STIMULATORY AND INHIBITORY EFFECTS OF UVA AND UVB RADIATION ON SOME PHYSIOLOGICAL AND PATHOGENIC CHARACTERISTICS OF FUNGAL BIOCONTROL AGENTS TO ENHANCE MYCOHERBISTAT EFFECTIVENESS by Feridon Ghasem Khan Ghajar B. Sc. University of Tehran, M. Sc. University of Western Sydney A thesis submitted in fulfilment of requirements for the degree of Doctor of Philosophy College of Science, Technology and Environment University of Western Sydney Penrith South DC NSW 1797 Australia February 2004 To Mehri and Farideh, whose love and support have made the completion of this project possible. i ACKNOWLEDGEMENTS I would like to gratefully acknowledge the supervisory panel, Associate Professor Paul Holford (UWS), Dr Eric Cother (Principal Research Scientist, NSW Agriculture, Orange Agricultural Institute, Orange) and Professor Andrew Beattie (UWS) for their supervision and encouragement throughout this study. I also grateful to: Dr Alison McInnes (School of Environment and Agriculture, UWS) for her assistance with the DNA analysis of D. avenacea isolates. Dr S. D. Hetherington (NSW Agriculture, Orange Agricultural Institute, Orange) for providing the isolates of D. avenacea. Mr Oleg Nicetic for UV measurements and providing meteorological data. Technical officers, present and past, Mr Jim Sheehy, Ms Liz Darley, Dr Sumedha Dharmaratne, Mrs Kim Northcott and Mr Gary Morgan for their technical assistance throughout this study. Mrs Kylie Kaszonyi and Mrs Robynne Warne for their assistance with propagating and maintaining the test plants. ii DECLARATION The work presented in this thesis is, to the best of my knowledge and belief, original except as acknowledge in the text. I hereby declare that I have not submitted this material, either in full or in part, for a degree at this or any other institution. Feridon Ghasem Khan Ghajar February 2004 iii TABLE OF CONTENTS Dedication i Acknowledgements ii Declaration iii Table of contents iv Abbreviations xi Abstract xii CHAPTER 1: GENERAL INTRODUCTION 1 1.1 WEEDS: DEFINITION AND IMPORTANCE 1 1.2 WEED MANAGEMENT 4 1.2.1 Cultural control method 5 1.2.2 Mechanical control methods 6 1.2.3 Chemical control methods 7 1.2.4 Biological control methods 9 1.2.4.1 Classical strategy 10 1.2.4.2 Inundative strategy 11 1.2.5 Integrated weed management (IWM) 14 1.3 BIOLOGICAL CONTROL OF WEEDS USING MYCOHERBICIDES 16 1.3.1 Creating mycoherbicides 16 1.3.1.1 Discovery phase 17 1.3.1.1.1 Selection of target weeds 17 1.3.1.1.2 Selection of fungal pathogens 18 1.3.1.2 Development phase 19 1.3.1.3 Deployment phase 20 1.3.2 Formulation of mycoherbicides 21 1.3.3 Products on the market 26 1.3.4 Benefits, constraints and prospects 28 iv 1.4 PLANT-PATHOGEN SYSTEMS STUDIED IN THIS PROJECT 32 1.4.1 Alisma lanceolatum and Damasonium minus- Rhynchosporium alismatis 32 1.4.1.1 Alisma lanceolatum and Damasonium minus 32 1.4.1.2 Rhynchosporium alismatis 35 1.4.2 Xanthium spinosum-Colletotrichum orbiculare 36 1.4.2.1 Xanthium spinosum 36 1.4.2.2 Colletotrichum orbiculare 40 1.4.3 Avena fatua-Drechslera avenacea 42 1.4.3.1 Avena fatua 42 1.4.3.2 Drechslera avenacea 46 1.5 EFFECTS OF ULTRAVIOLET (UV) RADIATION ON FUNGAL PATHOGENS 47 1.5.1 Ozone (O3), UV radiation and climate change 47 1.5.2 UVB and fungal pathogens 48 1.6 OBJECTIVES OF THIS THESIS 49 CHAPTER 2: OPTIMISING SPORULATION AND PATHOGENICITY IN DRECHSLERA AVENACEA 51 2.1 INTRODUCTION 51 2.2 MATERIALS AND METHODS 55 2.2.1 Fungal isolates 55 2.2.2 Data analysis 55 2.3 EXPERIMENTAL 56 2.3.1 Effect of agar media and temperature on conidium production 56 2.3.2 Effect of pH of the medium on conidium production 59 2.3.3 Effect of carbohydrate content on conidium production 60 2.3.4 Effect of NUV intensity on conidium production on ½OMA and CZA 61 2.3.5 Effect of agar media and light quality on conidium production 63 v 2.3.6 Effect of continuous dark and light, diurnal or combined light conditions and constant or alternating temperature on conidium production on ½OMA 64 2.3.7 Effect of photoperiodism on conidium production on ½OMA 66 2.3.8 Effect of media and light quality on subsequent virulence of conidia produced by D. avenacea 67 2.3.9 Applicability of culture conditions for other isolates 69 2.3.10 Effect of culture age on conidium production 70 2.4 DISCUSSION 72 CHAPTER 3: GENETIC VARIATION IN DRECHSLERA AVENACEA ISOLATES DETERMINED BY RAPD-PCR ANALYSIS 82 3.1 INTRODUCTION 82 3.2 MATERIALS AND METHODS 84 3.2.1 RAPD-PCR analysis 84 3.2.1.1 DNA extraction 84 3.2.1.2 Oligonucleotide primers, PCR amplification and electrophoresis 85 3.2.2 Fragment analysis 86 3.3 RESULTS 87 3.4 DISCUSSION 91 CHAPTER 4: EFFECTS OF ULTRA-VIOLET RADIATION, SIMULATED OR AS NATURAL SUNLIGHT, ON CONIDIUM GERMINATION AND APPRESSORIUM FORMATION OF FUNGI WITH POTENTIAL AS MYCOHERBISTATS 93 4.1 INTRODUCTION 93 4.2 MATERIALS AND METHODS 96 4.2.1 Fungal pathogens: maintenance and conidia description 96 4.2.2 Preparing conidium suspensions for exposure to UV radiation 97 4.2.3 Assessment of conidium germination and appressorium formation 97 vi 4.2.4 Suitability of different types of glassware for UV radiation studies 98 4.2.5 Exposure of suspensions of conidia to simulated UV radiation 98 4.2.6 Experiment 1. Determination of photomorphogenic and damaging wavelengths 99 4.2.7 Experiment 2. Effect of UVB radiation exposure at different periods of time (UVB doses) 100 4.2.8 Natural sunlight experiments 100 4.2.8.1 The effects of full-spectrum natural sunlight and sunlight without UVB and interactions with temperature 100 4.2.8.2 The effect of different levels of solar irradiation on germination of conidia 102 4.2.9 Meteorological measurements 103 4.2.10 Statistical analysis 104 4.3 RESULTS 105 4.3.1 Conidia description 105 4.3.2 Suitability of different types of filter for UV radiation studies 106 4.3.3 Experiment 1. Determination of photomorphogenic and damaging wavelengths 107 4.3.3.1 R. alismatis 107 4.3.3.2 C. orbiculare 108 4.3.3.3 D. avenacea 110 4.3.4 Experiment 2. Effect of UVB irradiation for different periods of time (UVB doses) 111 4.3.4.1 R. alismatis 111 4.3.4.2 C. orbiculare 112 4.3.5 Dose-response curves 113 4.3.5.1 R. alismatis 113 4.3.5.2 C. orbiculare 114 vii 4.3.6 Natural sunlight experiment 115 4.3.5.3 The effects of full-spectrum natural sunlight or sunlight without UVB and interactions with temperature 115 4.3.5.3.1 R. alismatis 115 4.3.5.3.2 C. orbiculare 120 4.3.5.3.3 D. avenacea 126 4.3.5.4 The effect of different levels of solar irradiation on germination of conidia 132 4.3.5.4.1 R. alismatis 132 4.3.5.4.2 Dose-response curves 136 4.3.5.4.3 C. orbiculare 139 4.3.5.4.4 Dose-response curves 141 4.4 DISCUSSION 144 4.4.1 Effects of simulated sunlight 144 4.4.2 Effects of natural sunlight 147 4.4.3 Practical implications 154 CHAPTER 5: PHOTOSTABILISATION OF FUNGI WITH POTENTIAL AS MYCOHERBITATS BY UV PROTECTANTS 155 5.1 INTRODUCTION 155 5.2 MATERIALS AND METHODS 158 5.2.1 Conidium suspensions 158 5.2.2 Mineral and plant oils 158 5.2.3 UV protectants 158 5.2.4 Solubility of UV protectants 158 5.2.5 Toxicity testing 159 5.2.5.1 R. alismatis 159 5.2.5.2 C. orbiculare 160 5.2.6 Selection of UV protectants 160 5.2.6.1 R. alismatis 160 5.2.6.2 C. orbiculare 161 5.2.7 Natural sunlight experiment 161 viii 5.2.8 Spectrophotometer studies 162 5.2.9 Statistical analysis 162 5.3 RESULTS 163 5.3.1 Solubility of UV protectants 163 5.3.2 Toxicity testing 163 5.3.2.1 R. alismatis 163 5.3.2.2 C. orbiculare 166 5.3.3 Selection of UV protectants 168 5.3.3.1 R. alismatis 168 5.3.3.2 C. orbiculare 173 5.3.4 Natural sunlight experiment 174 5.3.5 Spectrophotometer studies 176 5.4 DISCUSSION 179 CHAPTER 6: EFFECT OF UV PROTECTANTS AND UVB RADIATION, SIMULATED OR AS NATURAL SUNLIGHT ON PATHOGEN-HOST PLANT INTERACTIONS 187 6.1 INTRODUCTION 187 6.2 MATERIALS AND METHODS 190 6.2.1 Conidium suspensions 190 6.2.2 Water- and oil-compatible UV protectants 190 6.2.3 Leaf disc bioassays for anthracnose development 190 6.2.3.1 Exposure to simulated UVB radiation 190 6.2.3.2 Exposure to full-spectrum natural sunlight 192 6.2.4 Pot-in-field experiments 192 6.2.5 Statistical analysis 196 6.3 RESULTS 197 6.3.1 Leaf disc bioassays for anthracnose development 197 6.3.1.1 Exposure to simulated UVB radiation 197 6.3.1.2 Exposure to full-spectrum natural sunlight 199 6.3.2 Pot-in-field experiments 204 6.4 DISCUSSION 207 ix CHAPTER 7: GENERAL DISCUSSION AND CONCLUSIONS 210 7.1 R. alismatis 210 7.2 C. orbiculare 215 7.3 D. avenacea 219 7.4 Summary 225 REFERENCES 226 PUBLICATIONS 250 x Abbreviations AFLP amplified fragment length polymorphism ANOVA analysis of variance APPs aryloxyphenoxypropionates CA carrot agar CHDs cyclohexanediones CIE Commission Internationale d’Eclairage CMA cornmeal agar CTAB cetyl-trimethyl ammonium bromide CZA Czapek Dox agar DAI days after inoculation dATP deoxyadenosine-5' -triphosphate dCTP deoxycytosine-5' -triphosphate dGTP deoxyguanine-5' -triphosphate DNA deoxyribonucleic acid dTTP deoxythymine-5' -triphosphate EDTA ethylenediaminetetraacetic acid, disodium salt FOP aryloxyphenoxypropionate GBA green bean agar IWM integrated weed management LBA lima bean agar L/D alternating 12 h white light and dark LSD least significant difference MA malt extract agar MCPA monochloro phenoxy acetic acid MPDA malt extract-peptone-dextrose agar MSMA monosodium methylarsonate MYA malt and yeast extract agar NUV near-ultraviolet NUV/D alternating 12 h near-ultraviolet radiation and dark OMA oatmeal agar pABA p-aminobenzoic acid PCA potato-carrot agar PCR polymerase chain reaction PDA potato-dextrose agar RAPD random amplified polymorphic DNA RFLP restriction fragment length polymorphism RNA ribonucleic acid SST selective spray-topping UV ultraviolet V8JA V-8 juice agar VPD vapour pressure deficit WA water agar WAI weeks after incubation YDA yeast extract-dextrose agar xi ABSTRACT Many candidate mycoherbicides have shown promise in the laboratory or greenhouse, but most have been ineffective in the field.