Establishing the use of Pseudomonas spp. as biocontrol agents of fungal and nematode pathogens Dissertation Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of the Ohio State University By Rebecca B. Kimmelfield, B.S. Graduate Program in Translational Plant Sciences The Ohio State University 2020 Dissertation Committee: Associate Professor Christopher G. Taylor, Advisor Professor Michelle Jones, Member Professor Sally Miller, Member Associate Professor Joshua Blakeslee, Member Associate Professor Stephanie Strand, Member Copyrighted By Rebecca B. Kimmelfield 2020 Abstract The use of microbial inoculants to control plant disease is an increasingly used method in agriculture to mitigate damage caused by phytopathogens across a variety of systems. Best management practices to control many plant diseases can include use of multiple types of control measures in which biocontrol is one of a suite of tools used. One bacteria genus commonly investigated as biocontrol agents is Pseudomonas. These bacteria are known to be capable of promoting plant growth and reducing damage caused by disease though a variety of modes of action including nutrient competition and niche exclusion, secretion of antibiotic compounds including phenazines, DAPG, and pyoluteorin, and production of volatile organic compounds (VOCs). The primary focus of this dissertation, broadly, is the use of microorganisms (specifically Pseudomonas spp.) as biocontrol agents. Studies performed using these bacteria, both physically and conceptually, ranged from basic science, to small-scale microplot field trials, to applied market research. The focus of Chapters 2 and 3 of this work was investigating the role of VOCs in the biocontrol of nematodes (Caenorhabditis elegans) and fungi (Fusarium oxysporum) under in vitro conditions. The first objective of this work was to investigate how bacterial VOCs affected the growth and activity of other microorganisms, and determine what bioactive VOCs are produced by the bacteria. In shared-air indirect exposure assays using a diverse group of 20 bacteria (19 strains of Pseudomonas representing seven species and one of Pantoea agglomerans) we established that a majority the of the bacteria tested produced VOCs inhibitory ii to both C. elegans and F. oxysporum while other strains were only effective in inhibiting C. elegans. We performed VOC profiling using proton transfer reaction time-of-flight mass- spectrometry (PTR-ToF-MS) to compare differences in volatile production between the bioactive and non-bioactive strains. Hydrogen cyanide (HCN) and organosulfur compounds were associated with F. oxysporum inhibition, while only HCN was correlated with C. elegans inhibition. These findings generally confirmed our knowledge of antagonistic bacterial VOCs, as members of these categories of compounds have been shown to be antagonistic toward microorganisms. The second objective of this dissertation was to build off this knowledge, in which we sought to determine whether manipulation of the bacteria growth medium could selectively enhance production of specific VOCs. To test this, in vitro assays and VOC profiling were performed with three strains of Pseudomonas (P. chlororaphis, P. rhodesiae, P. protegens) on three media (minimal medium± glycine or L-methionine). These amino acids were selected because they are precursors to HCN (glycine) and methanethiol (methionine), two compounds highly produced by the inhibitory bacteria in the first objective. HCN is a VOC with well- established control potential, and methanethiol is the precursor to multiple organosulfur compounds, some of which have control potential. The addition of each amino acid affected bacterial VOCs to different extents. L-methionine could effectively prime the system. Bacteria grown on minimal media supplemented with L-methionine produced more organosulfur compounds and had greater bioactivity in bioassays compared to bacteria growth on minimal media. Of the three strains, the P. rhodesiae was enhanced by L-methionine to the greatest extent. In both in vitro agar-based and soil-based assays, P. rhodesiae inhibitory behavior against F. oxysporum was increased with the addition of L-methionine. We also found that addition of only L-methionine to a soil system was sufficient to prime the native microorganism population iii to produce inhibitory VOCs against F. oxysporum. The addition of glycine to the system promoted an increase in bacterial hydrogen cyanide but varied in increased efficacy against microorganisms; for the strains that produced hydrogen cyanide, the VOCs produced by bacteria grown on minimal medium±glycine had no differences in F. oxysporum inhibition; however, an increase was seen in C. elegans inhibition. Results from this study indicate that VOCs can contribute to the control potential of pseudomonads, and the compounds produced by the bacteria can be manipulated though the inputs added to the growth matrix. The third objective of this work was to investigate the potential for Pseudomonas, Bacillus, and Pantoea strains to control soybean cyst nematode (SCN) in the greenhouse and microplot (small-scale field trial) settings. These experiments move the biocontrol research from basic (in vitro laboratory assays) to more applied. In total, eight strains (six Pseudomonas spp., one Bacillus sp., and one Pantoea agglomerans) were used in these trials. Bacteria were tested using multiple formulations including individual strains and consortia of bacteria, and soil drench and seed treatments. In the microplot trials, nematode control efficacy investigated two parameters: end of season SCN egg counts and soybean yield. The results of the studies were varied. Efficacy of individual treatments was inconsistent from trial to trial. There were no treatments that either caused significantly lower SCN egg densities or significantly higher yield in all microplot experiments, however select treatments showed potential in at least one experiment. Finally, the last objective of this work was to investigate the current environment for biocontrol research and product development within the industry. This project, presented in Chapter 5, explored the potential for increased collaborations between university researchers and the microbial bioproducts industry. Companies are continuing to explore new actives for iv bioproducts, and professors continue to research potential beneficial microorganism and natural products. We interviewed professors from the state of Ohio who research in the fields of microorganism and natural products and learned while many professors want to commercialize their research, not all have gone through the necessary steps required to do so. We also interviewed companies that develop microbial inoculants, specifically individuals involved in research and development, and discovered that many companies obtain microorganisms and natural products from external sources. Companies and university faculty also collaborate in research projects. The results from this project indicated that there is no uniform method of collaborating between university and industry, but there is potential to streamline the process. In this way, bioproducts research could be done to meet the needs and wants of both the industry developing the products and university researchers performing the basic science. The results obtained in each chapter, from the basic science presented in Chapters 2 and 3 to the market research project in Chapter 5, present evidence that there is potential for Pseudomonas as biocontrol agents. The VOCs produced by the bacteria have significant inhibitory activity against F. oxysporum and C. elegans. While we saw varied efficacy of the bacteria against SCN, future studies can continue to explore the mode of action of these bacteria and determine whether there is applicability in other field and greenhouse systems. With sufficient in vitro and mode of action studies, paired with applied greenhouse and field trials, our best candidates can be further explored to determine whether they have potential from a commercial perspective. v This work is dedicated to the memory of my Grandma Sue Krolikowski. Thank you for being my most vocal cheerleader though all my studies. You have inspired me to continue on my quest of being a lifelong learner. vi Acknowledgements I am truly grateful for the tremendous support—both professionally and personally—that I have received from countless individuals over my time in this program. I would first like to thank my advisor, Dr. Chris Taylor, for his support and guidance over the past six years. Chris has encouraged me, in the best possible way, to forge my own path and tackle scientific queries as an independent, critical thinker. I am also grateful for the rest of my scientific advisory committee, both current and past members. Thank you for your patience and sharing of scientific knowledge over the years. The bulk of the research presented within this dissertation succeeded because I had many great people helping me along the way. The success of the science truly took a village. There are multiple people in particular I would like to thank. Department of Plant Pathology members Dr. Xiao-Yuan Tao, Rachel Kaufman, Dr. Anna Testen and Therese Miller for helping train me to work with the microorganisms and field work and Wanderson Bucker Moraes for his help and patience with statistics; Dr. Shauna Brummet was an invaluable mentor for the entrepreneurial study presented in