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Characterisation of rhizobia and studies on N2 fixation of common weed legumes in New Zealand ___________________________________ A thesis submitted in partial fulfilment of the requirements for the Degree of Doctor of Philosophy in Molecular Microbiology by Wendy Ying Ying Liu _______________________________ Lincoln University 2014 Abstract of a thesis submitted in partial fulfilment of the requirements for the Degree of Doctor of Philosophy (Molecular Microbiology) Characterisation of rhizobia and studies on N2 fixation of common weed legumes in New Zealand by Wendy Ying Ying Liu Most legume species can fix atmospheric N2 via symbiotic bacteria (collectively termed rhizobia) in nodules on their roots, thus allowing them to colonise marginal land with low soil N availability. Over the last 150 years, over 100 legume species from different continents have become naturalised in NZ and many of these are now common weeds. The major objective of this study was to genotypically characterise rhizobial isolates which produce N2-fixing nodules on common weed legumes in NZ soils via phylogenetic analyses of 16S rRNA, recA, nifH, nodA and/or nodC gene sequences to establish their identity, diversity and presumptive origin(s). In addition, detailed studies were carried out on particular legumes or rhizobia linked to their ‘novelty’ outside and/or inside NZ. All legume plants sampled in the field were nodulated. Phylogenetic analyses of housekeeping and symbiosis gene sequences of 70 rhizobial isolates recovered from 19 legume species indicated that there is a diverse range of rhizobia which effectively nodulate weed legumes in NZ soils (Burkholderia, Bradyrhizobium, Ensifer, Mesorhizobium and/or Rhizobium depending on plant species). Almost all legumes were only effectively nodulated by rhizobia in a single genus. Exceptions were: (i) Dipogon lignosus, which had both determinate and indeterminate nodules, and was effectively nodulated by species of Burkholderia, Bradyrhizobium and Rhizobium and, (ii) Medicago sativa which was effectively nodulated by Ensifer sp. and Rhizobium sp.. This is the first report of a beta-rhizobia in NZ soils. Many of the rhizobia are likely to have been introduced into NZ either via plant materials/soil or commercial inoculants used in the field. However, for six Mesorhizobium isolates, there is a possibility of lateral transfer of symbiosis genes from introduced to native strains. There is also evidence of the ii occurrence of indigenous bradyrhizobia that are not associated with NZ native legumes but which are capable of nodulating the genistoid legumes. Host-specificity work confirmed that both native and exotic weed legumes are nodulated by disparate rhizobial populations in NZ. Bradyrhizobia could effectively nodulate legumes in the tribes Acacieae (Acacia), Genisteae (Chamaecytisus, Cytisus, Lupinus and Ulex), Loteae (Lotus and Ornithopus) and Phaseoleae (Dipogon). The symbiosis genes of the rhizobial isolates recovered here were largely plant species/tribe specific. However, bradyrhizobial cross-nodulation studies on seven legume genera of the Acacieae, Genisteae and Loteae indicated that legume species of the tribe Genisteae formed effective nodules with all Bradyrhizobium spp. tested. Those of the tribes Acacieae and Loteae only formed effective nodules with isolates associated with plants of their respective tribes. These plants generally showed greatest total dry weight when inoculated with strains isolated from the same host plants/related plants of the same genus. Cross- nodulation studies on M. sativa and Melilotus indicus indicated that they could form N2-fixing nodules with Ensifer and Rhizobium isolates. However, these isolates showed differences in their ability to promote growth of M. sativa. Tolerance patterns of the rhizobial isolates to environmental stresses were largely based on the genus the isolates belonged to. The Burkholderia isolates were generally the most stress- tolerant and also showed phosphate solubilisation and siderophore production. However, the Ensifer isolates were the most salt tolerant. 15 N natural abundance analysis indicated that N2 fixation contributed substantially to the total N nutrition of Lupinus arboreus and Ulex europaeus sampled at sand dunes and hedges bordering intensive agricultural systems in Canterbury, respectively. Glasshouse studies via 15 N-isotope dilution analysis showed that U. europaeus is a facultative N2 fixer with an increased reliance on soil N in comparison to N2 fixation as soil N levels increase. In conclusion, a wide range of rhizobia form effective nodules with specific weed legumes in NZ soils and many are likely to have been transported from abroad via plant material, soil and/or field inoculant but there is a possibility of pre-existing bradyrhizobial population occurring in NZ soils. Generally, their symbiosis genes are plant species/tribe specific but some iii legumes are able to form effective nodules with a wider range of rhizobia than those recovered from them in the field. The ability to fix N2 could be an important factor in the establishment of these weed legumes in NZ. Keywords: rhizobia, weed legumes, genotypic characterisation, 16S rRNA, recA, nifH, nodA, nodC, Burkholderia, Bradyrhizobium, Ensifer, Mesorhizobium, Rhizobium, cross-nodulation, nitrogen fixation, 15N natural abundance, 15N-isotope dilution, phosphate solubilisation, siderophore production, gorse, tree lupin iv Acknowledgements As this important phase in my life draws to an end, I would like to acknowledge the many individuals who have contributed both directly and indirectly to the completion of this study. To my main supervisor, Dr Mitchell Andrews, who has been an incredible mentor to me. Words cannot express my gratitude to him for cultivating my interest in research 6 years ago and for giving me an opportunity to grow as a researcher. His steadfast guidance, patience and support throughout this journey have been priceless. To my associate supervisors, Dr Hayley Ridgway and Dr Trevor James for their valuable assistance, expertise and suggestions. To Lincoln University, AgResearch and Meadow Mushrooms for the research funds and scholarship. To the university academic, administrative and technical staffs for their valuable help, technical advice and support, especially Candice Barclay, Simon Hodge, Manjula Premaratne, Roger Creswell, Brent Richards, Norma Merrick, Stuart Larsen, Joshua Philips, Judith Kidd, Alan Marshall and Leona Meachen. To my friends and colleagues, especially Heng Wee Tan, Nalini Vijayan and Travis Ryan-Salter, for their assistance throughout this time. To friends of Lincoln Malaysian Students’ Society, Lincoln Crew and MicroB, for the great memories and for being my ‘home’ away from home. To my family, especially my mum and aunt, Josephine and Elizabeth Sim, for their unending love, care, patience and support. To my beloved late father, Stanley Liu, for inspiring me to be a better person. I dedicate this thesis to him and I hope that his one and only daughter has made him proud. To my partner, Han Sheng Lim, for being my pillar of strength, for making me more of an optimist whenever things go awry, and for his unwavering commitment despite being many, many miles away. Above all, I would like to thank God for showering his abundant blessings upon my family and I. v Table of Contents Abstract ....................................................................................................................................... ii Acknowledgements .................................................................................................................... v Table of Contents ....................................................................................................................... vi List of Tables ................................................................................................................................ x List of Figures ............................................................................................................................ xii Chapter 1 General Introduction ................................................................................................ 1 1.1 Legumes ................................................................................................................................ 1 1.2 Nitrogen fixation in legumes ................................................................................................ 3 1.3 Rhizobia ................................................................................................................................ 4 1.3.1 Characterisation and taxonomy of rhizobia .......................................................... 4 1.3.2 Specificity in symbiotic legume-rhizobia mutualism ............................................ 6 1.3.3 Effect of environmental stresses on rhizobia and N2 fixation ............................... 7 1.4 Nitrogen assimilation