Intercropping is a powerful practice to alter the allocation of photosynthetic carbon (C) to belowground ecosystems via promotion of diversified plant communities. The feedback of soil C stability to intercropping is controlled by microbial C use efficiency (CUE). Despite its significance, there is currently insufficient evidence to decipher how soil microbial CUE reacts to intercropping. By combining a 10-year-long intercropping experiment with a substrate-independent ¹⁸O-H₂O labelling approach and high-throughput sequencing, we elucidated the performance of intercropping on soil C pool and microbial metabolic traits as well as their relationships with soil microbial communities. Compared with monoculture, maize intercropping with peanut and soybean significantly increased soil C storage, soil mineral-associated organic C (MAOC), soil dissolved organic (DOC), and soil microbial biomass (MBC) contents at maize four growth stages. Soil microbial CUE increased significantly, especially at maize flowering and mature stages, as a consequence of enhanced microbial growth and biomass turnover rate after maize intercropping with peanut and soybean. Soil C storage and accessibility indicators (e.g., MAOC, DOC, and MBC contents) could significantly predict the changes of soil microbial diversity and core taxa. Meanwhile, the beta-diversity (community composition) of soil bacteria, fungi, saprotroph and protists, as well as rare fungal taxa were positively correlated with soil microbial CUE, and these indicators showed a high prediction of the microbial CUE. Soil C storage and accessibility indicators directly and indirectly influenced soil microbial CUE by regulating microbial diversity and key taxa. Soil microbial diversity and core taxa directly and indirectly influenced microbial CUE by mediating microbial respiration, growth, biomass, and enzyme activity, which mediated by soil C storage and accessibility. These findings provide an evidence for the associations between microbial diversity, CUE, and soil C stability, highlighting the importance of intercropping-driven soil microbiome to enhance soil microbial CUE.

间作是一种通过促进植物群落多样化来改变光合碳 (C) 向地下生态系统分配的有效做法。土壤碳稳定性对间作的反馈是由微生物碳利用效率（CUE）控制的。尽管其意义重大，但目前还没有足够的证据来解释土壤微生物 CUE 对间作的反应。通过长达10年的间作实验与底物无关的¹⁸ O-H ₂ O标记方法和高通量测序相结合，阐明了间作对土壤碳库和微生物代谢性状的影响及其与土壤微生物群落的关系。与单作相比，玉米与花生、大豆间作显着提高了玉米四个生育期的土壤碳储量、土壤矿物相关有机碳（MAOC）、土壤溶解性有机物（DOC）和土壤微生物量（MBC）含量。玉米间作花生和大豆后，由于微生物生长和生物量周转率增强，土壤微生物CUE显着增加，特别是在玉米开花和成熟阶段。土壤碳储量和可及性指标（如MAOC、DOC和MBC含量）可以显着预测土壤微生物多样性和核心类群的变化。同时，土壤细菌、真菌、腐生菌和原生生物以及稀有真菌类群的β多样性（群落组成）与土壤微生物CUE呈正相关，这些指标对微生物CUE具有较高的预测能力。土壤碳储存和可及性指标通过调节微生物多样性和关键类群直接和间接影响土壤微生物CUE。土壤微生物多样性和核心类群通过介导微生物呼吸、生长、生物量和酶活性（由土壤碳储存和可及性介导）直接和间接影响微生物 CUE。这些发现为微生物多样性、CUE 和土壤 C 稳定性之间的关联提供了证据，强调了间作驱动的土壤微生物组对增强土壤微生物 CUE 的重要性。