The effect of a mutation affecting flocculation, differentiation into cyst-like forms, and root colonization on nitrogenase expression by Azospirillum brasilense is described. The gene flcA of strain Sp7 restored these phenotypes in spontaneous mutants of both strains Sp7 and Sp245. Employing both constitutive pLA-lacZ and nifH-lacZ reporter fusions expressed in situ, the colony morphology, colonization pattern, and potential for nitrogenase activity of spontaneous mutants and flcA Tn5-induced mutants were established. The results of this study show that the ability of Sp7 and Sp245 mutant strains to remain in a vegetative form improved their ability to express nitrogenase activity in association with wheat in a hydroponic system. Restoring the cyst formation and colonization pattern to the spontaneous mutant Sp7-S reduced nitrogenase activity rates in association with plants to that of the wild-type Sp7. Although Tn5-induced flcA mutants showed higher potentials for nitrogenase expression than Sp7, their potentials were lower than that of Sp7-S, indicating that other factors in this strain contribute to its exceptional nitrogenase activity rates on plants. The lack of lateral flagella is not one of these factors, as Sp7-PM23, a spontaneous mutant impaired in swarming and lateralflagellum production but not in flocculation, showed wild-type nitrogenase activity and expression. The results also suggest factors of importance in evolving an effective symbiosis between Azospirillum and wheat, such as increasing the availability of microaerobic niches along the root, increased supply of carbon sources by the plant, and the retention of the bacterial cells in vegetative form for faster metabolism.

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Nitrogen-fixing bacteria were isolated from the rhizosphere of different crops of Korea. A total of 16 isolates were selected and characterized. Thirteen of the isolates produced characteristics similar to those of the reference strains of Azospirillum, and the remaining 3 isolates were found to be Enterobacter spp. The isolates could be categorized into 3 groups based on their ARDRA patterns, and the first 2 groups comprised Azospirillum brasilense and Azospirillum lipoferum. The acetylene reduction activity (ARA) of these isolates was determined for free cultures and in association with wheat roots. There was no correlation between pure culture and plant-associated nitrogenase activity of the different strains. The isolates that showed higher nitrogenase activities in association with wheat roots in each group were selected and sequenced. Isolates of Azospirillum brasilense CW301, Azospirillum brasilense CW903, and Azospirillum lipoferum CW1503 were selected to study colonization in association with wheat roots. We observed higher expression of beta-galactosidase activity in A. brasilense strains than in A. lipoferum strains, which could be attributed to their higher population in association with wheat roots. All strains tested colonized and exhibited the strongest beta-galactosidase activity at the sites of lateral roots emergence.

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