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Environmental and biogeographical drivers of the Leptospermum scoparium (mānuka) phyllosphere microbiome A thesis submitted in partial fulfilment of the requirements for the degree of Master of Science (Research) at The University of Waikato by Anya Sophia Noble 2018 ii Abstract A substantial body of research exists on the physiology and genetics of Leptospermum scoparium (mānuka); however, the mānuka phyllosphere has not yet been investigated. This research, therefore, provides the first exploration of the bacterial communities comprising the mānuka phyllosphere. In total, 89 samples were collected from five native mānuka forests during the November 2016 – January 2017 mānuka flowering season, and examined alongside spatial, environmental, and host- tree related metadata. Cultivation-independent methods, including DNA extraction and 16S rRNA gene PCR amplicon sequencing analysis, were used to characterise phyllosphere communities. Using a combination of statistical tests, the BLAST algorithm, and a PICRUSt analysis, a habitat-specific core microbiome comprising members of Acidobacteria, Alphaproteobacteria, Bacteroidetes, and Verrucomicrobia, was present in all samples. Distinct microbial biogeographical patterning was also identified in the mānuka phyllosphere whereby spatially structured gradients in environmental variables, such as temperature and precipitation, correlated with bacterial community structure. The results from this study provide new insight pertaining to the structure, composition, and driving factors of the bacterial communities of the mānuka phyllosphere and shed light on theories of microbial ecology, such as microbial biogeography and core microbiomes. This research has the potential to open new avenues in both mānuka and microbial ecology fields of investigation within New Zealand. iii Acknowledgements I would like to thank my supervisor Dr Charles Lee for the opportunity to complete this research project at Waikato University. In addition, I would like to thank Dr Michael Clearwater and Stevie Noe for arranging and managing sampling trips, as well as Steens Honey, Timberlands ltd, East Taupō Lands Trust, and Waipa District Council for providing sample sites. I would also like to thank my amazing TRU-family; Shaun, Shelly, Georgia, Maria, Mafalda, and Hugo, for their support, inspiration, and the endless adventures and laughs that we shared both in and out of the lab. Last, but not least, I would like to thank Liam and my family (my mum, my dad, and my brother) for their unconditional love, support, patience, and humour during this challenging but rewarding year. iv Table of Contents Abstract ......................................................................................................................... iii Acknowledgements ....................................................................................................... iv List of Figures...............................................................................................................vii List of Tables .................................................................................................................. x 1. Chapter One: Introduction .................................................................................... 12 1.1. Introduction to the phyllosphere.................................................................... 12 1.2. Early phyllosphere microbiology .................................................................. 14 1.3. Bacteria in the phyllosphere .......................................................................... 14 1.4. Structure and composition of bacterial communities .................................... 16 1.5. Microbial Biogeography ................................................................................ 19 1.6. Temporal variation in the phyllosphere microbiome .................................... 20 1.7. Dispersal-related drivers in the phyllosphere microbiome ........................... 21 1.8. Niche-based drivers in the phyllosphere microbiome ................................... 23 1.9. The core microbiome concept ....................................................................... 25 1.10. Summary of recent cultivation-independent phyllosphere research ......... 26 1.11. Mānuka (Leptospermum scoparium) ......................................................... 29 1.12. Phyllosphere research in New Zealand ..................................................... 31 1.13. Research aims ............................................................................................ 31 2. Chapter Two: Review of molecular techniques ................................................... 33 2.1. Introduction.................................................................................................... 33 2.2. 16S rRNA ...................................................................................................... 33 2.3. The chloroplast contamination conundrum ................................................... 34 2.4. Isolating phylloepiphytic bacteria ................................................................. 36 2.4.1. Sonication ............................................................................................... 36 2.4.2. Centrifugation versus filtration .............................................................. 37 2.5. Cross-contamination ...................................................................................... 38 3. Chapter Three: Materials and Methods ............................................................... 43 3.1. Study sites ...................................................................................................... 43 3.2. Sample and metadata collection .................................................................... 43 3.3. Isolation of microbial cells from the mānuka leaf surface ............................ 46 3.4. DNA extraction .............................................................................................. 48 3.5. PCR amplification of 16s rRNA genes ......................................................... 49 3.6. Next Generation Sequencing ......................................................................... 49 v 3.7. Data analyses ................................................................................................. 50 4. Chapter Four: Results and Discussion ................................................................. 52 4.1. General characteristics of bacterial communities ......................................... 52 4.2. The mānuka phyllosphere core microbiome ................................................. 55 4.2.1. Putative identities of the core microbiome ............................................ 59 4.3. Exploring biogeographical patterns in the mānuka phyllosphere microbiome 66 4.3.1. Distance, latitude, longitude, and elevation (space) .............................. 70 4.3.2. Climatic heterogeneity ........................................................................... 74 4.3.3. Host tree characteristics ......................................................................... 79 4.3.4. Relative importance of spatial, climatic, and host tree characteristics .. 83 4.4. The predicted mānuka phyllosphere metagenome ........................................ 85 4.4.1. Phyllosphere-enriched KEGG pathways ............................................... 87 4.4.2. KEGG pathways shared by phyllosphere and soil communities........... 90 4.5. Discussion ...................................................................................................... 95 5. Chapter Five: Conclusions and future directions .............................................. 105 References .................................................................................................................. 108 6. Appendices .......................................................................................................... 123 6.1. Supplementary material for Chapter Four ................................................... 123 vi List of Figures Figure 1.1: Schematic of an Arabidopsis thaliana leaf cross section displaying the characteristic epidermal and mesophyll cell layers. Scanning electron micrograph (A) shows a leaf edge with visible trichomes and (B) shows phylloepiphytic bacteria located in the epidermal cell grooves. Image sourced and modified from Vorholt (2012). ........................................................................................................................... 13 Figure 1.2: Scanning electron micrographs depicting the colonisation patterns of phylloepiphytic bacteria. Colonisation patterns include solitary cells (A & B), multicellular aggregates at epidermal cell wall junctions (C), and biofilms