Diversified cropping rotation increases soil carbon sequestration via fungal-mediated necromass production and mineral association

Crop diversification boosts soil organic carbon (SOC) storage, yet uncertainty persists regarding its effects on molecular SOC composition. Using a 43-year field trial in fluvo-aquic soil, we contrasted conventional wheat-rice (ConCrop) versus diversified rice/wheat – cotton/barley – maize/faba bean rotations (DivCrop). We demonstrated DivCrop elevated SOC by 9.6% (0–15 cm) and 15% (15–30 cm). DivCrop alleviated C-nitrogen co-limitation, stimulating 150% greater microbial growth and 47% higher C use efficiency. This coincided with restructured microbial communities shifting toward fungal dominance and increased functional capacity. Community transplantation assays confirmed increased adaptation for efficient C processing. Diversification accelerated lignin decomposition (plant-derived C reduced by 19–35%) and redirecting C flow toward fungal necromass (20–44% of total microbial necromass). Consequently, mineral-associated organic C increased 48–69%, with short-term formation efficiency rising 98–160% via reactive iron oxide interactions. Diversification thus shifts plant-derived recalcitrance towards mineral-stabilized microbial products in low-SOC soils, reshaping SOC composition toward climate-resilient agriculture. Crop diversification increased soil organic carbon by 10–15%, boosted microbial growth and carbon-use efficiency, shifted communities toward fungi, and raised mineral-associated carbon by 48–69%, based on a 43-year field trial and community transplantation assays.