GUS代謝産物のフェノール抽出による土壌中のgus導入根粒菌数の推定

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Bradyrhizobium japonicum strain 61 A 124 a, in which Escherichia coli gus A gene was introduced to Bradyrhizobium japonicum, absorbs and hydrolyzes GUS-substrate (X-Gluc), and precipitates an indigo pigment. We attempted to use the absorbance of the accumulated GUS-metabolite as an indicator of the rhizobial number in soil. The accumulated indigo blue metabolite in rhizobia were extracted by phenol-water after they are incubated with X-Gluc (20 μL of 1% X-Gluc solution in N,N-dimethylformamide was added to 1 mL of culture solution) for 4 d. The absorbance of the extracted blue pigment in the phenol layer was measured at 645 nm. The initial number of rhizobia and the absorbance of the GUS-metabolite was positively correlated either in cultured 61 A 124 a or in the various types of soil inoculated with 61 A 124 a strain. In the presence of soil, the absorbance per rhizobial number was low compared with the pure culture. Therefore, a standard curve between rhizobial number of 61 A 124 a and the absorbance should be made for every soil sample. During incubation with X-Gluc, rhizobia did not proliferate because of the depression effect on cell growth of N,N-dimethylformamide and the accumulation of indigo pigment. The proliferation and mobility of inoculated 61 A 124 a strain was examined in a rhizobox containing various soil types into which soybeans were planted. The distribution and movement of the marked strain depended on the soil type. In the absence or very low density of indigenous Bradyrhizobium japonicum, the nodule was exclusively formed by the strain 61 A 124 a. On the other hand, the major part of nodules were formed by indigenous strains in the soil types containing a high population of indigenous bradyrhizobia, although the inoculated strain proliferated very well.
社団法人日本土壌肥料学会の論文
1997-04-05

GUS代謝産物のフェノール抽出による土壌中のgus導入根粒菌数の推定

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