Aspects of soil ecology of rhizobia affecting nodule occupancy on lucerne and white clover

Successful establishment of white clover (Trifolium repens) and lucerne (Medicago sativa) in the field depends on the formation of effective symbioses with Rhizobium leguminosarum and Sinorhizobium meliloti, respectively. High numbers of naturalised rhizobia and the poor persistence of commercial inoculants has cast doubt on the need for commercial inoculation in New Zealand. This study investigated aspects of the soil ecology of R. leguminosarum and S. meliloti that affected the persistence and success of applied strains. In planta the commercial inoculant, strain S. meliloti strain RRI128, was present in 57-61% of lucerne nodules four years after sowing when applied as a peat slurry. In contrast, R. leguminosarum strain TA1 was only present in 8% of white clover nodules eight months after sowing. Naturalised strains of rhizobia were also present in the nodules of both species. The abundance of naturalised strains differed seasonally, from 5.2 x 104 cells g-1 soil in the winter to 200 cells g-1 soil in the summer. This was explained by increasing numbers at low (<10°C) temperatures and high (>20%) soil moisture. Additional bacteria, including a Pseudomonas sp. capable of forming nodules, were also isolated from both plant species. Illumina sequencing revealed that Rhizobium and Sinorhizobium were the dominant genera in the nodules of white clover and lucerne plants, respectively, regardless of inoculation treatment and made up 89-97% of all sequences. Sequencing confirmed the presence of other bacteria in the nodules of both host species with 408-879 genera identified, with each represented by <1% of the total sequences. The ecology of TA1 and other R. leguminosarum strains in their free living state in the soil was investigated using the γ-MicroResp™ assay. This was developed to measure carbon utilisation of individual rhizobia strains in a sterile soil environment. Using a panel of diverse strains, results demonstrated that strains of a R. leguminosarum utilised carbon compounds differently, with some strains (WU95, CC1047 and CC1049a) being high users of sugars while others (SU157 and TA24) used a wide of range of carbon sources. Some strains (CC275e, WSM1325 and SU157) retained their grouping when placed in a different soil background, whereas, others (WSM409 and TA1) altered their carbon utilisation patterns. γ-MicroResp was then used to determine the relative carbon utilisation of New Zealand naturalised and commercial R. leguminosarum strains in a range of soils. The link between carbon utilisation and nodulation success was explored with paired strains with different carbon utilisation profiles. A naturalised strain from New Zealand, ADWC3a, was a high user (average absorbance: >1.5) of many carbon sources and was more competitive for nodule occupancy compared with TA1, which utilised fewer carbon sources at lower amounts (average absorbance: <1.5). White clover plants inoculated with ADWC3a and TA1 at a ratio of 1:4 had ADWC3a present in 75% of the nodules. This work confirmed γ-MicroResp as a useful tool to screen for strains with increased competitive ability in the soil. To determine whether there was potential to select S. meliloti strains to improve establishment of lucerne in the NZ high country, the Al3+ tolerance of the current commercial strain (RRI128) against acid tolerant strains from Australia (SRDI736 and SRDI672) was investigated. The acid tolerance of SRDI736 was confirmed in a hydroponic experiment with higher nodulation (>70% plants nodulated) at pH 5.1 compared with RRI128 (<50% plants nodulated). However, the number of nodules per plant decreased from 2-4 to 0.5-2.5 as Al3+ increased from 0 to 8 μM, suggesting it had limited Al3+ tolerance. The potential of the acid tolerant strains to increase lucerne production in high Al3+ and low pH environments was assessed in a pot experiment. SRDI736 and SRDI672 again showed acid tolerance with increased number of nodules per plant at pH 5.8 and Al3+ level of 1.4 mg kg-1 soil compared with S. meliloti strain RRI128 (>100 vs. <50 nodules per plant). However, in both experiments increased nodulation under increased pH and Al3+ did not result in increased dry matter production. Overall, this research has highlighted differences in the success of the commercial inoculants R. leguminosarum and S. meliloti, as measured by nodule occupancy. For R. leguminosarum, strains better able to utilise common rhizosphere exudates were competitive for nodule occupancy. For S. meliloti, results showed that there was potential to select strains better able to drive nodulation of lucerne in the harsh New Zealand high country soils. Thus, the new tools and knowledge generated in this thesis have identified key traits for better selection of commercial inoculants.