Summary auto-generated
Researchers isolated nitrogen-fixing Azospirillum bacteria from roots of Miscanthus, a C4 grass cultivated as a potential bioenergy crop. Using polyphasic taxonomy including 16S rRNA sequencing, physiological tests, and nitrogen-fixation assays, they identified a new bacterial species. The isolates are curved rods or S-shaped cells, Gram-negative, motile with a single polar flagellum, and optimally grow at 30°C and pH 6.0-7.0. They fix nitrogen under microaerobic conditions and utilize several carbon sources including malate, mannitol, sorbitol, and glycerol as sole carbon sources. Phylogenetic analysis showed closest relationships to Azospirillum lipoferum, A. largimobile, and A. brasilense, with 96.6%, 96.6%, and 95.9% 16S rRNA sequence similarity respectively. The new species was named Azospirillum doebereinerae with strain GSF71T as the type strain. Two species-specific oligonucleotide probes were designed for identification by fluorescence in situ hybridization. The DNA G+C content was 70.7 mol%, consistent with the Azospirillum genus. Nitrogen fixation was confirmed through nifD gene amplification, immunodetection of dinitrogenase reductase, and acetylene-reduction assays.
Key findings
- A new nitrogen-fixing bacterial species, Azospirillum doebereinerae, was isolated from Miscanthus grass roots and characterized through polyphasic taxonomy
- Phylogenetic analysis using 16S rRNA sequencing showed closest relationships to A. lipoferum, A. largimobile, and A. brasilense
- The bacteria fix nitrogen under microaerobic conditions and preferentially utilize malate, mannitol, sorbitol, and glycerol as carbon sources
- Two species-specific oligonucleotide probes were developed enabling identification by in situ fluorescence hybridization
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Abstract
A new group of nitrogen-fixing Azospirillum sp. bacteria was isolated from the roots of the C(4)-gramineous plant Miscanthus. Polyphasic taxonomy was performed, including auxanography using API galleries, physiological tests and 16S rRNA sequence comparison. The ability of the isolates to fix dinitrogen was evaluated by amplification of the nifD gene, immunodetection of the dinitrogenase reductase and acetylene-reduction assay. On the basis of these 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 curved rods or S-shaped, 1.0--1.5 microm in width and 2.0--30 microm in length, Gram-negative and motile with a single polar flagellum. Optimum growth occurs at 30 degrees C and at pH values between 6.0 and 7.0. No growth takes place at 37 degrees C. They have a respiratory type of metabolism, grow well on arabinose, D-fructose, gluconate, glucose, glycerol, malate, mannitol and sorbitol. They differ from A. largimobile and A. lipoferum by their inability to use N-acetylglucosamine and D-ribose, from A. lipoferum by their ability to grow without biotin supplementation and from A. brasilense by their growth with D-mannitol and D-sorbitol as sole carbon sources. Nitrogen fixation occurs 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%.