Symbiotic Effectiveness, Phylogeny and Genetic Stability of Biserrula pelecinus-nodulating Mesorhizobium sp. isolated from Eritrea and Ethiopia Amanuel Asrat Bekuma A thesis submitted for the degree of Doctor of Philosophy Murdoch University, Perth Western Australia June 2017 ii Declaration I declare that this thesis is my own account of my research and contains as its main content work which has not previously been submitted for a degree at any tertiary education institution. Amanuel Asrat Bekuma iii This thesis is dedicated to my family iv Abstract Biserrula pelecinus is a productive pasture legume with potential for replenishing soil fertility and providing quality livestock feed in Southern Australia. The experience with growing B. pelecinus in Australia suggests an opportunity to evaluate this legume in Ethiopia, due to its relevance to low-input farming systems such as those practiced in Ethiopia. However, the success of B. pelecinus is dependent upon using effective, competitive, and genetically stable inoculum strains of root nodule bacteria (mesorhizobia). Mesorhizobium strains isolated from the Mediterranean region were previously reported to be effective on B. pelecinus in Australian soils. Subsequently, it was discovered that these strains transferred genes required for symbiosis with B. pelecinus (contained on a “symbiosis island’ in the chromosome) to non-symbiotic soil bacteria. This transfer converted the recipient soil bacteria into symbionts that were less effective in N2-fixation than the original inoculant. This study investigated selection of effective, stable inoculum strains for use with B. pelecinus in Ethiopian soils. Genetically diverse and effective mesorhizobial strains of B. pelecinus were shown to be present in Ethiopian and Eritrean soils. These strains were shown to belong to the genus Mesorhizobium and carry highly mobile symbiosis islands, with a novel integration hotspot (ser-tRNA). In vitro, the transfer of the symbiosis island from these strains to a non-symbiotic recipient strain resulted in novel bacteria with a poorly effective phenotype, except for one highly effective strain. By deleting a relaxase gene, which is involved in the conjugative transfer of the symbiosis island, a more stable strain was created containing an immobile symbiosis island. The study highlights the presence of taxonomically and symbiotically distinct B. pelecinus-nodulating Mesorhizobium strains in East African soils. In these Mesorhizobium strains, the rate of symbiosis island transfer was as high as 3x10-3 in vitro. It is suggested that island transfer has a significant role in the rapid evolution of poorly effective strains. Further, it is likely that this transfer contributes to one of the most intractable problems compromising N2-fixation in agricultural systems - that of poorly effective but competitive background rhizobia. In this study, the management of symbiosis island transfer through inactivation of the relaxase gene without affecting the symbiotic phenotype was found to be a viable approach for tackling this problem. v Acknowledgments I am deeply indebted to many people and organizations that have supported me faithfully to make this achievement possible and I wish to take this opportunity to thank them here. I acknowledge the generous financial support of Australian Award scholarship (AusAID) and Murdoch University for sponsoring my study. I also acknowledge and Curtin University for partially supporting my research. I would like to thank my supervisors, John Howieson, Graham O’Hara, Jason Terpolilli, Ravi Tiwari, Solomon Mengistu and Joshua Ramsay for all your advice and always making time for my meetings throughout my years at Murdoch. A special thank goes to John for sharing all your brilliant thoughts, guidance and encouragement from inception to completion of the thesis. I also thank Graham for your critical comments and handling of the side matters. I thank Ravi for not getting tired of teaching me the basics of molecular biology and being available almost any time for help. I also thank Jason for sharing your wisdom and upholding great supervision during my study period. I thank Solomon Mengistu for your guidance and assistance during the field research. I also thank Josh for the advices and constructive criticism that gave me an opportunity to excel. I also thank, Regina for assistance during glasshouse works, George for the invaluable discussions and support, Tim for sharing your witty thoughts and support during lab work, and Sofie for sharing your expertise in the phylogenetic analysis. All of you have made my work easier and productive. I would also like to thank CRS team members, my fellow students, and friends, Lambert, Anabel, Aliyu, Mazvita, Waynee, Rebecca, Tian, Ron, Julie, Wani, Liza, Tom, Rob, Jamba, Rachael, Tefera, Alemu, Yehualashet, Selam and others for helping me have a productive, yet enjoyable stay in Australia. A special thanks are also reserved for my parents, Asrat and Beletu, for raising me with love and bringing me to this level; and all my beloved family; Tady, Kidist, Hailu, Keneni, Yematawork, Bogale, Kedija, the late Mickey, Biruk, Misrak, Tesfa, Firehiwot, Kidus, Mekdes, Ashenafi, Yeabsira, Bahran, Hawi, Kiya, Natnael, Dagim, Selome, Markan, and Abem, for your encouragement and prayers from half way around the world. I miss you all. vi Finally, sincerest thanks to my loving wife Honey and son Akiya for your unwavering support of my work and for the patience that you have shown throughout my studies. I Love you both. Above all, thanks God! I couldn’t have done it without you. vii Table of Contents Declaration .............................................................................................................. ii Abstract .................................................................................................................. iv Acknowledgments .................................................................................................. v List of figures ......................................................................................................... xi List of Tables ....................................................................................................... xiii List of abbreviations ........................................................................................... xiv 1 Introduction and literature review ................................... 1 1.1 The significance of biological nitrogen fixation (BNF) ........................... 1 1.1.1 The process of nitrogen fixation in Rhizobia ..................................................... 2 1.2 The rhizobia-legume symbiosis ................................................................. 2 1.3 Host specificity in the rhizobia-legume symbiosis ................................... 5 1.4 Diversity of rhizobia ................................................................................... 6 1.4.1 Molecular techniques for elucidation of rhizobial diversity............................... 6 1.4.2 The current approach to rhizobial classification: polyphasic taxonomy ............ 7 1.4.3 Taxonomy of Mesorhizobium .......................................................................... 10 1.4.4 The genus Mesorhizobium ............................................................................... 12 1.5 The case for the introduction of B. pelecinus to Ethiopian agriculture 13 1.6 Biserrula pelecinus .................................................................................... 14 1.7 Is inoculation required to introduce B. pelecinus to a new area? ........ 15 1.8 Mobile genetic elements and their role in bacterial evolution and genetic stability ..................................................................................................... 16 1.8.1 Significance of symbiosis island transfer in Mesorhizobium ........................... 17 1.8.2 Evolution of poorly N2-fixing symbionts of B. pelecinus in Western Australian soils through SI transfer ................................................................................................ 17 1.8.3 The mechanism and regulation of symbiosis island excision and transfer ...... 18 1.8.4 Integration and excision ................................................................................... 18 1.8.5 Conjugative transfer ......................................................................................... 19 1.8.6 Quorum sensing ............................................................................................... 20 1.9 Aims of the study: .................................................................................... 21 2 Symbiotic effectiveness and promiscuity of indigenous B. pelecinus-nodulating rhizobial strains isolated from Ethiopian and Eritrean soil .................................................... 23 2.1 Introduction .............................................................................................. 23 2.2 Materials and Methods ............................................................................ 24 2.2.1 Strains and legume hosts .................................................................................. 24 2.2.2 Glasshouse screening for symbiotic effectiveness and host range ................... 26 2.2.3 Pot preparation ................................................................................................. 27 2.2.4 Germination and planting ................................................................................. 27 2.2.5 Inoculation