Studies On Polyphasic Taxonomy Of Rice Root-associated Bacteria And Description Of Rheinheimera Tangshanensis Sp.nov.

The bacterial diversity and some characters of forty two rhizobia-like strains isolated from the roots of fresh rice plants(Oryza sativa L.) in Luannan County,Hebei province,China were investigated with a polyphasic strategy.The 16S rRNA,23S rRNA,atpD,recA,rpoB and gyrB genes were PCR amplified and sequenced,then analysed by Neighbour-Joining, Minimum Evolution and Maximum Parsimony methods.Phenotypic characters were assessed by use of the Biolog and.FAME techniques.The G+C content was determined by thermal denaturation method.According to the study of phyiogenetics,these forty two strains were clustered in four rDNA groups,assigned to the genus of Agrobacterium,Allorhizobium,Sinorhizobium(Ensifer), Rhizobium.The results of 16S rDNA partial and whole sequences analysis,numerical taxonomy based on Biolog,composition analysis of FAME were quite agreed with each other.Sequences of genes of 16S rRNA,recA,atpD,gyrB,rpoB,23S rRNA of some representative strains were analyzed for the study of phyiogenetics.J3-46,J3-47-1,J3-44 etc standing for the rDNA groupâ… were located in the branch of E.adhaerens on basis of the sequence analysis of all six gene types,with closest similarity to E.adhaerens LMG 20216~T(97-99%).The phylogenetics results have very nice coherence with Bilog and FAME results.DNA content G+Cmol%values of these strains confirmed these results.It is showed that the twelve strains of 16S rDNA groupâ… belong to the species of E.adhaerens.The strains in other three rDNA groupâ…¡,â…¢,â…£formed three independent brand branches in the phylogenetics trees according the six conserved genes and phyiogenetically related to species of the genus in Agrobacterium,Allorhizobium,Rhizobium respectively.All the 16S rDNA sequence of strains of showed the highest similarity to the type strains of known species was below or close to 97%.The other house-keeping genes with closest similarity to known species was most below 90%.They were probably standed for new species in genus of Agrobacterium,Allorhizobium and Rhizobium respectively.The phenon results were consistent with the phylogenetics analysis.To determine the noduling and nitrogen-fixing ability with legumes of these strains,a nodulation test was carried out using four different legumes:soybean(Glycine max), kidney bean(Phaseolus vulgaris),clover(Trifolium repens),lucern(Medicago sativa).Eleven strains among them successfully formed nodules on M.sativa including three E.adhaerens strains.Four strains(J3-A127,J3-N43,J3-N19,J3-AN59) were marked with gfp gene successfully and root colonization of rice by these strains were monitored respectively.These strains were detected at the root tips,root cracks,and inner roots.We also report on the taxonomic characterization of a Rheinheimera-like bacterial strain, JA3-B52,isolated from the roots of fresh rice plants during the study of bacterial diversity in rice roots.According to physiological and biochemical characteristics and genotypic analysis, the bacterium represents a novel species of the genus Rheinheimera,and the name Rheinheimera tangshanensis sp.nov,is proposed.