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United States Department of Agriculture

Agricultural Research Service

Research Project: Detection and Management of Pathogens in Strawberry and Vegetable Production Systems
2013 Annual Report


1a.Objectives (from AD-416):
The overall objective of this project is to develop diagnostic tools for detection, quantification, and identification of plant pathogens currently controlled by chemicals including methyl bromide and to develop technically and economically feasible alternatives to chemical control for high value crops, such as strawberries and vegetables.

Objective 1: Optimize delivery and evaluate performance of cultural and biological control, management practices, and genetic alternatives for management of pathogens currently mediated by soil fumigation.

Objective 2: Develop rapid and accurate 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.


3.Progress Report:
This is the new bridging project for 5305-22000-012-00D (See for additional information). To identify genetic alternatives for management of pathogens currently mediated by soil fumigation, research is ongoing to evaluate the utility of a DNA-based assay for the quantification of Verticillium wilt disease resistance in spinach. Collaboration with the University of California, Davis, is ongoing to characterize the potential role of three plant genes in disease resistance and genes expressed in lettuce in response to Verticillium dahliae. Studies launched to determine the location of Verticillium dahliae in spinach were completed. Studies are underway to evaluate genes that regulate microsclerotial development in Verticillium dahliae.

To develop rapid and accurate molecular diagnostic tools for the identification of emerging diseases, correlations between the mitochondrial haplotype and nuclear genotype for 100+ isolates of Phytophthora cinnamomi representing a worldwide collection has been completed and coalescent analysis of the mitochondrial sequence data is on progress. The correlation between mitochondrial haplotype and nuclear genotype for 102 strains of Phytophthora infestans have been evaluated and coalescent analysis of the mitochondrial data completed to assess evolutionary relationships. The manuscript is in the final stages of completion. A new molecular diagnostic assay for Phytophthora that has both a genus and species specific detection capability multiplexed in the same tube has been developed and validated. The manuscript describing it is in the final stages of completion. Macrophomina phaseolina is an emerging pathogen of strawberry with the switch from methyl bromide to alternative fumigants. A collection of isolates recovered from strawberry state wide have been genotyped by Simple sequence repeats (SSR) analysis. Collaborative tests to determine correlations between genotype and host range or virulence are in progress. Research is ongoing to detect and quantify the spinach downy mildew pathogen, Peronospora effusa, in soil samples using molecular diagnostic tools.

Determined that Pseudomonas cannabina pv. alisalensis caused bacterial blight outbreaks on organic arugula in Minnesota, bacterial blight of black mustard used as a cover crop in organic production in California and broccoli stalk rot, thus expanding the geographic, symptom and host range of the pathogen and impacting crop rotation strategies. To identify genetic alternatives for management of a bacterial pathogen, a screening method was developed to evaluate arugula germplasm for resistance to bacterial blight of crucifers caused by P. cannabina pv. alisalensis. Screening for resistance was initiated on 236 accessions of arugula from the US National Germplasm System and commercially available cultivars. Demonstrated that although P. syringae pv. apii and P. syringae pv. coriandricola have broad experimental host ranges, they are the causal agents of bacterial leaf spot of celery and cilantro, respectively, in California. Experiments were initiated to identify pathogens or potential biological control agents from raspberry and cranberry.


4.Accomplishments
1. Determined where the fungal pathogen Verticillium dahliae is localized in spinach seeds. Verticillium dahliae is a fungus that causes vascular wilt in a number of plant species and crops like strawberry and lettuce. An ARS researcher in Salinas, California, in collaboration with researchers at the University of California, determined where Verticillium dahliae is located in infected spinach seeds. The results indicated that the thick fruit wall and seed coat are heavily infected with the fungus. Some fungal colonization of the more centrally located embryo tissue was observed, consistent with the finding that two fungicides applied to the seeds did not eliminate the pathogen in the seed. The information is useful for the preparation of effective seed treatments for the elimination or reduction of the pathogen in seeds.

2. Developed sensitive methods to detect and quantify the corky root pathogen Sphingobium suberifaciens of lettuce. Research on Sphingobium suberifaciens is limited because the pathogen is not amenable to classical detection and quantification methods. An ARS researcher in Salinas, California, identified DNA sequences specific to this pathogen. The primers were used to develop pathogen specific polymerase chain reaction (PCR) and real-time PCR protocols. The protocols specifically detect and quantify the pathogen from environmental samples including lettuce roots and naturally infested field soil at levels important for disease development (200 cells per PCR sample). These methods will facilitate research on the epidemiology of the disease and allow agriculturalists to detect and quantify the pathogen before planting susceptible crops.

3. Examination of historical strains of a pathogen. Phytophthora infestans has had a significant, adverse impact on potato and tomato production worldwide for the past 160 years. An ARS researcher in Salinas, California, collaborated with researchers in Germany and the United Kingdom to sequence a collection of potato tissue samples infected with the pathogen from herbarium collections dating back to 1845. The nuclear and mitochondrial genomes of these historical strains were assembled and compared to current pathogen genomes to identify differences between them; the early strains were different from current ones. Since the host genome was assembled and pathogen resistance genomes identified, these results provided insight to better understand not just the historical spread of the pathogen but also pathogen evolution following the deployment of host resistance genes in commercial cultivars around 1900.

4. Elucidated a relationship between genotypic and pathogenic diversity in a bacterial pathogen of lettuce. Xanthomonas campestris pv. vitians is an important pathogen of lettuce world-wide. ARS researchers in Salinas, California, described the genetic diversity of over 100 strains of the bacterium from a wide geographic range using a multilocus sequence typing approach. A relationship between pathogen genotypes and pathogenic response was elucidated. California genotypes of the pathogen elicited hypersensitive responses within 24 hours on lettuce cultivars previously identified as resistant. In contrast, genotypes of the pathogen from other geographic regions did not elicit hypersensitive responses and were pathogenic. This work enables USDA and public seed company breeders to develop lettuce cultivars resistant to strains that overcome the currently available resistance and allows for methods to distinguish California genotypes from others as they move into the state.

5. Improved identification and detection of Phytophthora species. There are approximately 120 species of Phytophthora, many of which are responsible for significant disease losses in commercial agriculture and natural ecosystems. In an effort to design a diagnostic tool that can be used to identify of wide range of species (including many that are under quarantine or not present in the United States) that may be present in an environmental sample, an ARS researcher in Salinas, California, collaborated with scientists at Agriculture and Agri-Food Canada, National Program on Environmental Health-Biodiversity, in Ottawa, Ontario and the University of California, Riverside, on development of macroarrays. This technique enables identification of an unknown by comparing it against examples of all described species. This provides an additional tool for researchers and regulatory personnel for identification of isolates to a species level.

6. Characterization of the transposons in Verticillium dahliae. Transposons are mobile DNA sequences, also sometimes referred to as jumping DNA, that are present in most organisms, including Verticillum dahliae. An ARS researcher in Salinas, in collaboration with researchers at Agriculture and Agri-Food, Canada and North Carolina State University, characterized transposable element sequences (TEs) in genome of Verticillum dahliae and those of related Verticillium species. These analyses indicate that certain strains of Verticillium dahliae contain more of some types of TEs and that certain types of TEs are lacking in some strains. The results indicate that TEs have played a role in generating genetic diversity within and among Verticillium species and may be useful to differentiate strains of the fungus.

7. Analysis of the population structure of Phytophthora nicotianae. Phytophthora nicotianae is a broad host range pathogen common throughout the world and can be transported across borders on plant material. In an effort to better understand the population structure of the pathogen and identify examples of pathogen movement, an ARS researcher in Salinas, California, collaborated with scientists at the Università degli Studi Mediterranea, Italy. The collaborative effort focused on investigating the correlation between mitochondrial haplotype and nuclear genotype of Phytophthora nicotianae. The results provided insight on the pathogen population structure of Phytophthora nicotianae. In addition, the research yielded tools that can be used to monitor movement of specific strains of the pathogen by analysis of mitochondrial haplotypes and nuclear genotypes.

8. 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.

9. Identified oospores of downy mildew in spinach leaves. Peronospora effusa causes downy mildew on spinach and can cause economic losses to this crop. The sexually produced survival structures, or oospores, that can persist in the soil between growing seasons, were identified in spinach leaf samples in California. The oospores had not been previously reported in California. These findings suggest genetic recombination between strains of P. effusa in California, which may lead to new races of that overcome resistance to downy mildew disease. Because oospores of the pathogen can serve as a primary inoculum source, soil treatments to eliminate these types of spores in the soil may help to slow the production of emerging races of the pathogen.


Review Publications
Amyotte, S.G., Tan, X., Pennerman, K., Del Mar Jimenez-Gasco, M., Klosterman, S.J., Ma, L., Dobinson, K.F., Veronese, P. 2012. Transposable elements in phytopathogenic Verticillium spp.: insights into genome evolution and inter- and intra-specific diversification. Biomed Central (BMC) Genomics. 13:314.

Schroeder, K., Martin, F.N., De Cock, A., Levesque, C.A., Spies, C., Okubara, P.A., Paulitz, T.C. 2013. Molecular detection and quantification of Pythium species: Evolving taxonomy, new tools and challenges. Plant Disease. 97(1):4-20.

Mammella, M., Martin, F.N., Cacciola, S.O., Coffey, M.D., Faedda, R., Schena, L. 2013. Analyses of the population structure in a global collection of Phytophthora nicotianae isolates inferred from mitochondrial and nuclear DNA sequences. Phytopathology. 103(6):610-622.

Chen, W., Robleh-Djama, Z., Coffey, M.D., Martin, F.N., Bilodeau, G.J., Radmer, L.E., Denton, G., Lévesque, C.A. 2013. Membrane-based oligonucleotide array developed from multiple markers for the detection of many Phytophthora species. Phytopathology. 103(1):43-54.

Maruthachalam, K., Klosterman, S.J., Anchieta, A.G., Mou, B., Subbarao, K.V. 2013. Colonization of spinach by Verticillium dahliae and effects of pathogen localization on the efficacy of seed treatments. Phytopathology. 103:268-280.

Sarris, P.F., Trantas, E.A., Baltras, D.A., Bull, C.T., Wechter, W.P., Yan, S., Ververidis, F., Almeida, N.F., Jones, C.D., Dangl, J.L., Panopoulos, N.J., Vinatzer, B.A., Goumas, D.E. 2013. Comparative genomics of multiple strains of Pseudomonas cannabina pv. alisalensis, a potential model pathogen of both Monocots and Dicots. PLoS One. 8(3):1-16.

Schofield, D., Bull, C.T., Rubio-Salazar, I., Wechter, W.P., Westwater, C., Molineux, I.R. 2013. "Light-tagged" bacteriophage as a diagnostic tool for the detection of phytopathogens. Bioengineered. 4:1-5.

Zacaroni, A.B., Koike, S.T., De Souza, R.M., Bull, C.T. 2012. Bacterial leaf spot of radicchio (Cichorium intybus) is caused by Xanthomonas hortorum. Plant Disease. 96:1820.

Yoshida, K., Schuenemann, V.J., Cano, L.M., Pais, M., Mishra, B., Sharma, R., Lanz, C., Martin, F.N., Kamoun, S., Krause, J., Thines, M., Weigel, D., Burbano, H.A. 2013. The rise and fall of the Phytophthora infestans lineage that triggered the Irish potato famine. eLife 2013. DOI: 10.7554/eLife.00731.001.

Martin, F.N. 2013. Molecular identification of Phytophthora. In: Lamour, K., editor. Phytophthora: A Global Perspective. Boston, MA: CABI. p. 19-27.

Last Modified: 12/21/2014
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