Progress 05/14/07 to 05/08/12
Outputs
Progress Report 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. 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. Important results that will enhance the quality and quantity of agricultural products were obtained during this 5 year project. Pink rot and late blight disease was suppressed using bacterial biocontrol agents on stored potato tubers in small pilot tests for 5 successive years. A unique, cost effective, concept of co-culturing several of these biocontrol strains in one fermentor was devised. The process stimulates inter-strain activities to boost biocontrol efficacy and consistency. Techniques for enhancing soil suppressiveness were developed and employed to discover new pink rot antagonists with a visiting Turkish scientist. In total, the consistency and breadth of problem control demonstrated in this work will enhance the marketability of consortia of biocontrol agents and benefit potato producers and storage operators. A yeast antagonist of Fusarium head blight (FHB) disease was shown to effectively colonize wheat heads. Fungicide tolerant variants of the yeast had enhanced efficacy and heavily colonized when combined with triazole fungicides. A co-cultured yeast biocontrol product reduced FHB and the mycotoxin deoxynivalenol in wheat in a 5 state, 2 year field project. ARS scientists in Crop Bioprotection Unit, National Center for Agriculture (NCAUR) in Peoria, IL, built a prototype liquid production medium for producing yeast antagonists and founded a method for evaluating the impact of stress on cell membranes of biocontrol agents. Antifungal secondary metabolites including lipopeptides that were produced by Bacillus and Paenibacillus strains were isolated, quantified, characterized, and bioassayed. In collaboration with a California- Berkeley researcher, methods were developed for identifying and quantifying surfactant-producing bacteria in different ecological niches. Collectively, this research provides new tools for use in promoting the integrated management of FHB and other diseases in traditional and organic farming systems. ARS scientists in the Crop Bioprotection Research Unit in Peoria, IL, discovered methods for producing durable and efficacious wettable granules of yeast antagonists via fluidized-bed and spray drying. The scientists at Peoria, IL, isolated dry-storage tolerant cultures of beneficial Gram negative bacteria and successfully produced a dried product that reduced a variety of potato storage maladies. Polysaccharides produced by one strain of a coculture protected both members from drying injury. These discoveries provide products and concepts for formulating effective biocontrol products. This new project�s objective of managing production and formulation to favorably influence biocontrol agent gene expression and interactions with the host and pathogen will continue to build on these discoveries. Accomplishments 01 Draft genome sequence constructed for bacterial biocontrol isolate. The complexity of interactions between biocontrol agents, host, plant, and environment contributes to the sometimes inconsistent efficacy of biocontrol agents in the field. ARS researchers in the Crop Bioprotectio Research Unit, National Center for Agricultural Utilization Research, Peoria, IL, sequenced and assembled a draft genome of a Bacillus isolate that reduces several diseases in wheat. These results demonstrate that these beneficial bacteria are able to biologically control plant pathoge The work provides a blueprint of the number and types of compounds thes beneficial bacteria can produce, such as antifungal compounds toxic to plant pathogens. This breakthrough improves our knowledge of the mode-of action of these beneficial bacteria, and improves their consistency and efficacy in reducing crop diseases in the field. 02 Mixes of co-cultured biocontrol agents and chemicals superior in reducin dry rot on stored potatoes. The variety of storage diseases that afflic potatoes complicates management decisions and can result in the use of multiple compounds to protect a stored potato crop. ARS researchers in t Crop Bioprotection Research Unit, National Center for Agricultural Utilization Research, Peoria, IL, developed co-cultures of biocontrol agents active against a variety of potato maladies, including dry rot. Combinations of the biocontrol treatment with a chemical not individuall active against dry rot resulted in a greater level of dry rot control th any other individual or combination treatment in laboratory tests on a small pilot scale. These results demonstrate that unique biocontrol/chemical combinations provide enhanced disease reduction whil reducing the number of types of treatments needed to manage potato stora diseases.
Impacts
(N/A)
Publications
- Schisler, D.A., Slininger, P.J., Boehm, M.J., Paul, P.A. 2011. Co-culture of yeast antagonists of Fusarium head blight and their effect on disease development in wheat. Plant Pathology Journal. 10:128-137. DOI: 10. 3923/ppj.2011.128.137.
- Burch, A.Y., Browne, P.J., Dunlap, C.A., Price, N.P., Lindow, S.E. 2011. Comparison of biosurfactant detection methods reveals hydrophobic surfactants and contact-regulated production. Environmental Microbiology. 13:2681-2691. DOI: 10.1111/j.1462-2920.2011.02534.x.
- Burch, A.Y., Shimada, B.K., Mullin, S.W., Dunlap, C.A., Bowman, M.J., Lindow, S.E. 2012. Pseudomonas syringae coordinates production of a motility-enabling surfactant with flagellar assembly. Journal of Bacteriology. 194(6):1287-1298.
- Dunlap, C.A., Schisler, D.A., Price, N.P., Vaughn, S.F. 2011. Cyclic lipopeptide profile of three Bacillus subtilus strains; antagonists of Fusarium head blight. Journal of Microbiology. 49:603-609. DOI: 10. 1007/s12275-011-1044-y.
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Progress 10/01/10 to 09/30/11
Outputs
Progress Report 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. 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. Newly discovered antagonists of Fusarium head blight on barley were assayed in multiple greenhouse tests and consistently reduced disease severity and were associated with grain weight increases compared to the control when assayed individually or in two strain mixtures. Under objective 2, we initiated studies to optimize the liquid culture production of pink rot antagonists, a necessary step to achieve economically feasible industrial production protocols, and conducted preliminary tests to determine if the strains also reduce dry rot, another common potato storage disease. Studies were initiated in the previous year with a novel cocultured product developed by ARS scientists in Peoria, IL, which consisted of two different yeast antagonists of Fusarium head blight. These studies were continued in five states and again demonstrated that a combination of fungicide and dual cultured yeast was the most effective treatment in reducing (37% on average) the mycotoxin deoxynivalenol produced by the pathogen in wheat kernels. Under objective 3, additional work with fungicide tolerant variants of our patented yeast biocontrol agent Cryptococcus flavescens OH 182.9 was completed. Phyllogenetic comparison of the variants and the original strain demonstrated all were cospecific and growth rate studies demonstrated shorter doubling times for the variants in liquid medium containing the fungicide. Field evaluation demonstrated that the variant yeast aggressively colonized wheat heads in the presence or absence of fungicide, indicating the yeast could successfully integrate with fungicide to combat Fusarium head blight. Collaborative studies with The Ohio State University initiated the process of sequencing the genome of C. flavescens OH 182.9. Novel sequences discovered were used to develop a first generation molecular- code-based assay for monitoring populations of OH 182.9 in the environment, especially on wheat heads where the Fusarium head blight pathogen causes primary infection. The accuracy of the assay for monitoring populations of OH 182.9 was perfected in greenhouse trials where molecular and traditional methods were compared. Wheat field trials to study antagonist colonization have been initialized. In collaborative studies with The University of California, Berkeley, a new assay was developed to screen microbes from different plant surface environments for their ability to produce surfactants, compounds that can have useful industrial and biopesticidal properties. Accomplishments 01 New dual cultured yeast biocontrol product reduces Fusarium head blight and one of the toxins it produces in wheat. The significant and consistent reduction of the devastating plant disease Fusarium head blig (FHB) and a deleterious product it produces (deoxynivalenol (DON)) in wheat and barley remains elusive though research indicates integrating several methods of control is the best approach to maximizing disease an DON control. USDA, ARS researchers in the Crop Bioprotection Research Un at the National Center for Agricultural Utilization Research (NCAUR), in Peoria, IL, discovered methods for culturing two and three antagonists o FHB simultaneously in liquid culture to produce a mixture of products th are more affordable to produce and process than products produced using standard methods. A two strain yeast coculture product consistently reduced Fusarium head blight severity in greenhouse trials. The same product was field tested with collaborators in 5 states in successive years and, when integrated with a fungicide treatment, was the most effective of multiple treatments, including fungicide alone, in reducing the deleterious product DON in wheat kernels. This discovery has potenti to aid agricultural producers and consumers alike by providing a new too for the integrated management of plant diseases like FHB which, when unchecked, increase food prices by reducing crop quality and yield. 02 Discovery and development of novel agents for biological control of new pink rot infections on stored potatoes. New infections that result when inoculum of the pink rot pathogen contaminates uninfected tubers during harvesting and storage operations has become an increasingly important a difficult to control problem with losses of over 50% of the total harves possible from pink rot and closely related pathogens. Selection techniqu were developed by USDA, ARS scientists in the Crop Bioprotection Researc Unit at the National Center for Agricultural Utilization Research (NCAUR Peoria, IL, and a visiting scientist from Turkey to find naturally occurring microorganisms with that possessed both efficacy in reducing disease and superior amenability to production using commercially feasib liquid culture growth media. Six strains of biocontrol agents were discovered that significantly reduced pink rot disease in potato storage assays by as much as 33% when only a modest amount of the biocontrol age was applied to tubers, and several of these agents were not previously described as species that demonstrate biological control attributes. Further development of these patent pending biocontrol strains will provide a new product for reducing potato storage disease which would ultimately economically benefit potato growers, storage operators and consumers. 03 Methodology developed for discovery and identification of a class of antifungal compounds on plant surfaces. Bacterial surfactants are a diverse group of compounds that have important biological and industrial properties. They play an important role in bioremediation, petroleum extraction, and they are useful as biopesticides. In this collaborative study with scientists from the University of California-Berkeley, USDA, ARS scientists in the Crop Bioprotection Research Unit at the National Center for Agricultural Utilization Research (NCAUR), Peoria, IL, screen bacteria from different ecological niches for surfactant production usin a new assay. The new methodology efficiently screened bacteria for novel surfactants and determined the distribution of surfactant-producing bacteria in different ecological niches. Mass spectroscopy was employed identify the specific surfactant compound associated with the unique functional and chemical attributes that identify the presence of surfactants on plant surfaces. This research benefits commercial interes seeking novel surfactants and benefits scientists by helping them understand the role these surfactants play in the bacterial colonization of different ecological niches. Ultimately, this work will contribute to understanding how these molecules are used by plant pathogens or plant- associated bacteria in colonizing agriculturally important plants.
Impacts
(N/A)
Publications
- Schisler, D.A., Janisiewicz, W.J., Boekhout, T., Kurtzman, C.P. 2011. Agriculturally important yeasts: Biological control of field and postharvest diseases using yeast antagonists, and yeasts as pathogens of plants. In: Kurtzman, C.P., Fell, J.W., Boekhout, T., editors. The Yeasts, a Taxonomic Study. Vol. 1, 5th Edition. London, England: Elsevier. p. 45- 52.
- Adiyaman, T., Schisler, D.A., Slininger, P.J., Sloan, J.M., Jackson, M.A., Rooney, A.P. 2011. Selection of biocontrol agents of pink rot based on efficacy and growth kinetics index rankings. Plant Disease. 95(1):24-30.
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Progress 10/01/09 to 09/30/10
Outputs
Progress Report 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. 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. 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. Accomplishments 01 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 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. PT 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 mor 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 confirme the trend of PTCT variants reducing FHB symptoms to a greater extent tha the wild-type OH 182.9 strain. Because fungicide use is restricted short after flowering, combinations of fungicides with selected PTCT variants are likely to reduce pathogen infection at both flowering, and important after flowering due to antagonist colonization of the wheat head. This discovery, along with recent licensing of the underlying technology by a 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. 02 New tool for broad spectrum protection of potatoes in storage. Late blig 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 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-yea 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. 03 Methodology developed to evaluate the influence of stress events on cell membranes of biological control agents. The ability of cells of biologic 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 environment fluctuations during production, formulation, and deployment. Scientists 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 fluorescen detector was used to quantify perturbations in the cell envelope followi a stress tolerance event (e.g., osmotic shock, pH shock) and permitted u 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. 04 Documentation of successful colonization of wheat heads by yeast biological control agent Cryptococcus flavescens OH 182.9 when mixed wit 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 plac 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 Cente for Agricultural Utilization Research in Peoria, IL, to quantify colonization of wheat head tissues by a PTC tolerant (PTCT) variant of O 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 PT 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 observati 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 tha 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 O 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 benefi of wheat growers and consumers.
Impacts
(N/A)
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.
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Progress 10/01/08 to 09/30/09
Outputs
Progress Report 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. 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. Significant Activities that Support Special Target Populations Researchers� efforts contributed to solving the need for producing stable, effective biocontrol products that reduce plant diseases and the environmental impact of chemical pesticide use. More than 100 putative antagonists of pink rot on stored potato tubers were isolated and evaluation of their commercial development potential was initiated. Culture nutrient compositions that promote dry storage stability of biocontrol strain mixtures were investigated. The sensitivity of the drying stability of five Pseudomonas fluorescens strains to culture nutrition was documented using a microplate-based high throughput screening method designed in our laboratory and will aid the production of dry storage stable mixtures of biocontrol agents. Research on biologically controlling Fusarium dry rot and pink rot on potato tubers was conducted under a Cooperative Research and Development Agreement. In laboratory tests, disease reduction for both diseases ranged from 20% to 77% using co-cultured microbial treatments. Small-pilot-scale tests in collaboration with the University of Idaho, demonstrated as much as 30% reduction of both diseases. In work on biocontrol of Fusarium head blight (FHB) of wheat using our patented strains, we began characterizing colonization of wheat heads by antagonist strain Cryptococcus flavescens OH 182.9 in greenhouse and field work where the antagonist and a fungicide were applied at various stages of wheat development. The chemical nature of the wheat head surfaces during maturation also was characterized. We determined methods of enhancing the stress tolerance of OH 182.9 and studied the physiochemical nature of the stress tolerant strains produced. Spray agglomeration of OH 182.9 in a fluidize-bed dryer was studied to promote the development of commercially feasible prototype products and glycerol was found to potentially contribute to the drying survival of OH 182.9. In research funded in part by a U.S. Wheat and Barley Scab Initiative grant, fungicide tolerant variants of OH 182.9 and mixed cultures of two yeast antagonists were tested in multiple states. Efforts continued to identify stress tolerance genes in OH 182.9. A cross species microarray was employed to identify about 27 putative genes that were differentially expressed under cold-shock conditions that promote stress tolerance. After sequence verification, two genes, G2 and G3, were identified as tentative target genes and a partial coding region of G3 was successfully cloned. Lastly, a discovery program for antagonists active in reducing FHB on barley was expanded by isolating organisms from barley heads and assaying the strains obtained in greenhouse and in field trials. Progress achieved in FY 2009 impacts private and public researchers and contributes to the development of biological control products that would improve plant disease control generally and reduce post harvest disease in potatoes and FHB in wheat specifically. Technology Transfer Number of New CRADAS: 1 Number of Active CRADAS: 1 Number of New/Active MTAs(providing only): 7 Number of Invention Disclosures submitted: 1
Impacts
(N/A)
Publications
- Schisler, D.A., Slininger, P.J., Miller, J.S., Woodell, L.K., Clayson, S., Olsen, N. 2009. Bacterial Antagonists, Zoospore Inoculum Retention Time, and Potato Cultivar Influence Pink Rot Disease Development. American Journal of Potato Research. 86:102:111.
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Progress 10/01/07 to 09/30/08
Outputs
Progress Report 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. 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. Significant Activities that Support Special Target Populations Projects designed to discover new strains of biological control agents were continued in parallel with studies designed to improve the efficacy of previously isolated strains in hand via proactive management of fermentation, drying, and formulation of microbial biomass. Studies were also continued with the longer range goal of determining gene expression patterns that can be promoted via management of biomass cultivation conditions to produce drying-tolerant phenotypes. Discovery programs for new biological control agents with potential to reduce Fusarium head blight (FHB) on wheat continued. Soils from potato-growing regions continue to be screened for suppressiveness to the pink rot pathogen, and antagonists are being isolated and characterized from superior suppressive soils. Studies are continuing to devise technologies needed to produce cells that are tolerant to the stresses of large-scale cultivation, separation, processing (drying or dewatering biomass), and storage with emphasis on antagonist strains patented in our previous work and effective against potato storage maladies or FHB of wheat. Specifically, work continues to devise liquid cultivation conditions for pure or mixed cultures of selected agents optimized for key parameters to biocontrol product success including yield, cell production rate, the production bioactive metabolites, efficacy, and survival of biomass. Work was initiated on use of inexpensive, commercially viable sources of carbon and nitrogen for the production of FHB antagonists. Biomass produced on the best alternative medium was successively tested in both the greenhouse and field. Fungicide tolerant variants of FHB antagonist Cryptococcus flavescens were isolated, characterized and tested for biocontrol efficacy. Field tests were conducted with C. flavescens in collaborative studies with University of Missouri, Michigan State University and The Ohio State University. Work continued to determine the basis of microbial stress tolerance by evaluating gene-expression networks and the physiology of stressed cells. Specifically, significant progress was made in developing a robust standard reference for quantitative gene expression analysis using qualitative real time polymerase chain reaction. Regarding studies on the physiology of stressed cells, storage-tolerant cultures of P. fluorescens were developed through the application of repetitive drying, storage, and growth cycles. The drying-tolerant strains will be useful in the comparative gene expression analysis of parent and adapted cultures to identify gene expressions that are involved in tolerance mechanisms. In other ongoing investigations, a polysaccharide isolated and identified from a biocontrol strain of P. fluorescens was shown to improve drying stress tolerance of the strain and other unrelated bacterial cells. NP 303, Component 4; and NP 306, Component 1. Technology Transfer Number of Active CRADAS: 1 Number of New/Active MTAs(providing only): 4
Impacts
(N/A)
Publications
- Zhang, S., Schisler, D.A., Boehm, M.J., Slininger, P.J. 2007. Utilization of chemical inducers of resistance and Cryptococcus flavescens OH 182.9 to reduce fusarium head blight under greenhouse conditions. Biological Control. 42:308-315.
- Kolombet, L., Zhigletsova, S.K., Kosareva, N.I., Bystrova, E.V., Derbyshev, V.V., Krasnova, S.P., Schisler, D.A. 2008. Development of an extended shelf-life, liquid formulation of the biofungicide Trichoderma asperellum. World Journal of Microbiology and Biotechnology. 24:123-131.
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Progress 10/01/06 to 09/30/07
Outputs
Progress Report 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. 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 (FHB) 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. Significant Activities that Support Special Target Populations This project officially began on May 14, 2007, and therefore projects that bridge the previous project (3620-22410-007-00D) and the current project have been carried out as well as the initiation of projects that will lead towards accomplishing milestones due for completion in future years. Studies were continued to discover new biocontrol agents with potential to control Fusarium head blight (FHB) on wheat. Of new strains tested in replicated greenhouse studies, 40% reductions in disease severity represented the best result obtained and this strain likely will be included in future field tests. Discovery programs have also been initiated in a quest for microbial antagonists with activity against FHB on barley. Several barley varieties have been compared in greenhouse and field evaluations for cultivars suitable for experimental purposes. Soils from potato-growing regions continue to be screened for suppressiveness to the pink rot pathogen, and antagonists are being isolated and characterized from superior suppressive soils. Studies are continuing to devise cultivation and formulation technologies needed to produce cells that are tolerant to the stresses of large-scale cultivation, separation, processing (drying or dewatering biomass), and storage with emphasis on antagonist strains patented in our previous work and effective against potato storage maladies or FHB of wheat. Specifically, work continues to devise liquid cultivation conditions for pure or mixed cultures of selected agents optimized for key parameters to biocontrol product success including yield, cell production rate, the production bioactive metabolites, efficacy, and survival of biomass. We also continue work to determine the basis of microbial stress tolerance by evaluating gene-expression networks and the physiology of stressed cells in order to facilitate and focus our work on developing formulations of our biocontrol agents that enhance convective drying, storage, rehydration, and deployment success. Accomplishments Efficacy of dry rot antagonists against pink rot established in laboratory assays. Pink rot of potato, incited primarily by Phytophthora erythroseptica, is a disease of increasing importance in many potato- growing regions of the world and control options for tubers in storage are limited. In previous research conducted at the Agricultural Research Services (ARS) National Center for Agricultural Utilization Research (NCAUR), Peoria, IL, we isolated 18 microbial strains from soils suppressive to dry rot and demonstrated the efficacy of strains of Pseudomonas, Enterobacter, and Pantoea in reducing one or more of dry rot, late blight, and sprouting on tubers in storage. We found several strains that reduced pink rot in laboratory studies by as much as 30%. Evidence that encysted zoospores incited more disease than motile zoospores and were more resistant to biocontrol was also obtained. These results point out the expanded utility of a potential biocontrol product for commercial storage operators to use in reducing maladies of potato tubers in storage and presents new information for researchers working on developing controls for pink rot. This research is concucted under National Program 303 Plant Diseases; Component 4--Biological and Cultural Strategies for Sustainable Disease Management; Problem Statement 4a: Biological and Cultural Control Technologies; and National Program 306 Quality and Utilization of Agricultural Products; Component 1--Quality, Characterization, Preservation, and Enhancement; Problem Area 1d: Preservation and/or Enhancement of Quality and Marketability. Potato-protective bacterium produces a polysaccharide that improves its dry storage tolerance. Numerous beneficial Pseudomonas bacteria strains have shown promise in naturally suppressing plant diseases. However, their ability to survive dry storage has been a bottleneck to their commercial use as alternatives to chemical pesticides. A polysaccharide was isolated and identified from a culture of P. fluorescens known to protect potatoes in storage from fungal diseases and sprouting. Subsequent drying studies showed that the accumulation of polysaccharide in cultures could benefit the efficacy of formulations of this biocontrol agent--and other bacterial populations combined with it--by significantly improving cell survival during desiccation. These discoveries will impact commercial culture production and formulation strategies to obtain a viable dry, shelf-stable biocontrol product to control maladies of potatoes in storage. This research is conducted under National Program 303 Plant Diseases; Component 4--Biological and Cultural Strategies for Sustainable Disease Management; Problem Statement 4c: Application of Sustainable Disease Management Tools; and National Program 306 Quality and Utilization of Agricultural Products; Component 1--Quality, Characterization, Preservation, and Enhancement; Problem Area 1d: Preservation and/or Enhancement of Quality and Marketability. Technology Transfer Number of New CRADAS and MTAS: 2 Number of Active CRADAS and MTAS: 2 Number of Invention Disclosures submitted: 1 Number of Non-Peer Reviewed Presentations and Proceedings: 6 Number of Newspaper Articles,Presentations for NonScience Audiences: 2
Impacts
(N/A)
Publications
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