**4.1 Sampling of soybean root nodules and isolation of nodulating bradyrhizobia**

The nodules used in the present experiment were collected from soybean roots at the flowering stage in 2017, as described in Section 3.2. The nodules were surface-sterilized with 70% ethanol for 3 min and diluted sodium hypochlorite solution (0.25% available chlorine) for 30 min, followed by washing with sterile distilled water. After washing, 24 nodules were randomly collected and transferred to 1.5 mL microcentrifuge tubes. Each nodule was homogenized in sterile distilled water and streaked onto a yeast extract– mannitol agar (YMA) plate [56]; to isolate a single colony per nodule, the plates were incubated for 5–7 days in the dark at 28°C. A total of 144 isolates were obtained from six soybean plants and used for PCR-restriction fragment length polymorphism (RFLP) analysis of the 16S–23S rRNA gene internal transcribed spacer (ITS) region.
