Long-term N and NPK fertilizations induce significant alterations in microbial compositions and co-occurrence patterns in saturated soil layers

The subsurface soil environment, spanning from the vadose zone to the saturated zone, serves as a critical zone where diverse soil processes and biogeochemical interactions take place. However, the specific influence of longterm fertilization on microbial interactions in this zone remains unclear. In this study, we conducted a five-year field experiment, collecting 76 soil samples from three 0-20 m boreholes to investigate the effects of fertilization (N-addition and NPK-addition) on bacterial, fungal and archaeal communities along the soil profile. Long-term fertilization significantly altered the composition, assembly processes, and inter-kingdom co-occurrence patterns of bacterial, archaeal and fungal communities. These changes were particularly pronounced in the saturated soil layer (5-20 m), where the network complexity increased (average degree rising by 5.19 and 8.05 times under Naddition and NPK-addition, respectively) and stability reduced (modularity decreased by 0.47 times and 0.43 times, respectively). Further analysis revealed that pH and nutrients were the main drivers of microbial communities and interactions under both fertilization treatments, explaining 97.20 % of variance in N-addition and 96.70 % in NPK-addition. Moreover, compared to N-addition, the NPK-addition treatment exhibited greater environmental friendliness, with enhanced carbon and nitrogen metabolic potential. This relatively milder impact could be attributed to the enhanced "bridge" role of fungi, acting as stabilizers in the inter-kingdom network. Our findings not only provided new insights into the influence of fertilization on microbial composition and inter-kingdom co-occurrence patterns in the saturated soil layer, but also highlighted the specific role of fungi in mitigating the disruption caused by fertilization.