Molecular Profiling of Fungal Functional Groups Associated With
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ECOLOGICAL AND MOLECULAR STUDIES OF FUNGAL COMMUNITIES ASSOCIATED WITH ROOTS OF SALIX SPP. GROWN UNDER HIGH DENSITY, SHORT ROTATION INTENSIVE CULTURE 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 Food & Bioproduct Sciences University of Saskatchewan Saskatoon By Aura Helena Corredor © Copyright, Aura Helena Corredor, January 2011. All rights reserved. PERMISSION TO USE In presenting this thesis in partial fulfilment of the requirements for a Postgraduate degree from the University of Saskatchewan, I agree that the Libraries of this University may make it freely available for inspection. I further agree that permission for copying 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 part 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 which may be made of any material in my thesis. Requests for permission to copy or make other use of material in this thesis in whole or part should be addressed to: Head of the Department of Food and Bioproduct Sciences University of Saskatchewan Saskatoon, Saskatchewan S7N 5A8 Canada i ABSTRACT In Canada, willow (Salix spp.) short rotation intensive cultures (SRIC) have been established to investigate their potential to produce biomass for bioenergy. Since root-associated fungal communities are involved in plant nutrition and disease susceptibility, it is relevant to understand their interactions with willows and their role on the sustainability of SRIC. In this project traditional methods together with molecular techniques were used to: i) assess the diversity of fungal communities in roots of willows and their relationship with major characteristics of SRIC ii) evaluate the effects on plant development of potentially beneficial and pathogenic fungi, and iii) identify arbuscular mycorrhizal fungal (AMF) species and their interaction with promising Salix clones. Potentially pathogenic fungi were more dominant in diseased and recently planted cuttings than in healthy older ones. This suggested the occurrence of a positive shift prompted by the establishment of the cuttings on the site. Gibberella/Fusarium sp. and Neonectria sp. were the most dominant taxa particularly in diseased plants. Under greenhouse conditions the inoculation with potentially beneficial and potentially pathogenic fungi induced significant differences in root biomass but not in overall aerial biomass production. PCR-denaturing gradient gel electrophoresis was successfully standardized and used to identify arbuscular mycorrhizal fungal species associated with different clones. The results indicated a degree of specificity between AMF species and plant genotype. Root-associated fungal communities appear to be useful to monitor the impacts of SRIC on soil ecology and their study may enlighten effective ways to increase the productivity of these biomass systems. ii ACKNOWLEDGEMENTS I would like to express my gratitude to my supervisor, Dr. Vladimir Vujanovic, who gave me the opportunity of joining his team and learning from his experience in mycology. I also appreciate the feedback of the members of my Advisory Committee: Dr. Ken van Rees (Dept. Soil Science), Dr. Darren Korber (Dept. of Food and Bioproduct Sciences) and Dr. Takuji Tanaka (Dept. of Food and Bioproduct Sciences). I am very grateful also to my external evaluator, Dr. Jeff Schoenau (Dept. of Soil Science), for his time and the valuable suggestions to improve the final document. This project was funded by a NSERC grant awarded to my supervisor. I also received financial support through the Dollie Hantelman Scholarship, the Devolved Scholarship of the Dept. of Food and Bioproduct Sciences, and the Molson Canada Scholarship. I want to acknowledge also the support of Dr Vujanovic’s team as well as the kindness of all my friends in the FABS department throughout this journey. Thanks also to my family for their infinite love and patience. iii TABLE OF CONTENTS ABSTRACT .................................................................................................................................... ii ACKNOWLEDGEMENTS ........................................................................................................... iii TABLE OF CONTENTS ............................................................................................................... iv LIST OF TABLES ......................................................................................................................... vi LIST OF FIGURES ...................................................................................................................... vii 1. INTRODUCTION .................................................................................................................. 1 2. LITERATURE REVIEW ....................................................................................................... 5 2.1. Willow Crops for Biomass Production .................................................................................... 5 2.2. Fungal Functional Groups Associated with Roots of Salix spp. .............................................. 7 2.2.1. Fungal Pathogens .................................................................................................................. 9 2.2.2. Mycorrhizas ...................................................................................................................... 10 2.2.3. Other Beneficial Endophytes .............................................................................................. 12 2.3. Denaturing Gradient Gel Electrophoresis (DGGE) in Fungal Studies .................................. 13 3. FUNGAL DIVERSITY ASSOCIATED WITH ROOTS OF WILLOWS GROWN UNDER SHORT ROTATION INTENSIVE CULTURE .................................................................... 17 3.1. Abstract ........................................................................................................................... 17 3.2. Introduction .................................................................................................................... 17 3.3. Objectives and Hypothesis ............................................................................................. 19 3.4. Materials and Methods ................................................................................................... 20 3.4.1. Study Sites ............................................................................................................ 20 3.4.2. Experimental Design and Sampling ..................................................................... 20 3.4.3. Data Collection .................................................................................................... 22 3.4.4. Identification of Fungal Taxa ............................................................................... 22 3.4.5. Statistical Analyses .............................................................................................. 23 3.5. Results ............................................................................................................................ 24 3.5.1. Fungal Diversity in the Roots of Willows Grown under SRIC ........................... 24 3.5.2. Taxonomical Groups of Fungi Associated with Willow Roots ........................... 27 3.5.3. Composition of Fungal Communities in Relation to Year of Establishment, Province and Plant Health Status ........................................................................ 28 3.5.4. Fungal Communities in Different Willow Clones ............................................... 35 iv 3.6. Discussion ......................................................................................................................... 38 4. EFFECTS OF POTENTIALLY BENEFICIAL AND PATHOGENIC FUNGI IN ROOTS OF WILLOW (SALIX PURPUREA) ..................................................................................... 47 4.1. Abstract ......................................................................................................................... 47 4.2. Introduction ................................................................................................................... 47 4.3. Objectives and Hypothesis ............................................................................................ 48 4.4. Materials and Methods .................................................................................................. 49 4.4.1. Pathogenicity Potential ....................................................................................... 49 4.4.2. Antagonistic Potential ........................................................................................ 49 4.4.3. Plant Material for Greenhouse Test .................................................................... 50 4.4.4. Inoculation of Cuttings and Experimental Design ............................................. 50 4.4.5. Harvesting .......................................................................................................... 51 4.4.6. Data Analyses ....................................................................................................