J. Pestic. Sci. 39(3), 115–120 (2014) DOI: 10.1584/jpestics.D14-003
Original Article
Effects of methyl viologen dichloride and other chemicals on nitrous oxide (N2O) emission and repression by pseudomonad denitrifiers isolated from corn farmland soil in Hokkaido, Japan Li Li,1 Mengcen Wang,1,2 Ryusuke Hatano1 and Yasuyuki Hashidoko1,* 1 Research Faculty of Agriculture, Hokkaido University, Sapporo 060–0808, Japan Present address: Institute of Pesticide and Environmental Toxicology, Zhejiang University, China
2
(Received January 13, 2014; Accepted May 11, 2014) The effects of some commercial herbicides and N-heterocyclic compounds structurally related to corn root constituents on N2O-emitting soil bacteria were examined. In the N2O emission assay, two N2O-emitting eubacteria, the incomplete denitrifier Pseudomonas sp. 10CFM5-1B and Pseudomonas sp. 10CFM5-2D (both isolated from Andisol corn farmland in Hokkaido), were used. We found that methyl viologen dichloride (Paraquat®) at 2 µM significantly repressed N2O emission by the active denitrifying bacteria. A corn antifungal secondary metabolite, 6-methoxy-2-benzoxazolone (MBOA), also repressed pseudomonad denitrifiers at a concentration of 10 µM. Other herbicides such as simazine (6-chloro-N,N′-diethyl-1,3,5-triazine-2,4-diamine) and amitrole (3-amino-1,2,4-triazole) accelerated N2O emission by Pseudomonas sp. 10CFM5-1B at 2 or 10 µM. It was thus shown that methyl viologen dichloride may have somewhat contributed to the repression of global warming by suppressing N2O production in farmland soils. Some herbicides, including amitrole and other triazole-type chemicals, may instead have the potential to activate soil N2O emission. © Pesticide Science Society of Japan Keywords: nitrous oxide emission, N-heterocyclic compound, repression of N2O emission, 1-hydroxy-1H-benzotriazole, methyl viologen dichloride, amitrole (3-amino-1,2,4-triazole).
Introduction Nitrous oxide (N2O) contributes to 7% of global warming1) and depletes the ozone layer in the atmosphere.2) On a global scale, anthropogenic nitrous oxide (N2O) emission is due to agriculture, industry, biomass burning, and indirect emission from reactive nitrogen leaching, runoff, and atmospheric deposition. Particularly, agricultural soil is a dominant source of N2O with widespread usage of nitrogen fertilizers and manure,3) and it is estimated that 57% of the annual N2O emitted is produced by agricultural farm soil and livestock manure.4,5) In the denitrification 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 N2O production, and it has been shown that almost 90% of N2O 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 * To whom correspondence should be addressed. E-mail:
[email protected] Published online July 17, 2014 © Pesticide Science Society of Japan
the composition and function of soil microoganisms.9,10) Studies on the positive and/or negative effects of pesticides on N2O 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 application of certain pesticides influences microbial and enzymatic reactions, including mineralization of organic matter, nitrification, denitrification, and ammonification.13–15) The potential agrochemical impact on N2O production by soil fumigation with chloropicrin and methyl isothiocyanate was separately examined, and it showed that both stimulated N2O production.16,17) It has also been reported that methyl parathion increases the emission of N2O13) and also reduces the diversity of the nirK gene, thus affecting N2O production.15) Conversely, the herbicides prosulfuron, glyphosate, and propanil and the fungicides mancozeb and chlorothalonil suppress N2O emission by soil due to the inhibition of nitrification and/or denitrification.14,18) In addition, the species of cultivated crops may affect the N2O emission of bacterial communities in farmland soil. Drury et al. have reported that farming soil used for corn monoculture emitted 3.1–5.1-fold higher N2O than that used for monoculturing winter wheat or soybeans,19) suggesting activation of N2O emission in the corn rhizosphere. In the present study, we aimed to determine the effects of chemicals (fungicides, herbicides, and a representative second-
116 L. Li et al.
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ary metabolite of corn) on N2O emitters from Andisol corn farmland.
Materials and Methods 1. Chemicals Six compounds were used in this study (Fig. 1). We obtained 6-methoxy-2-benzoxazolinone (MBOA), 2-benzoxazolinone (BOA), and 1-hydroxy-1H-benzotriazole (HOBt) from Wako (Osaka, Japan). N-heterocyclic herbicides methyl viologen dichloride (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 controls 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 histidine synthesis inhibitor.25) In a preliminary test, 10 µM of each test compound was exposed to N2O-emitting bacteria isolated from Andisol corn farmland (see the following subsection). Test compounds that showed an active repression of N2O emission at 10 µM were further investigated at lower concentrations ranging from 2.5 to 10 µM. Chemicals that showed accelerating activity toward N2O production of the denitrifier were tested using a culturing assay at concentrations of 2 and 10 µM. 2. Soil samples and denitrifiers isolated from soil Samples of post-harvest soil (approximately 10 g) were collected from Hokkaido University Shizunai Experimental Livestock Farm in Hokkaido, Japan (42°26′N, 142°28′E) in early November 2011. The isolation of Pseudomonas sp. 10CMF5-1B (accession 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 rhizosphere soil of corn farms and their identification and characteristics as culturable N2O 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 responses in the bioassay. Chemical compounds were tested for activity toward the N2O-emitting bacteria to inhibit and/or accelerate N2O productivity. 3. Culture of N2O-emittable bacteria and measurement of N2O For the N2O production assay, Winogradsky’s mineral solution containing 0.05% sucrose (0.5 g/L) and KNO3 (500 mg/L-N, as 3.6 g/L KNO3) as the carbon and nitrogen sources, respectively, was prepared; 0.3% gellan gum was added as the gelling 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, NO3− is utilized as an electron acceptor for nitrate respiration, leading to N2O production.28) After the incubation, N2O in the headspace gas was analyzed quantitatively by using an ECD (electron capture detector)gas chromatography (Shimadzu GC-14B, Kyoto, Japan) column equipped with an electron capture detector (Shimadzu ECD-2014). The column (1-m Porapak N column; Waters, Milford, MA, USA) was kept at 60°C by using a carrier gas of Ar with 5% CH4. A portion of headspace gas (from 50 µL to 1.0 mL) in the vials (22.5 mL) was analyzed by gas chromatography. 4. Preparation of the test medium The initial herbicide used in the present study was methyl viologen dichloride (Wako Pure Chemical Industries, Ltd., Osaka, Japan), an electron transport inhibitor known commercially as
Fig. 1. Chemicals used in this study.
Vol. 39, No. 3, 115–120 (2014)
Effect of chemicals on N2O emission by pseudomonad denitrifiers 117
Paraquat®. Methyl viologen dichloride was dissolved in sterilized water to 1.0 M, and then further diluted with sterilized water to a concentration of 10 mM (100-fold dilution). Ten microliters of each diluted solution was added aseptically to the bioassay medium, which was supplemented with 0.05% sucrose, autoclaved at 121°C for 15 min, and then cooled to room temperature. The final concentration of the test medium was 0.1–5 µM. At the same time, chemical-free medium was prepared as the control. Cell suspensions of two N2O-emittable bacteria (100 µL; 106 CFU/mL) were inoculated to the test and control mediums, vortexed, and then incubated at 20°C in the dark for 7 days. N2O produced in headspace of the cultured vial was shown by µg N2O per vial pea day (µg/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 µL 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 gas was used to assay N2O-reducing activity by the pseudomonad N2O emitters.29)
10CFM5-1B in 0.5% sucrose-supplemented medium during a 7-day incubation was more than 4-fold higher than that of the 0.05% sucrose-containing medium, at 40 µg N2O per vial/day in the headspace; 10CFM5-2D showed a similar tendency.27) Thus, the responses of the two Pseudomonas spp. were agreeable with those of typical denitrifying saprophytic bacteria. The addition of 10% C2H2 did not result in significant acceleration of N2O production by Pseudomonas spp. 10CFM5-1B and 10CFM5-2D, and thus the strains were characterized as incomplete denitrifiers.
Results 1. N2O emission assay results for pseudomonad denitrifiers isolated from Shizunai Andisol farmland Pseudomonas spp. 10CFM5-1B and 10CFM5-2D were isolated as candidates for active N2O emitters from post-harvest Andisol corn farmland soil at Shizunai Experimental Livestock Farm.7,27) N2O production was observed in the culture medium supplemented with 5 mM KNO3 but not with 5 mM of (NH4)2SO4, and N2O emission increased approximately 20-fold (approximately 10 µg N2O/vial/day) with the addition of 0.05% sucrose to the KNO3-containing medium. N2O emission of Pseudomonas sp.
2. Inhibitory effects of methyl viologen dichloride (Paraquat®) and other chemical compounds on bacterial N2O emission As it has been reported that methyl viologen radicals are powerful inhibitors against CH4 production by methanogens, such as Methanobacillus omelianskii,30) we first expected that methyl viologen dichloride would act as an inhibitor of nitrous oxide reductase to accelerate N2O production; therefore, we attempted to employ this herbicide for a positive control. Contrary to our speculation, methyl viologen dichloride showed high inhibitory activity against N2O emission by the tested denitrifier, with almost no emission at 10 µM (Fig. 2). Therefore, a more accurate test for the inhibition of N2O emission was performed for methyl viologen dichloride and two benzo-N-heterocyclic compounds in triplicate. Methyl viologen dichloride at 2.5 µM repressed N2O emission of Pseudomonas sp. 10CMF5-1B to less than 1/10 of that of the control (Fig. 3). At this concentration or even at 10 µM, methyl viologen dichloride showed no cellgrowth inhibition against the denitrifiers used for the N2O emission assay. The impact of other herbicides and chemicals related to corn antifungal metabolites on the denitrification process with pseudomonad denitrifiers was further investigated. MBOA, an allelochemical of corn, slightly repressed N2O emission of Pseudomonas sp. 10CMF5-1B at 10 and 100 µM without any inhibition of the bacterial cell growth, while BOA and HOBt did not show
Fig. 2. N2O production by N2O-emittable Pseudomonas spp. upon exposure to the chemical compounds methyl viologen dichloride, MBOA, BOA, and HOBt. The N2O emitters Pseudomonas spp. 10CFM5-1B and 10CFM5-2D were used in the presence of 10 µM of methyl viologen dichloride, MBOA, BOA, and HOBt (A), or 100 µM of BOA and HOBt (B). The culture medium was supplemented with 0.5 g/L N of NO3− form (36 mg KNO3 in 10 mL of medium) and 0.05% sucrose (5 mg in 10 mL). Culture condition: 25°C in the dark for 7 days. The values are means±SD (shown by error bars) (n=3). Methyl viologen dichloride showed statistically significant suppression of N2O production at 10 µM (*p
