Screening and Application of Highly Efficient Rhizobia for Leguminous Green Manure in Lyophilized Inoculants and Seed Coating.

, a key leguminous green manure widely cultivated in Southern China's rice-based cropping systems, plays a pivotal role in sustainable agriculture by enhancing soil organic matter sequestration, improving rice yield, and elevating grain quality. The symbiotic nitrogen-fixing association between and its matching rhizobia is fundamental to its agronomic value; however, suboptimal inoculant efficiency and field application methodologies constrain its full potential. To address these limitations, we conducted a multi-phase study involving (1) rhizobial strain screening under controlled greenhouse conditions, (2) an optimized lyophilization protocol evaluating cryoprotectant (trehalose, skimmed milk powder and others), and (3) seed pelleting trails with rhizobial viability and nodulation assessments over different storage periods. Our results demonstrate that CCBAU 33470 exhibits a superior nitrogen-fixing efficacy, significantly enhancing key traits in , including leaf chlorophyll content, tiller number, and aboveground biomass. Lyophilized inoculants prepared with cryoprotectants (20% trehalose or 20% skimmed milk powder) maintained >90% bacterial viability for 60 days and markedly improved nodulation capacity relative to unprotected formulations. The optimized seed pellets sustained high rhizobial loads (5.5 × 10 cells/seed) with an undiminished viability after 15 days of storage and nodulation ability after 40 days of storage. This integrated approach of rhizobial selection, inoculant formulation, and seed coating overcomes cultivation bottlenecks, boosting symbiotic nitrogen fixation for cultivation.