Phenotypic characterization of Astragalus glycyphyllos symbionts and their phylogeny based on the 16S rDNA sequences and RFLP of 16S rRNA gene

Abstract

In this study, the nitrogen fixing Astragalus glycyphyllos symbionts were characterized by phenotypic properties, restriction fragment length polymorphism (RFLP), and sequences of 16S rDNA. The generation time of A. glycyphyllos rhizobia in yeast extract mannitol medium was in the range 4-6 h. The studied isolates exhibited a low resistance to antibiotics, a moderate tolerance to NaCl, assimilated di-and trisaccharides, and produced acid in medium containing mannitol as a sole carbon source. In the cluster analysis, based on 86 phenotypic properties of A. glycyphyllos symbionts and the reference rhizobia, examined isolates and the genus Mesorhizobium strains were placed on a single branch, clearly distinct from other lineages of rhizobial genera. By the comparative analysis of 16S rRNA gene sequences and 16S rDNA-RFLP, A. glycyphyllos nodulators were also identified as the members of the genus Mesorhizobium. On the 16S rDNA sequence phylogram, the representatives of A. glycyphyllos nodule isolates formed a robust, monophyletic cluster together with the Mesorhizobium species at 16S rDNA sequence similarity of these bacteria between 95 and 99 %. Similarly, the cluster analysis of the combined RFLP-16S rDNA patterns, obtained with seven restriction endonucleases, showed that A. glycyphyllos rhizobia are closely related to the genus Mesorhizobium bacteria. The taxonomic approaches used in this paper allowed us to classify the studied bacteria into the genus Mesorhizobium.

Multilocus sequence analysis supports the taxonomic position of Astragalus glycyphyllos symbionts based on DNA–DNA hybridization

“…In all individual gene trees (Figs S1-S5), the A. glycyphyllos nodulators were consistently clustered with species of the genus Mesorhizobium (at 90-99 % similarity), in agreement with their grouping based on 16S rRNA gene sequences (Gnat et al, 2014). The species of Ensifer (Sinorhizobium) plus Rhizobium formed a well-supported sister clade to the monophyletic cluster formed by Mesorhizobium, whereas the species of the genus Bradyrhizobium and Azorhizobium caulinodans constituted a distinct lineage(s) on the outskirts of the phylograms.…”

“…1. There is strong agreement in the placement of the test A. glycyphyllos rhizobia, with respect to reference rhizobia, in the phylogram based on the five concatenated sequences and those based on the individual atpD, dnaK, glnA, recA and rpoB (Figs S1-S5) and 16S rRNA (Gnat et al, 2014) gene sequences. It is also worth underlining that, in the concatenated gene tree (Fig.…”

“…In our earlier studies, 28 A. glycyphyllos nodule isolates were classified into the genus Mesorhizobium, based on numerical analysis of phenotypic properties, almost fulllength 16S rRNA gene sequences and 16S rRNA genebased RFLP genomotyping (Gnat et al, 2014). The SSU rRNA gene sequence-based phylogenetic tree of six A. glycyphyllos symbionts, representing different phenons and genomotypes of these bacteria, clearly demonstrated that two of these strains, AG17 and AG22, are close phylogenetic neighbours of M. amorphae and M. septentrionale (98-99 % sequence similarity), whereas the four other strains, AG1, AG7, AG15 and AG27, showed a close phylogenetic relationship to M. ciceri (95-99 % sequence similarity).…”

“…For MLSA, DNA-DNA hybridization and analysis of DNA G+C content, A. glycyphyllos nodule isolates AG1, AG7, AG15, AG17, AG22 and AG27 were used (Gnat et al, 2014). Additionally, for the determination of DNA-DNA hybridization by a microplate method, Mesorhizobium amorphae ICMP 15022 T , M. ciceri USDA 3383 T , M. septentrionale SDW014 T , M. temperatum LMG 23931 T , M. loti USDA 3471 T , M. albiziae CCBAU 61158 T and M. huakuii USDA 4779 T were used as reference strains.…”

“…To clarify the phylogenetic relationship of A. glycyphyllos nodulators and establish their species position, strains AG1, AG7, AG15, AG17, AG22 and AG27, representing different bacterial clusters identified by numerical taxonomy (Gnat et al, 2014), were used for sequence analysis of five housekeeping genes. All PCR amplifications were performed in 25 ml PCR mixtures (ReadyMix Taq; Sigma) containing 50 ng DNA and 30 pmol of each primer using a Perkin-Elmer model 2400 thermocycler.…”