Do Mutualistic Organisms Associated with Wild Nicotiana Benthamiana Plants Influence Drought Tolerance?

Do mutualistic organisms associated with wild Nicotiana benthamiana plants influence drought tolerance? Khondoker M. G. Dastogeer B. Sc. Ag. (Hons.), M. S. (Plant Path.) This thesis is presented for the degree of Doctor of Philosophy at School of Veterinary and Life Sciences Murdoch University Perth, Western Australia Australia Statement of Contribution This thesis includes three original papers published in peer reviewed journals and two unpublished publications. The ideas, development and writing up of all the papers in the thesis were the principal responsibility of myself, the student, working within the school of veterinary and life sciences under the supervision of Prof Michael Jones, Dr Steve Wylie and Dr Hua Li. The inclusion of co-authors reflects the fact that the work came from active collaboration between researchers and acknowledges input into team-based research. My contribution to the work and those of the co-authors involved the following: Thesis Publication Title Status Nature and % Co-author name(s) Chapter of student Nature and % of Co- contribution author’s contribution Chapter 2 Differential Under review 65%, 1) Hua Li, Sample response of wild- Concept, collection, concept, collected Australian laboratory input into manuscript, Nicotiana species to works, data 10% water stress analysis and 2) Krishnapillai highlights natural writing Sivasithamparam, variation among the manuscript concept and input into accessions manuscript, 5% 3) Michael Jones, input into manuscript, 5% 4) Stephen Wylie, concept, sample collection and input into manuscript 15% Chapter 3 Host specificity of Published in 70%, 1) Hua Li, sample endophytic Microbial Concept, collection, input into mycobiota of wild Ecology. laboratory manuscript 10% Nicotiana plants DOI:10.1007/ works, data 2) Krishnapillai from arid regions of s00248- analysis and Sivasithamparam input northern Australia. 00017- writing into manuscript 5% 01020-00240. manuscript 3) Michael Jones, input into manuscript 5% iii 4) Stephen Wylie, concept, sample collection and input into manuscript 10% Chapter 4 A simple and rapid Published in 65%. 1) Hua Li, concept, in vitro test for Archives of Concept, input into manuscript large-scale Microbiology laboratory 10% screening of fungal DOI:10.1007/ works, data 2) Krishnapillai endophytes from s00203- analysis and Sivasithamparam, drought-adapted 00017- writing concept and input Australian wild 01411-00200. manuscript into manuscript 10% plants for 3) Michael Jones, input conferring water into manuscript 5% deprivation 4) Stephen Wylie, tolerance and concept, concept and growth promotion input into manuscript in Nicotiana 10% benthamiana seedlings. Chapter 5 Metabolic Published in 65%, 1) Hua Li, concept, responses of Environment Concept, input into manuscript endophytic al and laboratory 5% Nicotiana Experimental works, data 2) Krishnapillai benthamiana plants Botany 143, analysis and Sivasithamparam, and experiencing water 59-71. writing input into manuscript stress manuscript 5% 3) Michael Jones, input into manuscript 5% 4) Xin Du, laboratory works 5% 5) Yonglin Ren laboratory works 5% 6) Stephen Wylie, concept, concept and input into manuscript 10% Chapter 6 Fungal endophytes Unpublished 70%, 1) Hua Li, concept, and virus mediated Concept, input into manuscript drought tolerance of laboratory 5% Nicotiana works, data 2) Krishnapillai benthamiana plant analysis and Sivasithamparam, involves writing concept and input into accumulation of manuscript manuscript 5% iv osmolytes and 3) Michael Jones, input antioxidant into manuscript 5% enzymes and 4) Stephen Wylie, expression of host concept, and input into drought responsive manuscript 15% genes. Student signature: Date: The undersigned hereby certify that the above declaration correctly reflects the nature and extent of the student’s and co-authors’ contributions to this work. Hua Li: Date: 14 Nov 2017 Krishnapillai Sivasithamparam: Date: Michael Jones: Date: Xin Du: Date: 14 Nov 2017 Yonglin Ren: Date: Stephen Wylie: Date: 14 Nov 2017 v ACKNOWLEDGEMENTS This thesis is the result of approximately three and half years of work, during which I have been accompanied and supported by various people. It is with pleasure that I now have the opportunity to express my gratitude to all of these people. I would like to thank my PhD supervisors, Dr Steve Wylie, Dr Hua Li and Professor Mike Jones for supporting me during my PhD journey. I am deeply indebted to Steve for his fundamental role in my doctoral work. Steve is someone you will instantly love and never forget once you meet him. He is the most energetic supervisor and one of the smartest people I know. He has taught me, both consciously and unconsciously, how a sound experiment is done. I appreciate all his contributions of time, ideas, and support to make my PhD experience productive, stimulating and rewarding. The delight and enthusiasm he has for his research were contagious and motivational for me, even during difficult times in the PhD pursuit. With a little background in molecular biology, my work in the laboratory would not have been so easier without the continuous help of Hua Li. She has been an amazing and passionate guide throughout my PhD project. Mike has been supportive and has given me the freedom to pursue various projects without objection. I wish to extend my thanks and gratitude to Professor Krisnapillai Sivasithamparam for his scientific advice and instruction and many insightful discussions and suggestions. He was a prime resource for getting my science questions answered, and was instrumental in helping me write this thesis. I also have to thank my PhD committee chair, Ravi Tiwari, for his helpful suggestions in general. I wish to thank the other members of the plant virology group at Murdoch University, especially Shu Hui Koh, Jamie Ong, Duy Quang Nguyen, Thao Thi Tran, Rui Li, and Chao Zhang for their support any time I needed it. I wish to extend my gratitude to all my fellow lab mates and colleagues in the SABC and at Murdoch University for their all-out support, suggestions and optimism, which guided me along and made the whole PhD journey memorable and rewardable. Special thanks to David Berryman, John Fosu-Nyarko, Frances Brigg, Farhana Begum, Sharmin Rahman, Jebin Akter, Mirza Nazim-ud-doula, Mijan Uddin, Kabir Uddin, Roise Uddin Ahmed, Mahbubur Rahman, Moktadir Rahman, for their constant support and encouragement. I would like to thank the Australian Government and Murdoch University for awarding me a scholarship through the Research Training Program (RTP) scheme to enable me to carry out my studies. This accomplishment would not have been possible without their financial assistance. I take the opportunity to thank those to whom I am indebted forever, my parents, my siblings and my friends for their love, inspiration and sacrifice. I dedicate this thesis to my beloved parents, whose role in my life was, and remains, immense. The last word of acknowledgement I have reserved for my dear wife, Marufa Shamsy, who has been with me all these years and has made them the best years of my life. Her support, inspiration, patience and unwavering love were unquestionably the bedrock upon which the past few years of my life have been built. Her leniency of my occasional vulgar moods is a testimony in itself of her unyielding devotion and love. I wish to give her special thanks. vi ABSTRACT In arid regions, such as those in north-western Australia, plants survive under water deficit, high temperatures, intense solar radiation and nutrient-impoverished soils. They employ various morpho- physiological and biochemical adaptations including interaction with microbial symbionts. Seed from thirty-two accessions of four Nicotiana species (N. benthamiana, N. occidentalis, N. simulans, and N. umbratica) collected from wild plants from northern Australia, were grown and used to assess their responses to water stress. The original wild host plants were selected because they grow in an extremely hot environment where water supply is often unpredictable, and because they share a close genetic relationship to the international model plant N. benthamiana research accession 4 (RA-4). Under moderate water stress conditions, shoot fresh weight, shoot dry weight, root fresh weight, root dry weight, root/shoot ratio, and relative water content of leaves was significantly affected. However, the degree to which the accessions were affected varied considerably. Some accessions of N. simulans, N. benthamiana and N. occidentalis were significantly more affected by water stress than others. There was significant inherent variation between accessions in leaf and shoot tip wilting times. Initial symptom expression (leaf wilting) was significantly delayed in two accessions of N. benthamiana and in one accession of N. umbratica. The least water stress tolerant lines, three accessions each of N. occidentalis and N. simulans exhibited advanced symptoms of water stress (shoot tip wilting) within 14-17 days of cessation of watering. This stage was significantly delayed in three accessions of N. benthamiana and two accessions each of N. occidentalis and N. simulans, which exhibited tip wilting after only 21-24 days. There were variations among the accessions of Nicotiana species on their tolerance to water stress. Plant responses to water stress could not be predicted from their phenotype under well-watered conditions. We evaluated identity, host and tissue association, and geographical distribution of fungal endophytes isolated from above

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