2012 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 efficacy of pathogen management and modeling population dynamics of beneficial microbes.
Research is ongoing to examine the localization of the fungus Verticillium dahliae in spinach seeds, flowers, pollen, and other plant parts, in collaboration with the UC,Davis. Research was undertaken to characterize the functions of genes identified in the comparative analyses of the genomes of fungal vascular wilt pathogens. Research was initiated to evaluate the utility of a DNA-based assay for the quantification of disease resistance in spinach. DNA sequence analyses of lettuce gene homologs associated with plant resistance or genetic responses to the fungus, V. dahliae, and collaboration with the UC Davis is ongoing to further characterize the potential role of three of these plant genes in disease resistance. Research was conducted to assess the involvement of a gene from V. dahliae in pathogenicity, and experiments to assess plant gene expression in response to colonization by this particular mutant strain. Analyses of transposable elements in the fungal pathogen, V. dahliae is ongoing. Research was initiated on detection of Peronospora effusa, the downy mildew pathogen of spinach. Completed pathogenicity and host range testing needed to expand Pseudomonas cannabina to include Pseudomonas syringae pv. coriandricola and P. syringae pv. philadelphi. Began in situ testing of QPCR methods with molecular markers for S. suberifaciens. Bacterial leaf spot of radicchio (Cichorium intybus) is caused by isolates of Xanthomonas hortorum related to the bacterial leaf spot pathogen from lettuce which presents implications for disease management. Analyzed the comparative genomics of closely related pathogens in the species P. cannabina to provide insights into mechanisms of host-pathogen interaction, differential virulence factors, and pathogen evolution and find target genes for developing PCR-based detection and quantification protocols.
Identified a novel species causing bacterial blight on garlic in Brazil and identified potential sequences for the development of PCR-based detection and quantification protocols. Identified novel pathovars of Pseudomonas syringae causing bacterial blight and leaf spots on cucurbits. Demonstrated that leaf spot of Australian Cedar is caused by Xanthomonas axonopodis. Completed preliminary experiments that suggest that there are interactions between Xanthomonas campestris pv. vitians strains and lettuce cultivars for disease severity and pathogen growth/survival. Demonstrated that P. syringae pv. apii has a broader host range than previously reported and documented the occurrence of this pathogen causing disease in cilantro fields. Correlations between the mitochondrial haplotype and nuclear AFLP and SSR analysis of 100+ isolates of Phytophthora cinnamomi representing a worldwide collection is near completion. Finishing up analysis of field samples collected as part of the national survey for Phytophthora ramorum conducted through the USDA-FS using our Phytophthora real time PCR diagnostic markers. A single tube nested amplification that can be done directly from infected tissue is under development and validation for identification of Phytophthora ramorum mitochondrial haplotype.
Quantification of Verticillium dahliae in spinach seeds. The movement of the fungal plant pathogen in spinach seeds from seed production areas introduces pathogen inoculum and exotic strains to crop production areas. An ARS researcher in Salinas, in collaboration with researchers at the University of California, led the research for the development of an assay for the quantification of Verticillium dahliae in spinach seeds. The assay will be useful to quickly assess the level of seed infection in commercial seed lots and be helpful to limit the spread of the pathogen from seed production areas, to areas where susceptible crops are grown in rotation with spinach. The assay may also be useful as an additional tool to assess the level of plant resistance to V. dahliae.
Quantification of the pathogen Verticillium dahliae in the soil. V. dahliae causes vascular wilt in a number of plant species and crops like strawberry which are very sensitive to low levels of this pathogen, so having the ability to rapidly and accurately determine inoculum levels in the soil would help growers make planting decisions. In collaboration with a researcher at the University of California, ARS researchers in Salinas completed validation of a real time PCR TaqMan quantification assay for determining soil populations of V. dahliae. To help provide access to this assay for the growers, the specifics of the technique and materials needed to validate results were provided to labs that perform diagnostic services for the strawberry industry prior to manuscript publication. Prior techniques for soil quantification involved plating soil on culture medium but did not always provide accurate quantification and took between 6-8 weeks, so the development of this molecular assay should improve the ability of the growers to identify risk before planting.
Organic Strawberry Production Manual produced. Collaboration by ARS researchers in Salinas, CA, Co-Editors from the University of California and authors from various institutions led to the production of this useful manual. Producers had little scientific data to use for improving organic strawberry production. This manual provides a snapshot of organic strawberry research findings. The manual will allow growers to improve production and highlights gaps in knowledge as targets for future research projects.
Analyses of the function of a glucosyl transferase in Verticillium dahliae. The plant pathogenic fungus Verticillium dahliae causes disease in over 200 species of plants, including high value crops such as strawberry. An ARS researcher in Salinas, California, along with a team of researchers, deleted a gene encoding a glucosyl transferase in Verticillium dahliae and observed that the mutant strain was not pathogenic on one plant host. The glucosyl transferase is widely conserved among bacteria, but present in only a few genera of pathogenic fungi, suggesting that the gene may have been acquired through a mechanism involving a horizontal genetic transfer from bacteria to fungi. The bacterial glucosyl transferase is involved in responses to osmotic stress, and the fungal homolog of the gene may function similarly. 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.
Pseudomonas cannabina pv. alisalensis caused blight on radish in Germany. Pseudomonas cannabina pv. alisalensis had not previously been reported from continental Europe. Bacterial blight caused by P. cannabina pv. alisalensis causes significant economic damage. ARS researchers in Salinas, California, in collaboration with collaborators from The Netherlands expanded the known geographic range of this disease to continental Europe by documenting the first occurrence of this disease there. This information demonstrated the need for management practices and detection practices for this pathogen world-wide.
Pseudomonas cannabina pv. apii causes bacterial streak in fennel (Foeniculum vulgare). ARS researchers in Salinas, CA in collaboration with scientists from the University of California determined that bacterial streak in fennel was caused by an important pathogen of celery. Bacterial streak symptoms spread down the stems leading to unmarketable plants due to bulb decay. This pathogen also causes disease in parsley. This information was incorporated into recommendations for crop rotation practices to avoid other hosts of this pathogen.
Examination of a hybrid Phytophthora species. The ability of Phytophthora species to form interspecific hybrids and the impact these have on causing new disease has been of significant interest in the research and regulatory community. Phytophthora infestans has a significant impact on potato and tomato production worldwide and there is a new species, P. andina, that was believed to be a hybrid between P. infestans and a another unidentified species. A collaboration between ARS, University of California at Riverside and Franklin and Marshall College researchers examined a collection of these and closely related species using 11 genetic markers in an attempt to identify the other parental isolate and better understand the genetic background of the hybrid isolates. The results provided insight to better understand the process of species hybridization in the genus.
Development of web site to support Phytophthora research. The 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, California, 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.
“Light”- tagged reporter-phage detects and quantifies living bacterial plant pathogens on plants. ARS researchers from Salinas, California, and South Carolina in collaboration with a Cooperative Research and Development Agreement partner demonstrated proof of concept for a novel approach for detecting and quantifying bacterial plant pathogens. This method can be adapted to other plant pathogens. The phage will only infect and produce light in living cells. Thus, this method is highly specific and allows for detection and quantification of living cells of specific bacterial plant pathogens.
Atallah, Z.K., Maruthachalam, K.K., Vallad, G.E., Davis, M., Klosterman, S.J., Subbarao, K.V. 2011. Analysis of Verticillium dahliae suggests a lack of correlation between genotypic diversity and virulence phenotypes. Plant Disease. 95:1224-1232.
Martin, F.N., Coffey, M. 2012. Mitochondrial haplotype analysis for differentiation of isolates of Phytophthora cinnamomi. Journal of Phytopathology. 102:229-239.
Bilodeau, G.J., Koike, S., Uribe, P., Martin, F.N. 2012. Development of an assay for rapid detection and quantification of Verticillium dahliae in soil. Phytopathology. 102:331-343.
Jardini, T.M., Koike, S.T., Bull, C.T. 2011. First report of bacterial streak of fennel (Foeniculum vulgare) in California caused by Pseudomonas syringae pv. apii. Plant Disease. 96:285.
Duressa, D.O., Rauscher, G.M., Koike, S.T., Mou, B., Hayes, R.J., Maruthachalam, K., Subbarao, K.V., Klosterman, S.J. 2012. A real-time PCR assay for detection and quantification of verticillium dahliae in spinach seed. Phytopathology. 102:443-451.
Blair, J.E., Coffey, M.D., Martin, F.N. 2012. Species tree estimation for the late blight pathogen, Phytophthora infestans, and close relatives. PLoS One. 7(5):e37003.
Bull, C.T., Martin, F.N. 2012. Cultivar Selection for Pest Management in Organic Strawberry Production. In: Koike, S. T., Bull, C. T., Bolda, M., Daugovish, O., editors. Organic Strawberry Production Manual. University of California Division of Agriculture and Natural Resources. p. 15-24.
Bull, C.T., De Boer, S.H., Denny, T.P., Firrao, G., Fischer-Le Sau, M., Saddler, G., Scortichini, M., Stead, D.E., Takikawa, Y. 2012. List of New Names of Plant Pathogenic Bacteria (2008-2010). Journal of Plant Pathology. 94:21-27.
Klosterman, S.J., Subbarao, K.V., Kang, S., Veronese, P., Gold, S.E., Thomma, B.P.H.J, Chen, Z., Henrissat, B., Lee, Y., Park, J., Garcia-Pedrajas, M.D., Dez, B.J., Anchieta, A.G., De Jonge, R., Parthasarathy, S., Maruthachalam, K., Atallah, Z.K., Amyotte, S., Paz, Z., Inderbitizin, P., Hayes, R.J., Heiman, D.I., Young, S., Zeng, Q., Engels, R., Galagan, J., Cuomo, C., Dobinson, K.F., Ma, L-J. 2011. Comparative genomics yields insights into niche adaptation of plant vascular wilt pathogens. PLoS Pathogens. 7(7): e1002137.
Klosterman, S.J. 2012. Real-time PCR for the quantification of fungi in planta. In: Bolton, M.D., Thomma, B.P.H.J., editors. Plant Fungal Pathogens: Methods and Protocols. Series: Methods in Molecular Biology. Volume 835. New York, NY: Humana Press. p. 121-131.
Martin, F.N., Abad, Z., Balci, Y., Ivors, K. 2012. Identification and detection of Phytophthora: Reviewing our progress, identifying our needs. Plant Disease. 96(8):1080-1103.
Rubio, I., Hiddink, G., Asma, M., Bull, C.T. 2012. First report of the crucifer pathogen Pseudomonas cannabina pv. alisalensis causing bacterial blight on radish (Raphanus sativus) in Germany. Plant Disease. 96:904.
Schofield, D.A., Bull, C.T., Rubio, I., Wechter, W.P., Westwater, C., Molineux, I.J. 2012. Development of an engineered ‘bioluminescent’ reporter phage for the detection of bacterial blight of crucifers. Applied and Environmental Microbiology. 78:3592-3598.
Koike, S., Bull, C.T., Bolda, M., Daugovish, O. 2012. Organic strawberry production manual. Organic Strawberry Production Manual. 148 p.