Chemicals used in this study. 

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... oxide (N 2 O) contributes to 7% of global warming 1) and depletes the ozone layer in the atmosphere. 2) On a global scale, anthropogenic nitrous oxide (N 2 O) emission is due to agricul- ture, industry, biomass burning, and indirect emission from reactive nitrogen leaching, runoff, and atmospheric deposition. Particularly, agricultural soil is a dominant source of N 2 O with widespread usage of nitrogen fertilizers and manure, 3) and it is estimated that 57% of the annual N 2 O emitted is produced by agricultural farm soil and livestock manure. 4,5) In the denitrifica- tion process, denitrifying bacteria are ubiquitous and have been studied extensively in agricultural farmland soils, wastewater treatment systems, and natural environments, including marine and boreal ecosystems. 6,7) Biological denitrification is the most important process in global N circulation and N 2 O production, and it has been shown that almost 90% of N 2 O emitted from soil results from denitrification rather than nitrification. 8) In present-day soil, increasing use of pesticides and chemical fertilizers has become a cause for concern due to their effect on the composition and function of soil microoganisms. 9,10) Stud- ies on the positive and/or negative effects of pesticides on N 2 O emission by denitrifying soil bacteria have been performed to determine the effects of pesticides on soil microbial biomass and soil respiration. 1,11,12) Other studies have also shown that appli- cation of certain pesticides influences microbial and enzymatic reactions, including mineralization of organic matter, nitrifica- tion, denitrification, and ammonification. 13–15) The potential ag- rochemical impact on N 2 O production by soil fumigation with chloropicrin and methyl isothiocyanate was separately exam- ined, and it showed that both stimulated N 2 O production. 16,17) It has also been reported that methyl parathion increases the emission of N 2 O 13) and also reduces the diversity of the nirK gene, thus affecting N 2 O production. 15) Conversely, the herbi- cides prosulfuron, glyphosate, and propanil and the fungicides mancozeb and chlorothalonil suppress N 2 O emission by soil due to the inhibition of nitrification and/or denitrification. 14,18) In addition, the species of cultivated crops may affect the N 2 O emission of bacterial communities in farmland soil. Drury et al. have reported that farming soil used for corn monoculture emit- ted 3.1–5.1-fold higher N 2 O than that used for monoculturing winter wheat or soybeans, 19) suggesting activation of N 2 O emis- sion in the corn rhizosphere. In the present study, we aimed to determine the effects of chemicals (fungicides, herbicides, and a representative second- ary metabolite of corn) on N 2 O emitters from Andisol corn farmland. Six compounds were used in this study (Fig. 1). We obtained 6-methoxy-2-benzoxazolinone (MBOA), 2-benzoxazolinone (BOA), and 1-hydroxy-1 H -benzotriazole (HOBt) from Wako (Osaka, Japan). N -heterocyclic herbicides methyl viologen di- chloride (Paraquat ® ; reagent grade), simazine (reagent grade), and amitrole (reagent grade) were also purchased from Wako. MBOA is an allelochemical of corn, which shows antifungal and herbicidal activities. 20,21) Both BOA and HOBt commercially available as chemical reagents were expected to be negative con- trols without any significant repression or acceleration. BOA is often used as an oxidative-stress inducer, 22) and HOBt is a redox inhibitor and a coupling reagent for amide synthesis. 23) Simazine is known as a photosystem II-inhibitor, 24) while amitrole is a his- tidine synthesis inhibitor. 25) In a preliminary test, 10 μ M of each test compound was ex- posed to N 2 O-emitting bacteria isolated from Andisol corn farmland (see the following subsection). Test compounds that showed an active repression of N 2 O emission at 10 μ M were fur- ther investigated at lower concentrations ranging from 2.5 to 10 μ M. Chemicals that showed accelerating activity toward N 2 O production of the denitrifier were tested using a culturing assay at concentrations of 2 and 10 μ M. Samples of post-harvest soil (approximately 10 g) were collect- ed from Hokkaido University Shizunai Experimental Livestock Farm in Hokkaido, Japan (42 ° 26 ′ N, 142 ° 28 ′ E) in early Novem- ber 2011. The isolation of Pseudomonas sp. 10CMF5-1B (ac- cession no. AB856847, 100% agreeable with Pseudomonas sp. PAMC 26831 isolated from subarctic Alaskan grassland soil by 16S rRNA gene sequence) 26) and Pseudomonas sp. 10CMF5-2D (close to 10CMF5-1B with accession no. AB856848) from rhi- zosphere soil of corn farms and their identification and char- acteristics as culturable N 2 O emitters are described in another paper. 27) Both were used to examine responses to test chemicals. The incomplete denitrifiers Pseudomonas sp. 10CFM5-1B and Pseudomonas sp. 10CFM5-2D were used to show different re- sponses in the bioassay. Chemical compounds were tested for activity toward the N 2 O-emitting bacteria to inhibit and/or ac- celerate N O productivity. For the N 2 O production assay, Winogradsky’s mineral solution containing 0.05% sucrose (0.5 g/L) and KNO 3 (500 mg/L-N, as 3.6 g/L KNO 3 ) as the carbon and nitrogen sources, respec- tively, was prepared; 0.3% gellan gum was added as the gell- ing agent before preheating. Ten milliliters of the medium was poured into a 30-mL gas chromatography vial (Nichiden-Rika Glass Co., Kobe, Japan) and autoclaved at 121 ° C for 15 min. After the liquefied medium was cooled and gelled again, a loop of Pseudomonas sp. 10CFM5-1B or 10CFM5-2D was inoculated into the medium and allowed to incubate at 20 ° C in the dark for 7 days. In the cultured medium, NO 3 − is utilized as an electron acceptor for nitrate respiration, leading to N 2 O production. 28) After the incubation, N 2 O in the headspace gas was analyzed quantitatively by using an ECD (electron capture detector)- gas chromatography (Shimadzu GC-14B, Kyoto, Japan) col- umn equipped with an electron capture detector (Shimadzu ECD-2014). The column (1-m Porapak N column; Waters, Mil- ford, MA, USA) was kept at 60 ° C by using a carrier gas of Ar with 5% CH 4 . A portion of headspace gas (from 50 μ L to 1.0 mL) in the vials (22.5 mL) was analyzed by gas chromatography. The initial herbicide used in the present study was methyl vi- ologen dichloride (Wako Pure Chemical Industries, Ltd., Osaka, Japan), an electron transport inhibitor known commercially as Paraquat ® . Methyl viologen dichloride was dissolved in ster- ilized water to 1.0 M, and then further diluted with sterilized water to a concentration of 10 mM (100-fold dilution). Ten mi- croliters of each diluted solution was added aseptically to the bioassay medium, which was supplemented with 0.05% su- crose, autoclaved at 121 ° C for 15 min, and then cooled to room temperature. The final concentration of the test medium was 0.1–5 μ M. At the same time, chemical-free medium was pre- pared as the control. Cell suspensions of two N 2 O-emittable bac- teria (100 μ L; 10 6 CFU/mL) were inoculated to the test and con- trol mediums, vortexed, and then incubated at 20 ° C in the dark for 7 days. N 2 O produced in headspace of the cultured vial was shown by μ g N 2 O per vial pea day ( μ g/vial/day). Each assay was performed in triplicate. For other test compounds, a 1-M solution of the compound in dimethylsulfoxide was diluted with sterile water to the desired concentration. We then added 100 μ L of the diluted solution to 10 mL of the medium under aseptic conditions to prepare a 100-fold diluted medium. Subsequent procedures were the same as those performed for methyl viologen dichloride, including incubation and gas analysis. Each treatment was performed in triplicate. Acetylene inhibition with 10% acetylene ...