Abstract
pH-neutral soils, where mineral phosphorus (P) fixation is relatively limited and microbial activity is high, provide an ideal system for investigating microbial regulation of P cycling under long-term fertilization. Here, we combined P-33 isotope tracing with shotgun metagenomics and microbial network analysis to quantify soil P transformation in a 180-year fertilization experiment. Compared with the unfertilized control, P-only fertilization increased total P, PO43-, and microbial biomass P by 73%, 201%, and 227%, respectively. In contrast, N-only fertilization enhanced phosphatase activity and enriched genes involved in phosphate transport and polyphosphate synthesis, indicating greater microbial P acquisition and storage. These changes promoted organic P mineralization and microbial P immobilization simultaneously, resulting in no net accumulation of labile P. Microbial network analysis further revealed a shift in keystone taxa under N fertilization toward taxa associated with P immobilization and stabilization. Together, our results demonstrate that long-term N fertilization drives a conservative, microbially regulated P cycling strategy in which enhanced mineralization is tightly coupled with rapid immobilization, whereas P fertilization primarily increases soil P stocks. These findings highlight distinct microbial mechanisms regulating P cycling under contrasting nutrient inputs and underscore the importance of balanced fertilization for sustaining P availability while minimizing environmental losses.