Development of PCR primers targeting fungal nirK to study fungal denitrification in the environment

•Primer set to detect the fungal nirK was developed.•Congruence between phylogenies of the fungal 18S rRNA gene and nirK was identified.•Fungi responsible for N2O emissions in a surface-fertilized soil were identified.

Fungal denitrification in soils is receiving considerable attention as one of the dominant N2O production processes. However, because of the lack of a methodology to detect fungal denitrification-related genes, the diversity and ecological behavior of denitrifying fungi in soil remains unknown. Thus, we designed a primer set to detect the fungal nitrite reductase gene (nirK) and validated its sensitivity and specificity. Through clone library analyses, we identified congruence between phylogenies of the 18S rRNA gene and nirK of denitrifying fungal isolates obtained from the surface-fertilized cropland soil and showed that fungi belonging to Eurotiales, Hypocreales, and Sordariales were primarily responsible for N2O emissions in the soil.