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ARS Home » Midwest Area » Peoria, Illinois » National Center for Agricultural Utilization Research » Crop Bioprotection Research » Research » Research Project #432596

Research Project: Development of New Production Methodologies for Biocontrol Agents and Fastidious Microbes to Improve Plant Disease Management

Location: Crop Bioprotection Research

2018 Annual Report

Objective 1: Develop new microbial culturing and mass production technologies for biocontrol agents and nutritionally fastidious plant pathogens. Subobjective 1a: Develop new microbial culturing technologies for biocontrol agents. Subojective 1b: Develop new methodologies for culturing nutritionally fastidious plant pathogens. Objective 2: Define interactions between biocontrol agents, hosts, and pathogens using traditional and genomic approaches to increase disease management success.

Our approach will be to apply technologies allied with the fields of fermentation science, microbial physiology, metabolomics, genomics, and proteomics for two purposes: to enhance the efficacy and shelf-life of the antagonist biomass manufactured and to produce gnotobiotic (i.e., all of a limited number of organisms in a culture are known) or axenic cultures of nutritionally fastidious plant pathogens. More specifically, the shelf-life and efficacy of biocontrol strains will be improved by isolating efficacious stress tolerant variants of a yeast biocontrol agent and then testing the more promising strains isolated in small pilot tests against Fusarium head blight of wheat. Other studies will strive to discover cell production methodologies that promote the production of compounds that enhance cell stress tolerance. Strain transcriptional response to culture conditions will be determined to facilitate optimizing these cell production studies. This will include studies to elucidate the transcriptional response of a yeast biocontrol strain to cold-adaptation that improves cell survival and biocontrol efficacy. Gnotobiotic culturing studies will include establishing a selection of host plants in sterile tissue culture boxes or as callus cell cultures and evaluating methods for infecting these host tissues with axenic propagules of an obligate pathogen. The transcriptional response of gnotobiotic host cell tissue to infection by an obligate plant pathogen will then be determined as a prelude to attempting to grow one or more obligate plant pathogens in axenic culture.

Progress Report
Project 5010-22410-019-00D was initiated with ARS scientists at Peoria, Illinois on May 9, 2017. Significant progress has already been made in meeting the five-year project objectives which are to 1) Develop new microbial culturing and mass production technologies for biocontrol agents and nutritionally fastidious plant pathogens, and 2) Define interactions between biocontrol agents, hosts, and pathogens using traditional and genomic approaches to increase disease management success. First objective studies completed by ARS scientists at Peoria, Illinois, include work that demonstrated, for the first time, that economically important plant pathogen genus Fusarium contains many species that can produce microsclerotia in liquid culture when exacting environmental and nutritional conditions are employed. This discovery enhances the commercial potential of Fusarium-based bioherbicides given the enhanced shelf- life of products that are formulated using microsclerotia as the active ingredient. Collaborative research trials were conducted by ARS scientists at Peoria, Illinois, and university scientists in February 2018. The impact of rehydration protocols on the biocontrol efficacy of osmoprotected, dried formulations of tricultured dry tolerant variants of 3 bacterial strains was assayed against potato storage diseases “dry rot” and “Pythium leak”. Treatments composed of antagonists and chemicals were also conducted and results are pending. In research on culturing nutritionally fastidious plant pathogens, studies continued to determine the ability of several different culture media to produce callus of basil plant cells as a prelude to infecting callus with Peronospora belbahrii, the causal agent of the economically important downy mildew disease of basil. Establishing two component gnotobiotic systems composed only of host and pathogen would greatly facilitate conducting genomic, proteomic and metabolomic studies on this otherwise non-culturable pathogen. Work on characterizing the infection process of basil downy mildew under growth chamber conditions using light and scanning electron microscopy techniques was completed. Results of this work will assist ongoing gnotobiotic culturing work as well as controlling the disease in the field by providing definitive information on how to time control efforts based on when and how the pathogen infects basil plants. In work conducted on a different pathogen (Sclerophthora macrospora) that causes downy mildew (yellow tuft) on turf grass, samples of grass showing typical symptoms of the disease were obtained from collaborators at a university. ARS scientist in Peoria, Illinois, obtained spores of the pathogen from the samples and extracted DNA, which enabled a draft genome assembly to be produced. Studies to purify and identify metabolites produced by a bacterium that inhibit growth of a fungus that damages onions were also conducted. The genomes of these strains were sequenced and the bacteria determined to be new species. Plans are in place to test these metabolites against downy mildews to determine their potential for development as an agricultural pest control product. In research conducted in support of research project Objective 2, ARS researchers in Peoria, Illinois, made significant progress on a sub-project with collaborators from a university at Southern Queensland. In order to identify the causal agent of an economically important disease that causes shriveling of peanut kernels, samples were collected from diseased and disease-free plants across many growing locations. DNA sequencing methods were then employed to determine the total fungi and bacteria present in the samples. The results identified a fungus likely to be the causal agent. Additional sequencing was used to identify isolates of the fungus previously cultured from diseased plants. Greenhouse testing is currently underway to confirm the suspected fungus is the cause of the disease. In other studies, significant progress was made in understanding the transcriptional response to cold adaptation by a yeast antagonist. Data analysis has been completed for one transcriptomic experiment on the cold adaptation of Fusarium head blight antagonist P. flavescens grown under production conditions. The experiment has been repeated and the analysis of data initiated. Finally, additional progress has been made on research to understand the colonization of potato wounds by tricultured biocontrol strains of Pseudomonas spp using molecular characterization (biome analysis) of organisms present in potato wounds in the presence or absence of the pathogen (Fusarium sambucinum) over time. Results from these studies should improve the commercial development potential of this ARS-discovered biocontrol product.

1. Non-viable biocontrol cells nullify live cell efficacy in reducing plant disease. The use of naturally occurring microorganisms to biologically control diseases on agricultural crops continues to gain acceptance from producers and consumers but occasional inconsistent performance of biocontrol agents hampers their wholesale adoption. ARS research scientists in Peoria, Illinois, conducted experiments to determine if cells of the biocontrol yeast Papiliotrema flavescens OH 182.9 that die during product storage and/or during field application can counteract the beneficial effect of live antagonist cells. The scientists determined that live cells of the yeast caused spores of the pathogen that causes Fusarium head blight on wheat to be less effective in germinating and colonizing wheat tissue. However, this positive effect was largely negated when non-viable antagonist cells were mixed with the live cells, at least in part because nutrients from dead antagonist cells benefited pathogen growth. These results have broad implications in understanding why biocontrol of plant diseases can sometimes appear inconsistent and in directing biocontrol research towards discovering methods to preserve biocontrol agent viability during formulation and application to crops.

2. Genome characterization and first demonstration of antifungal activity by bacterial strain. Novel antifungal compounds are of critical need in agriculture and medicine alike as the resistance of pathogens to commonly utilized disease control compounds becomes more prevalent. ARS research scientists in Peoria, Illinois, collaborated with scientists from the University of Illinois to characterize the genome and secondary metabolites of a bacteria with high antagonistic activity against a wide variety of plant pathogens. The project team was the first to report antifungal activity for this species and to isolate bioactive compounds. The genes associated with the production of these compounds were also identified. Because the strain was shown to be nearly identical to a commercially produced probiotic strain of the same species, researchers were able to identify the active secondary metabolites produced by this important probiotic strain. These findings will help scientists to understand how this important class of bacteria adapt from living on a plant and controlling plant pathogens, to living in the human intestinal tract and controlling undesirable bacteria.

3. Evaluation of chemical and biological approaches to reducing basil downy mildew. The pathogen Peronospora belbahrii causes basil downy mildew, a destructive disease of sweet basil in the United States and globally. Foliar fungicide treatments show promise in reducing the severity of symptoms on basil leaves but repeated use of a fungicide can result in the pathogen population becoming resistant to the fungicide, limiting its effectiveness. Biocontrol agents have potential for reducing downy mildew development but few have been evaluated. ARS researchers in Peoria, Illinois, and collaborators at the University of Illinois found that weekly field applications of multiple fungicides, combinations thereof, application rates and sequences reduced downy mildew on basil by 80-95% when applied to basil foliage weekly over the course of a summer field season in Illinois. However, a previously untested fungicide product and two biocontrol agents discovered on potato tubers were not effective in reducing downy mildew in tests on greenhouse grown plants. These results demonstrate the effectiveness of new fungicide treatment combinations and sequences that have promise in reducing downy mildew on basil for basil producers and consumers without rapidly selecting for fungicide tolerant variants of the pathogen.

Review Publications
Johnson, E.T., Proctor, R., Dunlap, C.A., Busman, M. 2017. Reducing production of fumonisin mycotoxins in Fusarium verticillioides by RNA interference. Mycotoxin Research. 34:29-37.
Cao, W., Guo, L., Du, Z., Das, A., Saren, G., Jiang, M., Dunlap, C.A., Rooney, A.P., Yu, X., Li, T. 2017. Chengkuizengella sediminis gen. nov. sp. nov., isolated from sediment. International Journal of Systematic and Evolutionary Microbiology. 67:2672–2678. doi: 10.1099/ijsem.0.002006.
Cote, G.L., Dunlap, C.A., Vermillion, K.E., & Skory, C.D. 2017. Production of isomelezitose from sucrose by engineered glucansucrases. Amylase. 1(1):82-93. doi: 10.1515/amylase-2017-0008.
Dunlap, C.A., Lueschow, S.R., Carillo, D., Rooney, A.P. 2017. Screening of bacteria for antagonistic activity against phytopathogens of avocados. Plant Gene. 11:17-22.
Dunlap, C.A., Mascarin, G.M., Romagnoli, E.M., Jackson, M.A. 2017. Rapid discrimination of Isaria javanica and Isaria poprawskii from Isaria spp. using high resolution DNA melting assays. Journal of Invertebrate Pathology. 150:88–93.
Dunlap, C.A., Ramirez, J.L., Mascarin, G.M., Labeda, D.P. 2018. Entomopathogen ID: a curated sequence resource for entomopathogenic fungi. Mycologia. 111:897-904. doi:10.1007/s10482-017-0988-2.
Dunlap, C.A., Schisler, D.A., Perry, E.B., Connor, N., Cohan, F.M., Rooney, A.P. 2017. Bacillus swezeyi sp. nov. and Bacillus haynesii sp. nov., isolated from desert soil. International Journal of Systematic and Evolutionary Microbiology. 67:2720-2725. doi: 10.1099/ijsem.0.002007.
Lu, D., Xia, J., Dunlap, C.A., Rooney, A.P., Du, Z. 2017. Gracilimonas halophila sp. nov., isolated from a marine solar saltern. International Journal of Systematic and Evolutionary Microbiology. 67:3251-3255. doi: 10.1099/ijsem.0.002093.
Lu, D., Xia, J., Dunlap, C.A., Rooney, A.P., Du, Z. 2017. Salibacter halophilus gen. nov., sp. nov., isolated from a saltern. International Journal of Systematic and Evolutionary Microbiology. 67:1784–1788. doi: 10.1099/ijsem.0.001807.
Torres-Crus, T.J., Billingsley Tobias, T.L., Almatruk, M., Hesse, C.N., Kuske, C.R., Desiro, A., Benucci, G., Bonito, G., Stajich, J.E., Dunlap, C.A., Arnold, A., Porras-Alfaro, A. 2017. Bifiguratus adelaidae, gen. et sp. nov., a new member of Mucoromycotina in endophytic and soil-dwelling habitats. Mycologia. 109(3):363-378.
Xia, J., Xie, Z., Dunlap, C.A., Rooney, A.P., Du, Z. 2017. Rhodohalobacter halophila gen. nov., sp. nov., a moderately halophilic member of the family Balneolaceae. International Journal of Systematic and Evolutionary Microbiology. 67:1281–1287. doi: 10.1099/ijsem.0.001806.
Mascarin, G.M., Kobori, N.N., Jackson, M.A., Dunlap, C.A., Delalibera, I. 2018. Nitrogen source affects productivity, desiccation tolerance, and storage stability of Beauveria bassiana blastospores. Journal of Applied Microbiology. 124:810-820. doi: 10.1111/jam.13694.
Dunlap, C.A., Rooney, A.P. 2018. Acinetobacter dijkshoorniae is a later heterotypic synonym of Acinetobacter lactucae. International Journal of Systematic and Evolutionary Microbiology. 68:131-132. doi:10.1099/ijsem.0.002470.
Santos, V., Mascarin, G.M., Da Silva Lopes, M., Fregolente Alves, M.C., Rezende, J.M., Viccari Gatti, M.S., Dunlap, C.A., Delaliber Junior, I. 2017. Identification of double-stranded RNA viruses in Brazilian strains of Metarhizium anisopliae and their effects on fungal biology and virulence. Plant Gene. 11:49-58.