Bradyrhizobium occurrence in nodules of perennial horsegram

Abstract

The introduction of a forage legume into a tropical pasture should decrease the need for N fertilizer, provided biological N₂ fixation (BNF) contributes enough to compensate for exported N. Macrotyloma axillare (perennial horsegram) is a suitable legume for composing mixed pastures, and our hypothesis is that the isolation of indigenous rhizobia from roots and rhizosphere is the way of achieving an efficient inoculant to maximize BNF to the legume. Nodules and rhizosphere soil taken from M. axillare grown in a mixed pasture with palisade grass were sampled and used in a trap host assay using Leonard jars containing a mixture of vermiculite and sand. A total of ten bacteria were initially isolated using this technique. The isolates were then used in two experiments to evaluate the inoculation responses on the perennial horsegram in greenhouse conditions to which nodulation, plant growth, and shoot N accumulation were measured. Phylogenetic analyses based on 16S rRNA and recA placed all strains within genus Bradyrhizobium, some of them not previously described. The best strain provided more than 120 nodules and more than 65 mg of nodules per plant. Strain BR14182 was considered the most promising given the high dry matter and N accumulation in plant shoots. This study provides the first analysis of Bradyrhizobium diversity nodulating M. axillare in Brazil and provided evidence of the role of inoculation in incrementing the plant-rhizobium symbiosis in a forage legume.

Keywords: Macrotyloma axillare; Mixed pastures; Nodulation; Rhizobia inoculant.

Fig. 1

Phylogenetic tree based on 16S rRNA gene sequences (~ 1500 bp), including the bacterial type strain deposited in the NCBI database. Phylogeny was based on clustering of sequences according to the Neighbor-Joining algorithm and Kimura model using MEGA 6.1 program. Numbers located in the branches indicate the percentage of 1000 sub-samples (bootstrap). The scale represents the number of mutations per nucleotide position

Fig. 2

Phylogenetic tree based on recA rRNA gene sequences (~ 500 bp), including the bacterial type strain deposited in the NCBI database. Phylogeny was based on clustering of sequences according to the Neighbor-Joining algorithm and Kimura model using MEGA 6.1 program. Numbers located in the branches indicate the percentage of 1000 sub-samples (bootstrap). The scale represents the number of mutations per nucleotide position