Optimizing <i>Rhizobium-</i>legume symbioses by simultaneous measurement of rhizobial competitiveness and N₂ fixation in nodules.

Abstract

Legumes tend to be nodulated by competitive rhizobia that do not maximize nitrogen (N₂) fixation, resulting in suboptimal yields. Rhizobial nodulation competitiveness and effectiveness at N₂ fixation are independent traits, making their measurement extremely time-consuming with low experimental throughput. To transform the experimental assessment of rhizobial competitiveness and effectiveness, we have used synthetic biology to develop reporter plasmids that allow simultaneous high-throughput measurement of N₂ fixation in individual nodules using green fluorescent protein (GFP) and barcode strain identification (Plasmid ID) through next generation sequencing (NGS). In a proof-of-concept experiment using this technology in an agricultural soil, we simultaneously monitored 84 different <i>Rhizobium leguminosarum</i> strains, identifying a supercompetitive and highly effective rhizobial symbiont for peas. We also observed a remarkable frequency of nodule coinfection by rhizobia, with mixed occupancy identified in ∼20% of nodules, containing up to six different strains. Critically, this process can be adapted to multiple <i>Rhizobium</i>-legume symbioses, soil types, and environmental conditions to permit easy identification of optimal rhizobial inoculants for field testing to maximize agricultural yield.