Effects of spent mushroom substrate incorporation depth on soil microbial dynamics, carbon-nutrient cycling, and maize productivity in black soil ecosystems

In this study, we investigated the effects of spent mushroom substrate (SMS) incorporation at different depths—deep, shallow, surface, and control—on soil properties, microbial communities, and maize growth in black soil regions. Deep incorporation (DI) improved soil pH stability, enhanced enzyme activities, and promoted potassium release, but reduced nitrogen, organic carbon, and humic substances. Surface incorporation (SS) increased nitrogen-fixing and lignin-degrading microbes, boosting soluble organic carbon and nitrogen, though limited by phosphorus fixation. Metagenomic analysis revealed depth-specific functional genes, with DI enriched in carbohydrate-active enzymes and SS in nitrogen fixation and lignin degradation genes. While SMS improved maize height, reproductive-stage yield gains were limited by C–N imbalance. Importantly, this study uniquely integrates high-resolution metagenomics with soil enzymatic profiling to uncover depth-dependent mechanisms regulating nutrient cycling and microbial ecological functions, providing insights rarely addressed in previous SMS research. Given its high organic matter content, SMS also represents a biomass with latent energy potential, supporting its value in circular resource management. These findings highlight the novel role of SMS incorporation depth as a critical lever to optimize the balance between short-term soil fertility improvements and long-term carbon sequestration in black soil agroecosystems. • Depth-specific SMS application reshaped soil microbial networks and nutrient cycling pathways in black soils. • Deep SMS incorporation increased enzyme activities but reduced soil humus content and carbon sequestration potential. • Surface SMS application enriched nitrogen-fixing and lignin-degrading taxa, enhancing carbon–nitrogen coupling efficiency. • Trade-offs emerged between rapid soil fertility gains and long-term carbon stability under different SMS depths. • This work provides novel insights into sustainable SMS utilization for improving soil health and crop productivity.