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Molecular and Ecological Studies of Fungal Biodiversity on Durum Wheat Grown In View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by University of Saskatchewan's Research Archive Molecular and ecological studies of fungal biodiversity on durum wheat grown in rotation with pulses and canola 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 and Bioproduct Sciences University of Saskatchewan, Saskatoon By Delia Mavragani Copyright Delia Mavragani, July 2008. All Rights Reserved PERMISSION 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 this 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 which may be made of any material in my thesis. Requests for permission to copy or to 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) i ABSTRACT Fungi contribute to key processes in the sustainable function of terrestrial ecosystems including nutrient cycling and transport of water to plants. However, some fungal species are of interest because their infection of a susceptible host crop results in diseases negatively affecting food supply and quality. These diseases are expected to be influenced by rotation crops which could impact the development of plant pathogens and their genetic biodiversity. The objectives of this study were to assess the biodiversity of fungal pathogens in durum wheat, to identify the naturally occurring fungi that could have biocontrol potential, and to define the impact of crop rotation with non-cereal crops on fungal populations in durum wheat. For this purpose, in 2004, 2005, and 2006, soil and durum wheat plant samples were collected after preceding crops of Pisum sativum L. (pea), Lens culinaris Medik (lentil), Cicer arietinum L. (chickpea), Brassica napus L. (canola) and Triticum turgidum L. (durum) in a long term experimental site in Swift Current, Sakatchewan. Samples were analyzed using a combination of traditional cultivation techniques and polymerase chain reaction (PCR), sequencing, and denaturing gradient gel electrophoresis (DGGE) techniques. Fusarium species, known as the causal agent of Fusarium head blight (FHB) and Fusarium damaged kernels (FDK) were among the most ubiquitous and abundant in durum tissues. The most prevalent of all Fusarium at the study site were F. avenaceum, F. reticulatum, and F. tricinctum. Other recovered potential fungal pathogens belonged to the genera Bipolaris, Phaeosphaeria, Pyrenophora, Cladosporium, Epicoccum, Alternaria, Cladosporium, Arthrinium, Nigrospora, and ii Microdochium. Principal component analysis revealed negative correlations between Acremonium, Chaetomium, Penicillium, and pathogenic Fusarium, Bipolaris, Pyrenophora, and Alternaria. These isolates could be antagonistic, and their potential as biocontrol agents against pathogens colonizing durum wheat in the semiarid Saskatchewan should be assessed. Crop rotation had a limited impact on the abundance of fungal pathogens. Fusarium torulosum was less abundant in durum following canola while Bipolaris sorokiniana was less abundant in durum following pea. Even if no single crop rotation reduced significantly the prevalence of F. avenaceum in durum wheat, results suggest that a successful control of this important pathogen requires an integrated approach using diversified rotations. iii ACKNOWLEDGMENTS This dissertation is dedicated to my mother for her understanding and support of everything that I wanted to do in life. I would not have been able to complete this research without the guidance and encouragements of my supervisor Dr. Vladimir Vujanovic and co-supervisor Dr. Chantal Hamel and I thank them both for being wonderful mentors. I would also like to offer sincere thanks to my committee members Drs. Darren Korber, Xiao Qiu and George Khachatourians, your guidance was appreciated. I gratefully acknowledge financial assistance from Alberta Pulse Growers, Agriculture and Agri-Food Canada Swift Current Research Station and University of Saskatchewan. Technical directions from Dr. Prasad Daida were invaluable and appreciated. My fellow lab mates - Lobna Abdelatief and Yit Kheng Goh - deserve special mentions because they kept the lab in a friendly and enjoyable atmosphere. iv TABLE OF CONTENTS 1. INTRODUCTION.............................................................................................. 11 1.1 Study rationale…………………………………………………………………4 2. LITERATURE REVIEW ................................................................................. 15 2.1 The biodiversity of pathogenic fungi in wheat crops.................................... 15 2.1.1 Fungi causing crown and root rot diseases........................................... 15 2.1.2 Fungi causing leaf spotting diseases...................................................... 16 2.1.2.1 Biodiversity of Pyrenophora spp. on cereals............................. 17 2.1.3 Fungi causing kernel diseases ................................................................ 21 2.1.3.1 Biodiversity of Fusarium spp. on cereals .................................. 22 2.2 Methods to control pathogenic fungi in wheat crops.................................... 27 2.2.1 Crop rotation to control pathogens’ inocula ........................................ 28 2.2.2 Beneficial fungi........................................................................................ 33 2.3 Molecular tools to study the diversity of Fusarium and Pyrenophora spp. 36 3. FUNGAL DIVERSITY ASSOCIATED WITH DURUM WHEAT PRODUCTION CHRONOSEQUENCES, PLANT ORGANS AND PRECEDING CROPS........................................................................................... 42 3.1 Introduction...................................................................................................... 42 3.2 Objectives and hypothesis ............................................................................... 43 3.3 Materials and methods .................................................................................... 44 3.3.1 Site description........................................................................................ 44 3.3.2 Soil and plant sampling .......................................................................... 47 3.3.3 Determination of fungal biodiversity .................................................... 48 3.3.4 Statistical analyses .................................................................................. 50 3.4 Results ............................................................................................................... 51 3.4.1 Fungal biodiversity in soil and plant organs ........................................ 52 3.4.2 Fungal biodiversity as related to durum phenophases........................ 60 3.4.3 Fungal biodiversity as related to preceding crops ............................... 62 3.4.4 Relationships among fungi..................................................................... 63 3.5 Discussion.......................................................................................................... 67 3.5.1 Interactions among fungi ....................................................................... 67 3.5.2 Impact of preceding crops on pathogenic fungal species .................... 71 3.5.3 Fungal biodiversity ................................................................................. 71 v 4. DIVERSITY OF FUSARIUM SPECIES AND THE EFFECT OF PRECEDING CROPS OF PULSES AND CANOLA ON THEIR PREVALENCE IN DURUM WHEAT................................................................ 76 4.1 Introduction...................................................................................................... 76 4.2 Objectives and hypothesis ............................................................................... 77 4.3 Materials and methods .................................................................................... 78 4.3.1 Experimental design and sampling ....................................................... 78 4.3.2 Determining the biodiversity of Fusarium using a PCR-DGGE procedure.......................................................................................................... 80 4.3.3 Determining the biodiversity of Fusarium using culture-dependent, polymerase chain reaction, and sequencing methods................................... 81 4.3.4 Statistical analyses .................................................................................. 83 4.4 Results ............................................................................................................... 84 4.4.1 Impact of preceding crops...................................................................... 84 4.4.2 Fusarium biodiversity
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