Assessing the competitiveness of Rhizobium strains isolated from Manitoban agricultural soil on Phaseolus vulgaris

Abstract

Legumes typically obtain much of their nitrogen through a symbiotic relationship with rhizobia that can fix atmospheric nitrogen and supply it to the plant. While most agricultural legumes have a commercially available Rhizobium inoculant that is either applied to seeds or added during planting, an inoculant for dry beans (Phaseolus vulgaris) is not readily available. To address the lack of a viable inoculant for dry beans, a collection of 168 rhizobia capable of nodulating navy beans was previously assembled from across Manitoba. From this collection, 15 strains representing the top 90th percentile were chosen based on greenhouse trials to identify those with superior symbiotic nitrogen-fixation traits. In this study, we developed and refined a multi-iteration, competition-based screening method to identify the most competitive strains for nodule occupancy. Equal numbers of each of the 15 selected strains were combined into a consortium and used to inoculate bean seedlings. Three weeks after inoculation, 20 crown nodules were pooled, surface-sterilized, and the rhizobia were isolated for use in inoculating new plant sets. This process was repeated over three consecutive iterations. In the final round, rhizobia from 20 crown nodules per replicate were isolated, a single colony from each nodule was purified, and the whole genome was sequenced. To determine which of the original strains persisted through the selection process, average nucleotide identity (ANI) analysis was used. Across six independent competition experiments involving 120 crown nodules, an isolate of Rhizobium sophoriradicis (UMR 279) and an isolate of Rhizobium croatiense (UMR 297) showed the highest cumulative frequency, with UMR 297 appearing in 5 out of 6 replicates and occupying 41% of all nodules tested, while UMR 279 also appeared in 5 out of 6 replicates and accounted for 31% of all nodules tested. These strains were further characterized and are strong candidates for potential development as inoculants for dry beans.