[Differentiation among species of the genus Azospirillum]

To replace the Spirillum lipoferum denomination (2) for the nitrogen fixing group of soil spirilla, the Azospirillum genus has been created in recent years (12), which includes two species: A. lipoferum and A. brasilense. Both of them are nitrogen fixing bacteria able to induce a specific infection of grass roots (1, 6). Consequently it is important to differentiate correctly the species when plant-bacteria relationships must be studied or inoculation assays must be carried out. For this reason the response of 20 strains of Azospirillum to the classification tests will be described. Microorganisms were isolated from washed plant roots (Table 1) by similar methods to those described by Döbereiner et al. (5). NFb medium was used replacing the micronutrients with 0.05 g/l of yeast extract, which gave better results. For easily identification of Azospirillum colonies, Congo Red was added to the medium (11). Morphology, motility and type of colonies were typical of Azospirillum (1, 3, 4). Nitrogenase activity ranging from 7 to 57 nM C2H2/ml of culture medium/h, according to strain, was detected in all strains pure cultures, by a colorimetric ethylene determination (8). Differentiation tests were performed by Tarrand et al. method (12) (Table 2). Four strains were classified as A. lipoferum and 9 as A. brasilense. Five were A. brasilense-like organisms unable to grow in, a biotin-free medium. As these five strains have been isolated in a medium containing biotin (yeast extract), they might be A. brasilense mutants which have lost the biotin synthesizing capacity. The last two organisms could not be classified and a DNA study should be done to determine whether they belong to any of the Azospirillum species known (7, 10, 13). The results obtained show that to achieve a correct classification of Azospirillum strains (6, 9), all known tests should be performed.