**4.2 PCR-RFLP analysis of the 16S: 23S rRNA gene ITS region**

For DNA extraction, each isolate was cultured in 1.5 mL HM medium supplemented with 0.1% l-arabinose for 5–7 days in the dark at 28°C. Total DNA for use as the PCR template was extracted from the isolates in BL extraction buffer, as described previously [42] based on the method reported by Hiraishi *et al*. [57]. As reference strains, *B*. *japonicum* USDA 6T ; *B*. *diazoefficiens* USDA 110T ; and *B*. *elkanii* USDA 46, 76T , and 94 were used [16]. Total DNA of the reference strains for use as the PCR templates was also extracted using the same method [42, 57].

The 16S–23S rRNA gene ITS region was PCR-amplified using *TaKaRa Ex Taq*® Hot Start Version (TaKaRa Bio, Shiga, Japan) and the ITS primer set (BraITS-F: 5′-GACTGGGGTGAAGTCGTAAC-3′ and BraITS-R1: 5′-ACGTCCTTCATCGCCTC-3′) [58]. The PCR cycle comprised a pre-run at 94°C for 5 min, denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 1 min. This temperature control sequence was repeated for 30 cycles, followed by a final run at 72°C for 10 min. RFLP analysis of the 16S–23S rRNA gene ITS region was performed using the restriction enzyme *Msp*I (TaKaRa Bio, Shiga, Japan). The PCR product was digested with the restriction enzyme at 37°C for 16 h, and the restricted fragments were separated using 3% agarose gel electrophoresis.
