Diversity And Phylogeny Of Rhizobia And Endophytes Isolated From Glycyrrhiza SPP. Nodules In Northwestern China

To exploit rhizobia in agriculture, 159 endophytic bacteria were isolated from surface-sterilized root nodules of wild perennial Glycyrrhiza legumes growing on 40 sites in central and northwestern China. AFLP genomic fingerprinting and sequencing of partial 16S rRNA genes revealed that the collection mainly consisted of Mesorhizobium, Rhizobium, Sinorhizobium, Agrobacterium and Paenibacillus species.Based on symbiotic properties with the legume hosts G. uralensis and G. glabra, we divided the rhizobial species into true and sporadic symbionts. Five distinct Mesorhizobium groups represented true symbionts of the host plants, the majority of strains inducing N2-fixing nodules. Sporadic symbionts consisted of either of species with infrequent occurrence (R. galegae, R. leguminosarum) or species with weak (S. meliloti, R. gallicum) or no N2 fixation ability (R. giardinii, R. cellulosilyticum, Phyllobacterium sp.).Multivariate analyses applied displayed that the host plant species and geographic location explained only a small part of the total variation in bacterial AFLP patterns, the host plant (82%) explaining slightly more than the geography (74%). Discriminant analysis (DA) showed that the distribution of AFLP genotypes was associated with the host plant and geographic sampling region, strains isolated from G. glabra were clearly separated from those obtained from G. uralensis, strains obtained from central China were also well separated from those originating from Xinjiang, indicating host preference and regional endemism. Canonical correspondence analysis (CCA) suggested that the prevalence of Paenibacillus sp. and the poor discovery of symbiotic species among G. uralensis isolates originating in Xinjiang were associated with the low annual mean rainfall of the region.Phylogenetic analysis of nearly full-length 16S rRNA gene for Mesorhizobium grouped the test strains into five distinct clades separated from all currently recognized Mesorhizobium species. Grouping of the isolates under study based on the house-keeping genes recA and rpoB were consistent and in agreement with that of 16S rRNA. Similarly phylogenetic relationships based on the symbiosis-related genes nodC, nodA and nifH were generally similar to those shown by the core genes, suggesting that these Glycyrrhiza symbionts have a long history of separate evolution within Mesorhizobium. The study strongly suggests that the collection of Mesorhizobium strains comprises several new species, and also indicates the role of the symbiotic genes in determining the host range of these bacteria.