Unraveling biochar’s role: dosage-dependent impacts on continuous-cropping soil microbiome and metabolites

Although biochar is a promising soil amendment for shaping soil microbial communities and modulating allelochemical levels in continuous cropping soils, its effects on the soil microbiome -particularly through benzoic acid metabolism in tobacco continuous cropping systems-remains unclear. To elucidate these interactions, a pot experiment was conducted using soil collected from a three-year tobacco continuous cropping system. By employing high-throughput sequencing and non-targeted metabolomics, effects of various biochar application dosages (0, 1, 3, 5, and 7%) on soil microbial community structure and benzoic acid metabolic pathways were investigated. Results indicated that biochar significantly increased the contents of soil total carbon, nitrate nitrogen, ammonium nitrogen, and dissolved total nitrogen. Moreover, compared with the control (CK), CTB1% and CTB3% treatments significantly increased the numbers of bacterial and fungal amplicon sequence variants (ASVs). Bacterial Chao1 and Shannon indices were significantly reduced under CTB7%, while fungal Chao1 and Shannon indices significantly enhanced under CTB1%. Across all biochar treatments, 184 common metabolites were identified relative to CK. In addition, biochar affected enriched metabolic pathway associated with carbohydrates metabolism, lipid metabolism, and amino acids metabolism. Redundancy analysis (RDA) revealed that benzene and substituted derivatives played a key role in shaping the soil microbiome community. Notably, putatively annotated benzoic acid - positively related to soil polyphenol oxidase (PPO) and Desulfobacterota (R2 = 0.73 and 0.75; p < 0.01) - was significantly upregulated in the CTB 7% treatment. In contrast, the CTB3% treatment reduced soil dissolved inorganic carbon content and PPO activity relative to CTB7%, yet PPO remained above CK treatment, while promoting Benzenoids integration into the soil chemical-microbial network. In summary, CTB3% treatment established a suitable soil micro-environment that enhanced PPO and modulated putatively annotated benzoic acid level. These findings indicate that appropriate biochar dosages amendment can alleviate continuous-cropping obstacles by altering key soil enzyme activity and allelochemicals characterize, thus contributing to sustainable continuous-cropping practices. Biochar significantly affected the soil microbiome composition in continuous cropping, especially the CTB1% and CTB3%. The enriched metabolic pathways network showed Benzoic acid as the allelochemicals in continuous-cropping soil was significantly increased by CTB7%. Desulfobacterota exhibited a significant correlation with putatively annotated benzoic acid by extracted in the soil.