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United States Department of Agriculture

Agricultural Research Service


Location: Crop Bioprotection Research

2010 Annual Report

1a. Objectives (from AD-416)
Objective 1: Discover and develop new biocontrol agents that are active against an emerging disease, such as pink rot in stored potato tubers or sudden death syndrome of soybeans, and that possess superior potential for commercial development. Objective 2: For newly identified biocontrol agents and/or currently researched agents active against Fusarium head blight or storage maladies of potato tubers, devise methodologies that optimize cell production and efficacy. Objective 3: Develop and characterize the impact of culture variants, cultivation conditions or formulation protocols that proactively enhance convective drying, storage, rehydration, and deployment success of antagonist biomass. The encompassing objective of this project is to elucidate the impact of fermentation and formulation on the fundamental nature of biomass efficacy and tolerance of stress during the processing and deployment of biocontrol agents in order to facilitate overcoming this crucial hurdle in biocontrol product development. Two subobjectives for main objectives 2 and 3 further clarify our research strategy and are presented in the “Approach and Research Procedures” section. Our research team is pleased to possess a unique blend of expertise and interactive research experience in plant pathology, chemical engineering, biochemistry and molecular biology to draw on in advancing this research plan.

1b. Approach (from AD-416)
Assay whole microbial populations or individual candidate antagonist strains selected from key infection sites of pink rot on stored potato tubers against the pathogen using whole tuber bioassays that mimic natural infection conditions. Industrial economics and market needs will be considered in crafting novel screens to select optimal biocontrol products and manufacturing processes. Use batch liquid cultivation techniques to develop biomass production media and protocols to maximize biomass quantity and quality, efficacy, survival of processing (dewatering and/or drying processes such as freeze-, air-, or spray-drying), formulation and storage, and host compatibility. Selected Gram negative and positive bacterial strains shown to be superior in reducing Fusarium head blight of wheat and potato maladies will be screened for excretion of active antifungal compounds. Utilize DNA microarray technology to discover genes involved in biocontrol agent cell response to environmental stresses, especially those encountered during drying. Employ knowledge of stress-response genes to design novel reporter-gene technologies, microbial cultivation protocols, and down-stream processing systems to foster and retain desired gene expression for surviving stress.

3. Progress Report
ARS scientists discovered and characterized novel, microbial biocontrol agents for use alone or in combination with other tools for improving plant health. Research also emphasized expanding our knowledge of the basic and applied aspects of producing and formulating efficacious biomass of biocontrol agents. Antagonists of pink rot, a disease of potatoes in the field and tubers in storage, were isolated from suppressive soils, ranked for both effectiveness in reducing disease and ability to grow well in commercially relevant liquid culture media, and tested successfully in preliminary small pilot-scale studies using the best isolates discovered. In separate potato storage experiments conducted with a commercial partner, we tested the dose response of co-culture that was produced using an industrial-grade medium at the 400-liter pilot scale. Cultures stored at -20 F were applied to potatoes challenged with disease pathogens, and the laboratory scale test results showed consistent biocontrol, even at low dosages economical for commercial production and even using stored freeze-dried cultures. In pilot studies, we also observed significant biocontrol of both pink rot and dry rot, but at somewhat higher dosages still in the commercially feasible range. A new tool for combating Fusarium head blight disease of wheat and barley was discovered by isolating fungicide tolerant variants of previously patented yeast biocontrol agent Cryptococcus flavescens OH 182.9. Variants showed promise not only in being able to be combined with fungicide but also in colonizing wheat head tissues to high levels in the presence of fungicide and in possessing enhanced efficacy over the progenitor strain. Methods for culturing two strains of closely related yeasts in the same fermentation vessel were scaled up to 80 liters and the product shown to reduce Fusarium head blight in tests conducted with collaborators in multiple wheat-producing states. The physicochemical surface properties of wheat heads were characterized using contact angle methods in order to better understand the environment in which wheat tissues are colonized by strain OH 182.9 and the Fusarium head blight pathogen. Exposing strain OH 182.9 to colder temperatures during liquid culture enhances its biocontrol efficacy and possibly its tolerance to stress. A stopped flow instrument equipped with a fluorescence detector was used successfully to monitor perturbations in the cell envelope of OH 182.9 following a stress tolerance event (e.g., osmotic shock, pH shock) and provided evidence of the potential of the technique for predicting if cells of biocontrol agents would adequately tolerate drying and formulation, steps that would be crucial to developing a commercial biocontrol product. Lastly, antagonists discovered in previous studies that showed promise in first year field tests against Fusarium head blight in barley were studied in second year field tests. Progress achieved in FY 2010 contributes to the ultimate development of biological control products that would improve plant disease control in general and specifically reduce post harvest disease in potatoes and FHB in wheat.

4. Accomplishments
1. Fungicide tolerant yeast antagonist possesses enhanced efficacy against Fusarium headblight (FHB) of wheat. It is likely that no single control measure from among pesticides, biological control, cultural control, disease forecasting, and the use of resistant varieties will reduce FHB to economically acceptable levels. The suitability of a yeast antagonist Cryptococcus flavescens OH 182.9 for use in an integrated management strategy against FHB would be improved if the strain were tolerant of fungicides such as prothioconazole (PTC) that are active against FHB. PTC-tolerant (PTCT) variants of FHB antagonist OH 182.9 were selected by a scientist in the Crop Bioprotection Research Unit at the National Center for Agricultural Utilization Research in Peoria, IL, and shown to be more effective in reducing symptoms of FHB in greenhouse, and to a lesser extent, field trials than the progenitor wild-type strain with PTCT variants reducing FHB disease severity by as much as 83% in greenhouse studies compared to 36% for the wild-type strain. Field studies confirmed the trend of PTCT variants reducing FHB symptoms to a greater extent than the wild-type OH 182.9 strain. Because fungicide use is restricted shortly after flowering, combinations of fungicides with selected PTCT variants are likely to reduce pathogen infection at both flowering, and importantly, after flowering due to antagonist colonization of the wheat head. This discovery, along with recent licensing of the underlying technology by a U.S. agricultural company, represents a key step in the development of a biocontrol product and strategy for combating this intractable disease that costs wheat and barley growers in the U.S. an average of approximately 1 billion dollars per year.

2. New tool for broad spectrum protection of potatoes in storage. Late blight is considered to be the most significant disease of potatoes worldwide, and together with pink rot and dry rot, can cause losses of well over 50% of the total harvest in storage. Chemical fungicides traditionally used to control post harvest diseases of table stock potatoes are now of little use because of genetic resistance developed by causative pathogens. Additionally, the most common sprout inhibitor on the market is facing stricter regulation due to public health and safety concerns. Four beneficial bacteria, originally found in potato field soils, have been patented by the Agricultural Research Service and are able to suppress these diseases, as well as inhibit sprouting. As a result of a three-year study involving both laboratory and small pilot simulations of potato storages, scientists in the Crop Bioprotection Research and Bioenergy Research groups at the National Center for Agricultural Utilization Research in Peoria, IL, found that it is possible to cost effectively co-culture biocontrol strains together in one fermentor. This process stimulates inter-strain activities to boost biocontrol efficacy and consistency beyond that achievable by the more costly method of growing strains in separate fermentations then mixing just prior to addition to potatoes. These findings impact the potato industry and ultimately agricultural consumers by adding to the technology base needed to successfully manufacture a new tool for broad spectrum crop protection.

3. Methodology developed to evaluate the influence of stress events on cell membranes of biological control agents. The ability of cells of biological control agents of plant pathogens to survive short-term stress events is an important factor in determining the suitability of the strain for commercial development because cells are subjected to sudden environmental fluctuations during production, formulation, and deployment. Scientists in the Crop Bioprotection Research Unit at the National Center for Agricultural Utilization Research in Peoria, IL, examined new analytical methods for their ability to monitor changes on a cellular level that result when cells experience fluctuations that tax their ability to survive the stress. A stopped-flow instrument equipped with a fluorescence detector was used to quantify perturbations in the cell envelope following a stress tolerance event (e.g., osmotic shock, pH shock) and permitted us to monitor changes in cell size after rapid environmental changes on a time scale of milliseconds to several minutes. These initial experiments provided proof of concept for the technique and encouraging preliminary results. The novel application of this instrumentation allows scientists to monitor an additional microbial property that can contribute to microbial stress tolerance, and ultimately, may benefit agricultural consumers by contributing to the successful commercialization of more biocontrol agents when this methodology is used to identify traits that give rise to high levels of stress tolerance.

4. Documentation of successful colonization of wheat heads by yeast biological control agent Cryptococcus flavescens OH 182.9 when mixed with a fungicide that is active against Fusarium head blight (FHB) disease of wheat. Combining FHB biocontrol agent C. flavescens OH 182.9 with a fungicide such as prothioconazole (PTC) could be especially useful in limiting FHB disease and pathogen formation of the mycotoxin deoxynivalenol (DON) in grain since new pathogen infection can take place after fungicides can no longer be applied but when populations of the biocontrol agent could still be high. Experiments were conducted by scientists in the Crop Bioprotection Research Unit at the National Center for Agricultural Utilization Research in Peoria, IL, to quantify colonization of wheat head tissues by a PTC tolerant (PTCT) variant of OH 182.9 when the biocontrol agent was applied alone or in combination with PTC either at or seven days after wheat flowering. Populations of the PTCT variant were not affected by the presence of PTC; and after rain events, made up 40-95% of the total microbial population recovered from specific wheat head tissues from 8 to 12 days after flowering. FHB disease reduction associated with the various treatments supported the observation that the population of a PTCT variant of OH 182.9 on infection court tissues was not inhibited by the presence of PTC and that treatments that contained both the variant and PTC provided the greatest arithmetic reduction in FHB symptoms and DON. Basic information derived from this research provides further evidence for the feasibility of using strain OH 182.9 as part of an integrated disease management program against FHB on wheat and developing an OH 182.9-based commercial product for the benefit of wheat growers and consumers.

Review Publications
Slininger, P.J., Dunlap, C.A., Schisler, D.A. 2010. Polysaccharide Production Benefits Dry Storage Survival of the Biocontrol Agent Pseudomonas fluorescens S11:P:12 Effective Against Several Maladies of Stored Potatoes. Biocontrol Science and Technology. Biocontrol Science and Technology. 20(3):227-244.

Dunlap, C.A., Schisler, D.A. 2010. Fluidized-bed drying and storage stability of Cryptococcus flavescens OH 182.9, a biocontrol agent of Fusarium head blight. Biocontrol Science and Technology. 20(5):465-474.

Slininger, P.J., Schisler, D.A., Shea Andersh, M.A., Sloan, J.M., Woodell, L.K., Olsen, N.L., Frazier, M. 2010. Multi-strain co-cultures surpass blends for broad spectrum biological control of maladies of potatoes in storage. Biocontrol Science and Technology. 20(8):763-786.

Last Modified: 2/23/2016
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