Introduction

N is an essential element for the development and growth of rice plant. The application of N fertilizer can significantly increase rice yields and is widely practiced in rice cultivation (Cai et al. 2007; Wu et al. 2016). As the agricultural production in China increases to meet the food demands with population growth, N fertilizer use also increases (Chen et al. 2008; Geng et al. 2016). The average rate of N application in rice production was around 193 kg ha−1 in 2006 in China, approximately 90% higher than the world average rate (100 kg ha−1) (Peng et al. 2006; Wu et al. 2016). However, N fertilizer is not used efficiently, and rice plant can take up only 20% to 30% of the N applied (Cai 2002; Vlek and Byrens 1986; Xing and Zhu 2000). High N fertilizer input leads to low N use efficiency (NUE) due to denitrification and leaching losses (Jiang et al. 2016; Moir et al. 2007), causing environmental problems. Therefore, it is critical to increase the NUE and decrease the N losses for achieving high economic profit and low environmental impacts of N fertilizers in paddy field ecosystems.

The low NUE in modern production systems is largely due to the high-nitrifying activity of soil (Chen et al. 2015; Guo et al. 2010). During nitrification, the relatively immobile NH4 + is converted into highly mobile NO3 −, thus facilitating N losses (Lan et al. 2013, 2015). Both Fan et al. (2007) and Malla et al. (2005) showed that the NUE significantly increased by inhibiting nitrification. Factors affecting nitrification activity, such as root exudates, quantity and form of N fertilizers, and crop managements, can influence crop NUE (Aulakh et al. 2001; Jiang et al. 2016; Nicolaisen et al. 2004; Sun et al. 2014). In addition, deep placement of ammonium-based N fertilizers into the anaerobic zones is recommended as it reduces nitrification rate and thus increase the NUE (Cai 2002; Lan et al. 2013).

Denitrification is considered to be one of the main N losses in flooded paddy soils, accounting for more than 40% of the applied fertilizer N (Zhu et al. 2011; Zhu and Chen 2002) and depends on nitrification and thus indirectly affects rice growth and the NUE (Pasda et al. 2001). Therefore, nitrification process is regarded as an indirect driver of N loss via denitrification during the flooded rice growing season (Chen et al. 1998; Lan et al. 2013; Malla et al. 2005; Wang et al. 2015). Among the factors affecting denitrification loss in paddy soils, NO3 − concentration is the most important one (Galloway et al. 2004; Ishii et al. 2011; Li et al. 2014; Nicolaisen et al. 2004; Pina-Ochoa and Alvarez-Cobelas 2006; Zhou et al. 2012; Zhu et al. 2011; Zhu and Chen 2002), because denitrification is a substrate-dependent process under flooding conditions (Buresh and De Datta 1990; Reddy and Patrick 1975; Reddy and Patrick 1986; Van Lujien et al. 1996). Ammonium fertilizer is the most commonly used N fertilizer in paddy fields. Nitrification occurs in aerobic microzones such as the rhizosphere of rice plants and interface between soil and standing water. Both NO3 − and NO2 − undergo denitrification when they diffuse into anaerobic bulk soils (Cai 2002). Zhou et al. (2012) reported that the rate of NO3 − production through nitrification process controlled denitrification rate in flooded soils. To gain deeper insight, several studies concerning the relationship between nitrification activity and denitrification loss in paddy soils have been done, but most of them have been carried out in controlled environments or only by using soils with a high nitrification; whereas, reports about the effects of nitrification process on denitrification loss involving rice plants are scarce.

The objectives of this study were to (1) compare the differences of rice growth, NUE, and denitrification loss in paddy soils with different nitrification activities and (2) evaluate the influence of soil nitrification activity on rice NUE and denitrification loss in soils. Since denitrification is a substrate-dependent process and NUE is significantly affected by soil nitrification process, we hypothesized that denitrification loss and NUE of rice plant would substantially be affected by nitrification activity in paddy soils under flooding conditions.

Materials and methods

Soil samples

Soil pH is a key factor for soil nitrification activity, and soil texture influences oxygen diffusion (Che et al. 2015). Therefore, six paddy soils were screened for soil pH and texture. Two paddy soils with low soil pH (5.2) were collected from Jiangxi province, in Longhushan (JS) (28° 15′ N, 116° 55′ E) and Yingtang (JC) developed from sandstone and quaternary red clay, respectively. Two paddy soils with soil pH around 5.8 were collected from Yixing (YX) (31° 17′ N, 119° 54′ E) and Jurong (JR) (31° 56′ N, 119° 10′ E), Jiangsu province and developed from alluvial and Xiashu loess, respectively. The remaining two paddy soils with high soil pH (7.8) were collected, one from Huai’an (HA) (33° 43′ N, 118° 86′ E) Jiangsu province, developed from lake sediments and the other from Yanting (SC) (31° 16′ N, 105° 27′ E), and from Sichuan province, developed from purple rock. All the soil samples were collected from plow layer (0–20 cm) in March 2014 before rice transplantation, aired-dried at room temperature, sieved (<2 mm) and well mixed, and stored in plastic bags. Soil chemical properties and texture of the six paddy soils are given in Table 1.

Table 1 Properties of the paddy soils
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Pot experiment

The pot experiment was conducted in a greenhouse, located at Nanjing Normal University, Nanjing city, Jiangsu province (Southern China) in May 2015. The glass greenhouse had the sides open to facilitate ventilation, and the inside temperature was kept at 30 ± 3 °C during the day and at 20 ± 2 °C in the night; natural illumination and humidity conditions were maintained until the end of the pot experiment.

A total of 24 PVC pots (four replicates for each of the six paddy soil) with 6 soils were used in this study. Two hundred grams of air-dried paddy soils (oven-dry basis) were weighted and placed into each pot (depth, 12.5 cm; internal diameter, 8 cm), flooded with deionized water, and puddled to a depth of about 1.5 cm to remove native soil NO3 −-N via denitrification. After 2 weeks, the four replicated pots of each soil were thoroughly mixed and redistributed in four pots. Soil samples from one of the four pots were taken to determine the exchangeable NH4 +-N and NO3 −-N concentrations. The three other pots were transplanted with two rice plants at tillering stage, obtained from a wet bed nursery established in the artificial climate box. Deionized water was added to the paddy soil every 2 days to maintain a flooding condition (around a water depth of 2.0 cm) throughout the experiment. 15N-labeled (NH4)2SO4 (10.32 atom% excess) was used as the N source and added at the rate of 50 mg N kg−1 soil. 15N-labeled (NH4)2SO4 solution equivalent to 50 mg N kg−1 was injected into soil by a pipette in one dose, to a depth ranging from 3 to 5 cm at the 2.5th day after the rice plants were transplanted. The pots were arranged in a completely randomized design throughout the experiment.

Plant and soil sampling

The rice plants (including roots and shoots) were harvested 5 days after N fertilizer application. Rice plant height and tiller number were measured and recorded. The roots and shoots of rice plants were washed with distilled water and dried at 65 °C until constant weight, and the relative biomass was measured. Dried shoots and roots were mixed and pulverized in a mortar to pass through a sieve (0.10 mm) to be used to analyze their N concentration and 15N atom% enrichment. After rice harvest, to get a representative soil and flooded water sample, soil and water were thoroughly mixed, and 25 g of the soil slurry were transferred into a 250-ml flask. After the soil slurry sampling, 80 ml of 2.5 M KCl were added into the flask, which was shaken for 1 h at 200 rpm at 25 °C (the concentration of KCl after mixing with soil slurry was about 2 M). The extracts were filtered through a filter paper and the concentrations of exchangeable NH4 + and NO3 − in the filtrate were determined with a continuous-flow analyzer (Skalar, Breda, Netherlands). Exchangeable NH4 + and NO3 − were separated for isotopic composition and their 15N enrichment determined by using isotope ratio mass spectrometer (Sercon Ltd., Crewe, UK) as reported by Zhang et al. (2011). Briefly, a portion of the filtrate was steam-distilled with MgO to separate NH4 + on a stream distillation system, thereafter the sample in the flask was distilled again after addition of Devarda’s alloy to reduce NO3 − to NH4 +. The liberated NH3 was trapped in boric acid solution. The trapped N was acidified and converted to (NH4)2SO4 using 0.02 mol L−1 H2SO4 solution. The H2SO4 solution, which was then dried at 65 °C in an oven and analyzed for 15N abundance. The KCl-extracted soil was suspended with distilled water to remove residual mineral N. After 6 h, the soil suspension was filtered through a quantitative filter paper, rinsed another two more times with distilled water, and oven-dried at 55 °C to measure the soil insoluble organic N concentration and the relative 15N enrichment by mass spectrometry. All data from wet soil analysis were calculated on an oven-dry weight basis.

Plant and soil measurements

The methods for measuring soil NH4 +, NO3 −, and organic N concentrations in soil samples have previously been described (Lan et al. 2013; Yang et al. 2016). The measurement of 15N enrichment of rice plant samples was described by Zhang et al. (2014) and Wang et al. (2015).

The soil total N was determined by semi-micro Kjeldahl digestion with Se, CuSO4, and K2SO4 as catalysts; the soil organic C was determined by wet digestion with H2SO4-K2Cr2O7, and soil pH was measured with a DMP-2 mV-pH detector (Quark Ltd., Nanjing, China) in a soil/water ratio of 1:2.5 (V/V) slurry (Cheng et al. 2014). The net nitrification rate of soil was determined by incubation for 7 days at 25 °C and at 60% water holding capacity as described by Yang et al. (2016). Briefly, for each soil, six 250 ml Erlenmeyer flasks were prepared with 20 g air-dried soil (oven-dry basis) and pre-incubated for 4 days at 25 °C after adjusting the soil water content to 40% water holding capacity. One milliter of (NH4)2SO4 solution was added to each of flasks at a concentration of 50 mg NH4 +-N kg−1 soil. The soil was adjusted to 60% water holding capacity and incubated for 7 days at 25 °C. The soils were extracted at 1 and 7 days to determine the concentration of NO3 −. The net nitrification rates of soil were calculated by the increase in NO3 −-N concentration divided by the incubation time (as shown in Table 1).

Calculation and statistical analyses

N use efficiency of rice plant was calculated by Eq. (1).

$$ \mathrm{N}\kern0.5em \mathrm{use}\kern0.5em \mathrm{efficiency}\kern0.5em \left(\%\right)\kern0.5em =\kern0.5em \frac{\mathrm{rice}\kern0.5em \mathrm{plant}\kern0.5em {}^{15}\mathrm{N}}{\mathrm{added}\kern0.5em {}^{15}\mathrm{N}}\kern0.5em \times \kern0.5em 100\% $$
(1)

Since 15N-NH4 + was applied at 3–5 cm depth, ammonia volatilization could be ignored (Liu et al. 2015) and denitrification N loss was calculated by considering the un-recovery of 15N by Eq. (2).

$$ \begin{array}{l}\mathrm{Denitrification}\kern0.5em \mathrm{loss}\kern0.5em \left(\%\right)=\kern0.5em \frac{\mathrm{added}\kern0.5em {}^{15}\mathrm{N}\kern0.5em -\left({}^{15}\mathrm{N}{{\mathrm{H}}_4}^{+}\kern0.5em +\kern0.5em {}^{15}\mathrm{N}{{\mathrm{O}}_3}^{-}\kern0.5em +\kern0.5em {\mathrm{organic}}^{15}\mathrm{N}+\kern0.5em \mathrm{rice}\kern0.5em \mathrm{plant}\kern0.5em {}^{15}\mathrm{N}\right)}{\mathrm{added}\kern0.5em {}^{15}\mathrm{N}}\kern0.5em \\ {}\kern18.5em \times \kern0.5em 100\%\end{array} $$
(2)

where 15NH4 +, 15NO3 −, and organic 15N represented the 15N recovered as NH4 +, NO3 −, and insoluble organic N in soil, respectively, after rice harvest.

One-way ANOVA analysis was used to test the significant differences in rice height, tiller number, biomass, NUE, and denitrification loss among the paddy soils. The difference was considered significant for P < 0.05. Nonlinear analysis was applied to examine the correlation between soil nitrate ratio and net nitrification rate, soil native inorganic N (NH4 + and NO3 −) concentrations and rice height and biomass, nitrification activity and denitrification loss. Multiple linear regression was used to examine the relationship among the NUE, net nitrification rate, and the soil native exchangeable NH4 + content. All tests were conducted in SPSS 16.0.

Results

As shown in Table 1, the net nitrification rate determined at 60% water holding capacity varied greatly among the studied soils, ranging from 0.48 mg N kg−1 day−1 in JC soil to 5.72 mg N kg−1 day−1 in HA soil. Even after 2 weeks of pre-flooding, the soil NO3 − was not denitrified completely and NO3 −-N contents ranged from 5.67 to 39.3 mg N kg−1 soil. The nitrate ratio, defined as N-NO3 −/(NH4 + + NO3 −), correlated significantly with the net nitrification rate (P < 0.01) after 2 weeks flooding (Fig. 1).

Fig. 1
figure 1

The relationship between the nitrate ratio (N-NO3 −/N-(NH4 + + NO3 −)) after 2 weeks of pre-flooding and the net nitrification rate determined at 60% water-holding capacity

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Seven and half days after transplanting, height, tiller, and biomass of rice plant varied with the studied soils (Table 2). The rice plant in JS soil had the highest plant height (37.3 cm), biomass (1.52 g pot−1), and tiller number (4.7 pot−1), while the lowest values were observed in HA or SC soils. One-way ANOVA analysis showed significant differences in plant height and biomass (P < 0.05), while the difference in tiller number was not significant (P > 0.05, Table 2) among the different paddy soils.

Table 2 Rice plant height, tiller number, biomass, and N use efficiency
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The correlation analysis showed that both plant height and biomass of rice were significantly increased by increasing the native NH4 +-N content (P < 0.01, Fig. 2a, b). In contrast, by increasing soil native NO3 −-N content, both rice plant height and biomass were significantly decreased (P < 0.05) (Fig. 2c, d). The rice plant height, biomass, and soil net nitrification rate correlated negatively to the R 2 values of 0.822 (P < 0.01) and 0.947 (P < 0.01), respectively (Fig. 3).

Fig. 2
figure 2

The relationships between native NH4 +-N and NO3 − concentrations in soils and plant height (a, c) and biomass (b, d) at tillering stage (5 days)

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Fig. 3
figure 3

The relationships between net nitrification rate of soils with plant height (a) and biomass (b) at tillering stage (5 days)

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Similar to the growth pattern, the NUE of rice varied greatly, ranging from 54 to 82% (Table 2). The highest NUE was observed in JS soil, and the lowest in SC soil. ANOVA analysis showed that the differences in the NUE were significant among the tested soils (P < 0.05), but no correlation was observed between net nitrification rate and rice NUE. Considering that soil native NH4 +-N is also an important source for rice, the multiple linear regression analysis was used to analyze the correlation between net nitrification rate, the ratio of added fertilizer NH4 +-N content to soil native NH4 +-N content and rice NUE. The result showed the significant relationship between rice NUE with soil net nitrification rate (NR), and the ratio of added fertilizer NH4 +-N content to soil native NH4 +-N content (ratio). This relationship is represented by Eq. (3):

$$ \mathrm{N}\kern0.5em \mathrm{use}\kern0.5em \mathrm{efficiency}\kern0.5em =58.83+11.94\times \mathrm{ratio}-15.97\times \mathrm{N}\mathrm{R}\kern0.5em \left({R}^2=0.929, P<0.05\right) $$
(3)

The P values of the intercept, ratio and NR of the equation were 0.000, 0.011, and 0.009 respectively, revealing significant effects of all the equation parameters on the NUE.

Denitrification loss, expressed as 15N unrecovered at the end of the pot experiment, is shown in Fig. 4. The denitrification loss in the tested soils ranged from 5.3 to 17.4%. The denitrification losses of JS (5.3%) and JC soils (6.4%) were significantly lower than that in the HA soil, with YX and JR soil inbetween. A significant difference was detected in denitrification losses among the tested soils. The denitrification losses and net nitrification rates correlated significantly (R 2 = 0.896, P < 0.01, Fig. 5).

Fig. 4
figure 4

15N losses by denitrification in paddy soils 5 days after application as 15N-(NH4)2SO4 under flooded conditions

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Fig. 5
figure 5

The relationship between net nitrification rates and denitrification losses in paddy soils 5 days after 15N-(NH4)2SO4 application

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Discussions

In this study, soil native NO3 − of HA and SC soils were not completely denitrified after 2 weeks of pre-flooding incubation (Table 1). This may be due to the insufficient supplies of easily decomposable organic C required for denitrification. Soil denitrification is positively correlated with the amount of soil easily decomposable organic C (D’Haene et al. 2003; Lan et al. 2015; Xu and Cai 2007). The occurrence of nitrification under the flooding conditions would be another reason (Yang et al. 2016) explaining the high NO3 − concentration of HA and SC soils at the end of the pre-flooding incubation.

Our previous study showed that nitrification activities of paddy soils were not sensitive to oxygen concentrations probably due to the adaptation of nitrifiers to low oxygen concentration in flooded paddy soils (Yang et al. 2016). It was reported that nitrifiers are well adapted to local conditions (Myers 1975). For instance, AOA could adaptively grow in acid paddy soils (Huang et al. 2014) and the abundances of AOA were significantly higher in paddy soils than in upland soils (Alam et al. 2013; Wang et al. 2016). The adaption of AOA to low oxygen concentration could explain the high abundances of AOA amoA gene in flooded paddy soils (Huang et al. 2014; Song and Lin 2014). However, there are contradictory opinions (Lim et al. 2016; Yang et al. 2016), and the adaptation of nitrifiers to low oxygen concentration in paddy soil needs to be further investigated. The significant positive relationship between the nitrate ratio (N-NO3 −/N-(NH4 + + NO3 −)) after 2 weeks of pre-flooding and the net nitrification rate determined at 60% water holding capacity (Fig. 1) implied that the nitrification rates under flooded conditions paralleled those under aerobic conditions.

Rice is known as one of the NH4 +-favoring plants (Cai 2002; Chen et al. 2013; Sun et al. 2015). The occurrence of nitrification initiates a competition for NH4 + between rice plant and nitrifiers, leading to a decrease in NH4 + uptake by rice plants and thus to a decrease in the NUE. In this study, the decrease in the NUE was indeed significantly correlated with the increase in the nitrification rate (Eq. 3). The presence of soil native NH4 + decreased the chance of added NH4 + to be taken up by rice plants, thus the NUE also increased significantly with the ratio of added NH4 + to soil native NH4 + (Eq. 3).

In this study, the increase in soil native NO3 − concentration was significantly correlated with biomass and plant height of rice plant (Fig. 2c, d), probably due to the oxidation of NH4 + to NO3 −, which reduced the NH4 +, the most important nutrient for rice plant. Therefore, the negative relationships did not mean that NO3 − inhibited rice plant growth but confirmed that available N for plant uptake decreased by increasing nitrification rate because of the competition for NH4 + between nitrifiers and plant.

We observed that the NUE in the pot experiment (54 to 82%) was substantially higher than that measured in the field (20 to 30%) (Cai 2002; Xing and Zhu 2000). This could be attributed to the experimental conditions. Firstly, soil N supply was strictly limited by the low amounts of soil used for rice growth (200 g pot−1) than in the field. Secondly, the N fertilizer was added as solution and thus it was easily taken up by rice plants (Jiang et al. 2004). Thirdly, the N fertilizer was applied at the tillering stage when rice plants had the highest N uptake rate (Peng et al. 2006).

Because 15N-(NH4)2SO4 was injected into the soil at 3–5 cm soil depth, ammonia volatilization could be ignored (Liu et al. 2015) and the unrecovered 15N in the soil and rice plant system could be attributed to N losses via denitrification and anaerobic ammonium oxidation (anammox) processes. Denitrification was thought to be the sole driver of microbial N2 production process in paddy soils before the discovery of anammox, which has been detected recently in a wide range of ecosystems including river sediments (Zhu et al. 2013), marine ecosystems (Hu et al. 2011), and agricultural drainage ditches (Shen et al. 2016) by using 15N isotope technique. However, the potential contributions of anammox to total N2 production varied greatly, ranging from almost zero to 70% (Thamdrup and Dalsgaard 2002; Trimmer et al. 2003). Although anammox was detected in paddy fields in China (Li et al. 2016; Zhou et al. 2016), the contribution of denitrification was 21.0 nmol N g soil−1 h−1, higher than anammox in the surface paddy soils. Li et al. (2016) also demonstrated that denitrification might be regarded as the main N loss pathway in our paddy soils. Denitrification loss significantly increased by increasing the net nitrification rate of the studied soils (Fig. 5), and this depends on the fact that nitrification provides the substrate NO3 − for denitrification (Zhou et al. 2012; Lan et al. 2015). During the pre-incubation with rice plants, denitrification seemed to be limited by the supplies of easily decomposable organics, as shown by the high NO3 − contents after 2 weeks of flooding (Table 1). In contrast, the NO3 − contents in all the tested soils were lower than 1 mg N kg−1, and there were no significant differences in the NO3 − contents between the studied soils after the rice harvest, at 7.5 days (data not shown).

Most plants release roots exudates (sugars, amino acids, organic acids, etc.) that either stimulate or inhibit soil N reactions (Dendooven et al. 2010; Subbarao et al. 2015). The presence of rice plants and releases of root exudates might have stimulated denitrification potential, and nitrification might have been the limiting process.

Conclusions

In this study, rice NUE and N loss via denitrification varied greatly in the tested paddy soils and were significantly affected by nitrification activity confirming our hypothesis that the increase in the nitrification reduces rice NUE and increases N loss in rice paddy soils. Indeed the nitrification of NH4 + into NO3 − decreases the NH4 + uptake by rice plant and provides a substrate for denitrification. It should be emphasized that the pot experiment was conducted under conditions with soil native N supplies limited for rice plant uptake. Therefore, field investigations are needed to evaluate the role of nitrification in determining the NUE and denitrification losses in paddy field ecosystems.