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.

Plants growing in close association with N(2)-fixing bacteria are able to overcome growth limitations in N-depleted soils. The molecular mechanism by which free-living, N(2)-fixing bacteria promote plant growth is still a matter of debate. By inoculating N-depleted tomato (Lycopersicon esculentum Mill.) plants with Azospirillum brasilense or Azoarcus sp. we could demonstrate the induction of the root NH(+)(4)-transporter gene, LEAMT1;2 (L. esculentum ammonium transporter 1;2), indicating that bacterial NH(+)(4) might be used as an N source under these conditions. Azospirillum brasilense (nif(-)) mutants, which lack the structural nifDK genes, failed to induce LEAMT1;2 expression. This suggests that root-associated N(2)-fixing bacteria do excrete NH(+)(4) to levels that can be sensed by tomato roots and is in agreement with the induction of expression of LEAMT1;2 with as low as > or = 1 microM external NH(+)(4). While peak expression was obtained with 2-5 microM NH(+)(4), a further increase in NH(+)(4) reduced LEAMT1;2-mRNA levels in a concentration-dependent manner. The inhibition of LEAMT1;2 expression by glutamine and the glutamine synthetase blocker L-methionine sulfoximine (MSX) provided evidence for the control of LEAMT1;2 expression by cytoplasmic NH(+)(4) concentration or the plant N status. Since micromolar concentrations of NH(+)(4) strongly increased the LEAMT1;2-mRNA levels, the transported NH(+)(4) ion itself could represent a key signal in the associative interaction between higher plants and N(2)-fixing micro-organisms.

Azospirillum brasilense Sp 7 was grown in a nitrogen free medium in a chemostat with malate as the sole carbon source. Several steady states were established. Malate limitation occurred at dilution rates less than 0.05 (hr -1). Maximum growth rate and KS were calculated from Monod's model. Poly-~3-hydroxybutyrate content was found to decrease under malate limitation from 18.5% to about 2%. Protein exudation increases under these conditions. The acetylene reduction assay was shown to be not suitable for continuous culture systems. A model for the physiological properties of A. brasilense in a malate limited chemostat is developed.Azospirillum brasilense Sp 7 is the major organism responsible for nitrogenase activity in the rhizosphere of several tropical grasses (1). The rhizosphere microflora is mainly supported by carbonaceous compounds liberated by intact roots (2). In these experiments, the continuous system is used in order to understand and describe some processes in the rhizosphere (3). A chemostat has certain properties which reflect ecologically important characteristics. The microbial growth is limited by the availability of a selectable substrate (4).Due to its economic importance, work had been done previously on the physiology of A. brasilense in batch culture systems (S-7) and continuous culture systems (8-10). Carbon-limited continuous cultures of A, brasilense however have not yet been studied on N-free medium (8). For this reason, studies on the physiological properties of this organism in a carbon limited chemostat were undertaken. The effect of oxygen on the nitrogen fixing ability and the efficiency of the nitrogenase was the main objective of the studies (8-10). MATERIAL AND METHODSBacterial strain and inoculum. Azospirillum brasilense Sp 7 (DSM 1690, 447 ATCC