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United States Department of Agriculture

Agricultural Research Service

Research Project: DISCOVERY AND APPLICATION OF MICROBIAL PRODUCTION AND FORMULATION BIOTECHNOLOGIES TO ENHANCE BIOCONTROL OF FUNGAL PLANT DISEASES

Location: Crop Bioprotection Research

2011 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
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.


4.Accomplishments
1. 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 blight (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 and DON control. USDA, ARS researchers in the Crop Bioprotection Research Unit at the National Center for Agricultural Utilization Research (NCAUR), in Peoria, IL, discovered methods for culturing two and three antagonists of FHB simultaneously in liquid culture to produce a mixture of products that 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 potential to aid agricultural producers and consumers alike by providing a new tool for the integrated management of plant diseases like FHB which, when unchecked, increase food prices by reducing crop quality and yield.

2. 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 and difficult to control problem with losses of over 50% of the total harvest possible from pink rot and closely related pathogens. Selection techniques were developed by USDA, ARS scientists in the Crop Bioprotection Research 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 feasible 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 agent 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.

3. 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, screened bacteria from different ecological niches for surfactant production using 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 to 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 interests 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.


Review 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.

Last Modified: 7/22/2014
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