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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

2008 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
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.


4.Accomplishments
1. SIXTH AND SECOND CONSECUTIVE YEARS OF SUCCESSFUL LATE BLIGHT AND PINK ROT SUPPRESSION, RESPECTIVELY, WITH BENEFICIAL GRAM-NEGATIVE BACTERIA IN SMALL PILOT TESTS. Late blight is considered to be the most significant disease of potatoes worldwide and together with pink rot, have limited options for control in storage where together the diseases can cause losses of over 50% of the total harvest in storage. Several strains patented for biological control of Fusarium dry rot and sprouting were tested in collaboration with the University of Idaho and a cooperative research and development agreement partner in small pilot studies in Kimberly, ID. In this sixth consecutive season of testing, late blight reduction provided by the biological control treatments was again significant, ranging from 20 to 43% in the Fall ’07 trial and 37 to 78% in the Spring ’08 trial (compared to 20-62% in the third to fifth years, 35-91% in the second year of testing, and 20-90% the first season of pilot testing). Superior treatments also reduced the severity of new infections of pink rot by as much as 40% and incidence by 31%, levels that were similar to those obtained in first-year studies. Consistency of biocontrol is an important feature of these strains, especially when they are used in combination, as is their ability to simultaneously protect against four important potato storage problems--Fusarium dry rot, pink rot, late blight, and sprouting. This consistency and breadth of problem control is expected to enhance marketability and benefit to farmers. Patent license negotiations are underway. This research addresses NP 303, Component 4, Problem Statement 4c; and NP 306, Component 2, Problem Area 2b.

2. PRODUCTION OF A DRY, GRANULATED FORMULATION OF THE FUSARIUM HEAD BLIGHT ANTAGONIST CRYPTOCOCCUS FLAVESCENS. Cryptococcus flavescens OH 182.9 is effective in reducing Fusarium head blight in greenhouse and field studies. The unique physical properties of Cryptococcus flavescens, along with industry preferences, require it to be harvested as a high-viscosity cell slurry after fermentation. Converting this slurry to a dry granular product suitable for commercial distribution presents several obstacles. To overcome these obstacles, we evaluated spray agglomeration in a fluidized bed dryer as a potential process. This research evaluated the effect of different seed particles, drying temperature, and loading rates on cell survival. The research identified conditions that permitted high cell survival rates after drying. The results of this research can be applied to other organisms and lessens the technical risks for the companies looking to license this Cryptococcus flavescens technology. This research addresses NP 303, Component 4, Problem Area 4c; and NP 306, Component 1, Problem Area 1d.

3. ISOLATION, TESTING, AND QUANTIFICATION OF ANTIFUNGAL METABOLITES FROM BACILLUS AND PAENIBACILLUS. Bacillus and Paenibacillus strains are important biocontrol organisms and known to produce a variety of secondary metabolites with anti-fungal activity. This property has led to their use in suppressing a variety of important agricultural fungal diseases including Fusarium head blight of wheat. We have developed analytical methods to characterize and quantify these anti-fungal metabolites. We have isolated sufficient quantities of these metabolites from Bacillus subtilus strains for individual testing and for use as standards in assays for quantifying production levels under different conditions. Bioassays of anti-fungal activity and qualitative analysis have identified active metabolites in several Paenibacillus strains. This research provides basic technical tools needed to understand the role of secondary metabolites in the mode of action of these organisms. This research is conducted under NP 303, Component 4, Problem Area 4c; and NP 306, Component 1, Problem Area 1d.


5.Significant Activities that Support Special Target Populations
None.


6.Technology Transfer

Number of Active CRADAs1
Number of the New MTAs (providing only)4
Number of Non-Peer Reviewed Presentations and Proceedings1
Number of Newspaper Articles and Other Presentations for Non-Science Audiences1

Review 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.

Last Modified: 4/19/2014
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