Rapid identification of Rhizobium species based on cellular fatty acid analysis

As understanding of the evolutionary relationships between strains and species of root nodule bacteria increases the need for a rapid identification method that correlates well with phylogenetic relationships is clear. We have examined 123 strains of Rhizobium: R. fredii (19), R. galegae (20), R. leguminosarum (22), R. loti (17), R. meliloti (21), and R. tropici (18) and six unknowns. All strains were grown on modified tryptone yeast-extract (TY) agar, as log phase cultures, scraped from the agar, lysed, and the released fatty acids derivatized to their corresponding methyl esters. The methyl esters were analysed by gas-chromatography using the MIDI/Hewlett-Packard Microbial Identification System. All species studied contained 16:0, 17:0, 18:0 and 19cyclow9C fatty acids but only R loti and R tropici produced 12:0 3 OH, 13:0 iso 3 OH,18:1w9C and 15:0 iso 3 OH,17:0 iso 3 OH and 20:2w6,9C fatty acids respectively. Principal component analysis was used to show that strains could be divided into clusters corresponding to the six species. Fatty acid profiles for each species were developed and these correctly identified at least 95% of the strains belonging to each species. A dendrogram is presented showing the relationships between Rhizobium species based on fatty acid composition. The data base was used to identify unknown soil isolates as strains of Rhizobium lacking a symbiotic plasmid and a bacterium capable of expressing a symbiotic plasmid from R. leguminosarum as Sphingobacterium spiritovorum.