Characterisation of rhizobia associated with New Zealand native legumes (Fabaceae) and a study of nitrogen assimilation in Sophora microphylla : A thesis submitted in partial fulfilment of the requirements for the Degree of Doctor of Philosophy

Abstract

Many legume species have the capacity to fix atmospheric N₂ via symbiotic bacteria (generally termed “rhizobia”) in root nodules and this can give them an advantage under low soil N conditions if other factors are favourable for growth. There are four genera of native legumes, on the main New Zealand (NZ) islands. These are the closely related Carmichaelia, Clianthus and Montigena in the Carmichaelinae clade, tribe Galegeae, and Sophora, within the tribe Sophoreae: all are capable of nodulation. Little work has been done on the genotypic characterisation and host-range specificity of the rhizobia associated with NZ native legumes. Moreover, the ability of native legumes to assimilate soil N in comparison with their N₂ fixation has not been assessed. The primary objectives of this research were to 1) more fully characterise the rhizobia associated with the four genera of NZ native legumes, including their ability to cross nodulate different species and 2) assess the ability of Sophora microphylla to assimilate soil N in comparison with its N₂ fixation.

Gene sequencing results indicated that the bacterial strains isolated from NZ native legumes growing in natural ecosystems in the current and previous studies were of the genus Mesorhizobium. Generally, the Carmichaelinae and Sophora species were nodulated by two separate groups of Mesorhizobium strains. Ten strains isolated from the Carmichaelinae showed 16S rRNA and nifH similar to the M. huakuii type strain, but had variable recA and glnII genes, novel nodA and nodC genes and the seven strains tested could produce functional nodules over a range of Carmichaelinae species but did not nodulate Sophora species. Forty eight strains isolated from Sophora spp. showed 16S rRNA similar to the M. ciceri or M. amorphae type strains, variable recA, glnII and rpoB genes and novel and specific nifH, nodA and nodC genes which were different from those of the Carmichaelinae strains. Twenty one Sophora strains tested were able to produce functional nodules on a range of Sophora spp. but none nodulated C. australis. However, eighteen of the twenty one strains produced functional nodules on Cl. puniceus. These results indicate that, in general, the ability of different rhizobial strains to produce functional nodules on NZ native legumes is likely to be dependent on specific symbiosis genes. Clianthus puniceus appears to be more promiscuous in rhizobial host than the other NZ native legumes species tested.

Generally, strains isolated from NZ native Sophora spp. from the same field site grouped together in relation to their “housekeeping” gene sequences and ERIC-PRC fingerprinting banding patterns. Most strains were able to grow at pH 3 – pH 11 but only one showed phosphorus solubilisation ability and none showed siderophore production. The strains showed differences in their ability to promote the growth of S. microphylla under glasshouse conditions. DNA-DNA hybridisation tests indicated that strains isolated from New Zealand native Sophora spp. are of several new Mesorhizobium species.

The ability of S. microphylla to utilise soil NO₃⁻ and NH₄⁺ in comparison with its N₂ fixation was assessed under glasshouse conditions. N₂ fixing (nodulated) plants showed substantially greater growth and tissue N content than those relying solely on NH₄NO₃, NO₃⁻ or NH₄⁺ up to the equivalent of 200 kg N ha⁻¹ and N limitation is likely to have been the major cause of reduced growth of non-N₂ fixing (non-nodulated) plants. NO₃⁻ levels were negligible in plant tissues regardless of NO₃⁻ supply, indicating that virtually all NO₃⁻ taken up was assimilated. Thus, there appears to be a limitation on the amount of NO₃⁻ that S. microphylla can take up. However, it is possible that S. microphylla could not access NO₃⁻ in the potting mix and further work is required using different substrate and more regular NO₃⁻ applications to confirm this. Plants showed NH₄⁺ toxicity symptoms at 25 kg NH₄⁺-N ha⁻¹ and above. Nitrate reductase activity was not detected in roots or leaves of mature S. microphylla in the field: all plants were nodulated.

Overall, the two major findings of this research are 1) NZ native legumes are nodulated by diverse and novel Mesorhizobium species and 2) S. microphylla seedlings have limited ability to utilise soil inorganic N. Important future work based on the results obtained in this research is discussed.