Legume cover crops differentially alter soil organic carbon fractions across species and over time through microbial-mediated mechanisms

Legume cover crops are a promising agricultural practice for enhancing soil organic carbon (SOC) sequestration. However, their species-specific SOC sequestration potential and underlying mechanisms in orchard ecosystems remain unclear. In this study, we investigated high-altitude orchards in Tibet, China, using SOC fractionation, metagenomic sequencing, and biomarker analysis. The objectives of this study were to assess legume cover crop impacts on SOC fractions and microbial properties and to examine species-specific SOC sequestration mechanisms. Short-term cover by Trifolium repens L. and Vicia villosa R. significantly reduced particulate organic carbon (POC) but had a minimal effect on mineral-associated organic carbon (MAOC). This resulted in unchanged or reduced total SOC. Long-term legume cover crops enhanced SOC sequestration through concurrent increases in POC and MAOC, exhibiting species specificity. T. repens promoted greater SOC sequestration primarily through more efficient POC accumulation. Meanwhile, V. villosa enhanced SOC stability by facilitating the formation of MAOC. These differences between the two species were attributed to their distinct SOC transformation pathways. T. repens with a lower C/N ratio and higher non-structural carbohydrates stimulated soil microbial metabolism and hydrolase enzyme activity-this accelerated plant residue conversion to POC via the soil microbial ex vivo pathway. Under the V. villosa cover, elevated oxidative enzyme activities facilitated direct plant residue incorporation into MAOC, as shown by lower soil microbial necromass carbon (MNC) compared to T. repens. The results demonstrate time- and species-dependent heterogeneity in how legume cover crops mediate SOC sequestration and stabilization, providing a scientific basis for optimizing cover crop strategies in marginal orchards.