Azospirillum doebereinerae sp nov., a nitrogen-fixing bacterium associatedwith the C-4-grass Miscanthus

A new group of nitrogen-fixing Azospirillum sp, bacteria was isolated from the roots of the C-4-gramineous plant Miscanthus. Polyphasic taxonomy was p erformed, including auxanography using API galleries, physiological tests a nd 16S rRNA sequence comparison. The ability of the isolates to fix dinitro gen was evaluated by amplification of the nifD gene, immunodetection of the dinitrogenase reductase and acetylene-reduction assay. On the basis of the se results, the nitrogen-fixing isolates represent a new species within the genus Azospirillum. Its closest phylogenetic neighbours, as deduced by 16S rDNA-based analysis, are Azospirillum lipoferum, Azospirillum largimobile and Azospirillum brasilense with 96.6, 96.6 and 95.9% sequence similarity, respectively. Two 16S rRNA-targeting oligonucleotide probes were developed which differentiate the new species from the other Azospirillum species by whole-cell fluorescence hybridization. Strains of the new species are curve d rods or S-shaped, 1.0-1.5 mum in width and 2.0-30 mum in length, Gram-neg ative and motile with a single polar flagellum. Optimum growth occurs at 30 degreesC and at ph values between 6.0 and 7.0. No growth takes place at 37 degreesC. They have a respiratory type of metabolism, grow well on arabino se, D-fructose, gluconate, glucose, glycerol, malate, mannitol and sorbitol . They differ from A. largimobile and A. lipoferum by their inability to us e N-acetylglucosamine and D-ribose, from A. lipoferum by their ability to g row without biotin supplementation and from A. brasilense by their growth w ith D-mannitol and D-sorbitol as sole carbon sources. Nitrogen fixation occ urs in microaerobic nitrogen-limited conditions. For this species, the name Azospirillum doebereinerae sp. nov. is suggested, with strain GSF71(T) as the type strain (= DSM 13131(T): reference strain Ma4 = DSM 13400). Its G+C content is 70.7 mol%.