Nitrite induced transcription of p450nor during denitrification by Fusarium oxysporum correlates with the production of N2O with a high 15N site preference

Abstract

The greenhouse gas nitrous oxide (N2O) is produced in soil as a consequence of complex co-occurring processes conducted by diverse microbial species, including fungi. The fungal p450nor gene encodes a nitric oxide reductase associated with fungal denitrification. We thus hypothesized that p450nor gene expression is a marker for ongoing fungal denitrification. Specific PCR primers and quantitative PCR (qPCR) assays were developed targeting p450nor genes and transcripts. The novel PCR primers successfully amplified p450nor from pure cultures, and were used in an mRNA targeted qPCR to quantify p450nor gene transcription (i.e., gene expression) during denitrification activity in cultures of the fungal model denitrifier Fusarium oxysporum. Gene expression was induced by high (5 mM) and low (0.25 mM) nitrite concentrations. Nitrite stimulated N2O production rates by F. oxysporum, which correlated well with an up to 70-fold increase in p450nor gene expression during the first 12–24 h of anoxic incubation. The relative p450nor gene peak expression and peak N2O production rates declined 20- and 2-fold on average, respectively, towards the later phase of incubation (48–120 h). The 15N site preference of N2O (SP(N2O)) was high for F. oxysporum and independent of reaction progress, confirming the fungal origin of N2O produced. In conclusion, the developed fungal p450nor gene expression assay together with the analysis of SP(N2O) values provide a basis to improve current tools for the identification of fungal denitrification and/or N2O production in natural systems like soils.