Long-Term Effects of Straw-Biochar Application and Fertilization Gradients on Black Soil Carbon Sequestration via Prokaryote-Fungus-Protist Interactions and Metagenomic-Metabolite Linkages

Here, we conducted a seven-year field experiment in black soils of Northeast China to evaluate the effects of carbon (C) management, that is, control, straw return (SD), straw-biochar (BC), and a combined amendment (SDBC), with three fertilization levels (N0: unfertilized control, N60: 60% of conventional rates, N100: conventional rates) on soil microbiomes, metagenomics, and metabolomics. Results showed that BC significantly elevated soil total C (+15%), total N (+10%), and ${\mathrm{NH}}_4^{+}$ (+63%) relative to controls. Microbial community analyses revealed that SD increased prokaryotic richness but reduced protist diversity, whereas BC and SDBC suppressed fungal diversity. Integrated metagenomic and metabolomic profiling uncovered microbial functional adaptations to rich-C conditions under BC and SDBC, characterized by downregulated C metabolism-related genes and concurrent accumulation of lipid-associated metabolites. Crucially, BC decreased the abundance of bacterial virulence factors, contrasting with SD elevating pathogenic potentials. Among three fertilization levels, the reduced rates of N60 optimized microbial network complexity and minimized pathogen invasion risks more effectively than conventional rates of N100 without compromising soil fertility. Collectively, by deciphering prokaryote-fungus-protist interactions and metagenomic-metabolite linkages, our research highlights that straw-derived biochar application and optimized fertilization offers a sustainable strategy to foster beneficial microbial associations, suppresses pathogenic potential, and enhances carbon storage.