Polyphasic Taxonomy Of Five Novel Species Of The Genus Rhizobium

Rhizobia is one of the most important symbiotic partners of leguminous crops,pastures and trees,which provides available nitrogen for legumes through symbiotic nitrogen fixation to promote plant growth and crop yield increase.Taking full advantage of the symbiotic nitrogen fixation is of great significance to the sustainable development of agriculture.Phaseolus vulgaris?common bean?is one of the important introduced crops in leguminous crops.During the study on diversity and comparative genomics analysis of 226 strains of nodule bacteria isolated from the common bean,it was found that the number of rhizobia groups that had established symbiotic nitrogen fixation with P.vulgaris had increased after large-scale planting in China.Among them,7 strains were different from known species and were potential novel species.In this study,a polyphasic taxonomical method which synthesize phylogenetic analysis,genomic analysis,phenotypic and physiological characterization,chemical composition was employed to systematically analysis of these seven strains to determine their taxonomic status and named them.The results were as follows:Analysis of the 16S rRNA gene sequences revealed that the seven strains belonged to the Rhizobium genus.The phylogenetic tree of the housekeeping genes atpD,glnII,recA and concatenated atpD-glnII-recA gene sequences showed that the seven strains fell into five clades that were distinct enough from each other and from known species.The similatieties of concatenated housekeeping genes between the representative strains of the five clades and type strains of Rhizobium species ranged from 89.7⁹5.3%which were lower than the species threshold of 96%,indicating that these seven species represented five novel species of the genus Rhizobium,of which strains C5 and C16 represented the same novel species,strains M10 and M1 represented the same novel species,and strains L9,L18 and L43 each represented a novel species.Different from the results of housekeeping genes analysis,phylogenetic tree based on the symbiotic genes nodC and nifH showed that the symbiotic genes had a corresponding relationship with host plant of the strains but had little relationship with their taxonomic status.The calculation of ANI and dDDH based on the whole genome sequences showed that the ANI values between the representative strains of the five novel species and the related type strains of Rhizobium species ranged from 87.66⁹4.15%,and the dDDH values ranged from 31.8⁵4.5%,both lower than the species threshold?95%and 70%,respectively?.The UBCG set containing 92 core genes was used to construct the phylogenetic tree.The topology of the UBCG tree and the MLSA tree were similar.These results further confirmed these strains are novel species.The GC contents of the five novel strains were about 61%,which was within the range for Rhizobium strains?57⁶6%?.The morphology and culture characteristics,physiological and biochemical characteristics,cellular fatty acid profiles and cross-nodulation test of five novel species were carried out.It was found that their morphology and culture characteristics were similar,but there were differences in physiological and biochemical characteristics such as the utilization of sole carbon source,tolerance to antibiotics and enzyme activities.The results of the cross-nodulation test showed that these strains had a narrow range of nodulation and they could only nodulated the host plant Phaseolus vulgaris and the plant Glycine max.The cellular fatty profiles of these strains were similar to those reported for other Rhizobium strains,and C_18:08:0 and Summed Feature 8 were common major fatty acids,but there were differences among the strains in terms of other fatty acid compositions and ratios.In summary,five novel species of the genus Rhizobium are indentified in this study and they are Rhizobium leguminoides sp.nov.,the type strain is L43^T;Rhizobium phaseoloides sp.nov.,the type strain is L18^T;Rhizobium etloides sp.nov.,the type strain is L9^T;Rhizobium mazuyanense sp.nov.,the type strain is M10^T;Rhizobium chutanense sp.nov.,the type strain is C5^T?published?.This study not only enrichs the resource of rhizobia,but also provides more abundant strain resources for the inoculation of rhizobia to legumes.