Progress 03/01/11 to 02/28/15

Outputs
Target Audience: The target audience includes researchers, graduate students, and extension personnel The knowledge obtained from the project has been delivered to researchers, graduate students, and extension personnel through peer-reviewed publications, national, regional and local professional meetings, and formal classroom lectures. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Two Ph.D. students and a post-doctoral research associate have worked closely with the PD of the project. They have acquired a number of molecular, biochemistry and process-level techniques. They have been given a number of opportunities to present their work at national, regional and local professional meetings, to participate in professional development workshops, to collaborate with researchers outside of the PD's department, and to supervise or mentor visiting scholars and other students in the PD's research group. How have the results been disseminated to communities of interest? The results of the project have been disseminated to researchers, graduate students and extension personnel via peer-reviewed publications, formal and informal communications, professional meetings, classroom lectures and seminars. What do you plan to do during the next reporting period to accomplish the goals? A No-Cost Extension has been filed. If granted, we plan to complete the buildup of clone libraries and sequencing of metagenomic DNA. We also hope to have two manuscripts submitted.

Impacts
What was accomplished under these goals? Impacts and outcomes: Nitrous oxide, a trace gas for global warming and ozone depletion is mainly produced from agricultural soil. In USA, agriculture contributes approximately two third of total N2O emissions. It is believed that bacteria are the sole organisms mediating soil N2O emissions. However, increasing evidence suggests that fungi should not be ignored in mediating soil N2O emissions. This project aimed to provide comprehensive knowledge on N2O-producing fungal community and its significant contribution to soil N2O emissions. Fungi representing diverse taxonomic groups were isolated from agricultural soils and examined for the capability of N2O production. Thus-obtained N2O-producing fungal isolates were used as reference strains to help develop a PCR-based tool for cultivation-independent detection and characterization of soil N2O-producing fungal community. Direct soil measurements were then conducted to assess fungal and bacterial contributions to soil N2O emissions. Despite differences in vegetation types and management practices of agroecosystems, fungal contribution was comparable to or greater than the bacterial contribution. This work provides new evidence to support that (1) diverse N2O-producing fungi are prevalent in agricultural soil and (2) soil fungi are important bio-agents mediating soil N2O emissions. The data generated from this project improve our knowledge on biological sources and control factors of N2O emissions in agricultural systems and may have profound impacts on developing sound mitigation strategies for agricultural N2O emissions. What was accomplished under these goals? Accomplishments under the objective one:Soil samples were collected from five distinct agricultural soil systems at the Center for Environmental Farming Systems, Goldsboro, NC. Fungi were isolated from soil using a dilution plating method with a modified Czapek medium. Fungal isolates were tested for N2O-producing activity using a liquid Czapek medium containing streptomycin to prevent bacterial contamination. Further, ten fungal isolates were randomly selected to examine fungal N2O production in response to inorganic N type, pH, and O2 availability. Degenerate primers targeting both fungal nirK and P450nor were designed based upon the respective databases that were established by searching NCBI nucleotide database and HMMER protein database. These primers were tested against 28 fungal strains isolated from diverse agricultural soils. The successful PCR primers were further examined against multiple metagenomic DNAs. Data collected and results:Sixty-eight fungal isolates produced N2O in the nitrate-containing Czapek medium. These N2O-producing isolates represented at least 16 genera and 35 fungal species. Fusarium oxysporum was the only species that was isolated from all the ecosystems. Isolates of F. oxysporum sampled from soil in conventional farming and integrated crop and livestock systems had higher N2O-producing capability than those isolates from the non-agricultural systems. Effects of inorganic N substrate on fungal N2O production were species dependent, with nitrite being the preferred medium for most fungal isolates. About 70% of the tested fungal isolates had significantly higher N2O-producing activity at neutral pH than at acidic and alkaline pH. Furthermore, 80% of the tested fungal isolates produced a significantly greater amount of N2O when headspace O2 was below 5%. Four pairs of primers targeting nirK and P450nor were able to amply diverse fungal isolates and metagenomic DNA, with amplicon sizes from 233 bp to 543 bp. Sequences of nirK and P450nor gene fragments were made on both fungal isolates and metagenomic DNA. Key outcomes or other accomplishments realized:This work supports that diverse N2O-producing fungi are prevalent in agricultural soil. It also provides new knowledge on the impacts of abiotic factors on fungal N2O productions. The information obtained from this work has led to one peer-reviewed publication in Soil Biology & Biochemistry, five presentations at local, regional and national professional meetings, and one manuscript in preparation. Accomplishments under the objective two:Metagenomic DNA was extracted from 0.6 g soil in duplicate by the bead-beating method using a FastDNA Spin kit. For each soil sample, duplicate DNA extracts were combined and purified on a 2% agarose gel to remove PCR-inhibiting humic materials and cleaned from the gel slice. Purified DNA was amplified with designed forward and reverse primers targeting fungal nirK and P450nor genes. Amplicons from reconditioning PCR were used to build up clone libraries of both nirK and P450nor. Sequences of fungal nirK and P450nor gene fragments were then subjected to phylogenetic analyses. However, due to the low efficiency of cloning, we are not satisfied with the size of metagenomic clone libraries. We will continue to build up clone libraries for both fungal nirK and P450nor. Key outcomes or other accomplishments realized:Despite incomplete, cultivation-independent PCR techniques suggest that agricultural soils harbors N2O-producing fungi of diverse taxonomic groups. This work generates one review-type publication on phylogenetic and taxonomic diversity of fungal denitrifiers. We expect two more manuscripts will be produced following the completion of clone libraries and sequences of DNA fragments. In addition, we have also given three presentations at regional and national professional meetings. Accomplishments achieved under the objective three:For determining the relative contribution of fungi and bacteria to soil N2O flux, a laboratory microcosm experiment was made with four treatments, antibiotic-free soil and soil amended with streptomycin, cycloheximide or both. Soil moisture and pH effects were assessed under 65-90% water-filled pore space (WFPS) and pH 4.0-9.0, respectively. Effects of substrate quality on fungal and bacterial N2O production were examined after soils were amended with four different substrates, i.e., glucose, cellulose, winter pea, and switchgrass. To associate fungal and bacterial N2O production with the abundances of respective microbial functional groups, the abundances of soil fungi, bacteria, and bacterial denitrifiers were examined using a quantitative real-time PCR approach. Data collected and results:With the aid of antibiotic selective inhibition, we showed that fungi had a great potential of contributing soil N2O production. Regardless of differences in vegetation types and management practices of the five ecosystems, more than 40% production in soil N2O was made by the activity of soil fungi. The fungal contribution was comparable to or greater than the bacterial contribution across the five ecosystems. This new evidence supports that soil fungi are potentially important bio-agents for substantial amounts of soil N2O production. However, the degree of fungal dominance varied with soil moisture content/O2 availability as well as substrate complexity Key outcomes or other accomplishments realized:This is the first study comparing relative fungal and bacterial contribution to soil N2O production across diverse ecosystems that differed in vegetation types and management practices. By using antibiotic selective inhibition, we demonstrated that regardless of the types of ecosystems, fungal contribution accounted for a fairly large portion of total soil N2O production that should not be ignored. Our work also provides new insight into niche differences for fungal and bacterial N2O production. These finding may have profound impacts because current models for predicting soil N2O emissions are solely based on knowledge of bacterial denitrification. The information obtained from this work has led to four peer-reviewed publications, eight presentations at local, regional and national professional meetings, and one manuscript in preparation.

Publications

• Type: Journal Articles Status: Published Year Published: 2015 Citation: Chen H, Mothapo NV, Shi W, 2015. Fungal and bacterial N2O production regulated by soil amendments of simple and complex substrates. Soil Biology & Biochemistry 84, 116-126.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Mothapo NV, Chen H, Cubeta MA, Grossman JM, Fuller F, Shi W, 2015. Phylogenetic, taxonomic and functional diversity of fungal denitrifiers and associated N2O production efficacy. Soil Biology & Biochemistry 83, 160-175.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Chen H, Mothapo NV, Shi W, 2015. Moisture and pH control relative contributions of fungi and bacteria to N2O production. Microbial Ecology 69, 180-191.
• Type: Journal Articles Status: Published Year Published: 2014 Citation: Chen H, Mothapo NV, Shi W, 2014. The significant contribution of fungi to soil N2O production across diverse ecosystems. Applied Soil Ecology 73, 70-77.
• Type: Journal Articles Status: Published Year Published: 2013 Citation: Mothapo NV, Chen H, Cubeta MA, Shi W, 2013. Nitrous oxide producing activity of diverse fungi from distinct agroecosystems. Soil Biology & Biochemistry 66, 94-101.
• Type: Journal Articles Status: Published Year Published: 2013 Citation: Chen H, Li X, Hu F, Shi W, 2013. Soil nitrous oxide emissions following crop residue addition: a meta-analysis. Global Change Biology 19, 2956-2964.
• Type: Conference Papers and Presentations Status: Other Year Published: 2015 Citation: Chen H, Mothapo NV, Shi W, 2015. Substrate quality shifts microbial sources of soil nitrous oxide production: a microcosm study. Soil Science Society North Carolina 58th Annual Meeting, Raleigh, USA.
• Type: Conference Papers and Presentations Status: Other Year Published: 2015 Citation: Chen H, Shi W, 2015. Substrate complexity decides the main contributor to soil nitrous oxide emissions: bacteria or fungi? 4th Post-doctoral Research Symposium, North Carolina State University, Raleigh, NC.
• Type: Conference Papers and Presentations Status: Other Year Published: 2014 Citation: Chen H, Mothapo NV, Shi W, 2014. Substrate quality regulates fungal and bacterial contribution to soil nitrous oxide emissions. ASA/CSSA/SSSA Annual Meetings, Long Beach CA
• Type: Conference Papers and Presentations Status: Other Year Published: 2014 Citation: Mothapo NV, Chen H, Shi W, 2014. PCR primers targeting fungal P450nor gene for characterization of N2O producing fungi. ASA/CSSA/SSSA Annual Meetings, Long Beach CA.
• Type: Conference Papers and Presentations Status: Other Year Published: 2014 Citation: Chen H, Mothapo NV, Shi W, 2014. What controls fungal nitrous oxide production in agroecosystems? The 9th Annual North Carolina State University Graduate Student Research Symposium, Raleigh USA.
• Type: Conference Papers and Presentations Status: Other Year Published: 2014 Citation: Mothapo NV, Chen H, Cubeta MA, Shi W, 2014. Nitrous oxide producing activity of diverse fungi from distinct agroecosystems. 35th Annual Mid-Atlantic States Mycology Conference, Boone, NC.
• Type: Conference Papers and Presentations Status: Other Year Published: 2014 Citation: Chen H, 2014. Fungal nitrous oxide production in agro-ecosystems: Importance relative to bacteria and responses to abiotic factors. Soil Science Seminar Series, North Carolina State University.
• Type: Conference Papers and Presentations Status: Other Year Published: 2013 Citation: Chen H, Mothapo NV, Shi W, 2013. Fungal contribution to soil nitrous oxide production across diverse ecosystems. Annual Meeting Abstracts. ASA/CSSA/SSSA Annual Meetings, Tampa, Florida.
• Type: Conference Papers and Presentations Status: Other Year Published: 2013 Citation: Chen H, Li XC, Hu F, Shi W, 2013. Effects of crop residues on soil nitrous emissions: a meta-analysis. Annual Meeting Abstracts, Soil Science Society of North Carolina, Raleigh, NC.
• Type: Conference Papers and Presentations Status: Other Year Published: 2013 Citation: Chen H, 2013. Fungal denitrification: an overlooked source of soil N2O production. Soil Science Seminar Series, North Carolina State University.
• Type: Conference Papers and Presentations Status: Other Year Published: 2013 Citation: Mothapo NV, Chen H, Cubeta MA, Shi W, 2013. Nitrous oxide-producing activity of diverse soil fungi under imposed abiotic conditions. 34th Annual Meeting, Mid-Atlantic States Mycology Conference, Beltsville, MD.
• Type: Conference Papers and Presentations Status: Other Year Published: 2013 Citation: Mothapo NV, 2013. N2O-producing activity of diverse fungi from distinct soil systems. The 8th Annual North Carolina State University Graduate Student Research Symposium, Raleigh, USA.
• Type: Conference Papers and Presentations Status: Other Year Published: 2013 Citation: Mothapo NV, 2013. Detecting fungi with N2O-producing capability in managed ecosystems. Soil Science Seminar Series, North Carolina State University.
• Type: Theses/Dissertations Status: Published Year Published: 2014 Citation: Chen H, 2014. Fungal nitrous oxide production in agro-ecosystems: importance relative to bacteria and responses to abiotic factors. Ph.D. Dissertation. North Carolina State University.
• Type: Conference Papers and Presentations Status: Other Year Published: 2013 Citation: Shi W, 2013. Biodiversity and eco-physiology of nitrous oxide-producing fungi in distinct agroecosystems. USDA-NIFA Director meetings, Washington DC.
• Type: Conference Papers and Presentations Status: Other Year Published: 2013 Citation: Mothapo NV, Shi W, 2012. Nitrous oxide producing fungi isolated from agricultural soil. Annual Meeting Abstracts, ASA/CSSA/SSSA Annual Meetings, Cincinnati, Ohio.
• Type: Conference Papers and Presentations Status: Other Year Published: 2012 Citation: Shi W, 2012. Nitrous oxide-producing fungi in agricultural soil. USDA project director meetings, Plant Animal Genomics, San Diego, California.

Progress 02/28/13 to 02/27/14

Outputs
Target Audience: Target audience includes researchers, graduate students, and extension personnel. Knowledge gained from the project has been delivered to researchers, graduate students, and extension personnel through national, regional and local professional meetings. Also, the informaiton gained from the project has been added to the PD's graduate-level course, Soil Microbiology. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Two Ph.D. students have worked closely with the PD of the project. They have acquired a number of molecular, biochemistry and process-level techniques. They have been given a number of opportunities to present their work at national, regional and local professional meetings and to participate in professional development workshops. They have also been trained to review manuscripts for international journals. In addition, the information gained from this project has been incorporated into the PD’s graduate-level course, Soil Microbiology. How have the results been disseminated to communities of interest? The results of the project have been disseminated to researchers, graduate students and extension personnel via formal and informal communications, professional meetings, classroom lectures and seminars. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Accomplishments under the objective one: We have isolated nitrous oxide-producing fungi from distinct agroecosystems, including conventional farming, organic farming, integrated crop and livestock, plantation forestry, and an abandoned agricultural field subjected to natural succession. The assumption that diverse nitrous oxide-producing fungi are prevalent in agricultural soil is supported by isolation and screening of fungi from soil representing different agricultural and forestry-based systems. The number of nitrous oxide-producing fungal species detected in this project is greater than the number in any previous studies and has been expanded to other genera that include Actinomucor, Bionectria, Metarhizium, Mortierella, Mucor, Ophiocordyceps, and Phoma. The nitrous oxide-producing isolates are taxonomically distributed among Sordariomycetes (60%), Eurotiomycetes (27%), Dothideomycetes (4%), basal fungi (6%) and unidentified fungi (3%). While most fungi with high nitrous oxide activity belong to the Sordariomycetes class, Aspergillus spp. are the only isolates outside the Sordariomycetes. These fungal isolates have been examined for physiological responses to inorganic nitrogen species, pH and oxygen availability. Besides isolation of widely-identified nitrous oxide-producing fungi, our results provide convincing evidence that the majority of nitrous oxide-producing fungi prefer neutral than alkaline conditions. Our results also show that nitrous oxide-producing fungi of high activity are more prevalent in intensively managed systems. One fungal species identified in this project (Neocosmospora vasinfecta) possesses nitrous oxide-producing characteristics that differ from other fungi. These characteristics include several orders of magnitude greater nitrous oxide activity, nitrate preference, optimal activity under acidic conditions, and greater tolerance for oxygen. Further screening may be helpful in characterizing and identifying additional species of nitrous oxide-producing fungi with distinct physiological behavior. PCR primers have been designed and tested using obtained fungal isolates and soil DNA. Accomplishments under the objective two: We have used the primers targeting Fusarium and Aspergillus species to amply soil DNA obtained from diverse agroecosystems. Amplicons of the expected size have been sequenced. Currently, we are performing phylogenic analysis of DNA sequences. Accomplishments under the objective three: It is our working hypothesis that fungi may contribute to a great portion of soil nitrous oxide emission across diverse agroecosystems that differ in vegetation types and management practices. By using antibiotic selective inhibition, we have demonstrated that regardless of types of ecosystems, fungal contribution accounts for a large portion of total soil nitrous oxide production that should not be ignored. Our results have also shown that relative contribution of fungi versus bacteria to soil nitrous oxide emissions is dependent on soil pH as well as soil water content. Acidic soil pH favors nitrous oxide-producing fungi more than nitrous oxide-producing bacteria. In comparison to the bacterial counterpart, fungal contribution to soil nitrous oxide emissions is more favorable under aerobic than anaerobic conditions. The information gained from this project may help to understand controlling factors for soil nitrous oxide production and thus improve soil nitrous oxide modeling.

Publications

• Type: Journal Articles Status: Published Year Published: 2013 Citation: Mothapo NV, Chen H, Cubeta MA, Shi W, 2013. Nitrous oxide producing activity of diverse fungi from distinct agroecosystems. Soil Biology & Biochemistry 66, 94-101.
• Type: Journal Articles Status: Published Year Published: 2013 Citation: Chen H, Li X, Hu F, Shi W, 2013. Soil nitrous oxide emissions following crop residue addition: a meta-analysis. Global Change Biology 19, 2956-2964.
• Type: Journal Articles Status: Published Year Published: 2014 Citation: Chen H, Mothapo NV, Shi W, 2014. The significant contribution of fungi to soil N2O production across diverse ecosystems. Applied Soil Ecology 73, 70-77
• Type: Other Status: Other Year Published: 2013 Citation: Chen H, Mothapo NV, Shi W, 2013. Fungal contribution to soil nitrous oxide production across diverse ecosystems. Annual Meeting Abstracts. ASA/CSSA/SSSA Annual Meetings, Tampa, Florida.
• Type: Other Status: Other Year Published: 2013 Citation: Mothapo NV, Chen H, Cubeta MA, Shi W, 2013. Nitrous oxide-producing activity of diverse soil fungi under imposed abiotic conditions. 34th Annual Meeting, Mid-Atlantic States Mycology Conference, Beltsville, MD.
• Type: Other Status: Other Year Published: 2013 Citation: Shi W, 2013. Biodiversity and eco-physiology of nitrous oxide-producing fungi in distinct agroecosystems. USDA-NIFA Director meetings, Washington DC.
• Type: Other Status: Other Year Published: 2014 Citation: Chen H, Mothapo NV, Shi W, 2014. Relative importance and controlling factors of fungal nitrous oxide production in agro-ecosystems. Soil Science Society of North Carolina, Raleigh, NC
• Type: Other Status: Other Year Published: 2013 Citation: Chen H, 2013. Fungal denitrification: an overlooked source of soil N2O production. Soil Science Seminar Series, North Carolina State Univ.

Progress 03/01/12 to 02/27/13

Outputs
OUTPUTS: Experiments were conducted to develop molecular markers for PCR-amplification of fungal P450nor. Sequences of DNA for P450nor in Fusarium oxysporum, Cylindrocarpon tonkinense, Apsergillus oryzae, Neurospora crassa, and Trichosporon cutaneum were obtained from NCBI and used to design PCR primers. Specific, multiplex and degenerate primers for PCR-amplification of genes encoding P450nor in nitrous oxide-producing fungi were designed using Primer 3, MPprimer and iCODEHOP. Priming tests were then performed using pure genomic DNA (extracted from liquid culture using FastDNA Spin Kit according to manufacturer's protocol) of the 68 fungal strains shown to be capable of nitrous oxide production as well as metagenomic DNA extracted from soil using FastDNA Spit Kit for Soil. For all PCR reactions, negative controls included water and fungus isolates shown not to be capable of nitrous oxide production. PCR products were analyzed by 1% agarose gel stained with gelstar and visualized under UV. Experiments were also conducted to examine the effects of soil pH on fungal nitrous oxide production. Soil nitrous oxide flux was determined in a microcosm experiment with the aid of antibiotics. Soils (about 20 g) treated with streptomycin and cycloheximide for inhibiting bacterial and fungal activity, respectively, were placed into 125-ml jars fitted with rubber septa. Following overnight pre-incubation at a refrigerator, the microcosms were incubated at room temperature and 3-ml gas samples were withdrawn periodically using gas tight syringes. Soil without the addition of any antibiotic was used as the control. The concentration of nitrous oxide was analyzed using a gas chromatography. The relative contribution of fungi or bacteria to soil nitrous oxide production was estimated by the ratio of nitrous oxide flux from antibiotic-treated soil to that from the control. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
A total of three and two specific and multiplex primers (amplicon size 149-348 bp) designed for F. oxysporum were able to produce bands of expected size in five F. oxysporum and four G. fujikuroi species complex. However, these primers were not able to amplify one G. fujikuroi species, which was identified as G. thapsina with ITS, suggesting that the genes for P450nor in this species are divergent to those of F. oxysporum. Similarly, specific and multiplex primers designed for A. oryzae (amplicon size 225-401 bp) produced positive results for A. oryzae and A. terreus, but no amplification in other Aspergillus species with nitrous oxide activity. This was very interesting because Aspergillus phylogeny constructed with ITS shows A. terreus and A. oryzae to be distantly related. Thus, these primers could be optimized to be able to produce positive results in other Aspergillus species. Of the total 11 degenerate primers that were deemed probable for successful amplification of fungal P450nor, two (primer pairs C8/C26 and C8/D6 with amplicon sizes 227 and 319, respectively) produced bands of expected sizes for Aspergillus oryzae, F. oxysporum, G. fujikuroi species complex and Trichoderma piluliferum. The sequences of these primers, particularly the degeneracy of the 3' ends, are currently being manipulated in order to broaden the genera of nitrous oxide-producing fungi that can be detected with these primers. No positive results have been obtained with metagenomic DNA. The plausible cause for this negative outcome is that the copy numbers of P40nor genes are probably very low in soil. Overall, PCR with pure genomic DNA confirms that specific primers work effectively for the species they were designed for and close relatives. Thus, upon purification of soil DNA, F. oxysporum, A. oryzae primers and iCODEHOP primers C8/C26 and C8/D6 are expected to produce positive results. Simultaneously, PCR conditions are being optimized for other iCODEHOP primers.

Publications

• No publications reported this period

Progress 03/01/11 to 02/28/12

Outputs
OUTPUTS: Recent studies suggest that fungi can be important to denitrification and thus nitrous oxide production. However, little information is available on the distribution and abundance of nitrous oxide-producing fungi in agricultural soils. This project aims to use metagenomic and reference strain sequencing approaches to discover and characterize denitrifying fungi. We have evaluated the relative importance of fungi versus bacteria to soil nitrous oxide production and thereafter have isolated nitrous oxide-producing fungi from agricultural soils. Agricultural soils were collected from five farming systems (conventional farming, organic farming, integrated crop and livestock system, plantation forestry, and old agricultural field succession) in the Center for Environmental Farming Systems, located on the Upper Coastal Plain near Goldsboro, NC. Soil nitrous oxide flux was determined in a microcosm experiment with the aid of antibiotics. Approximately 20 g of soil treated with either streptomycin or cycloheximide for inhibiting bacterial or fungal activity, respectively, was placed into a 125-ml amber jar fitted with a rubber septum. Following overnight pre-incubation at 4 C, the microcosms were incubated at room temperature, and 3-ml gas samples were withdrawn periodically (i.e., 4 h, 12 h, 20 h, and 32 h) using gas tight syringes. Soil without the addition of any antibiotic was used as the control. The concentration of nitrous oxide was analyzed using a gas chromatography equipped with an ECD. The relative contribution of fungi or bacteria to soil nitrous oxide production was estimated by the ratio of nitrous oxide flux from antibiotic-treated soil to that from the control. Nitrous oxide-producing fungi were isolated from agricultural soils using Czapek media. First, soil solution made by a phosphate buffer was diluted and then an aliquot of dilution was inoculated to a Czapek medium containing an antibacterial agent, streptomycin by a plating method. The incubation was performed at aerobic conditions for over 5 days at 30 C. Then, different fungal colonies were randomly selected, purified, and preserved in slants at 4 C for later use. Second, these fungus isolates were detected for denitrifying activity using a Czapek liquid medium with 100 mg/L streptomycin. Each fungus isolate was inoculated into 25 ml of the liquid medium contained in a 50 ml Erlenmeyer flask sealed with a rubber stopper. The incubation was performed at 30 C for about 5 days. Periodically, 3 ml of the headspace gas was taken from each of the flasks for nitrous oxide analysis using a gas chromatography equipped with an ECD. Flasks without fungus inoculation were used as the controls for examining the emission of nitrous oxide from chemical reactions. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
The addition of antifungal antibiotic, cycloheximide into soil significantly reduced soil nitrous oxide production. This impact appeared to be more pronounced in the integrated crop and livestock system and plantation forestry than in conventional farming, organic farming, and old agricultural field succession. Total 151 fungal isolates were collected from the five farming systems and then tested for their abilities of nitrous oxide production. While nitrous oxide-producing fungal isolates were collected from each of the five farming systems, they differed significantly in the production rate of nitrous oxide. Our results highlight the significant role of fungi in producing nitrous oxide and add new knowledge for understanding and therefore managing nitrous oxide emission from agricultural soils.

Publications

• No publications reported this period