Pathogen Release Dynamics and Environmental Risk in Farmland Runoff Regulated by Organic–Inorganic Fertilizer Ratio

Runoff from manure-fertilized farmlands is an important pathway for microbial contamination of receiving waters within a One Health framework. Although organic fertilizers improve soil quality, their influence on pathogen release under mixed fertilization remains insufficiently resolved. This study aims to establish a quantitative framework linking fertilization intensity to pathogen export, which is essential for developing risk-informed nutrient management strategies. Therefore, controlled simulated rainfall experiments (60 mm·h−1) were conducted on soil plots receiving three nitrogen-equivalent fertilizer regimes: T1 (100% inorganic fertilizer), T2 (37.5% organic nitrogen replacement), and T3 (50% organic nitrogen replacement). Metagenomic sequencing and qPCR (targeting 16S rRNA, E. coli, Cryptosporidium, etc.) were used to characterize pathogen-marker sources, initial soil pathogen-marker burdens, and runoff dynamics during a 50 min rainfall event. A total of 29 pathogen-marker taxa were detected among the 41 targeted taxa. Higher proportions of organic fertilizer were associated with greater initial pathogen-marker loads in the soil and greater cumulative export in runoff, with T3 consistently exhibiting the highest levels across indicators. Based on the two-pool model developed in this study, the fitted total release potential (Total M) of Escherichia in T3 reached ~2.6 × 109 copies·m−2, compared with ~1.3 × 109 in T1 and ~6.8 × 108 in T2. Interestingly, the relative ranking between T1 and T2 varied among specific pathogen markers, suggesting a potential environmental buffering capacity at moderate organic substitution levels. Across all treatments, pathogen-marker concentrations peaked during the early stages of rainfall, indicating a pronounced first-flush effect. Furthermore, bacterial indicators were mobilized more readily than fungal and protozoan targets, the latter of which were more strongly retained by the soil matrix. Overall, the results suggest that optimized organic–inorganic fertilization ratios, combined with improved manure stabilization, are essential for mitigating downstream microbial contamination potential within a One Health framework.