PHYLOGENETIC ANALYSIS OF AZOSPIRILLUM BY DIRECT SEQUENCING OF PCR AMPLIFIED 16S RDNA

16S ribosomal (r) DNA from 9 members of the genus Azospirillum and the type strain of Aquaspirillum itersonii (NCIMB 9070) was amplified usi ng the PCR. Of the strains received as Azospirillum, five were the typ e strains for the validly described species whilst the remaining 4 str ains had been deposited as ''Azospirillum'' sp. The amplified rDNA was directly sequenced using the linear PCR reaction and the sequences an alyzed using a distance matrix method to determine the intergeneric an d intrageneric relationships of Azospirillum, All of the type strains of Azospirillum, together with 2 of the unassigned strains, formed a d eep branching clade within the alpha-proteobacteria in which Azospiril lum amazonense, A. brasilense, A. halopraeferens, A. irakense and A. l ipoferum constituted phylogenetically well defined species. A. brasile nse, A. lipoferum and A. halopraeferens were more closely related to e ach other than to A. amazonense or A. irakense. Two of the unassigned strains (DSM 4834, DSM 4835) were closely related to, but distinct fro m, A. irakense. The other two ''Azospirillum'' sp. (DSM 1726, 1727) we re misclassified. ''Azospirillum'' sp. DSM 1726 is closely related to Agrobacterium tumefaciens while DSM 1727 was recovered as a deep branc hing lineage within the gamma-proteobacteria. The anoxygenic phototrop h Rhodospirillum centenum was recoverd within the radiation defined by Azospirillum an close to A. irakense. The nearest relatives to the az ospirilla were Magnetospirillum magnetotacticum and Rhodospirillum ful vum. Aquaspirillum itersonii formed a distinct lineage within the alph a-proteobacteria but its exact position was unstable and influenced by the composition of the alignment.