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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

2016 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:
Sub-objective 1.a: Evaluate alternatives to replace current fumigation strategies for management of soilborne pathogens: Steam treatment and soil amendments of mustard seed meal were evaluated as alternatives for fumigation and shows effectiveness in the management of a soilborne pathogen in strawberry production fields. The treatment is administered by a tractor-drawn device as it passes through the planting beds. This research relates to project plan objective 1 to 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.b.1: Identify genes associated with pathogenicity of Verticillium dahliae based on a previous insertional mutagenesis study: We conducted screens of potential genetic mutants of the soilborne fungus Verticillium dahliae and had difficulty in this particular objective in confirming a gene deletion event. We increased the number of mutants screened to identify the appropriate gene deletion, and were able to find a gene deletion mutant strain that requires analysis. We will not pursue additional genes from this particular study. Progress in elucidating the genes responsible for pathogenicity of V. dahliae relates to project plan objective 1 to evaluate genetic alternatives for management of pathogens currently mediated by soil fumigation. Sub-Objective 1.b.2: Evaluate expression of candidate pathogenicity-related genes in resistant and susceptible lettuce-Verticillium dahliae interactions: Sensitive tests examining gene expression were validated for detection of pathogenicity-related genes in lettuce. Plants were inoculated with each race of Verticillium dahliae in growth chambers, and the tissue was analyzed for differential gene expression. Progress in this research area relates to project plan objective 1: to evaluate genetic alternatives for management of pathogens currently mediated by soil fumigation. Sub-objective 1.c.1: Evaluate spinach germplasm for new sources of Verticillium dahliae resistance using an enhanced screening technique: We were able to show clear correlation between the amount of DNA present and susceptibility to wilt disease. Progress in this research area relates to project plan objective 1: to evaluate genetic alternatives for management of pathogens currently mediated by soil fumigation. Sub-objective 1.c.2: Evaluate primary inoculum sources of Peronospora effusa the cause of downy mildew of spinach: Field soil samples combined with Peronospora effusa-infected leaf tissue were negative for detection of P. effusa months after collection, even with a very sensitive detection test. Additional experiments were initiated to grow out the pathogen from infested spinach seed. Biological staining were used for showing viability of the pathogen on seed. A manuscript was published on P. effusa in modern spinach seed lots. Research in these areas relates to project plan objective 1 to optimize delivery and evaluate performance of cultural and biological control, management practices, and genetic alternatives for management of pathogens. Sub-objective 1.c.3: Identify pathogens of emerging and established bacterial diseases of strawberry and vegetables: Characterizations of the genetic relationships of these pathogens in relation to other pathogens were completed and host range evaluations of plant pathogens are complete. The research supports project plan objective 2: develop rapid and accurate molecular diagnostic tools for the identification of emerging diseases of vegetables. Sub-objective 1.c.4: Identification and characterization of lettuce and crucifer germplasm resistant to bacterial pathogens: Methods were developed to screen arugula for resistance to the bacterial pathogen Pseudomonas cannabina pv. alisalensis and 100 arugula accessions were evaluated. Progress in identifying plant resistance to the bacterial pathogen P. cannabina pv. alisalensis relates to project plan objective 1 to evaluate genetic alternatives for management of pathogens currently mediated by soil fumigation. Sub-objective 1.d.1: Identify and evaluate microbial predators of fungal and bacterial pathogens for disease control: Inhibition of the growth of two different lifecycle stages of the fungus Verticillium dahliae by myxobacteria was evaluated in laboratory cultures. Biological control tests using bacteriophage and bacteriocins were conducted. Methods for microcosm studies to evaluate the killing of soilborne fungi were developed. Progress here relates project plan objective 1: evaluate performance of biological control for management of pathogens currently mediated by soil fumigation. Sub-objective 1.d.2: Functionally analyze candidate genes identified as up-regulated in microsclerotia development in Verticillium dahliae: We have deleted two genes in the fungus Verticillium dahliae that were previously identified as up-regulated in microsclerotia development. These genes are anticipated as important for the long term survival of this soilborne pathogen. We confirmed the deletion of these genes and evaluated the role of these in virulence, revealing that neither had an important role in pathogenicity. Two other genes were determined important for microsclerotia production in Verticillium dahliae and full virulence, relating to plan objective 1: Optimize delivery and evaluate performance of genetic alternatives for management of pathogens currently mediated by soil fumigation. Sub-Objective 2.a: Develop molecular markers for detection and quantification of bacterial plant pathogens: Primers available for detection and quantification of Pseudomonas cannabina pv. alisalensis have been evaluated for specificity and were not specific. Assembly was completed for all genomes sequenced. Additional strains were selected for sequencing based on plant reaction studies. This research supports project plan objective 2: develop rapid and accurate molecular diagnostic tools for the identification of emerging diseases of vegetables. Sub-objective 2.b: Develop new molecular diagnostic tools for detection, quantification, and characterization of isolates of Peronospora effusa: New assays were devised for a specific detection of the spinach pathogen Peronospora effusa, based on nuclear and mitochondrial targets. The assays were tested and validated on a range of different isolates of P. effusa and other Peronospora species, and the assay is under further development and validation. The 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 in a subordinate project to detect the lettuce downy mildew pathogen, Bremia lactucae. Progress here relates the project plan objective 2: develop rapid and accurate molecular diagnostic tools for the identification of emerging diseases of vegetables. Sub-objective 2.c.1: Molecular Diagnostics: A manuscript describing a real time polymerase chain reaction assay for Phytophthora that is multiplexed for genus and species-specific detection was published. Species-specific diagnostic markers for an additional 35 species (most of the species on the USDA Animal and Plant Health Inspection Service (APHIS) species of concern list) have been validated (for a total of 50 species) and the data suggest that markers for at least an additional 40 species can be developed. To support technology transfer, the details were deposited at a journal website. A unique gene order was identified using comparative mitochondrial genomics for the plant pathogenic genus Pythium that has been useful for development of 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 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. The progress in these research areas relates the project plan objective 2: develop rapid and accurate molecular diagnostic tools for the identification of emerging diseases of vegetables and strawberries. Sub-objective 2.c.3: Mitochondrial genomics for development of molecular markers and phylogenetic purposes. Collaborative projects have been established internationally where raw genomic data is provided and mitochondrial genomes are assembled in Salinas, California. Over 300 mitochondrial genomes have been assembled from a wide range of genera representing plant pathogenic oomycetes. Comparisons of these genomes has 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 provides insight on mechanisms driving mitochondrial genome evolution. This relates the project plan 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.


4. Accomplishments
1. Population analysis of Phytophthora nicotianae. P. nicotianae is a broad host range pathogen with a worldwide distribution capable of infecting a wide range of hosts. In an effort to develop techniques for monitoring different lineages of the pathogen, an ARS researcher in Salinas, California developed DNA fingerprinting markers that were used to analyze a worldwide collection of isolates. Isolates recovered from some hosts tended to have the same genotype, suggesting there may be some level of host specificity. Publication of the techniques and conclusions of the population analysis facilitates research on this economically important pathogen and provides tools for monitoring pathogen distribution.

2. Development of a detection assay for the lettuce downy mildew pathogen. Downy mildew of lettuce is a disease of economic importance on lettuce in central coastal California, where the disease development is favored by a cool coastal climate. Two ARS researchers in Salinas, California in collaboration with University of California personnel, and scientists abroad, developed and field tested a highly sensitive and species specific mitochondrial DNA-based detection assay for the downy mildew pathogen of lettuce, Beta lactucae. The assay was capable of detecting the equivalent of a single spore of the pathogen and enabled detecting downy mildew spores even during a two-week lettuce-free period in the Salinas Valley (required by Monterey County ordinance) for control of lettuce mosaic virus, suggesting the possibility of downy mildew inoculum contribution from wild lettuce. The assay can be useful to predict disease risk based on spore load in the environment, and therefore may be useful to reduce the number of fungicide applications for disease control.

3. Identification of genes important for microsclerotia production in the fungus Verticillium dahliae. Verticillium dahliae causes plant diseases, known commonly as Verticillium wilts, on economically important crops in the U.S. and abroad. The long-term survival of this fungus in the soil often means that rotating to non-host crops or other plants does not curtail Verticillium wilt disease development in subsequent crops. The long-term survival structures formed by Verticillium dahliae is known as a microsclerotia. An ARS researcher in Salinas California, in collaboration with two scientists in China, revealed the importance of two genes for the formation of the long-term survival structures. Knowledge on the types of genes and their protein products required for the formation of microsclerotia may lead to insights into molecular targets to specifically control the formation of microsclerotia and hence survival of the fungus in the field.

4. Web site to support Phytophthora research. The genus Phytophthora is responsible for many diseases of crop plants worldwide and consists of approximately 140 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. A section is devoted to a review of molecular diagnostic techniques. 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. Updating of content is a continuous process.

5. Molecular diagnostics of Phytophthora. ARS researchers at Salinas, California optimized a systematic approach for detection of the broad host range plant pathogen Phytophthora. A unique gene order present in Phytophthora has been used to design a genus and species specific detection assay and DNA sequence data has been collected from over 140 species. A total of 50 species-specific assays have been validated, many of these for species that are invasive to North America and of regulatory concern. Examination of the DNA sequence data suggests that specific markers can be developed for 89% of the species in the genus. Additional work is in progress to collect data from recently described species and facilitate technology transfer by posting results on a website devoted to research on Phytophthora.


5. Significant Activities that Support Special Target Populations:
An ARS scientist participated in presentations made to an adopted fifth grade classes at a local minority serving elementary school (Los Padres Elementary), to provide science field trips. One ARS scientist from Salinas, California mentored a minority intern from California State University, Monterey Bay (Hispanic Serving Institution, HSI.


Review Publications
Wang, Y., Tian, L., Xiong, D., Klosterman, S.J., Xiao, S., Tian, C. 2016. The mitogen-activated protein kinase gene, VdHog1, regulates osmotic stress response, microsclerotia formation and virulence in Verticillium dahliae. Fungal Genetics and Biology. 88:13–23.

Kunjeti, S.G., Anchieta, A.G., Subbarao, K.V., Koike, S.T., Klosterman, S.J. 2016. Plasmolysis and vital staining reveal viable oospores of Peronospora effusa in spinach seed lots. Plant Disease. 100:59-65.

Klimes, A., Dobinson, K.F., Thomma, B.P.H.J., Klosterman, S.J. 2015. Genomics spurs rapid advances in our understanding of the biology of vascular wilt pathogens in the genus Verticillium. Annual Review of Phytopathology. 53:181-198.

Mou, B., Klosterman, S.J., Anchieta, A.G., Wood, E.M., Subbarao, K. 2015. Characterization of spinach germplasm for resistance against two races of Verticillium dahliae. HortScience. 50:1631-1635.

Li, Z., Liu, Y., Feng, Z., Feng, H., Klosterman, S.J., Zhou, F., Zhao, L., Shi, Y., Zhu, H. 2015. VdCYC8, encoding CYC8 glucose repression mediator protein, is required for microsclerotia formation and full virulence in Verticillium dahliae. PLoS One. 10(12):e0144020. doi: 10.1371/journal.pone.0144020.

Koike, S., Arias De Ares, R.S., Hogan, C.S., Martin, F.N., Gordon, T. 2016. Status of Macrophomina phaseolina on strawberry in California and preliminary characterization of the pathogen. International Journal of Fruit Science. doi: 10.1080/15538362.2016.1195313.