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Study of Biocontrol Mechanisms Involved Chemical and Biological Control of SclerotiniaDiseases in Sunflower and Study of Biocontrol Mechanisms Involved by Sarangi Nirosha Priyajeevani Athukorala A Thesis Submitted to the Faculty of Graduate Studies of The University of Manitoba in partial fulfillment of the requirements of the degree of MASTER OF SCIENCE Department of Plant Science University of Manitoba Wirmipeg, Manitoba Copynght O 2008 by Sarangi Nirosha Priyajeevani Athukorala THE T]NIVERSITY OF MANITOBA FACULTY OF GRADUATE STIJDMS ¡k*tr*rt COPYRIGHT PERMISSION Chemical and Biological Control of Sclerotini¿ Diseases in Sunflower and Study of Biocontrol Mechanisms Involved BY Sarangi Nirosha Priyajeevani Athukorala A ThesislPracticum submitted to the Faculty of Graduate Studies of The University of Manitoba in partial fulfillment of the requirement of the degree of MASTER OF SCIENCE Sarangi Nirosha Priyajeevani Athukorala @ 2008 Permission has been granted to the University of Manitoba Libraries to lend a copy of this thesis/practicum, to Library and Archives Canada (LAC) to lend a copy of this thesiiipracticum, and to LAC's agent (IMlÆroQuest) to microfilm, sell copies and to publish an abstract of this thesis/practicum. This reproduction or copy of this thesis has been made available by authority of the copyright owner solely for the purpose of private study and research, and may only be reproduced and copied as permitted by copyright laws or with express written authorization from the copyright owner. ACKNOWLEDGEMENTS Firstly, I would like to be very grateful to my advisor, Dr. Dilantha Fernando, who took all the steps to give me this opportunity to study as a graduate student and plant pathologist under an advanced environment. I really appreciate the support, guidance he gave me and the faith he had in me during this two year period. I am also very thankful to my co-advisor, Dr. Khalid Rashid who also joined with Dr. Fernando to give me this opportunity and for the guidance and encouragements he gave throughout this project. I also thank my committee members, Dr. Teresa de Kievit and Dr. Mario Tenuta, who helped me in different ways especially in clarifying my results. My special gratitude goes to my former advisor Dr. Charmalie Abayasekara, who inspired and motivated me to continue higher studies and for her endless encouragements, friendship and love offering to me throughout my life. Without her this degree would not have been a reality. Secondly, I should be very thankful to my loving husband Thilanka Jayasekera, who went through so many difficulties and sacrificed his future and dreams for my success. Without his support and encouragements this degree would not have been possible. I cannot forget our families who allowed us to come here far away from home despite the strong bondage we have together as families. Special thanks should go to my loving old parents who always wanted to see me doing higher studies and for bearing my absence around them. I would also like to thank The National Sunflower Association of Canada, University of Manitoba Intemational Graduate Student Entrance Scholarship and Manitoba Graduate Scholarship for Master's students for the financial support they provided throughout this project. I should extend a special thanks to Paula Parks and Rajesh Ramarathnam. Paula's assistance in my field experiments is really appreciated and thank you for bearing with my inexperience in field work. Your motherly advice was really helpful in developing my research skills and I enjoyed your company and friendship in the lab. Raj, Thank you very much for answering endless questions I had and without your help in the lab and füendship my project would not have succeeded. I would also like to thank my lab colleagues, Ru Li, Xiaowei Guo and my office mates Benil Sabel, Hassna Alkher for sharing the difficult moments I have faced and for their friendship. Finallly, I am thankful to Wayne Buchanon for the GC-MS analysis, Qian Yuwei for the MALDI-TOF analysis, Tricia Cabernel and Maurice Penner for taking care of my field trials at Morden and all the summer sfudents for their help. TABLE OF CONTENTS Page ACKNOV/LEDGEMENTS.......... .........,.. ii LIST OF TABLES ................vii LIST OF FIGURES ..,.,..,.....viii GENERAL 48STR4CT............... ........... x FORW4RD............... ...,........ xii 1.0 INTRODUCTION ............ 1 2.0 LITERATURE REVIEW ...................6 2.1 Sunflower: Helianthus annuLts L............... .................... 6 2.1 . 1 History .............. ...........,..... 6 2.1.1.1 Sunflower in Canada ........,..,...... 10 2.1.2 Agronomy and Production........... .,.... 11 2.1.3 Uses. ....,..,....... 16 2.1.3.1 Oil-type Sunflower Cultivars .....17 2.l.3.2ConfectionaryTypeSunflowerCultivars ......................18 2.1.4 Diseases of Sunflower ..... 19 2.2 Sclerotinia sclerotíorum (Lib.) de Bary ......................20 2.2.7 Taxonomy and Nomenclature....... .....20 2.2.2Host Range........ ...............22 2.2.3 Geographical Distribution and Prevalence ......,...23 2.2.4 Life Cycle, Diseases and Epidemiology....... .......24 2.2.5 Economic Importance........ ........,....... 33 2.3 Management of Sclerotinia sclerotiorum........... ......... 35 2.3.1 Cultural Management......... ...,......,.,..35 2.3.2 Genetic Resistance.............. .......,.......38 2.3.3 Chemical Treatments........... .............. 39 Z.3.4Biological Control.............. .,.............41 2.3.4.1 Mechanisms of Control ..............43 2.3 .4.1. 1 Biosynthetic Genes. ...... ... .. 48 2.3.4.7.7.1 Pseudomones Species and Antibiotic Biosynthetic Genes .........49 2.3 .4.1 .l .2 B aci l/zzs Species and Antibiotic Biosynthetic Genes ,........50 2.3.4.2 Biological Control of S. sclerotiorum ..........5l 111 Page 2.3.4.3 Commercialization and Future Research Directions .....54 3.0 Field Investigation of the Efficacy of Two Biocontrol Agents and a Fungicide Against Sclerotinia Diseases of Sunflower in Canada................ ............ 57 3.1 Abstract...... .................57 3.2 Introduction. ................... 58 3.3 Materials and Methods ............62 3.3.1 Experimental Site, Design and Agronomy............ ................ 62 3.3,2 Biocontrol Agents and Culture Conditions .......... 63 3.3.3 Head rot Trial .................63 3.3.3.1 Preparation of Biocontrol Agents .............,.. 63 3.3.3.2 Preparation of Pathogen Inoculum .........,.....63 3.3.3.3 Application of Fungicide and Biocontrol 4gent.,...... ......................64 3.3.3.4lnoculation of the Pathogen .......64 3.3.3.5 Survival of Biocontrol Agents .,.,...........,.....65 3.3.3.6 Disease Assessment and Data Analysis ....... 65 3.3.4 Wilt Trial....... .......... ......66 3.3.4.1 Application of Biocontrol Agent and the Fungicide. .....66 3.3.4.2 Application of the Pathogen... ......................67 3.3.4.3 Survival of the Biocontrol Agents ...............67 3.3.4.4 Disease Assessment and Data Analysis .......68 3.3.4.5 Growth Promotion Trial.......... .....................68 3.4 Results .........69 3.4.7 Head Rot Trial .................69 3.4.2Wi1t Trial. ......70 3.4.3 Growth Promotion Trial ....................75 3.4.4 Survival of Antagonists at the Sites of Inoculation ...............79 3.5 Discussion... ...................79 4.0 Pseudomonqs chlororaph¿s Strain PA23 Antagonistic to Soil-Borne Plant Pathogens and the Role of Volatile and Non-Volatile Antibiotic Production in Its Root Colonization and Biocontrol Ability ...,............ 88 4.1 Abstract. .. ................. 88 4.2 Introduction. ...................89 4.3 Materials and Methods ............92 4.3.7 Bacterial Strains, Fungal Strain and Culture Conditions................ .........92 4.3.2 Laboratory Experiment................ ...... 93 4.3.2.1 Effect of Bacterial Volatiles on Mycelial Growth of S. sclerotiorum 1V Page 110 110 114 5.0 Identification of Antifungal Antibiotics of Bacil/øs Species Isolated from Different Microhabitats Using Polymerase Chain Reaction and MALDI-TOF Mass Spectrometry ............. ...125 5.1 4bstract................. ................. 125 5.2 Introduction ......... ..................126 5.3 Materials and Methods ..........129 5.3.1 Bacterial Isolates and Culture Conditions ............. .............. 129 5.3.2 DNA Extraction and Quantification ..................I29 5.3.3 PCR Analysis.... .............132 5.3.4 Purification, Sequencing and n-Blast Search of PCR Products............. 133 5.3.5 MALDI-TOF-MS Analysis of the Antibiotics Present in the Page Cell-surface Extracts of Bacterial Strains B. amy lo liquifaci ens B56, B. subtílis 3057 and B. mycoides 4079 ..............134 5.4 Results ................ 136 5.4.1 PCR Analysis and BLAST Search .................... 136 5.4.2MALDI-TOF MS Analysis............... ................. 138 5.5 Discussion............. .................144 6.0GENERALDISCUSSIONANDCONCLUSIONS............... ........,.....150 7.0 LITERATURE CITED ...................157 vi LIST OF TABLES Table Page 2.I Sunflower growth stages........ .......14 3.1 Disease severity index (DSD, AUDPC and yield from 2006 head rot trial and percent emetgence from wilt trial carried out at Morden Research Centre, MB, Canada ................'. .......13 3.2 Disease severity index (DSI), AUDPC and yield from 2007 head rot trial and percent emergence from wilt trial carried out at Morden Research Centre, MB, Canada '......74 3.3 Average growth parameters measured from 2007 trial carried out at Morden Research Centre, MB, Canad4................ .........77 3.4 Average growth parameters measured ftom 2007 trial
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