Effects of Nitrogen Forms on Root Morphology and Nitrogen Accumulation in Pinus tabuliformis carr. Seedlings under Exponential Fertilization

Abstract

In this study, the effects of different fertilization methods and nitrogen forms on the root morphology and nitrogen accumulation of Pinus tabuliformis carr. were investigated, and the interaction mechanism between roots and nitrogen fertilizer was analyzed to provide a theoretical basis for the rational fertilization of Pinus tabuliformis. The total amount of nitrogen fertilizer applied to seedlings was 100 mg·plant⁻¹; four nitrogen form treatments of ammonium nitrogen, nitrate nitrogen, ammonium nitrate 1:1, and amide nitrogen were set up; and two fertilization methods of conventional and exponential fertilization were applied, with a total of eight experimental treatments. By measuring root parameters, plant biomass, and nitrogen accumulation, the correlation between fertilization method and nitrogen form on the root index of seedlings was analyzed, and the effects of fertilization methods and nitrogen forms on the root growth of seedlings were discussed. Compared with conventional fertilization, exponential fertilization significantly promoted root growth and development, and amide nitrogen was the best nitrogen form. The total length, total surface area, total volume, average diameter, root tips, root/shoot ratio, root tissue density of seedlings’ roots, biomass, and nitrogen accumulation of seedlings in exponential fertilization with amide nitrogen EF₃ treatment all increased substantially compared with the others, The effects of fertilization mode, nitrogen form, and their interaction on the partial growth of Pinus tabuliformis seedlings were significant (p < 0.05). The correlation analysis of each index showed that the correlation between nitrogen accumulation and biomass was strongest. Therefore, considering the morphological and structural characteristics of the root system and the nitrogen accumulation in the root system, amide nitrogen in the experimental fertilization can better promote the growth and development of the root system of seedlings.

1. Introduction

Fertilization is the most direct way to replenish nutrient elements to promote the rapid growth of seedlings and improve the quality of seedlings, and reasonable fertilization methods can provide sufficient nutrients for each stage of plant growth and create a good nutritional environment [1,2]. Conventional fertilization utilizes only one fertilization application or applies the same amount of fertilizer from the beginning to the end of the seedlings, which ignores the nutrient demand of seedlings in each period and also leads to the waste of fertilizers and the occurrence of problems such as soil physicochemical properties. Exponential fertilization adopts an exponential incremental approach, which can satisfy the nutrient demand of seedlings at different growth stages, directly promote seedling growth, and improve resilience [3,4]. Studies have shown that Ormosia hosiei Hemsley & E. H. Wilson [5], Podocarpus macrophyllus [Thunb.] D.Don [6], Larix olgensis Henry [7], Hydrangea macrophylla [Thunb.] Ser. [8], and Pinus banksiana Lamb. [9] are more efficient in transporting nutrients under the exponential fertilization method.

Nitrogen (N) is the main exogenous nutrient element in the breeding and growth process of seedlings, and the uptake and utilization of different nitrogen forms vary among different tree species under the selection of different environmental conditions over a long period of time [10,11]. Nitrogen that can be absorbed by plants is mainly divided into inorganic nitrogen and organic nitrogen. Inorganic nitrogen is dominated by nitrate nitrogen (NO₃⁻-N) and ammonium nitrogen (NH₄⁺-N), and NO₃⁻-N and NH₄⁺-N have a certain interaction when mixed in a certain proportion, which can promote plant growth better than nitrogen in a single form. When plants absorb NH₄⁺-N, H⁺ will be excreted during the NH₄⁺-N assimilation process, thus maintaining soil charge balance. When there is too much NH₄⁺-N, ammonium toxicity will lead to the mechanism of plant acidification and inhibit plant growth [12]. NO₃⁻-N is usually reduced to NH₄⁺-N for absorption by plants, and excessive NO₃⁻-N alkalizes roots and reduces chlorophyll content [13]. Urea is converted to NH₄⁺-N by the urease of soil microorganisms and plants [14]. This study had shown that Cunninghamia lanceolata Lamb. is better suited to grow in soils containing ammonium nitrogen, and Schima superba Gardn. is better suited to grow in soils containing nitrate nitrogen [15].

In natural environments, plants have developed transport and signaling mechanisms specific to their respective nitrogen sources, and the root system, as a nutrient organ for the plant uptake and transport of nitrogen, is a hub connecting the plant to the soil, microorganisms, and the environment [16,17]. The development of the root system directly affects the vitality of the root system, which in turn affects the absorption and utilization of nitrogen by plants and affects the accumulation of dry matter. The total length, root surface area, total volume, average diameter, root tips, and specific root length of plant root morphology indicators can directly reflect the changes in plant root morphology and indirectly prove the strength of root absorption and nutrient transport capacity [18,19,20]. The change in root morphology will affect the ability of plant roots to absorb and transport nutrients, the specific root length can reflect the efficiency of root nutrient absorption to a certain extent [21,22], and root tissue density and average diameter reflect the potential absorption capacity of the root system. Therefore, the experiment of the morphological characteristics of the root system can visually reveal the nutrient uptake and growth status of the plant [23].

As a major tree species in arid and semi-arid areas, the Pinus tabuliformis has a strong adaptive capacity, and cultivating high-quality seedlings is the basis for successful afforestation. Therefore, in environmentally stressed sites, the transplant of containerized seedlings is often superior to bare-root seedlings. In the process of Pinus tabuliformis seedlings, conventional fertilization is usually adopted to improve the nutrient demand of Pinus tabuliformis seedlings, but the scientific application of the fertilization method and fertilization amount not only affects the growth and development of Pinus tabuliformis, but also affects the environment [24,25]. Therefore, rational fertilization is helpful to improve plant growth and development and enhance plant resistance to biotic and abiotic stresses [26,27]. Studies on the effects of nitrogen forms on plant roots mainly focus on herbs, crops, and fruit trees [28,29], and few studies have been conducted on conifer species. At present, the application rules of Pinus tabuliformis in China are mainly based on the seedling stage and mainly on the study of fertilization methods and fertilizer types. Some scholars have shown that exponential fertilization can effectively improve the nutrient accumulation of Pinus tabuliformis container seedlings [30,31], and the main fertilizers are nitrogen fertilizer, phosphate fertilizer, and potassium fertilizer [32], but the influence of the specific nitrogen form on the growth of Pinus tabuliformis is still unclear. It is worth studying whether nitrogen forms can increase nitrogen accumulation and improve the seedling quality of Pinus tabuliformis. In this experiment, we investigated the effects of nitrogen forms and fertilization methods on the root morphology and nitrogen accumulation of Pinus tabuliformis. We also set up two kinds of fertilization methods (conventional fertilization and exponential fertilization) and four nitrogen form conditions (ammonium nitrogen, nitrate nitrogen, amide nitrogen, and ammonium nitrate 1:1) to explore whether different nitrogen forms can improve the root growth and development of Pinus tabuliformis seedlings, and whether the effect of exponential fertilization is affected by nitrogen forms, to provide scientific nutritional management reference for Pinus tabuliformis seedling rearing technology.

2. Materials and Methods

2.1. Plant Materials and Environmental Conditions

The experiment was conducted from March 2022 to December 2022 in a greenhouse at the College of Forestry, Shenyang Agricultural University. The ambient temperature for growing seedlings indoors was 24 ± 2 °C, and relative humidity was 60%~70%. After soaking the Pinus tabuliformis seeds (Shenyang, China) at room temperature for 48 h in April 2022 to absorb sufficient moisture, the seeds were screened for empty shells and deflated seeds. Gauze-wrapped seeds were sown in 32-well (4 × 8) seedling hole trays after being sprayed daily with water to 30% dew. The hole tray substrate was sterilized with peat soil 60%, vermiculite 20%, and perlite 20%. After 6 months of cultivation in the greenhouse, 216 seedlings with a height of 7.5 ± 0.5 cm and a diameter of 0.8 ± 0.1 mm were selected for 10 weeks of the fertilization experiment. Specific fertilization methods are described below.

2.2. Fertilization Design and Nitrogen Application Methods

The experiment was set up with 4 nitrogen form treatments and 2 fertilization methods (a total of 8 treatments), with each treatment containing 9 replications, and samples were collected for 3 months, for a total of 216 seedlings. The experiment was conducted in a completely randomized block design.

2.2.1. Fertilization Method

Fertilization was applied every week from 3 October 2022, and the fertilization was continued 10 times, applying a total of 100 mg·plant⁻¹ of nitrogen fertilizers [31]. Fertilizers were applied using a water-soluble method by dissolving the fertilizers required for each treatment in 640 mL of water, and after weighing 20 mL in a beaker, it was slowly applied to the substrate of each container seedling. There were 2 types of fertilization methods, conventional fertilization and exponential fertilization, and the formula for weekly fertilizer application was calculated as follows:

Formula of conventional fertilization:

$$N_t=N_T/10$$

(1)

Formula of exponential fertilization model [33]:

$$N_t=N_S\left(-1\right)-N_{t-1};$$

(2)

$$\mathrm{r}=Ln(N_T/N_S+1)/t;$$

(3)

where N_t is the amount of fertilization applied for the tth time, N_T is the total amount of nutrients to be applied, N_t−1 is the total amount of fertilization applied including the t − 1 time, N_S is the initial nutrient content of the fertilization Pinus tabuliformis, and N_S was measured to be 4.97 mg·plant⁻¹. In this experiment, r is the relative growth rate. The specific amount of fertilization applied per week is shown in Figure 1.

2.2.2. Nitrogen Form Additions

In this experiment, four types of nitrogen fertilizers were used: ammonium nitrogen fertilizer—ammonium chloride (N 26%) (Shanghai Maclin Biochemical Technology Co. Ltd., Shanghai, China); nitrate nitrogen fertilizer—potassium nitrate (N 14%) (Longcheng bacterial fertilizer factory, Chaoyang, China), amide nitrogen—urea (N 46%) (Shanghai Maclin Biochemical Technology Co. Ltd., Shanghai, China); and NH₄⁺-N:NO₃⁻-N = 1:1. Each nitrogen form was added as shown in

Table 1. Nitrogen form additions under different treatments.

Treatment	Nitrogen Forms
CF1	Ammonium nitrogen
CF2	Nitrate nitrogen
CF3	Amide nitrogen
CF4	Ammonium nitrogen/nitrate nitrogen = 1:1
EF1	Ammonium nitrogen
EF2	Nitrate nitrogen
EF3	Amide nitrogen
EF4	Ammonium nitrogen/nitrate nitrogen = 1:1

.

2.3. Sample Collection and Indicator Measurement

Determination of root parameters: Root sampling was carried out in late December, and 9 Pinus tabuliformis seedlings with uniform growth were selected for each treatment to determine the root parameters of Pinus tabuliformis seedlings. The roots of each seedling were cut, rinsed with water to remove the residual soil from the roots, and then slowly rinsed with distilled water. And filter paper was used to absorb the excess water on the surface of the root system, which was then scanned with a root scanner (microtek HED-WinRHIZO) (Shandong Holder electronic Technology Co. Ltd., Weifang, China) to produce a picture with a resolution of 600 dpi, which was analyzed to obtain the total root length (TRL, cm), total root surface area (TRSA, cm²), total root volume (TRV, cm³), roots’ average diameter (RAD, mm), and root tips (RT, number). The root/shoot ratio, root tissue density, and specific root length were also calculated using the following equation:

$$\mathit{Root} \mathit{shoot} \mathit{ratio} = \mathit{Root} \mathit{biomass}/\mathit{Stem} \mathit{and} \mathit{leaf} \mathit{biomass};$$

(4)

$$\mathit{Root} \mathit{tissue} \mathit{density} (\mathrm{g}·{\mathrm{cm}}^{-3}) = \mathit{Root} \mathit{biomass}/\mathit{Total} \mathit{root} \mathit{volume};$$

(5)

$$\mathit{Specific} \mathit{root} \mathit{length} (\mathrm{g}·{\mathrm{cm}}^{-1}) = \mathit{Total} \mathit{root} \mathit{length}/\mathit{Root} \mathit{biomas};$$

(6)

Determination of biomass: At the end of October, November, and December, samples were collected from 9 seedlings of each treatment; the soil on the root surface of the seedlings was cleaned; and the roots, stems, and leaves of the seedlings were separated and put into envelopes with branch shears. After 15 min in the oven at 105 °C, the seedlings were dried at 75 °C to constant weight. After 48 h, the analytical balance (0.0001 g) was used to weigh and count.

Determination of nitrogen accumulation: The total nitrogen content of Pinus tabuliformis seedlings was measured using H₂SO₄-H₂O₂ decoction method, 0.3 g of Pinus tabuliformis seedlings weighed to constant weight were put into the decoction tubes on the previous day, and 5 mL of H₂SO₄ was added to each decoction tube and then placed in a fume hood overnight. On the day of the test, the decoction oven was set at 150 °C, the sample was decocted for 30 min with 20 drops of H₂O₂, the temperature of the decoction oven was raised to 550 °C and decocted for 10 min, H₂O₂ was added until a light orange color was obtained, the decoction was continued for 10 min, and H₂O₂ was added until the color became transparent and was then decocted for 2 h. The decocted samples were fixed into 50 mL volumetric flasks, and extracted portions of the solution were measured for plant nitrogen content using an AA3 continuous flow analyzer (Zhongke Ruijie Technology Co. Ltd., Tianjin, China) [34]. The amount of nitrogen accumulated in the root system of Pinus tabuliformis was calculated using the following formula [35].

$$\mathit{Nitrogen} \mathit{accumulattion} (\mathrm{mg}·{\mathrm{plant}}^{-1}) = \mathit{Nitrogen} \mathit{content} \times \mathit{biomass};$$

(7)

2.4. Data Analysis

SPSS26.0 software (IBM Corp., Armonk, NY, USA) was used for statistical analysis. One-way ANOVA and Duncan’s method were used for variance analysis and multiple comparison (α = 0.05), respectively, to analyze the differences in fertilization methods and nitrogen forms in Pinus tabuliformis seedlings. The interaction effects of fertilization methods and nitrogen forms were analyzed using two-factor ANOVA. Pearson correlation analysis was used to examine the correlation between root indexes of Pinus tabuliformis seedlings under different treatments. And Microsoft Excel 2021 (Microsoft Office, Redmond, WA, USA) and Origin 2021 (OriginLab, Northampton, MA, USA) were used to draw relevant charts. The data in the chart are the mean ± standard deviation.

3. Results

3.1. Effects of Different Nitrogen Forms on Root Parameters of Pinus tabuliformis Seedlings under Exponential Fertilization

The root parameters of Pinus tabuliformis seedlings were better in EF than in CF under the same nitrogen form conditions (Figure 2). In addition to the specific root length under the same fertilization conditions, the root parameters of Pinus tabuliformis seedlings under the amide nitrogen form were better than those of other forms. The total root surface area, total root volume, and roots’ average diameter under EF₃ treatment were significantly higher than those under other treatments. Compared to the more CF and EF methods, the total surface area increases of 66.0%~129.7% and 3.8%~104.8% (Figure 2B), respectively; the total volume increases of 91.4%~186.2% and 29.8%~64.5% (Figure 2C, respectively; the roots’ average diameter increases of 74.0%~122.5% and 29.8%~68.5% (Figure 2D), respectively; and the total root length, root tips, and root/shoot ratio were not significantly different from EF₄ but significantly higher than those of the other treatments. Compared to the more CF and EF methods, the total root length increases of 39.4%~71.5% and 2.3%~36.9% (Figure 2A), respectively; the root tips number increases of 14.5%~60.7% and 0.4%~28.0% (Figure 2E), respectively; the root/shoot ratio increases of 17.7%~29.9% and 4.3% to 39.3% (Figure 2F), respectively; and the root tissue density were not significantly different between EF₁ and EF₂, but significantly higher than those of the other treatments. Compared to the more CF and EF methods, the root tissue density increases were 27.6%~77.5% and 4.4%~15.8% (Figure 2G), respectively. The specific root lengths of nitrate nitrogen and ammonium nitrogen were better than those of the other treatments under the two fertilization methods (Figure 2H).

3.2. Effects of Different Nitrogen Forms on the Biomass of Pinus tabuliformis Seedlings under Exponential Fertilization

In October, the root biomass of Pinus tabuliformis seedlings under CF₃ treatment was significantly higher than those under other treatments and had no significant difference to the stem biomass of CF₄, EF₂, and EF₃, which was significantly higher than those treated with other treatments. The leaf biomass of Pinus tabuliformis seedlings treated with CF₂ and CF₃ had no significant difference, which was significantly higher than those treated with other treatments (Figure 3). In November, the root, stem, and leaf biomass of EF₃ treatment was significantly higher than those of other treatments. In December, the root and leaf biomass of Pinus tabuliformis seedlings under EF₃ was significantly higher than those of other treatments, while there was no significant difference between stem biomass and EF₄, which were significantly higher than those of other treatments. Under the same nitrogen condition, the biomass of roots, stems, and leaves under EF was better than that under CF. In the exponential fertilization mode, the root, stem, and leaf biomass of the amide EF₃ treatment was largest, with values of 0.7 g, 0.3 g, and 1.8 g, respectively. Compared with CF and EF, the increases were 36.2%~86.1% and 12%~57.2%, 9.1%~31.0% and 2.3%~14.2%, and 17.1%~42.3% and 8.8%~15%, respectively.

3.3. Effects of Different Nitrogen Forms on Nitrogen Accumulation in Pinus tabuliformis Seedlings under Exponential Fertilization

Under the same nitrogen form conditions, nitrogen accumulation in the roots, stems, and foliage of Pinus tabuliformis seedlings was higher under the EF method than under the CF method (Figure 4). Under the same fertilization method, nitrogen accumulation in the roots and leaves of amide CF₃ and EF₃ treatment was significantly higher than those of other treatments, and nitrogen accumulation in the stems was not significantly different from that of the EF₄ treatment, which was higher than those of other treatments. Among them, the highest nitrogen accumulation was found in the roots, stems, and leaves of EF₃ with 0.78 mg·plant⁻¹, 0.4 mg·plant⁻¹, 2.1 mg·plant⁻¹, respectively. The enhancements were 35.2%~78% and 11.3%~56.8%, 8.6%~28.7% and 1.8%~14%, and 16.2%~39.9% and 8.3%~14.8%, respectively, compared to the CF and EF methods.

3.4. Analysis of the Relationship between Nitrogen Accumulation and Root Indexes of Pinus tabuliformis Seedlings under Different Fertilization Methods and Nitrogen Forms

In the treatment of CF₁, root nitrogen accumulation (RNA) was found to have a significant positive correlation with root tissue density (RTD) and root biomass (RB) (0.71~0.77), and a significant negative correlation with specific root length (SRL) (−0.80) (Figure 5A). In the treatment of CF₂, RNA was found to be significantly correlated with total root volume (TRV), root/shoot ratio (RSR), and RB (0.80~0.91) and negatively correlated with SRL (−0.99) (Figure 5B). In the treatment of CF₃, RNA was found to be significantly positively correlated with total root length (TRL) and TRV (0.72~0.77), extremely significantly positively correlated with root tips (RT) and RB (0.84~0.91), and significantly negatively correlated with SRL (−0.70) (Figure 5C). In the CF₄ treatment, RNA showed a significant positive correlation with TRL and RSR (0.72~0.77); extremely significant positive correlation with total root surface area (TRSA), RT, and RB (0.81~0.98); and significantly negative correlation with SRL (−0.73) (Figure 5D). In the EF₁ treatment, RNA was positively correlated with TRSA and TRV (0.67~0.70); positively correlated with TRL, RB (0.94~0.95), and SRL (−0.75); and negatively correlated with SRL (−0.75) (Figure 5E). In the EF₂ treatment, RNA showed a significant positive correlation with RT, RSR, and RTD (0.70~0.79); extremely significant positive correlation with TRL and RB (0.85~0.88); significantly negative correlation with roots’ average diameter (RAD) (−0.67); and extremely significant negative correlation with SRL (−0.89) (Figure 5F). In the EF₃ treatment, RNA was significantly positively correlated with TRL, RSR, and RB (0.84~0.95); negatively correlated with RAD (−0.74); and negatively correlated with SRL (−0.97) (Figure 5G). In the EF₄ treatment, RNA showed a significant positive correlation with TRL, TRSA, RT, and RSR (0.71~0.77), and RB showed an extremely significant positive correlation (0.99). There was a significant negative correlation with SRL (−0.98) (Figure 5H).

3.5. Analysis of the Correlation between Different Nitrogen Forms on the Root System of Pinus tabuliformis Seedlings: A Two-Factor Analysis of the Effect of Fertilization Methods and Nitrogen Forms on the Growth of Pinus tabuliformis Seedlings

According to two-factor analysis of variance (

Table 2. A two-factor analysis of the effect of fertilization methods and nitrogen forms on the growth of Pinus tabuliformis seedlings.

Index	Source	Sum of Squares	df	Mean Square	F	P
TRL	FM	299,225.66	1	299,225.66	1230.34	<0.001
	NF	129,304.04	3	43,101.35	177.22	<0.001
	FM × NF	102,753.41	3	34,251.14	140.83	<0.001
TRSA	FM	36,217.00	1	36,217.00	2099.38	<0.001
	NF	28,504.26	3	9501.42	550.76	<0.001
	FM × NF	8473.57	3	2824.52	163.73	<0.001
TRV	FM	175.11	1	175.11	1052.23	<0.001
	NF	79.95	3	26.65	160.14	<0.001
	FM × NF	24.76	3	8.25	49.59	<0.001
RAD	FM	4.96	1	4.96	1318.15	<0.001
	NF	1.92	3	0.64	170.24	<0.001
	FM × NF	1.26	3	0.42	111.36	<0.001
RT	FM	219,784.50	1	219,784.50	321.44	<0.001
	NF	342,260.50	3	114,086.83	166.85	<0.001
	FM × NF	22,364.50	3	7454.83	10.90	<0.001
RSR	FM	0.01	1	0.01	5.34	0.024
	NF	0.04	3	0.01	14.36	<0.001
	FM × NF	0.02	3	0.01	6.93	<0.001
RTD	FM	299.57	1	299.57	183.01	<0.001
	NF	10.05	3	3.35	2.05	0.116
	FM × NF	56.17	3	18.72	11.44	<0.001
SRL	FM	102,443.95	1	102,443.95	10.98	0.002
	NF	288,349.04	3	96,116.35	10.31	<0.001
	FM × NF	125,161.86	3	41,720.62	4.47	0.006
RB	FM	0.18	1	0.18	74.93	<0.001
	NF	0.40	3	0.13	54.71	<0.001
	FM × NF	0.08	3	0.03	10.86	<0.001
SB	FM	0.01	1	0.01	38.54	<0.001
	NF	0.03	3	0.01	27.52	<0.001
	FM × NF	0.00	3	0.00	2.09	0.11
LB	FM	1.00	1	1.00	87.51	<0.001
	NF	0.62	3	0.21	18.24	<0.001
	FM × NF	0.05	3	0.02	1.49	0.225
RNA	FM	0.24	1	0.24	72.47	<0.001
	NF	0.52	3	0.17	52.71	<0.001
	FM × NF	0.10	3	0.03	10.37	<0.001
SNA	FM	0.02	1	0.02	35.33	<0.001
	NF	0.03	3	0.01	25.34	<0.001
	FM × NF	0.00	3	0.00	1.19	0.322
LNA	FM	1.29	1	1.29	80.08	<0.001
	NF	0.78	3	0.26	16.11	<0.001
	FM × NF	0.05	3	0.02	1.01	0.393

TRL: total root length. TSA: total root surface area. TRV: total root volume. RAD: roots’ average diameter. RT: root tips. RSR: root/shoot ratio. RTD: root tissue density. SRL: specific root length. RB: root biomass. SB: stem biomass. LB: leaf biomass. RNA: root nitrogen accumulation. SNA: stem nitrogen accumulation. LNA: leaf nitrogen accumulation. FM: fertilization method. NF: nitrogen form.

), FM had significant effects on the TRL, TRSA, TRV, RAD, RT, RSR, SRL, RTD, RB, stem biomass (SB), leaf biomass (LB), RNA, stem nitrogen accumulation (SNA), and leaf nitrogen accumulation (LNA) of Pinus tabuliformis seedlings. NF had significant effects on TRL, TRSA, TRV, RAD, RT, RSR, SRL, RB, SB, LB, RNA, SNA, and LNA, but had no significant effects on RTD. The synergistic effect of the two methods had significant effects on TRL, TRSA, TRV, RAD, RT, RSR, SRL, RB, and RNA, but had no significant effects on SB, LB, SNA, and LNA.

4. Discussion

4.1. Effects of Different Nitrogen Addition Methods on Root Parameters of Pinus tabuliformis Seedlings

Nutrient conditions in the soil environment can significantly affect the growth and development of the plant root system, as well as the root as the main nutrient organ of the plant to obtain nutrients, so the morphological characteristics of the root system to a certain extent can directly or indirectly reflect the plant’s ability to explore the soil. Therefore, root parameters can respond to the status and basis of the plant uptake of nutrients from the soil [36,37]. In this study, the overall root index of Pinus tabuliformis under the exponential fertilization method was better than that under the conventional fertilization method. Compared with the conventional fertilization method, the total root length increased by 13.6%~67.6%, root surface area increased by 12.1%~73.7%, root volume increased by 47.4%~111.9%, root tips increased by 14%~31.9%, and roots’ average diameter increased by 32%~102.1% under the exponential fertilization method. It may be that the nutrients absorbed by the seedlings in the early stage of conventional fertilization application inhibit the development of the root system [38], or the nutrients supplied in the later stage of seedling development cannot meet the needs of Pinus tabuliformis. The exponential fertilization method delivers nutrients to the seedlings in a gradual manner to meet the nutrient requirements of the Pinus tabuliformis seedlings at each stage. The results showed that there was a close relationship between nutrient uptake by Pinus tabuliformis and root growth and morphology [9]. Compared with the conventional fertilization method, exponential fertilization was more beneficial to cultivate a healthy and complete root system.

Nitrogen morphology has an important effect on plant growth and development; when there are sufficient nutrients, the plant root system grows vigorously, which can improve the ability of the root system to compete for nutrients in the soil ecosystem. The opposite leads to a reduction in the number of roots, a simplification of the spatial structure of the root system, and a reduction in the ability of the root system to compete for nutrients [39,40]. The results of this study showed that the TRL, TRSA, and TRV of Pinus tabuliformis seedlings were positively correlated under different nitrogen forms (Figure 5), suggesting that different nitrogen forms could promote the root growth of Pinus tabuliformis seedlings. It has been shown that nitrogen deposition leads to the growth of TRL, TRSA, TRV, and RAD in plants, with a tendency for the root system to elongate and expand [15]. The results are similar to those in this study. RSR, as a measure of biomass allocation, responds to the correlation between aboveground and belowground plant parts. In this study, compared with fertilization methods and nitrogen forms, nitrogen form has a direct effect on RSR (Table 2). RSR was positively correlated with RB and RNA among all treatments, which indirectly indicated that RSR could effectively express dry matter accumulation and nitrogen use efficiency (Figure 5). The RSR of Pinus tabuliformis seedlings was the most significant under the 1:1 treatment of amide nitrogen and ammonium nitrate (Figure 2F). Therefore, it is speculated that the changes in RSR in these two nitrogen forms will improve the nitrogen uptake strategies of each part of Pinus tabuliformis. RTD and SRL can reflect the plant’s own activity and respiratory capacity indicators; the greater the RTD value, the greater the tensile force of its root tissues, and the greater the SRL, the greater its absorption capacity. Amide nitrogen (urea) RTD is better than other treatments, with urea as an organic nitrogen, possibly through some series of hydrolysis reactions of ammonium, and soil binding can better promote the uptake of the root system of Pinus tabuliformis. Llebrés et al. [41] found that organic nitrogen was more effective in promoting root growth than inorganic nitrogen when they studied seedlings of Pinus strobus (L.) and Pinus wallichiana (A. B. Jacks.) hybrids in germination media with different nitrogen compositions, and the results of Wang et al. [42] and Zhang et al. [43] are similar to the results of the present study in which the root morphology indexes under ammonium nitrate 1:1 mixture treatment were superior to those of single ammonium and nitrate nitrogen.

4.2. Effects of Different Nitrogen Addition Methods on Seedling Biomass of Pinus tabuliformis

The root system can not only absorb water and mineral elements, produce hormones needed for plant growth, and regulate the growth of the above-ground part of the plant, but also provide a place for the assimilation of some substances and metabolic reactions, and biomass is an important indicator of the strength of the plant’s productivity. Therefore, the biomass accumulation and distribution of the plant will be affected by the nitrogen fertilizers, so the morphology of the root system and the physiological function of the whole plant are particularly important for the growth of the plant [44]. The overall superiority of the root biomass of Pinus tabuliformis under the conventional fertilization method over the exponential application in October in this study may be due to the adequate nutrients provided by the conventional fertilization method in the early stage, which enhanced the rate of accumulation of dry matter mass. From November to December, the biomass of all parts of Pinus tabuliformis seedlings under the exponential fertilization method was better than that under the conventional fertilization method. In December, compared with conventional fertilization, the root biomass, stem biomass, and leaf biomass increased by 4%~36.2%, 4%~13.6%, and 10.5%~23.8%, respectively, under exponential fertilization (Figure 3). It may be that the conventional fertilization method can provide sufficient nutrients to the plant in the early stage, but excess nitrogen fertilizer is toxic to the plant and inhibits the growth of the seedlings. The exponential fertilization method can better solve this phenomenon, which gradually increases the nutrients over time to better promote the growth of the plant root system, to better promote the synthesis of chlorophyll, to enhance the accumulation of the plant’s dry matter mass, and to improve the nitrogen accumulation of the seedlings. This is similar to the results of Springer et al. [45] and Wei et al. [46] who found that the exponential fertilization effect of Panicum virgatum L. and Podocarpus macrophyllus [Thunb.] D. Don was superior to the conventional fertilization effect. In this study, amide nitrogen (urea) was more conducive to dry matter accumulation in the roots of Pinus tabuliformis seedlings, which is similar to the results of Yan et al. [47]. The biomass of the roots, stems, and leaves in the mixed treatment of 1:1 nitrate and ammonium was higher than that of the single form of ammonium nitrogen and nitrate nitrogen. An excess concentration of single ammonium nitrogen can inhibit photosynthesis, while nitrate nitrogen can promote photosynthesis. The interaction relationship may be caused by the amide nitrogen and ammonium nitrate 1:1, which increases the transpiration rate and stomatal conductance of Pinus tabuliformis leaves, which in turn enhances the accumulation of dry matter. This is consistent with the findings of Boschiero et al. [48].

4.3. Effects of Different Nitrogen Addition Methods on Nitrogen Accumulation of Pinus tabuliformis Seedlings

The nitrogen content absorbed by plants from the soil fully reflects the strength of the plant’s own nitrogen absorption capacity, and the nitrogen accumulation of plants mainly depends on the dry matter accumulation and nitrogen content [48,49]. The nitrogen accumulation in the roots of Pinus tabuliformis seedlings under the exponential fertilization method was better than that under the conventional fertilization method, which may be due to the good root development of Pinus tabuliformis seedlings under the exponential fertilization method, which improved the nitrogen absorption capacity and increased the nitrogen content. Different plants have different absorption capacities for NO₃⁻-N, NH₄⁺-N, and NO₃⁻-N: NH₄⁺-N. Camellia sinensis is more inclined to absorb NH₄⁺-N [50]. The application of NH₄⁺-N is unfavorable to Buchloe dactyloides (Nutt.) Engelm. growth [29]. The highest nitrogen accumulation was observed in the root system of Pinus tabuliformis under amide nitrogen treatment in this study. This is similar to the results of the high nitrogen fertilizer utilization efficiency of amide nitrogen in the study of the effect of nitrogen form on the growth of Populus spp. by Du et al. [51]. It has been shown that root biomass and root morphology were the two factors that improve the nitrogen uptake efficiency of plants [52], which is consistent with the positive correlation between RB and RNA under different nitrogen addition conditions of the Pinus tabuliformis root system in this study (Figure 5). Nitrogen forms did not only affect the dry matter accumulation and nitrogen accumulation of Pinus tabuliformis, but also differed in the root form. A well-developed root system is the basis for efficient nutrient uptake by plants; the more developed the root system, the finer the average diameter of the roots, the greater the number of root tips, the longer the total root length, and the more favorable it is to the plant’s access to nitrogen [53]. Nitrogen accumulation in the root system of Pinus tabuliformis is not only related to root morphology, but also to the choice of fertilization method and nitrogen form, similar to the experimental results of Xiao et al. [54].

5. Conclusions

The results of this study showed that compared with the conventional fertilization method, the exponential fertilization method significantly improved the morphological indexes, biomass, and nitrogen accumulation of the root system of Pinus tabuliformis seedlings, indicating that the exponential fertilization method effectively improved the nutrient demand of Pinus tabuliformis and effectively promoted the growth of the root system of Pinus tabuliformis. In terms of different nitrogen forms, the ammonium nitrate 1:1 mixture and amide nitrogen provided relatively stable nutrients to the root growth of Pinus tabuliformis, which was better than single ammonium nitrogen and nitrate nitrogen, indicating that the ammonium nitrate 1:1 mixture slowed down the unfavorable effect of the single nitrogen form on the root system of Pinus tabuliformis through the combination of two forms. The amide nitrogen may be more suitable for the growth of the root system after a series of hydrolysis reactions. The correlation showed that root biomass was the index affecting the nitrogen accumulation of Pinus tabuliformis. Under the conditions of this experiment, amide nitrogen was the best fertilization treatment for the growth of Pinus tabuliformis roots under the exponential fertilization method.