UNIVERSITÉ DU QUÉBEC À MONTRÉAL THE MICROBIOME OF ACER SACCHARUM: ENVIRONMENTAL DRIVERS OF MICROBIAL COMMUNITIES IN DIFFERENT PLANT STRUCTURES THESIS PRESENTED AS PARTIAL REQUIREMENT OF THE MASTERS OF BIOLOGY BY JESSICA WALLACE AUGUST 2016 UNIVERSITÉ DU QUÉBEC À MONTRÉAL Service des bibliothèques Avertissement La diffusion de ce mémoire se fait dans le respect des droits de son auteur, qui a signé le formulaire Autorisation de reproduire et de diffuser un travail de recherche de cycles supérieurs (SDU-522 - Rév.0?-2011 ). Cette autorisation stipule que «conformément à l'article 11 du Règlement no 8 des études de cycles supérieurs, [l 'auteur] concède à l'Université du Québec à Montréal une licence non exclusive d'utilisation et de publication de la totalité ou d'une partie importante de [son] travail de recherche pour des fins pédagogiques et non commerciales. Plus précisément, [l 'auteur] autorise l'Université du Québec à Montréal à reprodu ire , diffuser, prêter, distribuer ou vendre des copies de [son] travail de recherche à des fins non commerciales sur quelque support que ce soit, y compris l'Internet. Cette licence et cette autorisation n'entraînent pas une renonciation de [la] part [de l'auteur] à [ses] droits moraux ni à [ses] droits de propriété intellectuelle. Sauf entente contraire, [l 'auteur] conserve la liberté de diffuser et de commercialiser ou non ce travail dont [il] possède un exemplaire.» UNIVERSITÉ DU QUÉBEC À MONTRÉAL LE MICROBIOME D'ACER SACCHARUM: L'INFLUENCE DES FACTEURS ENVIRONNEMENTAUX SUR LES COMMUNAUTÉS MICROBIENNES DES TISSUS VÉGÉTAUX MÉMOIRE PRÉSENTÉE COMME EXIGENCE PARTIELLE DE LA MAÎTRISE EN BIOLOGIE PAR JESSICA WALLACE AOÛT 2016 TABLE OF CONTENTS TABLE OF CONTENTS ......... .... .. .. .... .......... ............ .......................... ii LIST OF FIGURES ... ........ ........ .. ........................................ ..... .. ....... vi LIST OF TABLES ..... ..... ........................ .. .................. .. ... ................ viii ABSTRACT .......... ... .................. ................... .... ........ .... ... .... ... ...... .. x RÉSUMÉ ......... ........... ..... ....... .. .......... ..... ..... .. ........ ... ........ .... .... .. xi INTRODUCTION ................ ........................ ...................................... 1 CHAPTERI THE MICROBIOME OF ACER SACCHARUM SEEDLINGS: ELEVATIONAL RANGE LIMIT EFFECTS IN DIFFERENT PLANT STRUCTURES ......... ......... 9 1.1 Introduction .... .... ... ............. .. .. .... ..... ... ...... ... .............. .. .... ..... ....... 9 1.2 Materials and Methods .. ... .... ... ... ... ...... ... .... .. ......... ........ ........ ..... ... 14 1.2.1 Specimen collection .. .......................................... .... .. ............. 14 1.2.2 Functional traits and biometries ................................................. 15 1.2.3 Sample preparation and DNA extraction ................. ..... ... ...... .... .. ... 16 1.2.4 PCR and multiplexing for 16S rRNA gene sequencing .. .. .................. 18 1.2.5 PCR and multiplexing for ITS fungal sequencing ...... ............................... 19 1.2.6 DNA sequencing processing and data analysis .... .. .. .. .. .................... 21 1.2.7 Indicator species anal y sis ...... ... ................................................ 22 1.2.8 Statistical analysis ....... .. .. .... ...... .. ... .... .. .. ...... .... ..................... 23 1.3 Results .. .... .. .. ..... ...... .. ...... ... ....... ... .......... .. ... ......... ... ........ ......... 25 · 1.3 .1 Taxonomie composition of bacterial communities ..................... ....... 25 1.3 .2 Indicator species anal y sis of bac teri al taxa ........... .............. .. .. .... .. 26 1.3.3 Differences in bacterial community structure among structures and tissues .. .......... .... ..... .. ... ...... ..... .. ... .... ..... .... ... .. ... .. .... .. ................. ..... " 27 1.3.4 Differences in bacterial community phylogenetic structure .................. 28 1.3.5 Taxonomie composition offungal communities ... ............... ........ .... 28 1.3.6 Shannon diversity of bacterial communities ....... .............................. 29 111 1. 3.7 Correlations of microbial communities with functional traits of host plant ................ ....... ...... .. ............ .......... ... .. .... : ........ ..... ·········· ··· ······ 30 1.4 Discussion ............... ............ ... ................................. ................... 31 1.4.1 Taxonomie distribution of bacterial and fungal communities ............... 31 1.4.2 Microbial associations with different plant structures and tissues ........ .. 32 1.4.3 Analysis of variance between stmctures, tissues and enviromnents ........ 34 1.4.4 Microbial richness and diversity differed between plant structure and tissue ................. .. ........ ... .... ... .. .. .. .. ... ...... .... ......... .... ..................... ... 36 1.4.5 Correlations between microbial communities and host functional traits ···· ···· ···························· ............. ........ .................... ...... .. ..... .... 38 1.4.6 Comparison of bacterial and fungal results in root endophytic communities .... .... ..... ..... .... ... ..... ....... ... .... .. .. ... ...... ..... ..... .. ... .... ................... 39 1.5 Conclusion ... ... ....... ... .. .. .... ..... .. ................... ............. ... ............... 40 CHAPTER II THE MICROBIOME OF ACER SACCHARUM SAPLINGS: EFFECTS OF DIFFERE T SOILS AND CANOPY COVERS IN ENDOPHYTIC AND EPIPHYTie BACTERIAL AND FUN GAL COMMUNITIES ... ............. ......... 58 2.1 Introduction .. .... ............................. ............ ....... .... ........... ........... 58 2.2 Materials and Methods .................. .. ... ...... ..... .. .......... ... ..... ............. 62 2.2. 1 Specimen collection ................ .... ... .... .. ............... ............ ....... 62 2.2.2 Functional traits and biometries ......................... ... ... .. ....... ..... .. ... 63 2.2.3 Sample preparation and DNA extraction ... ... ...... .. ....... ..... .......... ... 64 2.2.4 PCR and multiplexing for 16S rRNA and ITS sequencing ........................ 66 2.2.5 DNA sequencing processing and data analysis ....... ............... .......... 67 2.2.6 Indicator species analysis of bacterial taxa .. ................. ....... ..... .. .... 69 - 2.2.7 Statistical analysis .... .. ......................................... ..... ........ ...... 70 2.3 Results ................... .... ..................................................... ......... 71 2.3 .1 Taxonomie composition of bac te rial cornmunities ........ ...... ............. 72 2.3 .2 lndicator species ana1ysis of bacterial taxa ... .. ....... ...... ...... .. ....... ... 73 lV 2.3 .3 Differences in bacterial community structure among structures and tissues .. .... .. ..... .................................... ... ..... .... .... ..... .. ... ......... ...... .. ... 74 2.3.4 Differences in bacterial commLmity phylogenetic structme .............. .... 75 2.3 .5 Taxonomie composition offungal communities ..... ... ...................... 75 2.3 .6 Differences in fungal cornmunity structure among tissues and soi! samples ·················· ·· ·· ··· ···· ·· ········ ······ ················· ···· .. ....... ... .... ....... .. .... 76 2.3.7 Shannon diversity offungal and bacterial communities .... ........ ... ... .. .. 76 2.3.8 Conelations of microbial communities with functional traits of host plant ·············· ········ ·· ············ ... ............... ........... .. .. .... .. ...... ........ ...... 77 2.4 Discussion .. .... .. ........... ..... .. ............... .................. ......... ............. 78 2.4. 1 Taxonomie distribution of bacterial and fungal communities ....... ..... .. 7~ 2.4.2 Microbial associations with different plant structures, tissues and envirorunents .. ............... .. ..................... ....... .... .. .. .. .. ... .......... .... .. 79 2.4.3 Analysis of variance between structures, tissues and envirorunents ... .. ............................ ... ...... ..... ..... .. .... .. .. ... .. ............... .. ..... ...... 81 2.4.4 Microbial richness and diversity differed between plant structure and tissue ......... ............... ... ... ...... ... .. .. .. ...... .............. ..... .... ......... .. .. .. ... ... 83 2.4.5 Conelations between microbial communities and host functional traits .... ... ........ ......... ................. .. ...... .... ............ .. ··· ········· ·· ·············· 84 2.4.6 Comparison of bacterial and fungal results in endophytic communities ... 85 2.5 Conclusion .............. ......... ........ ......... ... ............... ... ... ... .... .......... 86 CONCLUSION .............. .. ....... ... ....... ... ....... ... .. ...... .. .. .. ............. : ..... 102 BIBLIOGRAPHY ....... ..... ... ...... ... ..... ... .. ..... .... .... .. ... ... .... ............... 105 LIST OF FIGURES Figure Page 1.1 Relative abundances of bacteria and fungi ph y la in the different plant tissues and surfaces of sugar maple seedlings ................. 42 1.2 Cladograms ofLEfSe results showing bacterial indicator taxa at the phyla levels comparing epiphytie to endophytic and root- associated to leaf-associated communities .. .......... .... ......... .. 43 1.3 Non-metric multidimensional scaling (NMDS) ordination using Bray-Curtis distance of the dissirnilarity between bacterial communities from different plant surfaces and tissues