Symbiotic effectiveness, competitiveness and salt tolerance of lucerne rhizobia

With the increase of salinity in Australia, salt tolerance of rhizobia in salinised and nitrogen-deficient soils is crucial to legume plants. The isolated salt-tolerant rhizobia for lucerne were equivalent in nitrogen fixation and plant growth to the recommended agricultural inoculant. The nodule occupancy by the strain NN2-1(with gusA) was 60% greater than that of the commercial inoculant RR1128 at all NaCl concentrations, suggesting that NN2-1 was more competitive than RR1128. An RNA expression-based strategy was devised to further investigate salt tolerance genes. Degenerate primers were designed based on regions of the salt tolerance genes (bet and ndv genes) and specific primers were designed to amplify these DNA regions more precisely. The transposase sequences of the IS116/110/902 family protein of S. medicae WSM419 for strains NN2-1 and DS2 and the permease sequence from R. etli for for strain RR1128 were found in salt-tolerant but not non-salt-tolerant rhizobia. Similar results were also found with extracted RNA, suggesting those sequences were related to salt tolerance in rhizoiba. Quantitative PCR (qPCR) was used as a quantification method for expression of both sequences in strains NN2-1, RR1128 and DS2 grown with or without 3% NaCl. The transposase sequences in strains NN2-1 and RR1128 were up-regulated by factors of 6.45 and 3.73 respectively in 3% NaCl, but not in strain DS2. This demonstrated a possible role of transposase sequences in salinity tolerance. The permease sequences were expressed equally in all tested strains with and without NaCl. The DNA sequences of the transposase and permease fragments from strains NN2-1 and RR1128 were further amplified and cloned into Escherichia coli, and the E. coli transconjugants with one or both genes were mated with non-salt-tolerant rhizobia to transfer one or both of these sequences into these strains. There was no difference in growth between cloned and control E. coli strains up to 5% NaCl, whereas the non-salt-tolerant recipients of rhizobia with plasmids containing either one or both sequences increased their salt tolerance up to 3% NaCl, suggesting that these small sequences were important in salt tolerance in rhizobia.