Caracterización genética de Bradyrhizobium sp. cepa U531: Eficiencia simbiótica en Lotus angustissimus INIA Basalto

Lotus angustissimus is a legume that is increasingly used in Uruguay as a forage species for the improvement of natural fields. It is currently inoculated with Bradyrhizobium sp. strain U531, the commercial inoculant for Lotus subbiflorus. Under controlled conditions, the symbiotic efficiency of strain U531 in both hosts showed that in L. subbiflorus the biomass yielded 63% compared to the nitrogen control with 10 mM KNO₃, while in L. angustissimus the biomass of the inoculated treatment was 57% higher than the control with N. This suggests that strain U531 has an adequate symbiotic efficiency in L. angustissimus, but also raises questions about why the N control accumulates less biomass than the inoculated treatment. None of the treatments with different concentrations of KNO₃ and sources of N (urea) was able to overcome the production of aerial biomass generated by the nodulated plants. On the other hand, of 5 isolates with different ERIC profiles obtained from L. angustissimus nodules collected in Glencoe, GL2 and GL10 strains induced the same dry biomass as the treatment with the U531 strain, while GL5 strain induced more accumulation of biomass. To estimate the competitiveness of strain U531, the marking with the reporter gene gusA was attempted. The different conjugation times tested did not allow the transposon to be incorporated into the receiver, which indicates incompatibility between the donor and the receiver, so another type of marking should be evaluated, or an evaluation through the identification of ERIC or BOX-PCR profiles. Regarding the effect of the inoculant of L. corniculatus, Mesorhizobium huakuii strain U510 on L. angustissimus, at 21 days in-vitro conditions, efficient nodulation was not induced with this strain.

The genetic characterization of Bradyrhizobium sp. strain U531 included the analysis of the sequence of 16S RNA gene (1,245 nt), the housekeeping genes atpD (330 nt), glnII (525 nt) and recA (368 nt) and the symbiotic genes nodC (203 nt) and nifH (240 nt). The sequence of the 16S RNA gene places this strain within the genus Bradyrhizobium, but the sequence of the concatenated housekeeping genes does not group it with any of the described species, so it is proposed that it is a new species. According to the symbiotic genes, it groups together with a Bradyrhizobium strain isolated from Chamaecytisus proliferus in the Canary Islands and the CNL9 strain, isolated in Uruguay from Lotus uliginosus. Since the ability to denitrify is common among Bradyrhizobium, homologous sequences to the genes involved in denitrification were searched in the genome of strain U531. Through this analysis, sequences with homology to the nir, nor and nos genes, responsible for this pathway, were identified. The phenotypic analysis of the ability to denitrify in an anaerobic medium with NO₃^- does not allow us to ensure that the strain denitrifies.