Rhizobium Strain S2_8_1 Promotes Ryegrass Regrowth under Soil and Hydroponic Conditions

(1) Purpose: Sustainable forage production requires strategies that accelerate plant regrowth while reducing reliance on synthetic nitrogen fertilizers. This study aimed to evaluate the potential multifunctionality of a plant growth-promoting microorganism, strain S2_8_1, hypothesized to enhance ryegrass regrowth in association with increased rhizosphere nitrification and cytokinin-related plant responses. (2) Methods: Comparative experiments were conducted using S2_8_1 and a cytokinin producing Streptomyces strain (Shan2) under both soil and hydroponic conditions. Treatments were evaluated for biomass production, soil NO3−-N content, nitrification rate, and leaf zeatin riboside (ZR) levels. Multiple linear regression was applied to quantify the relative contributions of nitrification and cytokinin signaling to regrowth. (3) Results: S2_8_1 was associated with increased soil nitrification and leaf ZR accumulation, leading to a 52% improvement in total biomass after 14 days of regrowth. Regression analysis showed that nitrification rate and ZR content jointly explained over 90% of biomass variation (R2 = 0.91–0.92), indicating a strong joint statistical association. Under hydroponic conditions, S2_8_1 retained functional activity, increasing ZR by 47% and biomass by 41% within 10 days. By contrast, Shan2 promoted regrowth primarily through cytokinin production, with limited impact on soil nitrogen dynamics. (4) Conclusions: S2_8_1 is associated with coordinated nitrification- and cytokinin-related responses contributing to ryegrass regrowth. Its plant growth-promoting effects suggest its potential as a multifunctional biofertilizer for reducing fertilizer dependence and supporting sustainable forage production.