Abstract: Abstract Aims Establishing legume–grass mixed swards is an effective strategy for restoring degraded grasslands and enhancing productivity in Southwest China. Arbuscular mycorrhizal fungi (AMF) form extensive hyphal networks after colonizing host plant roots, thereby facilitating nutrient uptake and transfer. However, in legume – grass communities composed of species with contrasting root architectures, the mechanisms by which AMF regulate plant nitrogen uptake and nutrient allocation remain poorly understood. Methods In this study, we selected the deep-rooted legume Medicago sativa and the shallow-rooted legume Trifolium repens, based on their nitrogen fixation characteristics and root depth differences, to establish both mixed and monoculture systems with two grass species (Dactylis glomerata and Lolium perenne). Two arbuscular mycorrhizal fungi (AMF) treatments (inoculated vs. non-inoculated) were applied, and a 15N labeling experiment was conducted at 3 cm shallow and 25 cm deep soil layers to investigate the effects of AMF on nitrogen uptake and community functioning. Important findings The results showed that mixed sowing significantly increased total community biomass, with the combination of Trifolium repens + Dactylis glomerata + Lolium perenne performing the best, indicating that spatial complementarity of root systems enhanced resource use efficiency. AMF inoculation markedly promoted biomass accumulation in the highly mycorrhiza-dependent legumes (Trifolium repens and Medicago sativa), while reducing the competitive advantage of the less dependent grasses (Dactylis glomerata and Lolium perenne), thereby altering community structure and interspecific interactions.15N tracing further revealed that both mixed treatments enhanced nitrogen fixation capacity of the legumes compared to monocultures, and AMF inoculation further increased their fixation rate while decreasing 15N uptake. Moreover, AMF inoculation balanced the 15N uptake differences of Dactylis glomerata and Lolium perenne between soil layers. This study demonstrates that AMF promote resource complementarity and functional optimization in legume–grass mixed systems by modulating plant root architecture, nitrogen acquisition strategies, and interspecific relationships, providing a theoretical basis for constructing high-yield, sustainable artificial grasslands.

Key words: arbuscular mycorrhizal fungi (AMF), legume–grass mixture, deep and shallow root systems, nitrogen uptake, biological nitrogen fixation, 1?N labeling