Evidence for niche differentiation of nitrifying communities in grassland soils after 44 years of different field fertilization scenarios

Long-term nitrogen (N) fertilization imposes strong selection on nitrifying communities in agricultural soil, but how a progressively changing niche affects potentially active nitrifiers in the field remains poorly understood. Using a 44-year grassland fertilization experiment, we investigated community shifts of active nitrifiers by DNA-based stable isotope probing (SIP) of field soils that received no fertilization (CK), high levels of organic cattle manure (HC), and chemical N fertilization (CF). Incubation of DNA-SIP microcosms showed significant nitrification activities in CF and HC soils, whereas no activity occurred in CK soils. The 44 years of inorganic N fertilization selected only 13C-ammonia-oxidizing bacteria (AOB), whereas cattle slurry applications created a niche in which both ammonia-oxidizing archaea (AOA) and AOB could be actively 13C-labeled. Phylogenetic analysis indicated that Nitrosospira sp. 62-like AOB dominated inorganically fertilized CF soils, while Nitrosospira sp. 41-like AOB were abundant in organically fertilized HC soils. The 13C-AOA in HC soils were affiliated with the 29i4 lineage. The 13C-nitrite-oxidizing bacteria (NOB) were dominated by both Nitrospira- and Nitrobacter-like communities in CF soils, and the latter was overwhelmingly abundant in HC soils. The 13C-labeled nitrifying communities in SIP microcosms of CF and HC soils were largely similar to those predominant under field conditions. These results provide direct evidence for a strong selection of distinctly active nitrifiers after 44 years of different fertilization regimes in the field. Our findings imply that niche differentiation of nitrifying communities could be assessed as a net result of microbial adaption over 44 years to inorganic and organic N fertilization in the field, where distinct nitrifiers have been shaped by intensified anthropogenic N input.