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Thesis Submitted in Partial Fulfilment of the Requirements for the Degree of Doctor of Philosophy Expanding the host range of Rhynchosporium alismatis, a potential biological control agent for Alismataceae weeds in Australian rice fields. Wayne Maxwell Pitt BAppSci (Medical and Applied Biotechnology) Honours Class 1 A thesis submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy School of Agriculture Faculty of Science and Agriculture Charles Sturt University Wagga Wagga, NSW, Australia April 2003 Statement of Authenticity STATEMENT OF AUTHENTICITY HD7 CERTIFICATE OF AUTHORSHIP OF THESIS & AGREEMENT FOR THE RETENTION & USE OF THE THESIS DOCTORAL AND MASTER BY RESEARCH APPLICANTS To be completed by the student for submission with each of the bound copies of the thesis submitted for examination to the Centre of Research & Graduate Training. For duplication purpose, please TYPE or PRINT on this form in BLACK PEN ONLY. Please keep a copy for your own records. I Wayne Maxwell Pitt Hereby declare that this submission is my own work and that, to the best of my knowledge and belief, it contains no material previously published or written by another person nor material which to a substantial extent has been accepted for the award of any other degree or diploma at Charles Sturt University or any other educational institution, except where due acknowledgment is made in the thesis. Any contribution made to the research by colleagues with whom I have worked at Charles Sturt University or elsewhere during my candidature is fully acknowledged. Should this thesis be favourably assessed and the award for which it is submitted approved, I agree to provide at my own cost a bound copy of the thesis as specified in the Rules for the presentation of theses, to be lodged in the University Library. I also agree that the thesis be accessible for the purpose of study and research in accordance with the normal conditions established by the University Librarian for the care, loan and reproduction of thesis.* 4th November 2003 Signature Date * Subject to confidentiality provisions as approved by the University Acknowledgements ACKNOWLEDGEMENTS • Peter and Jan Pitt. • The Cooperative Research Centre for Sustainable Rice Production for the provision of funding for this study (Project No. 2401). • Dr Laurie Lewin. • Dr Gavin J. Ash and Dr Eric J. Cother. • Dr Janet L. Taylor, Janet Condie, Carla Barber and the staff of the Plant Biotechnology Institute, NRC, Canada. • Dr Harsh Raman (NSW Agriculture, Wagga Wagga), Dr Rob Loughman and Christy R. Grime (University of Western Australia) for the provision of R. secalis samples. • Megan, Wendy and the staff at the Office for Research and Graduate Studies. • Robyn Lonard at interlibrary loans. • Dr. Karen Bailey and Dr. C. Y. Chen of Agriculture and Agri-Food Canada for supplying the pABC vector. • Eric Hines from CSIRO department of Entomolgy. • Ian Mason for the provision of A. plantago–aquatica plants. • Dr Jim Virgona, Helen Nicol and Dr Neil Coombes for statistical advice and consultation.. i Table of Contents TABLE OF CONTENTS STATEMENT OF AUTHENTICITY .............................................................................................. i ACKNOWLEDGEMENTS ............................................................................................................... i TABLE OF CONTENTS ..................................................................................................................ii LIST OF FIGURES.......................................................................................................................... iv LIST OF TABLES............................................................................................................................. v LIST OF ABBREVIATIONS AND STATISTICAL SYMBOLS................................................ vi ABSTRACT....................................................................................................................................... ix 1 Introduction..................................................................................................................................... 1 1.1 INTRODUCTION ........................................................................................................................... 1 1.2 OBJECTIVES OF THIS STUDY ........................................................................................................ 2 2 Literature Review ........................................................................................................................... 3 2.1 THE AUSTRALIAN RICE INDUSTRY.............................................................................................. 3 2.2 WEEDS........................................................................................................................................ 4 2.3 WEED MANAGEMENT IN RICE .................................................................................................... 5 2.4 DEVELOPMENT OF THE MYCOHERBICIDE..................................................................................... 6 3 Phylogenetics................................................................................................................................. 12 3.1 INTRODUCTION ......................................................................................................................... 12 3.2 TAXONOMY AND NOMENCLATURE ........................................................................................... 13 3.3 CONCEPTS AND METHODOLOGY ............................................................................................... 14 3.4 MATERIALS AND METHODS ...................................................................................................... 16 3.4.1 Fungal isolate collection, isolation, cultivation, storage and maintenance: ................... 16 3.4.2 Culture preparation and DNA extraction: ....................................................................... 18 3.4.3 Amplifying the ITS region (ITS−PCR):............................................................................. 18 3.4.4 Sequencing the ITS region:............................................................................................... 19 3.4.5 Assembling the ITS database:........................................................................................... 20 3.4.6 DNA sequence alignment and phylogenetic analysis:...................................................... 23 3.5 RESULTS ................................................................................................................................... 24 3.6 DISCUSSION .............................................................................................................................. 28 4 Population Structure .................................................................................................................... 33 4.1 INTRODUCTION ......................................................................................................................... 33 4.2 CONCEPTS AND METHODOLOGIES ............................................................................................. 34 4.3 TOOLS AND TECHNIQUES .......................................................................................................... 36 4.4 MATERIALS AND METHODS ...................................................................................................... 38 4.4.1 Fungal isolates and DNA extraction:............................................................................... 38 4.4.2 Repetitive Repeat motif (ERIC/REP)−PCR:..................................................................... 40 4.4.3 Simple sequence repeat (SSR)−PCR: ............................................................................... 40 4.4.4 Data analysis:................................................................................................................... 41 4.5 RESULTS ................................................................................................................................... 45 4.6 DISCUSSION .............................................................................................................................. 50 ii Table of Contents 5 Infection Process ........................................................................................................................... 55 5.1 INTRODUCTION ......................................................................................................................... 55 5.2 FUNGAL ATTACHMENT AND/OR ADHERENCE TO THE PLANT SURFACE ...................................... 56 5.3 GERMINATION AND DIFFERENTIATION OF INFECTION STRUCTURES........................................... 57 5.4 FUNGAL PENETRATION AND PATHOGENESIS.............................................................................. 59 5.5 PLANT DEFENCES ...................................................................................................................... 62 5.6 MATERIALS AND METHODS ...................................................................................................... 67 5.6.1 Fungal isolates: ................................................................................................................ 67 5.6.2 Plant growth and leaf material preparation: ................................................................... 67 5.6.3 Pathogenicity and selection for virulence ........................................................................ 67 5.6.3.1. Inoculum preparation:..............................................................................................
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