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The Molecular Basis of Pasteuria-Nematode

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The Molecular Basis of Pasteuria-Nematode THE MOLECULAR BASIS OF PASTEURIA-NEMATODE INTERACTIONS USING CLOSELY RELATED BACILLUS SPP. Arohi Srivastava, B.Sc., M.Sc. Submitted to the University of Hertfordshire in partial fulfilment of the requirements of the degree of Doctor of Philosophy School of Life and Medical Sciences, University of Hertfordshire, Hatfield, Herts, AL10 9AB April 2017 To Mumma for being my first teacher and Papa for his strong believe in me. Acknowledgments “You can teach a student a lesson for a day; but if you can teach him to learn by creating curiosity, he will continue the learning process as long as he lives.” -- Clay P. Bedford I am thankful to Dr Keith G. Davies who, as my PhD supervisor, instilled curiosity in me and in the process, I learnt to learn. Whenever I went to him with questions, he often responded by saying: “It’s your PhD, I’ve got mine” and then asked me other questions relating to the questions I had. This inculcated in me more curiosity about the topic; the insightful discussions that followed helped me figure out the answers myself. I feel that ‘not providing ready-made answers’ has helped me a lot in the process of becoming an independent researcher and thinker. He was always very enthusiastic about my research and his warm and encouraging attitude kept me motivated throughout. Despite suffering from a heart attack last December, he continually bestowed his unwavering support and guidance on me through to the last phases of writing this manuscript. I thank my PhD co-supervisors, Drs Avice M. Hall and Kate Graeme Cook, for their competent guidance and untiring support, both academic and personal. The Christmas meals and pancakes parties at Avice’s place always made me feel homely in a foreign country. Thanks, Avice, these are indeed some of my ever-lasting memories in the UK. The supervision and suggestions given to me by Kate, before my first ever academic talk at a big international conference, helped me develop public speaking ability and improved my presentation skills. Thanks, Kate, I have so far done several talks at research conferences and symposia and I feel the first ‘practice session’ I had with you and Avice, has been very useful throughout and will always be in the long run of my career; in fact, it made me win the best student oral presentation at one of the international symposia during my studies. Special thanks are due to Dr Sharad Mohan, Principal Scientist at Indian Agricultural Research Institute, who taught me my first lessons on Pasteuria. What an amazing teacher, a motivating boss, and a passionate scientist! He has been a beacon of encouragement to me ever since I joined him at IARI as a research fellow in 2012. Although I quit my job just after one year to enrol for a PhD at the University of Hertfordshire, he continues to guide me, support me and most importantly help me realize my own potential. Working with him was a life-changing opportunity. Thanks to Dr Timothy Mauchline (Rothamsted Research, Harpenden) for providing the Pasteuria Res148 contig sequences which were used for in silico analyses; and Dr. Alla Mashanova, whose expertise in statistics was invaluable during the analysis, interpretation and presentation of my data. I am grateful to the Indian Council of Agricultural Research (ICAR) which provided funding for this work through its ICAR-International Fellowship scheme. There are so many people who deserve to be acknowledged, without whom this piece of work would not have been possible. My doctoral studies at the University of Hertfordshire were transformational for me and I will fondly remember the time spent here. I am grateful to the excellent technical team at School of Life and Medical Sciences. Thank you, Di, Kishore, Sue, Nathan, Ravi, Noelia and Mansukh for your untiring support during my lab work. Thanks to my incredible office mates; to Thomas for many ‘real scientific discussions’ we had during our respective PhDs and to Coretta for the interesting and engaging conversations on different realms of life. I extend my thanks to my friends, colleagues, and lab mates: Unnati, Bo, Asna, Sudhir, Georgia and Chinthani who formed a supportive network of friends. My warmest thanks must be to my parents for making me what I am and to my parents-in-laws for their unfailing support and understanding. Special thanks to my elder sister, Rangoli, for always inspiring me and for whom I will always be a little sister. Lastly, I thank Ankit for being a fantastically supportive partner. He is a protein scientist by profession and this was an added advantage. The intellectual discussions we had when he reached back home, after a tiring day at work, helped me a lot in my thought process. During those ‘temporary periods of uncertainties’ when my confidence slumped, he was always there to fire fight my worries and anxieties. For the final phase of my studies, to save me time for writing, he took cooking completely off my plate and made me the world’s most delicious and healthiest meals. In fact, his contributions are countless. Thank you, Ankit, to the moon and back; we did it finally! --Arohi Srivastava Contents ABSTRACT........................................................................................................................................... i-ii LIST OF FIGURES ............................................................................................................................. iii-v LIST OF TABLES .................................................................................................................................. vi LIST OF APPENDICES ........................................................................................................................ vii LIST OF ABBREVIATIONS................................................................................................................. viii CHAPTER 1: INTRODUCTION .............................................................................................................. 1 1.1 FOOD SECURITY ....................................................................................................................... 1 1.2 NEMATODES: OFTEN UNNOTICED AGRICULTURAL PESTS .............................................. 1 1.3 SEDENTARY ENDOSPARASITIC PHYTONEMATODES ........................................................ 2 1.3.1 LIFE CYCLE AND DEVELOPMENT ................................................................................................. 3 1.3.2 HOST RANGE .............................................................................................................................. 5 1.4 NEMATODE MANAGEMENT .................................................................................................... 7 1.4.1 TRADITIONAL METHODS OF NEMATODE CONTROL ....................................................................... 7 1.4.2 NEMATODE RESISTANT GM CROPS ............................................................................................ 8 1.4.3 NEMATODE SUPPRESSIVE SOILS AND BIOCONTROL OF PPN...................................................... 8 1.4.4 PASTEURIA SPP. – POTENTIAL BIOCONTROL AGENTS AGAINST NEMATODES ............................. 10 1.5 THE MEMBERS OF THE GENUS PASTEURIA ...................................................................... 10 1.5.1 LIFE CYCLE AND MORPHOLOGICAL STAGES .............................................................................. 13 1.5.2 PASTEURIA ENDOSPORES AND THEIR ATTACHMENT TO THE NEMATODE CUTICLE ...................... 15 1.6 CONSTRAINTS TO THE SUCCESSFUL DEPLOYMENT OF PASTEURIA SPP. AS A BIO- NEMATICIDE ........................................................................................................................................ 17 1.6.1 MASS PRODUCTION IN VIVO AND IN VITRO ................................................................................. 17 1.6.2 HOST SPECIFICITY ..................................................................................................................... 17 1.6.3 DNA ISOLATION OF PASTEURIA – A MAJOR CONSTRAINT IN GENOMIC STUDIES ........................ 18 1.7 BACILLUS SPP. – A MODEL TO STUDY PASTEURIA BIOLOGY ....................................... 19 1.7.1 PHYLOGENETIC CLOSENESS OF BACILLUS AND PASTEURIA ..................................................... 19 1.7.2 BACILLUS ENDOSPORES AND ATTACHMENT MECHANISMS ........................................................ 21 1.8 AIMS AND OBJECTIVES ......................................................................................................... 22 1.8.1 HYPOTHESIS ............................................................................................................................. 22 1.8.2 AIMS AND OBJECTIVES BY CHAPTER ........................................................................................ 22 CHAPTER 2: PHYLOGENETIC PLACEMENT OF PASTEURIA SPP. WITHIN FIRMICUTES ......... 24 2.1 INTRODUCTION ....................................................................................................................... 24 2.1.1 GROEL AND GYRB AS EMERGING CANDIDATES FOR PHYLOGENY RECONSTRUCTION ................ 24 2.1.2 THE GENE SPO0A AS AN ENDOSPORE-SPECIFIC CANDIDATE FOR PHYLOGENY RECONSTRUCTION ................................................................................................................................................. 25 2.1.3 AIMS AND OBJECTIVES .............................................................................................................. 26 2.2 MATERIALS AND METHODS ................................................................................................
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