Soil Property Alterations and Nitrogen Use Dynamics of Hemarthria altissima Under Distinct Nitrogen Forms

Abstract

Atmospheric nitrogen deposition is increasing worldwide, with profound implications for plant nitrogen acquisition and ecosystem nutrient cycling, particularly in nitrogen-limited systems. In this study, we investigated how inorganic nitrogen form regulates nitrogen uptake in H. altissima through pot experiments by applying ammonium nitrogen, nitrate nitrogen, mixed nitrogen, and a nitrogen-free control in Songnen grassland ecosystems at the eastern end of Eurasia. Soil abiotic properties, root morphological traits, and nitrogen uptake dynamics were jointly quantified using integrative modeling in combination with ¹⁵N stable isotope tracing. Relative to the no-nitrogen control, both ammonium and nitrate nitrogen significantly altered soil physicochemical conditions and stimulated root development, with ammonium consistently exhibiting stronger effects. Ammonium and nitrate applications reduced soil pH by 4.83% and 6.25%, increased electrical conductivity by 2.01% and 1.17%, and enhanced inorganic nitrogen pools by 115.84% and 45.69%, respectively. Root morphological traits were significantly enhanced under ammonium, nitrate, and mixed nitrogen treatments. ¹⁵N tracing further demonstrated that ammonium nitrogen significantly increased root ¹⁵N uptake compared with the no-nitrogen control (p < 0.05) and promoted a 20.10% greater allocation of absorbed nitrogen to aboveground biomass than nitrate nitrogen. Collectively, these findings highlight nitrogen form as a key regulator of soil–plant nitrogen coupling, with ammonium nitrogen more effectively enhancing nitrogen acquisition and internal translocation than nitrate.

1. Introduction

Nitrogen (N) deposition has increased markedly over the past decades as a consequence of intensified anthropogenic activities, including fossil fuel combustion, agricultural fertilization, and industrial emissions [1]. This persistent enhancement of reactive nitrogen input is projected to continue under future global change scenarios, profoundly altering biogeochemical processes and ecosystem function. Grasslands, which constitute nearly one third of the global terrestrial surface, are particularly sensitive to changes in nitrogen availability because of their long-term evolution under nutrient-limited conditions [2]. Thus, understanding how nitrogen deposition influences soil physicochemical properties, plant nutrient uptake strategies, and ecosystem nutrient cycling has become a central topic in global change ecology. Previous studies have shown that elevated nitrogen deposition can substantially reshape soil chemical environments, including shifts in soil pH, electrical conductivity (EC), mineral nitrogen pools, and ratios reflecting the balance of C, N, and P [3]. In many temperate grasslands, long-term N addition tends to acidify soils and enhance inorganic N availability, which may further alter microbial processes, nutrient turnover, and plant nutrient acquisition [2,4]. However, the response magnitude and pathways are not universal across grassland types. For instance, temperate grasslands often exhibit rapid increases in soil nitrate and declines in pH, semi-arid grasslands display stronger N retention but limited change in soil acidity due to low buffering capacity, whereas alpine grasslands are constrained by low temperature and short growing seasons, resulting in slow N transformation and unique plant adaptation strategies [5,6]. These biome-specific responses highlight the need to investigate nitrogen effects under different chemical forms and ecological contexts, particularly considering the increasing complexity of atmospheric nitrogen composition, which includes ammonium (NH₄⁺), nitrate (NO₃⁻), and mixed reactive nitrogen [1,7,8].

Different nitrogen forms can markedly alter soil physical and chemical properties in grassland ecosystems, thereby influencing root morphology and biomass accumulation of dominant herbaceous species [8,9]. NH₄⁺ inputs often increase soil acidity and modify cation exchange capacity, potentially enhancing the solubility of certain nutrients while affecting soil aggregate stability [10]. In contrast, NO₃⁻ fertilization tends to increase soil solution mobility and can accelerate leaching processes, shaping soil moisture distribution and nutrient availability in deeper layers [11]. These soil property shifts drive distinct root morphological responses: ammonium frequently promotes denser, more branched root systems concentrated in upper soil horizons, whereas nitrate encourages deeper rooting and greater root elongation due to its vertical mobility [12,13]. As a result, grassland biomass accumulation reflects the interplay between nitrogen form and root foraging strategies, with balanced N inputs enhancing both aboveground productivity and belowground biomass, while excessive or unbalanced nitrogen supply may reduce root-shoot coordination, alter species composition, and ultimately affect grassland ecosystem functioning.

Distinct nitrogen deposition forms exert strong control over soil processes and the pathways through which plants acquire nitrogen [14]. Ammonium usually promotes soil acidification through nitrification, while nitrate is more mobile and easily leached, leading to distinct impacts on soil nitrogen pools and stoichiometric balance. Mixed nitrogen inputs may further generate nonlinear interactions between ammonium and nitrate transformation pathways [8]. Despite extensive research on the effects of nitrogen addition on grassland soils, fewer have simultaneously compared the effects of ammonium nitrogen, nitrate nitrogen, and mixed nitrogen forms under controlled field conditions, limiting our understanding of how nitrogen composition modulates soil–plant feedbacks, especially in dominant grass species such as Leymus chinensis and Hemarthria altissima [1,8]. H. altissima, as a widely distributed perennial grass in temperate and semi-arid regions, plays a critical role in maintaining ecosystem stability, soil conservation, and grassland productivity [15,16,17]. This species exhibits strong adaptability to nutrient-poor soils, yet its physiological and morphological strategies under differential nitrogen forms remain inadequately understood. Root morphology is a crucial determinant of nitrogen acquisition, while ¹⁵N stable isotope tracing offers a powerful means to track nitrogen allocation patterns in plant tissues and soils [18,19]. Integrating root traits, above- and below-ground biomass production, and ¹⁵N accumulation provides a comprehensive framework to evaluate plant nitrogen utilization dynamics and to identify preferential uptake of specific nitrogen forms.

Previous studies have shown that different nitrogen forms can differentially affect soil acidification, microbial activity, and plant growth [7,17]; however, the mechanisms linking soil biochemical changes with plant nitrogen uptake and allocation remain insufficiently resolved, particularly in perennial grassland species. By integrating soil biochemical responses, root morphological traits, and ¹⁵N tracer-based assessments of plant nitrogen uptake and allocation [20], this study aims to clarify how nitrogen form regulates soil nutrient availability, plant nitrogen assimilation, and soil–plant nitrogen coupling. The novelty of this research lies in its process-based comparison of ammonium, nitrate, and mixed nitrogen inputs, explicitly linking changes in soil nitrogen pools and stoichiometry with root-mediated nitrogen acquisition and biomass production. This integrated approach moves beyond assessments of biomass responses alone and provides mechanistic insight into how distinct reactive nitrogen forms drive ecosystem-level nitrogen cycling. Overall, our findings advance the mechanistic understanding of nitrogen form-specific effects on soil–plant interactions in temperate grasslands and provide a theoretical basis for predicting grassland responses to future increases in atmospheric nitrogen deposition, particularly under global change scenarios.

2. Materials and Methods

2.1. Site Description

The experiment was conducted at the Songnen Grassland Ecosystem National Observation and Research Station, Jilin Province, China (44°34′ N, 123°31′ E). The region experiences a typical temperate continental monsoon climate, with warm and humid summers contrasted by long, cold, and dry winters. The surface soil layer (0–20 cm) is characterized as shown in

Table 1. Chemical and physical properties of the basic soil (mean ± SE, n = 3).

Soil Properties	Soil pH	Total Nitrogen (g·kg−1)	Total Phosphorus (g·kg−1)	Total Organic Carbon (g·kg−1)	NH4+-N (mg·kg−1)	NO3−-N (mg·kg−1)	Soil EC (μS·cm−1)
Content	8.68 ± 0.026	1.02 ± 0.004	0.66 ± 0.002	6.37 ± 1.206	1.23 ± 0.051	1.89 ± 0.063	78.76 ± 1.352

. The site has a mean annual temperature ranging from 4.6 to 6.5 °C, and annual precipitation varies between 280 and 620 mm, with the majority occurring during the growing season. For the pot experiment, each container was filled with 3.5 kg of wind-deposited sandy soil collected from the grassland. Pots were arranged in a completely randomized block design with six replicates per treatment. The test species was H. altissima, a perennial C₄ grass widely distributed across the Songnen Plain, with six seedlings transplanted into each pot. Four nitrogen treatments were established and applied in two equal installments on May 30 and June 15. These treatments included a no-nitrogen control (N0), ammonium nitrogen supplied as (NH₄)₂SO₄ (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) (AN), nitrate nitrogen supplied as Ca(NO₃)₂ (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) (NN), and a mixed nitrogen treatment consisting of equimolar NH₄⁺-N and NO₃⁻-N (ANN). All nitrogen treatments were applied at a rate equivalent to 10 g N m⁻². To suppress nitrification, dicyandiamide (DCD) (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) was added at 10 mg m⁻² in the AN treatment and 5 mg m⁻² in the ANN treatment. To minimize confounding effects unrelated to nitrogen treatments, all pots were maintained under standardized management conditions throughout the experimental period. No chemical pesticides, fungicides, or herbicides were applied at any stage. Weed control was conducted manually at weekly intervals by visual inspection, and any emerging weeds were removed immediately by hand. Plant health status was monitored regularly, and no visible symptoms of pest infestation or disease were observed during the experiment. To avoid potential limitations of other macro- and micronutrients that could interfere with nitrogen uptake and allocation, each pot received 200 mL Hoagland nutrient solution once per month. This solution supplied non-nitrogen nutrients at balanced and non-limiting concentrations, while nitrogen availability was strictly controlled by the experimental nitrogen treatments. The application rate and frequency were identical across all treatments, ensuring that differences in plant responses could be attributed to nitrogen form rather than to other nutrient constraints. Water availability was primarily regulated by natural precipitation. During periods of insufficient rainfall, supplemental irrigation was applied uniformly to all pots to maintain soil moisture near field capacity. Specifically, irrigation was provided when surface soil moisture declined visibly or when prolonged dry periods (>7 consecutive days without precipitation) occurred, with approximately 500 mL per pot per event applied to avoid water stress. No waterlogging was observed during the experiment. Importantly, irrigation frequency and volume were consistent across treatments, thereby minimizing potential interactions between water availability and nitrogen uptake. Plants were harvested on September 15, corresponding to the post-fruiting stage, when biomass accumulation and nitrogen allocation patterns had stabilized. This timing allowed for an integrated assessment of cumulative nitrogen uptake, root morphological development, and soil–plant nitrogen dynamics over the entire growing season.

2.2. Determination of Root Morphology and Plant Biomass

Root samples of H. altissima were collected for analysis. For each treatment, six pots were selected, with three plants of similar growth vigor chosen from each pot. The root systems were thoroughly washed and then imaged using an Epson 11000 scanner (Epson America, California, Inc., USA). The root characteristics, including root length, surface area, volume, and tip number were quantified using the WinRHIZO 2016b root analysis system (Regent Instrument, Quebec, Canada). The aboveground biomass of the plants was harvested and placed into envelopes. The belowground samples were washed with distilled water, placed in envelopes, and brought to the laboratory. The samples were then blanched at 105 °C for 30 min, followed by drying at 65 °C until a constant weight was achieved. The dry weight of the aboveground tissue was recorded as the aboveground biomass (AGB, g), while the weight of the underground tissue was recorded as the belowground biomass (BGB, g).

2.3. Soil Sample Collection and Physicochemical Property Determination

Soil samples were collected using a composite design, with five random points in each plot. After collection, the samples were sieved through a 100-mesh screen to remove large debris, then further sieved through a 2 mm mesh and divided into two portions. One portion was stored at 4 °C and the other portion was air-dried, ground, and sieved through a 1 mm mesh for subsequent analysis. SWC was calculated as the difference between fresh and dry weights. Soil pH and EC were measured in aqueous extracts [2]. The supernatant was analyzed using a pH meter (PHS-3C, Shanghai Leici Instrument Factory, Shanghai, China) and conductivity meter (DDSJ-318, Shanghai Leici Instrument Factory, Shanghai, China).

Extractable inorganic nitrogen was determined using potassium chloride extraction. NH₄⁺-N and NO₃⁻-N concentrations were quantified using a continuous flow analyzer (Futura, Alliance Flow Analyzer, Frassaillon, France) [1]. Total carbon and nitrogen contents were measured with an elemental analyzer (vario EL cube, Elementar, Langenselbold, Germany). Total phosphorus was quantified by molybdenum–antimony colorimetric detection. The C, N, and P contents and their stoichiometric ratios were calculated from the measured values [8].

2.4. ¹⁵N Stable-Isotope Labeling

To evaluate the preferential uptake of different nitrogen forms by H. altissima, a δ¹⁵N-labeling tracer experiment was conducted during the heading stage in late June, following which an analysis was conducted using a modified version of the method proposed by Zhang et al. [21]. Two ¹⁵N-labeled nitrogen treatments were applied, (¹⁵NH₄)₂SO₄ and Ca(¹⁵NO₃)₂ (Shanghai Chemical Industry Research Institute Co., Ltd., Shanghai, China), with ¹⁵N abundances of 9.18% and 9.24%, respectively. Four pots were randomly selected for each treatment to determine the initial ¹⁵N abundance before labeling. Subsequently, isotopically labeled nitrogen was supplied by (¹⁵NH₄)₂SO₄ or Ca(¹⁵NO₃)₂ to each flask, resulting in equivalent application rates of ¹⁵NH₄⁺-N 20 mg N m⁻² soil and ¹⁵NO₃⁻-N 20 mg N m⁻² soil, respectively. Soil samples (four replicates per ¹⁵N treatment) were collected 7d after nitrogen application to determine the concentrations and isotopic compositions of NH₄⁺ and NO₃⁻. These measurements were then used to assess differences in δ¹⁵N values of plant tissues, soil, and soil-to-plant transfer among the different nitrogen-form treatments. On the 7th day following ¹⁵N application, plant samples (leaf, stem and root components) were collected from both labeled and control treatments within each plot. Immediately after harvest, tissues were subjected to enzymatic deactivation at 105 °C for 30 min and subsequently dried at 65 °C for 48 h. Dried materials were weighed and finely homogenized using a ball mill (MM 400; Retsch, Haan, Germany). About 3 mg of finely homogenized sample was sealed in tin capsules and subjected to determination of total nitrogen content and isotopic ratios using an isotope ratio mass spectrometer (Isoprime 100) coupled with an elemental analyzer (vario EL cube; Elementar, Langenselbold, Germany). The δ¹⁵N values were calculated using the following equation [19,20,21]:

$${\mathsf{\delta }}^{15}\mathrm{N}(‰) ({\displaystyle \frac{R_{sample}}{R_{standard}}}-1) \times 1000$$

(1)

where R_sample refers to the ¹⁵N/¹⁴N ratio measured in the sample, whereas R_standard corresponds to that of atmospheric N₂, which has a fixed ratio of 0.00368.

2.5. Statistical Analysis

Data were processed and analyzed using R software (version 4.5.1). Assumptions of normality and homogeneity of variances were assessed using the Kolmogorov–Smirnov test and Levene’s test, respectively. For post hoc comparisons, Tukey’s honestly significant difference (HSD) test was applied, with a significance level set at p < 0.05. Graphical representations of the data were generated using the ggplot2 package. All analyses were conducted to determine significant treatment effects.

3. Results

3.1. Soil Chemical Properties

Nitrogen addition significantly altered several soil chemical properties, especially pH and EC (p < 0.05; Figure 1). Relative to the control, soil pH was markedly reduced under ammonium nitrogen, nitrate nitrogen, and mixed nitrogen treatments, with decreases of 4.81%, 6.25%, and 5.62%, respectively (Figure 1A). Electrical conductivity showed a distinct response to nitrogen form, with the ammonium nitrogen treatment exhibiting significantly higher EC values than control as well as nitrate nitrogen and mixed nitrogen treatments (p < 0.05; Figure 1B). In contrast, SWC remained statistically unchanged across all nitrogen treatments (Figure 1C).

3.2. Soil Stoichiometric Characteristics

Soil inorganic nitrogen and total nutrient concentrations varied significantly among the nitrogen-form treatments (p < 0.05; Figure 2). NO₃⁻–N concentrations were greatest under the ammonium nitrogen treatment, exceeding those in the control, nitrate nitrogen, and mixed nitrogen treatments (Figure 2A). A comparable trend occurred for NH₄⁺–N, with the ammonium nitrogen treatment showing substantially higher levels than the other treatments, 167.01%, 73.83%, and 61.88% higher than those of the control, nitrate nitrogen, and mixed nitrogen treatments, respectively (Figure 2B).

Total nitrogen (TN) was highest under the mixed nitrogen treatment, with values significantly surpassing those of the control, ammonium nitrogen, and nitrate nitrogen treatments (Figure 2C). Soil TC also peaked in the mixed nitrogen treatment, increasing by 16.92%, 15.69%, and 15.92% relative to the control, ammonium nitrogen, and nitrate nitrogen treatments, respectively (Figure 2D). In contrast, TP levels showed no significant response to the different nitrogen forms (Figure 2E).

The control, ammonium nitrogen, nitrate nitrogen, and mixed nitrogen treatments exerted statistically significant effects on soil stoichiometric characteristics (p < 0.05; Figure 3). Specifically, soils receiving ammonium nitrogen or mixed nitrogen inputs exhibited markedly higher C/N and C/P ratios than those under the control and nitrate-N treatments (p < 0.05). Relative to the control, the C/N ratio increased by 11.28% and 9.24% under ammonium nitrogen and mixed nitrogen treatments, respectively (Figure 3A). A comparable pattern was observed for the C/P ratio, which rose by 25.43% in the ammonium nitrogen treatment and by 29.24% in the mixed nitrogen treatment (Figure 3B). In contrast, the N/P ratio remained statistically unchanged across all nitrogen forms, indicating a limited sensitivity of this metric to nitrogen speciation (Figure 3C).

3.3. Morphological Characteristics of Root Systems

Root morphological characteristics responded distinctly to variations in nitrogen form (p < 0.05; Figure 4). Among all treatments, ammonium nitrogen application resulted in the greatest total root length, significantly surpassing those observed under the control, nitrate nitrogen, and mixed nitrogen conditions (Figure 4A). Nitrogen enrichment generally promoted root surface development, as evidenced by the substantially larger root surface area in all nitrogen-supplemented treatments compared with the unfertilized control (Figure 4B). Furthermore, total root volume and the number of root tips were both markedly elevated under ammonium nitrogen treatment relative to the control and nitrate nitrogen treatments (Figure 4C,D), indicating a pronounced stimulatory effect of ammonium nitrogen on root system proliferation.

3.4. Absorption and Utilization of ¹⁵N in Different Forms

Significant differences in δ¹⁵N values of leaves, stems, roots, and soil were observed among the control, ammonium nitrogen, nitrate nitrogen and mixed nitrogen treatments (p < 0.05;

Table 2. Comparison of the leaf δ¹⁵N, stem δ¹⁵N, root δ¹⁵N and soil δ¹⁵N in H.altissima under different N form treatments (means ± SE, n = 4). Control(N0), ammonium nitrogen (AN), nitrate nitrogen (NN), mixed nitrogen (ANN). Statistically significant differences between treatments are indicated by different lowercase letters based on the Tukey test (p < 0.05; n = 6).

Variable	N0 Control	AN 15N Labeling	NN 15N Labeling	ANN 15N Labeling
Leaf δ15N (‰)	4.96 ± 0.64 c	14.18 ± 1.52 ab	11.93 ± 0.78 b	17.83 ± 1.05 a
Stem δ15N (‰)	6.79 ± 0.78 b	26.3 ± 4.28 a	29.1 ± 1.81 a	36.65 ± 3.49 a
Root δ15N (‰)	143.07 ± 1.35 d	8278.78 ± 59.21 a	6020.23 ± 276.73 c	7212.62 ± 129.14 b
Soil δ15N (‰)	3.92 ± 0.04 c	145.48 ± 1.32 b	157.40 ± 1.20 a	145.17 ± 0.48 b

). Compared with N0, leaf δ¹⁵N was markedly elevated under ammonium nitrogen, nitrate nitrogen and mixed nitrogen treatments. Stem δ¹⁵N showed higher values in mixed nitrogen and ammonium nitrogen treatments than in control and nitrate nitrogen treatments. Root δ¹⁵N reached its highest level under the ammonium nitrogen treatment and was significantly greater than those of the other treatments. In contrast, soil δ¹⁵N was highest in the nitrate nitrogen treatment, exceeding the values measured in control, ammonium nitrogen and mixed nitrogen treatments.

3.5. Biomass Accumulation in Different Forms

Aboveground and belowground biomass responded significantly to the different nitrogen form treatments (p < 0.05). Compared with the unfertilized control, aboveground biomass was substantially enhanced by nitrogen addition, with increases of 92.09%, 59.94%, and 66.55% under ammonium nitrogen, nitrate nitrogen and mixed nitrogen treatments, respectively (Figure 5A). Similarly, belowground biomass was significantly lower in the control treatment than in all nitrogen-fertilized treatments (p < 0.05; Figure 5B).

Results from the Mantel test revealed that both aboveground and belowground biomass were significantly associated with a broad range of abiotic variables as well as root system traits (Figure 6). Notably, indices related to plant nitrogen acquisition—represented by δ¹⁵N signatures in roots, stems, and leaves—exhibited strong positive correlations with biomass allocation in both plant compartments. In addition, key root morphological attributes, including total root length, root surface area, root volume, and root tip number, were also positively linked to aboveground and belowground biomass of H. altissima, highlighting the close coupling between nitrogen utilization, root architecture, and plant growth.

4. Discussion

4.1. Nitrogen Form-Induced Changes in Soil Properties

Soil chemical properties responded sensitively to nitrogen form, with all N additions (ammonium nitrogen, nitrate nitrogen and mixed nitrogen) significantly decreasing soil pH compared with the control. Nitrogen-induced soil acidification is a well-documented phenomenon and is primarily attributed to proton release during nitrification of ammonium and rhizosphere acidification associated with inorganic N uptake [22,23]. Although nitrate is often considered less acidifying than ammonium under field conditions, the observed pH decline under nitrate nitrogen treatment in this study suggests that constrained buffering capacity and limited solute transport in pot systems can amplify acidification responses regardless of N form [11,24]. In contrast to the consistent pH decline, EC increased significantly only under ammonium supply. Soil EC is widely used as an integrative indicator of soluble ion concentration and fertilizer-derived salts in the soil solution [22]. The elevated EC under ammonium nitrogen treatment likely reflects higher ionic strength associated with ammonium fertilizers and enhanced mobilization of exchangeable cations under acidifying conditions. Because soil water content did not differ among treatments, the observed EC increase can be attributed primarily to nitrogen-driven ionic processes rather than moisture effects. Together, these results indicate that nitrogen form creates distinct belowground chemical environments that may differentially constrain or facilitate root functioning and nutrient acquisition.

Changes in soil nitrogen pools and macronutrient stocks (TC, TN, TP) provide essential context for interpreting plant nitrogen uptake and soil–plant coupling [25]. Nitrogen additions commonly increase soil inorganic nitrogen (NH₄⁺-N and NO₃⁻-N) directly through fertilizer inputs and indirectly by stimulating microbial turnover and N transformations, although the relative dominance of NH₄⁺ versus NO₃⁻ in soil depends strongly on N form supplied and subsequent nitrification/immobilization processes [4,7]. Soil C:N:P stoichiometry offers a mechanistic lens to interpret how N form reshapes nutrient balance and potential limitation [3,26]. Soil C/N, C/P, and N/P ratios are widely used to indicate the relative availability of carbon, nitrogen, and phosphorus, and shifts in these ratios can signal changes in microbial nutrient demand, decomposition processes, and plant nutrient limitation status. In many ecosystems, nitrogen addition tends to increase soil N/P (and sometimes decrease C/N) by enriching N relative to P, which can intensify P limitation over time and reconfigure nutrient cycling pathways [27]. The consistent pH decline across N treatments suggests that N-induced acidification transformations likely occurred concurrently, which can influence the partitioning between NH₄⁺-N and NO₃⁻-N and their persistence in soil.

4.2. Ammonium Enhanced Root Morphological Plasticity as a Mediator of N Acquisition

Root systems of H. altissima exhibited strong morphological plasticity in response to nitrogen form, with ammonium supply inducing the greatest increases in total root length, mean root diameter, and root surface area. Root morphological adjustments are widely recognized as an adaptive strategy that allows plants to optimize nutrient foraging under variable soil conditions [28]. Increased root length and surface area enhance the soil–root contact interface and nutrient interception capacity, while greater root diameter may support higher transport capacity and structural investment [29]. The pronounced root development under ammonium supply may reflect a coordinated response to the acidifying and ion-enriched rhizosphere created by ammonium nitrogen treatment. In such environments, expanding the absorptive interface can improve nutrient capture efficiency and buffer plants against localized nutrient depletion or ionic stress [30]. These findings support the view that root traits act as a critical mediator linking nitrogen form-induced soil chemical changes to plant nutrient acquisition strategies.

4.3. ¹⁵N Tracer Evidence: Nitrogen Form Regulates Acquisition Magnitude and Within-Plant Partitioning

Stable isotope ¹⁵N labeling provided direct mechanistic evidence that nitrogen form regulates both nitrogen acquisition and internal partitioning in H. altissima. Compared with the control, all N forms treatments markedly increased δ¹⁵N values in plant organs and soil, confirming effective uptake of applied nitrogen. Across treatments, ¹⁵N enrichment consistently followed the hierarchy root > stem > leaf, indicating a pronounced root-centered retention strategy for newly acquired nitrogen.

Nitrogen form strongly influenced the magnitude of ¹⁵N enrichment and its distribution among organs. Ammonium treatment resulted in the highest ¹⁵N recovery and the greatest allocation of ¹⁵N to roots, coinciding with the strongest stimulation of root morphological traits. This convergence between morphological and isotopic responses supports a coherent mechanism whereby ammonium supply enhances root foraging capacity, leading to greater nitrogen capture and preferential retention in belowground tissues [18]. Similar root-centered nitrogen retention patterns under ammonium supply have been reported in other grass species using ¹⁵N tracer approaches [21,31].

In contrast, nitrate supply tended to promote greater ¹⁵N enrichment in stems, suggesting a higher propensity for upward translocation of absorbed nitrogen, consistent with the higher mobility of nitrate within the soil–plant continuum [32]. The mixed nitrogen treatment produced the highest root δ¹⁵N values, indicating potential complementarity between ammonium and nitrate that supports both efficient acquisition and subsequent redistribution of nitrogen within the plant. Such synergistic effects of mixed N supply have been observed in forage grasses and are often interpreted as reflecting reduced physiological constraints compared with single-form supply [15,18,21].

4.4. The Nitrogen Absorption and Utilization Effects of Soil-Root-Aboveground Parts Under Different Nitrogen Forms

Integrating soil chemical responses, root morphological traits, and ¹⁵N evidence reveals a clear cascade by which nitrogen form regulates nitrogen use dynamics in H. altissima. Nitrogen addition first altered soil chemical conditions, as reflected by universal pH decline and ammonium-specific EC increase. These changes were accompanied by strong root morphological plasticity, particularly under ammonium supply, which expanded the effective absorptive interface. Enhanced root development subsequently translated into higher nitrogen recovery and preferential retention of newly acquired nitrogen in roots [7,28,30].

The nitrogen uptake regulation model emphasizes that nitrogen form affects not only the quantity of nitrogen taken up, but also the transport of that nitrogen within the plant (Figure 7). Root traits emerge as a central mediator linking soil processes to whole-plant nitrogen use efficiency, highlighting the importance of considering soil–root interactions when evaluating nitrogen management strategies. Although pot experiments may limit root expansion and differ from fully natural field conditions, the controlled setting allowed us to disentangle nitrogen form-specific effects on soil properties, root traits, and plant nitrogen uptake by minimizing environmental heterogeneity and uncontrolled nutrient and water variability.

From a forage production perspective, the higher δ¹⁵N recovery and stronger root-centered nitrogen retention under ammonium supply suggest improved short-term nitrogen capture by H. altissima, potentially enhancing fertilizer nitrogen retention within the plant–soil system. However, the concurrent soil acidification across all N treatments and the ammonium-induced increase in EC point to potential trade-offs for long-term soil sustainability. Ammonium-based fertilization is widely recognized as a major driver of agricultural soil acidification, with implications for nutrient availability and soil health [7,31,33]. Mixed nitrogen supply may represent a practical compromise by maintaining efficient nitrogen acquisition and redistribution while potentially moderating some adverse soil chemical effects associated with single nitrogen forms. These results underscore the need to balance short-term gains in nitrogen use efficiency with long-term soil chemical stability in intensively managed forage systems.

5. Conclusions

Nitrogen addition exerted strong, form-dependent effects on soil properties, root traits, and plant nitrogen allocation. Compared with the control, all nitrogen treatments reduced soil pH (4.81–6.25%), while ammonium nitrogen caused the greatest increase in electrical conductivity (2.00%), indicating stronger ionic effects. Nitrogen form distinctly reshaped soil nitrogen pools: ammonium nitrogen increased NH₄⁺-N and mineral nitrogen availability (167.01%), whereas mixed nitrogen inputs maximized total nitrogen and carbon accumulation (15.8% and 26.83%) and increased C/N and C/P ratios (9.33% and 29.23%). Plant responses closely tracked these soil changes. Biomass production increased by 48.20–55.82% across nitrogen treatments, with ammonium nitrogen inducing the strongest aboveground growth (92.09%). Root morphological traits were consistently enhanced, particularly under ammonium nitrogen, which increased root length, surface area, volume, and tip number by up to 180.04%, 81.12%, 58.71% and 386.85%, thereby strengthening nutrient foraging capacity. ¹⁵N tracing revealed divergent nitrogen pathways: ammonium nitrogen promoted plant nitrogen uptake and translocation (root δ¹⁵N 57.86 times), whereas nitrate nitrogen showed greater short-term soil retention (soil δ¹⁵N 40.15 times). Mantel tests confirmed that biomass gains were positively associated with root traits and ¹⁵N uptake. Overall, ammonium-dominated nitrogen inputs most strongly promoted root development and nitrogen acquisition, while mixed nitrogen inputs preferentially enhanced soil carbon and total nitrogen accumulation. These findings underscore the critical role of nitrogen form in regulating soil–plant nitrogen coupling and provide mechanistic insight for predicting grassland responses to future nitrogen enrichment under global change.