Molybdenum-mediated nitrogen accumulation and assimilation in legumes stepwise boosted by the coexistence of arbuscular mycorrhizal fungi and earthworms

Molybdenum (Mo) is a critical micronutrient for nitrogen (N) metabolism in legumes, yet the impact of Mo on legume N metabolism in the context of natural coexistence with soil microorganisms remains poorly understood. This study investigated the dose-dependent effect of Mo on soil N biogeochemical cycling, N accumulation, and assimilation in alfalfa under conditions simulating the coexistence of arbuscular mycorrhizal fungi (AMF) and earthworms. The findings indicated that Mo exerted a hormetic effect on alfalfa N accumulation, facilitating it at low concentrations (below 29.98 mg/kg) and inhibiting it at higher levels. This inhibition was attributed to Mo-induced constraints on C supply for nitrogen fixation. Concurrently, AMF colonization enhanced C assimilation in Mo-treated alfalfas by promoting nutrients uptake, particularly Mg, which is crucial for chlorophyll synthesis. This effect was further amplified by earthworms, which improved AMF colonization (p < 0.05). In the soil N cycle, these organisms exerted opposing effects: AMF enhanced soil nitrification and earthworms reduced soil nitrate (NO₃⁻-N) reduction to jointly increase soil phyto-available N content (p < 0.05). Their combined action improved alfalfa N assimilation by restoring the protein synthesis pathway that is compromised by high Mo concentrations, specifically the activity of glutamine synthetase. These findings underscored the potential for soil microorganisms to mitigate N metabolic stress in legumes exposed to elevated Mo levels.