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

Members of the Fungi convert nitrate (NO 3 -) and nitrite (NO 2 -) to gaseous nitrous oxide (N 2O) (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 N 2O from added NO 3 - or NO 2 - 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 N 2O from NO 2 -. Novel PCR primers targeting the p450nor gene that encodes the nitric oxide (NO) reductase responsible for N 2O 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 N 2O from nitrite, whereas nirK (encoding the NO-forming nitrite reductase) was amplified in only 13-74%more » of the N 2O-forming isolates using two separate nirK primer sets. Altogether, our findings demonstrate the value of p450nor-targeted PCR to complement existing approaches to assess the fungal contributions to denitrification and N 2O formation.« less
Authors:
ORCiD logo [1] ;  [2] ;  [3] ;  [3] ;  [1] ;  [2] ;  [4] ;  [4]
  1. Univ. of Tennessee, Knoxville, TN (United States)
  2. Univ. of Illinois, Urbana, IL (United States)
  3. Georgia Inst. of Technology, Atlanta, GA (United States)
  4. Univ. of Tennessee, Knoxville, TN (United States); Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)
Publication Date:
Grant/Contract Number:
AC05-00OR22725
Type:
Accepted Manuscript
Journal Name:
Applied and Environmental Microbiology
Additional Journal Information:
Journal Volume: 82; Journal Issue: 10; Journal ID: ISSN 0099-2240
Publisher:
American Society for Microbiology
Research Org:
Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)
Sponsoring Org:
USDOE Office of Science (SC)
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES
OSTI Identifier:
1326528

Higgins, Steven A., Welsh, Allana, Orellana, Luis H., Konstantinidis, Konstantinos T., Chee-Sanford, Joanne C., Sanford, Robert A., Schadt, Christopher W., and Löffler, Frank E.. Detection and diversity of fungal nitric oxide reductase genes (p450nor) in agricultural soils. United States: N. p., Web. doi:10.1128/AEM.00243-16.
Higgins, Steven A., Welsh, Allana, Orellana, Luis H., Konstantinidis, Konstantinos T., Chee-Sanford, Joanne C., Sanford, Robert A., Schadt, Christopher W., & Löffler, Frank E.. Detection and diversity of fungal nitric oxide reductase genes (p450nor) in agricultural soils. United States. doi:10.1128/AEM.00243-16.
Higgins, Steven A., Welsh, Allana, Orellana, Luis H., Konstantinidis, Konstantinos T., Chee-Sanford, Joanne C., Sanford, Robert A., Schadt, Christopher W., and Löffler, Frank E.. 2016. "Detection and diversity of fungal nitric oxide reductase genes (p450nor) in agricultural soils". United States. doi:10.1128/AEM.00243-16. https://www.osti.gov/servlets/purl/1326528.
@article{osti_1326528,
title = {Detection and diversity of fungal nitric oxide reductase genes (p450nor) in agricultural soils},
author = {Higgins, Steven A. and Welsh, Allana and Orellana, Luis H. and Konstantinidis, Konstantinos T. and Chee-Sanford, Joanne C. and Sanford, Robert A. and Schadt, Christopher W. and Löffler, Frank E.},
abstractNote = {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. Altogether, our findings demonstrate the value of p450nor-targeted PCR to complement existing approaches to assess the fungal contributions to denitrification and N2O formation.},
doi = {10.1128/AEM.00243-16},
journal = {Applied and Environmental Microbiology},
number = 10,
volume = 82,
place = {United States},
year = {2016},
month = {3}
}