny using seven nuclear and four mitochondrial genes, a sequence database with over 6,900 entries of that is searchable by Basic Local Alignment Search Tool analysis, and a section on molecular identification and detection. This database will serve as a resource for researchers working on the genus, a means to simplify sequence based identification of species, as well as a repository for future work.
Molecular quantification of Verticillium dahliae from soil. The pathogen V. dahliae has a broad host range and can survive for long periods of time in the soil. Although soil plate assays can be used to quantify the pathogen populations in the soil, they are not always accurate and can take six to eight weeks to complete. The real time polymerase chain reaction (PCR) assay that was developed by ARS researchers is able to accurately determine population densities as low as 1 to 2 propagules/g soil within a day or two. This assay has the potential to significantly reduce the time needed for growers of susceptible crops to get the results they need with greater accuracy than current techniques.
Insight into niche adaptation of fungal vascular wilt pathogens. Two soilborne, fungal plant pathogens, Verticillium dahliae and V. albo-atrum cause vascular wilt diseases on over 200 plant species worldwide, and costly soil fumigants are sometimes deployed to kill these fungal pathogens in the soil. To gain insight into niche adaptation, ARS researchers in Salinas, California, along with a team of international researchers, completed analyses of these genomes and extended the comparison with other fungi. The research discovered a set of proteins shared among three wilt pathogens, and an abundance of enzymes that degrade pectin, that may enable the two fungi to infect their host crops and allow ecological adaptation. Identification of the genetic basis for pathogenicity or host range expansion may lead to alternative strategies for their control in high value crops such as lettuce.
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Bull, C.T., Clarke, C.R., Cai, R., Vinatzer, B.A., Jardini, T.M., Koike, S.T. 2011. Multilocus sequence typing of Pseudomonas syringae sensu lato confirms previously described genomospecies and permits rapid identification. Journal of Phytopathology. DOI: 10.1094/PHYTO-11-10-0318.
Bull, C.T., Mauzey, S.J., Koike, S.T. 2010. First report of bacterial blight of Brussels sprouts (Brassica oleracea L. var. gemmifera) caused by Pseudomonas cannabina pv. alisalensis in California. Plant Disease. 94:1375.
Grunwald, N.J., Martin, F.N., Larsen, M.M., Sullivan, C.M., Press, C.M., Coffey, M.D., Hansen Everett, M., Parke, J.L. 2011. Phytophthora-ID.org: a sequence-based Phytophthora identification tool. Plant Disease. 95(3):337-342.
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Mammella, M., Cacciola, S., Martin, F.N., Schena, L. 2011. Genetic characterization of Phytophthora nicotianae by the analysis of polymorphic regions of the mitochondrial DNA. Fungal Biology. 115:432-442.
Paz, Z., Garcia-Pedrajas, M.D., Andrews, D.L., Klosterman, S.J., Montanez, B., Gold, S.E. 2011. One step construction of agrobacterium recombination-ready plasmids (OSCAR), an efficient and robust tool for ATMT based gene deletion construction in fungi. Fungal Genetics and Biology. 48:677-684.
Mauzey, S., Koike, S.T., Bull, C.T. 2011. First report of bacterial blight of cabbage (Brassica oleracea var. capitata L.) caused by Pseudomonas cannabina pv. alisalensis in California. Plant Disease. 95(1):71.
Bull, C.T., Rubio, I. 2011. First report of bacterial blight of crucifers caused by Pseudomonas cannabina pv. alisalensis in Australia. Plant Disease. 95(8)1027.
Klosterman, S.J., Anchieta, A.G., Garcia-Pedrajas, M.D., Maruthachalam, K., Hayes, R.J., Subbarao, K.V. 2011. SSH reveals a linkage between a senescence-associated protease and Verticillium wilt symptom development in lettuce (Lactuca sativa). Physiological and Molecular Plant Pathology. 76:48-58.
Martin, F.N. 2010. Mitochondrial haplotype analysis as a tool for differentiating populations of Verticillium dahliae. Phytopathology. 100(11):1231-1239.
Maruthachalam, K., Klosterman, S.J., Kang, S., Hayes, R.J., Subbarao, K.V. 2011. Identification of pathogenicity-related genes in the vascular wilt fungus verticillium dahliae by agrobacterium tumefaciens-mediated t-DNA insertional mutagenesis. Molecular Biotechnology. PMID:21424547.
Maruthachalam, K., Atallah, Z.K., Vallad, G.E., Klosterman, S.J., Hayes, R.J., Davis, R., Subbarao, K.V. 2010. Molecular variation among isolates of verticillium dahliae and PCR-based differentiation of races. Phytopathology. 100(11):1222-30.