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Article

Effects of Multiple Global Change Factors on Symbiotic and Asymbiotic N2 Fixation: Results Based on a Pot Experiment

by
Zhenchuan Wang
1,2,3,†,
Xibin Sun
4,†,
Hao Chen
4 and
Dejun Li
1,3,*
1
Key Laboratory of Agro-Ecological Processes in Subtropical Region, Institute of Subtropical Agriculture, Chinese Academy of Sciences, Changsha 410125, China
2
Key Laboratory of Environment Change and Resources Use in Beibu Gulf, Ministry of Education, Nanning Normal University, Nanning 530001, China
3
Guangxi Key Laboratory of Karst Ecological Processes and Services, Huanjiang Observation and Research Station for Karst Ecosystems, Chinese Academy of Sciences, Huanjiang 547100, China
4
State Key Laboratory of Biocontrol, School of Ecology, Sun Yat-sen University, Shenzhen 518107, China
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to the work.
Nitrogen 2023, 4(1), 159-168; https://doi.org/10.3390/nitrogen4010011
Submission received: 7 January 2023 / Revised: 3 March 2023 / Accepted: 6 March 2023 / Published: 17 March 2023
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Abstract

:
Biological N2 fixation, a major pathway for new nitrogen (N) input to terrestrial ecosystems, largely determines the dynamics of ecosystem structure and functions under global change. Nevertheless, the responses of N2 fixation to multiple global change factors remain poorly understood. Here, saplings of two N2-fixing plant species, Alnus cremastogyne and Cajanus cajan, were grown at rural and urban sites, respectively, with the latter representing an environment with changes in multiple factors occurring simultaneously. Symbiotic N2 fixation per unit of nodule was significantly higher at the urban site than the rural site for A. cremastogyne, but the rates were comparable between the two sites for C. cajan. The nodule investments were significantly lower at the urban site relative to the rural site for both species. Symbiotic N2 fixation per plant increased by 31.2 times for A. cremastogyne, while that decreased by 88.2% for C. cajan at the urban site compared to the rural site. Asymbiotic N2 fixation rate in soil decreased by 46.2% at the urban site relative to the rural site. The decrease in symbiotic N2 fixation per plant for C. cajan and asymbiotic N2 fixation in soil was probably attributed to higher N deposition under the urban conditions, while the increase in symbiotic N2 fixation per plant for A. cremastogyne was probably related to the higher levels of temperature, atmospheric CO2, and phosphorus deposition at the urban site. The responses of N2 fixation to multiple global change factors and the underlying mechanisms may be divergent either between symbiotic and asymbiotic forms or among N2-fixing plant species. While causative evidence is urgently needed, we argue that these differences should be considered in Earth system models to improve the prediction of N2 fixation under global change.

1. Introduction

Biological dinitrogen (N2) fixation (BNF or N2 fixation hereafter), a process by which diazotrophs convert atmospheric N2 into ammonia catalyzed by nitrogenase, is a major pathway of external N input to terrestrial ecosystems [1]. As N is often the major limiting nutrient for net primary production, BNF is thus crucial in determining the structure and function of terrestrial ecosystems via its influence on soil N availability [2,3]. N2 fixation can be divided into symbiotic N2 fixation (SNF hereafter) and asymbiotic N2 fixation (ANF hereafter). SNF is performed by diazotrophs residing in nodules of N2-fixing plants, while ANF is conducted by diazotrophs freely distributed in ecosystem compartments such as soil [1]. Considering its key role in determining soil N availability, how N2 fixation changes is tightly related to the responses of ecosystem structure, process, and function to multiple global change factors, including warming, altered precipitation, CO2 enrichment, and atmospheric N or phosphorus (P) deposition.
Over the past few decades, a few studies have been conducted to explore the effects of global change factors on N2 fixation [4,5]. Among these factors, warming, increased precipitation, and CO2 enrichment generally benefit N2 fixation, since N2 fixation is an enzymatic and energetically expensive process [4,5]; however, drought and increased N deposition substantially inhibit N2 fixation [4,5,6]. However, contrasting results have often been observed. For example, CO2 enrichment suppressed nitrogenase activity in the nodules of Alfalfa (Medicago sativa) regardless of temperature and moisture conditions based on a pot experiment [7]. For another, ANF rates might not be suppressed by atmospheric N deposition in two subtropical forests [6,8]. Furthermore, very limited studies have explored the impacts of multiple global change factors on SNF or ANF, especially the former, probably owing to the difficulty in experimental layout with multiple factors [4]. Though quite a few studies include two factors, only one study has explored the response of SNF [7] or ANF [9] to three or more factors. Based on the limited studies, there are strong interactive effects of the global change factors on ANF rate or indices of SNF [5,7,9,10]. In a laboratory incubation experiment, the patterns of soil ANF response to moisture varied with temperature [5]. In a pot experiment with Alnus hirsuta and Alnus maximowiczii, the response of nitrogenase activity to CO2 enrichment was insignificant, but the nodule biomass responses to CO2 enrichment were divergent under high and low P availability [11]. Nasto et al. [12] reported that CO2 enrichment significantly promoted SNF rates of four N2-fixing plant species, but the positive effect was diminished or disappeared under N addition. Due to the complex interactive effects, results from studies with single or limited factors may not be suitable for predicting the responses of N2 fixation to a global change in reality. As a matter of fact, the mechanistic representation of N2 fixation is very weak in Earth system models [13,14], resulting in their poor performance in predicting the responses of N2 fixation to global change in the boreal region [14]. It is hence urgently needed to investigate the responses of N2 fixation to multiple global change factors using any suitable approaches.
The SNF of different N2-fixing plant species may respond divergently to global change factors due to various N2-fixation strategies or plant physiology. N2-fixing plant species with a facultative N2 fixation strategy down-regulate SNF as soil N availability increases, while those with an obligate N2 fixation strategy would maintain SNF as soil N availability increases [15,16]. Menge et al. [16] explored the responses of SNF of eight N2-fixing herbaceous species to N addition; they found that the SNF rates of six species decreased, and those of the other two species did not change under N addition. The response of SNF to CO2 enrichment may also vary among N2-fixing plant species. For example, West et al. [17] investigated the effects of CO2 enrichment on SNF rates of four N2-fixing plant species; they found that SNF rates of two species were stimulated, but those of the other two species were suppressed by CO2 enrichment. Therefore, it is possible that SNF rates of different N2-fixing plant species may respond divergently to multiple global change factors.
Rural–urban environmental gradients have often been used to simulate multiple global change factors, since cities experience higher levels of global change factors compared to the global average, including CO2 concentration, temperature, N deposition and others [10,18]. Since actinorhizal and leguminous plants are the two main types of vascular plants that form symbioses with diazotrophs, two N2-fixing plant species, i.e., Alnus cremastogyne (an actinorhizal species) and Cajanus cajan (a leguminous species), were included in the current study. The saplings of the two pant species were grown under rural and urban conditions, respectively. For comparison, SNF and soil ANF were determined. The major objectives are to address the following: (1) how would SNF and soil ANF respond to multiple global change factors, and (2) is there difference in the responses of SNF to multiple global change factors between the two N2-fixing plant species?

2. Materials and Methods

2.1. Experimental Design

Huanjiang observation and research station for karst ecosystems in Huanjiang County (24°44′20″ N, 108°19′34″ E) and Guangxi University in Nanning City in Guangxi Zhuang Autonomous Region (22°51′19″ N, 108°17′13″ E) were used as rural and urban sites, respectively (Figure S1). For the rural site, mean annual temperature (MAT) is 20.1 °C with the lowest monthly temperature in January (9.4 °C) and the highest monthly temperature in August (27.1 °C); and mean annual precipitation (MAP) is 1603.3 mm with a wet season from April to September and a dry season from October to March. For the urban site, MAT is 21.9 °C with the lowest monthly temperature in January (15.5 °C) and the highest monthly temperature in September (28.1 °C); and annual average precipitation is 1548.7 mm with 80% contributed by wet season from April to September (Figure S2).
A pot experiment was conducted with saplings of two N2-fixing plant species, i.e., A. cremastogyne and C. cajan. In March 2017, seeds of the two N2-fixing plant species were sown separately in 0.3 L seedling bags. In April 2017, limestone soil (Luvisols) from the surface layer (0–30 cm) of a karst shrubland in Huanjiang observation and research station for karst ecosystems was collected and sieved to 1 cm, and then it was put into 20 pots with a volume of 13 L each after mixed thoroughly. Soil physicochemical properties are shown in Table 1. Specifically, soil pH was 7.80, and SOC, total N, and total P were 19.04, 1.68, and 0.81 g kg−1, respectively. At the end of April, seedlings of the two N2-fixing plant species were transplanted into the pots, keeping a single plant in each pot, with five replicates of each treatment. Each seedling was inoculated with a homogenate of nodules and rhizosphere soil of the corresponding plant species from the nearby forests to ensure inoculation, as performed by others [19,20]. At the beginning of May, five pots of each species were deployed to rural and urban sites, respectively. Plants were extirpate weed and desinsectization regularly during the experiment to ensure that their growth was not limited by other factors.

2.2. Sample Collection and Analysis

Rainwater and air samples were collected from May 2017 to April 2018. Rainwater was collected once per rainfall event for the measurement of nitrate (NO3−), ammonium (NH4+), dissolved organic N (DON), and dissolved P. The rainwater sample was filtered for the direct measurement of NO3−, NH4+, DON, and dissolved P using an auto-analyzer (Fiastar 5000; Foss Tecator AB, Höganäs, Sweden). Air samples were collected weekly for the determination of CO2 concentration using an Agilent 7890A gas chromatograph (Agilent Technologies, Santa Clara, CA, USA) equipped with a thermal conductivity detector.
All N2-fixing plants were harvested in October 2017 after five months’ growth, and they were then divided into shoots, roots, and nodules for each plant. A portion of the nodules with a few fibrous roots were used for the measurement of SNF rate in situ. The shoots, roots, and nodules were dried at 65 °C to constant weight to determine plant biomass and nodule biomass. Nodulation investment (mg nodule g−1 plant) was calculated by the ratio of nodule biomass to whole-plant biomass. Fresh soils surrounding the roots were randomly collected and mixed thoroughly, and they were then divided into three parts. One part was used for the determination of ANF rates in situ, one part was stored at 4 °C for the measurement of soil available nutrients, and the third part was air dried for the analysis of soil physicochemical properties.
The fresh soil was sieved to 2 mm for the determination of soil NO3− and NH4+, and the air-dried soil was sieved to 0.15 mm for the measurement of available P [21]. Soil NO3− and NH4+ were extracted with 2 M KCl solution and analyzed by an auto-analyzer (Fiastar 5000; Foss Tecator AB, Höganäs, Sweden). Soil available P was extracted with 0.5 M NaHCO3 at a pH of 8.5, and P contents were analyzed using the ascorbic acid molybdate method.

2.3. Determination of Biological N2 Fixation Rate

BNF rates were determined by the acetylene reduction method [22]. Fresh samples (10~15 nodules or 10 g soil) were put into 125 mL glass flasks with rubber stoppers. Then, 10% of the headspace in the flasks was replaced with high purity acetylene (99.99% purity). The flasks with sample only and acetylene only were also set up as references. All flasks were placed under the field conditions but away from direct sunlight. After incubation (0.5 h for nodule and 24 h for soil), 30 mL of gas sample from each flask was extracted with a syringe and subsequently injected into a pre-vacuumed 12 mL glass vial (Labco Exetainer, Labco Limited, Ceredigion, UK). The ethylene concentration in each gas sample was determined by an Agilent 7890A gas chromatograph (Agilent Technologies, Santa Clara, CA, USA) equipped with a flame ionization detector. The specific SNF (μmol C2H4 g−1 nodule h−1) or ANF (nmol g−1 soil d−1) rate was represented by acetylene reduction rate (ARA). The SNF rate per plant (or PNF hereafter; μmol C2H4 plant−1 h−1) was calculated by multiplying specific SNF rate by nodule biomass per plant. Since the current study aimed to investigate the response of BNF rate to multiple global change factors, the conversion factor which was used to transfer ARA to BNF was not determined.

2.4. Statistical Analysis

All data were tested for normality and homogeneity of variances before the analysis. Two-way analysis of variance (ANOVA) was used to test the effects of site, species, and their interaction on indices of N2 fixation and soil properties. t-test was used to examine the differences in atmospheric CO2 concentration and deposition rates of N and P between the rural and urban sites. Pearson correlation analysis was adopted to analyze the relationship between N2 fixation rates and soil physicochemical properties. The above analyses were performed using SPSS 19.0 (SPSS Inc., Chicago, IL, USA).

3. Results

3.1. Biological N2 Fixation

Specific SNF rate was significantly affected by site and its interaction with plant species; nodulation investment and PNF were significantly affected by site, plant species and their interaction, but specific soil ANF was only affected by site (Figure 1 and Figure 2, Table 2). Specific SNF rate increased by 58.6 times at the urban site compared to the rural site for A. cremastogyne, but the rates were comparable between the two sites for C. cajan. Nodulation investment of A. cremastogyne and C. cajan decreased by 36.0% and 91.6% at the urban site relative to the rural site, respectively. The PNF of A. cremastogyne increased by 31.2 times, while that of C. cajan decreased by 88.2% at the urban site compared to the rural site. Specific soil ARA decreased by 46.2% at the urban site relative to the rural site.

3.2. Environmental Variables and Their Correlations with Biological N2 Fixation

Environmental variables differed significantly between the rural and urban sites (Table 3). MAT and atmospheric CO2 concentration increased by 1.84 °C and 76 ppm, respectively, at the urban site relative to the rural site. The atmospheric deposition rates of NO3−, DON, total N, and P increased by 2.3, 1.4, 1.0, and 10.0 times at the urban site compared to the rural site, respectively, but NH4+ deposition was comparable between the two sites. Site had significant effects on soil NO3−, NH4+, and available P, but significant effects of plant species or the interaction between site and plant species were not found (Figure 3; Table S1). Soil NO3−, NH4+, and available P increased by 0.6, 0.6, and 1.4 times at the urban site compared to the rural site, respectively. Pearson correlation analysis showed that nodulation investment was significantly and negatively correlated with soil NO3− (r = −0.48, p = 0.03), NH4+ (r = −0.59, p < 0.01), and available P (r = −0.60, p < 0.01) (Table 4). Soil ARA was negatively correlated with NH4+ (r = −0.52, p = 0.02) and available P (r = −0.49, p = 0.03). Neither nodule ARA nor plant ARA was significantly correlated with soil physicochemical properties (Table 4).

4. Discussion

4.1. Response of Symbiotic N2 Fixation to Multiple Global Change Factors Depends on N2-Fixing Plant Species

Our results show that the responses of PNF to multiple global change factors were divergent between the two plant species. Since PNF is determined by both specific SNF rate and nodulation investment, we would discuss the responses of the two indices separately. The specific SNF rate is influenced by a few factors, including N deposition, soil P availability, atmospheric CO2 concentration, temperature, etc. [11,19,23,24]. Specific SNF rate is usually suppressed or not altered by N deposition depending on N2 fixation strategies of the N2-fixing plants [20,25]. Specific SNF rates of plants with a facultative N2 fixation strategy are often suppressed by N deposition, whereas those with obligate N2 fixation strategy are less sensitive to N addition [16]. Therefore, the higher N deposition at the urban site would theoretically not alter specific SNF rate of A. cremastogyne, but it would inhibit that of C. cajan in the current study, since actinorhizal plants are usually obligate, while leguminous plants are usually facultative in their N2 fixation strategies [15]. The increase in specific SNF rate for A. cremastogyne or unaltered specific SNF rate for C. cajan at the urban site suggests that the effect of N deposition may have been overridden or offset by the positive effects of other factors.
Promotion of specific SNF rate by increased soil P availability or atmospheric CO2 has been demonstrated in several studies [19,24,26]. P is a necessary element since N2 fixation needs large amount of adenosine triphosphate (ATP), and P plays a key role in regulating O2 near nitrogenase, which catalyzes N2 fixation, but it depends on legume species [27,28]. P may also regulate SNF via its fertilization role in the growth of N2-fixing plants, which supply carbon to diazotrophs residing in nodules in the exchange of N [26]. This mechanism is also applicable to the regulation of atmospheric CO2 on SNF, since atmospheric CO2 enrichment usually stimulates plant growth given that there is sufficient P availability [11,19]. In a pot experiment, the specific SNF rate of Inga punctata increased by 3.5-fold under P addition [19]. In the current study, the higher P deposition and atmospheric CO2 level at the urban site was probably responsible for the higher specific SNF rate of A. cremastogyne relative to the rural site. Meanwhile, a recent study revealed that the optimum temperature ranged from 29.0 °C to 36.9 °C for SNF conducted by Rhizobia-type or Frankia-type diazotrophs [29]. Correspondingly, the higher MAT could be another reason for the higher specific SNF of A. cremastogyne at the urban site. Nevertheless, the aforementioned explanations are plausible, and further investigations are needed to unravel the underlying mechanisms.
The nodulation investment was decreased under environmental change for both plant species in the current study. Previous studies showed that the nodulation of N2-fixing plants could be influenced by atmospheric CO2 concentration, soil P availability, and N deposition [19,20,30]. Positive effect of CO2 enrichment on nodulation has been demonstrated by several studies [7,11,30], which reported that CO2 enrichment significantly improved the nodule number and nodule biomass. In the current study, the higher atmospheric CO2 concentration at the urban site should theoretically enhance the nodulation investment of the two plant species. Therefore, the decrease in nodulation investment was probably caused by other factors. Many previous studies have reported the promotion of nodulation by P addition [19,28,31,32]. For example, Wurzburger and Hedin [19] found that nodule biomass of Inga punctata increased by 1.8-fold under P addition. However, nodulation investment decreased with soil available P in the current study. This negative relationship should be apparent, and other factors might have a stronger negative effect on nodulation. A few studies have reported the N addition suppressed nodulation investment [20,33,34,35]. For example, Batterman et al. [20] reported that N addition suppressed the nodulation investment Inga punctata by 85% based on a pot experiment. Lin et al. [35] revealed that high NO3− levels suppressed the nodulation investment by inhibiting the biosynthesis of cytokinin required for nodulation. Similarly, the decreased nodulation investment was accompanied by higher soil NO3− and NH4+ levels at the urban site of the current study. Therefore, the decreased nodulation investment of both N2-fixing plant species was probably due to the higher N deposition at the urban site.

4.2. Decrease in Soil N2 Fixation in Response to Multiple Global Change Factors

In the current study, specific ANF rate in soil was reduced under the urban conditions. Specific ANF rate in soil can be influenced by temperature, soil available P, and N deposition [5,27]. A meta-analysis revealed that warming promoted soil ANF [4]. Our previous study showed that soil ANF rate increased with temperature with the optimum temperature being higher than 35 °C based on a laboratory experiment using soils collected at a nearby karst forest [5]. Therefore, the higher temperature may have stimulated specific soil ANF rate at the urban site of the current study. P usually benefits soil ANF via its key role in ATP synthesis and maintenance of nitrogenase activity [27]. The promotion of P on soil ANF has been shown in many previous studies [36,37,38]. Thus, higher P deposition may also have promoted specific soil ANF rate at the urban site in the current study. The decreased specific soil ANF rate at the urban site indicates that the positive effects of temperature and P deposition may have been overridden by the negative effects of other factors. The inhibition of N deposition on soil ANF has been frequently demonstrated [6,27,39,40]. For example, our previous study found that high N addition decreased soil ANF by 17.1% in a karst forest [6]. Zheng et al. [40] found that soil ANF decreased by 33.7% under N addition based on a global data synthesis. This is because N2 fixation consumes large amount of C and energy, so that soil diazotrophs prefer to obtain N directly from soils and hence down-regulate N2 fixation if there is an abundance of available N [41]. In the current study, specific soil ANF rate was negatively correlated with soil N availability, corroborating that higher N deposition at the urban site was probably responsible for the lower specific soil ANF rate.

5. Conclusions

Our results suggest that the responses of biological N2 fixation to multiple global change factors may be divergent either between symbiotic and asymbiotic forms or among N2-fixing plant species. It should be noted that uncertainties exist in the current study. First, our study was based on a short-term experiment, so we are not sure whether the patterns from long-term response of N2 fixation to multiple global change factors are similar. Second, the findings were obtained from a pot experiment, so we are not sure whether they are applicable to field conditions. Third, altered precipitation pattern is one major aspect of global change, but it was not considered in the current study. Fourth, the patterns of global change or the combinations of multiple global change factors may vary among regions, so the rural–urban environmental gradient in the current study may only represent one scenario of global change. Considering the uncertainties, more investigations are urgently needed in order to obtain a better understanding of the responses of N2 fixation to multiple global change factors.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/nitrogen4010011/s1, Figure S1: Schematic showing the geographic locations of the rural and urban sites; Figure S2: Daily and monthly air temperature and cumulated precipitation at rural and urban sites in 2017. (a) Daily mean air temperature and daily precipitation at rural site; (b) daily mean air temperature and daily precipitation at urban site; (c) monthly air temperature and cumulated precipitation at rural site; (d) monthly air temperature and cumulated precipitation at urban site. Data were obtained from Chinese National Meteorological Centre (https://data.cma.cn/) in 20 May 2020; Table S1: Results of two-way ANOVA showing the effects of site (ST), species (SP), and their interaction on soil NO3−, NH4+, and available P.

Author Contributions

Conceptualization, D.L.; methodology, Z.W. and X.S.; software, Z.W. and X.S.; validation, D.L., Z.W., X.S. and H.C.; formal analysis, Z.W. and X.S.; investigation, Z.W. and X.S.; resources, D.L., Z.W., X.S. and H.C.; data curation, Z.W. and X.S.; writing—original draft preparation, D.L., Z.W. and X.S.; writing—review and editing, D.L., Z.W. and X.S.; visualization, Z.W. and X.S.; supervision, D.L.; project administration, D.L. and Z.W. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the National Natural Science Foundation of China (grant numbers 41877094, 42007086), and Department of Science and Technology of Guangxi Autonomous Region (Guangxi Bagui Scholarship Program to D.L.).

Data Availability Statement

The datasets generated during the current study are available from the corresponding author on reasonable request.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Indices of symbiotic N2 fixation at the rural and urban sites: (a) specific rate of symbiotic N2 fixation, (b) nodulation investment, and (c) symbiotic N2 fixation for per plant (PNF). Bars represent mean values with standard errors (n = 5). * and ** denote significant difference at p < 0.05 and p < 0.01 between the rural and urban sites, respectively.
Figure 1. Indices of symbiotic N2 fixation at the rural and urban sites: (a) specific rate of symbiotic N2 fixation, (b) nodulation investment, and (c) symbiotic N2 fixation for per plant (PNF). Bars represent mean values with standard errors (n = 5). * and ** denote significant difference at p < 0.05 and p < 0.01 between the rural and urban sites, respectively.
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Nitrogen 04 00011 g002 550
Figure 2. Specific rate of asymbiotic N2 fixation in soil at the rural and urban sites. Bars represent mean values with standard errors (n = 10). ** denotes significant difference at p < 0.01 between the rural and urban sites.
Figure 2. Specific rate of asymbiotic N2 fixation in soil at the rural and urban sites. Bars represent mean values with standard errors (n = 10). ** denotes significant difference at p < 0.01 between the rural and urban sites.
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Nitrogen 04 00011 g003 550
Figure 3. Soil N and P availability at the rural and urban sites. Bars represent mean values with standard errors (n = 10). * and ** denote significant difference at p < 0.05 and p < 0.01 between the rural and urban sites, respectively.
Figure 3. Soil N and P availability at the rural and urban sites. Bars represent mean values with standard errors (n = 10). * and ** denote significant difference at p < 0.05 and p < 0.01 between the rural and urban sites, respectively.
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Table
Table 1. Soil physicochemical properties before treatment. Values represent means ± standard errors (n = 3).
Table 1. Soil physicochemical properties before treatment. Values represent means ± standard errors (n = 3).
Soil ParametersValues
pH7.80 ± 0.04
SOC (g C kg−1)19.04 ± 3.32
Total N (g N kg−1)1.68 ± 0.21
Total P (g P kg−1)0.81 ± 0.08
Exchange Ca2+ (coml kg−1)20.98 ± 0.61
Exchange Mg2+ (coml kg−1)6.61 ± 0.07
Available P (mg P kg−1)6.80 ± 1.44
Available Mo (μg kg−1)24.96 ± 12.70
Table
Table 2. Results from two-way ANOVA showing the effects of site (ST), species (SP), and their interaction on indices of biological N2 fixation including specific rate of symbiotic N2 fixation (SNF), nodulation investment, symbiotic N2 fixation for per plant (PNF), and specific rate of asymbiotic N2 fixation (ANF) in soil.
Table 2. Results from two-way ANOVA showing the effects of site (ST), species (SP), and their interaction on indices of biological N2 fixation including specific rate of symbiotic N2 fixation (SNF), nodulation investment, symbiotic N2 fixation for per plant (PNF), and specific rate of asymbiotic N2 fixation (ANF) in soil.
FactorsSpecific SNF RateNodulation InvestmentPNFSpecific Soil ANF Rate
F Valuep ValueF Valuep ValueF Valuep ValueF Valuep Value
ST5.150.0443.36<0.014.380.0518.07<0.01
SP0.270.619.18<0.016.770.020.420.53
ST × SP4.540.047.940.0114.25<0.010.210.65
Table
Table 3. Atmospheric environmental variables at the rural and urban sites. Values represent means ± standard errors (n = 20 for CO2 concentration and 49 for precipitation properties) except for annual average temperature and precipitation.
Table 3. Atmospheric environmental variables at the rural and urban sites. Values represent means ± standard errors (n = 20 for CO2 concentration and 49 for precipitation properties) except for annual average temperature and precipitation.
Environmental VariablesRuralUrbanp Value
Annual average temperature (°C)20.1021.94−
Annual precipitation (mm)1603.31548.70−
Atmosphere CO2 (ppm) 499.5 ± 10.4575.5 ± 22.1<0.01
NO3− deposition (kg N ha−1 yr−1)4.16 ± 0.5013.89 ± 1.09<0.01
NH4+ deposition (kg N ha−1 yr−1)15.92 ± 2.9217.55 ± 1.910.64
DON deposition (kg N ha−1 yr−1)10.17 ± 2.1024.75 ± 2.79<0.01
Total N deposition (kg N ha−1 yr−1)30.24 ± 4.5859.86 ± 4.54<0.01
P deposition (kg P ha−1 yr−1)0.33 ± 0.103.64 ± 1.050.01
Table
Table 4. Relationships between soil available nutrients and indices of biological N2 fixation including specific rate of symbiotic N2 fixation (SNF), nodulation investment, symbiotic N2 fixation for per plant (PNF), and specific rate of asymbiotic N2 fixation (ANF) in soil.
Table 4. Relationships between soil available nutrients and indices of biological N2 fixation including specific rate of symbiotic N2 fixation (SNF), nodulation investment, symbiotic N2 fixation for per plant (PNF), and specific rate of asymbiotic N2 fixation (ANF) in soil.
Soil NutrientsSpecific SNF RateNodulation InvestmentPNFSpecific Soil ANF Rate
rp Valuerp Valuerp Valuerp Value
Soil NO3−0.340.14−0.480.03−0.330.16−0.300.20
Soil NH4+0.080.73−0.59<0.01−0.250.28−0.520.02
Soil available P0.310.19−0.60<0.01−0.300.19−0.490.03
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Wang, Z.; Sun, X.; Chen, H.; Li, D. Effects of Multiple Global Change Factors on Symbiotic and Asymbiotic N2 Fixation: Results Based on a Pot Experiment. Nitrogen 2023, 4, 159-168. https://doi.org/10.3390/nitrogen4010011

AMA Style

Wang Z, Sun X, Chen H, Li D. Effects of Multiple Global Change Factors on Symbiotic and Asymbiotic N2 Fixation: Results Based on a Pot Experiment. Nitrogen. 2023; 4(1):159-168. https://doi.org/10.3390/nitrogen4010011

Chicago/Turabian Style

Wang, Zhenchuan, Xibin Sun, Hao Chen, and Dejun Li. 2023. "Effects of Multiple Global Change Factors on Symbiotic and Asymbiotic N2 Fixation: Results Based on a Pot Experiment" Nitrogen 4, no. 1: 159-168. https://doi.org/10.3390/nitrogen4010011

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