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Diversity of Methylobacterium Species Associated with New Zealand Native Plants A thesis submitted in partial fulfilment of the requirements for the degree of Masters of Science in Biological Sciences at the University of Waikato by Rowshan Jahan _________ The University of Waikato 2013 Abstract The genus Methylobacterium are pink-pigmented facultative methylotrophs (PPFMs), and are abundant colonizers of the phyllosphere, due to the availability of methanol, a waste product of pectin metabolism during plant cell division. Besides methanol cycling, Methylobacterium has important effects on plant health. The phyllosphere is an extreme environment with a landscape that is heterogeneous, continuously changing as the plant grows, and is exposed to very high ultra violet irradiation. Geographically, New Zealand has been isolated for over a million years, has a biologically diverse group of species, and is considered a biodiversity hotspot, with most of the native plants being endemic. We hypothesize that NZ native plants harbor diverse groups of Methylobacterium species, and to test this we aimed to isolate Methylobacterium species from the phyllosphere of native New Zealand plants. A leaf imprinting technique using methanol supplemented AMS agar media was used to isolate bacteria and diversity was determined using a combination of ARDRA, 16S rRNA gene sequencing and phylogenetic analysis. Methylobacterium spp. were successfully isolated from 19 of the 21 plant species used in this study. Eleven Methylobacterium species have been identified in association with the phyllosphere of native NZ plants: M. adhaesivum, M. brachiatum, M. komagatae, M. marchantiae, M. mesophilicum, M. phyllosphaerae, M. fujisawaense, M. oryzae, M. radiotolerans, M. tardum and M. zatmanii, with the first six being the most frequently isolated from more plant species. In this study other α, β, γ-proteobacterial species were also isolated: Hyphomicrobium, Methylopila, Rhizobium, Achromobacter, Methylophilus, Ramlibacter and Xanthomonas; Janibacter melonis (Actinomycetes); Niastella populi (Bacteroidetes) and Paenibacillus lautus (Firmicutes), highlighting the presence of potential novel methanol utilizer within the ecosystem. Results from this study indicate that Methylobacterium are abundant and dominant members of the NZ phyllosphere environment, with species diversity and composition dependent on the host plant species. Abstract ii Acknowledgements All praises are to the Almighty Allah who enabled me to complete this thesis. I’d like to express my heartfelt respect and gratitude to my supervisor Associate Professor Ian McDonald for his guidance, constructive suggestions and encouragement during the entire period of research and in preparing this thesis. His door was always open for me for help and advice, many thanks. I have found a warm, friendly and collaborative environment within the Thermophile Research Unit throughout of my research. In particular I’d like to thank Stephen Archer for help with many technical problems, giving good advice and teaching me the basics. Thanks to Chelsea Vickers for her assistance in the laboratory, especially with PCR. Thanks to Charles Lee for giving suggestions when I have presented my research in the TRU lab meeting. Thanks to Craig Herbold for his advice, and Eric Bottos for help with making primers and with technical problems arises during electrophoresis and visualization. I have found Professor Craig Cary is a superb mentor. I can’t forget his supportive sentence “One of us has to guide you to complete your masters and this is your masters, you have to achieve this” – he replied when I approached him to take me under his wing. Special thanks go to Craig Cary and Ian McDonald for allowing me to conduct my research in the TRU. I have found Professor Hugh Morgan has always been supportive. Special thanks also go to Hugh Morgan for his everyday looking after when Ian McDonald was away for study break. Thanks are extended to Lynne Parker, Colin Monk and Roanna Richards-Babbage for their support and assistance in my every mechanical problem to fix. I can’t finish this research without their help. I’d like to thank John Longmore in the Waikato DNA Sequencing unit, who has seen my sad face several times and helped me to successfully complete the sequencing. I am thankful to Andrew Grilli, for allowing me to spend time at his nursery, to know about native plants and take samples of them. I am thankful to my mother, brothers and sisters and special thanks to my children Nazifa and Rayyed for their sacrifices and day to day encouragement at every down moment through the entire period of the research for success. Acknowledgements iii Dedication I’d like to dedicate this thesis to my late youngest brother Nasim and late father Alhaj Md Abu Zahed Talukder, who I believe always, expects more from me. Dedication iv Table of Contents Abstract ........................................................................................................................ ii Acknowledgements ..................................................................................................... iii Dedication ................................................................................................................... iv Table of Contents ........................................................................................................ v List of Figures ............................................................................................................ vii List of Tables ............................................................................................................ viii List of Abbreviations ................................................................................................. ix Chapter 1: Background and Literature Review ...................................................... 1 1.1 The Genus Methylobacterium ...................................................................... 2 1.1.1 Species of Methylobacterium .................................................................. 2 1.1.2 Habitat ..................................................................................................... 3 1.2 The Phyllosphere .......................................................................................... 5 1.2.1 What is the source of methanol in leaves? .............................................. 8 1.2.2 How does methanol exit plant surfaces? ................................................. 9 1.3 Phyllosphere Microbiology ........................................................................ 11 1.3.1 The Association between Methylobacterium and Plants ...................... 14 1.3.2 The Role of Methylotrophy in Plant Colonization ................................ 15 1.3.3 Synthesis of Bioactive Substances by Epiphytic Methylobacteria ....... 17 1.3.4 Biotechnological Potential of Methylobacteria ..................................... 19 1.3.5 Phyllosphere Methylotrophic Bacterial Diversity ................................. 20 1.4 Molecular Approaches ............................................................................... 21 1.4.1 16S rRNA is the molecular marker to study biodiversity ..................... 23 1.4.2 Restriction Fragment Length Polymorphism (RFLP) ........................... 25 1.4.3 Aims, approach and outline of this research ......................................... 26 Table of Contents v Chapter 2: Diversity of Methylobacterium Species Associated with New Zealand Native Plants ........................................................................................... 29 2.1 Introduction ................................................................................................ 29 2.2 Materials and Methods .............................................................................. 33 2.2.1 Sample collection and isolation of Methylobacterium species ............. 33 2.2.2 Extraction of chromosomal DNA from pure cultures ........................... 36 2.2.3 PCR amplification of 16S rRNA genes ................................................ 37 2.2.4 Agarose gel electrophoresis .................................................................. 37 2.2.5 Amplified ribosomal DNA restriction analysis (ARDRA) ................... 38 2.2.6 Sequencing of 16S rRNA genes ............................................................ 38 2.2.7 DATA analysis ...................................................................................... 39 2.3 Results .......................................................................................................... 39 2.3.1 Isolation of Methylobacterium species.................................................. 39 2.3.2 PCR amplification and ARDRA analysis ............................................