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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
2009 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
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.


4.Accomplishments
1. DRY-STORAGE TOLERANT CULTURES OF BENEFICIAL BACTERIA PROTECT POTATOES FROM POSTHARVEST STORAGE MALADIES. The continued development of drying and storage-tolerant cultures by the Crop BioProtection Research Unit scientists is a critical advancement toward the commercial use of these gram-negative bacteria to control maladies of potatoes in storage--late blight, pink rot, Fusarium dry rot, and sprouting. Cultures of four strains of Pseudomonas fluorescens enriched in drying and storage tolerance were developed through the application of repetitive drying, storage, and growth cycles. A library of single-cell derived colonies varying in dry storage tolerance was prepared for each strain. 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. Dry storage tolerant colonies were also evaluated and confirmed to retain biocontrol efficacy, and will in future work be studied for commercial promise compared to wild-type strains. Basic information derived from the research will potentially speed the commercial development of a large number of beneficial Pseudomonas strains that have shown promise as new biological pest control products to suppress plant diseases.

2. ISOLATION AND CHARACTERIZATION OF THE ANTIFUNGAL ACTIVITY OF METABOLITES OF BACILLUS SUBTILUS AGAINST THE CAUSATIVE AGENT OF FUSARIUM HEAD BLIGHT (FHB). We have previously identified different classes of lipopeptides from Bacillus subtilus that demonstrated antifungal activity. Lipopeptides are a class of molecules that all possess alkyl chains of variable length, an important role in the antifungal activity of the molecules since alkyl chains interact directly with the outer membrane of fungal cells. We have now quantified and isolated individual alkyl chain isomers from two lipopeptides classes: iturins and fengycins. The minimum inhibitory concentration of these compounds against Gibberella zeae, the causative agent of FHB of wheat, was determined. Our results demonstrate a strong correlation between increasing alkyl chain length and higher antifungal activity. This research improves our understanding of the mode of action of these secondary metabolites and should be useful in helping select lipopeptide compounds with enhanced bioactivity.

3. DEVELOPMENT OF AN ECONOMICALLY FEASIBLE, INDUSTRIAL-GRADE LIQUID MEDIUM PROTOTYPE THAT FOSTERS HIGH YIELDS OF EFFECTIVE CELLS OF FUSARIUM HEAD BLIGHT (FHB) ANTAGONIST CRYPTOCOCCUS FLAVESCENS OH 182.9. The production of affordable biocontrol products for the agricultural marketplace requires the development of liquid culture production media that are economical to produce and support the production of high quantities of efficacious cells of biocontrol agents. We developed a medium that featured an industrial source of casein digest as well as carbon loading and C:N ratios that mimicked those used in our semi-defined laboratory medium. The new medium produced OH 182.9 inoculum that was indistinguishable in quantity and effectiveness from that produced in the semi-defined laboratory medium. The improved economics that result from producing inoculum of FHB antagonist OH 182.9 in our newly devised, commercially feasible liquid medium enhances the likelihood of bringing an OH 182.9-based, FHB biocontrol product to the marketplace.

4. POLYSACCHARIDE PRODUCED IN A CULTURE OF A POTATO-PROTECTIVE BACTERIUM IMPROVES ITS DESICCATION TOLERANCE AND THAT OF OTHER BACTERIA STRAINS COMBINED WITH IT. In previous research, a polysaccharide was isolated and identified from a culture of Pseudomonas fluorescens known to protect potatoes in storage from fungal diseases and sprouting. Subsequent drying studies showed that the accumulation of polysaccharide in cultures could potentially benefit the efficacy of formulations of this biological control agent as well as others combined with it by significantly improving cell survival during desiccation. The impact of the polysaccharide on the viability and growth kinetics of the producing strain and other non-producing strains was additionally evaluated in fermentor cultures. Such data will be incorporated into kinetic models to design the fermentation/formulation of co-cultures of our four most efficacious strains for biocontrol of potato storage maladies. Together with our previous work which established that strain mixtures achieved more consistent biocontrol of potato storage maladies than single strain formulations, these results increase the likelihood of developing a successful biological control product for the agricultural marketplace.


6.Technology Transfer

Number of New CRADAS1
Number of Active CRADAs1
Number of the New/Active MTAs (providing only)7
Number of Invention Disclosures Submitted1

Review 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. DOI: 10.1007/s12230-008-9066-7

Last Modified: 12/18/2014
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