Location: Crop Bioprotection Research2015 Annual Report
1a. Objectives (from AD-416):
Objective 1: Optimize fermentation and formulation protocols for selected biocontrol strains to produce products with enhanced field efficacy, consistency, and compatibility with integrated pest management methodologies. Objective 2: Elucidate selected interactions among host, biocontrol agent, and pathogen on plant surfaces using physiochemical, proteomic, and/or genomic characterization of the host or agent to guide development of effective agent production and formulation technologies. Objective 3: Develop methods and technologies to isolate and identify fastidious microorganisms that threaten U.S. agriculture and determine characteristics that contribute to pathogenicity. The encompassing goal of this project is to improve biocontrol agent product performance by both optimizing agent fermentation, formulation, and deployment protocols; and by elucidating agent, pathogen, and host interactions on plant surfaces in order to optimize antagonist colonization of host surfaces, efficacy in reducing disease and utility in integrated pest management environments. Results from research conducted in each of these key areas will be of value in meeting each individual objective as well as providing data that reciprocally provides leads and concepts that enhance the advancement of the science conducted under the other individual objective. Our Agricultural Research Service (ARS) research team possesses a unique blend of expertise and interactive research experience in plant pathology, biochemistry, and molecular biology to draw on in completing this research. Additionally, collaborators within ARS, at Universities, and within the agricultural business community have committed to providing critical skill-sets that will be instrumental in meeting our overall research goal.
1b. Approach (from AD-416):
Objective 1: Optimize fermentation and formulation protocols for selected biocontrol strains to produce products with enhanced field efficacy, consistency, and compatibility with integrated pest management methodologies. Objective 2: Elucidate selected interactions among host, biocontrol agent, and pathogen on plant surfaces using physiochemical, proteomic, and/or genomic characterization of the host or agent to guide development of effective agent production and formulation technologies. Objective 3: Develop methods and technologies to isolate and identify fastidious microorganisms that threaten U.S. agriculture and determine characteristics that contribute to pathogenicity.
3. Progress Report:
ARS scientists in Peoria, Illinois, conducted independent and collaborative research with university faculty and industry personnel in FY15 in support of five-year project objectives. Studies that contributed to project objective 1, included ARS scientists independently developing and determining that a frozen, flowable, concentrated formulation of cells of patented Cryptococus flavescens strain 3C can maintain biocontrol efficacy against Fusarium head blight (FHB) of wheat and greater than 50% viability after storage for 80 weeks. In collaboration with The Ohio State University, scientists isolated and identified a broad collection of strains of the yeast C. flavescens from multiple fields in Eastern and Midwestern states. The genetic diversity of the strains was determined using molecular techniques, and it was found that a small but significant proportion of all wheat samples contained this yeast. Additional ARS research demonstrated that growing two strains of a variety of isolates of C. flavescens together (co-cultures) resulted in products that were occasionally more effective in reducing FHB than component strains tested individually in greenhouse trials. Collectively, results from these studies promote the development of improved C. flavescens-based biocontrol products active against FHB. Also in support of Project Objective 1, the Peoria-based scientists made important advances in preserving the viability and efficacy of Gram negative bacterial biocontrol agents grown in co-culture. The inert ingredients and chemical supplements that protected individual strains during drying also preserved the viability and efficacy of each component strain of co-cultures active against a wide range of maladies affecting potato tubers in storage. Collaborative small pilot-scale storage studies of these products are ongoing under a Specific Cooperative Agreement (SCA) with researchers at a University. Together with a foreign visiting scientist who collaborated on the project, the Peoria-based scientists developed inexpensive, prototype alternative microbiological media made from extracts of readily available plant materials Considerable progress also was made in meeting Project Objective 2, ARS scientists in Peoria, Illinois, made considerable progress in understanding the genomics and phylogeny of biocontrol agents in the bacterial genus Bacillus. The Peoria scientists sequenced more than twenty genomes of agriculturally important strains. This research identified gene clusters of novel putatively bioactive metabolites. The products of these gene clusters are currently being isolated and assayed for biological activity. In additional studies, the Peoria, Illinois, scientists collaborated with university scientists in Argentina on characterizing the genomes of Bacillus species isolated from wheat heads that were identified to have biocontrol potential. Analysis of the genomes also resulted in the Peoria scientists enhancing and improving Bacillus taxonomy and species identification capability of this important group of agricultural and biocontrol species. A visiting scientist from Korea worked with the Peoria, Illinois scientists to characterize the diversity of Bacillus strains that ferment soybeans. This research identified two novel species of bacteria that were formally described and accessioned into the ARS culture collection where their potential for biocontrol applications will be assessed in future studies. Finally, as an extension of the work on the genomics of biocontrol strains of Bacillus, the Peoria scientists characterized the phytobiome of raspberry plants under different growing conditions. The research characterized the microbial communities in the soil and roots of plants provided different fertilizers. The results showed significant changes in the microbial communities are associated with fertilizer inputs. Future studies will address how to use this knowledge to improve overall plant performance. ARS scientists in Peoria, Illinois, initiated two new studies to control important plant pathogens. One study (Objective 2) aims to develop biocontrol agents that target the fungal pathogens that cause laurel wilt on avocado in Florida and Fusarium dieback on avocado in California. Current efforts for this study focus on isolation and identification of bacteria and yeast that can either kill the avocado pathogens outright or serve as antagonists to suppress pathogen growth. The other new study (Objective 3) focuses on development of new methods and technologies to isolate and identify fastidious and/or uncultivable plant pathogens. For the initial work on this project, downy mildews were chosen as the target pathogens due to their inability to be cultured on artificial media and their economic importance worldwide. To date, studies have focused primarily on Peronospora belbahrii the causal agent of downy mildew on basil. ARS scientists in Peoria, Illinois, have used light and scanning electron microscopy to determine that sporangia of the pathogen germinate after 24 hours and preferentially gain entrance to leaves via stomata. After 6 days sporangiophores emerge, again through stomata. Scientists then developed a first generation tissue culture medium for producing callus of basil, a medium for growing microbe-free basil seedlings, and a method of producing microbe-free sporangia of the pathogen. Studies to produce basil and downy mildew binary systems in otherwise sterile environments can now be initiated which should enable genomic characterization of the pathogen and, ultimately, the discovery of improved methods for controlling this and other downy mildew pathogens.
1. Development of a new dried, multi-strain bacterial biocontrol product. Gram negative bacteria have enormous potential as biocontrol agents against plant pathogens but they rarely become commercial products due to difficulties in producing effective, viable, dried bacterial cells. To solve problems in drying Pseudomonas fluorescens biocontrol strains, ARS scientists in Peoria, Illinois, invented a high-throughput microtiter plate assay to discover compounds that protected Gram negative bacterial cells during drying up to 10 times better than unprotected cells. Researchers then identified inert materials that also improved the viability and efficacy of dried strains produced in consistently effective three-strain co-cultures. The dried, co-culture product reduced post-harvest disease of potatoes in small-pilot scale tests conducted in collaboration with the University of Idaho. These results enhance the likelihood of commercially developing these and other effective Gram negative bacterial biocontrol agents into plant disease control products that benefit producers and consumers of agricultural crops.
2. Determination of the genome sequence and characterization of bioactive metabolites produced by the type strain of Bacillus methylotrophicus. Regulatory guidelines governing the use of bacterial biocontrol agents is often times confounded due to problems in correctly identifying the species to which a particular strain belongs. Bacillus amyloliquefaciens is an agriculturally and industrially important bacteria species, and many of its strains are under development by researchers in academia, government and private industry for use in biocontrol efforts against a number of plant pathogens. However, due to problems in correct species identification, approval and subsequent field deployment of these agents can be delayed for a substantial period of time. ARS scientists in Peoria, Illinois, resolved a major hurdle in the taxonomy and identification of these strains through the discovery that the species B. methylotrophicus is the same as B. amyloliquefaciens subspecies plantarum. The resulting clarification of the species taxonomy and improvement of identification methods ends much of the scientific and regulatory confusion in interpreting previously published investigations which will reduce the time needed for the development of new biological control products.
Carrillo, D., Dunlap, C.A., Avery, P.B., Navarrete, J., Duncan, R.E., Jackson, M.A., Behle, R.W., Cave, R., Crane, J., Rooney, A.P., Pena, J.E. 2014. Entomopathogenic fungi as a biological control for the vector of the laurel wilt disease: The redbay ambrosia beetle. Biological Control. 81:44-50.
Dunlap, C.A., Bowman, M.J. 2014. The use of genomics and chemistry to screen for secondary metabolites in bacillus spp. biocontrol organisms. In: Gross, A.D., Coats, J.R., Duke, S.O., Seiber, J.N., editors. Biopesticides: State of the Art and Future Opportunities. Washington, D.C.: American Chemical Society. p. 95-112.
Dunlap, C.A., Schisler, D.A., Bowman, M.J., Rooney, A.P. 2015. Genomic analysis of Bacillus subtilis OH 131.1 and coculturing with Cryptococcus flavescens for control of fusarium head blight. Plant Gene. 2:1-9.
Kobori, N.N., Mascarin, G.M., Jackson, M.A., Schisler, D.A. 2015. Liquid culture production of microsclerotia and submerged conidia by Trichoderma harzianum active against damping-off disease caused by Rhizoctonia solani. Fungal Biology. 119:179-190.
Patt, J.M., Chow, A., Meikle, W.G., Gracia, C., Jackson, M.A., Flores, D., Setamou, M., Dunlap, C.A., Avery, P., Hunter, W.B., Mafra-Neto, A., Adamczyk Jr, J.J. 2015. Efficacy of an autodisseminator of an entomopathogenic fungus, Isaria fumosorosea, to suppress Asian citrus psyllid, Diaphorina citri, under greenhouse conditons. Biological Control. 88:37-45.
Dunlap, C.A., Kim, S., Kwon, S., Rooney, A.P. 2015. Phylogenomic analysis shows that Bacillus amyloliquefaciens subsp. plantarum is a later heterotypic synonym of Bacillus methylotrophicus. International Journal of Systematic and Evolutionary Microbiology. 65:2104-2109. DOI: 10.1099/ijs.0.000226.
Schisler, D.A., Boehm, M.J., Paul, P.A., Rooney, A.P., Dunlap, C.A. 2015. Reduction of Fusarium head blight using prothioconazole and prothioconazole-tolerant variants of the Fusarium head blight antagonist Cryptococcus flavescens OH 182.9. Biological Control. 86:36-45.