A Gram-negative, rod-shaped, motile, aerobic bacterial strain, W3(T), was isolated from hexachlorocyclohexane (HCH)-contaminated groundwater from Lucknow, India, and its taxonomic position was determined using a polyphasic approach. Strain W3(T) shared highest 16S rRNA gene sequence similarity of 97.8 % with Rhizobium selenitireducens B1(T), followed by Rhizobium daejeonense L61(T) (97.7 %), Rhizobium radiobacter ATCC 19358(T) (97.5 %) and Blastobacter aggregatus IFAM 1003(T) (97.2 %). Strain W3(T) formed a monophyletic clade with Blastobacter aggregatus IFAM 1003(T) (= DSM 1111(T)) in the cluster of species of the genus Rhizobium. Phylogenetic analyses of strain W3(T) using atpD and recA gene sequences confirmed the phylogenetic arrangements obtained by using 16S rRNA gene sequences. Hence, the taxonomic characterization of B. aggregatus DSM 1111(T) was also undertaken. Strains W3(T) and B. aggregatus DSM 1111(T) contained summed feature 8 (18 : 1�s7c and/or 18 : 1�s6c; 65.4 and 70.8 %, respectively) as the major fatty acid, characteristic of the genus Rhizobium. DNA-DNA relatedness of strain W3(T) with Rhizobium selenitireducens LMG 24075(T), Rhizobium daejeonense DSM 17795(T), Rhizobium radiobacter DSM 30147(T) and B. aggregatus DSM 1111(T) was 42, 34, 30 and 34 %, respectively. The DNA G+C contents of strain W3(T) and B. aggregatus DSM 1111(T) were 62.3 and 62.7 mol%, respectively. A nifH gene encoding dinitrogenase reductase was detected in strain W3(T) but not in B. aggregatus DSM 1111(T). Based on the results obtained by phylogenetic and chemotaxonomic analyses, phenotypic characterization and DNA-DNA hybridization, it is concluded that strain W3(T) represents a novel species of the genus Rhizobium, for which the name Rhizobium rosettiformans sp. nov. is proposed (type strain W3(T) = CCM 7583(T) = MTCC 9454(T)). It is also proposed that Blastobacter aggregatus Hirsch and M?ller 1986 be transferred to the genus Rhizobium as Rhizobium aggregatum comb. nov. (type strain IFAM 1003(T) = DSM 1111(T) = ATCC 43293(T)).

The genera Agrobacterium, Allorhizobium, and Rhizobium belong to the family Rhizobiaceae. However, the placement of a phytopathogenic group of bacteria, the genus Agrobacterium, among the nitrogen-fixing bacteria and the unclear position of Rhizobium galegae have caused controversy in previous taxonomic studies. To resolve uncertainties in the taxonomy and nomenclature within this family, the phylogenetic relationships of generic members of Rhizobiaceae were studied, but with particular emphasis on the taxa included in Agrobacterium and the "R. galegae complex" (R. galegae and related taxa), using multilocus sequence analysis (MLSA) of six protein-coding housekeeping genes among 114 rhizobial and agrobacterial taxa. The results showed that R. galegae, R. vignae, R. huautlense, and R. alkalisoli formed a separate clade that clearly represented a new genus, for which the name Neorhizobium is proposed. Agrobacterium was shown to represent a separate cluster of mainly pathogenic taxa of the family Rhizobiaceae. A. vitis grouped with Allorhizobium, distinct from Agrobacterium, and should be reclassified as Allorhizobium vitis, whereas Rhizobium rhizogenes was considered to be the proper name for former Agrobacterium rhizogenes. This phylogenetic study further indicated that the taxonomic status of several taxa could be resolved by the creation of more novel genera.

Genomic ANI (Average Nucleotide Identity) has been found to be able to replace DNA-DNA hybridization in prokaryote taxonomy. The ANI of each of the core genes that has a phylogeny congruent with the reference species tree of rhizobia was compared to the genomic ANI. This allowed us to identify three housekeeping genes (SMc00019-truA-thrA) whose ANI reflected the intraspecies and interspecies genomic ANI among rhizobial strains, revealing an ANI gap (?2%) between the inter- and intra-species comparisons. The intraspecies (96%) and interspecies (94%) ANI boundaries calculated from three genes (SMc00019-truA-thrA) provided a criterion for bacterial species definition and confirmed 621/629 of known interspecies relationships within Bradyrhizobium, Mesorhizobium, Sinorhizobium and Rhizobium. Some widely studied strains should be renamed. The SMc00019-truA-thrA ANI also correlates well with the genomic ANI of strains in Agrobacterium, Methylobacterium, Ralstonia, Rhodopseudomonas, Cupriavidus and Burkholderia, suggesting their wide applicability in other bacteria.