Long-term N fertilization imbalances potential N acquisition and transformations by soil microbes.

Nitrogen (N) fertilization in agricultural soils has been receiving worldwide attention due to its detrimental effects on ecosystem services, particularly on microbial N transformation. However, few studies provide a complete picture of N-fertilization effects on the N transformation cycle within a single agricultural ecosystem. Here, we explored the main steps of the microbial N cycle, using targeted gene abundances as proxies, in relation to soil properties, following 35 years of N-fertilization at increasing rates (0, 202 and 269 kg N/ha) in continuous corn (Zea mays L.) and corn-soybean [Glycine max (L.) Merr.] rotations. We used real-time quantitative polymerase chain reaction (qPCR) for the quantification of phylogenetic groups and functional gene screening of the soil microbial communities, including genes encoding critical enzymes of the microbial N cycle: nifH (N fixation), amoA (first step of nitrification), nirK and nirS (first step of denitrification), and nosZ (last step of denitrification). Our results showed that long term N-fertilization increased the abundance of fungal communities likely related to decreases in pH, and an enrichment of Al and Fe in exchange sites at the expense of critical macro and micronutrients. At the same time, long term N-fertilization damaged potential biological N fixation by significantly reducing the abundance of nifH genes in both continuous and rotated corn systems, while accelerating potential nitrification activities under continuous corn by increasing the abundance of bacterial amoA. Fertilization did not affect the abundance of denitrifying groups. Altogether, these results suggest that N fertilization in corn crops potentially decreases N acquisition by free-living soil microbes and stimulates nitrification activities, thus creating a vicious loop that makes the overall agricultural system even more dependent on external N inputs.