紫云英根瘤菌胞外多糖缺失变种及其结瘤性状的变化

Exopolysaccharide-deficient Mutants of Rhizobium astragalus and Their Nodulation on the Host Plants

根据紫云英根瘤菌在寄主豆科植物紫云英上的结瘤能力,经转座子Tn5诱变获得的18株Exo^-变种可分为4种结瘤类群(A-D):A类变种诱导植物产生小的瘤状突起,不具固氮能力;B类变种形成无效根瘤;C类变种产生固氮效率降低的根瘤;D类变种丧失了结瘤能力。电镜分析显示:无效瘤和瘤状突起中不存在类菌体区,根瘤细胞均为不含细菌的空细胞,侵染线不能穿透到根瘤细胞中。说明紫云英根瘤菌胞外多糖很可能参与有效根瘤的形成。

Like most species of Rhizobium, R. astragalus cells can produce large quantities of exopolysaccharides (EPS) and the colonies they form are mucoid (Exo^+) in appearance (Fig. la). By screening random Tn5 insertion mutants on minimal agar, 18 mutants that formed non-mucoid colonies (Exo^-) were isolated from R. astragalus strain 107 (Fig. 1b). All the mutants had similar colony morphologies and their phenotypes were not altered by different kinds and concentrations of carbohydrates supplemented to the growing media. The EPS yields of these mutants were dramatically reduced to levels of about 0. 9%～10. 5% that of the wild bacteria, and the mutants acquired K_m resistance, but not C_m resistance, indicating that Tn5 had been inserted into Rhizobium genetic sequence, while the vector plasmid did not exist in the Rhizobium. Moreover, every Exo^-mutant had two plasmids identical to those of the parental strain(Fig. 2), demonstrating that all Exo^- mutants were derived from the parental strain.The wild type strain 107 cells induced on its host, Astragalus sinicus L. , brown, round nodules within a week after inoculation and three weeks later, approximately 7 green and cylindrical nodules were formed per plant, which were capable of fixing nitrogen. On the other hand, when the plants were inoculated with Exo^- mutants,the symbiotic phenotype of these bacteria changed in color, shape, weight, and acetylene reduction ability of the nodules formed (Table 1). According to these differences, 18 mutants were classified into 4 groups(A,B,C,D).The bacterial isolates recovered from both Fix^+ and Fix^- nodules and also those from callus-like structure showed exclusively non-mucoid colony appearance. All the isolates showed K_m resistance(conferred by Tn5 insertion) and their symbiotic phenotypes were the same as the inoculum strains. Under microscopic examination the Fix^-nodules appeared to contain no 'bacteroid zone' but form infection threads packed with bacteria (Plate Ⅱ-2 ), while the suggest that EPS of R. astragalus is intimately involved in the development of effective nodules.