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A Survey of the Bacterial Root Endophytes Associated with the Natural Vegetation at the Bitumount Provincial Historic Site, Alberta, Canada

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A Survey of the Bacterial Root Endophytes Associated with the Natural Vegetation at the Bitumount Provincial Historic Site, Alberta, Canada A SURVEY OF THE BACTERIAL ROOT ENDOPHYTES ASSOCIATED WITH THE NATURAL VEGETATION AT THE BITUMOUNT PROVINCIAL HISTORIC SITE, ALBERTA, CANADA A thesis submitted to the College of Graduate Studies and Research in Partial Fulfillment of the Requirements for the Degree of Master of Science in the Department of Soil Science University of Saskatchewan Saskatoon By Natalie Pierrette Blain © Copyright Natalie Pierrette Blain, September, 2016. All rights reserved PEMISSION TO USE In presenting this thesis in partial fulfillment of the requirements for a Postgraduate degree from the University of Saskatchewan, I agree that the Libraries of the University may make it freely available for inspection. I further agree that permission for copying of this thesis in any manner, in whole or in part, for scholarly purposes may be granted by the professor or professors who supervised my thesis work or, in their absence, by the Head of the Department or the Dean of the College in which my thesis work was done. It is understood that any copying or publication or use of this thesis or parts thereof for financial gain shall not be allowed without my written permission. It is also understood that due recognition shall be given to me and to the University of Saskatchewan in any scholarly use that may be made of any material in my thesis. Request for permission to copy or to make other use of material in this thesis in whole or in part should be addressed to: Head of the Department of Soil Science University of Saskatchewan Saskatoon, Saskatchewan S7N 5A8 i ABSTRACT The Bitumount Provincial Historic site is the location of two of the world’s first oil extracting and refining operations. Bitumount, located in the Athabasca oil sands of Alberta, is thought to be impacted by hydrocarbons through both natural and human activity. Plants have been able to recolonize the site in spite of varying hydrocarbon levels, through means of natural revegetation. Due to the apparent resilience of these plant species, Bitumount offers a unique opportunity to study the root-associated bacterial communities. This study was designed to achieve a better understanding of the root-associated partnerships occurring within naturally revegetated hydrocarbon contaminated soils. Plant and soil samples were collected in June 2014. Six representative plant species were identified and collected based on abundance on site, including smooth brome (Bromus inermis, SB), horsetail species (Equisetum spp., HT), slender wheatgrass (Agropyron trachycaulum, SW), Kentucky bluegrass (Poa pratensis, KB), an unspecified member of the pea family (Fabaceae, PF), and wild strawberry (Fragaria virginiana, WS). Population abundance of rhizosphere and root endosphere bacteria was significantly influenced (p<0.05) by plant species and sampling location. The vegetation was found to support diverse root endophytic communities despite hydrocarbon contamination. Culture dependent techniques were able to identify some of the more abundant bacteria characterized by high-throughput sequencing. In general, members of the Actinomycetales, Rhizobiales, Pseudomonadales, Burkholderiales, and Sphingomonadales orders were the most commonly identified via both techniques. Community structure of root-associated bacteria was found to be influenced by both plant species and sampling location. Quantitative real-time polymerase chain reaction was used to determine the potential functional diversity of the root endophytic bacteria. The gene copy numbers of 16S rRNA and two hydrocarbon degrading genes (CYP153 and alkB; both of the alkane hydroxylase family) were quantified. The gene copy abundance of 16S rRNA, CYP153, and alkB was significantly affected by the interaction of plant species and sampling location. The increased colonization of hydrocarbon degrading bacteria within grass species emphasizes their ability to be used for reclamation efforts. Overall, it was found that the endosphere was able to support diverse bacterial communities with known plant growth promoting abilities. In addition, the diversity and abundance of the endophytic bacteria was influenced by many different factors instead of one ii sole dominant one. The findings of this study provide insight into the root-associated bacterial communities occurring within natural revegetated soils. iii Acknowledgments First and foremost, I would like to thank my supervisors Dr. Jim Germida and Dr. Bobbi Helgason for their guidance and support over the last few years. I am very fortunate to have been mentored by such knowledgeable scientists. I also greatly appreciate the time and advice given by the members of my committee, Dr. Fran Walley and Dr. Ken Van Rees. And a special thanks to my external examiner Dr. Chris Yost for his comments and suggestions. I would like to thank my friends in the Soil Microbiology lab for not only their technical support but also for all the great stories. A very special thanks to Jorge Cordero, Eduardo Mitter, Panchali Katalunda, and Sarah Kuzmicz for always having time to answer my many questions. A special thanks to everyone associated with the Forest Watershed and Riparian Disturbance (FORWARD) Project and to Alberta Culture for allowing me access to the Bitumount Provincial Historic site. Lastly, I would like to thank my family and friends for their support in this endeavour. This research is funded through a collaborative research and development grant between the Natural Sciences and Engineering Research Council of Canada (NSERC) and the FORWARD project based out of Lakehead University, ON. Partners and supporting organizations of the FORWARD project include Canada’s Oil Sands Innovation Alliance (COSIA), Suncor Energy Inc., Canadian Natural Resources Limited, Total E&P Canada Ltd., Syncrude Canada Ltd., Alberta Newsprint Company, Alberta Pacific Forest Industries, Hinton Pulp, Millar Western Forest Products, Slave Lake Pulp, Tervita Corporation, Environment Canada, Oil Sands Research and Information Network, and Alberta Innovates Technology Futures. iv TABLE OF CONTENTS PEMISSIONTO USE ................................................................................................................... i ABSTRACT .................................................................................................................................. ii ACKNOWLEDGMENTS ........................................................................................................... iv TABLE OF CONTENTS ............................................................................................................. v LIST OF TABLES .................................................................................................................... viii LIST OF FIGURES ...................................................................................................................... ix LIST OF ABBREVIATIONS ...................................................................................................... x 1.0 INTRODUCTION ................................................................................................................. 1 2.0 LITERATURE REVIEW ....................................................................................................... 5 2.1 Oil Sands ..................................................................................................................... 5 2.1.1 Athabasca oil sands ...................................................................................... 5 2.1.2 Bitumount Historic Site ............................................................................... 6 2.2 Contaminated Soils .................................................................................................... 8 2.2.1 Hydrocarbon contaminated soils ................................................................. 8 2.2.2 Reclamation ................................................................................................ 9 2.3 Endophytes ................................................................................................................. 10 2.3.1 Root microbiome ........................................................................................ 10 2.3.2 Endophyte colonization mechanisms ........................................................... 11 2.3.3 Diversity ...................................................................................................... 12 2.3.4 Abundance .................................................................................................. 13 2.3.5 Plant growth promoting abilities ................................................................. 14 2.3.6 Microbial community structure in contaminated soils ................................ 15 2.3.7 Hydrocarbon degradation ............................................................................ 17 2.3.8 Diversity of hydrocarbon degrading bacteria ............................................. 18 2.4 Scope of Work ........................................................................................................... 19 3.0 ENDOPHYTIC ROOT BACTERIA ASSOCIATED WITH THE VEGETATION GROWING ON THE BITUMOUNT HISTORIC SITE ............................................................. 20 3.1 Preface ........................................................................................................................ 20 3.2 Abstract ...................................................................................................................... 21 3.3 Introduction ...............................................................................................................
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