Azospirillum halopraeferens sp. nov., a Nitrogen-Fixing Organism Associated with Roots of Kallar Grass (Leptochloa fusca (L.) Kunth)

Among the nitrogen-fixing bacteria associated with the roots of Leptochloa fusca (L.) Kunth in salt-affected soils in the Punjab region of Pakistan, we found a homogeneous group of eight diazotrophs. Cells are vibrioid to S shaped, are motile by one polar flagellum, and produce granules of poly-β-hydroxybutyrate. They have a respiratory type of metabolism, show microaerophilic growth when fixing nitrogen, grow well on salts of organic acids, and can also use fructose and mannitol. On nitrogen-free semisolid media, they require biotin, utilize mannitol, but not glucose or sucrose, and cannot acidify glucose aerobically or anaerobically. Optimal growth occurs at 0.25% NaCl and 41°C. Deoxyribonucleic acid (DNA)-ribosomal ribonucleic acid (rRNA) hybridizations show that the organisms belong to the Azospirillum rRNA branch, where they cluster together with Azospirillum amazonense. They form a phenotypically and protein electrophoretically homogeneous group of bacteria, clearly distinct from Azospirillum amazonense, Azospirillum lipoferum, and Azospirillum brasilense. As no DNA-DNA binding was found with any of the three Azospirillum species, we propose a fourth Azospirillum species for this group of isolates. Because of better growth at increased NaCl concentrations, we named the new species Azospirillum halopraeferens. Strain Au 4 (= LMG 7108) is the type strain, which has been deposited at the Deutsche Sammlung von Mikroorganismen, Gottingen, Federal Republic of Germany, as DSM 3675.