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-P-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 41OC. Deoxyribonucleic acid (DNA)-ribosomal ribonucleic acid (rRNA) hybridizations show that the organisms belong to the Azospirilhm rRNA branch, where they cluster together with Azospirilhm 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 Azospirilhm 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.

The populations of diazotrophic and nondiazotrophic bacteria were estimated in the endorhizosphere and on the rhizoplane of Kallar grass (Leptochloafusca) and in nonrhizosphere soil. Microaerophilic diazotrophs were counted by the most-probable-number method, using two semisolid malate media, one of them adapted to the saline-sodic Kallar grass soil. Plate counts of aerobic heterotrophic bacteria were done on nutrient agar. The dominating N2-fixing bacteria were differentiated by morphological, serological, and physiological criteria. Isolates, which could not be assigned to a known species, were shown to fix nitrogen unequivocally by 15N2 incorporation. On the rhizoplane we found 2.0 x 107 diazotrophs per g (dry weight) of root, which consisted in equal numbers of Azospirillum lipoferum and Azospirillum-like bacteria showing characteristics different from those of known Azospirillum species. Surface sterilization by NaOCl treatment effectively reduced the rhizoplane population, so that bacteria released by homogenization of roots could be regarded as endorhizosphere bacteria. Azospirillum spp. were not detected in the endorhizosphere, but diazotrophic, motile, straight rods producing a yellow pigment occurred with 7.3 x 107 cells per g (dry weight) of root in the root interior. In nonrhizosphere soil we found 3.1 x 104 nitrogen-fixing bacteria per g. Diazotrophs were preferentially enriched in the Kallar grass rhizosphere. In nonrhizosphere soil they made up 0.2% of the total aerobic heterotrophic microflora, on the rhizoplane they made up 7.1 %, and in the endorhizosphere they made up 85%. Owing to high numbers in and on roots and their preferential enrichment, we concluded that diazotrophs are in close association with Kallar grass. They formed entirely different populations on the rhizoplane and in the endorhizosphere.

Chemotactic responses of three Azospirillum strains originating from different host plants were compared to examine the possible role of chemotaxis in the adaptation of these bacteria to their respective hosts. The chemotaxis to several sugars, amino acids, and organic acids was determined qualitatively by an agar plate assay and quantitatively by a channeled-chamber technique. High chemotactic ratios, up to 40, were obtained with the latter technique. The chemotactic response did not rely upon the ability of the bacteria to metabolize the attractant. Rather, it depended on the attractant concentration and stereoconfiguration. Chemotaxis was found to be strain specific. Differences were particularly observed between a wheat isolate and strains originating from the C4-pathway plants maize and Leptochloa fusca. In contrast to the other two strains, the wheat isolate was strongly attracted to D-fructose, L-aspartate, citrate, and oxalate. The other strains showed maximal attraction to L-malate. The chemotactic responses to organic acids partially correlate with the exudation of these acids by the respective host plants. Additionally, a heat-labile, high-molecular-weight attractant was found in the root exudates of L. fusca, which specifically attracted the homologous Azospirilum strain. It is proposed that strain-specific chemotaxis probably reflects an adaptation of Azospirillum spp. to the conditions provided by the host plant and contributes to the initiation of the association process.

By a polyphasic approach (DNA:rRNA and DNA:DNA hybridizations, and computer assisted analysis of auxanographic results) it was shown that Herbaspirillum seropedicae, EF group I strains and the generically misnamed [Pseudomonas] rubrisubalbicans and [Aquaspirillum] autotrophicum constitute one genus. Within this genus we delineated four groups that each deserve species rank. Species I contains EF group I strains and [Pseudomonas] rubrisubalbicans strains; species 2 is composed only of [Pseudomonas] rubrisubalbicans strains including the type strain. Species 3 contains Herbaspirillum seropedicae and one [Pseudomonas] rubrisubalbicans strain. Species 4 corresponds to [Aquaspirillum] autotrophicum.

The effect of oxygen on [Formula: see text] growth of two Azospirillum strains and two diazotrophic rods closely associated with roots of Kallar grass (Leptochloa fusca) and one Azospirillum brasilense soil isolate as reference was studied. To enable precise comparison, bacteria were grown in dissolved oxygen controlled continuous cultures at a constant dilution rate. Similar O2 responses for all strains were found. Steady states were achieved from about 1 to 190 μM dissolved O2, all of them carbon limited. The response of all steady-state cultures to increasing O2 concentrations suggested that neither oxygen limitation nor oxygen toxicity occurred within the range of O2 concentrations applied. Growth of the plant-associated diazotrophs on combined nitrogen did not explain the colonization pattern of the different root zones of Kallar grass.