Identification of Agrobacterium and Rizobium species based on cellular fatty acid composition

The increasing number of phylogenetically defined species in the genera Agrobacterium, Rhizobium and Sinorhizobium suggests a need for a rapid identification method which will distinguish between these species. We have examined 65 strains of Agrobacterium representing: A. tumefaciens, (34); A. rhizogenes, (16) A. vitis, (10) Al rubi (2) and some unclassified strains, and 150 strains of Rhizobium and Sinorhizobium representing: R. etli (21); R. galegae (20); R. huakuii (17); R, leguminosarum (20); R. loti (16); R. topici (18); S. fredii( 19); and S. meliloti (20). Fatty acid methyl esters (FAME) were obtained from each strain, as previously described, and analysed by gas-chromatography using the MIDI Hewlett-Packard Microbial Identification System (MIS). Fatty acid profiles were recorded, characteristic fatty acids noted and the overall similarities between fatty acid profiles for each species calculated. Relationships between species were also derived from the fatty acid data by principal component analysis. This showed overlapping clusters for strains of R, leguminosarum and R, etli, R. topici and A. rhizogenes and S. fredii and S. meliloti within one supercluster. Strains of A. tumefaciens, A I nbi, A. vitis and R. galegae formed a second supercluster while R. loti and R. huakuii strains formed a third cluster well separated from all the other strains. The fatty acid profiles were used to correctly identify at least 94% of the strains representing each species in the collection except R. etli. R. etli strains (23.8%) were misidentified as R. leguminosarum. This was attributed to the high similarity (44.7%) between R. etli and R. leguminosarum. It is concluded that whole cell fatty acid analysis should form part of the polyphasic description of new species of root nodule bacteria, with the proviso that growth conditions and analytical methods be carefully standardized. It is suggested that FAME-MIS system and the database we have compiled provide a basis for future development.