PHENAZINE-1-CARBOXYLIC ACID-PRODUCING PSEUDOMONAS SPP. OF THE INLAND PACIFIC NORTHWEST (U.S.) By JAMES A. PAREJKO A dissertation submitted in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY IN MICROBIOLOGY WASHINGTON STATE UNIVERSITY School of Molecular Biosciences DECEMBER 2012 To the Faculty of Washington State University The members of the Committee appointed to examine the dissertation of James A. Parejko find it satisfactory and recommend it be accepted. Linda S. Thomashow, Ph.D., Chair David M. Weller, Ph.D. Michael L. Kahn, Ph.D. William B. Davis, Ph.D. ii Acknowledgments I am endlessly grateful to the support that the USDA-ARS Root Disease and Biological Control Unit has provided for me in my years as a graduate student. I am thankful for the advisement and independence that Dr. Thomashow has given me and her unparalleled eye for the details has taught me to make every word and revision count. The encouragement from Dr. Weller to keep the big picture in mind has provided me with new perspectives on the microbial world and the shown me the importance of ‘keeping one foot in the lab and one foot firmly planted in the field’. The help, support and advice that Dr. Dmitri Mavrodi and Dr. Olga Mavrodi have given me over the last few years has helped me not only grow as a scientist, but also as a person. I would also like to thank Dr. Kahn and Dr. Davis for serving on my thesis committee and providing me with advice and encouragement. Their help was crucial to successfully traversing the rolling hills of graduate school (metaphorically-speaking) from my first proposal as a second-year Ph.D. student to the Ph.D. candidate I am today. I am extremely appreciative for the help provided by Karen Hansen, Dr. A. Kamil Mohd Jaaffar, Jennifer Apple, Emilia Gan, Chelsea Stone, Irina Mavrodi and Nathalie Walter in completing the work described in this dissertation. I am also grateful to the support provided by the School of Molecular Biosciences and Department of Plant Pathology. Without the intellectual and finanacial support from both departments I wouldn’t have been able to complete the work described in this dissertation. iii PHENAZINE-1-CARBOXYLIC ACID-PRODUCING PSEUDOMONAS SPP. OF THE INLAND PACIFIC NORTHWEST (U.S.) Abstract by James A. Parejko, Ph.D. Washington State University December 2012 Chair: Linda S. Thomashow Phenazine antibiotic-producing rhizobacterial strains belonging to the genus Pseudomonas are effective biocontrol agents against soilborne fungal plant pathogens. Beyond the well-studied model phenazine-producing (Phz+) biocontrol strain Pseudomonas fluorescens 2-79, there existed few studies demonstrating phenazine production in root-colonizing members of the ‘P. fluorescens’ lineage. Furthermore, our collective knowledge of these important biological control rhizobacteria lacked a comprehensive analysis of representatives of Phz+ populations indigenous to intensive agricultural regions, most notably the Inland Pacific Northwest (U.S.) dryland agroecosystem. In the first of the studies within this dissertation, a collection of 412 Phz+ Pseudomonas spp. isolates from three cereal fields of east-central Washington State was genotypically and phenotypically characterized. The results revealed four new groups of Phz+ strains, three of which were divergent from P. fluorescens 2-79. These groups were associated iv with dryland cereal crops with seven different cropping histories, however cropping history shaped the community of genotypes present. Another collection of 497 Phz+ isolates from throughout the Inland Pacific Northwest dryland agroecosystem was taxonomically described by multi-locus sequence analysis and the phenazine biosynthesis gene phzF was cloned from winter wheat at nine sites. Using this approach, it was determined that the four groups of Phz+ strains from the first study are separate Pseudomonas species, two of which have never been described, and agroclimatic conditions influence Phz+ community diversity. The influence of phenazine-1- carboxylic acid (PCA) on biofilm formation under water limitation and biocontrol characteristics against Rhizoctonia solani AG-8 were also determined for selected strains from the Phz+ species. Generally the strains developed high levels of biofilm and responded uniquely to simulated matric or osmotic stress. The importance of PCA production to biofilm formation was variable by strain, although biofilm architecture was significantly impacted in most Phz- mutants. Representative Phz+ strains protected wheat plants from the Rhizoctonia root rot causal agent R. solani AG-8 in greenhouse bioassays with reduced plant protection from Phz- mutants of the same strains. These findings significantly increase our understanding of indigenous Phz+ populations in dryland agriculture and their involvement in an effective cropping system for a more sustainable dryland agroecosystem. v Table of Contents Page Acknowledgments.......................................................................................................................... iii Abstract .......................................................................................................................................... iv List of Tables ...................................................................................................................................x List of Figures .............................................................................................................................. xiii General Introduction Sustainable dryland cereal production and root-associated bacteria and fungi ...............................1 The ‘Pseudomonas fluorescens’ species-complex.........................................................................12 Phenazines and the ecological implications for phenazine-producing Pseudomonas spp. in the rhizosphere micro-environment .....................................................................................................14 Microbial population genetics and the emerging biogeography of indigenous phenazine- producing P. fluorescens strains ....................................................................................................25 Goals of this dissertation ................................................................................................................29 References cited .............................................................................................................................32 Specific contributions to this work ................................................................................................45 vi Chapter 1. Population structure and diversity of phenazine-1-carboxylic acid producing fluorescent Pseudomonas spp. from dryland cereal fields of central Washington State (U.S.) Abstract ..........................................................................................................................................47 Introduction ....................................................................................................................................48 Materials & methods ......................................................................................................................49 Results ............................................................................................................................................60 Discussion ......................................................................................................................................82 References cited .............................................................................................................................89 Chapter 2. Taxonomy and distribution of phenazine-1-carboxylic acid-producing Pseudomonas spp. in the dryland agroecosystem of the Inland Pacific Northwest (U.S.) Abstract ..........................................................................................................................................98 Introduction ....................................................................................................................................99 Materials & methods ....................................................................................................................103 Results ..........................................................................................................................................113 Discussion ....................................................................................................................................127 References cited ...........................................................................................................................135 vii Chapter 3. Significance of phenazine-1-carboxylic acid (PCA) to biofilm production and colony biofilm morphology of phenazine-producing Pseudomonas species under stress Abstract ........................................................................................................................................142 Introduction ..................................................................................................................................143 Materials & methods ....................................................................................................................147 Results ..........................................................................................................................................154 Discussion ....................................................................................................................................167