Perennial grassland led to more complex bacterial communities as well as inter-domain networks across three continuous monoculture systems.

Long-term continuous monoculture is a common practice in modern agricultural production, yet its effects on soil microbial communities-particularly the differences between annual crops and perennial forages-remain poorly understood. Here, we investigated the diversity, composition, and co-occurrence networks of soil bacterial and fungal communities under three long-term continuous monoculture systems in Northeast China: corn (annual crop, with fertilizer application), alfalfa (perennial legume forage, without fertilizer), and sheepgrass (perennial grass forage, without fertilizer). Fungal α- and β-diversity indices differed significantly across the three systems, whereas bacterial α-diversity showed no significant differences among them. The dominant bacterial phyla were Proteobacteria, Acidobacteria, Actinobacteria, and Gemmatimonadetes, while Ascomycota, Basidiomycota, and Glomeromycota were the most abundant fungal taxa. Co-occurrence network analysis revealed system-specific patterns: the bacterial network exhibited the highest number of nodes and edges in alfalfa, whereas the fungal network was most complex in sheepgrass. The ratio of positive to negative edges in the bacterial network was more than three times higher in alfalfa than in the other systems, while the fungal network showed the highest positive-to-negative link ratio in sheepgrass. Inter-domain associations were predominantly characterized by a higher number of edges in sheepgrass. Our findings demonstrate that perennial forage systems (alfalfa and sheepgrass) promote more complex bacterial and inter-domain microbial networks compared to the annual corn system. These differences in network may have important implications for soil ecosystem functions mediated by microbial communities, highlighting the potential benefits of incorporating perennial forages into long-term monoculture systems to enhance soil microbial interactions and ecosystem sustainability.