Effects of nitrogen and phosphorus addition on soil respiration in a soybean cropping system

Abstract

Soil respiration is an important pathway of carbon release from the terrestrial biosphere to the atmosphere, which plays a key role in ecosystem carbon cycling. However, the response and mechanism of soil respiration to nitrogen and phosphorus addition in legume plants are still unclear. Here, a pot experiment planted with soybean (Glycine max (L.) Merr.) was conducted to investigate the effects of nitrogen (N) and phosphorus (P) addition on soil respiration. Four treatments were designed: control, N addition, P addition, and both N and P addition. Soil respiration was measured twice a month from June to September in 2022. Our results showed that nutrient addition treatments presented significantly negative effects on soil respiration. In particular, nitrogen addition not only directly affected soil respiration, but also indirectly impacted soil respiration by altering soil nitrate nitrogen content. Elevated soil nitrate nitrogen content could inhibit soybean root nodule number and reduce biomass allocation to roots, thereby decreasing soil respiration. Furthermore, phosphorus addition and nitrogen–phosphorus co-addition strongly inhibited soybean nodulation by changing soil pH value, thus inhibiting soil respiration of soybean. The findings provide baseline information for optimizing nutrient management in legume crops.

摘要

氮磷添加对大豆种植系统中土壤呼吸的影响

土壤呼吸是陆地生物圈向大气释放碳的重要途径，在生态系统碳循环中发挥着关键作用。然而，豆科植物土壤呼吸对氮磷添加的响应机制尚不清楚。为此，我们进行了一项种植大豆(Glycine max (L.) Merr.)的盆栽实验，以研究氮(N)和磷(P)添加对土壤呼吸的影响。该实验共涉及4个处理：对照、添加N、添加P以及同时添加N和P，实验期间每月测量两次土壤呼吸。实验结果表明，养分添加处理显著抑制土壤呼吸。其中，N添加不仅直接影响土壤呼吸，还通过改变土壤硝态氮含量间接影响土壤呼吸。土壤硝态氮含量升高抑制了大豆根瘤数量，减少了根部生物量分配，从而降低了土壤呼吸。此外，P添加和N、P同时添加通过改变土壤pH值显著抑制了大豆的根瘤形成，从而抑制了大豆的土壤呼吸作用。上述研究结果为优化豆科作物的养分管理提供了基础信息。

Introduction

Nitrogen (N) and phosphorus (P) are essential nutrients for plant growth, which are limiting factors for ecosystem productivity (Fay et al. 2015; Zheng et al. 2023). Anthropogenic activities such as fossil fueling combustion and fertilizer application have significantly increased the inputs of N and P in terrestrial ecosystems (Wei et al. 2020). Sustained input of N and P significantly can strongly affect the carbon cycle of terrestrial ecosystems, such as soil carbon storage and microbial biomass carbon (Ren et al. 2016; Zeng et al. 2018b). Soil respiration is the second largest terrestrial carbon flux from the biosphere to the atmosphere (Liu et al. 2021; Zheng et al. 2022), and plays a crucial role in regulating terrestrial carbon cycling and atmospheric CO₂ concentration (Lei et al. 2021; Yang et al. 2022b; Zhang et al. 2023). The influencing factors of soil respiration include biotic and abiotic factors, such as plant productivity, microbial activity, soil temperature and moisture (Zheng et al. 2022, 2023). However, the response and mechanism of soil respiration to nitrogen and phosphorus additions remains elusive.

Fertilization is the primary method to increase crop yield in agricultural production, it has an important effect on soil carbon flux. Previous studies have shown that the N addition usually stimulates plant growth, which in turn enhances plant root respiration and increases soil respiration rates (Wei et al. 2020; Xiao et al. 2020a). However, prolonged or excessive N additions may also lead to negative effects such as soil acidification and nutrient imbalance, which can adversely affect the soil microbial community, thereby inhibiting microbial heterotrophic respiration and complicating changes in soil respiration rates (Tian and Niu 2015; Zhou et al. 2016). The influence of N addition is still debated despite many experiments being carried out to examine how ecosystem soil respiration responds to the N application in farmland (Dong et al. 2022; Kou et al. 2023; Zhong et al. 2016). Previous results of a meta-analysis showed that the N addition increased soil respiration to a different degree in farmland (Feng et al. 2017; Xiao et al. 2020a). This result was different from the meta-analysis conducted by Ngaba et al. (2023), who found that the N addition can inhibit soil respiration by reducing soil microbial biomass. This difference may be due to differences in scale, different types, amounts, and rates of N addition.

Given that phosphorous (P) can interact with N to regulate soil respiration. Previous studies have shown that P addition increased soil respiration by changing microbial biomass carbon and fine root biomass (Song et al. 2011; Wei et al. 2020). However, a meta-analysis found that P addition did not significantly change soil respiration across all ecosystems (Feng and Zhu 2019). These differences may be caused by differences in factors such as environmental conditions, timing and type of P application and land use type. However, the combined effect of N and P additions on soil respiration is more complex. On the one hand, the synergistic effect of N and P may promote plant growth and further enhance root respiration (Liu et al. 2021; Zeng et al. 2018b). On the other hand, an imbalance in the ratio of N and P may lead to changes in the structure of the soil microbial community and affect microbial respiration (Widdig et al. 2020). Therefore, in agricultural production, reasonable control of N and P addition is important for maintaining soil health and promoting soil respiration balance. Therefore, understanding the mechanisms of biological and abiotic factors affecting soil respiration under N and P inputs is critical to predicting the future responses of carbon cycling to agricultural fertilization.

Symbiotic nitrogen fixation has received increasing attention from researchers in advancing the sustainable development of agricultural soils. A previous study suggests that soil respiration can be affected by mycorrhizal respiration and inter-root microbial respiration in both the medium-nitrogen (10 g N m⁻² yr⁻¹) and high-nitrogen (15 g N m⁻² yr⁻¹) zones in forest ecosystems (Du et al. 2014). Soybean, as a crop with nitrogen fixation, few studies have combined biological nitrogen fixation with soil respiration to investigate the effects of nutrient addition on soil respiration in soybean crops. Researchers found that the N application could significantly inhibit nodulation amount and soil respiration amount of soybean compared with N reduction and no N application (Peng et al. 2022). However, there are few studies investigating the effects of different nutrients and their interactions on soybean nitrogen fixation and soil respiration. Here, we conducted a pot experiment planted with soybeans to examine the effects of N and P additions on soil respiration. Two specific questions are shown as follows: (i) How do N and P and both of them interactively impact soil respiration? (ii) which factors drive the changes in soil respiration under N and P addition?

Materials and methods

Site description

The experiment was performed at Henan University (34° 81ʹ N, 114° 30ʹ E, 50 m a.l.s.), Kaifeng, Henan Province, China. It belongs to the warm temperate continental monsoon climate, and the average annual rainfall is about 625 mm, 87% of which is concentrated from April to October. Mean annual temperature was 14 °C, with the highest monthly mean temperature at 27.1 °C (in July) and the lowest monthly mean temperature at -0.16 °C (in January) (Zhang et al. 2024).

Total carbon content of soil before planting in 2022 (in situ soil plot, three repetitions, 0–20 cm soil layer mean, the same below) is 2.22 g kg⁻¹, the total nitrogen content is 0.35 g kg⁻¹, the total phosphorus content is 0.31 g kg⁻¹, the soil total organic carbon is 0.62 g kg⁻¹, the nitrate nitrogen content is 2.93 mg kg⁻¹, the ammonia nitrogen content is 0.27 mg kg⁻¹, and the available phosphorus (AP) content is 3.20 mg kg⁻¹.

Experimental design

The experiment followed a two-factor (nitrogen and phosphorus addition) design. Twenty potted plants were randomly assigned to a control group and three treatment groups, with a total of four treatments: control (CK), nitrogen addition (N addition), phosphorus addition (P addition), and nitrogen and phosphorus addition (N and P addition). The total amount of fertilizer applied was urea 10 g m⁻² and superphosphate 112.5 g m⁻² (equivalent to adding P₂O₅ 13.5 g m⁻²), that is CK (no nutrient addition), N addition (urea 10 g m⁻²), P addition (superphosphate 112.5 g m⁻²), N and P addition (urea 10 g m⁻² and superphosphate 112.5 g m⁻²). According to the pot surface area, urea was calculated by applying 0.7 g per pot and superphosphate was 7.95 g per pot, and fertilization was averaged applied at the branching stage and the first flowering stage of soybean. Select soybean seed with full grains and the same size for sowing, and set three plants in each pot after seedling emergence. Before the experiment, five pots were randomly selected for the basement soil experiment.

Soil respiration measurement

One PVC collar (5.5 cm radius, 10 cm height) was placed in the center of each pot. The depth of the PVC soil breathing rings inserted into the soil was 8 cm, with 2 cm remaining above ground. Soil respiration was measured twice a month from June to September 2022 using a Li-8100 automated soil CO₂ efflux system (LI-COR Inc.,), which should be shown in Fig. 1. All living plants and litter inside the collars were removed 24 h before soil respiration measurement. Soil respiration was measured in the sealed chamber for 1.5 min. All measurements were taken between 9:00 and 11:00 a.m. Soil temperature and volumetric soil moisture were measured at 10 cm depth concurrently with soil respiration (TR-6D).

Soil sampling and analysis

Soil samples were collected at soybean maturity. Soil samples from each pot were mixed to generate composite samples. The sample was passed through a 2 mm soil sieve after collection. Some of the sieved soil samples were dried to analyze the physical and chemical properties of the soil. Some were stored in ice packs and brought back to the laboratory for soil microbial index analysis. Three soybean plants were selected and taken from each plot, treated at 105 °C for 2 h, and then baked to a constant weight at 65 °C to measure the aboveground biomass. The roots were washed with distilled water and dried at 65 °C for 48 h, then weighed to calculate the root biomass. All soybean plants were selected and cleaned with tap water, and the nodules larger than 2 mm in diameter (nodules with nitrogen fixation activity) were removed with scalpels and counted (Zhao et al. 2022).

Soil pH was measured by the potential method, and soil carbon and nitrogen content were determined by a semi-constant element analyzer (Elementar vario MACRO CUBE, Elementar Co., Hanau, Germany) after soil was finely ground and homogenized using a ball mill (Retsch MM400, Hanau, Germany). Ammonium and nitrate were extracted from 10 g of field-moist soil with a mixture of 1 mol L^-1 KCl and 0.5 mol L^-1 HCl and measured by a continuous-flow auto-analyzer (Skalar San++, Breda, the Netherlands). AP was extracted with 0.5 mol L^-1 NaHCO₃, soil total phosphorus was extracted by HClO₄–H₂SO₄ method, and soil phosphorus content was measured calorimetrically. Microbial biomass carbon (MBC) (Vance et al. 1987) and microbial biomass nitrogen (MBN) using the chloroform fumigation–extraction method and determined by total organic carbon analyzer.

Statistical analysis

Before statistical analysis, the variables were tested for normality and homoscedasticity. When these assumptions were not met, the data were log-transformed. The main and interactive effects of N and P addition on soil respiration were analyzed using repeated measures ANOVA (SPSS 26.0, SPSS Inc., Chicago, IL, USA). The individual and interactive effects of N and P addition on response variables that were measured only once (soil physical and chemical properties and microbial biomass) were analyzed using two-way ANOVAs. One-way ANOVA with Duncan testing (P < 0.05) was used to evaluate the significant differences in soil properties and CO₂ emission among the four treatments. Associations of soil respiration with soil and plant variables were tested using Pearson correlations and stepwise multiple linear regression (SPSS 26.0, SPSS Inc.). Based on the results of stepwise multiple linear regression, the dominant factors that drove the variances in soil respiration were incorporated in the structural equation modeling (SEM) to determine direct and indirect paths of controlling factors to soil respiration. SEM analyses were performed using the SPSS AMOS 26.0 modeling software (SPSS AMOS 26.0, SPSS Inc.). Chi-square (%) tests, degrees of freedom (df), comparative fit index (CFI), and approximate root-mean-square error (RMSEA) were used to evaluate the fitness of structural equation models. The figures were generated using R (version 4.3.2).

Result

Soil respiration and Soil microclimate

There was a significant time fluctuation in soil respiration, with higher fluxes in August and September than in other months (P < 0.001, Table 1; Fig. 2a). Soil respiration was significantly reduced by the three nutrient addition treatments compared to the control treatments (N addition by 6.7%, P addition by 6.4%, and N and P addition by 9.1%; P < 0.05, Fig. 2b).

Soil temperature and moisture showed significant seasonal variability (Table 1; Fig. 3a and b). However, the addition of N and P did not interact with soil temperature (Table 1; Fig. 3a). The N and P addition alone did not significantly affect soil moisture, but their interaction significantly affected soil moisture (P < 0.05, Table 1; Fig. 3b).

Soil and plant properties

Compared to the control, soil pH was significantly decreased under all three nutrient addition treatments: by 1.3% with N addition, 3.7% with P addition, and 2.7% with combined N and P addition (P < 0.05, Fig. 4a). Neither N nor P addition affected soil carbon or soil nitrogen (Table 1). Nitrogen addition increased soil nitrate by 103.6% (P < 0.01, Fig. 4c). Phosphorus addition increased available P and ammonium by 290.1% (P < 0.01, Fig. 4b) and 216.4% (P < 0.05, Fig. 4d), respectively. Nitrogen and P addition interactively affected soil pH and soil ammonium (Table 1). However, no interactive effects of N and P addition were observed on other soil chemical variables (Table 1).

The result showed that neither N nor P addition affected microbial biomass carbon (MBC) (Table 2; Fig. 5a) or microbial biomass nitrogen (MBN) (Table 2; Fig. 5b). Phosphorus and N and P addition interaction affected above-ground biomass. Phosphorus addition increased above-ground biomass by 39.4% (P < 0.05, Table 2; Fig. 5d). Nitrogen or P addition did not affect soybean root biomass (Table 2; Fig. 5e). The number of root nodules of soybean was reduced by all three nutrient addition treatments (N addition by 33%, P addition by 36%, and N and P addition by 29.5%; P < 0.05, Table 2; Fig. 5c). Nitrogen addition decreased the root–shoot ratio by 29.4%, and P addition reduced the root–shoot ratio in potted soybeans by 44.1% (P < 0.05, Table 2; Fig. 5f).

Factors influencing soil respiration

The stepwise multiple linear regression showed that number of nodules and root biomass were the most dominant drivers of soil respiration (Supplementary Fig. S2). During the experiment, soil respiration was negatively dependent upon soil nitrate (P < 0.05, Table 3; Fig. 6c, R² = 0.29), soil available P (P < 0.05, Table 3; Fig. 6a, R² = 0.20), whereas positively dependent upon soil pH (P < 0.05, Table 3; Fig. 6b, R² = 0.20), the number of root nodules (P < 0.01, Table 3; Fig. 6d, R² = 0.69), root biomass (P < 0.01, Table 3; Fig. 6f, R² = 0.45). Soil respiration was not correlated with microbial biomass carbon (Table 3; Fig. 6e).

The structural equation model (SEM) explained 82 % of the variation in soil respiration (Fig. 7). Nitrogen addition could directly affect soil respiration. Nitrogen addition reduced the number of root nodules and root biomass by stimulating soil nitrate increase. Phosphorus addition leads to a decline in soil pH, subsequently resulting in reduced soybean nodules. Through these pathways, the number of root nodules and root biomass decreased, which reduced soil respiration. The model demonstrated N or P addition altered soil respiration via influencing soil nitrate and soil pH, with ensuing consequences on the number of root nodules and root biomass.

Discussion

Our findings highlighted that soil respiration was significantly reduced in the three nutrient addition treatments. The N addition not only directly affected soil respiration, but also showed indirect effects by altering soil nitrate nitrogen content, and the growth of soybean nodulation. Phosphorus additions indirectly affected soil respiration by changing soil pH and soybean nodule formation.

Effects of nitrogen addition on soil respiration

Our results showed that the N addition treatment significantly reduces soil respiration. However, Zhao et al. found that the N application increased soil respiration by stimulating root biomass (Zhao et al. 2020), which is inconsistent with our results. In our study, soil respiration was positively correlated with root biomass (Fig. 4f). It has been suggested that increased nutrient availability may reduce the allocation of plant biomass to the roots, thereby reducing root respiration (Wang et al. 2020). We found N addition significantly reduced the root–shoot ratio by 29.5 % compared to the control, this can help explain the effect of the N application on soybean biomass after increasing soil available nutrients. Therefore, we speculate that the N addition increased soil nutrient availability and reduced biomass allocation to roots, thereby reducing root respiration.

In this study, the N addition had no effect on MBC. Soil microbial biomass carbon (MBC) is an important factor that might have an impact on soil CO₂ emission (Iqbal et al. 2010). Some studies reported that N fertilization may directly or indirectly limit soil microbial biomass and activity. Our results were not consistent with previous studies. For example, Dong et al. found that the N addition induced the reduction in microbial activity which decreased soil respiration (Dong et al. 2022; Xiao et al. 2020b; Xing et al. 2022). However, Wang et al. showed that the N addition did not have a significant effect on the growth season flux of MBC, which attributed to the N addition did not alleviate carbon limitation for microbes (Wang et al. 2018). Soil microorganisms are generally carbon-limited (Fleischer et al. 2019; Soong et al. 2020), and N addition did not change the root biomass of soybean (Fig. 3f), meaning that the N addition did not alleviate the carbon limitation of microorganisms and therefore N addition did not significantly affect the microbial activity of soybean. Furthermore, the result of Jiang et al. in the Northeast Plains demonstrated that MBC increased with higher levels of N addition, but there was no significant difference in MBC at low nitrogen levels (from 5 g m⁻² to 10 g m⁻²) (Jiang et al. 2024). Therefore, insufficient N addition may be responsible for the lack of a significant response of microbial biomass carbon to N addition in this study.

In our study, soil respiration was negatively dependent upon soil nitrate, but was positively dependent upon the number of root nodules (P < 0.01, Fig. 5d, R² = 0.69). Coincidently, soil nitrate content was significantly increased by the N addition (Table 1; Fig. 3c). Nitrate depletion reduced the transport of photosynthetic products to the nodules, delaying formation and growth of the nodules, further inhibiting the nitrogen fixation (Fujikake et al. 2002). Increased soil nitrate nitrogen inhibited the number of potted soybean root nodules (Supplementary Fig. S1) which is in agreement with previous studies (Gan et al. 2004; Saito et al. 2014). Saito et al. also found that the N addition inhibited nodule formation, and the stronger inhibition occurred with the N addition amount increasing thus inhibiting soil respiration (Saito et al. 2014). Due to the nitrogen fixation capabilities of leguminous crops, the respiration of rhizobium represents an important component of heterotrophic respiration in soybean croplands. Therefore, the decrease in the number of root nodules could significantly reduce soil respiration.

Effect of phosphorus addition on soil respiration

Our results showed P addition significantly increased aboveground biomass of soybean and root-shoot ratio, but did no significant affect root biomass. Mori et al. concluded that P addition could increase autotrophic respiration in tropical acacia forests due to the alleviation of P limitation and the increase of fine root yield (Mori et al. 2013). However, other studies found that increased soil phosphorus availability reduced soil respiration. This reduction is suggested to be due to the increase of soil AP inhibits total root biomass and root organic carbon allocation in plants (Li et al. 2016; Shi et al. 2021). Previously, studies (Heyburn et al. 2017; Müller et al. 2000) demonstrated that increasing soil nutrient availability could alter plant resource allocation and reduce total root biomass. These results indicate that P addition did not affect soil respiration by changing root biomass, but rather influenced the carbon cycling process through its impact on plant root-shoot ratio.

Our results demonstrated that P addition significantly decreased the soil pH values, consistent with most previous studies (Du et al. 2021; Wei et al. 2011; Yang et al. 2022a). Soil pH could positively impact soybean root nodule number. The effect of soil pH on symbiotic nitrogen fixation is very wide and significant, and the effect on legume nodulation and growth varies between plants. For example, soil acidification can significantly reduce soil and root nitrogen accumulation in peanuts, subsequently inhibiting peanut nodulation and nitrogen fixation (Zhao et al. 2022). In addition, other studies have also reported that nodulation of some legumes was reduced under low soil pH conditions (Ferguson et al. 2013; Macció et al. 2002). Therefore, it is speculated that P addition can inhibit the nodulation process of soybeans by changing the soil pH environment. Therefore, the decreased pH values under P addition led to the reduction of soybean nodulation and finally reduced soil respiration.

Effects of nitrogen and phosphorus additions on soil respiration

It is well known that the synergistic effect of N and P can promote plant growth and further enhance root respiration (Liu et al. 2021; Zeng et al. 2018a). In our results, the results of the two-factor analysis of variance showed no interaction between N and P additions. However, our study found that compared to the control (one-way ANOVA), the N and P co-addition has a significant negative impact on soil respiration. Previous studies have shown that soil respiration increases significantly when N and P are added together (Lu et al. 2023; Wei et al. 2020; Zhang et al. 2021; Zheng et al. 2023). In contrast, other studies have shown that N and P addition inhibit soil respiration (Wang et al. 2017; Zhou et al. 2014). The reduction of soil CO₂ efflux was attributed to the decrease in RB and MBC as the result of increase in soil nutrient availability. In this study, the simultaneous addition of N and P enhanced the inhibition effect of single nutrient addition on soil respiration. One possible explanation is that N and P addition change resource allocation during soybean growth and influence the carbon cycling process. On the other hand, soil pH changes with N and P additions, which affects soil microbial activity. Consistent with adding P alone, the addition of N and P reduced the soil pH value, affecting soybean nodulation and ultimately inhibiting soil respiration.

Conclusions

Our results suggest that N and P had different impact mechanisms on soil respiration. Nitrogen addition increased soil nutrient availability and reduced biomass allocated to roots, thereby reducing root respiration. Nitrogen addition inhibited the number of root nodules of soybean by increasing soil nitrate nitrogen content and thus inhibiting soil respiration of soybean. Phosphorus addition and N and P interaction inhibited soybean nodulation by changing soil pH value, thereby inhibiting soil respiration of soybean. These findings enhance understanding of soybean carbon cycling mechanisms under nutrient addition and provide scientific value for optimizing nutrient application in leguminous crops.

Supplementary Material

Supplementary material is available at Journal of Plant Ecology online.

Figure S1: Relationships of number of nodules with NO₃⁻–N.

Figure S2: The standardized coefficients of stepwise regression analysis of soybean nodule number and root biomass as the main driving factors of soil respiration.

Figure S3: Mean values of soil organic carbon under the four treatments (mean ± SE, n = 5).

Figure S4: Mean values of soil total carbon under the four treatments (mean ± SE, n = 5).

Figure S5: Mean values of microbial biomass phosphorus under the four treatments (mean ± SE, n = 5).

Authors’ Contributions

Jingyuan Yang conceived and designed the experiments, performed the experiments, analyzed the data, prepared figures and/or tables, authored or reviewed drafts of the article, and approved the final draft. Qi Xu performed the experiments, authored or reviewed drafts of the article, and approved the final draft. Yuxuan He performed the experiments, authored or reviewed drafts of the article, and approved the final draft. Meiguang Jiang performed the experiments, authored or reviewed drafts of the article, and approved the final draft. Minglu Ji performed the experiments, authored or reviewed drafts of the article, and approved the final draft. Linyu Qi performed the experiments, authored or reviewed drafts of the article, and approved the final draft. Huan Qi performed the experiments, authored or reviewed drafts of the article, and approved the final draft. Cancan Zhao authored or reviewed drafts of the article, and approved the final draft. Yuan Miao conceived and designed the experiments, authored or reviewed drafts of the article, and approved the final draft. Shasha Liu authored or reviewed drafts of the article, and approved the final draft. Yanfeng Sun reviewed drafts of the article, and approved the final draft.

Funding

This work was supported by grants from the National Natural Science Foundation of China (42107225), Xinyang Academy of Ecological Research Open Foundation (2023XYQN15), Natural Resources Research Project of Henan Province (Grant No. 2021-157-9), Henan Province natural science Foundation project (242300420139), Kaifeng science and technology development plan project (2202007) and Key Scientific Research Project Plan of Colleges and Universities in Henan Province (23A180013).

Acknowledgements

The authors are grateful to Xinlei Fu, Fan Yang and Kaixin Yan giving help in the field work. The authors are indebted to the editors and reviewers for their constructive comments and suggestions during the review phase of this paper.

Conflict of interest statement. The authors declare that there is no conflict of interest regarding this manuscript.

References