DIFFERENTIAL CHARACTERIZATION OF AGROBACTERIUM SPECIES USING CARBON-SOURCE UTILIZATION PATTERNS AND FATTY-ACID PROFILES

Pathogenic and nonpathogenic strains of Agrobacterium tumefaciens (i.e., biovar 1), A. rhizogenes (i.e., biovar 2), A. vitis (i.e., biovar 3), and A. rubi were tested for their ability to utilize 95 different carbon sources available in the GN Microplate system and analyzed for fatty acid content using the MIDI gas-liquid chromatography system. Several carbon sources were identified as being of diagnostic value. With the exception of the A. rubi strain, hydroxy-L-proline and i-erythritol were exclusively metabolized by A. tumefaciens and A. rhizogenes, respectively. Analysis of fatty acid composition confirmed that cis-vaccenic acid (18:1omega cis) is the predominant acid in all the Agrobacterium strains. The amount of 3-hydroxypalmitic acid (16:0 3-OH) differed in A. rhizogenes, A. tumefaciens, and A. vitis, providing a quantitative measure of differentiation; this acid was not detected in A. rubi ICPB TR2. Another fatty acid with diagnostic value was 15:0 iso 3-OH, which was found exclusively in A. rhizogenes cells. Cluster analyses based on fatty acid composition and metabolic fingerprints confirmed the phenotypic differences between members of the four Agrobacterium species investigated; both systems grouped the strains according to their respective species. These rapid and simple methods are major improvements over the classical methods of identification of Agrobacterium species and should prove useful for the rapid classification of large numbers of bacterial strains, such as those isolated in ecological studies.