Subsurface split manure application increased soil organic carbon accumulation and stability by enhancing aggregation and recalcitrant carbon fractions with depth-dependent mechanisms.

Increasing soil organic carbon (SOC) is crucial for soil quality and fertility. Subsurface manure application has been identified as an important strategy to improve soil properties and enhance SOC sequestration. However, how do SOC accumulation and stabilization response to manure application frequency remain unclear. To solve this knowledge gap, we comprehensively explored the underlying mechanisms of multiple SOC fractions in response to subsurface manure application, based on a two-year field experiment in the North China Plain subjected to three treatments, including a control without manure application (T0); single application of manure every two years (T1); split applications of manure (once a year) with the same total amount (T2). Across 0-40 cm, compared to T0, T1 and T2 increased SOC by 23-28%, particulate organic carbon (POC) by 57-118%, and mineral-associated organic carbon (MAOC) by 7-16%, with greater increases by T2 in topsoil (0-20 cm). Compared with T0, T2 increased SOC in macroaggregates and increased the mean weight diameter (MWD). Moreover, T2 increased the chemical stability of SOC by enriching recalcitrant organic C content (aromatic-C, aliphatic-C, and phenol-C) and reducing labile organic C content (carboxyl/amidogen-C and polysaccharide-C) in soil aggregates of all sizes. Compared with topsoil, subsoil exhibited greater increases in POC, aggregate stability, and chemical recalcitrance, indicating depth-dependent responses to subsurface manure application. Moreover, chemical recalcitrance exhibited the strongest direct positive effect on SOC content in both soil layers. Overall, these findings reveal that subsurface manure application has the potential to enhance stable SOC accumulation via synergistic physical and chemical stabilization pathways, with a better performance of split manure application, aiding SOC sequestration and soil fertility improvement.