Reforestation with broadleaf tree species in degraded Pinus massoniana plantations is a widely adopted strategy to enhance soil organic carbon (SOC) sequestration in subtropical China. However, the specific microbial processes that mediate this effect, particularly regarding nutrient cycling and microbial physiological efficiency, remain poorly understood. Here, we compared single pine stands with stands where broadleaf species were introduced, across a restoration chronosequence (10, 20, and 41 years). We aimed to elucidate how tree species mixing influences SOC storage through its effects on soil properties, microbial communities (with a focus on arbuscular mycorrhizal fungi, AMF), extracellular enzyme activities, and microbial carbon use efficiency (CUE). We found that broadleaf introduction increased SOC, total nitrogen, and AMF biomass, while stimulating the activity of nitrogen-acquiring enzymes (leucine aminopeptidase). In contrast, mixed stands showed reduced activity of phosphorus-acquiring enzymes, indicating an alleviation of soil phosphorus (P) limitation. Changes in microbial carbon metabolism accompanied these shifts, reflected in the altered CUE patterns. Structural equation modeling confirmed that the introduction of broadleaf promoted SOC accumulation primarily by increasing AMF abundance and alleviating P limitation, which collectively reshaped microbial community structure and function. This multi-pathway process enhanced the transformation of organic matter and microbial residue formation, contributing to greater SOC stabilization. Our results demonstrate that rehabilitating degraded pine forests with broadleaf species enhances carbon sequestration by modifying microbial nutrient limitation and metabolic strategies. Microbial CUE in this context reflects microbial metabolic status rather than functioning as a direct driver of SOC accumulation, providing a critical mechanistic basis for targeted forest management in subtropical region.
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