Impact of soil warming on the activity and abundance of nitrifiers under nitrogen fertilization conditions*

Tatoba R Waghmode, ZHANG Xinyuan, DONG Wenxu, ZHANG Chuang,2, HU Chunsheng**

Impact of soil warming on the activity and abundance of nitrifiers under nitrogen fertilization conditions*

Tatoba R Waghmode1†, ZHANG Xinyuan1,2†, DONG Wenxu1, ZHANG Chuang1,2, HU Chunsheng1**

(1. Key Laboratory of Agricultural Water Resources, Chinese Academy of Sciences / Hebei Laboratory of Agricultural Water-Saving / Center for Agricultural Resources Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Shijiazhuang 050022, China; 2. University of Chinese Academy of Sciences, Beijing 100049, China)

The first step of nitrification (i.e., the oxidation of ammonia to nitrate) is catalyzed by nitrifiers, such as ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA). However, the impact of soil warming on the activity and abundance of nitrifiers under different nitrogen (N) fertilization conditions remains poorly understood. A long-term field warming experiment has been conducted since October 2008 at the Luancheng Agro-Ecosystem Experimental Station of Chinese Academy of Sciences in the North China Plain, during which soil temperature was increased by 1.5 ℃ using infrared heaters (power, 1 000 W) placed 2 m above the soil surface. In 2018, we investigated soils from the control (no warming) and warming treatment plots for potential nitrification rate (PNR), abundance of AOB and AOA at 10 cm and 20 cm soil depth under two N fertilization conditions: without N fertilization (N0) and with 240 kg(N)∙hm-2∙a-1fertilization (N1). Soil PNR, nitrate (NO3−-N), and ammonium (NH4+-N) contents were spectrophotometrically assessed, and the abundance of functional genes was investigated via real-time quantitative PCR. Warming increased PNR and NO3−-N content under N1 treatment and decreased them under N0 treatment (< 0.05). Moreover, warming significantly increased AOB abundance under N1 treatment (< 0.05), whereas it decreased the abundance of both AOA and AOB under N0 treatment, at both soil depths. Compared with N0, N1 exhibited substantial decrease in AOA/AOB ratio, suggesting that compared with warming without N fertilization, warming with N fertilization exhibited higher stimulation of AOB growth than of AOA growth. Conclusively, this study suggests that AOB significantly and positively responded to warming with N fertilization, whereas both AOA and AOB significantly and negatively responded to warming without N fertilization.This study provides an understanding of nitrifier activity and the response of ammonia-oxidizing microorganisms to warming conditions and N availability.

Climate warming; Nitrifier activity; Ammonia-oxidizing microorganism; N availability

Nitrification is one of the fundamental steps in the nitrogen (N) cycle. It is responsible for the oxidation of ammonia to nitrite via nitrate catalyzed by nitrifiers, such as ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA) (Kowalchuk and Stephen, 2001). The oxidation of ammonia is considered the rate-limiting step in nitrification, which has significant control over the balance between immobile ammonium and more mobile nitrite and nitrate, and is therefore critical for plant N availability and groundwater nitrate leaching (Qian and Cai, 2007; Singh et al., 2010; Cui et al., 2016). Therefore, the understanding of nitrification is essential, considering its impact on environmental quality and the efficiency of N fertilizer used (Kowalchuk and Stephen, 2001; Zhou et al., 2013).

Potential nitrification rate (PNR), an index of nitrification activity for nitrifiers, such as AOA and AOB, is significantly influenced by climate change (in terms of temperature), vegetation type, soil properties (such as soil moisture, pH, organic matter, and mineral N content), and microbial community (Sahrawat, 2008). Similarly, a previous study found that elevated temperatures caused an increase in nitrification rate (Grundmann et al., 1995). So far, bacteria (AOB) have been considered the dominant taxa responsible for ammonia oxidation; however, recently a study found that ammonia-oxidizing activity was more abundant in archaea (AOA) than in bacteria (AOB) present in agricultural soils (Leininger et al., 2006). Moreover, there is increasing evidence suggesting that there are differences between AOA and AOB at the cellular, genomic, and physiological levels and these differences lead to different responses to resource partitioning, which has been reduced owing to climate change (Jia and Conrad, 2009; Hu et al., 2016). Compared with AOB, AOA prefer ammonia-poor and acidic environments owing to their high affinity for ammonia (Martens-Habbena et al., 2009; He et al., 2012); however, AOB play a significant role in nitrification under N-rich and alkaline environments (Verhamme et al., 2011; Xia et al., 2011). Further understanding of the nitrification process is needed with respect to ammonia oxidation (in terms of nitrification rate), functional microbial guilds involved in N cycling, and nutrient interactions in agricultural ecosystems (Kowalchuk and Stephen, 2001).

Currently, the mean global surface temperature has increased by 0.85 ℃ from the year 1880 to 2012 and is estimated to increase by 1.5-3.6 ℃ by the end of the 21stcentury (IPCC, 2013). Temperature is a key factor that regulates many terrestrial biogeochemical processes, such as N mineralization, nitrification, and denitrification, and soil warming can strongly impact N cycling (Smith, 1997; Rustad et al., 2001). A number of studies found that the rise in soil temperature has changed ammonia (Rustad et al., 2001), nitrate (Rinnan et al., 2009), and total nitrogen (Patil et al., 2010) contents in soils. Moreover, this temperature change has impacted the nitrification activity and abundance of ammonia oxidizers; however, differential responses have been observed in different soils. For example, the increase in temperature has shown to either increase, decrease, or have no effect on the community structure and abundance of AOB or AOA in grasslands (Horz et al., 2004), forests (Horz et al., 2004), dryland forests (Hu et al., 2016), and maize () cropping soils (Cui et al., 2016). However, these studies were limited to microcosm and growth chamber at monolith levels and mainly focused on forest, dryland forest, grassland, and maize cropping soils. Thus, the impact of simulated warming on the nitrification activity and abundance of AOA and AOB under different N fertilization conditions in wheat () field soil remain poorly understood.

This experiment aimed to assess the impact of soil warming (by 1.5℃) on the nitrification activity and abundance of AOA and AOB under different N fertilization conditions. We tested the following hypotheses: i) nitrification activity differs under warming conditions with different fertilization treatments; and ii) both AOA and AOB differently respond to soil warming, N availability, and different soil depths.

1 Materials and methods

1.1 Experimental design

Soil sampling field was located at the Luancheng Agro-Ecosystem Experimental Station of Chinese Academy of Sciences (37°53′N, 114°41′E) in the North China Plain, China. The field was planted in October 2017 with the local winter wheat cultivar ‘Shixin 828’. Wheat rows were spaced out in 20 cm intervals. The soil texture was sandy loam, and the soil bulk density was 1.27 g∙cm−3with a pH of 8.1, total C of 25.9 g∙kg−1, and total N of 1.1 g∙kg−1in the top 0-20 cm of soil (Liu et al., 2016). In warming plots, six pairs of infrared heaters (size 2 m × 0.02 m with rated power of 1 000 W) were installed 2 m above ground at the center of the plots in October 2008. The heater power was continuously turned on. The plot dimension was 4 m × 4 m, and the successive radiation region was 2 m × 2 m. In the control plot, six pairs of heaters (without power) with a similar framework were installed to mimic the shadowing effect of the heating frames.

A two-factor design was applied using two warming treatments (warming: expected warming of soil by 1.5 ℃ on average over 10 years at 5 cm soil depth; control: no warming) and two N fertilization treatments (N0: without N fertilization; N1: 240 kg(N)∙hm−2∙a−1fertilization). This resulted in four separate treatments: control without N fertilization, warming without N fertilization, control with N fertilization, and warming with N fertilization. Each treatment had three replicates. Furthermore, irrigation was applied twice, in April and May. Soil sampling was performed in May 2018, one week after irrigation and N fertilizer application, at wheat heading stage from 0-10-cm (10 cm) and 10-20-cm (20 cm) soil depth. Four soil cores were obtained from each triplicate plot and mixed together to prepare composite samples. The soil samples were carried in an icebox to the lab and sieved through a 2 mm sieve to remove stones. The sampled soil was divided into two parts: one part was stored at 4 ℃ for PNR measurement and mineral N analysis, and the other was stored at −80 ℃ for functional (AOB and AOA) gene analysis.

1.2 Soil analysis

The mean monthly soil temperature, obtained during May at the time of soil sampling, (0-5-cm soil depth) and soil moisture (0-10-cm and 10-20-cm soil depth) were measured using an automated data logger (CR 10X, Campbell, CA, United States) and time-domain reflectometry from experimental plots, respectively. Temperature and moisture were monitored on a daily basis (each reading was taken at 1 h interval for 24 h). Soil ammonium (NH4+-N) and nitrate (NO3−-N) were extracted with 2 mol∙L−1KCl by shaking the samples for 30 min at 150 rpm, and filtrates were measured using spectrophotometry (UV-2450, Shimadzu, Japan), as per previously reported methods (Page et al., 1982). PNR was measured using a previously published method (Kurola et al., 2005). In brief, 5.0 g of soil samples in triplicate was incubated in 20 mL phosphate buffer containing ammonium sulfate and potassium chlorate at 25 ℃ for 24 h in the dark; after filtration, PNR was spectrophotometrically measured (540 nm) by estimating the nitrite produced in the soil.

1.3 DNA extraction and qPCR

We used 0.5 g of soil samples in triplicates to extract total genomic DNA using an E.Z.N.A.® Soil DNA Kit (Omega Bio-tek, Inc., Norcross, GA, United States), according to the manufacturer’s instructions. DNA quality was checked using 1% agarose gel electrophoresis, and DNA was quantified using NanoDrop (NanoDrop- 2000C Tech, United States). Then, DNA samples were stored at −20 ℃ until qPCR was performed. The qPCR amplification of AOB and AOA genes was performed with primer pairs asA- 1F/A-2R (Jin et al., 2010) and Arch_AF/Arch_AR (Francis et al., 2005), respectively, using protocols described in a previous study (Waghmode et al., 2018).

1.4 Statistical analysis

Student’s t-test (< 0.05) was performed to examine the effect of different treatments (warming and N fertilization) on soil biochemical parameters. Two-way ANOVA combined with a post-hoc test (HSD,< 0.05) was performed to assess the main and interactive effects of warming and fertilization on mineral N (NO3−-N and NH4+-N) content and abundance of AOB and AOA. Pearson’s analysis was conducted to assess the significance and strength of correlations among PNR, mineral N content, and abundance of AOB and AOA. All statistical analyses were performed using SPSS, and three replicates were used for each treatment for the statistical analyses.

2 Results

2.1 Soil temperature, soil moisture, and mineral N (NO3−-N and NH4+-N)

Warming increased the soil temperature by 1.31-1.37 ℃, which is very close to expected warming temperature, and decreased soil moisture content under the N1 treatment (Fig. 1a & b). Warming decreased soil NO3−-N content compared with the control under N0 treatment (< 0.05); however, warming increased soil NO3−-N under the N1 treatment at both soil depths (< 0.05). The NO3−-N content was considerably higher in the N1 treatment than in the N0 treatment and was considerably higher in the surface layer (10 cm) soil than in the deep layer (20 cm) soil (Fig. 1c). Soil warming only significantly affected NH4+-N content under N0 treatment at both soil layers, and it decreased NH4+-N content compared with the control in 20 cm soil depth (Fig. 1d).

Fig. 1 Effect of soil warming on temperature (a), moisture (b), and contents of nitrate (c) and ammonium (d) under different N fertilization conditions: N0 (without N fertilization) and N1 (240 kg(N)∙hm-2∙a-1) at 0-10-cm (10 cm) and 10-20-cm (20 cm) soil depth.

Different letters indicate significant differences between warming and control at< 0.05 (Student’s t-test). Error bars indicate standard deviation of the mean (= 3).

2.2 Potential nitrification rate and abundance of AOB and AOA

Potential nitrification rate (PNR) was higher under the N1 treatment than under the N0 treatment (Fig. 2a). Warming under the N1 treatment, as opposed to the control, led to significantly higher PNR (< 0.05); however, PNR was significantly lower under the N0 treatment in both soil layers. The application of N fertilizer significantly stimulated AOB growth at both soil depths, and compared with the control under the N1 treatment, the warming treatment resulted in higher AOB abundance (< 0.05; Fig. 2b). In contrast, AOA abundance was lower in the warming treatment than in the control treatment (< 0.05), except at 20 cm soil depth under the N1 treatment (Fig. 2c). AOB and AOA abundance was higher in the surface layer (10 cm) than in the deep layer (20 cm). AOB abundance was much higher under the N1 treatment than under the N0 treatment. The AOA/AOB ratio was lower under warming conditions and substantially lower under the N1 treatment than under the N0 treatment (Fig. 2d). Two-way ANOVA showed that warming conditions or fertilization alone and their interaction had a significant correlation with AOB abundance (< 0.001) and NO3−-N content (< 0.01), whereas fertilization, rather than warming, had a strong correlation with AOA (< 0.01; Supplementary Table 1).

2.3 Relationship between nitrification activity, abundance of AOA and AOB, and mineral N content

In the N0 treatment, a positive correlation was found between AOB and NO3−-N in the 0-10-cm soil depth (< 0.05) and between PNR, NO3−-N, and NH4+-N in the 10-20-cm soil depth (< 0.01). Although the relationship of PNR with AOA or AOB was not significant, it was slightly stronger with AOA than with AOB (Table 1). In the N1 treatment, PNR showed a significant positive correlation with NO3−-N (< 0.05) and AOB (< 0.01). Moreover, PNR showed a more significant positive correlation with AOB than with AOA under the N1 treatment at both soil depths. These correlations suggested that lower N fertilization had a stronger effect on AOA than on AOB under warming conditions.

3 Discussion

The quantification of functional genes involved in nitrification provides useful information on the dynamics of N-cycling microbial population abundance in response to warming under different N fertilization conditions. The NO3−-N content was significantly higher under warming with N fertilization than under warming without N fertilization owing to higher nitrification activity (< 0.05). These results are in accordance with those of a previous study conducted by Xu et al. (2016). They found significantly higherNO3−-N content under warming with N fertilization than under warming without N fertilization in an open-top chamber experiment on vegetable soil. PNR measured in our experiment was significantly influenced by warming under both fertilization treatments (Fig. 2a) and was significantly higher under warming with N fertilization than under warming without N fertilization, which could be because of the higher N substrate availability for nitrifier activity and could explain the significant interactive effect of warming and fertilization on nitrifier among numerous climatic and environmental factors of soil nitrification, which is mainly controlled by nitrifiers. Similarly, nitrifier contribution to nitrification is determined by enzyme activity, which is sensitive to temperature and exhibits a direct response through acclimation and thermal adaptation to warming conditions (Karhu et al., 2014; Tourna et al., 2008). In this study, we concluded that the growth of AOB population stimulated by warming was much higher under the warming conditions than under the control conditions (Fig. 2b), implying that elevated soil temperature may provide beneficial conditions for nitrifiers by promoting cell survival and maintenance and reducing the lag time for cell growth (Avrahami and Bohannan, 2007). Additionally, previous studies reported higher AOB abundance in vegetable field soils under warming with N fertilization than under warming without N fertilization (Xu et al., 2016). Furthermore, Long et al. (2012) reported that compared with warming alone, a combined treatment of soil warming and N fertilization had a more profound and significantly increasing effect on AOB abundance in forest soils. Our results indicate that fertilizer application could magnify climate warming effects on the N-cycling processes through the stimulation of PNR to a higher rate as observed under N fertilization condition, and lower PNR under warming without N fertilization condition may be because of the limited availability of N substrate for nitrifier activity.

Fig. 2 Effect of warming on the potential nitrification rate (PNR, a), abundance of AOB (b), AOA (c), and AOA/AOB ratio (d) under different N fertilization conditions: N0 (without N fertilization) and N1 (240 kg(N)∙hm-2∙a-1) at 0-10-cm (10 cm) and 10-20-cm (20 cm) soil depth

Different letters indicate significant differences between warming and control at< 0.05 (Student’s t-test). Error bars indicate standard deviation of the mean (3).

Table 1 Pearson’s correlation (r values) analyses between PNR (nitrification activity), nitrifier abundance (AOA and AOB), and mineral N (NO3--N and NH4+-N) content under N fertilization conditions: N0 (without N fertilization) and N1 (240 kg(N)∙hm-2∙a-1) at 0-10-cm (10 cm) and 10-20-cm (20 cm) soil depth

*, **: correlation coefficient is significant at the< 0.05,< 0.01 levels, respectively (2-tailed).

Compared with the control, warming without N fertilization exhibited significantly low AOA abundance. AOA and PNR were significantly higher under the control treatment than under warming (< 0.05), and AOA showed a stronger and more positive correlation with PNR and NO3−-N under the N0 treatment (Table 1). A previous study reported that AOA’s high metabolic efficiency enables it to thrive in oligotrophic environments (Konneke et al., 2014). However, the lower AOA abundance under the N0 treatment in our study may be because of competition for N acquisition between plants and nitrifiers aggravated by warming conditions under limited N availability (Andresen et al., 2010). The marginal reduction in AOA/AOB ratio under the N1 treatment in warming conditions was mainly because of the significant growth of AOB, as opposed to AOA. A previous study also reported similar significant decrease in AOA/AOB ratio under warming and N fertilization conditions (Xu et al., 2016), indicating that N availability could be a determining factor for the sensitivity of AOB and AOA abundance to artificial climate changes. The AOA/AOB ratio was lower in the surface layer than in the deep layer under both fertilization conditions, possibly owing to a decrease in AOB over AOA in the deep layer. These results were supported by those of the study by Leininger et al. (2006), who found that this ratio increased with soil depth owing to a decrease in AOB over AOA in forest soil. Two-way ANOVA showed that both fertilization and warming had a significant impact on AOB compared with AOA; however, compared with warming, only fertilization had a strong impact on AOA (Supplementary Table 1). N fertilization significantly enhanced AOB, as opposed to AOA, under warming conditions. AOA abundance decreased under warming conditions; however, AOA abundance was substantially higher than AOB abundance under the N0 treatment. Previous studies reported that archaea dominate over bacteria in unfertilized soils; however, bacteria seem to thrive in fertilized soils (Leininger et al., 2006; Valentine, 2007). The significant positive correlation found between AOB and PNR under fertilization suggests that the AOB community dominates under the conditions of sufficient N availability (Table 1). The results of this study indicated that artificial warming affected the nitrification activity and abundance of both AOB and AOA, particularly under N fertilization, suggesting an integrated impact of artificial climate change and N fertilization.

4 Conclusion

This study demonstrated that experimental warming magnifies PNR and AOB abundance under N fertilization, suggesting the considerable impact of warming on the N-cycling process in N-fertilized soils. Warming increased PNR and NO3−-N content and decreased soil moisture; AOB abundance was significantly higher than AOA abundance under warming conditions with N fertilization. However, AOA abundance was marginally higher than AOB abundance but significantly lower in warming conditions without N fertilization. In conclusion, warming conditions with N fertilization have a significant impact on nitrification activity and AOB abundance, whereas warming conditions alone (i.e., without N fertilization) affected AOA abundance. This alteration would affect the selectivity of ammonia-oxidizing microorganisms involved in the N-cycling process under different warming and fertilization conditions.

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氮肥施加条件下增温对硝化菌活性和丰度的影响*

Tatoba R Waghmode1†, 张新媛1,2†, 董文旭1, 张 闯1,2, 胡春胜1**

(1. 中国科学院遗传与发育生物学研究所农业资源研究中心/中国科学院农业水资源重点实验室/河北省节水农业重点实验室 石家庄 050022; 2. 中国科学院大学 北京 100049)

温度在多种生物地球化学过程中起到关键的调节作用, 是影响土壤硝化作用和微生物分布的重要因素之一。硝化过程的第1个步骤由氨氧化细菌(AOB)和氨氧化古菌(AOA)催化, 然而, 不同施氮量下, 增温对硝化菌活性和丰度的影响尚不清楚。本研究基于2008年10月起设立于太行山山前平原的长期增温试验平台(高于地表2 m的红外加热器使土壤温度升高1.5 ℃), 于2018年5月对不施氮(N0)和施氮[N1, 240 kg(N)∙hm-2∙a-1]下增温分别对0~10 cm和10~20 cm土壤硝化潜势(PNR)、AOA和AOB丰度的影响进行了研究。硝态氮(NO3--N和铵态氮(NH4+-N)含量用分光光度法测量, 应用缓冲液培养法测定土壤PNR, 提取土壤DNA后用实时荧光定量PCR技术测定功能基因AOA和AOB的丰度。结果表明: 温度升高显著增加N1条件下PNR和NO3--N含量(<0.05), 降低了N0条件下PNR和NO3--N含量, 但差异不显著。N1条件下, 增温土壤AOB丰度显著提高(<0.05); N0条件下, 增温土壤AOA丰度显著降低(<0.05)。与N0相比, N1条件下的AOA/AOB比值明显降低, 表明增温加氮肥处理对AOB的生长刺激更强烈。在增温加施氮条件下, 细菌(AOB)表现显著的正反应, 在增温不施氮条件下, 古菌(AOA)和AOB表现显著的负反应。本研究结果可为全球增温背景下进一步了解硝化活性和氨氧化微生物对增温和氮有效性的响应提供科学依据。

气候变暖; 硝化菌活性; 氨氧化微生物; 氮有效性

WAGHMODE T R, ZHANG X Y, DONG W X, ZHANG C, HU C S. Impact of soil warming on the activity and abundance of nitrifiers under nitrogen fertilization conditions[J]. Chinese Journal of Eco-Agriculture, 2019, 27(11): 1649-1655

* This study was supported by the National Natural Science Foundation of China (41530859, 31850410480), the National Key R & D Program of China (DQGG0208-4) and the Key Program of the Chinese Academy of Sciences (ZDRW-ZS-2016-5-1).

HU Chunsheng research interests are carbon, nitrogen, water cycle and soil ecological processes in agroecosystem. E-mail: cshu@sjziam.ac.cn.

† Equal contributors. Tatoba R Waghmode, the main research direction is soil warming effects on nitrifier, denitrifiers and plant-microbe interaction. E-mail: tatobawaghmode@yahoo.com. ZHANG Xinyuan, the main research direction is nitrogen cycle in agroecosystem. E-mail: zhangxy20160101@163.com.

Mar. 5, 2019;

Jun. 25, 2019

* 国家自然科学基金项目(41530859, 31850410480)、空气污染原因及治理重点项目(DQGG0208-4)和中国科学院重点项目(ZDRW-ZS-2016-5-1)资助

胡春胜, 主要从事农田生态系统碳氮水循环和土壤生态过程研究。E-mail: cshu@sjziam.ac.cn

† 同等贡献者

2019-03-05

2019-06-25

S154.1

2096-6237(2019)11-1649-07

10.13930/j.cnki.cjea.190166