Characterisation of rhizobia associated with Sophora, Trifolium ambiguum and Genisteae legumes in New Zealand and assessment of their importance to nitrogen assimilation at Lincoln University of European Broom and Sophora microphylla : A thesis submitted in partial fulfilment of the requirements for the Degree of Doctor of Philosophy

A review of the literature on legumes and their associated rhizobia in New Zealand resulted in four objectives being set for this project. These were to 1) characterise rhizobia associated with endemic Sophora in natural ecosystems in New Zealand with a focus on the North Island; 2) determine if rhizobial inoculum strains used on hexaploid Caucasian clover are persistent in NZ low and high fertility soils; 3) characterise the rhizobia associated with NZ exotic Genisteae legumes across the NZ South Island with the specific objective of determining their origin; 4) determine if European broom (Cytisus scoparius) and Sophora microphylla can utilise soil NO3- and if so, does this impact on their N2 fixation. Housekeeping gene sequences indicated that all 44 rhizobial isolates from NZ native Sophora sampled at four sites in the South Island and seven in the North Island were Mesorhizobium. Twenty six of these isolates grouped with Mesorhizobium spp. previously isolated from Sophora in NZ based on their concatenated recA, glnII and rpoB gene sequences but 18 isolates separated into two specific groups and a pair of isolates clearly separated from all Mesorhizobium type strains. DNA-DNA hybridisations indicated that the two groups of isolates could belong to novel Mesorhizobium species. The nifH and nodC gene sequences of all isolates grouped closely with those for Mesorhizobium previously isolated from NZ endemic Sophora spp. indicating that the various species within the group obtained their symbiosis genes via lateral gene transfer. Rhizobial isolates with different housekeeping gene sequences gave similar increases in dry weight (DW) and total nitrogen (N) content of four Sophora species under glasshouse conditions. Hexaploid Caucasian clover was grown in soil sampled at three South Island high country sites to which specific rhizobium inoculum had been added with sowing of the legume in 1975, 1992 and 1997; two sites on the Lincoln University farm sown with inoculated Caucasian clover in 2012 and 2013 and six sites not sown with the crop. Caucasian clover nodulated in soil from all sites sown with inoculated Caucasian clover but did not nodulate in soils from the other sites. Rhizobial isolates from plants in each soil where nodulation occurred showed the same genetic profile and gave a similar increase in growth of Caucasian clover in low nitrogen soil, as the recommended inoculum for hexaploid Caucasian clover. This indicates that Caucasian clover specific rhizobia can persist and retain their effectiveness for at least 42 years on New Zealand South Island low fertility, high country soils and at least 5 years in high fertility soils. Root nodules of Genisteae species were sampled along an eleven site ‘transect’ from north to south in the South Island of NZ. Sixty eight bacterial isolates from these nodules, 22 from European broom (Cytisus Scoparius), 19 from gorse (Ulex europaeus) and 27 from Lupinus polyphyllus, along with 3 isolates from European broom sampled in Belgium, and 1 isolate from European broom, and 2 from gorse sampled in the UK, were characterised on the basis of their recA, glnII, atpD and nodA genes. Sequences of recA, glnII and atpD genes indicated that all 74 isolates belonged to the genus Bradyrhizobium. Forty representative isolates from different groupings on the concatenated recA, glnII and atpD genes tree were shown to be able to form effective nodules on their host plants. Sequences for the recA, glnII, atpD and nodA genes showed high similarity to B. japonicum and B.canariense strains from the Mediterranean region and Europe. It is concluded that bradyrhizobia nodulating European broom, gorse and Lupinus polyphyllus throughout the South Island are not native to NZ but were in some way introduced to NZ with their host plants. Total plant DW and N for European broom increased two to three fold with increased NO3- supply from 0 to 100 kg NO3--N ha-1 under glasshouse conditions. Also, acetylene reduction activity (ARA) for nodulated root systems of European broom decreased linearly with applied NO3--N indicating a decrease in N2 fixation. 15N-labelled NO3- indicated that the proportion of N derived from N2 fixation (%Ndfa) of European broom decreased with increased NO3- supply from around 98% at zero N to around 25% at 100 kg N ha-1. It is concluded that European broom is a facultative N2 fixer and at high soil NO3- levels (100 kg N ha-1) obtains the bulk of its N requirements from the soil. Total plant DW and N for nodulated S. microphylla changed little with NO3- supply from 0 to 100 kg N ha-1. The ARA values for nodulated root systems of S. microphylla decreased with applied NO3- but values were always relatively high. Similarly, the 15NO3- analysis indicated that even at 100 kg N ha-1, S. microphylla obtained the major proportion of its N requirement (76%) from N2 fixation. Total plant DW and N were similar for inoculated plants and uninoculated plants supplied 50 or 100 kg N ha-1 as NO3-. This shows that S. microphylla can utilise soil NO3- if not nodulated. Total plant DW and total plant N were greater for plants supplied 50 kg N ha-1 as NH4+ or 50 and 100 kg N ha-1 as urea than for inoculated plants or those supplied 50 or 100 kg N ha-1 as NO3-. The urea treatments gave two to three times greater total plant DW and total plant N in comparison with inoculated plants. Areas for further research are highlighted.