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Control of Fungal Pathogens of Triticum Aestivum Using Endophytic Fungi and Non-Conventional Fungicides

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Control of Fungal Pathogens of Triticum Aestivum Using Endophytic Fungi and Non-Conventional Fungicides CONTROL OF FUNGAL PATHOGENS OF TRITICUM AESTIVUM USING ENDOPHYTIC FUNGI AND NON-CONVENTIONAL FUNGICIDES by Abdurraouf Sadek Abaya A Thesis presented to The University of Guelph In partial fulfilment of requirements for the degree of Doctor of Philosophy in Environmental Sciences Guelph, Ontario, Canada © Abdurraouf Abaya, April, 2020 ABSTRACT CONTROL OF FUNGAL PATHOGENS OF TRITICUM AESTIVUM USING ENDOPHYTIC FUNGI AND NON-CONVENTIONAL FUNGICIDES Abdurraouf Sadek Abaya Advisor: University of Guelph, 2020 Dr. Tom Hsiang Wheat diseases result in major losses worldwide. The purpose of this research was to evaluate the effects of endophytic fungi or non-conventional fungicides on suppressing wheat pathogens. Endophytic fungal isolates recovered from wheat were found to be antagonistic to the fungal pathogens Fusarium graminearum, Microdochium majus, or Waitea circinata in dual culture experiments. A hyphal plug sandwich of three selected antagonistic endophytic fungi inhibited the severity of diseases caused by Fusarium graminearum (36-80%) and Waitea circinata (31-80%) on wheat plants in growth room tests. Agar inoculum of Simplicillium lamellicola or Clonostachys rosea was applied to an upper or lower leaf four days before agar inoculum of F. graminearum, resulted in systemically induced disease resistance in the pathogen-inoculated leaf, and reduced lesion lengths by 68-70% in growth room tests and 43- 49% in outdoor garden tests. S. lamellicola or C. rosea added to soil before seeding or seeds soaked in biocontrol increased the growth of Triticum aestivum at the seedling stage compared with untreated control by up to 22%. Spore suspensions of S. lamellicola or C. rosea significantly reduced Fusarium head blight (FHB) in the growth room tests (58-66%), outdoor garden tests (67-74%), and field tests (71-76%). Treatments with 1% humic acid, 1% phosphite, 30 mM para-aminobenzoic acid, 10 mM salicylic acid, or 2% Civitas + 0.2% Harmonizer on five-week-old plants significantly reduced foliar disease in growth room tests by 67-96%. For adult stage tests, these non-conventional fungicide treatments were sprayed three hours before the spore suspension of F. graminearum was applied to wheat spikes in the growth room, and these compounds significantly reduced FHB by 27-92% on the moderately resistant cultivar ‘Glenn’ and by 40-72% on the susceptible cultivar ‘Wilkin’. For outdoor garden tests, non- conventional fungicides were sprayed three days before inoculating the spike with the F. graminearum and significantly reduced FHB by 54-95%. In field trials, 1% humic acid significantly reduced FHB index % on three wheat cultivars, ‘Wilkin’, ‘Glenn’, and ‘AAC Scotia’ by 54%, 69%, and 80%, respectively. S. lamellicola and non-conventional fungicide treatments are promising for control of diseases caused by F. graminearum and other wheat pathogens. ACKNOWLEDGEMENTS First of all, I would like to express my honest gratitude to my advisor Prof. Tom Hsiang for the continuous support of my Ph.D. study and research, for his patience, motivation, enthusiasm, and immense knowledge. His guidance helped me in all the time of research and writing of this thesis. It was a great privilege and honor to work and study under his guidance. I am extremely grateful for what he has offered me. I would also like to thank him for his friendship, empathy, and great sense of humor. I am also very thankful for the opportunities I have had to participate in several conferences throughout my time as a Ph.D. student. Besides my advisor, I would like to thank the rest of my thesis committee: Dr. Allen Xue, Dr. Ali Navabi, and Dr. Mitra Serajazari, for their encouragement, insightful comments, and guidance. Many thanks again to Mitra Serajazari, and Nicholas Wilker for their assistance in the field studies. I am also grateful to undergraduate student assistants in the Hsiang Lab, including Hyo Choi, Mina Nasar-Sharif, Aysu Gundogan, and Vivian Forte-Perri for their assistance inside and outside the lab. I am very much thankful to my wife and my sons for their love, understanding, prayers and continuing support to complete this research work. Thank you to my sons Sadek and Mohamed for their help in the field. I am extremely grateful to my brothers and sisters for their support and valuable prayers. I also owe a great debt of gratitude to the Ministry of Education in Libya for their support and sponsorship throughout my study. Finally, my thanks go to all the people who have supported me to complete the research work directly or indirectly. iv TABLE OF CONTENTS ABSTRACT .................................................................................................................................... ii ACKNOWLEDGEMENTS ........................................................................................................... iv LIST OF TABLES ....................................................................................................................... viii LIST OF FIGURES ..................................................................................................................... xiv LIST OF ABBREVIATIONS AND ACRONYMS .................................................................... xxi Chapter 1: Literature Review .......................................................................................................... 1 1.1. Introduction .......................................................................................................................... 1 1.1.1. Wheat and its importance .............................................................................................. 1 1.1.2. Major diseases and pathogens of wheat ........................................................................ 2 1.1.3. Fusarium graminearum ................................................................................................. 3 1.1.4. Microdochium majus ..................................................................................................... 4 1.1.5. Waitea circinata ............................................................................................................ 5 1.2. Biological control of plant diseases using beneficial fungi .................................................. 5 1.2.1. Endophytes .................................................................................................................... 6 1.3. Non-Conventional fungicides .............................................................................................. 8 1.4. Hypotheses and objectives ................................................................................................... 9 Chapter 2: Selection and Screening of Endophytes and Non-Conventional Fungicides Against Wheat Pathogens ........................................................................................................................... 11 2.1. Introduction ........................................................................................................................ 11 2.2. Objectives ........................................................................................................................... 14 2.3. Materials and methods ....................................................................................................... 15 2.3.1. Fungal isolates ............................................................................................................. 15 2.3.2. Effect of endophytic fungi against wheat pathogens in dual culture plates ................ 15 2.3.3. Potential phytopathogenicity of endophytes ............................................................... 16 2.3.3.1. Plant preparation and seedling inoculation ........................................................... 16 2.3.4. Molecular identification .............................................................................................. 16 2.3.4.1. Polymerase chain reaction (PCR) ......................................................................... 17 2.3.5. Biological activity against wheat diseases on plants in the growth room ................... 17 2.3.5.1. Wheat planting and seedling inoculation .............................................................. 17 2.3.6. Non-Conventional fungicides to control wheat diseases in the growth room ............. 18 2.3.7. Sensitivity test of wheat pathogens to non-conventional fungicides ........................... 18 2.3.8. Hyphal staining for wheat-biocontrol-pathogen interactions ...................................... 18 v 2.3.8.1. Plant material and inoculations ............................................................................. 18 2.3.8.2. Sample collection, staining, and scoring ............................................................... 19 2.3.9. Data analyses ............................................................................................................... 19 2.4. Results ................................................................................................................................ 19 2.4.1. Isolation of endophytic fungi ....................................................................................... 19 2.4.2. Screening of fungal isolates in dual culture against selected wheat pathogens........... 20 2.4.3. Molecular characterization and identification of endophytic fungal isolates .............. 21 2.4.4. Biological or chemical activity against wheat diseases on plants in the growth room 21 2.4.5. Direct sensitivity test ..................................................................................................
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