Primer Development to Target N2O-Producing Fungal Community

H. Chen, W. Shi

Author address: 

North Carolina State Univ., Raleigh, NC

Abstract: 

Background: Fungal denitrification has been increasingly investigated as an important biological source of N2O, an important greenhouse gas and ozone-depleting substance. But the community ecology of N2O-producing fungi is still poorly understood due to the lack of cultureindependent tools, such as PCR primers.Methods: Degenerate primers that target fungal nitrite reductase gene (nirK) and nitric oxide reductase gene (P450nor), the two important genes for regulating fungal N2O-production were designed based on the conservative protein and DNA regions in public databases and evaluated for primer specificity and efficiency using a number of N2O-producing fungal cultures and agricultural soil samples.Results: Four pairs of fungal nirKtargeting primers (FnirK_F1/R1, F1/R2, F2/R2, and F3/R2) were developed, among which FnirK_F3/R2 appeared to be more efficient as this pair amplified ~ 80% of tested fungal cultures, including Aspergillus, Fusarium, Penicillium, and Trichoderma, etc. The soil clone sequences amplified by FnirK_F3/R2 were distributed not only in the clusters of fungal cultures isolated from the soil sample (e.g., Fusarium and Aspergillus), but also in the clusters of Chaetomium, Metarhizium, and Myceliophthora, which were failed to be isolated from the soil sample in our previous work. However, some bacterial nirK could also be amplified due to high similarities between fungal nirK and bacterial type II nirK. A pair of degenerate primers that target P450nor genes was also proved to be able to amplify diverse groups of N2O-producing fungi from fungal cultures and soil samples. The amplicons were shown to phylogenetically relate to known denitrifying fungi.Conclusions: Our primers that target fungal nirK and P450nor genes were proved to have the wide-range capability of amplifying diverse denitrifying fungi from the environment. However, the P450nor-targetting primers may be better than nirKtargeting primers to explore N2O-producing fungal community in the environment, given that high similarities between fungal nirK and bacterial type II nirK could limit the specificity of fungal nirK primers.
2016

abstract No: 

SUNDAY-045

Full conference title: 

ASM Microbe 2016
    • ASM microbe 1st (2016)