Nitrous oxide (N2O) emissions from arable soils are predominantly caused by denitrifying microbes, of which
fungal denitrifiers are of particular interest, as fungi, in contrast to bacteria, terminate denitrification with N2O.
Reduced tillage has been shown to increase gaseous nitrogen losses from soil, but knowledge of how varying
tillage regimes and associated soil physical and chemical alterations affect fungal denitrifiers is limited. Based on
results from a long-term (>40 years) tillage experiment, we show that non-inversion tillage resulted in increased
potential denitrification activity in the upper soil layers, compared to annual or occasional (every 4–5 years)
conventional inversion tillage. Using sequence-corrected abundance of the fungal nirK gene, we further identified
an increased genetic potential for fungal denitrification, compared to that caused by bacteria, with decreasing
tillage intensity. Differences in the composition and diversity of the fungal nirK community imply that different
tillage regimes select for distinct fungal denitrifiers with differing functional capabilities and lifestyles, predominantly
by altering carbon and nitrogen related niches. Our findings suggest that the creation of organic
hotspots through stratification by non-inversion tillage increases the diversity and abundance of fungal denitrifier
communities and modifies their composition, and thus their overall relevance for N2O production by
denitrification, in arable soils.