Detection of Nitrous Oxide-Producing Fungi in the Environment Using Nirk Gene-Targeting Primers.

Fungal denitrification has been increasingly investigated as an important biological source of N₂O, but the community ecology of N₂O-producing fungi are still poorly understood due to the lack of culture-independent based tools. In this work, four pairs of nirK gene-targeting primers (FnirK_F1/R1, F1/R2, F2/R2, and F3/R2) were designed and evaluated for primer specificity and efficiency using 30 N₂O-producing fungal cultures and an agricultural soil sample. All four primer sets amplified nirK genes from fungal cultures and soil genomic DNA, but efficiency and specificity differed.  FnirK_F3/R2 appeared to be more efficient as this pair successfully amplified ~ 80% of tested fungal cultures, including Aspergillus, Fusarium, Penicillium, and Trichoderma, as compared to ~ 40-70% for other three primers. Rarefaction analysis, based on libraries of 16-27 clones constructed from the soil sample, also showed that FnirK_F3/R2 amplified diverse nirK genes from the environment. The nirK gene fragments of fungal cultures and soil sample amplified by FnirK_F3/R2 were phylogenetically related to known denitrifying fungi in the orders Eurotiales, Hypocreales, and Sordariales; and a large fraction of clone sequences were clustered with nirK genes of the genera Chaetomium, Metarhizium, and Myceliophthora, which were not isolated previously. This further confirmed the capability of newly-designed primers for amplifying diverse denitrifying fungi from the environment. However, our primers amplified some bacterial nirK genes, suggesting primer specificity was not strong. The theoretical basis for this issue was discussed by comparing nirK similarity between denitrifying fungi and bacteria.