Chronic Inorganic Fertilization Shifts Evolutionary Trajectories and Induces a Regulatory Shield in Maize Rhizosphere Bacteria

Long-term agricultural fertilization is a major driver of structural shifts in the soil microbiome, yet traditional metagenomic analyses often fail to capture the population-level evolutionary dynamics and heritable genetic adaptations that dictate species survival in the rhizosphere. Here, we built and utilize a high-resolution, gene-centric population-genomic framework to resolve the microdiversity and selective landscapes shaping maize rhizosphere bacteria across a chronic management gradient including Inorganic NPK fertilization, Organic compost amendment, and an Unfertilized Control. We observed a distinct decoupling between total mutational volume and adaptive fixation rates. Inorganic fertilization acts as a massive genotoxic hotspot generating 5,544 variants but maintaining a baseline intra-population variants consensus rate of 37.3% due to intense purifying selection. Whereas organic management restricts global mutational volume but drives relatively high rate of 49.6%, signaling a highly coordinated, directional selection where transposable elements function as bridges for structural adaptations. Crucially, we uncover that regulatory shield mechanism that protects the core genome from deleterious mutations while core metabolic and structural genes exhibit absolute sequence stasis. Additionally, we report that mutations were precisely accumulated within the immediate 0–200 bp promoter region of key stress-defense and metabolic genes. This targeted regulatory remodeling is chemically fingerprinted by a depressed transition-to-transversion ratio and structurally supported by Mobile Fortress model that significantly expands the repository of Highly Conserved Mobile Loci sequestered on cryptic circular plasmids. Together, these evolutionary trajectories indicate that long-term inorganic fertilization forces rhizosphere bacteria to prioritize cellular repair and resource hoarding over host interactions. By demonstrating how high-input management shifts selection from cooperative pathways toward internal defense, these findings provide a population-genomic explanation for the suppression of biological nutrient-use efficiency observed in intensive agroecosystems.