A MUTANT OF AZOSPIRILLUM-BRASILENSE SP7 IMPAIRED IN FLOCCULATION WITHA MODIFIED COLONIZATION PATTERN AND SUPERIOR NITROGEN-FIXATION IN ASSOCIATION WITH WHEAT

We report here significant phenotypic and genetic differences between Azospirillum brasilense Sp7 and spontaneous mutant Sp7-S and their rel ated properties in association with wheat. In contrast to the wild-typ e strain of Sp7, colonies of Sp7-S stained weakly with Congo red when grown on agar media containing the dye and did not flocculate in the p resence of fructose and nitrate. Scanning and transmission electron mi crographs showed clearly that the Sp7-S strain lacked surface material s present as a thick layer on the surface of the wild-type Sp7 strain. Different patterns of colonization on wheat roots between Sp7 and Sp7 -S, revealed by in situ studies using nifA-lacZ as a reporter gene, we re related to a large increase in nitrogenase activity (acetylene redu ction) with Sp7-S in association with normal and 2,4-dichlorophenoxyac etic acid-treated wheat for assays conducted under conditions in which the nitrogenase activity of free-living Azospirillum organisms was in hibited by an excess of oxygen. Randomly amplified polymorphic DNA ana lysis indicated the close genetic relationship of Sp7-S to several oth er sources of Sp7, by comparison to other recognized strains of A. bra silense. Genetic complementation of Sp7-S was achieved with a 9.4-kb f ragment of DNA cloned from wild-type Sp7, restoring Congo red staining and flocculation.