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Biocontrol of Sclerotinia Stem Rot of Canola by Bacterial Antagonists and Study of Biocontrol Mechanisms Involved

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Biocontrol of Sclerotinia Stem Rot of Canola by Bacterial Antagonists and Study of Biocontrol Mechanisms Involved BIOCONTROL OF SCLEROTINIA STEM ROT OF CANOLA BY BACTERIAL ANTAGONISTS AND STUDY OF BIOCONTROL MECHANISMS INVOLVED by Yilan Zhang A Thesis Submitted to the Faculty of Graduate Studies In Partial Fulfillment of the Requirements for the Degree of MASTER OF SCIENCE Department of Plant Science University of Manitoba Winnipeg, Manitoba, Canada © Yilan Zhang 2004 THE UNIVERSITY OF MANITOBA FACULTY OF GRADUATE STUDIES ***** COPYRIGHT PERMISSION PAGE Biocontrol of Sclerotinia Stem Rot of Canola by Bacterial Antagonists and Study of Biocontrol Mechanisms Involved By Yilan Zhang A Thesis/Practicum 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 Yilan Zhang © 2004 Permission has been granted to the Library of the University of Manitoba to lend or sell copies of the this thesis/practicum, to the National Library of Canada to microfilm this thesis and to lend or sell copies of the film, and to the Univesity Microfilm Inc. to publish an abstract of this thesis/practicum. The author reserves other publication rights, and neither this thesis/practicum nor extensive extracts from it may be printed or otherwise reproduced without the author’s written permission. ACKNOWLEGEMENTS Fist of all, I would like to express my appreciation to my supervisor Dr. Dilantha Fernando for his guidance in my experiments, and support given at conference presentations and applying for several travel awards. Only with your patience, kindness and encouragement I could grow as a student and plant pathologist. I also appreciate the help of my co-supervisor Dr. Fouad Daayf, for the advice and support for my project during my Masters program. I would also like to thank both of you for your great input on my thesis writing. To two of my other committee members, Dr. Annemieke Farenhorst and Dr. Georg Hausner, thank you very much for your great effort on advising me in my project and thesis. Special thanks to Paula Parks, Alvin Iverson and Lorne Adam for your technical support. Also a big thank you to my lab mates for your continuous support and help. Special thanks to Clinton Jurke for providing breeder tents and some of the pictures. A very special thank you to Dr. Teresa de Kievit and Chrystal Berry for the work on southern blots. I thank Dr. Tim Paulitz for giving me the idea to investigate bacterial antibiotic-related genes using PCR. I also appreciate the staff and fellow graduate students in Plant Science who helped me on my project and shared the fun at BBQ’s and parties… I would like to thank my parents for their never changed love and support. The mountains and oceans would never separate our hearts being close to each other. Only when I live far away from you, I realize how much you sacrificed in making me an independent person. I also would like to thank the life itself. I had fully experienced the interesting, exciting and frustrations in life in Canada in the past two years. I survived and I did a good job! Nothing to regret! II TABLE OF CONTENTS ACKNOWLEDGEMENTS ………………………………………………………… II TABLE OF CONTENTS ...…………………………………………...…………… III LIST OF TABLES .………………..………………………………….…………….VI LIST OF FIGURES ………………………………………………………….……..VII ABSTRACT ……………………………………………………………….……..…IX FORWARD ……………………………………………………………….………...XI 1.0 INTRODUCTION ……………………………………………….…………...1 2.0 LITERATURE REVIEW ……………………………………………….…….4 2.1 Canola………………………………………………………………………4 2.1.1 Crop History……………………………………………………...4 2.1.2 Crop usage…….………………...…………………………….….6 2.1.3 Transgenic canola………………………………………………...7 2.1.4 Production and economic importance……..……………………..8 2.1.5 Growth stages and condition……………………………………..9 2.1.6 Diseases on canola……………………………………………….10 2.2 Sclerotinia sclerotiorum ……………………………………………….….11 2.2.1 Introduction………………………………………………………11 2.2.2 Taxonomy………………………………………………………..12 2.2.3 Host range..………………………………………………………12 2.2.4 Economic importance……………………………………………13 2.2.5 Disease cycle, infection and symptomology…………………….13 2.2.6 Epidemiology……………………………………………….……17 2.3 Management of S. sclerotiorum on canola…………………….…………..18 2.3.1 Host resistance…………………………………………………...18 2.3.2 Cultural management…………………………………………….18 2.3.3 Disease forecasting…..………………………………………..…19 2.3.4 Chemical control…………………………………………………20 2.3.5 Biological control……………………………………………….. 20 2.4 Biological control……………………………………………………….….20 2.4.1 Introduction…………………………………………….…………20 2.4.2 Biocontrol mechanisms…………………………………………..21 2.4.2.1 Antibiosis……………………………………………….21 2.4.2.1.1 Introduction……….….…………………………21 2.4.2.1.2 Major bacterial antibiotics in biological control..22 2.4.2.1.3 Identification and characterization of antibiotic- related genes / gene clusters…………………….25 2.4.2.2 Plant induced resistance………………………….….…26 III 2.4.2.2.1 Introduction…………………………………….26 2.4.2.2.2 Induced systemic resistance…………….….…..27 2.4.2.2.3 Systemic acquired resistance…………………...28 2.4.2.2.4 Plant defence-related secondary metabolites…...28 2.4.2.2.5 Plant phenolics………………………………….30 2.4.2.3 Plant growth promoting rhizobacteria …………………31 2.4.2.4 Competition…………………………………………….31 2.4.2.5 Parasitism and predation……………………………..…32 2.4.2.6 Other mechanisms……………………………………...33 2.4.3 Biocontrol of S. sclerotiorum…………………………………….34 3.0 EVALUATION OF BIOLOGICAL CONTROL OF SCLEROTINIA SCLEROTIORUM ON CANOLA (BRASSICA NAPUS) BY BACTERIA THROUGH IN VITRO SCREENINGS, GREENHOUSE EXPERIMENTS, AND A FIELD STUDY……….……………………………………………….38 3.1 Abstract……………………………………………………………………..38 3.2 Introduction…………………………………………………………………39 3.3 Materials and methods……………………………………………………...41 3.3.1 Bacillus spp. isolation and storage……………………………...41 3.3.2 Evaluation of bacterial antagonism through general plate inhibition assays…………………………………………………42 3.3.3 Antagonism through production of volatile compounds………..43 3.3.4 Testing for the presence of the oxalate oxidase enzyme………..44 3.3.5 Bacterial indentification………………………………………...44 3.3.6 Production of rifampicin-resistant strains and establishing of growth curves…………………………………………………..45 3.3.7 Evaluation of biocontrol agents against sclerotinia on canola in the greenhouse………………………………………………..46 3.3.8 Evaluation of the effect of time of application of Bacillus spp. BS6 against sclerotinia on canola and bacterial survival in the greenhouse………………………………………………………47 3.3.9 Evaluation of biocontrol agents under field conditions…………48 3.4 Results………………………………………………………………………51 3.4.1 Bacterial isolation and identification…………………………..51 3.4.2 Evaluation of bacterial antagonism through general plate inhibition assays, inhibitory volatile production and oxidase enzyme presence……………………………………………….51 3.4.3 Production of rifampicin-resistant strains and establishing of growth curves………………………………………………….55 IV 3.4.4 Evaluation of biocontrol agents against sclerotinia on canola in the greenhouse……………………………………………..….55 3.4.5 Effect of the time of application of strain BS6 against sclerotinia on canola in the greenhouse……………………….56 3.4.6 Evaluation of biocontrol agents under field conditions…….…63 3.5 Discussion………………………………………………………...………...64 4.0 DETECTION OF ANTIBIOTIC-RELATED GENES FROM BACTERIAL BIOCONTROL AGENTS USING POLYMERASE CHAIN REACTION ……………………………………………………………………………….….74 4.1 Abstract……………………………………………………………………...74 4.2 Introduction………………………………………………………………….75 4.3 Materials and methods………………………………………………………77 4.3.1 Genomic DNA extraction…………………………………..……79 4.3.2 DNA quantification……………………………………….……..80 4.3.3 PCR reactions……………………………………………………80 4.3.4 Sequencing and BLAST search…………………….……………82 4.4 Results……………………………………………………………………….82 4.4.1 Genomic DNA…………………………………………….……..82 4.4.2 PCR reactions………………………………………………...….83 4.4.3 Sequencing and BLAST search………………………………….84 4.5 Discussion……………………………………………………………………88 5.0 PLANT INDUCED RESISTANCE MEDIATED BY SCLEROTINIA SCLEROTIORUM AND BACTERIAL BIOCONTROL AGENT BACILLUS AMYLOLIQUEFACIENS BS6 ………………………………………………....91 5.1 Abstract………………………………………………………….………..….91 5.2 Introduction………………………………………………………………..…92 5.3 Materials and methods……………………………………………………….93 5.3.1 Greenhouse experiment………………………………………….93 5.3.2 Phenolics extraction and fractionation…………………………...96 5.3.3 HPLC analysis…………………………………………………...97 5.4 Results…………………………………………………………………..……98 5.4.1 Greenhouse experiment…………..…………………..……….…98 5.4.2 HPLC analysis………………………………………..……….…99 5.5 Discussion……………………………………………………………….....106 6.0 GENERAL DISCUSSION AND CONCLUSIONS..….………………………110 7.0 REFERENCE LIST………………..………………………………….…….….115 V LIST OF TABLES Tables Pages 2.1 Fungal diseases and pathogens on canola (Martens et al. 1994)…….………..….11 3.1 Nineteen bacterial strains isolated from canola leaves…………………………...52 3.2 Bacterial sources and identifications……………………………………………..53 4.1 Bacterial antibiotic-gene-specific primers used in this study…………………….78 . VI LIST OF FIGURES Figures Pages 2.1 Life cycle of Sclerotinia sclerotiorum (Lib.) de Bary on canola……………...…14 3.1 S. sclerotiorum growth inhibition by strain BS6 on PDA and LBA……………..54 3.2 S. sclerotiorum growth inhibition by inhibitory volatiles produced by strains H and E12………….…………………………………………….……………….54 3.3 Bacterial growth curves for rifampicin-resistant strains PA-23, #41, BS8, BS6, H, and E16….……………..……………………………………………………...57 3.4 Sclerotinia disease incidence (DI) in the greenhouse test using five bacterial antagonists….……………………………………………………………………..58 3.5 Inhibition of sclerotinia disease progression in the greenhouse by using five bacterial antagonists.…………….………………………………………………..59 3.6 Canola yield
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