Estimation of gus marked bradyrhizobial number by phenol extraction of GUS metabolite.

摘要

Bradyrhizobium japonicum strain 61 A 124 a, into which the Escherichia coli gus A gene was introduced, absorbed and hydrolysed GUS [beta-glucuronidase]-substrate (X-Gluc), and precipitated an indigo pigment. The absorbance of the accumulated GUS-metabolite was used as an indicator of the number of rhizobia in the soil. The accumulated indigo blue metabolite in rhizobia was extracted by phenol-water after incubation with X-Gluc (20 鎙 of 1% X-Gluc solution in N,N-dimethylformamide was added to 1 mlof culture solution) for 4 d. The absorbance of the extracted blue pigment in the phenol layer was 645 nm. The initial number of rhizobia and the absorbance of the GUS-metabolite were positively correlated both in cultured 61 A 124 a and in the various types of soil inoculated with strain 61 A 124 a. In the presence of soil, the absorbance per number of rhizobia was low compared with the pure culture. A standard curve between number of rhizobia of 61 A 124 a and the absorbance is required for every soilsample. During incubation with X-Gluc, rhizobia did not proliferate because of the depressive effect on cell growth of N,N-dimethylformamide and the accumulation of indigo pigment. The proliferation and mobility of inoculated strain 61 A 124 a was examined in a rhizobox containing various soil types in which soyabeans were planted. The distribution and movement of the marked strain depended on the soil type. In the absence or very low density of indigenous B. japonicum, the nodule was exclusively formed by the strain 61 A 124 a. Most nodules were formed by indigenous strains in the soil types containing a high population of indigenous bradyrhizobia, although the inoculated strain proliferated very well.