title: Detection of N2O-producing fungi in environment using nitrite reductase gene (nirK)-targeting primers.
abstract: Fungal denitrification has been increasingly investigated, but its community ecology is poorly understood due to the lack of culture-independent tools. In this work, four pairs of nirK-targeting primers were designed and evaluated for primer specificity and efficiency using thirty N2O-producing fungal cultures and an agricultural soil. All primers amplified nirK from fungi and soil, but their efficiency and specificity were different. A primer set, FnirK_F3/R2 amplified ∼80 % of tested fungi, including Aspergillus, Fusarium, Penicillium, and Trichoderma, as compared to ∼40-70 % for other three primers. The nirK fragments of fungal and soil DNA amplified by FnirK_F3/R2 were phylogenetically related to denitrifying fungi in the orders Eurotiales, Hypocreales, and Sordariales; and clone sequences were also distributed in the clusters of Chaetomium, Metarhizium, and Myceliophthora that were uncultured from soil in our previous work. This proved the wide-range capability of primers for amplifying diverse denitrifying fungi from environment. However, our primers and recently-developed other primers amplified bacterial nirK from soil and this co-amplification of fungal and bacterial nirK was theoretically discussed. The FnirK_F3/R2 was further compared with published primers; results from clone libraries demonstrated that FnirK_F3/R2 was more specifically targeted on fungi and had broader taxonomical coverage than some others.
authors.affiliation: Department of Soil Science, North Carolina State University, Raleigh, NC 27695, USA.
authors.forename: Huaihai
authors.initials: H
authors.lastname: Chen
authors.affiliation: Department of Soil Science, North Carolina State University, Raleigh, NC 27695, USA; School of Environment and Resource Sciences, Zhejiang Agriculture and Forestry University, Linan, Zhejiang 311300, China.
authors.forename: Fangbo
authors.initials: F
authors.lastname: Yu
authors.affiliation: Department of Soil Science, North Carolina State University, Raleigh, NC 27695, USA. Electronic address: wei_shi@ncsu.edu.
authors.forename: Wei
authors.initials: W
authors.lastname: Shi
mesh_terms: D016000:Cluster Analysis; D017931:DNA Primers / Q000235:genetics*; D004783:Environmental Microbiology; D005658:Fungi / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D056186:Metagenomics / Q000379:methods*; D009572:Nitrite Reductases / Q000235:genetics*; D009609:Nitrous Oxide / Q000378:metabolism*; D010802:Phylogeny; D017422:Sequence Analysis, DNA
chemical_list: D017931:DNA Primers; D009572:Nitrite Reductases; D009609:Nitrous Oxide
keywords: Bacteria; Denitrification; Fungi; Nitrous oxide; Soil