Arbuscular mycorrhizal fungi and rhizobia synergistically enhance alfalfa productivity and alleviate nutrient limitations, particularly under low-input conditions.

Arbuscular mycorrhizal fungi (AMF) and rhizobia enhance the ability of crops to access nitrogen (N) and phosphorus (P) and are keystone microorganisms that engage in reciprocal interactions with plants. However, their contributions and impacts in alleviating plant nutrient dependency and mitigating N deposition and P limitation under various N and P additions remain unclear. We hypothesized that co-inoculation with AMF and rhizobia would synergistically enhance crop nutrient absorption and stabilize N:P stoichiometry for mitigating N deposition and P limitation and enhancing biomass. Here, two experimental systems were established: (1) constant low P with three increasing N levels, and (2) constant low N with three increasing P levels. Within each system, we examined the effects of inoculation with AMF alone, rhizobia alone, both AMF and rhizobia, and no inoculation on alfalfa biomass, photosynthesis, nutrient content, and N:P stoichiometry. Results revealed that co-inoculation with AMF and rhizobia significantly increased alfalfa aboveground biomass (AGB) and belowground biomass (BGB) by 88.54 % and 236.96 %, respectively, under low N and low P conditions. Additionally, co-inoculation significantly enhanced the content of photosynthetic pigments, N and P. However, co-inoculation did not significantly affect photosynthetic rate (Pn) or transpiration rate (Tr), while significantly reducing stomatal conductance (Gs). Furthermore, co-inoculation alleviated N limitation in alfalfa, shifting its limitation from N to P by increasing N:P stoichiometry. Notably, co-inoculation buffered leaf N:P stoichiometry against nutrient change, while maintaining the maximum leaf N:P stoichiometry. In contrast, as N or P levels rose, alfalfa BGB, photosynthetic pigments content, Pn, and N content, and P content were inhibited to varying degrees across all inoculation treatments, with increased N limitation. Moreover, partial least squares path modeling (PLS-PM) demonstrated a unique mechanism in the co-inoculation treatment whereby alfalfa AGB and BGB was enhanced through the root N:P stoichiometric regulation. Overall, our findings highlight multifaceted benefits of co-inoculation under low-input conditions, including increased alfalfa biomass, improved nutrient use efficiency, and reduced reliance on exogenous N. These results suggest the potential of co-inoculation with AMF and rhizobia to reduce fertilizer dependency and enhance alfalfa productivity in low-input agricultural ecosystems, promoting sustainable forage crop production.