Field-grown soybean nodules harbor multiple Bradyrhizobium species

Background Bradyrhizobium contribute to soybean yield by fixing nitrogen. Bradyrhizobium populations in nodules are widely viewed to be clonal, but their diversity in rhizosphere suggests the possibility of mixed occupancy. We hypothesized that individual nodules host more than one species of Bradyrhizobium. Methods To characterize bacterial communities in individual nodules versus rhizosphere, we obtained full 16S rRNA gene sequences using Oxford Nanopore Technologies (ONT) long read sequencing. Recent advances in ONT provided us with higher fidelity and increased number of reads which enabled us to assign Bradyrhizobium populations to the species level. Results Our results revealed the presence of multiple Bradyrhizobium species within individual soybean nodules grown under field conditions. This observation challenges the current paradigm that a single Bradyrhizobium strain exclusively occupies a nodule. Due to inability to distinguish some species of 16S sequence, B. liaoningense and B. diversitatis , and B. americanum and B. daqingense were treated as species groups. The most predominant taxa found in the nodules were B. liaoningense/B. diversitatis (51.02%), and B. americanum/B. daqingense (20.79%). Of the commonly reported soybean nodulators, only B. diazoefficiens was predominant, while B. japonicum was present at 1%, and B. elkanii was not detected. Conclusions Our results indicate presence of multiple Bradyrhizobium species in individual soybean nodules under field conditions. This questions the stringency of gatekeeping of entry into and population establishment in soybean nodules. Future work is needed to characterize entry of multiple Bradyrhizobium into the infection thread, and population development in nodules.