2007 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 (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. 3.Progress Report 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. 4.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. 5.Significant Activities that Support Special Target Populations None. 6.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 and other presentations for non-science audiences 2