Interactive comment on “ The combined effects of nitrification inhibitor and biochar incorporation on yield-scaled N 2 O emissions from an intensively managed vegetable field in southeastern China ”

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Introduction
Nitrous oxide (N 2 O) is an important greenhouse gas and also contributes to ozone depletion in the stratosphere (IPCC, 2007;Saggar et al., 2007;Ravishankara et al., 2009).Global N 2 O emissions increased from 10 Tg to 12 Tg N 2 O-N yr −1 between 1900 and 2000 and may reach 16 Tg N 2 O-N yr −1 by 2050 (Bouwman et al., 2013).The increase in nitrogen (N) fertilizer application in agricultural ecosystems has been recognized as a major source of N 2 O, representing approximately 60% of the global anthropogenic emission rates in 2005 (Smith et al., 2007;Park et al., 2012).Emissions from soils increase markedly following the application of N fertilizers and animal manures for the purpose of increasing crop production (Mosier et al., 1998;Davidson, 2009).
Increasing grain yields is a boundary condition for 'greening' Chinese agriculture in light of the increasing food demand in China.The high N fertilizer application rate contributes to high crop yields (Qin et al., 2010) but, inevitably, also to high N 2 O emissions (Ding et al., 2007) and nitrate leaching (Li et al., 2007).To minimize the overall greenhouse gas (GHG) impact of agriculture while increasing crop production, the amount of N 2 O emitted per unit of crop production (yield-scaled N 2 O emissions) needs to be considered (Van Groenigen et al., 2010).The yield-scaled N 2 O emissions approach has been suggested as a more comprehensive index to assess N 2 O emissions in agricultural ecosystems (Van Groenigen et al., 2010;Grassini and Cassman, 2012).This approach is attractive in view of the need to balance crop production with the mitigation of N 2 O emissions from agriculture.Only a few studies have directly addressed yield-scaled emissions in agriculture cropping systems (Halvorson et al., 2010;Wei et al., 2010;Gagnon et al., 2011;Zhou et al, 2014), particularly intensively managed vegetable systems.
Intensively managed vegetable cultivation represents a major source of N 2 O emissions in the agricultural sector in China (Zheng et al., 2004;Wang et al., 2011) as a result of the practical characteristics of high N fertilization, intensive irrigation and favorable environmental conditions that are associated with this type of agriculture.Previous studies have shown that annual N fertilizer inputs are extremely high for certain intensively managed vegetable fields (Ju et al., 2006;Xiong et al., 2006;He et al., 2009).These levels are almost 3-4 times the fertilizer levels used for non-vegetable crops in fields where 2 crops are grown per year (Zheng et al., 2004).As a consequence, N 2 O emissions from N fertilizer in vegetable ecosystems represent 20% (Zheng et al., 2004) or 21.4% (Wang et al., 2011) of the total emissions from China's farmland.
Alternative practices that could reduce N 2 O emissions without necessarily reducing N inputs or crop yields have also been considered, such as nitrification inhibitor (NI) application (Zaman et al., 2009;Ji et al., 2011) and biochar amendment (Zhang et al., 2010;Jia et al., 2012;Wang et al., 2013) in agricultural soils.NIs have been used in the field to improve the efficiency of fertilizers and to reduce both nitrate leaching and denitrification by maintaining the N in the soil as NH 4 + (Majumdar et al., 2000;Pathak and Nedwell, 2001;Malla et al., 2005;Chen et al., 2010), thus mitigating N 2 O emissions and increasing the crop yield from the agricultural ecosystem.A newly developed urea mixed with NI of nitrapyrin as chlorinated pyridine (CP) has been used in agricultural ecosystems to mitigate GHG emissions and simultaneously increase crop yield (Ma et al., 2013;Xiong et al., 2013;Zhang et al., 2015).Biochar amendment of soils is currently being considered as a means of mitigating climate change by sequestering carbon (C) while concurrently improving soil properties and functions (Lehmann, 2007;Woolf et al., 2010;Zhang et al., 2012a).However, the effects of biochar amendment induced by biochar addition on N 2 O emissions may be either positive or negative depending on the inherent characteristics of biochar, the addition of exogenous N and the soil water regime (Spokas and Reicosky, 2009;Xie et al., 2013;Cayuela et al., 2013Cayuela et al., , 2014)).The soil pH of intensively managed vegetable fields has been found to be much lower than that of other agricultural ecosystems due to the input of large amounts of N. Most likely, this lower soil pH will cause a negative effect if biochar amendment is used to mitigate N 2 O emissions because it would affect activity of N 2 O reductase in soil (Cayuela et al., 2014).Overall, based on previous results, both nitrapyrin application and biochar amendment could serve to decrease yield-scaled N 2 O emissions in various agricultural ecosystems.As nitrapyrin application and biochar amendment could both significantly affected the soil pH, the combined use of the two practices may have different effects on N transformation processes and NH 3 volatilization, and thus affect the N 2 O emissions and crop yield in intensively managed vegetable field (Zhu et al., 2011;Soares et al., 2012;Gong et al., 2013).However, information for investigating the combined effects of nitrapyrin application and biochar amendment incorporation on yield-scaled N 2 O in intensively managed vegetable agriculture is limited.
Accordingly, we quantified the effect of nitrapyrin application and biochar amendment incorporation on yield-scaled N 2 O emissions in intensively managed vegetable agriculture in southeastern China.The objective of the study was to find appropriate practices for increasing vegetable yield and mitigating N 2 O emissions from intensively managed vegetable fields.

Experiment site and biochar properties
A field experiment was conducted at a suburban site (31°59′N, 118°51′E) in Nanjing City, Jiangsu Province, China, from 2012 to 2014.This area has a subtropical monsoon climate with an annual mean rainfall of 1,107 mm and an annual mean air temperature of 15.34°C (Nanjing Meteorology).The selected site had been conventionally continuously cultivated with vegetables for approximately 10 years and is a typical vegetable field.The studied soil was classified as Fimi-Orthic Anthrosols (RGCST, 2001), with a bulk density of 1.2 g cm -3 , a total porosity of 51%, a clay (<0.002 mm diameter) fraction of 30.1%, a silt (0.002-0.02 mm diameter) fraction of 64.7% and a sand (0.02-2 mm diameter) fraction of 5.2%.The main properties of this soil are as follows: pH, 5.52; total N, 1.90 g kg -1 ; organic carbon, 15.6 g C kg -1 ; and CEC (cation exchange capacity), 31.2 cmol kg -1 .
For the field experiment, biochar was produced from wheat straw at the Sanli New Energy Company in Henan, China by pyrolysis and thermal decomposition at 400 °C.The biochar had a carbon content of 467 g C kg -1 and an N content of 5.9 g N kg -1 .The initial values of pH, CEC and ash content were 9.4, 24.1 cmol kg -1 and 20.8%, respectively.

Treatments and vegetable management
There were 6 treatments with the same amount of total N in triplicate for 7 consecutive vegetable crops from Apr. 12, 2012 to Jun. 12, 2014 in Nanjing, China.Each plot had an area of 7.5 m 2 and measured 3 m × 2.5 m.Biochar was applied at rates of 0, 20 and 40 t ha −1 (C0, C1 and C2, respectively) with compound fertilizer (CF) or urea fertilizer mixed with nitrapyrin (CP).All treatments received the same amount of N fertilizer based on the local practices during the experimental period.The total N application rate for each treatment was equal, 1217 kg N ha -1 yr -1 across the experimental period, of which 312.5 kg N ha -1 was applied for Amaranth (Amaranthus mangostanus L.) and Coriander (Coriandrum sativum L.), 600 kg N ha -1 was applied for Tung choy (Ipomoea aquatica Forssk.) and 250 kg N ha -1 for Baby bok choy (Brassica chinensis L.).Compound fertilizer with an m(N):m(P 2 O 5 ):m(K 2 O) ratio of 15:15:15 was used for the CF group treatments and the N form of the compound fertilizer is ammonium fertilizer, while the corresponding P and K fertilizers were broadcast in the form of calcium phosphate and potassium chloride, respectively, in addition to the nitrapyrin urea for the CP group treatments.All fertilization occurred before transplanting and as the base fertilizer for each vegetable crop except for Tung choy, which had 312.6 kg N ha -1 as basal fertilization and 287.4 kg N ha -1 as top-dressing according to the local farmers' practice.Additionally, both P and K fertilizers for the CP group treatments were applied as top-dressing for the Tung choy growing period as well.
Biochar was added once to the vegetable fields before sowing of the first vegetable crop (Amaranth) on Apr. 8, 2012 and was incorporated with the soil by hand plowing at a depth of 20 cm.There were 7 vegetable crops grown successively during the entire observation period (Apr. 12, 2012to Jun. 12, 2014).Each type of vegetable was seeded by hand and harvested at the appropriate mature stage according to the local farmers' practice.Furthermore, a short fallow period was imposed after fresh biomass was harvested from each vegetable crop.Soon after harvesting each vegetable crop, the field was tilled to a depth of approximately 12-15 cm.A protective plastic film was used to cover the crops according to the growth requirements of each vegetable crop, i.e., from Apr. 12, 2012to May 25, 2012and Mar. 15, 2014to May 12, 2014for Amaranth and from Nov. 20, 2012, to Feb. 24, 2013and Nov. 5, 2013to Mar. 14, 2014 for Baby bok choy, as Amaranth and Baby bok choy require relatively warm weather conditions for growth.All the other management procedures, including crop species, tillage, irrigation, and pesticide followed the local farmers' practices are presented in Table 1.

Measurements of N 2 O fluxes, soil samples and environmental factors
A static opaque chamber method was used to collect air samples from the experimental sites from 3 replicates for each treatment.Each chamber was made of PVC and consisted of a chamber body (50×50×50 cm 3 ).The outside of the chamber was coated with sponge and aluminum foil to prevent the effects of high temperatures on the chamber.The chamber was installed on a frame.The frames were inserted 0.1 m deep in the soil in each plot and filled with water to make the chamber gas-tight.
Sampling was conducted between 8:30 and 10:30 in the morning every other day for 1 week after fertilizer application and then once per week thereafter.Gas fluxes were measured on 121 occasions over the 2-year period.On each sampling occasion, air samples were taken 0, 10, 20 and 30 min after chamber closure.The samples, collected in 20-mL syringes, were returned to the laboratory, and the N 2 O was determined on the same day with a gas chromatograph (Agilent 7890A, Agilent Ltd, Shanghai, China) equipped with an electron capture detector (ECD).The carrier gas was argon-methane (5%) at a flow rate of 40 mL min -1 .The column and ECD temperature were maintained at 40°C and 300°C, respectively.Concentrations of N 2 O were quantified by comparing the peak area with those of reference gases (Nanjing special gas factory, Nanjing, China).N 2 O fluxes were calculated by using the linear increases in gas concentration with time.The mean flux for 1 vegetable crop was calculated as the average of all measured fluxes.The measured fluxes were weighted by the interval between 2 measurements (Xiong et al., 2006).The cumulative seasonal N 2 O was calculated as the product of the mean flux and the seasonal duration.
Except for the soil that was analyzed immediately before the experiment in April 2012, another batch of soil samples for each treatment was collected on Jun. 12, 2014 and stored at -20 °C for laboratory analysis.In accordance with Lu (2000), the soil texture was measured using pipette analysis, the total soil organic carbon (SOC) was analyzed by wet digestion with H 2 SO 4 -K 2 Cr 2 O 7 , and the TN was determined by semi-micro Kjeldahl digestion using Se, CuSO 4 and K 2 SO 4 as catalysts.The soil pH was measured at a volume ratio of 1:2.5 (soil to water ratio) using a PHS3C mv/pH detector (Shanghai, China).The soil temperature was measured at a depth of 15 cm beneath the collection point when the gas samples were collected.
Simultaneously with the determination of the trace gas fluxes, soil sampling at 0-15 cm depth was conducted for the determination of soil mineral N and soil water content.Soil mineral N was determined at approximately 7-15 days interval.The soil NH 4 + -N and NO 3 --N were extracted by shaking for 1 h on a rotary shaker with 2 mol L −1 KCl solution.According to Lu (2000), soil NH 4 + -N and NO 3 --N contents were measured following the two wavelength ultraviolet spectrometry and indophenol blue methods, respectively, using an ultraviolet spectrophotometer (HITACHI, U-2900, Japan).The soil moisture content obtained by oven drying was converted to water-filled pore space (WFPS) using the following equation: WFPS = volumetric water content (cm 3 cm -3 ) / total soil porosity (cm 3 cm -3 ). (1) Here, total soil porosity = [1 -(soil bulk density (g cm -3 ) / 2.65)] with an assumed soil particle density of 2.65 (g cm -3 ).The total soil bulk density was determined with the cutting ring method to 010 cm depth according to Lu (2000).

Estimation of vegetable yields and yield-scaled N 2 O emissions
The fresh vegetable yields were measured after each vegetable growth period by weighing all of the above-ground vegetable parts that were grown in each plot.
The yield-scaled N 2 O emissions were related to crop yield as in Van Groenigen et al. (2010) and Grassini and Cassman (2012) and were calculated as follows: (2)

Data processing and statistics
The values presented are given as arithmetic means ± standard error (SE

Soil properties and soil microclimate
Nitrapyrin had no significant influence on soil TN or SOC during the experimental period.However, significant increases in soil TN were observed in treatments with biochar amendments ( No statistical differences in WFPS and soil temperature were detected among all the treatments over the whole experimental period (data not shown).Dynamic variation of soil temperature was detected with seasonal change of outside temperature although plastic film was set sometimes in low-temperature seasons in the vegetable field.As shown in Figure 1a and 1b, significant differences were found between different vegetable crops, with high temperature in summer and low in winter season (p<0.001).Moreover, soil WFPS rates ranged from 31.9-76.0%across all the experimental period.No significant difference on WFPS rates were detected among vegetable crops (Fig. 1c).Soil NH 4 + -N and NO 3 --N contents ranged from 58.0-413.9mg N kg -1 and 36.4-279.1 mg N kg -1 across the 7 vegetable crops growth period, respectively as shown in Figure 3 and Figure 4, respectively.The fertilization events considerably increased soil mineral N (NO 3 -+ NH 4 + ) content.As the nitrapyrin limited the nitrification process, significant higher soil NH 4 + -N contents were observed for the treatments with nitrapyrin compared to the treatments with compound fertilizer at the same N rate (Fig. 3).In contrast to the NH 4 + -N, a significant lower content of soil NO 3 − -N was observed in the plots applied with nitrapyrin (Fig. 4).In addition, biochar increased the soil NH 4 + -N content while decreased the soil NO 3 --N contents throughout the intensive vegetable growth period, but not significantly (Fig. 3, Fig 4).

Cumulative N 2 O emissions, vegetable yield and yield-scaled N 2 O emissions
The cumulative N 2 O emissions for each treatment across the entire experimental period are shown in Table 3a.These emissions varied widely among different crops during the individual vegetable crop-growing season.Additionally, the cumulative N 2 O emissions showed significant differences among all the treatments.The greatest N 2 O-N flux was observed in the CF-C0 treatment (54.6±1.5 kg N ha -1 ), whereas the lowest flux was in the CP-C0 treatment (37.1±4.4 kg N ha -1 ).As shown in Table 3a and, Table 4 significant decreases in the cumulative N 2 O emissions were detected in the treatments with nitrapyrin (p<0.001).The decreases ranged from 15.9%-32.1% compared with the treatments without nitrapyrin application (p<0.001).In contrast, biochar amendment had no significant influence on cumulative N 2 O emissions in both CF and CP group treatments (Table 4).However, as shown in Table 3a, slight increases in cumulative N 2 O emissions were observed.These increases were 7.9% and 18.3% for CP-C1 and CP-C2, respectively, but were not significant compared with CP-C0, which also indicated that the biochar amendment rates would not result in a significant difference on the cumulative N 2 O emissions.Furthermore, no significant interaction between nitrapyrin and biochar amendment was observed to affect cumulative N 2 O emissions throughout the intensive vegetable experimental period (Table 4).
Table 3b shows details of the fresh weight for each vegetable crop.The highest fresh weight yield for the 7 consecutive vegetable crops was 535.4±16.7 t ha -1 for CP-C1, an increase of 71.7% compared with CF-C0.Both nitrapyrin application (p<0.001) and biochar amendment (p<0.05)significantly increased the yield in the intensively managed vegetable system across the experimental period (Table 4).As shown in Table 3b, nitrapyrin significantly increased vegetable yield compared with the treatments without nitrapyrin.In addition, the vegetable yield was significantly enhanced by 9.8%-41.9%with the increase of the biochar amendment rates in CF group treatments (Table 3b).
However, a decrease of vegetable yield was observed in CP-C2 treatment compared with CP-C0.
Moreover, significant interactions between nitrapyrin and biochar amendment were observed to affect vegetable yield throughout the intensive vegetable experimental period (Table 4, p<0.05).(Ma et al., 2013;Xiong et al., 2013;Zhang et al., 2015).Different types of NI that were effective in reducing N 2 O have been reported by previous studies (Xu et al., 2000;Boeckx et al., 2005;Zaman et al., 2008Zaman et al., , 2009;;Zaman and Blennerhassett, 2010).Nitrapyrin could efficiently inhibit the activity of the ammonia oxidase and reducing the abundance of nirK gene in vegetable soil, and thus inhibit the nitrification process while regulating the NH 4 + and NO 3 − content of soil (Fig. 3, Fig. 4) , which is highly related to mitigating N 2 O emissions (Di et al. 2009(Di et al. , 2014)).Moreover, nitrapyrin application in vegetable soil resulted in a significant increase in soil pH though insignificant in soil TN and SOC across the 7 consecutive vegetable growing periods ( A previous study has shown that N fertilizers combined with NI such as DCD and DMPP may improve the yield and quality of agricultural and horticultural crops (Pasda et al., 2001).Similarly, nitrapyrin produced a significant increase in vegetable yield in our study across the experimental period (Table 3b, Table 4, p<0.001).Possible explanations for higher crop yields obtained with NH 4 + -containing fertilizers supplemented with nitrapyrin (Fig. 3) include the reduction of N losses by leaching and volatilization, and improved bio-availability of N and N uptake of the crops (O'Connor et al. 2012) in the vegetable soil.Ma et al. (2013) reported that 2 types of NIs (nitrapyrin and DCD) increased average wheat yield by 9.7% under conventional and no-till practices during the winter wheat-growing season.In addition, since nitrapyrin could significantly increase soil pH, the uptake rates of the inorganic N would increase due to the increasing effect of nitrapyrin on soil pH in vegetable field (Jampeetong et al., 2013).Moreover, Zhang et al. (2015) find CP significantly increased vegetable yield by 12.6% in an intensively managed vegetable field which may due to CP was beneficial for the growth and N assimilation of the crops (Liu et al., 2013).

Effects of biochar on N 2 O emissions and vegetable yield
As shown in Table 3a and Table 4, biochar had no significant influence on cumulative N 2 O during the experimental period.Decreases in the net emissions of N 2 O from certain agricultural ecosystems as a result of soil amendment with biochar have been well documented by previous studies (Cayuela et al., 2014;Felber et al., 2014).Additionally, a meta-analysis of 30 papers (published from 2007 to 2013) by Cayuela et al. (2014) found that soil N 2 O emissions, which were affected by biochar characteristics, soil characteristics and N fertilizer type, were reduced by 54% in both laboratory and field studies.
However, biochar amendment had no significant influence on cumulative N 2 O emissions during the experimental period and even slightly increased cumulative N 2 O by 7.9%-18.3% in the CP group treatments (Table 3a, Table 4).Thus, the mitigating effect of biochar amendment on N 2 O emissions did not work in the intensively managed vegetable field in this study, which is in consistent with previous short-term laboratory incubation results in acidic soils (Yuan and Xu, 2011;Wang et al., 2014).This finding may firstly be due to the decrease in soil pH in the treatments amended with biochar (Table 2).
In contrast with the significant increase in soil pH due to the liming effect of biochar generally reported in previous studies (Biederman andHarpole 2012, Zhang et al., 2010), soil pH decreased significantly in the plots amended with biochar (Table 2, p<0.001).With such a high amount of N fertilization application, biochar may lose the buffering effects of soil pH changes.Luo et al (2011) reported that biochar with low pyrolysis temperature had more water extractable organic carbon.Although considered to be stable in soil, biochar brings extra carbon source for heterotrophic nitrification process, which may also cause an increase in the H + content due to nitrification processes in the soil (Schmidt, 1982;De Boer and Kowalchuk, 2001;Wrage et al., 2001) and thus decrease the soil pH significantly.
In addition, the decrease in soil pH may, most likely, be attributed to the weathering effect of biochar after 2 years of incorporation into fields (Jones et al., 2012;Spokas, 2013), particularly in intensively managed vegetable fields.Yao et al. (2010) reported that the pH of biochar samples decreased from 8.4 to 7.5, primarily due to the loss of base cations through leaching and probable carbonation during the weathering process, and biochar offers the practical benefits of high N fertilization input and may also be weathered more easily in vegetable soils.Cayuela et al. (2014) also reported that the effectiveness of biochar application on mitigating N 2 O emissions was significant in neutral and alkaline soils but not in acidic soils with pH < 5, which probably due to that low soil pH may result in adversely affecting the activity of N 2 O reductase in vegetable field (Liu et al., 2010).The decrease in soil pH may also increase the heterotrophic nitrification rate, which may cause the increase of N 2 O emissions in vegetable field (Zhu et al., 2011) though heterotrophic nitrification is generally considered to be a minor source of N 2 O (Anderson et al., 1993).Moreover, vegetable field had a high amount of N input and may expect ammonia oxidation and linked nitrifier-denitrification (ND) being important processes generating N 2 O (Wrage et al., 2001;Huang et al., 2014).Sánchez-García et al. (2014) found that biochar increased cumulative N 2 O emissions in the soil when ammonia oxidation and were the ND major processes generating N 2 O emissions, whereas it decreased N 2 O emissions in the soil when denitrification is the main pathway leading to N 2 O emissions under the same experimental conditions.
Biochar amendment significantly increased SOC and soil TN (Table 2, p<0.001) and thus significantly improved vegetable yield compared with the treatments that received no biochar addition (Table 4, p<0.05), which agree well with previous reported benefit of adding biochar to soils (Major et al. 2010;Jia et al., 2012).In addition, vegetable yield was significantly increased with the increase of biochar amendment rates in the CF group treatments, which is in agree with previous results (Zhang et al., 2010).In our study, SOC increased significantly by 66.9%-85.1% in the treatments amended with biochar over the experimental period (Table 2, p<0.001).This result is in consistent with the finding that average SOC increased by 61% due to biochar addition, reported in a meta-analysis by Biederman and Harpole (2012).Biochar amendment significantly increased SOC, most likely due to its inert recalcitrant C component, which can contribute to soil carbon sequestration, at least over periods of decades to millions of years (Kuzyakov et al., 2009(Kuzyakov et al., , 2014;;Lehmann et al., 2011).Additionally, biochar amendment also significantly increased soil TN (Table 2, p<0.001), which is in consistent with previous study in paddy rice (Zhang et al., 2012b).Most likely, this difference in soil TN is probably due to the release of N in soil from the biochar (Singh et al., 2010;Schouten et al., 2012).The N content of the biochar used in our experiment was 5.9 g N kg -1 , and this potential N source may also have increased the soil TN.Additionally, the amendment of biochar would offer a further opportunity to achieve N fertilizer savings in vegetable soil, which may have resulted in an increase of soil TN due to the higher inorganic N absorption effects, as seen in comparison with treatments without biochar amendment in an intensively managed vegetable field (Ding et al. 2010).Furthermore, vegetable yield enhancement effect of biochar may also be associated with increases in root exudation in the plots amended with biochar (Gregory, 2006).Biochar amendment in agricultural soil may stimulate microbial activity, resulting in nutrient release (Steinbeiss et al., 2009), reducing nutrient leaching (Laird et al., 2010), and improving crop nutrient availability and plant N uptake (Saarnio et al., 2013) in the intensively managed vegetable field.Moreover, as shown in Table 3b, a significant difference in vegetable yield among the treatments was found under different biochar application rates with compound N fertilization (CF-C0, CF-C1 and CF-C2), in agreement with the results reported by Jeffery et al. (2011) although the plots amended with biochar showed a significantly lower soil pH (Table 2, p<0.001).

The combined effects of nitrapyrin and biochar incorporation on yield-scaled N 2 O emissions
Analyzing N 2 O emissions on a yield basis provides interesting information for estimating the environmental impacts of intensive agricultural production systems.As shown in Table 3c, yield-scaled N 2 O emissions ranged from 0.074±0.004-0.175±0.017kg N 2 O-N t −1 yield, much lower than previously reported values (Van Groenigen et al., 2010;Ma et al., 2013).Ordinarily, vegetable crops require higher N application rates than staple food crops such as rice, wheat and maize (Li and Wang, 2007).Moreover, the leafy vegetables in this study differed from other crops and all aboveground portions of the vegetable plants were considered as the yield, resulting in low values of yield-scaled N 2 O in our vegetable field.
Overall, nitrapyrin significantly decreased yield-scaled N 2 O emissions across the experimental period (Table 3c, Table 4, p<0.001), attributing to the N 2 O reducing and vegetable increasing effects of nitrapyrin in intensively managed vegetable field.Our results indicate that the yield-scaled N 2 O emissions were minimal in the CP-C0 treatment (0.074±0.004 kg N 2 O-N t −1 yield).This treatment showed the lowest cumulative N 2 O emissions (37.1±4.4 kg N ha -1 ) and the second highest vegetable yield (500.3±34.9t ha -1 ).Under the application of equal amounts of N, nitrapyrin application was a more efficient way to reduce the yield-scaled N 2 O emissions in our case.This approach may significantly improve vegetable yield while causing a decrease in N 2 O emissions and, thus, improved agronomic N use efficiency (NUE) in intensively managed vegetable agriculture (Li et al., 2007;Asing et al., 2008).Overall, nitrapyrin application without biochar amendment (CP-C0) can serve as an appropriate way of mitigating N 2 O emissions while increasing vegetable yield in intensively managed vegetable agriculture.
Although biochar amendment did not significantly decreased the cumulative N 2 O emissions, it significantly decreased the yield-scaled N 2 O emissions across the experimental period (Table 3c, Table 4, p<0.05), mainly due to the increasing effect of vegetable yield with biochar.Obvious interactions in yield-scaled N 2 O emissions were observed between nitrapyrin and biochar addition (Table 4, p<0.05).
However, no significant interactions between nitrapyrin and biochar in N 2 O emissions were observed in the current study.Treatments with nitrapyrin and biochar incorporation slightly increased the cumulative N 2 O by 7.9%-18.3%,but not significantly (Table 3a, Table 4), which indicated that biochar could be able to diminish the mitigating effect of nitrapyrin, namely, the effect of inhibiting the nitrification process in vegetable soil (Fig. 3,Fig 4).Vegetable soil in our study is acid due to large N application in which biochar would increase the heterotrophic nitrification rate in the treatments applied with both nitrapyrin and biochar (CP-C1 and CP-C2) compared with CP-C0 treatment, and thus increase the N 2 O emissions in vegetable field.Additionally, with such low soil pH, the denitrification process and the ND process may also become the major contributors to the N 2 O pool (Zhu et al., 2011;Zhu et al., 2013).Although biochar had no significant effect on soil inorganic N content, it decreased the soil NH 4 + -N content and increased the soil NO 3 --N content in the treatments amended with biochar in both CF and CP group (Fig. 3,Fig 4), which may simultaneously increase the NO 2 -content in the vegetable soil, and thus increase the cumulative N 2 O emissions linked with other N 2 O generating processes in vegetable field across the experimental period.Moreover, Di et al. (2014) reported that DCD was highly effective in inhibiting the growth of AOB communities, and reducing N 2 O emissions under high soil moisture, whereas Yanai et al., (2007) found that when the soils were rewetted at 83% WFPS, the suppressive effects of charcoal addition on N 2 O emissions were not observed.Thus, biochar addition diminished the mitigation effect of nitrapyrin on the cumulative N 2 O emissions in vegetable soil under the high WFPS condition due to frequent irrigations (Fig. 1).
Significant interactions in yield were observed though no significant differences in vegetable yield were found among different combinations of nitrapyrin and biochar as CP-C0, CP-C1 and CP-C2 (Table 3b), indicating biochar incorporation rate did not increase vegetable yield when combined with nitrapyrin application.Most likely, the vegetable yields were relatively high compared with other ecosystems although both nitrapyrin and biochar separately can significantly increase vegetable yield, which can also explain the fact that increasing biochar amendment rates did not resulted in significant increase in vegetable yield across the experimental period (        *** p<0.001; n.s.not significant.
Figure 2. The pattern of these fluxes was relatively consistent and was sporadic and pulse-like.N 2 Ofluxes showed a similar trend during the same period in both years.Following basal fertilization, tillage and irrigation, N 2 O emissions ranged from 17-3406 μg N m -2 h -1 were observed in all treatments across the experimental period.N 2 O emissions primarily occurred with the increase in soil temperature during the summer, from May to October.However, as shown in Figure2dand Figure2g, no significant N 2 O peaks were found in certain vegetable crops to which basal or top-dressing fertilization was added because of the low soil temperature after fertilization in the vegetable field.

FFigure 1
Figure 1 Dynamics of soil temperature (T) and WFPS across the experimental period (a).Box plots for soil temperature (b) and WFPS (c) in different vegetable crops.The 1st to 7th mean different vegetable crops in rotation across the experimental period.Different letters indicate significant difference among all treatment medians (p<0.05).The plus mark in the box represents the medians of all data.

Figure 2
Figure 2 Dynamics of soil N 2 O emissions fluxes under different treatments in vegetable fields with 7 consecutive vegetable crops from 2012 to 2014 in southeastern China.The solid and dashed arrows indicate basal fertilization and top-dressing, respectively.The dashed vertical line in each sub figure separates the vegetable growing and fallow periods.The bars indicate the standard error of the mean (+SE) for the 3 replicates of each treatment.See Table 2 for treatment codes.

Figure 3
Figure 3 Dynamics of the soil NH 4 + -N concentrations within the 0-15 cm soils under different treatments in vegetable fields with 7 consecutive vegetable crops from 2012 to 2014 in southeastern China.The solid and dashed arrows indicate basal fertilization and top-dressing, respectively.The dashed vertical line in each sub figure separates different vegetable growth periods.The bars indicate the standard error of the mean (+SE) for the 3 replicates of each treatment.See Table 2 for treatment codes.***p<0.001; n.s.not significant.

Figure 4
Figure 4 Dynamics of the soil NO 3 --N concentrations within the 0-15 cm soils under different treatments in vegetable fields with 7 consecutive vegetable crops from 2012 to 2014 in southeastern China.The solid and dashed arrows indicate basal fertilization and top-dressing, respectively.The dashed vertical line in each sub figure separates different vegetable growth periods.The bars indicate the standard error of the mean (+SE) for the 3 replicates of each treatment.See Table 2 for treatment codes.
).All figures in this study were plotted in Microsoft Excel 2003.Significant differences on soil temperature and WFPS among different vegetable crops were determined by the nonparametric Kruskal-Wallis test.A 2-way ANOVA was used to analyze the effects of nitrapyrin, biochar and their interactions on soil TN, SOC, soil pH, soil mineral 2 O emissions, and yield-scaled N 2 O emissions throughout the experimental period.A Tukey's multiple range tests was used to determine if significant differences occurred between the treatment means at a significance level of 0.05.All statistical analyses were performed using JMP, ver.7.0 (SAS Institute

Table 2
p<0.001).Compared with the treatments without biochar amendments, soil TN was increased by 81.5%-99.3%and44.8%-63.2%for the CF and CP group treatments, respectively.Similar to soil TN, SOC increased significantly in the treatments with biochar amendment (Table2, p<0.001).SOC was enhanced by 66.9%-85.1% and 80.4%-81.3%for the CF and CP group treatments, respectively, compared with the treatments without biochar amendment.Moreover, nitrapyrin increased soil pH Table3cshows the yield-scaled N 2 O emissions, which were related to the cumulative N 2 O emissions and the fresh weight yield ranged from 0.074±0.004to0.175±0.017kgN 2 O-N t −1 yield over the entire experimental period.The lowest value of yield-scaled N 2 O emissions was 0.074±0.004kgN 2 O-N t −1 yield for CP-C0, namely, the treatment with nitrapyrin application and without biochar amendment.As shown in Table4, both nitrapyrin application (p<0.001) and biochar amendment (p<0.05)significantlydecreased the yield-scaled N 2 O emissions during the entire experimental period.Effects of nitrapyrin as nitrification inhibitor on N 2 O emissions and vegetable yield N 2 O emissions are directly related to the amount of mineral N available in the soil.A two-way ANOVA indicated that seasonal N 2 O emissions during the vegetable-growing periods were significantly affected by nitrapyrin application (Table4, p<0.001), in agreement with previous results

Table 2
(Singh et al., 2008)crease effect may have been a result of the production of hydroxyl (OH -) ions during urea hydrolysis(De Boer and Kowalchuk, 2011).Similarly,Zaman et al. (2008)have reported a low rise in soil pH after applying Agrotain-treated urea to pasture soil.A laboratory incubation of DCD decomposition also showed a soil pH increase with the addition of urine with or without DCD(Singh et al., 2008).Zhu et al. (2011)reported a negative correlation between soil pH and N 2 O emission rate in vegetable soils, which may indicate that the increasing of soil pH is another factor mitigating N 2 O emissions.
Table3b).Since we did not measure the individual N transformation process and the microbe in vegetable soil responding to N 2 O emissions and vegetable yield, the combined effects of nitrapyrin and biochar incorporation on N 2 O emissions in intensively managed vegetable fields need further study.SOC and vegetable yield but had no significant influence on the cumulative N 2 O emissions, whereas this amendment significantly decreased soil pH and yield-scaled N 2 O emissions.Nitrapyrin and biochar incorporation into vegetable soil slightly increased yield-scaled N 2 O emissions during the experimental period.Yield gains were the most important factor for lower yield-scale N 2 O emissions in our case compared with previous studies.Overall, taking environmental and economic benefits into consideration, nitrapyrin application in the vegetable field was the best procedure for reducing the

Table 2
Influence of biochar amendment on soil total nitrogen (TN), soil organic carbon (SOC), and soil pH in 6 different treatments over the entire experimental period.Values represent means±SD (n=3).
*** p<0.001P value: the index of differences between the control group and the experimental group.If p<0.05 and p<0.01, significant differences exists between the control group and the experimental group

Table 3
Cumulative N 2 O emissions, vegetable yield and yield-scaled N 2 O emissions under different treatments over the entire experimental period.See Table2for treatment codes.The total range of observation dates includes sampling that occurred during vegetable planting or not during vegetable planting.The values indicate (mean ± SD).Data in the brackets indicate the vegetable growing days/total days including the following fallow period for each vegetable crop in rotation.Different letters indicate a significant difference between treatments (p<0.05).

Table 4
Two-way ANOVA for the effects of nitrapyrin (CP) and biochar (Bc) on the cumulative N 2 O emissions, vegetable yield and yield-scaled N 2 O emissions over the entire