Location: Crop Bioprotection Research2016 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:
Substantial research progress was made in FY 16 by ARS scientists in Peoria, Illinois, in support of all three project objectives. Accomplishments were obtained in work conducted with each other, and colleagues in ARS, universities, and industry. In studies conducted under Objective 1 with scientists at a university, cells of patented Fusarium head blight (FHB) biocontrol agent Cryptococcus flavescens strain 3C were assayed in greenhouse and field tests on wheat. One combination reduced FHB symptoms to a greater extent than the control. Collectively, these results demonstrate the feasibility of using an eventual commercial product composed of C. flavescent cells in an integrated control program against FHB. Also in studies that supported research Objective 1, patented Gram negative bacterial strains discovered by ARS scientists were assayed in laboratory studies against the pathogens that cause Pythium leak and Fusarium dry rot on stored potato tubers. A putative biocontrol product composed of dried cells of all three strains grown in one culture was also tested against these same pathogens in collaborative small pilot-scale storage studies with scientists at a university under a Non Assistance Cooperative Agreement (NACA). We demonstrated, in studies conducted in collaboration with a visiting Ph.D. student, that hot water extraction of a variety of plant fruits, pods, or seeds can provide essential nutrients and minerals for an inexpensive biocontrol agent growth medium. This medium produced cells in high titer that were equivalently effective in reducing dry rot on potatoes to those produced in powered commercially available media. In collaboration with other ARS scientists tests were also initiated on the effect of newly developed starch complexes with potential for inhibiting selected plant pathogens and being an effective water holding formulation for antagonists, depending on how the starch complex was produced. Progress in research conducted in support of Objective 2 was also made. ARS scientists in Peoria, Illinois, identified new biocontrol agents that target the fungal pathogens that cause laurel wilt on avocado in Florida, and Fusarium dieback on avocado in California. The scientists isolated bacteria and yeast from the bark of commercial avocado trees, developed an in vitro bioassay for screening candidate strains for antagonism against two Fusarium spp. pathogens that infect avocado, and identified 5 bacterial strains that were effective against both pathogens. Future work will address how best to produce and deliver the beneficial strains to infected trees in the field. In other work conducted under Objective 2, ARS scientists in Peoria, Illinois, sequenced more than forty genomes of agriculturally important microorganisms. Among other benefits from this work, the taxonomy of several species of the bacterium Bacillus was clarified which will enable scientists to clearly assign species traits and impacts of species isolates on agriculture without the ambiguity that was previously inherent in this process. We continued to progress in developing new methods and technologies to isolate and identify fastidious and/or uncultivable plant pathogens under Objective 3 research. Scientists made additional improvements to tissue culture medium for producing callus of basil, a host for the downy mildew pathogen Peronspora belbahrii. Studies were initiated to infect basil callus with the downy mildew fungus, with the goal of being able to periodically transfer infected callus to fresh medium to maintain the pathogen in gnotobiotic (known microbial components only) culture which would greatly facilitate conducting genomic and metabolomics studies on this otherwise non-culturable pathogen. Studies are also nearly completed on testing a wide variety of fungicides against the pathogen on basil and determining how combining the best fungicides identified with biocontrol agents would influence overall control of the disease.
1. Plant extract-based microbiological media development for economical production of biocontrol agents. Costs of commercially prepared nutrient media represent a significant hurdle to conducting plant pathology and biological control research. ARS scientists and a visiting exchange scientist prepared 13 plant-based liquid media from hot water extracts of plant fruits, pods, or seeds. Cells of a Fusarium dry rot biocontrol agent discovered by ARS scientists in Peoria, Illinois, grew to densities equivalent to those reached in commercially available powdered media and were as effective as cells produced on commercial liquid media in reducing dry rot on stored potato tubers. Studies using the best plant extract media and high efficiency equipment for measuring cell growth showed that growth rates and antagonist cell densities depended on medium composition. The discovery of less costly alternatives to commercially produced liquid growth media provides opportunity for scientists here and abroad to conduct additional research to discover environmentally friendly alternatives for plant disease control.
2. Genomes of multiple microbial strains of agricultural importance sequenced. Being able to assign an unambiguous taxonomic classification to microbial isolates is crucial for allowing scientists to accurately collect knowledge on a microbial species and predict its behavior in the environment. ARS scientists in Peoria, Illinois, sequenced more than forty genomes of agriculturally important microbial strains. This research identified gene clusters coding for novel bioactive metabolites that are implicated in strain behavior including biological control activity. Analysis of the genomes of a range of Bacillus isolates also resulted in the Peoria, Illinois, scientists enhancing and improving Bacillus taxonomy and species identification capability of this important group of agricultural and biocontrol species. These studies also show that these widely commercialized strains have been described in the literature under at least five different species names. With these results, a new accurate framework has been provided for the identification and taxonomic classification of one of the most important groups of bacterial biocontrol products in the world.
5. Significant Activities that Support Special Target Populations:
Dunlap, C.A. 2015. Phylogenomic analysis shows that ‘Bacillus vanillea’ is a later heterotypic synonym of Bacillus siamensis. International Journal of Systematic and Evolutionary Microbiology. 65(10):3507-3510.
Kim, S.J., Dunlap, C.A., Kwon, S.W., Rooney, A.P. 2015. Bacillus glycinifermentans sp. nov., isolated from fermented soybean paste. International Journal of Systematic and Evolutionary Microbiology. 65(10):3586-3590.
Dunlap, C.A., Kwon, S.W., Rooney, A.P., Kim, S.J. 2015. Bacillus paralicheniformis sp. nov., isolated from fermented soybean paste. International Journal of Systematic and Evolutionary Microbiology. 65(10):3487-3492.
Khalil, S., Ali, T.A., Skory, C., Slininger, P.J., Schisler, D.A. 2016. Evaluation of economically feasible, natural plant extract-based microbiological media for producing biomass of the dry rot biocontrol strain Pseudomonas fluorescens P22Y05 in liquid culture. World Journal of Microbiology and Biotechnology. 32(2):25. doi: 10.1007/s11274-015-1984-1.
Schisler, D.A., Slininger, P.J., Olsen, N.L. 2016. Appraisal of selected osmoprotectants and carriers for formulating Gram-negative biocontrol agents active against Fusarium dry rot on potatoes in storage. Biological Control. 98:1-10. doi: 10.1016/j.biocontrol.2016.03.009.
Dunlap, C.A., Kim, S.J., Kwon, S.W., Rooney, A.P. 2016. Bacillus velezensis is not a later heterotypic synonym of Bacillus amyloliquefaciens; Bacillus methylotrophicus, Bacillus amyloliquefaciens subsp plantarum and ‘Bacillus oryzicola’ are later heterotypic synonyms of Bacillus velezensis based on phylogenomics. International Journal of Systematic and Evolutionary Microbiology. 66(3):1212-1217. doi: 10.1099/ijsem.0.000858.