The pot experiment was conducted in a greenhouse at agricultural farm of Gyeongsang National University, Jinju, South Korea. Soil was collected from rice field (0–15 cm depth) in the spring of 2013. The soil sample was air dried, sieved (<10 mm) and packed into Wagnor pot (25 cm in diameter and 30 cm in height, 13 kg dried soil pot^-1). The soil collected for this experiment was fine silty, mixed, mesic Typic Endoaquept [11]. The soil had following characteristics: organic matter, 10.88±1.61 g kg^-1; total N, 0.74±0.42 g kg^-1; soil pH, 6.68±0.26 (soil: H₂O = 1:5, w/v), available phosphate, 45.06±0.49 mg kg^-1 and exchangeable cations Ca²⁺, Mg²⁺ and K⁺, 3.58±0.33, 0.60±0.04 and 0.35±0.03 cmol⁺ kg^-1, respectively. Pots were then flooded with water and allowed to stand for stabilization (filling up of capillary pores with water). After 1 week of flooding, chemical fertilizer and 2-bromoethanesulfonate (BES) were applied and 25 days old 3 seedlings of Korean rice cultivar ‘Dongjinbyeo’ (Oryza sativa, Japonica type) was transplanted (June 20, 2013) in each pot.

The chemical fertilizers were applied at the rates of 90 kg N ha^-1, 45 kg P₂O₅ ha^-1, and 58 kg K₂O ha^-1 as per the Korean recommended fertilization levels for rice cultivation [12], using urea, fused superphosphate and potassium chloride. The basal chemical fertilizer applied before transplanting were: 45 kg N ha^-1, 45 kg P₂O₅ ha^-1 and 40.6 kg K₂O ha^-1. Tillering fertilizer (18 kg N ha^-1) was broadcasted approximately 2 weeks after rice transplanting and panicle fertilizer (27 kg N ha^-1, 17.4 kg K₂O ha^-1) was broadcasted 6 weeks after rice transplanting. BES was applied at different levels as 0 (control), 20, 40 and 80 mg kg^-1 of soil. The BES concentration selected in this experiementis on the basis of incubation test results, where 80 mg kg^-1 BES (soil weight basis) showed ca. 50% inhibition of CH₄ production in rice soil (data not shown). The ‘bases’ of cylindrical chambers were permanently fixed in each pot and then the pots were arranged in the greenhouse following completely randomized design. Each treatment had three replicates. The water level was maintained at 5–6 cm above the soil surface during cropping season and then drained 2 weeks before rice harvesting. The harvesting of rice was carried out after 120 days after transplanting (hereafter, DAT).

A closed-chamber method was used to measure CH₄ emissions from rice planted pots during rice cultivation [13]. The gas collection chambers having a diameter of 24 cm and height of 100 cm with a circulating fan for gas mixing and thermometers to monitor inside temperature were placed on bases of rice planted pots during gas sampling. The air gas samples were collected from chambers using 50 ml air-tight syringes at 0, 15 and 30 min intervals after chamber placement and transferred into pre-evacuated 20 ml glass vials fitted with butyl rubber stoppers for analysis in the laboratory. Gas sampling was carried out once a week and three times (0800, 1200 and 1600 h) in day to get the average CH₄ emission flux during cropping season. Gas sampling and air temperature measurements were simultaneously carried out.

CH₄ concentrations in the collected air samples were measured by gas chromatography (Shimadzu, GC-2010, Japan) packed with a Porapak NQ column (Q 80–100 mesh) and a flame ionization detector (FID). The temperatures of column, injector and detector were adjusted at 70°C, 150°C and 200°C, respectively. Helium and hydrogen were used as carrier and burning gases, respectively. Average fluxes and standard deviations were calculated from triplicate pots.

Methane emission from soil was calculated as the increase in CH₄ concentrations per unit surface area of the chamber for a specific time interval. A closed-chamber equation was used to estimate CH₄ fluxes from each treatment [13]. F=p×VA×ΔcΔt×273T Where, F was the CH₄ flux (mg CH₄ m^-2 hr^-1), ρ was the gas density (0.714 mg cm^-3), V was the volume of the chamber (m³), A was the surface area of the chamber (m²), Δc/Δt was the rate of CH₄ gas accumulation in the chamber (mg m^-3 hr^-1), and T (absolute temperature) was calculated as 273 + mean temperature in (°C) of the chamber.

Total CH₄ flux for the entire cultivation period was calculated using following equation [14]. TotalCH4flux(gm−2d−1)=∑in(Ri×Di) Where, R_i was the CH₄ emission flux (g m^-2 d^-1) in the i^th sampling interval, D_i was the number of days in the i^th sampling interval, and n was the number of sampling intervals.

To determine Co-M concentration, the fresh soil collected on 30 DAT (active tillering), 60 DAT (Booting), 80 DAT (Heading) and 120 DAT (Harvesting) was homogenized with lysis buffer (100 mM Tris-HCl solution (pH 8.0), 100 mM EDTA solution (pH 8.0) and 1.5 M NaCl solution) (Soil: buffer = 1: 2, w/v basis) and sonicated for 2 min (1 min sonication followed by 10 sec vortex and then 1 min sonication again). The soil suspension was centrifuged at 4000 rpm for 10 min. The required amount of absolute ethanol was added to the 2 ml supernatant to make it 80% ethanol solution. The solution mixture was allowed to stand for 2 h at 4°C and centrifuged again at 4000 rpm for 10 min. The precipitate was dissolved in deionized water and diluted to a suitable volume for high performance liquid chromatography (HPLC) analysis. 10 μl of the serially diluted standard solutions were injected into the column (Agilent Eclipse XDB—C₁₈, 4.6 x 250 mm) of HPLC (Agilent DE/1200, 5 μm) and the data were analyzed at 270 nm wavelength using UV detector. The mixture of acetonitrile and 50 mM trichloroacetic acid solution (30:70, v/v) was used as mobile phase for Co-M quantification.

The soil samples collected at 30, 60, 80 and 120 DAT during rice cultivation were immediately lyophilized by Pilot Lyophilizer (PVTFD50A, Ilsin, Korea) and then sieved through 2-mm size. The DNA was extracted from the lyophilized soil samples by using FastDNA SPIN Kit for Soil (MP Biomedical, CA, USA) following the manufacturer’s instructions. The extracted DNA was used as a template for PCR to amplify mcrA gene (alpha subunit of methyl coenzyme M reductase) using suitable primers [15], mlas_forward (5’-GGTGGTGTMGGDTTCACMCARTA-3’) and mcrA_reverse (5-CGTTCATBGCGTAGTTVGGRTAGT-3’). The PCR amplification was performed with a Takara Extaq (Takara biotechnology, Japan) using 1 μl of a DNA template in 25 μl of reaction mixture. The PCR amplification was performed with the following reaction conditions: initial denaturation at 95°C for 3 min, 34 cycles of 95°C for 45 sec, annealing at 55°C for 45 sec and 72°C for 45 sec, followed by a final extension at 72°C for 7 min. The PCR product was analyzed by electrophoresis on a 1.2% agarose gels to verify the extraction and amplification. DNA concentrations were quantified by Nanodrop 2000 spectrophtometer (Thermo Scientific, USA).

The quantitative PCR of mcrA gene copy numbers were analyzed by BioRad CFX96 real-time thermocycler (BioRad Laboratories, Hercules, CA, USA). The reaction mixture (SYBR Green Real-time PCR Master Mix, Toyobo, Japan) was composed of 10 pmol of each primer [15], 1 μl template DNA (10 ng μl^-1) and sterilized distilled water added to make the final volume up to 40 μl. The initial denaturation was done at 95°C for 3 min, followed by 40 cycles at 95°C for 45 sec, 55°C for 45 sec and 72°C for 45 sec. The DNA standard was prepared from the purified plasmid DNA of mcrA clone after 10-fold serial dilutions of plasmids containing a sequence of mcrA gene from Methanosarcina mazei. The amplification efficiency of the PCR was calculated using standard curves with the following formula: Efficiency=[10(−1/slope)]−1

The amplifications of serial diluted standards were performed for samples of each pot to minimize the inhibitory effect exerted by substances co-extracted with DNA. The quality of the amplification was evaluated by the generation of a melting curve for the PCR product.

In order to determine the effect of BES on methanogenesis, methanogens activity was carried following method of Pramanik and Kim [16]. The soil samples were collected at 30, 60, 80 and 120 DAT in each treatment pot in triplicate during rice cultivation. Ten gram of fresh soil was mixed with 25 ml distilled water in 115 ml serum bottle and incubated under anaerobic condition at 30±0.5°C for 5 h. The methanogen activity was measured by estimating CH₄ concentration in the headspace of the bottles and the values were expressed as ng of CH₄-C produced g soil^-1 hr^-1.

In order to check the effect of BES application on soil enzyme activity (i.e. soil biological activity) other than methanogenesis, the soil dehydrogenase activity was monitored during rice cultivation. The soil dehydrogenase activity was determined using the reduction of 2,3,5-triphenyltetrazolium chloride (TTC) method [17]. A sample of 6 g soils and 60 mg CaCO₃ were mixed thoroughly and then were transferred into each of three glass vails (20 ml). To each vial with stopper, 1 ml of 3% TTC and 2.5 ml of deionized water were added. The samples were mixed on a vortex and incubated at 37°C. After 24 h, the triphenylformazan (TPF), a product from the reduction of TTC, was extracted by adding 10 ml methanol and shaken for 1 min. The samples were collected in a volumetric flask. The vial was washed with methanol until the red color disappeared. The filtrate was then diluted with additional methanol to a final volume of 100 ml. The color intensity was measured at 485 nm with methanol as a blank.

Soils were collected in triplicate (from each treatment) from the 0–15 cm depth at the harvesting stage, air dried, and passed through a 2-mm size sieve for chemical analysis. The soil chemical properties were analyzed using the Korean standard method [18]: pH (1:5 with H₂O), available phosphate (Lancaster method), organic matter content (Walkley and Black method; [19]) and total N [18]. At harvesting stage, the whole rice plant was cut from the 1–2 cm above soil surface from each pot and transferred to the lab for yield parameter measurement. A rice plant growth parameters like plant height, tiller numbers, straw yield and rice yield characteristics like ripened grains %, 1000 grain weight and grain yield and total biomass were investigated at a harvesting stage of rice plant. Yield components were determined by following Korean standard rice cultivation guidelines [20].

Statistical analyses were conducted using SPSS 11.5 software for windows. A one-way analysis of variance (ANOVA) was carried out to compare the means of the different treatments. Tukey’s post-hoc test was used to separate treatment means when the F-test showed to be significant at the P<0.05 probability level. Linear regression analysis was performed to evaluate relationships between response variables.

CH₄ emissions were gradually increased after transplanting and showed first peak at 63 DAT followed by second peak at 91 DAT, thereafter decreased prior to the rice harvest (Fig 1A). The highest CH₄ emission was recorded in control treatment and BES application effectively (P<0.001) reduced rate of CH₄ emission during rice cultivation. The rate of CH₄ emissions were inversely proportional to the doses of BES application. The total seasonal CH₄ flux from rice planted soils were significantly affected by BES applicaiton. The total CH₄ flux in control soil was 39.2 g m^-2, which was significantly decreased by 17–49% after BES application (Fig 1B).

Coenzyme M concentrations in soil varied depending on the applied treatments and rice cultivation period. Irrespective of the time of rice cultivation, the highest Co-M concentrations were observed in control soil and BES application significantly (P<0.001) reduced Co-M concentration in soil (Fig 2A). At active tillering stage, Co-M concentrations in 20 and 40 mg mg kg^-1 treament soils were statistically at par with 80 mg kg^-1 BES treatments, which was significantly (P<0.05) increased at booting stage. At booting stage, Co-M concentration in the control soil was 482.1 ± 6.07 μmoL g soil^-1, which was decreased to 394.1 ± 5.15, 356.4 ± 5.94 and 259.7 ± 9.4 μmol g soil^-1 in 20, 40 and 80 mg kg^-1 BES treatment soils, respectively.

Irrespective of rice cultivation, the highest mcrA genes abundance was observed in the control and the application of BES significantly (P<0.001) decreased mcrA genes in soil (Fig 2B). The mcrA genes of 20 and 40 mg kg^-1 BES treatment soils were higher than that of 80 mg kg^-1 treated soil, but lower than the control soil during rice cultivation. At booting stage, the mcrA gene copy numbers of 80 mg kg^-1 treated BES soil were significantly lower (P<0.05) when compared to the control soil.

Methanogen activity in rice paddy soils also varied with the BES application levels and period of rice cultivation (Table 1). BES application significantly (P<0.001) reduced methanogen activity in soil and recorded lower methanogen activity at all rice cultivation stages. At booting stage, methanogen activity was highest in control soil and the BES application significantly (P<0.05) reduced in it.

In case of soil dehydrogenase activity, the dehydrogenase activity was not affected by BES application (Table 1). The dehydrogenase activity were significantly (P<0.05) lower at active tillering stage than the booting stage among all treatments, but statistically at par among treatments at all stages of rice cultivation.

Soil chemical properties were not affected by BES application in paddy soil (Table 2). Also, BES application did not affect plant and yield characteristics, except for plant hight and straw yield.