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View of 3C As a New Biopesticide Ingredient

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View of 3C As a New Biopesticide Ingredient Genomic Analysis, Population Quantification and Diversity Characterization of Cryptococcus flavescens DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Xiaoqing Rong, M.S. Graduate Program in Plant Pathology The Ohio State University 2013 Dissertation Committee: Dr. Brian B. McSpadden Gardener, Advisor Dr. Thomas K. Mitchell Dr. Pierce A. Paul Dr. Christopher G. Taylor Copyrighted by Xiaoqing Rong 2013 Abstract Cryptococcus flavescens strain OH182.9_3C (3C) has been shown to have biocontrol efficacy against Fusarium head blight (FHB) of wheat, and, 3C is currently under development as a biopesticide. The research described in this dissertation advanced that commercial development of this agent by providing new information about the biology, genetics, diversity, and ecology of this species for the control of Fusarium Head Blight. Such data may be used by the US EPA and the company developing the strain for the regulatory review of 3C as a new biopesticide ingredient. A draft genome sequence of 3C was obtained through high-throughput sequencing and short read assembly. Ab initio gene prediction and sequence similarity search were conducted to annotate the genome. Nine conserved regions were identified from the assembled genome sequence to develop molecular markers for population quantification and diversity characterization. Putative non-ribosomal peptide synthetase and glucanase genes were also found in the genome and may contribute to the biocontrol activity previously noted for the strain. A quantitative PCR (qPCR) assay was developed to quantify the abundance and population dynamics of 3C-like C. flavescens in wheat production systems. Following application of 3C to flowering wheat spikes, the population of 3C-like C. flavescens in the inoculated areas remained relatively stable for the remainder of the growing season. The populations of 3C-like C. flavescens seem to have spread from inoculated areas to non-inoculated areas and increased over time. Populations decreased on threshed grains during storage, but persisted in the postharvest field during the winter and early spring following the growing season. Detailed study on diversity within C. flavescens species has not been reported before. Multilocus sequence typing was performed on strain 3C and 12 other C. flavescens ii strains using six apparently unlinked loci: rDNA-ITS, β-tubulin, chitin synthase 1, elongation factor 1, heat shock protein 70 kDa and a mostly anonymous region. The rDNA-ITS sequences were identical across all the 13 strains. The other five loci consistently revealed two genotypes, A (6 strains, including 3C and type strain NRRL Y- 1401T) and B (7 strains), with high bootstrap support. This genotypic split was congruent to the mating type previously performed on some of the tested strains. Phenotypic variation noted using BIOLOG assays supported the grouping by genotype and revealed the slow assimilation of several carbon sources by two isolates of genotype A. In green- house bioassays, the strains of both genotypes showed biocontrol efficacy against Fusarium head blight, but genotype A as a whole showed slightly greater biocontrol efficacy than genotype B strains. This study provided insight into the phylogenetic relationships within C. flavescens species and resources for developing genetic and phenotypic markers to differentiate subspecies of C. flavescens. This research paves the way for additional studies of C. flavescens and their use as biopesticidal agents. Genome-wide discovery of genes involved in biocontrol of FHB by 3C may help reveal the biocontrol mechanisms expressed by C. flavescens. The developed qPCR assay and MLST can be used to further characterize the abundance and diversity of natural and inoculated populations of C. flavescens. Collectively, such studies will further facilitate the development and registration of 3C and possibly other C. flavescens strains as biopesticides for use against FHB. iii Acknowledgements I would like to acknowledge my Ph.D. advisor Dr. Brian McSpadden Gardener for the mentoring and support during my graduate career. He gave me me great amounts of advice that was very helpful for my career development and personal growth. I also thank him for giving me opportunities for working on the genomic and molecular sides of microbiology which I am most interested in. I thank my Student Advisory Committee members for advising me throughout the Ph.D. program. I thank Dr. Paul for his advice and help on the projects we collaborated on, as well as his thorough comments on my thesis. I thank Dr. Mitchell and Dr. Taylor for all their advice and suggestions on my projects. I thank Dr. Paul and Dr. David Schisler from USDA-ARS for the collaboration on my dissertation projects, especially their kind help and readily offers of collaboration efforts. I thank Dr. Michael Boehm for his efforts in discovering and initially characterizing the biocontrol activities of Cryptococcus flavescens OH182.9, work that laid the foundation for my thesis research. I also thank all the past and current members in McSpadden Gardener lab and collaboration labs who have assisted with my projects. iv Vita 2009 .................................................................. B.S. Honors Program of Life Sciences, China Agricultural University 2012 .................................................................. M.S. Plant Pathology, The Ohio State University 2009 to present ............................................... Graduate Research Associate, Department of Plant Pathology, The Ohio State University Publications Rong, X.; Schisler, D.A.; McSpadden Gardener, B.B. 2013. Genotypic and phenotypic characterization of Cryptococcus flavescens, biocontrol agents for Fusarium head blight of wheat. Submitted for publication. Rong, X.; Paul, P.A.; Schisler, D.A.; McSpadden Gardener, B.B. 2013. A quantitative PCR assay useful for monitoring the field dispersal and persistence of Cryptococcus flavescens, a biocontrol yeast applied to wheat. Submitted for publication. Rong, X.; Baysal Gurel, F.; Meulia, T.; McSpadden Gardener, B.B. 2012. Draft Genome Sequences of the Pseudomonas fluorescens Biocontrol Strains Wayne1R and Wood1R. Journal of Bacteriology 194(3):724-725. Rong, X.; Baysal Gurel, F.; Meulia, T.; McSpadden Gardener, B.B. 2012. Draft Genome Sequences of the Biocontrol Bacterium Mitsuaria sp. Strain H24L5A. Journal of Bacteriology 194(3):734-735. v Kim, H.J.; Lee, J.H.; Kang, B.R.; Rong, X.; McSpadden Gardener, B.B.; Ji, H.J., et al.. 2012. Draft Genome Sequence of Pantoea ananatis B1-9, a Nonpathogenic Plant Growth-Promoting Bacterium. Journal of Bacteriology 194(3):729-729. Kim, H.J.; Park, J.Y.; Han, S.H.; Lee, J.H.; Rong, X.; McSpadden Gardener, B.B., et al. 2011. Draft genome sequence of the biocontrol bacterium Chromobacterium sp. strain C-61. Journal of Bacteriology 193(23):6803-6804. Park, J.Y.; Han, S.H.; Lee, J.H.; Han, Y.S.; Lee, Y.S.; Rong, X., et al. 2011. Draft genome sequence of the biocontrol bacterium Pseudomonas putida B001, an oligotrophic bacterium that induces systemic resistance to plant diseases. Journal of Bacteriology 193(23):6795-6796. Fields of Study Major Field: Plant Pathology vi Table of contents Abstract .....................................................................................................................................ii Acknowledgements ................................................................................................................. iv Vita ............................................................................................................................................ v Table of contents ....................................................................................................................vii List of Tables ........................................................................................................................... xi List of Figures.........................................................................................................................xii Chapter 1: Literature review – Fusarium head blight and Cryptococcus flavescens OH182.9_3C, a biological control agent against this disease ............................................... 1 Fusarium head blight ...................................................................................................... 1 Overview of Fusarium head blight ....................................................................... 1 Biology of pathogen infection and mycotoxin production ................................. 2 Economic loss due to Fusarium head blight ........................................................ 7 Need for biological control of FHB ............................................................................... 7 Inadequacy of cultural, chemical, and host resistance in controlling FHB ....... 8 Biocontrol as a promising approach to FHB control .......................................... 9 C. flavescens OH182.9 as a biocontrol agent against Fusarium head blight (FHB) of wheat ................................................................................................................................ 9 Discovery, isolation and selection of a fungicide-resistant variant .................... 9 Reduction of FHB damages by OH182.9 and OH182.9_3C ............................ 11 Taxonomy, physiology and sexual reproduction of C. flavescens species ............... 12
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