COMPOSITION AND DIVERSITY OF MEDICAGO TRUNCATULA ROOT BACTERIAL ENDOPHYTE POPULATIONS RESULTING FROM GROWTH IN DIFFERENT OKLAHOMA SOILS By JAMES NEIL ENIS Associate of Science in Agronomy Eastern Oklahoma State College Wilburton, Oklahoma 1999 Bachelor of Science in Plant & Soil Science Oklahoma State University Stillwater, Oklahoma 2001 Submitted to the Faculty of the Graduate College of the Oklahoma State University in partial fulfillment of the requirements for the Degree of MASTER OF SCIENCE July, 2008 COMPOSITION AND DIVERSITY OF MEDICAGO TRUNCATULA ROOT BACTERIAL ENDOPHYTE POPULATIONS RESULTING FROM GROWTH IN DIFFERENT OKLAHOMA SOILS Thesis Approved: _____________Michael P. Anderson_______________ Thesis Advisor _______________ Shiping Deng__________________ _____________ Robert V. Miller_________________ _____________ A. Gordon Emslie________________ Dean of the Graduate College ii ACKNOWLEDGEMENTS I would like to offer my sincere gratitude to my major advisor and committee chair, Dr. Michael P. Anderson, and to the members of my committee, Drs. Shiping Deng and Robert V. Miller for their guidance, support, and constructive criticism. The Department of Plant and Soil Sciences and the Oklahoma Biotechnology Network are gratefully acknowledged for their financial support. I would also like to thank Dr. Bruce A. Roe of the University of Oklahoma Department of Chemistry and Biochemistry and the members of his research team for their invaluable contribution to this research in the form of contig construction and sequencing of the M. truncatula endophyte clonal library. I gratefully acknowledge Dr. Stephen Marek’s support, guidance, and willingness to share equipment and valuable bench space for this work while I was employed as a technician in his laboratory. Carole Anderson, M.S. is also gratefully acknowledged for her support and assistance as technician for the Anderson laboratory. Above all, I would like to thank my wife, Sahar for her support, encouragement, understanding, and patience through all of the late nights, weekends, and holidays spent at the bench and at the computer. While too young to read this quite yet, my twin daughters Sierra and Sonora are also gratefully acknowledged for providing the motivation to see this work through to the end. iii I would also like to thank my parents, Jim and Linda, my brother, Brad, grandparents Evelyn, Onie, and Vestal, as well as my in-laws, Stephen, Giti, Emmanuel, Daniel, Ammid, and Sarah for their support and encouragement. Without the network of assistance from all those mentioned above, this work would not have been possible. iv PREFACE The endophytic bacterial populations of surface disinfected M. truncatula root tissues collected from plants grown in a commercial growing medium and six diverse soils from across the state of Oklahoma were evaluated for diversity and differences in composition resulting from plant growth in dissimilar soils by cloning and sequencing of near full-length 16S rDNA and temperature gradient gel electrophoresis (TGGE) of 16S rDNA fragments. Cloning and sequencing of 16S rDNA revealed 36 genera of bacteria encompassing five phyla as putative M. truncatula root endophytes. Large differences in diversity were observed between endophyte populations originating from plants grown in different soils, with differences becoming increasingly pronounced at lower taxonomic levels. At the genus level, two acidic soils with a forest background and a commercial growing medium containing 15-25% ground pine bark yielded the highest endophyte diversity, while moderate diversity was observed in plants grown in managed agricultural soils. Root bacterial endophyte diversity was lowest in plants grown in soil collected from an undisturbed native tallgrass prairie. The TGGE technique failed to adequately resolve the complex endophytic bacterial 16S rDNA fragments with respect to the level of diversity revealed by the cloning approach and 16S rDNA bands on the silver-stained TGGE gel were unable to be subsequently sequenced or cloned into plasmid vectors for identification. v TABLE OF CONTENTS Chapter Page I. REVIEW OF LITERATURE Introduction.................................................................................................................. 1 Defining the Term “Endophyte” ............................................................................. 1 Biodiversity of Bacterial Endophytes and Their Plant Hosts.................................. 4 Evaluation of Endophytic Bacterial Populations..................................................... 6 Origin of Bacterial Endophytes............................................................................... 8 Plant Colonization by Bacterial Endophytes......................................................... 10 Beneficial Aspects of Endophytic Associations ........................................................ 13 Plant Growth Promotion........................................................................................ 13 Biological Pest Control ......................................................................................... 17 Biosynthesis of Natural Products .......................................................................... 19 Phytoremediation................................................................................................... 20 Endophytes as Emerging Pathogens .......................................................................... 22 Research Objectives................................................................................................... 25 Bibliography .............................................................................................................. 27 II. IDENTIFICATION OF ROOT BACTERIAL ENDOPHYTES FROM MEDICAGO TRUNCATULA GROWN IN DIVERSE SOILS BY CLONING AND SEQUENCING OF PCR-AMPLIFIED 16S rRNA GENES Abstract...................................................................................................................... 35 Introduction................................................................................................................ 36 Materials and Methods............................................................................................... 40 Development of Surface Disinfection and Total DNA Extraction Protocol ......... 40 Surface Disinfection Experiment 1 ....................................................................... 40 Surface Disinfection Experiment 2 ....................................................................... 43 Surface Disinfection Experiment 3 ....................................................................... 45 Surface Disinfection Experiment 4 ....................................................................... 47 Verification of Surface Disinfection Efficacy by Culturing ................................. 49 Identification of Unknown Bacteria Surviving Surface Disinfection ................... 53 Soil Collection and Analysis ................................................................................. 55 Growth of Medicago truncatula ............................................................................ 56 Collection of Root Tissue Samples ....................................................................... 57 Surface Disinfection of Root Tissue ..................................................................... 58 Extraction of Total DNA from Surface Disinfected Root Tissue ......................... 59 Verification of Surface Disinfection Efficacy by PCR ......................................... 59 vi Chapter Page Amplification of Full-Length 16S rDNA from Surface Disinfected Roots .......... 61 Production of Competent Cells for Cloning.......................................................... 66 A-Tailing of PCR Amplified Full-Length 16S rRNA Gene Inserts...................... 66 Ligation of A-Tailed Inserts to the pGEM ®-T Easy Cloning Vector.................... 67 Transformation of E. coli XL-10 Gold ® with pGEM ®-T Easy Constructs ........... 68 Blue/White Screening of E. coli XL-10 Gold ® pGEM ®-T Easy Clones............... 69 Verification of Successful Ligation and Transformation by PCR ........................ 70 Clonal Library Construction.................................................................................. 73 Sequencing of Full-Length 16S rDNA Inserts ...................................................... 74 Putative Identification of 16S rDNA Inserts ......................................................... 76 Results and Discussion .............................................................................................. 78 Physical and Chemical Properties of Experimental Soils ..................................... 78 Identity of Unknown Bacteria Surviving Surface Disinfection ............................ 87 Efficacy of the Surface Disinfection Protocol....................................................... 88 Identity of M. truncatula Root Bacterial Endophytes ........................................... 90 Genera of Putative Endophytic Bacteria Identified in Multiple Soils................. 103 Soil-Dependant Variation in M. truncatula Root Endophyte Diversity.............. 107 Taxonomic Composition of M. truncatula Endophyte Populations.................... 108 Bibliography ............................................................................................................ 122 III. EVALUATION OF ROOT BACTERIAL ENDOPHYTE DIVERSITY IN MEDICAGO TRUNCATULA GROWN IN DIVERSE SOILS BY TEMPERATURE GRADIENT GEL ELECTROPHORESIS