2014 Volume 39 Issue 3 Pages 115-120
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.
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 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 secondary metabolite of corn) on N2O 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-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 soilSamples 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 N2OFor 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 mediumThe 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 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)
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. 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.
2. Inhibitory effects of methyl viologen dichloride (Paraquat®) and other chemical compounds on bacterial N2O emissionAs 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 cell-growth 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 any statistically significant inhibitory effects. HOBt performed as a negative control (Fig. 2). Therefore, we examined more accurate dose responses of MBOA, HOBt, and BOA toward N2O-emitting bacteria.
MBOA showed a statistically significant repression of N2O emission at 10 and 50 µM (Fig. 4). In addition, HOBt at 150–500 µM reduced N2O production to almost null, but a culture medium containing 150 µM or higher concentration of HOBt inhibited the cell growth of Pseudomonas sp. 10CMF5-1B (Fig. 5). Repression of N2O production by exceptionally high chemical doses with the inhibition of bacterial cell growth thus resulted in disturbance of soil ecosystems. BOA also showed toxicity similar to that of HOBt (data not shown).
Amitrole (3-amino-1H-1,2,4-triazole) at 2 µM showed significant acceleration of N2O production. This acceleration effect, 2.5-fold higher than that of the control, was observed only in the incomplete denitrifier Pseudomonas sp. 10CFM5-1B (Fig. 6). Importantly, the incomplete denitrifying N2O emitter showed a clear response to amitrole at a low concentration (2 µM), while the accelerating activity of amitrole in N2O production was apparent at 2 µM. At 10 µM, however, amitrole suppressed N2O emission of the incomplete denitrifier.
The aim of this study was to demonstrate the inhibitory effects of chemical pesticides toward soil denitrifiers of root-associating saprophytic pseudomonads.31,32) Acetylene is the most potent chemical that selectively inhibits nitrous oxide reductase (N2OR),29,33) while other chemicals including azide anion, thiocyanate, carbon monoxide, and cyanide, are also known as significant N2OR inhibitors.34) However, those selective inhibitors are highly toxic to mammals and difficult to handle due to their instability or gaseous states. Denitrification inhibitors hydroxamine (against nitrate reductase),35) methanol, and allylthiourea (both in the anammox process),36) have also been reported but are not acceptable for agricultural farmland applications.
In a preliminary study, we had hypothesized that methyl viologen dichloride (Paraquat®) would accelerate N2O production, as it is a known competitive substrate of N2OR.34) However, this herbicide completely repressed N2O production of pseudomonads at 5 µM (Fig. 3). It is likely that methyl viologen dichloride nonselectively inhibits redox enzymes associated with the denitrification process.37) Even at 1 µM, equivalent to approximately 200 µg/L, this herbicide resulted in a 50% inhibition of N2O production. The allowable dose limit of residual Paraquat® in many crops is 0.5 ppm (2 µM), which is probably a level similar to the adsorbed Paraquat® in the soil. Although Paraquat® is not currently approved as a herbicide in Japan, this chemical continues to be used widely in developing countries and likely contributes to N2O repression.
Conversely, 6-methoxy-2-benzoxazolone (MBOA), an antifungal corn compound, also repressed pseudomonad denitrification at 10 µM; however, its inhibitory effect on N2O emission was not significant at 5 µM (Fig. 4). In contrast, the structurally similar benzoxazoline derivatives 2-benzoxazolinone (BOA) and 1-hydroxy-1H-benzotriazole (HOBt) were both inactive against N2O emission by the denitrifying pseudomonads. At concentrations greater than 150 µM, both chemicals exhibited a remarkable repression of N2O production, but the effects of BOA and HOBt on the repression of N2O emission were mainly due to the repression of bacterial cell growth (Fig. 5). Other herbicides, such as simazine, trifluralin, and amitrole, accelerated N2O emission by Pseudomonas sp. 10CFM5-1B at 2 or 10 µM. Particularly, 2 µM of amitrole led to a 2.5-fold increase in N2O emission, suggesting that farm soil sprayed with amitrole became an active N2O efflux source (Fig. 6). The possibility of the selective inhibition of N2OR by amitrole, in contrast to other reductoxidases in the denitrification process, should be further investigated.
N2O emission from farm soil is correlated with the soil type, nitrogen application, crop rotation, and climatic and seasonal soil conditions.38–41) In corn farmland, nitrogen fertilization of Andisol resulted in a high accumulation of NO3− by nitrifying bacteria and Archaea, and growing corn actively provides organic carbon to the rhizosphere soil. Subsequently, denitrifying saprophytic microbial communities in the soil effectively convert NO3− to N2O gas. In the soil of Shizunai Experimental Livestock Farm, where manure and chemical fertilizers have been applied together since 2010, the most active annual N2O efflux was observed,42) similar to those reported at another volcanic Andisol farm in Nasu, Japan.43)
The culturing assay to evaluate the N2O emitting potential of soil microbial communities reliably reflected the activities of the bulk soils as N2O efflux sources as shown in our previous report.44) Hence, certain fungicides and bacteriocides can greatly impact soil microbial communities and their functions.45,46) In this study, we demonstrated via a culturing assay that the herbicide methyl viologen dichloride has the potential to repress N2O emission from farm soils by its unexpected activity to inhibit N2O production in fertilized farmland soils. Conversely, other herbicides, including amitrole and other triazole-type chemicals, may activate N2O efflux from farm soils. Further field investigation in fertilized farmlands should be conducted to estimate the practical effects of herbicide spraying on N2O emission from the soils. Thus, a global balance of pesticide chemicals that minimize or maximize N2O emission should be evaluated in detail for eco-friendly soil management. Beyond the environmental safety issues, next-generation herbicides and other agricultural chemicals must be more environmentally remediable via the regulation of denitrification in soil microbial communities.
This research was supported by a Grant-in-Aid for Scientific Research A (20248033 to YH) from the Japan Society for the Promotion of Science (JSPS) and a scholarship to LL from The Chinese Scholarship Council (CSC 2011491196).
