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Aims To identify the rhizobia nodulating Vicia sativa in Northwestern China and to estimate their geographic distribution. Methods Rhizobia trapped with V. sativa plants from soils at six sites in the northwest of China were classified into genotypes by PCR-based restriction fragment length polymorphism (RFLP) of 16S-23S rRNA intergenic spacer (IGS) and 16S rRNA genes, and phylogenetic analyses of housekeeping (16S rRNA, recA, atpD) and symbiotic genes were performed for the representative strains. Soil physicochemical characteristics were recorded and canonical correlation analysis was performed to examine the correlations between soil features and distribution of rhizobial genotypes. Results A total of 202 rhizobial isolates were discriminated by RFLP analyses into 15 IGS types and a single 16S rRNA type, which were identified as Rhizobium by 16S rRNA gene phylogeny and as four clusters by multilocus sequence analysis (MLSA). Cluster 1 covering 86 strains and 7 IGS types prevalent in all sites was identified as Rhizobium laguerreae; cluster 2 was R. sophorae with 18 strains in 2 IGS types found in the site Q-GD. Each of cluster 3 and cluster 4 contained three IGS types representing two novel Rhizobium genospecies specific to Gansu Province and Shanxi Province, respectively. Four nodC phylogenetic clades were defined among the isolates. Conclusions R. sophorae, R. laguerreae, and two novel Rhizobium genospecies with diverse symbiotic genotypes are associated with Vicia sativa L. in Northwest China. Their biogeographic patterns are mainly directed by soil pH and salinity. This is the first study on the diversity of rhizobia nodulating Vicia sativa in China.

Aims To investigate the succession of chickpea rhizobia over years and sampling sites. Methods Rhizobia were sampled four times from nodules of chickpea grown in four sites of Mulei County and Qitai County in Xinjiang, China during 2009-2016. The recA genes of rhizobia representing different populations were phylogenetically compared for their genotypic differentiation. Soil properties were examined to explore their hypothetical incidence on the rhizobial succession. Results The 628 isolates were all identified as Mesorhizobium muleiense, belonging to 28 genotypes distinguished by recA phylogeny. For each site, 15, 17, 10 and 7 genotypes were detected during the whole sampling period; while 4, 15, 12 and 17 genotypes were evidenced in each of the 4 sampling years (2009, 2013, 2015 and 2016) from all the four sites. Four major genotypes (T12 through T15) were found throughout the four years in the four sites at various frequencies. While the other genotypes were site and/or year specific. Both increased genetic diversity as revealed by Shannon-Weaver index and shifts in the population composition as revealed by genotyping were detected in the chickpea rhizobial populations during the study period. Nitrogen content, available phosphorus and potassium, and pH were the main factors correlated to the genotype distribution of chickpea rhizobia in the soil over years. Conclusions The genetic diversity of M. muleiense increased and the population composition shifted over years and at different sampling sites. The recA gene is an adequate molecular marker to estimate the intraspecific variation and succession of M. muleiense genotypes. The rhizobial genotype succession/evolution in the fields might be driven by the fluctuations of soil characteristics like nitrogen content, available phosphorus and potassium, and pH.

Aims To identify the rhizobia nodulating Astragalus sinicus in the acid soils at Xinyang, China and to estimate the effects of fertilization strategies on the rhizobial community. Methods Soil samples were collected from treatments submitted to different fertilization strategies (chemical fertilizers, A. sinicus green manure, various input levels). Nodules obtained from the A. sinicus trapping plants grown in the soil samples were used for rhizobial isolation. The isolates were grouped into PCR-RFLP genotypes based on analyses of 16S rRNA gene and IGS, and representatives for each genotype were further subjected to phylogenetic analyses of housekeeping genes (16S rRNA gene, atpD, glnII, and rpoB), as well as symbiotic genes (nodC and nifH). Soil physicochemical characteristics were also estimated and redundancy analysis was performed to examine the relations between soil features and distribution of rhizobial genotypes across different fertilization treatments. Results A total of 257 rhizobial isolates were obtained and discriminated into 12 IGS types in a single 16S rRNA type by RFLP analyses. Further 16S rRNA gene sequence-based phylogeny and multilocus sequence analysis (MLSA) distinguished a minor group (10 strains, 4 IGS types) affiliated to Mesorhizobium huakuii and a main group (247 strains, 8 IGS types) to Mesorhizobium jarvisii, for which a novel symbiovar, sv. astragali, was described. The long-term input of A. sinicus green manure increased rhizobial diversity and modified rhizobial community structures, via the increase of organic matter and decrease of soil pH, available phosphate, available potassium, and total salt. Conclusions M. jarvisii sv. nov. astragali was the predominant microsymbiont of A. sinicus in the tested acidic soils. The long-term input of green manure modified the soil features, enhancing the genetic diversity of rhizobia associated with A. sinicus and alternating the rhizobial community structure.