Multiphase characterization of wild <i>Vigna</i> associated root nodule bacteria from Japanese subtropical islands unveiled novel high temperature resistant <i>Bradyrhizobium</i> strains having high symbiotic compatibility with soybean and mungbean

<i>Vigna riukiuensis</i> plant – a rare type of vigna, found only in Taiwan and the islands of Okinawa prefecture, Japan – possesses intrinsic property of high level of salt and heat tolerance. To understand the diversity and identify suitable rhizobia, multiphase characterization of root nodule bacteria associated with <i>V. riukiuensis</i> grown in Ishigaki and Iriomote Islands of Okinawa prefecture was performed. Multigene phylogenetic analysis of housekeeping genes based on 16S rRNA gene sequences, 16S-23S rRNA gene internal transcribed spacer (ITS) and 23S rRNA gene sequences identified three main groups closely similar to <i>Bradyrhizobium japonicum, B. elkanii</i> and <i>B</i>. <i>jicamae</i> family. However, analysis of symbiotic <i>nifH</i> and <i>nodD1</i> genes and their phylogenetic trees showed similar topology, having only few discrepancies in comparison to the housekeeping gene phylogeny. Interestingly, for some of the isolates having similarity with <i>B. elkanii</i>, growth was observed at 40°C, which exceed the highest record for <i>B. elkanii</i> to the best of our knowledge. All the isolates were observed to have the capability of forming root nodules and fix nitrogen in their original host plant <i>V. riukiuensis</i> and two other crops: soybean and mungbean. Most of the isolates showed similar or higher nitrogen-fixing capability in comparison with <i>B</i>. <i>diazoefficiens</i> USDA110 in <i>V. riukiuensis</i> and <i>V. radiata</i> (mungbean), and Iri 5/6 in <i>V. riukiuensis</i>, Iri 5/12 in soybean and Ishi 7/2 in mungbean showed highest acetylene reduction assay (in µmol/h/gm nodule dry weight) activity, which was significantly higher than <i>B. diazoefficiens</i> USDA110. In addition, six isolates attained higher soybean biomass production compared with <i>B. diazoefficiens</i> USDA110, suggesting high symbiotic compatibility with soybean. Among them, Iri 5/7 of <i>B. elkanii</i> group contributed 29% higher soybean biomass production than <i>B. diazoefficiens</i> USDA110 and could grow at 40°C, hence it could be a promising soybean inoculant in the tropics. ARA: acetylene reduction assay, ISR: intergenic spacer regions, ITS: internal transcribed spacer