Study Of Endophytic Rhizobium Smilacinae Sp. Nov In Smilacina Japonica A. Gray

Traditional medicinal plants contained a vast of unknown microorganism resources and bioactive functional strains are excellent materials for looking for new potential microbial species and natural products. During a study of diversity of endophytic bacteria from traditional Chinese medicinal plants collected from TaiBai mountain, a white bacterial strain, designated PTYR-5T, was isolated from the leaf of Smilacina japonica A. Gray and subjected to a taxonomic study by using polyphasic approaches.Phylogenetic analysis of 16 S rRNA gene sequences showed that strain PTYR-5T is a member of the genus Rhizobium and the similarity of 16 S rRNA gene with all the reported species in genus Rhizobium was very low, reporting the highest 16 S rRNA gene sequence similarity to R. cellulosilyticum LMG23642T(97.2 %), R. huautlenseLMG18254T(97.2 %) and R. alkalisoli CCBAU 01393T(97.1 %). The phylogeny of 16 S rRNA gene sequences and housekeeping genes showed that the strain maybe a novel species of Rhizobium.Phenotypic analysis showed significance difference between strain PTYR-5T and its reference strains in nitrate reduction capability, growth temperature range, resistance to antibiotic and salts, utilization of carbon and nitrogen source and enzyme activity. The main fatty acid composition of strain PTYR-5T was summed feature 8(C18:1ω7c /C18:1ω6c; 56.5 %) and C16:0(16.7 %), which fit for the characteristic of Rhizobium but differ from above three reference strains in the kinds and proportions. In addition, strain PTYR-5T was unable to induce any nodule formation on Medicago sativa, Vigna radiata and Trifolium repens, and no PCR product was obtained using nodC and nifH primers, suggesting that it was without the capability of plant nodulation and nitrogen fixation, which differ from R. cellulosilyticum LMG 23642T、R. huautlense LMG 18254 T and R. alkalisoli CCBAU 01393 T too.The analysis of DNA homology showed that the DNA G+C mol% of the novel strain PTYR-5T was 61.3 mol%, within the scale reported for the genus Rhizobium(57-66 mol%), but it differed from the DNA G+C mol% of reference strains R. cellulosilyticum LMG 23642 T, R. huautlense LMG 18254 T and R. alkalisoli CCBAU01393 T,suggesting there were large difference between their genome constitutions. Besides, the DNA–DNA relatedness between the novel strain PTYR-5T and reference strains R. cellulosilyticum LMG 23642T、R. huautlense LMG 18254 T and R. alkalisoli CCBAU 01393 T were 33.6%,21.4% and 29.5%, respectively. which was lower than the standard(70%) for reporing new species.Based on the morphological features, physiological biochemical characteristics, chemotaxonomy, phylogenetic analysis and genome information, we consider that the strain represent a novel species of Rhizobium and we propose the name Rhizobium Smilacinae sp. nov. for it. PTYR-5T(=CCTCC AB2013016T=KCTC 32300T=LMG 27604T) is designated as the type strain.