Numerical analysis of phenotypic properties, genomic fingerprinting, and multilocus sequence analysis of Bradyrhizobium strains isolated from root nodules of Lembotropis nigricans of the tribe Genisteae

Abstract

Purpose: The aim of this study was to estimate the level of genomic and phenotypic diversity as well as the genus and species position of bacterial strains isolated from root nodules of Lembotropis nigricans (family Fabaceae). Methods: The genomic diversity of studied L. nigricans nodule symbionts was examined by using BOX-PCR and AFLP (amplified fragment length polymorphism) fingerprinting techniques. To assign bacteria to the genus, numerical analysis of phenotypic features and comparative analysis of 16S rDNA sequences were performed. The comparative analysis of combined atpD, dnaK, gyrB, and rpoB gene sequences (multilocus sequence analysis, MLSA) was used to determine the most closely related species to the studied bacteria. Results: Both BOX-PCR and AFLP techniques revealed a high level of genomic heterogeneity of L. nigricans nodulators. Among 33 studied bacteria, 32 genotypes were delineated by the AFLP method and 27 genotypes were identified by the BOX-PCR fingerprinting. The numerical analysis of 86 phenotypic characteristics of L. nigricans nodule isolates and reference rhizobia showed that studied bacteria belong to the genus Bradyrhizobium. Affiliation of L. nigricans nodule isolates to the genus Bradyrhizobium was supported by comparative analysis of 16S rDNA sequences and the concatenation of atpD, dnaK, gyrB, and rpoB gene sequences. MLSA indicated also that L. nigricans microsymbionts are members of Bradyrhizobium japonicum. Conclusion: L. nigricans root nodule symbionts are members of Bradyrhizobium japonicum and exhibit high phenotypic and genomic diversity important for their survival in soil.