UC Merced UC Merced Electronic Theses and Dissertations Title Bacterial endophytes of California conifers: cultures, genomes, and community analysis. Permalink https://escholarship.org/uc/item/0p54q4mt Author Wilson, Emily Catherine Publication Date 2015 Peer reviewed|Thesis/dissertation eScholarship.org Powered by the California Digital Library University of California UNIVERSITY OF CALIFORNIA, MERCED Bacterial endophytes of California conifers: cultures, genomes, and community analysis. A dissertation submitted in partial satisfaction of the requirements for the degree Doctor of Philosophy in Quantitative and Systems Biology by Emily Catherine Wilson Committee in Charge: J. Michael Beman, Chair Miriam Barlow A. Carolin Frank Clarissa Nobile Chapters 1- 3 © Emily Catherine Wilson, 2015 All rights reserved The dissertation of Emily C. Wilson is approved, and it is acceptable in quality and form for publication on microfilm and electronically: _____________________________________ Miriam Barlow _____________________________________ A. Carolin Frank _____________________________________ Clarissa Nobile _____________________________________ J. Michael Beman, Chair University of California, Merced 2015 iii Dedication William Shakespeare once wrote: “Some are born great, some achieve greatness, and some have greatness thrust upon them.” - Twelfth Night Shakespeare also wrote: “Sweet are the uses of adversity, Which, like the toad, ugly and venomous, Wears yet a precious jewel in his head; And this our life, exempt from public haunt, Finds tongues in trees, books in the running brooks, Sermons in stones, and good in every thing.” - As You Like It I dedicate this dissertation to those adversities which have helped me realize the greatness to which I was born, that which I have striven to achieve, and that which has been thrust upon me in this struggle. I have indeed found good in every thing. TGBTG. iv Table of Contents List of Tables .............................................................................................. viii List of Figures............................................................................................... ix Acknowledgements.................................................................................... viii Curriculum Vita............................................................................................. xi Abstract of the Dissertation ...................................................................... xiii 1 Introduction.............................................................................................. 1 1.1 Organization of the Dissertation ...................................................... 2 1.2 References......................................................................................... 2 2 Isolation and identification of bacterial endophytes from buds of lodgepole pine, ponderosa pine and European black pine....................... 5 2.1 Abstract ............................................................................................. 5 2.2 Introduction........................................................................................ 6 2.3 Methods.............................................................................................. 8 2.3.1 Sample collection and sterilization ................................................ 8 2.3.2 Tissue preparation......................................................................... 8 2.3.3 Culturing and isolation................................................................... 8 2.3.4 DNA extraction .............................................................................. 9 2.3.5 DNA amplification.......................................................................... 9 2.3.6 Sequence processing and identification........................................ 9 2.4 Results................................................................................................ 9 2.5 Discussion........................................................................................ 10 2.6 Concluding Remarks....................................................................... 12 2.7 References ....................................................................................... 12 2.8 Tables ............................................................................................... 19 2.9 Figures.............................................................................................. 22 3 Bacterial endophytes of Monterey pine: community analysis and culture-based identification........................................................................ 23 3.1 Abstract ........................................................................................... 23 3.2 Introduction...................................................................................... 24 3.3 Methods............................................................................................ 25 3.3.1 Sample collection and sterilization .............................................. 25 3.3.2 Bacterial Community Analysis..................................................... 25 3.3.2.1 Tissue Preparation and DNA extraction .................................. 25 3.3.2.2 DNA amplification .................................................................... 26 v 3.3.2.3 Sequence processing and identification .................................. 27 3.3.3 Bacterial Culture Isolation ........................................................... 27 3.3.3.1 Tissue processing and Culture Conditions .............................. 27 3.3.3.2 Isolate DNA Extraction............................................................. 28 3.3.3.3 Isolate DNA Amplification ........................................................ 29 3.3.3.4 Isolate Sequence Processing and Identification...................... 29 3.4 Results.............................................................................................. 30 3.4.1 Community Analysis.................................................................... 30 3.4.2 Isolates ........................................................................................ 31 3.5 Discussion........................................................................................ 31 3.6 Concluding Remarks....................................................................... 33 3.7 References ....................................................................................... 33 3.8 Tables ............................................................................................... 39 3.9 Figures.............................................................................................. 44 4 Draft genome analysis of Monterey pine endophytes suggests that the aboveground endosphere is colonized by host-adapted bacteria and is a source of previously uncultured bacterial diversity. ........................ 46 4.1 Abstract ........................................................................................... 46 4.2 Introduction...................................................................................... 47 4.3 Methods............................................................................................ 48 4.3.1 DNA extraction ............................................................................ 48 4.3.2 DNA shearing .............................................................................. 48 4.3.3 Size-select processing ................................................................ 49 4.3.4 Shearing quality control............................................................... 49 4.3.5 Library prep ................................................................................. 49 4.3.6 Library quality control .................................................................. 50 4.3.7 Library Pooling and Sequencing ................................................. 50 4.3.8 Sequence Processing and Assembly.......................................... 50 4.3.9 Genome BLAST searches........................................................... 50 4.3.10 Ribsomal Database Project 16S rRNA comparison to isolates and uncultured bacteria................................................................................. 51 4.4 Results.............................................................................................. 51 4.4.1 Genome assembly statistics........................................................ 51 4.4.2 Identity of the isolates.................................................................. 51 4.4.3 Presence of known endophyte genes ......................................... 52 4.4.3.1 Motility and colonization............................................... 52 4.4.3.2 Secretion systems ........................................................ 52 4.4.3.3 Quorum sensing ........................................................... 53 4.4.3.4 Growth Promotion......................................................... 53 4.4.3.5 Drought Tolerance and Stress tolerance...................... 53 4.5 Discussion........................................................................................ 54 4.6 Concluding Remarks....................................................................... 55 vi 4.7 References ....................................................................................... 55 4.8 Tables ............................................................................................... 60 4.9 Figures.............................................................................................