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<P>The overall goal of the proposed project will be to provide fundamental and applied information that supports the useful integration of microbial inoculants into agricultural production, especially certified organic farming systems. Emphasis will be placed on the study of plant-associated microorganisms that can enhance plant health and food quality, e.g. through the suppression of plant pathogens and the diseases they cause. This will be accomplished by pursuing the following specific objectives. Objective 1: Identify new and useful plant-associated microorganisms. Studies will focus on microbial populations obtained from different plant parts with the marker-assisted selection methods developed previously in my laboratory. Once isolated, potentially useful strains will be tested to establish their effects on plant hosts and pathogens. Additional characterization of their ecological functions may involve some mix of genomic, metabolomic, and phenotypic testing. Objective 2: Define the diversity and biogeography of beneficial plant-associated microorganisms. In order to fully understand the natural range and preferred niches of microorganisms used as inoculants, studies of agricultural environments will be used to characterize the natural distribution, population dynamics, and spread of select microbial populations. These studies will also determine which environmental variables most strongly constrain the abundance and activities of microbial inoculants, especially those that enhance plant health. Objective 3: Develop effective formulation and application strategies for microbial inoculants. Studies will include assessments of how different material components, their preparation, and their organization affect cell viability and activity in vitro and in planta. Such efforts will focus on establishing new and effective microbial inoculants that provide value to agricultural producers, processors, and consumers. </P>
<P>NON-TECHNICAL SUMMARY: Humanity needs to further develop environmentally sound and sustainable technologies that maintain or enhance agricultural production efficiency in the face of a changing climate. Although chemical inputs have a significant role to play, the over-dependence of conventional farming systems on synthetic fertilizers and pesticides presents economic and environmental problems, both locally (e.g. contamination of drinking water) and globally (e.g. through increased emissions of greenhouse gases) (Robertson 2000). Despite such problems, it is well recognized that high productivity systems will be needed to meet the material, energy, and food demands of the coming century (Ragauskas 2006, Godfray 2010). One approach to reducing agricultural dependence on synthetic chemicals while maintaining high levels of plant productivity is to use microorganisms to provide similar services. Because of this, there has been a dramatic increase in the application of plant-associated microbial bioproducts in agriculture during the past ten years, especially in organic systems and throughout the developing world (Glare et al 2012). Through fundamental studies of the ecology of microorganisms in agricultural systems, new opportunities for integrating microbial products in to agriculture will be developed. Microbial inoculants, encompassing a wide range of activities, are playing an increasing role in plant production, and now constitute a billion-plus dollar industry that is expected to grow dramatically in the next five years (Piper Jaffray 2013). Of particular interest are biopesticides, whose use has steadily grown in recent years throughout North America (Bailey et al 2010, Glare et al 2012). In principle, most biopesticide, biofertilizer, and biostimulant products that contain microorganisms should impact plant productivity, health, or quality in some measurable way on some farms; however, there is a remarkable lack of publicly available data that substantiates the efficacy and return on investment for most of these inputs. This fact highlights a critical need to evaluate and disseminate science-based information that will promote value-based development and adoption of such inputs. The proposed research will contribute to the sustainability and productivity of U.S. agriculture by providing relevant information regarding the identity, activities, and relative value of various microbial inoculants, particularly those that enhance plant health. Furthermore, the research will provide useful new information, ingredients, formulations, and innovative delivery systems to the rapidly growing microbial inoculant industry. </P>
<P>APPROACH: A variety of procedures will be used to meet project objectives. These will include using the evolving tools of microbiology, plant biology, and plant pathology research, and they will involve collaborative efforts that will take the research across multiple levels of scale, from subcellular to ecosystem. Specifically, the lab will collaborate with biochemists and plant molecular biologists at the finest levels and extension specialists and industry partners on the most expansive levels. The following approaches are planned to meet the stated objectives, but may be amended in response to the development of new technologies. Objective 1: Identify new and useful species of plant-associated microorganisms. In order to further expand the inventory of agriculturally relevant microbial inoculants, I propose to screen different environments for new and useful species of microorganisms. Previously, we developed different marker-assisted approaches for identifying novel biocontrol microbes based on known markers for biocontrol activities (McSpadden Gardener et al 2001, Joshi and McSpadden Gardener 20006, Park et al 2013a). We will continue to use similar approaches, but alter the specific molecular targets and use newer genomics-enabled procedures to increase productivity. Specifically, we will apply metagenomic sequencing to plant production systems where differentials in plant health and quality can be identified. Additionally, we will use refined and targeted sampling procedures to recover novel isolates based on variation in phylogenetic markers. By applying both nonparametric and multivariate statistical analyses of molecular profiles of plant-associated microorganisms, we will identify new and different populations associated with plant disease suppression. Once recovered, multiple isolates can be evaluated for the expression of disease and pathogen-suppressing activities in controlled conditions as well as field bioassays. For the most active isolates, genomic and metabolomic analyses will be used to further define the molecular bases for plant disease suppression. Emphasis will be place on characterizing novel species, because basic studies are more likely to result in the discovery of previously uncharacterized metabolites and/or mechanisms of biological control, opening up new avenues for investigation and, potentially, commercial development. Objective 2: Define the diversity and biogeography of several plant-associated microorganisms. In order to more effectively implement biocontrol with microbial inoculations, the natural range and niches of beneficial microorganisms must be better characterized. Thus, we will undertake studies of the diversity and distribution of select beneficial microorganisms, such as those currently under commercial application and/or development as microbial inoculants. Because the statistical associations between individual populations and specific plant habitats and micro-habitats are expected to vary with environmental conditions (e.g., Rotenberg et al 2007), studies will involve diverse measurements related to both geography and local conditions (e.g., cultivar, temperature, moisture, available nutrients, etc). Combining both biological and environmental measurements will provide new insights into the factors restricting the range and activities of plant-associated microorganisms that promote plant health. Objective 3: Develop effective formulation and application strategies for microbial inoculants. In order to further the expansion of the microbial inoculant industry, studies related to the composition of commercially-relevant formulations will be undertaken. Specifically, strains obtained from previous studies in our laboratory and our commercial partners will be studied for their compatibility with various formulation strategies, particularly those that are suited to certified organic production. Much of the formulation work conducted in the biopesticide industry has focused on relatively few organically-acceptable formulants or ingredients that are not allowed under the National Organic Program standards (Burgess 1998, Hynes and Boyetchko 2006). Therefore, we proposed to conduct research to develop new microbial formulation ingredients and strategies that are suitable for certified organic as well as chemical-intensive agriculture. Such work will involve commercial partners and lead to new and innovative intellectual property. The proposed work will be conducted in laboratory, greenhouse, and growth chamber space provided to Dr. McSpadden Gardner in and adjacent to Selby Hall. In addition, the proposed project will rely heavily on the equipment resources present in the Molecular Cellular Imaging Center (MCIC) and the Soil Testing and Research Laboratory (STAR lab) as well as central service labs on the Columbus campus, such as The Metabolomics Laboratory (TML). Field research will be conducted annually at the Ohio Agricultural Research and Development Center (OARDC). Specifically, we will conduct trials at the Wooster and Outlying Branch Research Stations on a mixture of organically- and conventionally- managed fields. Technical and equipment support for planting, inoculating, maintaining, and harvesting plots at those locations will be required for the successful completion of the proposed research. </P>