Assessment of a novel delivery system for microbial inoculants and the novel microbe Mitsuaria spp. H24L5A THESIS Presented in Partial Fulfillment of the Requirements for the Degree Master of Science in the Graduate School of The Ohio State University By Donald Patriq Bruce Gillis Graduate Program in Plant Pathology The Ohio State University 2016 Master's Examination Committee: Dr. Christopher G. Taylor, Advisor Dr. Joshua Blakeslee Dr. Michelle Jones Copyrighted by Donald Patriq Bruce Gillis 2016 Abstract The demand for alternatives to chemical based products has increased along with the popularity of natural products. Microbial based products including biopesticides and biofertilizers are being used for the promotion of plant growth and control of pests. Delivery of these products has improved in recent years; many formulations and delivery methods have been examined and tested to determine the optimal conditions for growth and delivery of microbial products. In this study, a quality control assessment was carried out on a novel fermentation system created by an Ohio based company. This product utilizes actively growing bacterial strains to directly inoculate seeds or soil with beneficial microbes. The microbes used in this system are Pseudomonas brassicacearum strains. Additionally, field and greenhouse studies were conducted that examined the efficacy of microbes in this novel fermentation system as well as a novel microbe isolated from Ohio soils, Mitsuaria spp. H24L5A compared to industry standards and other know beneficial microbes. Small increases were seen in bacterial numbers during increasing days of storage while detection of inoculants was possible early after initial inoculation of the system. Mitsuaria spp. H24L5A showed a small increase in stand and stand and yield ii on corn and soybean plots when used as a seed treatment. A trend of small increases in plant growth was also noted with soil inoculation of the novel microbe. This data indicates that there is potential for the novel fermentation system to be used to increase bacterial biomass soon after introduction of the target microbe for future delivery as a seed or soil treatment. The plant growth promoting activity of Mitsuaria spp. H24L5A is also suggested by the increases seen in both field and greenhouse experiments. iii Dedication To my ever supportive family: Mom and dad, I thank you for your endless support over the years since I realize my passion for science. David and Maya, thank you for your patience; even when you were ready for me to come home you always supported me from 600 miles away. And to my two angels in heaven, Eva and Elmer, this is for you. iv Acknowledgments I would like to thank: My advisory committee members Dr. Christopher Taylor, Dr. Joshua Blakeslee and Dr. Michelle Jones. Your support over the last year has made this moment in time possible. All members of the McSpadden Gardener and Taylor labs. All of you have been my family away from home and an amazing support system both in and out of the lab. I would not have made it through the program without friends like you. The Department of Plant Pathology and everyone in Selby and Kottman; thank you for letting me be me and accepting me and allowing me to be a part of your family. My best friends: Ashley, Loren, Vashti, Kandyce, Adrienne, Sue, Martavius, Bianca, Corey, Fred, Brooke, Taylor, Terrance and Samantha. You all have been my main support system these last few years and I thank God that I have you all in my corner. You all definitely kept me grounded and focused on the end goal and I thank you. My line brothers for being supportive and patient with the distance as I pursued my passion. Finally, my family, for their support and understanding during this time v Vita 2009................................................................B.S. Biology, Morehouse College 2013 to present ..............................................Graduate Student, Department of Plant Pathology, The Ohio State University Fields of Study Major Field: Plant Pathology vi Table of Contents Abstract ............................................................................................................................... ii Dedication .......................................................................................................................... iv Acknowledgements ............................................................................................................. v Vita ..................................................................................................................................... vi Table of Contents .............................................................................................................. vii List of Tables ..................................................................................................................... ix List of Figures .................................................................................................................... xi Chapter 1. Introduction ....................................................................................................... 1 Chapter 2: Formulation, stability and purity of a bacterial inoculant in a novel delivery sytem ................................................................................................................................. 34 Abstract ......................................................................................................................... 34 Introduction ................................................................................................................... 36 Materials and Methods .................................................................................................. 40 Results ........................................................................................................................... 45 vii Discussion ......................................................................................................................... 49 Chapter 3: Assessment of Mitsuaria spp. H24L5A plant growth promotion, pathogen suppression activity and mode of action ........................................................................... 74 Abstract ......................................................................................................................... 74 Introduction ................................................................................................................... 76 Materials and Methods .................................................................................................. 80 Results ........................................................................................................................... 89 Discussion ..................................................................................................................... 92 List of References ........................................................................................................... 123 Appendix: Bioinformatic analysis of Mitsuaria spp. H24L5A for select biocontrol enzymes........................................................................................................................... 137 viii List of Tables Table 1.1. List of commercially available microbial based products. .............................. 32 Table 1.1 Continued. List of commercially available microbial based products .............. 33 Table 3.1. List of treatments used in field assays on corn and soybean seeds and greenhouse assays on ...................................................................................................... 106 Table 3.2. Average stand and yield across 10 counties for corn and 6 counties for soybean ......................................................................................................................................... 107 Table 3.3. Average stand and yield per treatment for soybeans across six counties in Ohio ......................................................................................................................................... 108 Table 3.4. Stand and yield data from a soybean field site in Mercer County, OH (Site Code: C1). No significant differences were seen across treatments ............................... 109 Table 3.5. Stand and yield data from a soybean field site in Clark County, OH (Site Code: C2). No significant differences were seen across treatments ......................................... 110 Table 3.6. Stand and yield data from soybean field site in Henry County, OH (Site code: N1). No significant differences were seen across treatments ......................................... 111 Table 3.7. Stand and yield data from soybean field site in Erie County, OH (Site code: N2). No significant differences were seen across treatments ......................................... 112 Table 3.8. Stand and yield data from soybean field site in Preble County, OH (Site code: S1). No significant differences were seen across treatments .......................................... 113 ix Table 3.9. Stand and yield data from soybean field site in Clinton County, OH (Site code: S2). No significant differences were seen across treatments .......................................... 114 Table 3.10. Summary of disease suppression assays from Summer 2015 greenhouse trials ......................................................................................................................................... 115 x List of Figures Figure 2.1. Bioreactord developed