Long-term conservation tillage enhances microbial carbon use efficiency by altering multitrophic interactions in soil.

Microbial carbon (C) use efficiency (CUE) plays a key role in soil C storage. The predation of protists on bacteria and fungi has potential impacts on the global C cycle. However, under conservation tillage conditions, the effects of multitrophic interactions on soil microbial CUE are still unclear. Here, we investigate the multitrophic network (especially the keystone ecological cluster) and its regulation of soil microbial CUE and soil organic C (SOC) under different long-term (15-year) tillage practices. We found that conservation tillage (CT) significantly enhanced microbial CUE, turnover, and SOC (P < 0.05) compared to traditional tillage (control, CK). At the same time, tillage practice and soil depth had significant effects on the structure of fungal and protistan communities. Furthermore, the soil biodiversity of the keystone cluster was positively correlated with the microbial physiological traits (CUE, microbial growth rate (MGR), microbial respiration rate (Rs), microbial turnover) and SOC (P < 0.05). Protistan richness played the strongest role in directly shaping the keystone cluster. Compared with CK, CT generally enhanced the correlation between microbial communities and microbial physiological characteristics and SOC. Overall, our results illustrate that the top-down control (the organisms at higher trophic levels affect the organisms at lower trophic levels) of protists in the soil micro-food web plays an important role in improving microbial CUE under conservation tillage. Our findings provide a theoretical basis for promoting the application of protists in targeted microbial engineering and contribute to the promotion of conservation agriculture and the improvement of soil C sequestration potential.