Investigations on the early stages of interactions between the nematodes Meloidogyne javanica and Pratylenchus thornei and two of their plant hosts SOSAMMA PAZHAVARICAL Doctor of Philosophy August 2009 University of Western Sydney “O LORD, How great are your works!” Psalm 92:5 Dedicated to My father the late P. V. Easow, who passed away in 1976 when I was still in high school; y brother the late P. Vergis, who passed away in early 2009 and y loving other Maria a Easow, who is, always, y ain source of inspiration . ACKNOWLEDGEMENTS This thesis was one which was almost never written but for the grace of God. I wish to express my gratitude to the many who directly and indirectly contributed towards the completion of this thesis from the beginning, eight years ago. First and foremost I would like to thank the University of Western Sydney for the Post Graduate Award Scholarship, and the Australian National University for the additional funding to enable me to realise my research dreams at the UWS Hawkesbury and ANU Canberra. I express my deep appreciation for the understanding and support of my supervisory panel, especially my Principal Supervisor Associate Professor Robert Spooner-Hart, without whose infinite patience, dedication, lateral thinking and valuable time which he spent editing, this thesis would never have been realised. My heartfelt thank you goes to Associate Professor Tan Nair for his valuable guidance during the initial stages of my candidature, and his advice in all academic matters up until recent retirement. I would like to thank Professor Dr. Geoff Wasteneys for his kindness and support in providing all laboratory facilities for my cytoskeletal research at the Research School of Biological Sciences, Australian National University, Canberra. I consider it a privilege to have worked with Dr Wasteneys on my project. I want to thank Dr. David Collings for sharing with me his technical expertise in confocal microscopy procedures, his knowledge and enthusiasm, patience and generosity through the years, and for his valuable time editing the cytoskeleton chapter. Dr Collings was the best mentor I could have ever wished to work with. My gratitude goes to Dr Zhaohui Wang, Murdoch University Perth, WA for graciously teaching me the aseptic culturing technique of tomato and Meloidogyne javanica and also supplying me with aseptic Meloidogyne javanica inoculum; Dr Sharyn Taylor, SARDI, SA for supplying the Pratylenchus thornei inoculum; Dr Jennifer Cobon, DPI, QLD for providing additional Meloidogyne javanica inoculum, and tomato seeds for my experiment and Daigo Takemoto and Dr David Jones at RSBS, ANU for kindly supplying GFP-hTalin transformed Arabidopsis thaliana seeds for the GFP experiment. Thank you to all other academic, technical and administrative staff from UWS, Hawkesbury and RSBS, ANU especially Oleg Nicetic, Liz Kabanoff, Rosalie Laing, Gillian Wilkins, Christina Harvey and Dr. Paul Holford at UWS and everyone from the Wasteneys’ and David Jones’ laboratories at RSBS, ANU. I would like to thank Dr Shelley Burgin for her understanding and compassion and providing me with many extensions of candidature so that I could finish the thesis. I would like to express my deep love and unending gratitude to my mother, Mariamma Easow, a retired science teacher, for sparking my interest in science from a very early age, my late father, P. V. Easow, a Finance Accountant, and my late brother, P. Vergis, who always emphasised the importance of a good education in my life. I would like to thank my sister, Dr Marykutty Samuel, for her unwavering support through all the trials and tribulations of the darkest times in my life which I faced during the past few years, for encouraging me to persist in my studies when I was ready to give up on several occasions, for several reasons. I would like to thank my sons Arun and Ashish, who have supported me and always encouraged me to finish my studies in spite of the amount of time I spent away from them for the sake of research and suffering my ill temper at times because of the stress. I also want to convey my deepest gratitude to my sister-in-law, Lysamma Vergis, and my nieces Sherly and Sherin in India, for their love and hospitality during my visits. I would like to express my sincere appreciation and also a very belated thank you to Shah who greatly assisted me through my Masters Degree in India and who I, regretfully, failed to acknowledge at that time. I want to express my warm gratitude to Dr. Ahmed Regina and her family for their hospitality, friendship and kindness especially during the time of my stay in Canberra. Thanks also to my many relatives, friends and acquaintances, too numerous to mention by name, who have knowingly or unknowingly given me strength to face difficult situations in life and an ability to see the bright side of all things And I thank God for RG, who has shown me positivity, hope and a new perspective. STATEMENT OF AUTHENTICATION The work presented in this thesis is, to the best of my knowledge and belief, original except as acknowledged 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. …………………………………………………………………. (Signature) TABLE OF CONTENTS Page No i Table of Contents List of Tables vii List of Figures viii Abbreviations xii Summary xiv Chapter 1 General introduction 1.1 Choosing nematodes for the study 1 1.1.1 Economic importance of Meloidogyne spp. 1 and Pratylenchus spp. in crop production 1.1.2 Control of plant parasitic nematodes 20 1.2 Choosing model host plants for this study 24 1.3 Aims and Objectives 26 1.3.1 Aim of this study 26 1.3.2 Rationale behind the aims and objectives 26 of this study i Chapter 2 Changes in the cytoskeleton in root cells of Arabidopsis thaliana due to infection by Meloidogyne javanica and Pratylenchus thornei 2.1 Introduction: The cytoskeleton in animals and 28 plants 2.1.1 Functions of the plant cytoskeleton 32 2.1.2 Dynamics and organisation of the 34 microtubules within plant cells 2.1.3 Interaction between actin and 35 microtubules in plant cells 2.1.4 Changes in the plant cytoskeleton due to 36 external factors 2.1.5 Immunofluorescence and the study of 39 plant cytoskeleton 2.1.6 GFP in the study of plant cytoskeleton 40 2.1.7 Nematodes and the plant cytoskeleton 42 2.2 Materials and Methods 44 2.2.1 Culture of Meloidogyne javanica 44 inoculum 2.2.2 Pratylenchus thornei inoculum 45 2.2.3 Culture of Arabidopsis thaliana ecotype 46 Columbia as host 2.2.4 Immunolabelling for confocal 46 microscopy of whole roots 2.2.5 Embedding and sectioning of whole roots 47 in BMM 2.2.6 Immunolabelling BMM sections for 48 ii confocal microscopy 2.2.7 Visualisation of the actin cytoskeleton in 48 living root cells of Arabidopsis thaliana using GFP-hTalin fusion protein 2.3 Results 2.3.1 Uninfected Arabidopsis thaliana roots 49 2.3.2 Arabidopsis thaliana infected by 62 Meloidogyne javanica 2.3.3 Arabidopsis thaliana infected by 89 Pratylenchus thornei 2.4 Discussion 92 2.5 Conclusion 98 Chapter 3 Effect of the nematodes Meloidogyne javanica and Pratylenchus thornei on growth and yield of tomato, Solanum lycopersicum 3.1 Introduction 99 3.2 Materials and Methods 100 3.2.1 Statistical analysis 101 3.3 Results 102 3.3.1 Observations on the growth stages of 102 tomato, Solanum lycopersicum, in the greenhouse 3.3.2 Meloidogyne javanica 102 iii 3.3.3 Pratylenchus thornei 108 3.3.4 Comparison of growth and yield of 114 tomato, Solanum lycopersicum, inoculated with Meloidogyne javanica and Pratylenchus thornei 3.4 Discussion 116 3.5 Conclusions 122 Chapter 4 Movement of Meloidogyne javanica and Pratylenchus thornei through sandy, sandy clay loam and clay soils to the roots of tomato, Solanum lycopersicum 4.1 Introduction 4.1.1 Soil types and soil texture 124 4.1.2 Organic matter in soils 125 4.1.3 Pore space and aeration in soils 126 4.1.4 Nematode morphology 126 4.1.5 Relationship between soil and movement 127 of nematodes 4.2 Materials and Methods 129 4.2.1 Statistical analysis 131 4.3 Results 131 4.3.1 Movement of Meloidogyne javanica to 131 the roots of tomato, Solanum lycopersicum, through sandy, sandy clay loam and clay soils iv 4.3.2 Movement of Pratylenchus thornei to the 133 roots of tomato, Solanum lycopersicum, through sandy, sandy clay loam and clay soils 4.4 Discussion 135 4.5 Conclusions 139 Chapter 5 Temporal study of the early infection of tomato, Solanum lycopersicum, by Meloidogyne javanica and Pratylenchus thornei 5.1 Introduction 141 5.2 Materials and methods 143 5.3 Results 144 5.3.1 Early infection of tomato, Solanum 144 lycopersicum root by Meloidogyne javanica 5.3.2 Early infection of tomato, Solanum 157 lycopersicum, root by Pratylenchus thornei 5.4 Discussion 164 5.5 Conclusions 167 Chapter 6 General discussion 6.1 Major findings / outcomes 168 6.1.1 Comparison between the effects of 171 Meloidogyne javanica and Pratylenchus v thornei on Arabidopsis thaliana and Solanum lycopersicum 6.1.2 Understanding the effects of nematode 172 and plant interactions 6.1.3 Soil characteristics and plant growth 176 parameters 6.1.4 Temporal study of nematode-plant 178 parasite-host relationships 6.2 Usefulness / implications of the work to others 179 6.3 Integrated management of nematode pests 180 6.4 Impact of climate change on plant-parasitic 181 nematodes 6.5 Future research work 182 6.6 Final conclusion 183 Chapter 7 References 184 Appendix 1 Results of analysis of soils used in pot 221 culture trial vi LIST OF TABLES Page Number