DISCOVERY AND APPLICATION OF MICROBIAL PRODUCTION AND FORMULATION BIOTECHNOLOGIES TO ENHANCE BIOCONTROL OF FUNGAL PLANT DISEASES
Location: Crop Bioprotection Research
Title: Fermentation: Prelude to success in the formulation of bioactive agents
Submitted to: Symposium Proceedings
Publication Type: Proceedings
Publication Acceptance Date: December 5, 2007
Publication Date: December 6, 2007
Citation: Schisler, D.A., Dunlap, C.A., Jackson, M.A., Slininger, P.J., Zhang, S., Boehm, M.J. 2007. Fermentation: Prelude to success in the formulation of bioactive agents [abstract]. First International Domestication of Microorganism (DOM) Symposium on Microbial Formulation, Uppsala, Sweden. p. 6.
In spite of the enormous potential of biological control for solving intractable pest control problems, relatively few biocontrol products have been commercialized. Our research addresses a serious impediment to bringing effective biocontrol products to the marketplace; the lack of adequate methodologies for selecting efficacious strains and producing biomass with enhanced amenability to the stresses of commercial-scale production and formulation. To select microbial strains more suited for commercial development, we designed screening techniques that were sensitive to both liquid culture process economics and biological function of the end product. This allowed us to focus limited resources only on agents that were superior performers under prevailing conditions of commercial production. Once putative biocontrol agents have been selected, fermentation protocols can have significant ramifications regarding the quantity, quality, and type of biomass produced. Our studies with Colletotrichum truncatum NRRL 13737, a filamentous fungal pathogen of the noxious weed hemp sesbania (Sesbania exaltata), showed that lowering the carbon-to-nitrogen (C:N) ratio of the liquid, conidiation medium influenced spore yield, morphology, and efficacy in inciting disease in S. exaltata. Because maintaining the conidial shelf-life of C. truncatum proved problematic, fermentation protocols were modified toward high aeration and carbon loading which resulted in the production of microsclerotia-like mycelial aggregates (MS). Unlike conidia, MS had exceptional shelf-life in an air-dried, diatomaceous earth formulation. Microsclerotia killed hemp sesbania seedlings, when incorporated into soil, by germinating sporogenically to produce conidia that in turn germinated to infect root systems.
Management of the fermentation environment also impacts the performance and characteristics of biomass of yeast biocontrol agents. Yeast Cryptococcus flavescens OH 182.9 reduces Fusarium head blight (FHB) severity on wheat by as much as 60 percent and deoxynivalenol in grain by nearly 30 percent in field experiments. The carbon loading and C:N ratio of fermentation media were optimized to enhance the efficacy and drying tolerance of OH 182.9 biomass. We further discovered that prolonged cold adaptation (28 hours) of colonized culture broth at 15 C enhanced the storage stability and efficacy of an OH 182.9 product. Cold adaptation of biomass improved the liquid hyperosmotic shock tolerance of cells and altered the temperature dependence of their osmotic shock tolerance. Fluorescence anisotropy and force curves from atomic force microscopy suggested that cold adaptation significantly altered the cell membranes of OH 182.9. In other studies, the feasibility of obtaining the efficacy advantages of microbial mixture formulations by conducting bi- and tripartite, mixed-strain yeast fermentations was assessed. Growth curves of each component strain of mixed cultures were determined by plating on a melezitose-based medium. A co-culture of OH 182.9 and C. aureus OH 71.4 that reached equivalent cell densities significantly reduced (32 percent) FHB disease severity in multiple greenhouse experiments. The possibility of obtaining superior efficacy and cost benefits with mixed-strain fermentation products justifies further evaluation of this approach.