The mechanisms of soil organic carbon accumulation in the intercropping systems of alfalfa and ryegrass

Background and Aims In the process of soil ecological restoration, balancing the accumulation of soil organic carbon (SOC) in terms of particulate organic carbon (POC) and its stable sequestration as mineral-associated organic carbon (MAOC) presents a critical challenge. Legume-grass intercropping is recognized as an effective strategy for enhancing soil carbon pools, yet the underlying microbial mechanisms remain incompletely understood. Methods This study systematically compared the effects of ryegrass monoculture (Lp), alfalfa monoculture (Ms), and their intercropping system (LM) on SOC fractions through a 90-day microcosm cultivation experiment, integrating metagenomics with partial least squares path modeling. Results Results indicate that Lp and LM system exhibit a “high-input, fast-turnover” strategy, significantly promoting active POC accumulation primarily through high biomass input and enrichment of fermentation genes and organic carbon oxidation genes ( β-glucosidase , fdhB ). In contrast, Ms exhibited a “resource-conservative-stable carbon” strategy, effectively maintaining a stable MAOC proportion by alleviating nitrogen limitation and enriching autotrophic carbon fixation genes ( cdhD , aclB ). PLS-PM analysis further revealed that intense interspecific competition in the intercropping system caused a deficit in soil total nitrogen (TN), thereby limiting microbial biomass accumulation (MBC) and hindering the conversion of microbial residues into MAOC. Additionally, the study confirmed that chemoautotrophic carbon fixation genes directly and positively contribute to soil MAOC. Conclusion In summary, interspecific plant competition reshapes soil carbon cycling trajectories by altering nitrogen availability and microbial metabolic strategies. When designing intercropping systems, constraints imposed by belowground nutrient competition on microbial carbon pump (MCP) efficiency should be fully considered to maximize soil carbon sequestration benefits.