Detection and diversity of fungal nitric oxide reductase genes (p450nor) in agricultural soils

Authors: HIGGINS, S - UNIVERSITY OF TENNESSEE; WELSH, A - UNIVERSITY OF ILLINOIS; ORELLANA, L - GEORGIA TECH; KONSTANTINIDIS, K - GEORGIA TECH; Chee Sanford, Joanne; SANFORD, R - UNIVERSITY OF ILLINOIS; SCHADT, C - UNIVERSITY OF TENNESSEE; LOEFFLER, F - UNIVERSITY OF TENNESSEE

Submitted to: Applied and Environmental Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/1/2016
Publication Date: 5/1/2016
Citation: Higgins, S.A., Welsh, A., Orellana, L.H., Konstantinidis, K.T., Chee-Sanford, J.C., Sanford, R.A., Schadt, C.W., Loeffler, F.E. 2016. Detection and diversity of fungal nitric oxide reductase genes (p450nor) in agricultural soils. Applied and Environmental Microbiology. 82(10):2919-2928.

Interpretive Summary: Fungi are common and abundant in soil environments and among the many species present, fungal denitrifiers are known to reduce nitrate to nitrous oxide. Little is known about their contribution as a significant source of this important greenhouse gas in soil environments. Knowledge about the diversity of fungal denitrifiers are limited to cultivation strategies that are biased or our inability to detect key genes involved in the pathway. In this study, the gene p450nor that encodes the nitric oxide (NO) reductase responsible for nitrous oxide production in fungi was targeted for new design of molecular primers to be used to detect a broader range of fungal denitrifiers from soils. Additionally, a cultivation strategy utilizing nitrite yielded 214 isolates placed into 15 morphological groups, of which151 produced nitrous oxide. The p450nor genes were detected in all the isolates and further, new p450nor genes were retrieved from two agricultural soils. In contrast, nirK, the gene encoding nitrite reductase and previously used in fungal denitrifier studies, were unreliable for detecting cultures active in denitrification. The signficance of these findings demonstrate the value of p450nor-targeted PCR to complement existing approaches to assess the fungal contributions to denitrification and N2O formation. Further, the results show that fungi likely play an important role in the production of nitrous oxide in agricultural soil environments and their activity should be considered in the fate of N-fertilizer and in greenhouse gas budgets.

Technical Abstract: Members of the Fungi convert nitrate (NO3-) and nitrite (NO2-) to gaseous nitrous oxide (N2O) (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 N2O from added NO3- or NO2- in the presence of antibiotics to inhibit bacteria. Cultivation efforts yielded 214 fungal isolates belonging to at least 15 distinct morphological groups, of which 151 produced N2O from NO2-. Novel PCR primers targeting the p450nor gene that encodes the nitric oxide (NO) reductase responsible for N2O 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 54-98% amino acid identity to reference P450nor sequences within the phylum Ascomycota, and expand the known fungal P450nor sequence diversity. p450nor was detected in all fungal isolates that produced N2O from nitrite, whereas nirK (encoding the NO-forming nitrite reductase) was amplified in only 13-74% of the N2O-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 N2O formation.