Rhizobium ciceri sp. nov., consisting of strains that nodulate chickpeas (Cicer arietinum L.)

The taxonomic status of 16 collection strains of chickpea (Cicer arietinum L.) rhizobia which were previously determined to belong to two groups (groups A and B) were compared with reference strains belonging to different genera and species of the family Rhizobiaceae. We used the following taxonomic, phylogenetic, and phenotypic characteristics and approaches to study these organisms: DNA homology, guanine-plus-cytosine content, restriction fragment length polymorphism of the amplified 16S-intergenic spacer rRNA gene, partial 16S rRNA sequencing, and auxanographic tests performed with 147 carbon sources. Similar groups of chickpea strains were identified by the different approaches. The chickpea strains were found to belong to the genus Rhizobium regardless of the phylogenetic group to which they belonged (group A or B). All strains fell into a tight cluster which included Rhizobium loti and Rhizobium galegae, and the group B strains were closely related to R. loti. An analysis of partial 16S ribosomal DNA sequences revealed identical nucleotide sequences for the slowly growing strains and fast-growing strains that were used as representatives of groups A and B, respectively, and these organisms fell into the Rhizobium-Agrobacterium lineage. When the sequences of these organisms were compared with the partial sequences of Rhizobium huakuii and R. loti, one- and two-nucleotide mismatches were observed, respectively, indicating that the chickpea rhizobia are closely related to these two species. The DNA-DNA hybridization data revealed that the chickpea rhizobia exhibited low levels of homology (less than 17%) to previously described Rhizobium and Bradyrhizobium species. Moreover, when we compared chickpea strains to R. loti and R. huakuii, the most closely related species as determined by the partial 16S rRNA sequence analysis, the homology values ranged from 21 to 52% and the delta Tm values were greater than 5 degrees C (delta Tm is the difference between the denaturation temperatures of the heterologous and homologous duplexes). These results confirmed that the rhizobia that nodulate chickpeas cannot be assigned to a previously described species. Within the chickpea rhizobia, the DNA homology values obtained when members of groups A and B were compared were less than 38%, indicating that the group A and group B organisms belong to different species. Furthermore, these organisms can be distinguished from each other by the results of phenotypic tests. We propose that the group B chickpea rhizobia should be assigned to a new species, Rhizobium ciceri; UPM-Ca7 is the type strain of R. ciceri.