*2.3. Measurements of N2O Emissions*

Soil N2O emissions were continuously monitored using the static chamber-gas chromatography technique as described by Zhou et al. [5] and Zheng et al. [31] from November 2016 to October 2017 (a whole wheat-maize rotation season). Briefly, prior to the measurements, stainless-steel base collars with a uniform area of 0.25 m<sup>2</sup> were inserted into topsoil (10 cm in depth) and kept in place throughout the whole measurement period. The equipped chambers, with a circulating fan and an adjustable height according to the crop growth, can guarantee the chamber headspace uniformly mixed and minimize temperature changes when conducting the measurements. After tillage or fertilization practices, soil N2O emissions were continuously observed for 7 days, and then were measured every other day of the following week. For the remaining experimental period, the measurements were conducted twice a

week. For each measurement, five gas samples were collected using 50-mL volume plastic syringes after the chamber closure. Considering the low N2O flux in the forest, the sampling intervals were 7 min in the cropland treatments (0, 7, 14, 21 and 28 min) and 15 min in the forest treatment (0, 15, 30, 45 and 60 min). The measurements were uniformly performed between 9:00 and 10:00 am local time to calculate a daily average N2O flux. To minimize the enclosure effects of the chambers on plant growth and environmental conditions, they were immediately removed after gas sampling.

Immediately after sampling at each site, the collected gas samples were analyzed to obtain N2O concentrations using a gas chromatograph (Agilent-7890A; Agilent Technologies, Palo Alto, CA, USA) rigged with an electron capture detector (ECD) at the research station. The soil N2O fluxes were determined by the linear or nonlinear relationships between the gas concentration and the chamber closure time, as described in detail by Wang et al. [32]. Seasonal and annual cumulative N2O fluxes were calculated by linear interpolation of the daily fluxes between the gas sampling dates [25]. Yield-scaled N2O emissions (kg N Mg−<sup>1</sup> grain) were calculated using annual cumulative N2O emissions (kg N ha−<sup>1</sup> ) divided by the mean grain yield (Mg grain ha−<sup>1</sup> ) [10].

#### *2.4. Crop Yield Measurements*

For each cropland plot, three quadrats, 0.5×0.5 m<sup>2</sup> for wheat and 1×1 m<sup>2</sup> for maize, were randomly selected to measure crop yields. After the crop harvesting, the grains were collected separately and then oven dried at 70 ◦C for 48 h to constant weight to calculate the grain yield (Mg ha−<sup>1</sup> ).

#### *2.5. Auxiliary Measurements of Soil Parameters*

Throughout the experimental period, soil moisture, temperature, inorganic N (NO<sup>3</sup> <sup>−</sup> and NH<sup>4</sup> +), and dissolved organic C (DOC) concentrations were simultaneously measured for all the plots when gas samples were collected. The measurement procedures strictly followed the previous study of Zhou et al. [17]. For each plot, the topsoil moisture and temperature (5 cm in depth) were measured by a portable frequency domain reflector probe (RDS Technology Co. Ltd., Nanjing, Jiangsu, China) and a manual thermocouple thermometer (JM624, Tianjin Jinming Instrument Co. Ltd., Tianjin, China) with three replicates, respectively. Then the water-filled pore space (WFPS) was calculated based on the measured soil volumetric water content, bulk density and particle density (2.65 g cm−<sup>3</sup> ). At each plot, three soil cores (0–20 cm) were also randomly collected and completely mixed into one bulk sample. Then a 20 g fresh soil sample and 100 mL of 0.5 M K2SO<sup>4</sup> were used to extract soil NH<sup>4</sup> <sup>+</sup>, NO<sup>3</sup> −, and DOC, and an AA3 continuous flow analyzer (Bran + Lubbe, Norderstedt, Germany) was employed to colorimetrically analyze the filtered extracts. During the entire experiment period, the daily rainfall and mean air temperature were automatically observed using a meteorological station.

After the maize season, for each plot, topsoil samples (0–20 cm) were also collected to measure soil properties (soil pH, total N content [TN], soil organic carbon content [SOC], soil bulk density [BD], soil particle composition), following soil agro-chemical analysis procedures [33]. In detail, soil pH was measured in a 1:2.5 (soil-to-water [*w*/*v*]) water suspension using a DMP-2 mV/pH detector (Quark Ltd., Nanjing, China). SOC content was determined by wet digestion with H2SO4–K2Cr2O7, and TN content was determined by semi-micro Kjeldahl digestion using Se, CuSO<sup>4</sup> and K2SO<sup>4</sup> as catalysts. Soil BD was determined by the volumetric ring method. The pipette method was used to determine soil texture. Furthermore, soil aggregates were measured according to the methods reported by Six et al. [34], in which soils were separated into four aggregate size classes (<0.053, 0.053–0.25, 0.25–2, and >2 mm) by wet sieving, and the aggregate stability was quantified by the mean weight diameter (MWD) [35].

## *2.6. Contribution Rates of Tillage and Fertilization to Increased Soil N2O Emissions*

To quantify the effects of tillage and fertilization on increasing soil N2O emissions after land use conversion, the contribution rates of tillage and fertilization to increased soil N2O emissions were calculated as follows.

$$\text{CR}\_{\text{illage}} = (\frac{\text{NE}\_T - \text{NE}\_0}{\text{NE}\_{\text{TF}} - \text{NE}\_0}) \times 100\% \tag{1}$$

where *CR*tillage and *CR*fertilization are the relative contributions of tillage and fertilization to increased soil N2O emissions (%), respectively. *NE*0, *NET*, and *NETF* are the measured soil N2O emissions from the baseline forestland (CK) and the tillage without fertilization (NC-T and LC-T) and with fertilization (NC-TF and LC-TF) treatments, respectively. where *CR*tillage and *CR*fertilization are the relative contributions of tillage and fertilization to increased soil N2O emissions (%), respectively. *NE*0, *NET*, and *NETF* are the measured soil N2O emissions from the baseline forestland (CK) and the tillage without fertilization (NC-T and LC-T) and with fertilization (NC-TF and LC-TF) treatments, respectively.

#### *2.7. Statistical Analysis 2.7. Statistical Analysis*

The differences in soil N2O fluxes and soil environmental factors (i.e., soil moisture and temperature, NO<sup>3</sup> <sup>−</sup>, NH<sup>4</sup> <sup>+</sup> and DOC concentrations) between the different treatments were detected using one-way ANOVA analysis, followed by Duncan's range test (*p* < 0.05). The potential relationships between soil N2O fluxes and environmental factors were evaluated using Pearson's correlation analysis. However, before the correlation analysis, the soil N2O fluxes and environmental factors were primarily normalized by the ranked cases approach due to the original datasets not being normally distributed [17]. Moreover, multiple stepwise regression analysis was conducted to identify the key factors controlling soil N2O emissions from croplands after land use conversion. All statistical analyses were performed using the SPSS 20.0 software (SPSS Inc., Chicago, IL, USA) and OriginPro 2015 software (OriginLab Corp., Northampton, MA, USA). The differences in soil N2O fluxes and soil environmental factors (i.e., soil moisture and temperature, NO3−, NH4+ and DOC concentrations) between the different treatments were detected using one-way ANOVA analysis, followed by Duncan's range test (*p* < 0.05). The potential relationships between soil N2O fluxes and environmental factors were evaluated using Pearson's correlation analysis. However, before the correlation analysis, the soil N2O fluxes and environmental factors were primarily normalized by the ranked cases approach due to the original datasets not being normally distributed [17]. Moreover, multiple stepwise regression analysis was conducted to identify the key factors controlling soil N2O emissions from croplands after land use conversion. All statistical analyses were performed using the SPSS 20.0 software (SPSS Inc., Chicago, IL, USA) and OriginPro 2015 software (OriginLab Corp., Northampton, MA, USA).

#### **3. Results 3. Results**

#### *3.1. Environmental Conditions and Soil Properties 3.1. Environmental Conditions and Soil Properties*

forestland (average 52.6%) (*p* < 0.05).

During the whole experimental period, the total precipitation was 639.6 mm, and 67% occurred in the summer maize season (from June to October) (Figure 1). The daily mean air temperature changed from 4 to 31.2 ◦C with a mean of 16.9 ◦C. During the whole experimental period, the total precipitation was 639.6 mm, and 67% occurred in the summer maize season (from June to October) (Figure 1). The daily mean air temperature changed from 4 to 31.2 °C with a mean of 16.9 °C.

**Figure 1.** Air temperature and precipitation from November 2016 to October 2017. **Figure 1.** Air temperature and precipitation from November 2016 to October 2017.

Forestland conversion to cropland significantly influenced soil WFPS but not soil temperature at 5 cm depth (Figure 2). The WFPS values in the cropland sites (average 41.9%, 42.3%, 47.3% and 47.6% for NC-T, NC-TF, LC-T and LC-TF, respectively) were significantly lower than that in the Forestland conversion to cropland significantly influenced soil WFPS but not soil temperature at 5 cm depth (Figure 2). The WFPS values in the cropland sites (average 41.9%, 42.3%, 47.3% and 47.6%

for NC-T, NC-TF, LC-T and LC-TF, respectively) were significantly lower than that in the forestland (average 52.6%) (*p* < 0.05). *Sustainability* **2020**, *12*, x FOR PEER REVIEW 6 of 18

**Figure 2.** Temporal variations in soil temperature (**a**) and WFPS (**b**) at 5 cm depth. Abbreviations: WFPS, water-filled pore space; CK, control forestland; NC-TF and NC-T, newly converted cropland under tillage with and without fertilization, respectively; LC-TF and LC-T, long-term cropland under tillage with and without fertilization, respectively. **Figure 2.** Temporal variations in soil temperature (**a**) and WFPS (**b**) at 5 cm depth. Abbreviations: WFPS, water-filled pore space; CK, control forestland; NC-TF and NC-T, newly converted cropland under tillage with and without fertilization, respectively; LC-TF and LC-T, long-term cropland under tillage with and without fertilization, respectively.

Compared to forestland, tillage in the croplands significantly decreased the average soil NH4+ concentration (average 1.79 and 1.52 mg N kg−1 for NC-T and LC-T, respectively) but significantly increased the average soil NO3− concentration (average 7.23 and 5.54 mg N kg−1 for NC-T and LC-T, respectively) (*p* < 0.05; Figure 3a,b). However, tillage with fertilization not only significantly increased the average soil NH4+ concentration (average 16.15 and 12.55 mg N kg−1 for NC-TF and LC-TF, respectively) but also significantly increased the average soil NO3− concentration (average 27.74 and 31.10 mg N kg−1 for NC-TF and LC-TF, respectively) compared to forestland (*p* < 0.05; Figure 3a,b). Following mineral N fertilizer application, the soil NH4+ concentration in the NC-TF and LC-TF treatments quickly reached peaks of 203.14 and 146.16 mg N kg−1 in the wheat season and 23.51 and 27.64 mg N kg−1 in the maize season (Figure 3a), while the soil NO3− concentration in the NC-TF and LC-TF treatments quickly reached peaks of 165.80 and 154.45 mg N kg−1 in the wheat season and 55.19 and 45.70 mg N kg−1 in the maize season (Figure 3b). Compared to forestland, tillage in the croplands significantly decreased the average soil NH<sup>4</sup> + concentration (average 1.79 and 1.52 mg N kg−<sup>1</sup> for NC-T and LC-T, respectively) but significantly increased the average soil NO<sup>3</sup> <sup>−</sup> concentration (average 7.23 and 5.54 mg N kg−<sup>1</sup> for NC-T and LC-T, respectively) (*p* < 0.05; Figure 3a,b). However, tillage with fertilization not only significantly increased the average soil NH<sup>4</sup> <sup>+</sup> concentration (average 16.15 and 12.55 mg N kg−<sup>1</sup> for NC-TF and LC-TF, respectively) but also significantly increased the average soil NO<sup>3</sup> − concentration (average 27.74 and 31.10 mg N kg−<sup>1</sup> for NC-TF and LC-TF, respectively) compared to forestland (*p* < 0.05; Figure 3a,b). Following mineral N fertilizer application, the soil NH<sup>4</sup> <sup>+</sup> concentration in the NC-TF and LC-TF treatments quickly reached peaks of 203.14 and 146.16 mg N kg−<sup>1</sup> in the wheat season and 23.51 and 27.64 mg N kg−<sup>1</sup> in the maize season (Figure 3a), while the soil NO<sup>3</sup> − concentration in the NC-TF and LC-TF treatments quickly reached peaks of 165.80 and 154.45 mg N kg−<sup>1</sup> in the wheat season and 55.19 and 45.70 mg N kg−<sup>1</sup> in the maize season (Figure 3b).

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**Figure 3.** Temporal variations in soil NH4+ (**a**), NO3<sup>−</sup> (**b**) and DOC (**c**) concentrations. Vertical bars represent standard errors. Abbreviations: CK, control forestland; NC-TF and NC-T, newly converted cropland under tillage with and without fertilization, respectively; LC-TF and LC-T, long-term cropland under tillage with and without fertilization, respectively. **Figure 3.** Temporal variations in soil NH<sup>4</sup> <sup>+</sup> (**a**), NO<sup>3</sup> − (**b**) and DOC (**c**) concentrations. Vertical bars represent standard errors. Abbreviations: CK, control forestland; NC-TF and NC-T, newly converted cropland under tillage with and without fertilization, respectively; LC-TF and LC-T, long-term cropland under tillage with and without fertilization, respectively.

The soil DOC concentration significantly decreased after the conversion of forestland to cropland (*p* < 0.05, Figure 3c). In particular, long-term cultivation resulted in much lower soil DOC concentration (mean 51.30 and 50.54 mg C kg−1 for LC-T and LC-TF) than that in the short-term croplands (mean 66.37 and 68.30 mg C kg−1 for NC-T and NC-TF) (*p* < 0.05). The soil DOC concentration significantly decreased after the conversion of forestland to cropland (*p* < 0.05, Figure 3c). In particular, long-term cultivation resulted in much lower soil DOC concentration (mean 51.30 and 50.54 mg C kg−<sup>1</sup> for LC-T and LC-TF) than that in the short-term croplands (mean 66.37 and 68.30 mg C kg−<sup>1</sup> for NC-T and NC-TF) (*p* < 0.05).

Compared to forestland, land use conversion significantly decreased soil SOC, TN, C/N ratio, and bulk density (*p* < 0.05, Table 1). Moreover, compared to the newly converted croplands, the longterm croplands had significantly lower SOC and TN contents and C/N ratio but a higher bulk density (*p* < 0.05, Table 1). Conversion did not induce significant changes in soil texture in the short term; Compared to forestland, land use conversion significantly decreased soil SOC, TN, C/N ratio, and bulk density (*p* < 0.05, Table 1). Moreover, compared to the newly converted croplands, the long-term croplands had significantly lower SOC and TN contents and C/N ratio but a higher bulk density (*p* < 0.05, Table 1). Conversion did not induce significant changes in soil texture in the

however, long-term conversion increased the clay and silt contents and decreased the sand content (*p* < 0.05, Table 1). Furthermore, forestland conversion to cropland significantly decreased the short term; however, long-term conversion increased the clay and silt contents and decreased the sand content (*p* < 0.05, Table 1). Furthermore, forestland conversion to cropland significantly decreased the proportion of soil macroaggregates (0.25–2 mm and 2–8 mm) and the mean weight diameter (MWD) (*p* < 0.05, Table 2).

**Table 1.** Topsoil properties (mean ± SE) determined after maize harvest.


BD, bulk density; CK, control forestland; NC-TF and NC-T, newly converted cropland under tillage with and without fertilization, respectively; LC-TF and LC-T, long-term cropland under tillage with and without fertilization, respectively. a, b, c A different letter in the same row indicates a significant difference among different treatments (*p* < 0.05).

**Table 2.** Aggregate size distribution and mean weight diameter (MWD) (mean ± SE) determined after maize harvest.


CK, control forestland; NC-TF and NC-T, newly converted cropland under tillage with and without fertilization, respectively; LC-TF and LC-T, long-term cropland under tillage with and without fertilization, respectively. a, b, c A different letter in the same column indicates a significant difference among different treatments (*p* < 0.05).

## *3.2. Soil N2O Emissions*

Distinct temporal variations in N2O fluxes were observed in both forestland and cropland (Figure 4a). During the summer season, the N2O fluxes were higher than those during the other seasons. Compared to forestland, the N2O fluxes showed much greater temporal variations in the croplands. In the initial period of the wheat and maize season, tillage and fertilization practices induced pulse emissions of N2O that lasted several weeks and then decreased to base levels. For the croplands with only tillage, the peak N2O fluxes were 8.72 and 8.40 µg N m−<sup>2</sup> h −1 in the winter wheat season and 43.58 and 33.10 µg N m−<sup>2</sup> h −1 in the summer maize season for the NC-T and LC-T treatments, respectively. For the croplands with tillage and fertilization, the peak N2O fluxes were 21.56 and 56.99 µg N m−<sup>2</sup> h −1 in the winter wheat season and 185.61 and 152.00 µg N m−<sup>2</sup> h −1 in the summer maize season for the NC-TF and LC-TF treatments, respectively. This result indicates that fertilization could induce much higher N2O pulse emissions than tillage after land use conversion from forestland to cropland.

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**Figure 4.** Temporal variations in soil N2O emissions (**a**) and cumulative N2O fluxes (**b**). Vertical bars represent standard errors. Abbreviations: CK, control forestland; NC-TF and NC-T, newly converted cropland under tillage with and without fertilization, respectively; LC-TF and LC-T, long-term cropland under tillage with and without fertilization, respectively. **Figure 4.** Temporal variations in soil N2O emissions (**a**) and cumulative N2O fluxes (**b**). Vertical bars represent standard errors. Abbreviations: CK, control forestland; NC-TF and NC-T, newly converted cropland under tillage with and without fertilization, respectively; LC-TF and LC-T, long-term cropland under tillage with and without fertilization, respectively.

The N2O fluxes from forestland changed from 0.80 to 7.70 μg N m−2 h−1 over the whole experimental period, with a mean of 2.70 μg N m−2 h−1 (Figure 4a). For the croplands, the mean N2O fluxes were 6.49 and 12.44 μg N m−2 h−1 for NC-T and NC-TF, and 5.76 and 21.59 μg N m−2 h−1 for LC-T and LC-TF, respectively. Forestland conversion to cropland significantly increased the mean N2O fluxes (*p* < 0.05). Moreover, the average N2O fluxes were significantly greater from tillage with fertilization treatments than those from tillage without fertilization treatments (*p* < 0.05). This result shows again that tillage with fertilization had a much greater effect on increasing N2O emissions than tillage alone after land use conversion. The N2O fluxes from forestland changed from 0.80 to 7.70 µg N m−<sup>2</sup> h <sup>−</sup><sup>1</sup> over the whole experimental period, with a mean of 2.70 µg N m−<sup>2</sup> h −1 (Figure 4a). For the croplands, the mean N2O fluxes were 6.49 and 12.44 µg N m−<sup>2</sup> h −1 for NC-T and NC-TF, and 5.76 and 21.59 µg N m−<sup>2</sup> h −1 for LC-T and LC-TF, respectively. Forestland conversion to cropland significantly increased the mean N2O fluxes (*p* < 0.05). Moreover, the average N2O fluxes were significantly greater from tillage with fertilization treatments than those from tillage without fertilization treatments (*p* < 0.05). This result shows again that tillage with fertilization had a much greater effect on increasing N2O emissions than tillage alone after land use conversion.

The annual cumulative N2O emissions significantly increased after forestland conversion to cropland (*p* < 0.05, Table 3 and Figure 4b). Compared to forestland, the annual cumulative N2O emissions increased by 124% and 334% in the short-term croplands (NC-T and NC-TF) and by 76% and 491% in the long-term croplands (LC-T and LC-TF), respectively. Moreover, tillage with fertilization (NC-TF and LC-TF) significantly increased cumulative soil N2O emissions by 94% and 235%, compared to those from tillage without fertilization (NC-T and LC-T), respectively (*p* < 0.05, Table 3). Compared to the short-term conversion (NC-T and NC-TF), long-term tillage (LC-T) significantly decreased cumulative soil N2O emissions by 21%, while long-term tillage with fertilization (LC-TF) greatly increased cumulative soil N2O emissions by 36% (*p* < 0.05, Table 3). The annual cumulative N2O emissions significantly increased after forestland conversion to cropland (*p* < 0.05, Table 3 and Figure 4b). Compared to forestland, the annual cumulative N2O emissions increased by 124% and 334% in the short-term croplands (NC-T and NC-TF) and by 76% and 491% in the long-term croplands (LC-T and LC-TF), respectively. Moreover, tillage with fertilization (NC-TF and LC-TF) significantly increased cumulative soil N2O emissions by 94% and 235%, compared to those from tillage without fertilization (NC-T and LC-T), respectively (*p* < 0.05, Table 3). Compared to the short-term conversion (NC-T and NC-TF), long-term tillage (LC-T) significantly decreased cumulative soil N2O emissions by 21%, while long-term tillage with fertilization (LC-TF) greatly increased cumulative soil N2O emissions by 36% (*p* < 0.05, Table 3).



CK, control forestland; NC-TF and NC-T, newly converted cropland under tillage with and without fertilization, respectively; LC-TF and LC-T, long-term cropland under tillage with and without fertilization, respectively. a, b, c d, e A different letter in the same column indicates a significant difference among different treatments (*p* < 0.05).

Yield-scaled N2O emissions from the short-term converted croplands (NC-T and NC-TF) were significantly higher than those from the long-term converted croplands (LC-T and LC-TF) (*p* < 0.05, Table 3). After long-term plantation, the yield-scaled N2O emissions under tillage with fertilization treatment (LC-TF) were significantly greater than that from only tillage practice (LC-T) (*p* < 0.05, Table 3).

## *3.3. Relationships between N2O Fluxes and Soil Environmental Variables*

The correlations between N2O fluxes and soil environmental variables are presented in Figure 5. The soil N2O fluxes were significantly positively related to soil temperature, WFPS, NH<sup>4</sup> <sup>+</sup> and NO<sup>3</sup> − concentrations but significantly negatively related to DOC concentration for both forestland and cropland (*p* < 0.05). The further stepwise regression analysis indicated that variations in N2O fluxes from forestland were mainly regulated by soil WFPS and temperature (82%) (Table 4). However, after land use conversion, soil NO<sup>3</sup> <sup>−</sup> and NH<sup>4</sup> <sup>+</sup> availability and soil WFPS were the main factors influencing soil N2O emissions from croplands with only tillage, which explained 78% and 90% of the variations in N2O fluxes from the NC-T and LC-T treatments, respectively (Table 4). For the short-term tillage with fertilization treatment (NC-TF), soil DOC and NH<sup>4</sup> <sup>+</sup> availability and soil WFPS explained 74% of the variation in N2O fluxes (Table 4). While for the long-term tillage with fertilization treatment (LC-TF), N2O emissions were mainly regulated by soil NH<sup>4</sup> <sup>+</sup> and NO<sup>3</sup> − availability and WFPS, which explained 81% of the variation in N2O fluxes (Table 4).


**Table 4.** Stepwise multiple linear regressions between the soil N2O emissions and environmental factors.

ST, soil temperature; CK, control forestland; NC-TF and NC-T, newly converted cropland under tillage with and without fertilization, respectively; LC-TF and LC-T, long-term cropland under tillage with and without fertilization, respectively. Due to the original data not normally distributed, all the data sets were normalized before analysis.

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**Figure 5.** The correlations between soil N2O emissions and soil temperature (**a**,**b**), soil WFPS (**c**,**d**), soil NH4+ (**e**,**f**), NO3<sup>−</sup> (**g**,**h**) and DOC (**i**,**j**) concentrations for forestland (n = 87) and cropland (n = 348). **Figure 5.** The correlations between soil N2O emissions and soil temperature (**a**,**b**), soil WFPS (**c**,**d**), soil NH<sup>4</sup> <sup>+</sup> (**e**,**f**), NO<sup>3</sup> − (**g**,**h**) and DOC (**i**,**j**) concentrations for forestland (n = 87) and cropland (n = 348).

#### **4. Discussion**
