Effects of Exponential N Application on Soil Exchangeable Base Cations and the Growth and Nutrient Contents of Clonal Chinese Fir Seedlings

Nitrogen (N) is an essential macronutrient for plant function and growth and a key component of amino acids, which form the building blocks of plant proteins and enzymes. However, misuse and overuse of N can have many negative impacts on the ecosystem, such as reducing soil exchangeable base cations (BCs) and causing soil acidification. In this research, we evaluated clonal Chinese fir (Cunninghamia lanceolata (Lamb.) Hook) seedlings grown with exponentially increasing N fertilization (0, 0.5, 1, 2 g N seedling−1) for a 100-day trial in a greenhouse. The growth of seedlings, their nutrient contents, and soil exchangeable cations were measured. We found that N addition significantly increased plant growth and N content but decreased phosphorous (P) and potassium (K) contents in plant seedlings. The high nitrogen (2 g N seedling−1) treated seedlings showed a negative effect on growth, indicating that excessive nitrogen application caused damage to the seedlings. Soil pH, soil exchangeable base cations (BCs), soil total exchangeable bases (TEB), soil cation exchange capacity (CEC), and soil base saturation (BS) significantly decreased following N application. Our results implied that exponential fertilization resulted in soil acidification and degradation of soil capacity for supplying nutrient cations to the soil solution for plant uptake. In addition, the analysis of plants and BCs revealed that Na+ is an important base cation for BCs and for plant growth in nitrogen-induced acidified soils. Our results provide scientific insights for nitrogen application in seedling cultivation in soils and for further studies on the relationship between BCs and plant growth to result in high-quality seedlings while minimizing fertilizer input and mitigating potential soil pollution.


Introduction
Nitrogen (N) plays a key role in plant growth and N fertilizer is used to enhance the growth and productivity of crops or trees [1][2][3] but is also a limiting nutrient for plants [4]. Nutrient loading techniques based on exponential fertilization have shown that exponential fertilization was more consistent with the N required for plant growth patterns than conventional fertilization [5][6][7]. Compared to constant-rate nutrient loading of conventional fertilization, the rate of nutrient supply increases exponentially and exceeds the rate of seedling growth, minimizing the risk of nutrient toxicity in seedlings [8,9]. It is important to note that N additions may negatively impact ecosystem functions [10,11].
A common indicator for determining the availability of most plant nutrients is the measurement of the cation exchange capacity (CEC) in the soil [12]. The cation exchange capacity, or exchange rate, describes the number of cations in the soil solution that are 22 • 55 N). Circular pots with inner diameter of 25 cm and a height of 35 cm were filled with clay loam red soil (about 27 kg, 1/3 water content) derived from local Chinese fir (Cunninghamia lanceolata (Lamb.) Hook) forest. One-year-old clonal Chinese fir seedlings from the same nursery site with similar height and base diameter were used for the experiments. The Chinese fir seedlings were transplanted into pots and one seedling kept in each pot and the pots were randomized each week to prevent the effects of light.

Experimental Design
A complete randomized design (CRD) with four levels of exponential N application treatment was performed in the experiment. The four treatment levels of exponential N addition treatments were the control (without N addition, CK), a low level of N addition (0.5 g N sapling −1 , L1), a medium level of N addition (1 g N sapling −1 , L2), and a high level of N addition (2 g N sapling −1 , L3) with 10 replicates (10 pots for each treatment level), so that a total of 40 pots (4 × 10) was used in the experiment. The first fertilization was carried out on 15 May 2021, after the seedlings in the pots had grown steadily (no seedling died within three days). N fertilization was then applied every 10 days for a total of 10 applications (last fertilization on 22 August). Sample saplings were collected and measured ten days after the end of the exponential fertilization (1 September). Exponential fertilization was based on an exponential model to determine the amount of nitrogen per application [5], which was calculated by the following formula: where r is the relative rate of addition required to increase Ns (the initial level of the nutrient), to the final level N T + Ns, and N t-1 is the cumulative amount of fertilizer added including the last application. The amount Ns (72.19 mg N) was determined by chemical analysis of saplings 3 days before the start of fertilization. Knowing N T (0.5, 1, or 2 g N) and N S , r by Equation (1) to determine the number of fertilizations (t = 10), we can then calculate the exact amount of each fertilization by Equation (2). N was added in the form of urea (CH 4 N 2 O) solution, and the details of the fertilization process are shown in Figure 1.

Study Areas
The pot experiments were conducted in a cultivated greenhouse located in Guangxi Forestry Research Institute, Nanning city, Guangxi Zhuang Autonomous Region (108°21′ E, 22°55′ N). Circular pots with inner diameter of 25 cm and a height of 35 cm were filled with clay loam red soil (about 27 kg, 1/3 water content) derived from local Chinese fir (Cunninghamia lanceolata (Lamb.) Hook.) forest. One-year-old clonal Chinese fir seedlings from the same nursery site with similar height and base diameter were used for the experiments. The Chinese fir seedlings were transplanted into pots and one seedling kept in each pot and the pots were randomized each week to prevent the effects of light.

Experimental Design
A complete randomized design (CRD) with four levels of exponential N application treatment was performed in the experiment. The four treatment levels of exponential N addition treatments were the control (without N addition, CK), a low level of N addition (0.5 g N sapling −1 , L1), a medium level of N addition (1 g N sapling −1 , L2), and a high level of N addition (2 g N sapling −1 , L3) with 10 replicates (10 pots for each treatment level), so that a total of 40 pots (4 × 10) was used in the experiment. The first fertilization was carried out on 15 May 2021, after the seedlings in the pots had grown steadily (no seedling died within three days). N fertilization was then applied every 10 days for a total of 10 applications (last fertilization on 22 August). Sample saplings were collected and measured ten days after the end of the exponential fertilization (1 September). Exponential fertilization was based on an exponential model to determine the amount of nitrogen per application [5], which was calculated by the following formula: where r is the relative rate of addition required to increase Ns (the initial level of the nutrient), to the final level NT + Ns, and Nt-1 is the cumulative amount of fertilizer added including the last application. The amount Ns (72.19 mg N) was determined by chemical analysis of saplings 3 days before the start of fertilization. Knowing NT (0.5, 1, or 2 g N) and NS, r by Equation (1) to determine the number of fertilizations (t = 10), we can then calculate the exact amount of each fertilization by Equation (2). N was added in the form of urea (CH4N2O) solution, and the details of the fertilization process are shown in Figure  1.

Measurements
The seedling height from ground to apical meristem was measured using linear tape and the base diameter was measured in two vertical directions with a vernier caliper; the mean was calculated and used for analysis [32]. The measurements were taken on the first day and the last day of the experiment. In addition, the weekly measurements were taking between 8 am to 10 am during the experiment. The increments in height and base diameter of seedlings were calculated from the differences of two measurements (the first day and the last day of the experiment). At the end of the experiment, all seedlings were harvested to measure biomass and nutrient content. Each individual seedling was divided into root, stem and leaves for biomass measurements and chemical analysis in the laboratory. Plant samples were oven dried at 105 • C for 15 min and then at 75 • C for 48 h until constant weight for determining the biomass using an analytical balance. All leaves were ground and passed through a 40-mesh sieve for nitrogen (N), phosphorus (P), and potassium (K) analysis in the laboratory. Total N and P were determined according to the standard methods and the total potassium (K) was analysis by plasma emission spectrophotometry after dry ashing and extraction with HCl-HNO 3 [33].
Three soil samples in each pot were collected after harvesting of the plant seedlings. Each soil sample was passed through a 2-mm sieve to remove roots and stones, and then airdried. Soil pH was measured with a glass electrode at a water: soil ratio of 1:2.5 (w/v), and soil organic carbon (SOC) was measured by the potassium dichromate oxidation heating method. Total nitrogen (TN) was determined by spectrophotometry after digestion with K 2 Cr 2 O 7 -H 2 SO 4 using an automatic Kieldahl instrument (K9860, Hanong, Kaiyuan, China). The NH 4 + -N and NO 3 --N were measured by extraction using an automatic continuous flow analyzer (Autoanalyzer 3, Seal analytical, Norderstedt, Germany). Determination of total phosphorus (TP) was carried out by the molybdenum blue colorimetric method, after hydrolysis and diffusion of NaOH, using a UV-Vis spectrophotometer (UV-6800A, ZhuoGuang, Jinan, China). Total potassium (TK) was determined by spectrophotometry after wet digestion with HF-HClO 4 by an atomic absorption spectrophotometer (AA-7000, Shimadzu, Kyoto, Japan). The exchangeable acid (EA) was measured by potassium chloride exchange-neutralization titration. The exchangeable BCs (K + , Ca 2+ , Mg 2+ and Na + ) were measured by atomic absorption spectrophotometry using the NH 4 Cl-C 2 H 5 OH method (AA-7000, Shimadzu, Kyoto, Japan) [34].

Statistical Analyses
One-way analysis of variance (ANOVA) was used to assess the effects of different levels of exponential N application on increments of height, base diameter, and nutrient element content of each organ (root, stem, leaf) of seedlings and soil physicochemical properties. Tukey's HSD was used to test significant differences among different N addition treatments (p < 0.05). The Shapiro-Wilk test was used for the data analysis, and the original data were log-transformed to satisfy the normality and homoscedasticity assumptions of ANOVA. Pearson's correlation test was used to analyze the relationship among different parameters and the results were displayed by R package 'ggcorrplot' (version: 0.1.3). Considering collinearity, soil TEB, CEC, and BS were not included in the correlation analysis. The means of N application effects were compared by a Tukey-Kramer test using the SAS program. Multiple comparisons were conducted to test the differences among treatments in response to plants growth and nutrient content of plant seedlings including N, P, and K (p < 0.05). To examine the relationship between BCs and plant growth, a stepwise regression model (SRM) was used to select factors affecting plant growth with the best-fit model chosen using Akaike's Information Criterion (AIC). Statistical analyses were conducted using the SAS statistical package [35], and the statistical programming language R [36].

Effects of N Addition on Seedling Growth and Nutrient Content of Plants
Compared with CK (no N fertilization), N addition was beneficial to plant growth ( Table 1). The increments of ground diameter and height increased with increasing N fertilization concentrations, except in L3. Biomass of root, stem, and leaf and the total biomass of seedlings showed an increasing tendency with N addition. The biomass of each component of the plant seedlings had significant effects in response to the N application treatments (p < 0.05) except for root biomass. In general, seedlings grew best in L2 treatments. A similar growth state of the seedlings was observed between CK and L3 treatments, indicating the negative effect of high N addition. Nutrient elements of seedlings were significantly different between CK and N addition treatments (p < 0.05; Figure 2). With increasing N fertilization concentrations, the N content in the organs of the seedling increased, especially in the roots and leaves. The N content in roots at L3 reached 43.21 ± 0.15 mg·g −1 , while the N in stems decreased to 21.50 ± 0.14 mg·g −1 (Figure 2a). Conversely, P and K contents decreased after N addition, except for K in the roots. The content of P was the highest in roots at CK (3.30 ± 0.06 mg·g −1 ), and the lowest was in leaves at L3 (1.65 ± 0.02 mg·g −1 ) (Figure 2b). Both the minimum and maximum K contents occurred at CK, which were 7.95 ± 0.09 mg·g −1 in roots and 13.85 ± 0.09 mg·g −1 in leaves, respectively (Figure 2c). mass of seedlings showed an increasing tendency with N additi component of the plant seedlings had significant effects in respo treatments (p < 0.05) except for root biomass. In general, seedling ments. A similar growth state of the seedlings was observed be ments, indicating the negative effect of high N addition.
Nutrient elements of seedlings were significantly different b tion treatments (p < 0.05; Figure 2). With increasing N fertilizatio content in the organs of the seedling increased, especially in the content in roots at L3 reached 43.21 ± 0.15 mg·g −1 , while the N in s ± 0.14 mg·g −1 (Figure 2a). Conversely, P and K contents decreased for K in the roots. The content of P was the highest in roots at CK the lowest was in leaves at L3 (1.65 ± 0.02 mg·g −1 ) (Figure 2b). maximum K contents occurred at CK, which were 7.95 ± 0.09 mg 0.09 mg·g −1 in leaves, respectively (Figure 2c).

Effect of N Addition on Soil Physicochemical Properties
Soil physicochemical properties showed a significant differe and CK treatments (p < 0.05) (Figure 3

Effect of N Addition on Soil Physicochemical Properties
Soil physicochemical properties showed a significant difference between N addition and CK treatments (p < 0.05) (Figure 3). Soil pH, BCs, TEB, CEC, and BS decreased, but SOC, TN, NO 3 − , NH 4 + , and EA increased with increasing N fertilization concentrations. No significant differences of EA and CEC were observed among all treatments, with the corresponding maximum values of 145.73 ± 0.22 cmol·kg −1 and 157.00 ± 0.67 cmol·kg −1 , and minimum values of 142.26 ± 0.85 cmol·kg −1 and 154.40 ± 0.27 cmol·kg −1 , respectively. BCs were greatly reduced with increasing N fertilization (except for Ca 2+ and Na + in L1), which led to an increase of EA and a stable status of CEC. Compared to CK, N fertilization resulted in decrease of TP and increase of K, but there were no significant differences between these two elements among N addition treatments (Figure 3).
responding maximum values of 145.73 ± 0.22 cmol·kg and 157.00 ± 0.67 cmol·kg minimum values of 142.26 ± 0.85 cmol·kg −1 and 154.40 ± 0.27 cmol·kg −1 , respectivel were greatly reduced with increasing N fertilization (except for Ca 2+ and Na + in L1), led to an increase of EA and a stable status of CEC. Compared to CK, N fertilizati sulted in decrease of TP and increase of K, but there were no significant differenc tween these two elements among N addition treatments (Figure 3).

Relationships among Different Soil Parameters
The four types of BCs (Ca 2+ , Mg 2+ , K + , and Na + ), pH, and TP showed a significant positive correlation with each other, while BCs showed a significant negative correlation with SOC, TN, NO3 -, and NH4 + (Figure 4). pH was negatively correlated with SOC, TN, NO3 -, NH4 + , and EA, which were directly influenced by N addition. NO3 -had a closer relation (high coefficient) with other soil parameters than NH4 + . Soil K was not significantly correlated with any other soil parameters except soil pH, SOC, and TN.

Relationships among Different Soil Parameters
The four types of BCs (Ca 2+ , Mg 2+ , K + , and Na + ), pH, and TP showed a significant positive correlation with each other, while BCs showed a significant negative correlation with SOC, TN, NO 3 − , and NH 4 + (Figure 4). pH was negatively correlated with SOC, TN, NO 3 − , NH 4 + , and EA, which were directly influenced by N addition. NO 3 − had a closer relation (high coefficient) with other soil parameters than NH 4 + . Soil K was not significantly correlated with any other soil parameters except soil pH, SOC, and TN.

The Correlations between Plant Growth and BCs
In the SRMs of diameter, K + , Na + , and Mg 2+ were the fixed variables in the optimiza tion model (Table 2), and only Na + was significantly positively correlated with diamete (p = 0.04). K + , Na + , and Mg 2+ were also selected as the fixed variables in the optimizatio SRM of height, and K + was a significant factor negatively affecting plant height (p = 0.001 while Na + was significantly positively correlated with plant height (p = 0.01). There wer two fixed variables (K + and Na + ) in the optimization SRM of biomass, which were signif cantly negatively and positively correlated with biomass, respectively ( Table 2). Amon the exchangeable BCs, the cations of K + , Na + , and Mg 2+ may be the important BCs affectin the growth of seedlings under N fertilization, especially Na + .

The Correlations between Plant Growth and BCs
In the SRMs of diameter, K + , Na + , and Mg 2+ were the fixed variables in the optimization model (Table 2), and only Na + was significantly positively correlated with diameter (p = 0.04). K + , Na + , and Mg 2+ were also selected as the fixed variables in the optimization SRM of height, and K + was a significant factor negatively affecting plant height (p = 0.001), while Na + was significantly positively correlated with plant height (p = 0.01). There were two fixed variables (K + and Na + ) in the optimization SRM of biomass, which were significantly negatively and positively correlated with biomass, respectively ( Table 2). Among the exchangeable BCs, the cations of K + , Na + , and Mg 2+ may be the important BCs affecting the growth of seedlings under N fertilization, especially Na + .

Growth and Nutrient Contents of Seedlings
Our results showed that exponential N addition enhanced the growth of Chinese fir saplings. The diameter, height, and biomass of the saplings were significantly higher in N adding treatment than in control but the enhanced effect was weaker in high N treatment (L3). The high nutrient availability reduced the root biomass as well in the research. This is because roots confine their growth with sufficient availability of nutrients as they do not have to penetrate deeply in search of the nutrients [37][38][39]. Previous studies have demonstrated that exponential fertilization improved plant growth by nutrient loading [2,5,8,13]. N fertilization increased N content, but not P and K contents in leaves. It was clear that the N addition led to an increase in N content in plants. In this study, P generally showed a decreasing trend with N addition, which was likely attributable to the following two points. One was that the N addition led to the imbalance of the N:P ratio in soil, thus affecting the uptake of P by plants [31,40]. The other was that the addition of N caused a dilution effect on P, leading to a decrease of its content in plants. The K content increased with N addition in root but decreased in stem and leaf, suggesting absorption and accumulation of K element in the root. However, not all the studies showed plant growth was positively associated with N addition. Experiments of western hemlock [9] and Douglas-fir [6] showed that exponentially fertilized seedlings did not differ significantly in growth and nutrient contents. We also found that the growth of seedlings was influenced in the high N addition treatments (L3). This was because higher nutrient levels in seedlings may increase their susceptibility to moisture stress, frost damage, and herbivory [9,41], while N addition-induced soil acidification also influences seedling growth.

Response of Soil Parameters to N Addition
In this study, there were significant differences in soil physicochemical properties between the N addition treatment and the control treatment CK (p < 0.05). Soil pH, BCs, TEB, CEC, and BS decreased with increasing N fertilization, but SOC, TN, NO 3 − , NH 4 + , and EA increased under N addition treatments. Soil TP reduced and K increased in the N addition treatments compared to CK. Many studies have illustrated that N fertilization can result in decreasing soil pH [3,13,17,20,22]. Previous studies showed that N addition could lead to increasing hydrolysis of BCs and release of H + in the soil solution [42,43]. H + had more proton competitiveness to BCs for cation exchange sites which resulted in lower pH and soil BC depletion [13,44]. Decreasing BC content could cause lower TEB, CEC, and BS, and a low BS (<10%) also indicated an acidified context [13,45]. These processes might be the reason why the pH showed significant positive correlation with four BCs. On the other hand, N fertilization increased the nitrification process, which caused a decrease in pH and an increase in EA [46]. Excess of N input led to an increase of NO 3 − leaching from bulk soils into soil solution [10,47,48]. However, leaching loss of NO 3 -N was often accompanied by leaching of Ca 2+ , Mg 2+ , and other cations [47]. In addition, studies demonstrated that increase of NH 4 + would give a stronger bond strength with soil and could exchange the BCs adsorbed by the soil (primarily for K + and Na + ) and increase the loss of soil BCs [49]. Our results showed both NO 3 − and NH 4 + increased by N addition, which further caused the decrease of soil BCs. These phenomena of small change of CEC under different N concentrations were ascribed to (1) the magnitude of change in BCs being much smaller than that in CEC and (2) the cations of Al 3+ and Fe 2+ making major contribution to soil CEC [17], which were restricted in the pot experiments.
For other nutrients, SOC increased after N fertilization, which promoted plant productivity. Stimulation of microbial activity in soil by N addition may play a key role in this process [50,51]. There was a coupling relationship between N and P resulting in N:P imbalance due to N deposition [31,40]. Similar results were also observed in other studies [13,46]. We noticed inconsistent changes of K content in soil and plants as well in our research. Although the decrease in K + resulted in decrease in the K content in stems and leaves, the K content increased in roots after N addition which was consistent with the changes in soil K. This may be related to the complex conversion between K and K + [52] while our research might explain the interrelationships among the soil parameters.

Effects of BCs on Chinese Fir Seedling Growth
Three SRMs of plant growth, K + , Na + , and Mg 2+ were set as fixed variables in the diameter and height optimization model. Only K + , and Na + were in the biomass optimization model. Na + was significantly positively correlated with both diameter (p = 0.04) and height (p = 0.01) indicating that Na + may be a key cation for plant growth in acidified soil. Na + is not a nutrient directly involved in plant metabolism but plays a key role in soil acidity buffering along with the other BCs [13,53]. Soil acidification can lead to an imbalance of soil elements that ultimately affects plant growth [1,20]. Therefore, Na + may be more closely related to plant growth in the pot experiment in acidic soil (lower pH and BS) induced by nitrogen addition. Both N and K + can increase plant productivity, but N addition resulted in the decrease in K + in this study. In addition, K + was reduced more than Na + in our study because K + was easily taken up by the plant roots rather than remaining in the soil [52]. Therefore, our results suggest a negative correlation between K + and plant growth. Further research should focus more on how BCs directly affect plant growth.

Conclusions
In summary, our study demonstrated the exponential application of N fertilizer significantly affected the Chinese fir seedlings growth and nutrient concentrations in the plant organs. When compared to the control, the low N addition (0.5 and 1 g N seedling −1 ) significantly increased the Chinese fir seedlings growth and the N content in plants but decreased phosphorous (P) and potassium (K) contents in plant seedlings. However, the high N addition (2 g N seedling −1 ) decreased the growth of Chinese fir seedings because the excessive N application damaged the metabolism of the seedlings. N application significantly decreased soil physicochemical properties, including soil pH, soil exchangeable base cations (BCs), soil total exchangeable bases (TEB), soil cation exchange capacity (CEC), and soil base saturation (BS). Our hypothesis was supported by the results. In addition, the N exponential fertilization can lead to acidification and degradation of the cation exchange capacity in the soil while Na + was the most important base cation for soil BCs and for plant growth in N-induced acidified soils. Our results provide a solid scientific basis for improved understanding of N fertilizer application in plant seedling cultivation.
Author Contributions: R.W., X.D. and Y.P. conceived and designed the experiments; Z.Z., H.P. and L.L. performed the experiments; R.W. and R.H. analyzed the data; Y.W. and X.D. contributed technical advice. R.W. and Y.W. wrote the paper. All authors have read and agreed to the published version of the manuscript.

Data Availability Statement:
The data presented in this article are available on request from the corresponding authors.