Combined effects of earthworms and Bacillus spp. enhance soil ecosystem multifunctionality and reshape microbial communities

Long-term physicochemical agricultural management in tobacco systems has impaired the soil ecosystem multifunctionality, disrupted the soil microbial community structure, and affected the long-term health of farmland ecosystems. As key beneficial soil macrofauna and bacteria, earthworms and Bacillus spp., respectively, contribute independently to the improvement of soil quality and enhance plant growth. However, their synergistic effects on soil ecosystem multifunctionality and microbial communities remain unclear, particularly in tobacco systems. Therefore, we tested their individual and interactive effects in a tobacco pot experiment with four treatments: control (CK), earthworm inoculation alone (E), Bacillus spp. inoculation alone (B), and co-inoculation of earthworms and Bacillus spp. (EB). Earthworm inoculation alone significantly increased soil alkaline-hydrolyzable nitrogen and total nitrogen contents, as well as catalase activity, while co-inoculation further enhanced alkaline-hydrolyzable nitrogen, available phosphorus, and catalase activity. Earthworm activity, whether applied individually or in combination with Bacillus spp., significantly altered the β-diversity of soil bacterial communities. Both Bacillus spp. and earthworm inoculation independently increased the relative abundance of Actinobacteriota and decreased that of Fusarium . Moreover, earthworm inoculation alone significantly increased the relative abundance of Penicillium and Mortierella , whereas co-inoculation significantly increased the relative abundance of Proteobacteria, Bacteroidota, and Verrucomicrobiota. Earthworm inoculation alone and co-inoculation significantly enhanced soil ecosystem multifunctionality. In addition, all biotic treatments (B, E, EB) promoted the complexity of bacterial and fungal co-occurrence networks. Random forest analysis showed that catalase activity and total nitrogen were the key factors affecting soil ecosystem multifunctionality; available phosphorus was the main factor influencing soil bacterial network complexity; and dissolved organic carbon and total nitrogen were the key factors affecting fungal network complexity. In summary, this study demonstrates that earthworms are the core drivers of enhanced soil ecosystem multifunctionality and microbial community structure in tobacco systems, while Bacillus spp. exert synergistic effects by strengthening the ecological foundation established by earthworms.