Objectives Green manure (cover crops) can curb the degradation of black soil and simultaneously sequester carbon and improve soil fertility. This study aimed to clarify the carbon sequestration effects and underlying mechanisms in topsoil (0−20 cm) and subsoil (20−40 cm) under a maize-sesbania (Sesbania cannabina) intercropping system, from the perspectives of the microbial carbon pump and organic carbon fractions.

Methods A field experiment was established in Harbin, Heilongjiang province, in 2021. Maize monoculture (MM) and maize-sesbania intercropping (MG) treatments were included in the experiment. At the maize jointing stage in 2025, soil samples were collected from 0−20 cm and 20−40 cm layers. Basic physicochemical properties, soil organic carbon (SOC) and its physical fractions, and the accumulation of microbial- and plant-derived necromass carbon were compared between the two systems.

Results Compared to the MM treatment, the MG treatment showed no significant change in topsoil SOC, but subsoil SOC, microbial biomass carbon (MBC), and dissolved organic nitrogen (DON) increased by 14.4%, 135.0%, and 88.3%, respectively. The contents of subsoil mineral-associated organic carbon (MAOC) and particulate organic carbon (POC) increased by 32.9% and 40.8%, respectively, and both MAOC and POC showed a significant positive correlation with SOC content. The microbial carbon use efficiency in subsoil was significantly increased by 19.6%, and the activities of carbon-, nitrogen-, and phosphorus-cycling related hydrolases (β-glucosidase, β-cellobiosidase, N-acetylglucosaminidase, phosphatase) increased by 86.9% to 145.0%. The plant residue carbon (PRC) content in subsoil was significantly elevated by 31.1%, while the microbial necromass carbon content showed no significant change, resulting in a 56.9% reduction in the microbial necromass carbon accumulation coefficient. The syringyl phenol acid-to-aldehyde ratio (Ad/Al)s in both topsoil and subsoil remained unchanged, while the vanillyl phenol acid-to-aldehyde ratio (Ad/Al)v decreased by 69.2% and 33.6%, respectively, and the cinnamyl-to-vanillyl phenol ratio (C/V) decreased by 26.3% and 19.5%, respectively. Mantel analysis revealed that POC, MAOC, and SOC were all significantly correlated with PRC, indicating that subsoil PRC is a key factor in SOC accumulation.

Conclusions Leguminous green manure, Sesbania cannabina, is a deep-rooted crop , which can significantly increase the input of root-derived residue carbon in the subsoil. Its low C/N ratio can promote microbial carbon utilization efficiency and enhance the activities of carbon-, nitrogen- and phosphorus-cycling related hydrolases in the subsoil. The increase in soil microbial hydrolase activity accelerates the extracellular modification” process in the microbial carbon pump, promoting the oxidation of lignin in plant residue carbon and its transformation into POC and MAOC. This ultimately leads to the accumulation of soil organic carbon in the subsoil. Therefore, plant residue carbon enrichment in subsoil drives SOC accumulation in maize-sesbania intercropping on black soil.