Detection and diversity of fungal nitric oxide reductase genes (p450nor) in agricultural soils

Members of the Fungi convert nitrate (NO₃ ^-) and nitrite (NO₂ ^-) to gaseous nitrous oxide (N₂O) (denitrification), but the fungal contributions to N loss from soil remain uncertain. Cultivation-based methodologies that include antibiotics to selectively assess fungal activities have limitations, and complementary molecular approaches to assign denitrification potential to fungi are desirable. Microcosms established with soils from two representative U.S. Midwest agricultural regions produced N₂O from added NO₃ ^- or NO₂ ^- in the presence of antibiotics to inhibit bacteria. Cultivation efforts yielded 214 fungal isolates belonging to at least 15 distinct morphological groups, 151 of which produced N₂O from NO₂ ^-. Novel PCR primers targeting the p450nor gene, which encodes the nitric oxide (NO) reductase responsible for N₂O production in fungi, yielded 26 novel p450nor amplicons from DNA of 37 isolates and 23 amplicons from environmental DNA obtained from two agricultural soils. The sequences shared 54 to 98% amino acid identity with reference P450nor sequences within the phylum Ascomycota and expand the known fungal P450nor sequence diversity. p450nor was detected in all fungal isolates that produced N₂O from NO₂ ^-, whereas nirK (encoding the NO-forming NO₂ ^- reductase) was amplified in only 13 to 74% of the N₂O-forming isolates using two separate nirK primer sets. Collectively, our findings demonstrate the value of p450nor-targeted PCR to complement existing approaches to assess the fungal contributions to denitrification and N₂O formation.