Beneficial Effects of Nickel on Pseudomonas saccharophila under Nitrogen-Limited Chemolithotrophic Conditions.

Addition of nickel stimulated growth and nitrogenase activity of Pseudomonas saccharophila under nitrogen-limited chemolithotrophic conditions, apparently because of a significant increase in expression of uptake hydrogenase activity. Inhibition of hydrogenase expression by 50 muM EDTA was relieved by nickel over a wide concentration range (1 to 200 muM). Co, Zn, Mn, and Cu stimulated expression of hydrogenase activity, but to a much lesser degree than nickel, and Fe, Mg, SeO(4), and SeO(3) did not increase expression. Nickel in individual combination with Mg, Fe, SeO(3), and SeO(4) resulted in activities that were essentially the same as that with nickel alone. Hydrogenase synthesis required the presence of nickel, and repression by O(2) was alleviated by increasing the concentration of added nickel. Cells placed under hydrogenase derepression conditions showed progressive incorporation of radioactive nickel to a much greater extent than did cells which were not derepressed.

Enumeration of free-living aerobic n(2)-fixing h(2)-oxidizing bacteria by using a heterotrophic semisolid medium and most-probable-number technique.

A heterotrophic semisolid medium was used with two sensitive assay methods, C(2)H(2) reduction and O(2)-dependent tritium uptake, to determine nitrogenase and hydrogenase activities, respectively. Organisms known to be positive for both activities showed hydrogenase activity in both the presence and absence of 1% C(2)H(2), and thus, it was possible to test a single culture for both activities. Hydrogen uptake activity was detected for the first time in N(2)-fixing strains of Pseudomonas stutzeri. The method was then applied to the most-probable-number method of counting N(2)-fixing and H(2)-oxidizing bacteria in some natural systems. The numbers of H(2)-oxidizing diazotrophs were considerably higher in soil surrounding nodules of white beans than they were in the other systems tested. This observation is consistent with reports that the rhizosphere may be an important ecological niche for H(2) transformation.

Isolation and identification of N2-fixing Pseudomonas associated with wetland rice.

Semisolid yeast extract medium amended with glucose and tryptic soy agar were used to isolate aerobically N2-fixing (C2H2-reducing) heterotrophic bacteria from the root of wetland rice. The isolates were identified as Pseudomonas by gel immunodiffusion and fluorescent antibody techniques in combination with their morphological, cultural, and biochemical characteristics. The N2-fixing H2-utilizing Pseudomonas described in this paper is a new species.

Immunological techniques to identify Azospirillum associated with wetland rice.

Azospirilla associated with wetland rice were isolated and characterized by employing immunodiffusion and immunofluorescence techniques. Antisera against two strains belonging to Azospirillum lipoferum produced at least one heat-labile precipitation band with most isolates of A. lipoferum and A. brasilense. Antisera against two strains belonging to A. lipoferum and one strain belonging to A. brasilense produced one band only with strains of their respective species. Fluorescent antibody reactions with Azospirillum were species specific. Specificity of these antisera and fluorescent antibodies was further demonstrated with bacteria other than Azospirillum.

Population of aerobic heterotrophic nitrogen-fixing bacteria associated with wetland and dryland rice.

Nitrogen-fixing activity and populations of nitrogen-fixing bacteria associated with two varieties of rice grown in dryland and wetland conditions were measured at various growth stages during the dry season. Acetylene reduction activities were measured both in the field and for the hydroponically grown rice, which was transferred from the field to water culture 1 day before assay. The activities measured by both methods were higher in wetland than in dryland rice. The population of nitrogen-fixing heterotrophic bacteria associated with rhizosphere soil, root, and basal shoots was determined by the most probable number method with semisolid glucose-yeast extract and semisolid malate-yeast extract media. The number of nitrogen-fixing bacteria was higher in wetland conditions than in dryland conditions. The difference between two conditions was most pronounced in the population associated with the basal shoot. The glucose medium gave higher counts than did the malate medium. Colonies were picked from tryptic soy agar plates, and their nitrogen-fixing activity was tested on a semisolid glucose-yeast extract medium. The incidence of nitrogen-fixing bacteria among aerobic heterotrophic bacteria in association with rhizosphere soil, root, and basal shoots was much lower in dryland rice than in wetland rice.

Nitrogen-fixing (acetylene redution) activity and population of aerobic heterotrophic nitrogen-fixing bacteria associated with wetland rice.

Nitrogen-fixing activity associated with different wetland rice varieties was measured at various growth stages by an in situ acetylene reduction method after the activities of blue-green algae (cyanobacteria) in the flood water and on the lower portion of the rice stem were eliminated. Nitrogen-fixing activities associated with rice varieties differed with plant growth stages. The activities increased with plant age, and the maximum was about at heading stage. The nitrogen fixed during the whole cropping period was estimated at 5.9 kg of N per ha for variety IR26 (7 days) and 4.8 kg of N per ha for variety IR36 (95 days). The population of aerobic heterotrophic N(2)-fixing bacteria associated with rice roots and stems was determined by the most-probable-number method, using semisolid glucose-yeast extract and semisolid malate-yeast extract media. The addition of yeast extract to the glucose medium increased the number and activity of aerobic heterotrophic N(2)-fixing bacteria. The glucose-yeast extract medium gave higher counts of aerobic N(2)-fixing bacteria associated with rice roots than did the malate-yeast extract medium, on which Spirillum-like bacteria were usually observed. The lower portion of the rice stem was also inhabited by N(2)-fixing bacteria and was an active site of N(2) fixation.