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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

2015 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. Identification and characterization of 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 the identification of 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 detection and quantification of 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.


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 to control diseases. Identifying useful biological agents 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 shaped 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 an insertional mutagenesis study. We conducted screens of potential genetic mutants of the soilborne fungus V. dahliae and had difficulty in identifying the appropriate gene deletion for one particular gene. For the next step, we will increase the number of mutants screened to identify the appropriate gene deletion. 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 were further analyzed and validated for detection of pathogenicity-related genes in a lettuce cultivar resistant to one race but susceptible to another race of the fungus V. dahliae. This cultivar was inoculated with each race in growth chambers, and tissue stored for analyses. For the next step, we will inoculate lettuce with mutant strains of V. dahliae. 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: Completed analysis of spinach germplasm for resistance to Verticillium wilt and identified accessions with resistance using an enhanced screening technique. 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.2: Evaluate primary inoculum sources of Peronospora effusa the cause of downy mildew of spinach: Field soil samples combined with P. effusa-infected leaf tissue were negative for detection of P. effusa months after collection, even with a very sensitive detection test. As a next step, experiments were initiated to grow the pathogen from infested spinach seed, and assess the seedlings for P. effusa infection. 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, and objective 2: develop rapid and accurate molecular diagnostic tools for the identification of emerging diseases of vegetables. Sub-objective 1.c.3: Identify pathogens of emerging and established bacterial diseases of strawberry and vegetables: We have determined the etiology of several bacterial diseases of 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 and the manuscripts are in preparation. This 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 developed to screen arugula for resistance to the bacterial pathogen P. cannabina pv. alisalensis were deployed and 100 accessions of arugula germplasm were screened. In continuing work, the potential resistant selections are being evaluated in replicated studies. 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 the fungus Verticillium dahliae by myxobacteria was evaluated in cultures in the laboratory. Biological control tests using bacteriophage and bacteriocins have been conducted. Methods for microcosm studies to evaluate the killing of soilborne fungi were developed and are being initiated this summer. Progress in this area relates to 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. Next, we are evaluating their role in survival and conducting pathogenicity analyses to determine whether these strains are compromised in their ability to cause disease on lettuce. This research relates to project 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 P. cannabina pv. alisalensis have been evaluated for specificity and were not specific. De novo 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 diagnostic assays were devised for a highly specific detection of the spinach pathogen P. effusa, based on 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. A next step includes the quantification of the asexual spores of P. effusa to more precisely determine the amount of the pathogen present in environmental samples. Progress in this area relates to 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. Grant funding was recently obtained to support collecting sequence data from species described in the past three to four years and designing specific assays for their detection. Prior to publication the technology is being transferred to California Department of Food and Agriculture (CDFA) and USDA-APHIS Plant Protection and Quarantine (PPQ) Center for Plant Health Science and Technology (CHPST) for their use (we have a close working relationship with both labs). Comparative mitochondrial genomics enabled identification of a unique gene order for the plant pathogenic genus Pythium 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 oomycete deoxyribonucleic acids. Initial data indicate this locus can also be useful for design of species-specific diagnostic markers, the potential of which is currently under evaluation. The progress in these research areas relates to 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. Sub-objective 2.c.3: Mitochondrial genomics is useful 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 230 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 to 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. Identification of new bacterial races pathogenic on lettuce. Three races of bacterial leaf spot, caused by Xanthomonas campestris pv. vitians were identified and plant resistance to two of the races was identified by ARS researchers at Salinas, California. Bacterial leaf spot of lettuce is an economically important disease of lettuce worldwide. Resistant varieties are needed to sustainably control this disease. Lettuce breeders need to understand the genetic diversity of the bacteria so that they can identify and breed the necessary resistance genes into new varieties. In this research, three races of the pathogen and lettuce germplasm with resistance to two races were identified. Introgression of resistance into new varieties will provide resistance to a broad spectrum of bacterial strains.

2. Discovery of transmission of Verticillium dahliae from spinach to lettuce. Verticillium wilt, caused by the soilborne fungal pathogen, Verticillium dahliae, is a disease that often requires preplant fumigation for effective control. The transmission of viable V. dahliae on spinach seed is well known, but less is known on whether these strains of V. dahliae carried on these seeds impacts subsequent crops grown in rotation with spinach. An ARS Researcher at Salinas, California, provided evidence that two successive plantings of V. dahliae-infected spinach seeds led to detectable levels of the pathogen in V. dahliae-susceptible lettuce plants that were subsequently planted in the same soil. These results stress the importance of using V. dahliae-free spinach seed when spinach is grown in rotation with lettuce.

3. Detection of viable oospores of Peronospora effusa in modern spinach seed lots. Downy mildew on spinach, caused by the oomycete Peronospora effusa, is the major disease constraint on spinach production, and especially in organic spinach production, where effective fungicides are unavailable for downy mildew control. P. effusa produces long-lived oospores that are carried on seed and survive in plant debris in the soil. An ARS researcher at Salinas, California, led experiments to demonstrate that viable oospores of P. effusa are commonly present on modern spinach seed lots. Since oospores of P. effusa may serve as the primary inoculum in the field to initiate new downy mildew infections in spinach, the findings suggest the use of pathogen-free seed may be especially important in the management of this disease.

4. A new isothermal diagnostic marker for Phytophthora. An ARS researcher at Salinas, California, developed and published a new isothermal diagnostic marker system that can detect the pathogen fungal plant pathogen Phytophthora at a genus and species-specific level directly in the field in as little as 15 minutes without the need for DNA extraction. 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. DNA sequence data used for designing the markers system for nearly all Phytophthora spp. and a list of putative primers for additional species have been posted on a journal website to facilitate adaption of the technology by other labs. Salinas has worked closely with State and Federal regulatory labs on technology transfer, and publication of this research facilitates diagnostic efforts with this pathogen and provides tools for Animal and Plant Health Inspection Service (APHIS) to identify invasive species.

5. Characterization of Peronospora species in relation to the spinach downy mildew pathogen, Peronospora effusa. Downy mildew on spinach is the major disease constraint on spinach production, especially in organic production, where effective fungicides are unavailable. An ARS researcher at Salinas, California, along with international team of scientists determined the genetic relationship of P. effusa with closely allied Peronospora species obtained from related plant hosts. The results illustrate the close relationship between downy mildew pathogens that infect beet and spinach. The findings emphasize the importance of thoroughly testing molecular diagnostic markers against a range of closely related pathogens.


Review Publications
Short, D.P., Gurung, S., Koike, S.T., Klosterman, S.J., Subbarao, K.V. 2015. Frequency of Verticillium species in commercial spinach fields and transmission of V. dahliae from spinach to subsequent lettuce crops. Phytopathology. 105:80-90.

Miles, T.D., Martin, F.N., Coffey, M. 2015. Development of rapid isothermal amplification assays for Phytophthora species from plant tissue. Phytopathology. 105:265-278.

Miller, T.C., Martin, F.N., Broome, J., Dorn, N., Fennimore, S.A. 2014. Evaluation of a mobile steam applicator for soil disinfestation in California strawberry. HortScience. 49:1542-1549.

Choi, Y.-J., Klosterman, S.J., Kummer, V., Voglmayr, H., Shin, H.-D., Thines, M. 2015. Multi-locus tree and species tree approaches toward resolving a complex clade of downy mildews (Straminipila, Oomycota), including pathogens of beet and spinach. Molecular Phylogenetics and Evolution. 86:24–34.

Biasi, A., Martin, F.N., Schena, L. 2015. Identification and validation of polymorphic microsatellite loci for the analysis of Phytophthora nicotianae populations. Journal of Microbiological Methods. 110:61-67.

Bull, C.T., Ortiz-Lytle, M., Ibarra, A.G., du Toit, L.J., Reynolds, G.J. 2015. First report of bacterial blight of crucifers caused by Pseudomonas cannabina pv. alisalensis in Minnesota on arugula (Eruca vesicaria subsp. sativa). Plant Disease. 99:415.

Bull, C.T., Koike, S. 2015. Practical benefits of knowing the enemy: Modern molecular tools for diagnosing the etiology of bacterial diseases and understanding the taxonomy and diversity of plant pathogenic bacteria. Annual Review of Phytopathology. doi: 10.1146/annurev-phyto-080614-120122.

Bull, C.T., Gebben, S.J., Goldman, P.H., Trent, M., Hayes, R.J. 2015. Host genotype and hypersensitive reaction influence population levels of Xanthomonas campestris pv. vitians in lettuce. Phytopathology. 105:316-324.

Simko, I., Hayes, R.J., Bull, C.T., Mou, B., Luo, Y., Trent, M.A., Atallah, A.J., Ryder, E.J., Sideman, R.G. 2014. Characterization and performance of 16 new inbred lines of lettuce. HortScience. 49:679-687.

Hayes, R.J., Trent, M., Truco, M., Antonise, R., Michelmore, R.W., Bull, C.T. 2014. The inheritance of resistance to bacterial leaf spot of lettuce caused by Xanthomonas campestris pv. vitians in three lettuce cultivars. Horticulture Research. 1:14066.

Danies, G., Myers, K., Mideros, M.F., Restrepo, S., Martin, F.N., Cooke, D.E., Smart, C.D., Ristaino, J.B., Seaman, A.J., Gugino, B.K., Grunwald, N.J., Fry, W.E. 2014. An ephemeral sexual population of Phytophthora infestans in the northeastern United States and Canada. PLoS One. 9(12):e116354. doi: 10.1371/journal.pone.0116354.

Inderbitzin, P., Thomma, B.P.H.J., Klosterman, S.J., Subbarao, K.V. 2014. Verticillium alfalfae and V. dahliae, agents of Verticillium wilt diseases. In: Dean, R.A., Lichens-Park, A., Kole, C., editors. Genomics of Plant-Associated Fungi and Oomycetes: Dicot Pathogens. Berlin Heidelberg, Germany: Springer-Verlag. p. 65-97.

Hyde, K.D., Nilsson, R.H., Alias, S.A., Ariyawansa, H.A., Blair, J.E., Cai, L., De Cock, A.W., Dissanayake, A.J., Glockling, S.L., Goonasekara, I.D., Gorczak, M., Hahn, M., Jayawardena, R.S., Van Kan, J.A., Laurence, M.H., Levesque, C.A., Li, X., Liu, J., Maharachchikumbura, S.S., Manamgoda, D.S., Martin, F.N., Mckenzie, E.H., Mctaggart, A.R., Mortimer, P.E., Nair, P.V., Pawlowska, J., Rintoul, T.L., Shivas, R.G., Spies, C.F., Summerell, B.A., Taylor, P.W., Terhem, R.B., Udayanga, D., Vaghefi, N., Walther, G., Wilk, M., Wrzosek, M., Xu, J., Yan, J., Zhou, N. 2014. One stop shop: Backbones trees for important phytopathogenic genera. Fungal Diversity. doi: 10.1007/s13225-014-0298-1.

Subbarao, K.V., Sundin, G.W., Klosterman, S.J. 2015. Focus issue articles on emerging and re-emerging plant diseases. Phytopathology. 105:852-854.