Characterization and performance evaluation of rhizobial symbionts of L. tenuis from different soils of the Salado River Basin

F PIECKENSTAIN, MJ ESTRELLA, P UCHIYA, ME ETCHEPARE, ESCARAY, F., A SANNAZZARO, A MENÉNDEZ, OA RUIZ

Chascomus & Buenos Aires

Workshop; LOTus Adaptation and Sustainability in South-America Final Partners Meeting; 2009

LOTASSA

A high genetic diversity was found among the 103 isolates after REP-PCR analysis. Then, bacteria showing non-redundant REP-PCR profiles were examined for infectivity by re-inoculation on L. tenuis, and only 76% of the isolates analysed were infective.  Subsequently, the taxonomic position of the isolates was estimated. The 16S ribosomal RNA gene was characterized by restriction fragment length polymorphisms (RFLP). The full-length sequence of 16S rDNA of all different isolates was amplified by PCR, yielding a single band of the expected size. PCR products were digested using the  restriction enzymes Hinf I and Msp I and the combined Hinf I and Msp I restriction patterns were compared with those of reference strains and examined by cluster analysis. The analysis revealed 17 distinct RFLP patterns or groups, which were separated into two well-defined clusters (A and B). Cluster A comprised groups with restriction patterns similar to Mesorhizobium spp, while groups in cluster B were similar to Rhizobium spp. In order to determine their precise taxonomic position, representative strains of each RFLP group were chosen and the full 16S rDNA gene sequence was determined. 16S rRNA sequence and phylogenetic analysis further supported the distribution of most of the symbiotic isolates in either  Rhizobium  or  Mesorhizobium: the only exception was isolate BA135, whose 16S rRNA gene was closely related to that of the genus Aminobacter. Most Mesorhizobium-like isolates were closely related to M. amorphae, M. mediterraneum,  M. tianshanense, or the broad-host-range strain NZP2037, but surprisingly few isolates grouped with M. loti  type strain NZP2213.  Rhizobium-like strains were related to R. gallicum, R. etli, or R. tropici, for which Phaseolus vulgaris is a common host. In order to complete the characterization of isolates, the symbiotic genes nifH and nodC were sequenced and a phylogenetic tree was constructed.  nodC  genes from the Mesorhizobium  and  Aminobacter  strains were closely related to  nodC  genes from narrow-host-range  M. loti  strains. Likewise,  nifH  gene sequences were very highly conserved among the Argentinean isolates and reference  Lotus  rhizobia. The high levels of conservation of the nodC  and  nifH genes suggest that there was a common origin of the symbiosis genes in narrow-host-range  Lotus  symbionts, supporting the hypothesis that both intra and  intergeneric horizontal gene transfer are important mechanisms for the spread of symbiotic capacity in the SRB. Symbiotic performance of the isolates was evaluated on L. tenuis and compared with reference strain M. loti NZP2213. Rhizobia exhibiting the best symbiotic performance mainly fell within the group of isolates  obtained from non-saline cuvettes and high plains. Free-living salt-tolerance of the infective isolates was evaluated, half of them tolerating up to 200 mM NaCl. Isolates tolerating up to 1 M NaCl were found among those obtained from alkaline-saline cuvettes. Isolates were also tested for their ability to grow in vitro at alkaline pHs. All the 67 isolates evaluated were able to grow at neutral pH, while 88, 82, 68 and 55% of them tolerated pHs up to 8, 9, 10 and 11, respectively. All the alkalinity-tolerant isolates were native from alkaline-saline lowlands. An evaluation of symbiotic performance of  L. tenuis- nodulating rhizobia under salinity and alkalinity conditions is in progress.