Detection and Diversity of Fungal Nitric Oxide Reductase Genes ( p450nor ) in Agricultural Soils

ABSTRACT Members of the Fungi convert nitrate (NO 3 − ) and nitrite (NO 2 − ) to gaseous nitrous oxide (N 2 O) (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 N 2 O from added NO 3 − or NO 2 − 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 N 2 O from NO 2 − . Novel PCR primers targeting the p450nor gene, which encodes the nitric oxide (NO) reductase responsible for N 2 O 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 N 2 O from NO 2 − , whereas nirK (encoding the NO-forming NO 2 − reductase) was amplified in only 13 to 74% of the N 2 O-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 N 2 O formation. IMPORTANCE A comprehensive understanding of the microbiota controlling soil N loss and greenhouse gas (N 2 O) emissions is crucial for sustainable agricultural practices and addressing climate change concerns. We report the design and application of a novel PCR primer set targeting fungal p450nor , a biomarker for fungal N 2 O production, and demonstrate the utility of the new approach to assess fungal denitrification potential in fungal isolates and agricultural soils. These new PCR primers may find application in a variety of biomes to assess the fungal contributions to N loss and N 2 O emissions.