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ARS Home » Pacific West Area » Salinas, California » Crop Improvement and Protection Research » Research » Research Project #426096

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

Location: Crop Improvement and Protection Research

2017 Annual Report

1a. Objectives (from AD-416):
The overall objective of this project is to develop diagnostic tools for detection, quantification, and identification of soilborne 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. Sub-objective 1.a. Evaluate alternatives to replace current fumigation strategies for management of soilborne pathogens. Sub-objective 1.b. Identify genes associated with pathogenicity of Verticillium dahliae to support the development of alternative control procedures. Sub-objective 1.b.1. Identify genes associated with pathogenicity of Verticillium dahliae based on a previous insertional mutagenesis study. Sub-objective 1.b.2. Evaluate expression of candidate pathogenicity-related genes in resistant and susceptible lettuce-Verticillium dahliae interactions. Sub-objective 1.c. Identify emerging diseases and their etiology, and evaluate germplasm for resistance to diseases of strawberry, lettuce, and vegetables. Sub-objective 1.c.1. Evaluate spinach germplasm for new sources of Verticillium dahliae resistance using an enhanced screening technique. Sub-objective 1.c.2. Evaluate primary inoculum sources of Peronospora effusa, the cause of downy mildew of spinach. Sub-objective 1.c.3. Identify pathogens of emerging and established bacterial diseases of strawberry and vegetables. Sub-objective 1.c.4. Identify and characterize lettuce and crucifer germplasm resistant to bacterial pathogens. Sub-objective 1.d. Identify factors associated with production, maintenance, and the degradation of inoculum to elucidate potential alternatives for pathogen control. Sub-objective 1.d.1. Identify and evaluate microbial predators of fungal and bacterial pathogens for disease control. Sub-objective 1.d.2. Functionally analyze candidate genes identified as up-regulated in microsclerotia development in Verticillium dahliae. Objective 2: Develop rapid and accurate molecular diagnostic tools for identifying emerging diseases of vegetables and strawberries, and use these tools in the development of management strategies as alternatives to methyl bromide. Sub-objective 2.a. Develop molecular markers for detecting and quantifying bacterial plant pathogens. Sub-objective 2.b. Develop molecular diagnostic tools for detection, quantification, and characterization of isolates of Peronospora effusa. Sub-objective 2.c. Develop molecular tools for identification and detection of Pythium and Phytophthora species. Sub-objective 2.c.1. Molecular diagnostics. Sub-objective 2.c.2. Improved identification of Phytophthora species. Sub-objective 2.c.3. Mitochondrial genomics project. Objective 3: Develop disease management strategies for the soil-borne fungal pathogens Macrophomina and Fusarium on strawberry (strawberry collapse disorder) and integrate these strategies into sustainable crop production systems.

1b. Approach (from AD-416):
Develop integrated management approaches including crop rotation, biological control, selection of disease resistant varieties, organic production, other biological practices, and combinations of biological practices to control diseases. Evaluate responses of the root biome to these practices through metagenomics analyses. Identifying useful biological agents through traditional microbiological methods or soil metagenomics, as well as new agents, that will help to improve disease management. Molecular tools will be developed for plant pathogen identification, evaluation of the efficacy of pathogen management and modeling population dynamics of plant pathogens and beneficial microbes.

3. Progress Report:
This is the final report for Project 2038-22000-015-00D, which expired in March 2017 and has been replaced by new project 2038-22000-016-00D, "Management of Pathogens for Strawberry and Vegetable Production Systems." Substantial progress was made in FY 2017 in support of Objective 1. Methyl bromide has been phased out for control of soilborne diseases of strawberry and other high value crops; hence, effective and economical non-chemical fumigant alternatives are still being sought. Steam treatment and soil amendments of mustard seed meal showed effectiveness for management of a soil borne pathogen in strawberry production fields. Verticillium wilt of lettuce is a devastating disease that does not manifest until just before harvest. Breeding for resistance is ongoing in conjunction with the lettuce breeding project at Salinas. The pathogen is also a target of research on the molecular interactions of the pathogen and host. Screening of Verticillium wilt pathogen mutants revealed a gene deletion mutant strain of interest for further analysis. Sensitive gene expression tests for detection of pathogenicity-related genes in lettuce were validated. The amount of Verticillium dahliae DNA present in infected plants was significantly correlated with susceptibility to Verticillium wilt. Deletion of two genes in Verticillium dahlia were confirmed, which were previously identified as up-regulated in microsclerotia development and regarded as potentially important for the long-term survival of this soilborne pathogen, and evaluated their roles in virulence (infection)-neither had an important role in pathogenicity. Three other genes were determined important for microsclerotia production in Verticillium dahliae and full virulence on lettuce. Downy mildew of spinach is becoming more of a problem with the increased production of baby spinach, whereby millions of seeds are planted per acre, and new pathogenic races of the pathogen have been reported with increased frequency. The pathogen can reportedly survive in the soils, but field soil samples amended with Peronospora effusa-infected leaf tissue were negative for the pathogen within a few months after collection, even with a very sensitive detection test procedure. Commercially available spinach seed lots were shown infected with the pathogen through grow-outs of and biological stains of the seeds for viability. Bacterial infection of arugula leaves renders them unsalable, as they lead to decay during shipment and storage. Methods to screen arugula for resistance to the bacterial pathogen Pseudomonas cannabina pv. alisalensis were developed, and 100 arugula accessions were evaluated for resistance to the pathogen. Biological control is regarded as an important part of integrated pest management but has proven difficult to consistently demonstrate. Inhibition of the growth of two different life stages the fungus Verticillium dahliae by myxobacteria was evaluated in cultures in the laboratory. Biological control tests using bacteriophage and bacteriocins were conducted. Methods for microcosm studies to evaluate the killing of soilborne fungi were developed. Molecular marker-based diagnostic tools offer the promise of rapid and accurate detection of pathogens and their quantification. In support of Objective 2, primers available for detection and quantification of P. cannabina pv. alisalensis were determined to not be specific for either purpose. Assembly was completed for all genomes sequenced and additional strains were selected for sequencing. Molecular diagnostic tools for detection, quantification, and characterization of isolates of P. effusa, which incites downy mildew on spinach, would help reduce application of chemicals for control. Assays for specific detection of the spinach downy mildew pathogen were devised, based on nuclear and mitochondrial targets, and validated them on a range of different isolates of P. effusa and other Peronospora species. One of the assays was validated in the field and deployed for routine pathogen detection. Quantification of the asexual spores of P. effusa was performed to more precisely determine the amount of the pathogen present in environmental samples in relation to the amounts of DNA present. An additional assay was developed and deployed to detect the lettuce downy mildew pathogen, Bremia lactucae. Molecular diagnostic tools for Oomycte genera Phytophthora and Pythium are of great interest to growers and regulators. Three manuscripts were published describing a real time polymerase chain reaction assay for Phytophthora that is multiplexed for genus and species-specific detection. Species-specific diagnostic markers for a total of 50 species were validated (most of the species on the USDA Animal and Plant Health Inspection Service (APHIS) species of concern list); the data suggest that markers for 89% of the genus can be developed. A unique gene order was identified for the plant pathogenic genus Pythium that has been useful for development of a genus-specific diagnostic assays. The genus-specific nature of this marker has been validated against a range of plant and related oomycetes. Initial data indicate this locus should also be useful for design of species-specific diagnostic markers, the potential of which is currently under evaluation. A new isothermal diagnostic marker system was published that can detect the fungal plant pathogen Phytophthora at a genus and species-specific level directly in the field without the need for DNA extraction, with a level of sensitivity approaching previously applied but more costly lab techniques. The system allows for a systematic development of species-specific markers for over 85% of described species (markers have been validated for five species, including the quarantine species P. ramorum and P. kernoviae), and will facilitate detection of the broad host range of Phytophthora. Mitochondrial genes are more conserved than nuclear genes and have great potential for development of molecular markers and phylogenetic purposes. Collaborative projects have been established internationally where raw genomic data are provided and mitochondrial genomes are assembled. Over 500 mitochondrial genomes have been assembled from a wide range of genera representing plant pathogenic oomycetes. Comparisons of these genomes enabled identification of particular sequences useful for development of diagnostic markers for Phytophthora, Pythium and downy mildews, genes for broader scale phylogenetic analysis, mitochondrial sequences for population studies, and provided insight on mechanisms driving mitochondrial genome evolution. Objective 3 was added to this project in June 2016, and work is now underway. Development of an integrated management program for control of Macrophomina phaseolina and Fusarium oxysporum f. sp. fragariae on strawberry was initiated in collaboration with other researchers. The approach is to use molecular soil quantification assays, remote sensing and detailed yield data collection to evaluate how field management practices affect pathogen populations, disease incidence and yield.

4. Accomplishments
1. Protein that inhibits growth of the plant pathogenic fungus Verticillium dahliae identified. Verticillium wilt adversely affects many crops worldwide and is caused by the fungus Verticillium dahliae. The fungus survives in the soil even when non-susceptible crops are grown. An ARS researcher in Salinas, California, in collaboration with scientists in China, discovered a gene that produces a protein that inhibits growth of the fungus. Knowledge of genes and their protein products may be useful to devise environmentally friendly strategies to inhibit the fungus in the field.

2. Determining patterns in the airborne dispersal of spinach downy mildew. Spinach downy mildew is caused by the airborne plant pathogen Peronospora effusa. This disease threatens U.S. production of organic spinach. An ARS researcher in Salinas, California, and colleagues from the University of California, determined patterns in the airborne dispersal of the spinach downy mildew pathogen in the Salinas Valley of California, where much of the U.S. organic spinach is grown. Knowledge acquired in these studies will be used to more accurately predict periods in which the pathogen is most prevalent.

3. Improved management of cucurbit downy mildew. Downy mildew can cause devastating losses to cucurbit and hops production. Development of an effective pathogen monitoring program would enable a more efficient spray program to prevent disease. An ARS researcher in Salinas, California and collaborators at North Carolina State University, developed a molecular marker-based system for detecting and measuring the amounts of downy mildew pathogens on cucurbits and hops. These markers will provide a means for rapid identification of the pathogen and improved management of fungicide spray programs for enhanced disease control.

4. Diagnostics for Macrophomina root and crown rot. Macrophomina phaseolina is an emerging pathogen in California strawberry production with the loss of methyl bromide for soil fumigation. An ARS researcher in Salinas, California and collaborators at University of California and University of Georgia, developed a molecular diagnostic test to detect and measure the amounts of the fungus in plant tissue and soil that is specific for the Macrophomina types capable of infecting strawberry. Isolates of the fungus that are highly infectious on strawberry are, with a few exceptions, genetically identical. This information has been useful for selecting appropriate isolates to use when testing strawberry for genetic resistance to the fungus.

Review Publications
Fan, R., Klosterman, S.J., Wang, C., Subbarao, K.V., Xu, X., Shang, W., Hu, X. 2017. The Vayg1 gene controls microsclerotia development and melanin production in Verticillium dahliae. Fungal Genetics and Biology. 98:1–11.

Choudhury, R.A., Koike, S.T., Fox, A., Anchieta, A.G., Subbarao, K.V., Klosterman, S.J., McRoberts, N. 2017. Spatiotemporal patterns in the airborne dispersal of spinach downy mildew. Phytopathology. 107(1):50-58.

Cheng, X., Zhao, L., Klosterman, S.J., Feng, H., Feng, Z., Wei, F., Shi, Y., Li, Z., Zhu, H. 2017. The endochitinase VDECH from Verticillium dahliae inhibits spore germination and activates plant defense responses. Plant Science. 259:12–23.

Strausbaugh, C.A., Eujayl, I.A., Martin, F.N. 2016. Pathogenicity, vegetative compatibility, and genetic diversity of verticillium dahliae isolates from sugar beet. Canadian Journal of Plant Pathology. 38(4):492-505.

Miles, T.D., Martin, F.N., Robideau, G., Bilodeau, G., Coffey, M.D. 2017. Systematic development of Phytophthora species-specific mitochondrial diagnostic markers for economically important members of the genus. Plant Disease. doi: 10.1094/PDIS-09-16-1224-RE.

Rojas, A., Miles, T.D., Coffey, M.D., Martin, F.N., Chilvers, M. 2017. Development and application of qPCR and RPA genus and species-specific detection of Phytophthora sojae and Phytophthora sansomeana root rot pathogens of soybean. Plant Disease. doi: 10.1094/PDIS-09-16-1225-RE.

Makkonen, J., Vesterbacka, A., Martin, F.N., Jussila, J., Dieguez-Uribeondo, J., Kortet, R., Kokko, H. 2016. Mitochondrial genomes and comparative genomics of Aphanomyces astaci and Aphanomyces invadans. Scientific Reports. 6:36089. doi: 10.1038/srep36089.