Bacterial Endophytes: Exploration of Methods and Analysis of Community Variation by Shu Yi Shen A thesis submitted in conformity with the requirements for the degree of Master of Science Ecology and Evolutionary Biology University of Toronto © Copyright by Shu Yi Shen 2013 Bacterial Endophytes: Exploration of Methods and Analysis of Community Variation Shu Yi Shen Master of Science Ecology and Evolutionary Biology University of Toronto 2013 Abstract Bacterial endophytes, bacteria residing within plants, play an important role in the growth and development of plants and their ability to thrive under adverse conditions. The endophytes of Acer negundo, Ulmus pumila and Ulmus parvifolia trees sampled from a hydrocarbon- contaminated site were analyzed for variation between seasons and plant species. Branches from the same trees over a span of 3 seasons were collected and analyzed via culture dependent and culture independent methods. Numerous culture independent approaches were tested, culminating in the development of a new method for the amplification of endophytic bacterial ribosomal DNA that excludes plastid DNA. Community analyses using this new method in combination with T-RFLP showed significant differences between the endophytic communities of different plants species and of the same species growing in different seasons. The proposed technique can be used for the future study of endophytic communities of plants. ii Acknowledgments I would like to first and foremost thank my supervisor, Dr. Roberta Fulthorpe, for allowing me to carry out this project under her supervision. I am very thankful for your constant motivation, support and guidance throughout this project and through my moments of panic and irrationality. I would like to all the past and present members of the Fulthorpe Lab who have helped me along the way: Tony Qian for all your help with programming and being a sounding board for ideas, providing an analytical view to everything in life; Nicole Ricker for your great life advice, knowledge of almost everything there is to know about anything and the calmness you bring to the lab; Rosemary for the fun you brought into the lab and to Rhea Lumactud for all the help you have provided me with everything from going to the field and sampling, helping me process my samples, keeping me company in the late lab nights and helping me understand stats. I will miss the talks and lab karaoke that got me through the long days and nights in the lab. Specials thanks goes out to other people who have helped with various aspects of this project: Richard Dickinson for helping me identify all the trees species, you still amaze me with your knowledge and ability to identify plants in general and Thanushiga Theivendram for helping me streak out bacteria, I know it wasn't easy but thanks for doing it. Lastly I would like to thank my family for being there as a constant source of support and encouragement through the ups and downs of this project and my life. iii Table of Contents Acknowledgments ........................................................................................................................ iii Table of Contents ......................................................................................................................... iv List of Tables ............................................................................................................................... vii List of Figures............................................................................................................................... ix List of Appendices....................................................................................................................... xx Chapter 1 An Introduction to Bacterial Endophytes ................................................................ 1 1.1 General Introduction.......................................................................................................... 1 1.2 Current Culture Independent Methods for Studying Endophytes ................................ 4 Chapter 2 Analysis of Molecular Methods for Post Extraction Exclusion of Plastid Amplicons and Analysis of Endophytic Community................................................................. 9 2.1 Methods................................................................................................................................ 9 2.1.1 Database Analysis of Primers ...................................................................................................9 2.1.2 Surface sterilization of Acer negundo and Ulmus spp. Branches ........................................10 2.1.3 Isolation of DNA from Acer negundo and Ulmus spp. Branches.........................................11 2.1.4 Isolation of Plastid 16S rRNA Amplicons..............................................................................11 2.1.5 Primer Testing on Extracted Plastid Amplicons ..................................................................12 2.2 Results ................................................................................................................................ 13 2.2.1 Database Analysis of Primers .................................................................................................13 2.2.2 Isolation of Plastid Amplicons ................................................................................................15 2.2.3 Analysis of Primer 783R..........................................................................................................15 2.2.3 Primer Testing on Extracted Plastid Amplicons ..................................................................16 2.2.4 Preliminary Testing of Plastid Excluding Primers on Extracted Plant Samples ..............17 2.3 Discussion.......................................................................................................................... 18 Chapter 3 Physical Separation of Plastid Cells via Differential Centrifugation and Density Gradient Centrifugation............................................................................................................. 21 3.1 Introduction....................................................................................................................... 21 3.2 Method .............................................................................................................................. 22 iv 3.2.1 Preparation of Plant Macerate ...............................................................................................22 3.2.2 Differential Centrifugation of the Macerate .........................................................................23 3.2.3 Sucrose Density Gradient Centrifugation .............................................................................23 3.3 Results ................................................................................................................................ 25 3.3.1 Differential Centrifugation of the Macerate .........................................................................25 3.3.2 Sucrose Density Gradient Centrifugation .............................................................................26 3.4 Discussion........................................................................................................................... 29 Chapter 4 Subtractive Hybridization........................................................................................ 32 4.1 Introduction....................................................................................................................... 32 4.2 Methods.............................................................................................................................. 34 4.2.1 Designing Plastid Specific Probes...........................................................................................34 4.2.2 Binding of Magnetic Beads to Phosphorus Dendrimers ......................................................34 4.2.3 Immobilization of Oligonucleotide Probes ............................................................................35 4.2.4 Subtractive Hybridization.......................................................................................................36 4.3 Results ................................................................................................................................ 39 4.3.1 Plastid Specific Probe ..............................................................................................................39 4.3.2 Subtractive Hybridization.......................................................................................................40 4.4 Discussion.......................................................................................................................... 42 Chapter 5 Enzymatic Digestion of Genomic DNA samples .................................................... 46 5.1 Introduction....................................................................................................................... 46 5.2 Methods.............................................................................................................................. 47 5.2.1 Database Analysis of Restriction Enzymes............................................................................47 5.2.2 Enzymatic Digest of DNA and Amplification of Bacterial DNA .........................................47 5.2.3 Amplification using DGGE primers ......................................................................................48