Microbial inoculants that promote crop yield through biostimulation and biofertilization are an important tool to advancing sustainable agricultural practices. The most prevalent commercial inoculants are rhizobia, which provide nitrogen fixation capabilities to pulse and soybean crops. A key challenge in the market is to provide pulse and soybean seed suppliers and growers with rhizobial inoculants that remain viable on the plant seed, during extended storage prior to seeding, and perform optimally when planted many months later. This inoculant performance measurement is termed On-seed Survival. To a limited extent, inoculant producing companies can improve on-seed survival by formulation changes, but a ground-breaking improvement in rhizobial inoculant capability to survive in high population numbers during on seed application has not been readily achieved in the industry.  A true performance enhancement is imminently marketable in Canada and globally. This research project sought to use genomics techniques to advance our knowledge to produce improved on-seed survival soybean inoculant that could benefit the large soybean production in North America and globally. The Canadian soybean inoculant market is valued at $20M and the USA market is valued at $80M and can grow significantly by displacing chemical N fertilizer used by many US farmers who are not investing in inoculant technology due to inconsistent performance.

A technology known as genome shuffling was explored to determine its suitability to improving desiccation stress tolerance/resistance in Bradyrhizobium inoculants as well as comparative transcriptomics (RNAseq) was used to identify key genetic traits that can be exploited to improve desiccation stress tolerance/resistance. Lastly the project explored co-culture strategies which demonstrated increased desiccation tolerance resulting from growth synergies between the co-cultured Bradyrhizobium strains.

Despite significant efforts in experimentation and technical optimization we were unable to achieve success in developing a genome shuffling technology for Bradyrhizobium. Achieving successful regeneration of fused protoplasts has been a significant challenge reported in the literature for other Gram negative bacteria and we faced similar challenges. Therefore, genome shuffling technology remains unattained for use as a technology to improve rhizobial inoculants. Despite this technology set-back we have made substantial gains in understanding the mechanisms of desiccation tolerance/resistance in Bradyrhizobium inoculants for soybean, including new approaches for formulation of inoculants that may be of commercial value in improving inoculant performance for soybean growers.

Specific advancements and outcomes from this study include:

I). The first comparative gene expression profiling experiment and gene expression compendium to identify genetic traits that correlate with stronger desiccation tolerance in soybean nodulating strains of Bradyrhizobium. Almost 40% of the common upregulated genes were involved in carbohydrate metabolism and transport. Carbohydrate biosynthesis and transport has been known to be an important factor contributing to desiccation tolerance with genes involved in biosynthesis and transport of the disaccharide trehalose playing a major role. The differential transcriptional responses highlighted strains and genetic regulatory networks that can be valuable in considering development of superior inoculant strains.

II). The study demonstrated there is a significant pool of genetic potential within Bradyrhizobium strains that can be used to accessed to improve adaptation to desiccation stress within Bradyrhizobium inoculants. The study’s genomic datasets represent a valuable genetic resource that can be used for future bioprospecting of superior inoculant strains and can also be used in synthetic biology approaches for the rational design of inoculants with increased desiccation resistance and on seed survival.

III).  An additional outcome from the study was the discovery that co-culturing of Bradyrhizobium strains could be a promising approach towards addressing desiccation tolerance. Subsequently, the results from this study establish a foundation and premise for inoculant companies to explore co-culturing as a solution to increasing on seed survival of soybean microbial inoculants.

Taken collectively, the outcomes of this study will help to advance innovation in inoculant formulation to provide inoculants with improved performance for soybean growers.