Quantifying NH₄⁺ and NO₃⁻ uptake by wheat under microbial competition in acid and calcareous soils

Nitrogen (N) is a crucial nutrient that limits ecosystem productivity, and its availability directly influences plant growth^[1]. Plants primarily acquire N from soil in the form of inorganic N, specifically ammonium (NH₄⁺) and nitrate (NO₃⁻)^[2]. Microorganisms also require the same N sources for maintenance, growth, and reproduction^[3]. Therefore, competition occurs between plant N uptake and microbial N immobilization, both of which are essential pathways of N retention in soil^[3−6]. However, these two processes have often been studied independently in the past, despite their essential interactions in influencing N cycling in terrestrial ecosystems^[7].

Studies have shown that microbial competitiveness is a crucial factor affecting plant N uptake efficiency, regardless of N form^[6]. Microorganisms often possess a competitive advantage in rapidly acquiring most of the available N, which may lead to N limitation becoming a major constraint on plant productivity^[8]. Previous experiments using ¹⁵N labelling have demonstrated that microorganisms can assimilate more than 60% of added N^[9,10], and a field study reported that up to 46% of added ¹⁵N was immobilized by microorganisms^[11]. Competition between plants and microorganisms is particularly intense in the rhizosphere, where high plant N demand reduces soil N concentration, while root exudates stimulate rhizosphere microbial activity, leading to high microbial N immobilization^[3,5]. Therefore, a deeper understanding of plant-microbial competition is essential for elucidating the mechanisms controlling plant N acquisition.

Soil pH is a key environmental factor influencing chemical, physical, and biological soil processes, thereby affecting microbial and plant growth, soil structure, and nutrient availability^[12,13]. Soil pH not only regulates nutrient availability and N transformation processes but also alters the rhizosphere environment, thereby shaping N competition dynamics between plants and microorganisms^[3]. First, soil pH influences inorganic N availability and transformation, thereby modifying plant N uptake strategies^[14−16]. Nitrification is inhibited in acid soils, causing NH₄⁺ accumulation, whereas in alkaline soils, which typically exhibit higher nitrification rates, result in greater NO_{3^_} availability^[17−19]. Soil pH also profoundly affects the rhizosphere microbial community composition and activity^[3], influencing chemical niche partitioning^[5]. Specifically, plants can adjust rhizosphere carbon exudation and root exudate composition to shape specific microbial communities, thereby altering the intensity of N competition^[20−23]. While many studies have examined N or carbon availability as drivers of plant-microbial N competition^[24−26], the regulatory role of soil pH remains underexplored.

More importantly, plant species differ in their preferences for specific N forms, which represents an important strategy for N acquisition^[27]. Previous studies have shown that plant N uptake preferences often align with the dominant N forms present in soils^[26,28,29]. For example, some crop plants, such as rice, preferentially absorb NH₄⁺ rather than NO₃⁻; whereas most dryland crops, including wheat, maize, and various vegetables, prefer NO₃^−[29,30]. Because soil pH affects the prevailing NO₃⁻ to NH₄⁺ ratio by regulating nitrification, plant species with specific N preferences may exhibit different N competition dynamics with microorganisms under varying soil pH conditions^{[26,28,31,32]}. However, it remains unclear how pH-mediated changes in N forms influence N competition between crop plants and microorganisms.

To address this knowledge gap, a controlled experiment was conducted using soils of contrasting pH and wheat (Triticum aestivum), a significant global crop known to take up NO₃⁻ preferentially^[30]. A ¹⁵N-paired labelling (¹⁵NH₄NO₃ or NH₄¹⁵NO₃) approach was used to trace N uptake in acid and calcareous soil-wheat systems. Samples were collected at 4, 24, and 48 h after labelling. It was assumed that: (1) ¹⁵N recovery by both wheat and microbes would be higher in the calcareous soil-wheat system compared to the acid soil-wheat system; (2) soil microbes would compete with wheat and assimilate more inorganic N in the short term; and (3) ¹⁵NO₃⁻ uptake by wheat would be higher in the calcareous soil than in the acid soil, whereas the opposite would be occur for ¹⁵NH₄⁺ uptake rate.

The acid soil was collected from Pingxing Township, Leshan City, Sichuan province, China (103°35'60" E, 29°34'13" N, average elevation 500 m), a typical agricultural area characterized by acidic purple soil. The region has a mid-subtropical humid climate, and the traditional cropping pattern includes a rice/corn–wheat/rapeseed double rotation. The calcareous soil was collected from Yufeng Town, Suining City, Sichuan province, China (105°30'39" E, 30°23'38" N, average altitude 362 m), a region dominated by calcareous purple soil derived from purple-red mudstone and exhibiting a strong lime reaction. Traditional cropping in this area includes wheat/maize/peanut intercropping.

In September 2022, soil samples were collected from both locations at five to eight sampling points, with surface soil (0–20 cm depth) collected. The collected soil samples were thoroughly mixed to create composite samples. Upon arrival at the laboratory, the soil samples were processed promptly. Larger soil clumps were broken down into smaller pieces approximately 1 cm in diameter, and any remaining plant or animal residues and small stones were removed. The soil samples were then stored in a cool, well-ventilated environment to reduce moisture content. A portion of the soil was used to analyze the basic physicochemical properties, while the remaining soil was used for cultivation experiments. Key soil properties are presented in Table 1.

The ¹⁵N paired labelling experiment in the soil-wheat systems

An incubation experiment was conducted in the laboratory using calcareous and acid soils with wheat as the crop plant. The ¹⁵N paired-labelling approach enables the simultaneous calculation of multiple soil N transformation processes, including the uptake rates of ¹⁵NH₄⁺ and ¹⁵NO₃⁻ by plants and soil microorganisms. Soil samples were passed through a 2 mm sieve. Cultivation tubes, with a capacity of 100 mL, were filled with 90 g of soil (based on dry weight). Soil moisture was adjusted to 70% of the water holding capacity (WHC). Six treatments, each with four replicates, were established:

(1) ¹⁵NH₄NO₃-labeled calcareous soil-wheat system

(2) NH₄¹⁵NO₃-labeled calcareous soil-wheat system

(3) ¹⁵NH₄NO₃-labeled acid soil-wheat system

(4) NH₄¹⁵NO₃-labeled acid soil-wheat system

(5) Unlabeled NH₄NO₃ calcareous soil-wheat system

(6) Unlabeled NH₄NO₃ acid soil-wheat system

Cultivation tubes were then incubated for 3 d to restore microbial activity. A circular opening approximately 1.3 cm in diameter was created in the center of each centrifuge tube cap. A plastic tube with an inner diameter of 1 cm was inserted through this opening to support plant growth securely. Three holes, each 1 mm in diameter, were drilled on the left side of every centrifuge tube for injecting ¹⁵N-labeled N fertilizer. Once injected, these holes were sealed with silicone plugs to prevent soil loss. Wheat seeds were soaked in distilled water for 24 h and subsequently germinated on moistened gauze at 22.5 °C. After 3 d of pre-cultivation, seedlings showing uniform growth were transplanted into individual centrifuge tubes, with one seedling per tube. Growth chamber conditions were set to a 15 h light/9 h dark cycle, with temperatures of 22 °C (day) to 18 °C (night), and a light intensity of 100 μmol m⁻² s⁻¹. Daily watering was performed by weight to ensure consistent soil moisture in each centrifuge tube. Fertilization was applied twice (3 d before sowing and 5 d after sowing) at rates of 2 mg N, 1.1 mg P, and 2.8 mg K per 90 g of soil.

Approximately 1 month after planting, the ¹⁵N paired-labelling experiment was initiated. Each labeled sample received 30 mg kg⁻¹ of ¹⁵NH₄⁺ or ¹⁵NO₃⁻. Equivalent amounts of NH₄NO₃ were added to the unlabeled controls. Destructive sampling was conducted at 4, 24, and 48 h after labelling. Plant and soil samples were collected for ¹⁵N abundance analysis. Plant samples were promptly divided into above-ground and below-ground portions. Root samples were immersed in a 0.05 M KCl solution for 10 min to remove surface-adhered ¹⁵N, followed by rinsing with sterile water. The cleaned plant samples were subsequently sectioned into small pieces and stored at –80 °C for later measurement of ¹⁵N content. Soil samples were collected and divided into three portions. One portion was immediately subjected to immersion extraction for NH₄⁺ and NO₃⁻ concentration and abundance. The second portion was stored at 4 °C for microbial biomass nitrogen analysis. And the third portion was used to determine moisture content. Microbial biomass ¹⁵N content was extracted using the chloroform fumigation method^[33]. Briefly, 10 g of fresh soil samples were fumigated with chloroform for 24 h in the dark, and then all the samples were extracted with 0.05 M K₂SO₄. One part of the extract solution was immediately freeze-dried for analysis of ¹⁵N : ¹⁴N ratios by isotope-ratio mass spectrometry (Europa Scientific Integra, Crewe, UK). The other part of the solution was determining dissolved organic N (DON) by using a TOC-TN analyzer (Thermo Scientific, Dionex, USA). Soil ¹⁵NH₄⁺ and ¹⁵NO₃⁻ were enriched using the microdiffusion method^[17] and quantified by isotope ratio mass spectrometry.

Calculations

Soil gross N transformation rates, including gross mineralization rate as in Eq. (1), mineralization rate, Eq. (2), and immobilization rates, Eqs (3) and (4) were calculated by following equations^[34] based on the 'isotope dilution method'. Microbial immobilization of NH₄⁺ was calculated as the difference between ¹⁵NH₄⁺ consumption and the gross nitrification rate. This calculation assumed gaseous losses (e.g., from nitrification, denitrification, or volatilization) and heterotrophic nitrification of organic N had negligible impacts on the ¹⁵NH₄⁺ pool. In contrast, microbial NO₃⁻ immobilization was assumed to equal NO₃⁻ consumption^[35]. The equations for Eqs (1)–(4) are as follows:

Soil gross mineralization rate (µg g⁻¹ d⁻¹):

Soil gross nitrification rate (µg g⁻¹ d⁻¹):

Soil NH₄⁺ immobilization rate (µg g⁻¹ d⁻¹):

Soil NO₃⁻ immobilization rate (µg g⁻¹ d⁻¹):

where, [NH₄⁺]₀ and [NH₄⁺]_t represent the initial and ending NH₄⁺ concentrations (mg kg⁻¹), ¹⁵[NH₄⁺]₀, and ¹⁵[NH₄⁺]_t denote the initial and ending ¹⁵NH₄⁺ abundances (atom%), [NO₃⁻]₀ and [NO₃⁻]_t denote the initial and ending NO₃⁻ concentrations (mg kg⁻¹), ¹⁵[NO₃⁻]₀ and ¹⁵[NO₃⁻]_t denote the initial and ending ¹⁵NO₃⁻ abundances (atom%), m_{_gross} represents the gross mineralization rate, n_{_gross} represents the gross nitrification rate, i_NH4+ represents the NH₄⁺ immobilization rate and $\rm i_{NO_3^-} $ represents the NO₃⁻ immobilization rate.

The uptake of ¹⁵N by plants and soil microorganisms was calculated using Eqs (5)–(11)^[5,36]. The ¹⁵N atom% excess of plant and soil samples was determined as the difference in ¹⁵N atom% between the labeled and unlabeled control samples in Eq. (5). The ¹⁵N content in root and shoot tissues (μg) was calculated by multiplying N content in roots or shoots (μmol N g⁻¹) by the corresponding ¹⁵N atom% excess, biomass (g), and the molecular mass of ¹⁵N as in Eq. (6)^[5]. Soil ¹⁵N biomass was calculated by multiplying the soil N content (μmol N g⁻¹) by ¹⁵N atom% excess, soil mass (g), and the corresponding molecular mass of ¹⁵N in Eq. (7).

Plant and soil microorganisms ¹⁵N atom % excess:

¹⁵N content of root/shoot (μg):

Soil ¹⁵N biomass:

The plant ¹⁵N uptake rate (µg N g⁻¹ soil DW d⁻¹) was calculated by summing the ¹⁵N content of shoots and roots, and dividing this sum by the soil dry weight and the corresponding incubation time (day). Plant ¹⁵N recovery (%) was calculated as the ratio of the ¹⁵N content of shoots and roots to the added ¹⁵N concentration (µg g⁻¹), the weight of the soil (g), and the atom% of labelled ¹⁵NH₄NO₃ or NH₄ ¹⁵NO₃ as in Eqs (8) and (9).

Plant ¹⁵N uptake rate (µg N g⁻¹ soil DW d⁻¹):

Plant ¹⁵N uptake recovery (%):

Microbial ¹⁵N uptake rate (µg N g⁻¹ soil DW d⁻¹) was determined as the differences between the ¹⁵N mass of fumigated and unfumigated soils (µg), divided by the soil dry weight (g) and the corresponding incubation time (day) as in Eq. (10). Microbial ¹⁵N uptake recovery (%) was determined by calculating the microbial ¹⁵N content of microbial biomass divided by the added ¹⁵N (µg g⁻¹) and the ¹⁵N abundance of labelled ¹⁵NH₄NO₃ or NH₄¹⁵NO₃ (i.e., 10.13%) in Eq. (11). The result of recovery was multiplied by 100.

Microbial ¹⁵N uptake rate (µg N g⁻¹ soil DW d⁻¹):

Microorganism ¹⁵N uptake recovery (%):

Statistical analysis

Repeated measures analysis of variance to compare changes over time in NH₄⁺ and NO₃⁻ concentrations, as well as their ¹⁵N enrichment during the aerobic incubation period. One-way ANOVA was used to assess differences in plant traits across various growth stages and multiple N transformation rates. Linear regression was used to examine the relationship between wheat N uptake rates and soil microbial N assimilation rates. All statistical analyses were performed using SPSS 20.0 (IBM Corp., Armonk, NY, USA), with a significance level set at p < 0.05. Figures were drawn using GraphPad Prism 9.5 (GraphPad Software, San Diego, CA, USA).

The gross mineralization and gross nitrification rates were significantly higher in the calcareous soil-wheat system compared to the acid soil-wheat system, with increases of nine-fold and two-fold, respectively (Fig. 1a, b, p < 0.05). Correspondingly, exchangeable NH₄⁺ concentrations were lower in the calcareous soil-wheat system, while NO₃⁻ concentrations did not differ significantly between the two systems (Table 2). In the acid-soil wheat system, inorganic N immobilization rates were higher than those in the calcareous soil-wheat system, measuring 10.41 and 6.92 mg kg⁻¹ d⁻¹, respectively (Fig. 1c, p < 0.05). It is important to note that the NH₄⁺ immobilization rates were calculated assuming that volatilization, heterotrophic nitrification, and denitrification did not affect ¹⁵NH₄⁺, and that the NO₃⁻ immobilization rates were calculated assuming that denitrification and dissimilatory reduction of NO₃⁻ to NH₄⁺ (DNRA) were negligible. Thus, the immobilization rates may have been overestimated.

Figure 1. 

(a) The gross mineralization rates, (b) nitrification rates, and (c) immobilization rates in the acid and calcareous soil-wheat systems. Differences between the soil-wheat systems are indicated by *, * p < 0.05, ** p < 0.01, and *** p < 0.001.

¹⁵N uptake rates of wheat and soil microbes

In the calcareous soil-wheat system, the ¹⁵N uptake rate of ¹⁵NH₄⁺ labelling increased over time and became significantly higher than the uptake rate of ¹⁵NO₃⁻ after 48 h (Fig. 2a, p < 0.05). In contrast, in the acid soil-wheat system, there was no significant difference in the ¹⁵N uptake rate between ¹⁵NH₄⁺ and ¹⁵NO₃⁻, which were approximately equal (around 1:1). However, at 48 h, the uptake rate of ¹⁵NO₃⁻ was significantly higher than that of ¹⁵NH₄⁺ (p < 0.05).

Figure 2. 

The ¹⁵NH₄⁺ and ¹⁵NO₃⁻ uptake rates of (a) wheat and (b) soil microorganisms with time-varying conditions in calcareous and acid soil-wheat systems. Differences between ¹⁵NH₄⁺ and ¹⁵NO₃⁻ uptake/assimilation rates are indicated by *, * p < 0.05, ** p < 0.01, and *** p < 0.001.

Microbial assimilation rates of inorganic ¹⁵N declined over time in both systems. In the calcareous soil-wheat system, at first 4 h, microorganisms showed a significantly higher assimilation rate of ¹⁵NO₃⁻ than of ¹⁵NH₄⁺ (p < 0.05). Beyond this time point, the microbial assimilation rates of the two ¹⁵N forms did not differ significantly. Overall, in the calcareous soil-wheat system, microorganisms did not exhibit a clear preference for either NH₄⁺ or NO₃⁻ throughout the experiment (Fig. 2).

When comparing uptake rates between microbes and wheat, after 4 h of labelling, the inorganic N uptake rate of wheat was only half that observed in the acid soil, indicating that microbes exerted intense N competition in the early stage. As time progressed, the wheat uptake rate did not exceed that of microorganisms in either system.

In summary, total wheat ¹⁵N uptake rates were slightly lower than microbial ¹⁵N assimilation rates in the acid soil-wheat system (0.42 vs 0.40 µg g⁻¹ d⁻¹), but higher in the calcareous soil-wheat system (0.46 vs 0.19 µg g⁻¹ d⁻¹). However, there was no remarkable difference in either the ¹⁵N uptake rates between the calcareous soil-wheat system and the acid soil-wheat system (Table 3). Thus, soil pH-dependent differences in gross N transformation rates significantly changed microbial ¹⁵N assimilation, but not wheat ¹⁵N uptake.

¹⁵N recovery of wheat and soil microbes

Wheat ¹⁵N recovery increased progressively with incubation time, ranging from 1.0% to 30.2% in the calcareous soil-wheat system and from 2.3% to 28.0% in the acid soil-wheat system (Fig. 3). The preference for ¹⁵NH₄⁺ or ¹⁵NO₃⁻, as indicated by the uptake rates, was consistent at each time point (Fig. 3a). In the calcareous soil-wheat system, microbial ¹⁵N recovery gradually declined over time, with a significantly higher recovery of ¹⁵NO₃⁻ compared to ¹⁵NH₄⁺ (p < 0.05). In contrast, no clear decreasing trend was observed for microbial ¹⁵N recovery in the acid soil-wheat system (Fig. 3b, Table 3).

Figure 3. 

The % ¹⁵N recovery of (a) wheat and (b) soil microorganisms of calcareous and acid soil-wheat systems. The values at the top represent the ratios of total ¹⁵N recovery (¹⁵NH₄⁺ + ¹⁵NO₃⁻) in calcareous soil and that of acid soil at each time point.

Furthermore, regardless of whether ¹⁵NH₄⁺ or ¹⁵NO₃⁻ was labelled, microbial ¹⁵N assimilation rates were significantly negatively correlated with plant ¹⁵N uptake rates (Fig. 4), highlighting the competitive relationship between plants and microorganisms. Specifically, 4 h after labelling, microorganisms assimilated 22.1% of ¹⁵N in the calcareous soil-wheat system and 13.3% acid soil-wheat system, demonstrating a pronounced competitive advantage at the early stage over wheat. However, at 48 h after labelling, ¹⁵N recovery by wheat exceeded that of microorganisms (Table 3), independent of soil pH (i.e., calcareous or acid soil).

Figure 4. 

The regression analyses of N assimilation rates of wheat and soil microorganisms of calcareous and acid soil-wheat systems under (a) ¹⁵NH₄⁺ and (b) ¹⁵NO₃⁻ labelling.

First hypothesis mentioned that ¹⁵N recovery would be higher in the calcareous soil-wheat system, was partially supported for wheat but refuted for microbes, as microbial ¹⁵N recovery was higher in the acid soil after 48 h. For the second hypothesis, microbes would outcompete plants in the short term, was strongly supported only during the first 24 h. The third hypothesis stated that wheat would take up more ¹⁵NO₃⁻ and less ¹⁵NH₄⁺ in the calcareous soil than in the acid soil, was supported only during the first 24 h and reversed after 48 h.

The result showed that wheat ¹⁵N uptake rates, which preferentially absorb NO₃⁻, were higher in calcareous soil than in acid soil. In addition, gross nitrification rates were higher in calcareous soil (Table 3). Previous studies have confirmed that crop N preferences (NO₃⁻ vs NH₄⁺) are correlated with soil N transformation dynamics^[26,28]. The soil NH₄⁺ : NO₃⁻ ratio is a crucial determinant of plant N preference^[14,37,38]. For example, a pot experiment^[39] demonstrated that wheat preferentially absorbs NO₃⁻ under normal conditions, the application of a nitrification inhibitor (e.g., nitrapyrin) significantly increased NH₄⁺ uptake while reducing NO₃⁻ uptake, bringing their relative uptake rates into balance.

Although distinct gross N transformation rates influenced wheat ¹⁵N uptake patterns, wheat preferential NO₃⁻ uptake in the calcareous soil occurred only during the first 24 h (Figs 1b and 2a). At 48 h after labelling, the ¹⁵NH₄⁺ uptake rate of wheat exceeded NO₃⁻, despite abundant NO₃⁻ remaining in the calcareous soil. One explanation is that plant N uptake is highly plastic and flexible^[40,41], facilitating adjustments in the uptake of different N chemical forms to optimize resource partitioning. For instance, alpine plants increase NH₄⁺ absorption under competitive conditions^[42]. Because NH₄⁺ uptake requires less energy than the assimilation of organic N compounds^[11], wheat may simultaneously utilize both N sources under these conditions.

Moreover, the result showed that the decline in NH₄⁺ concentration in calcareous soil (Table 2) supports the increased NH₄⁺ uptake along with active nitrification (Table 3), indicating a dynamic adjustment in N uptake strategy of wheat. In fact, plant uptake of NO₃⁻ and NH₄⁺ is regulated by the dynamics of rhizosphere pH. NO₃⁻ uptake releases OH⁻ and increases pH, whereas NH₄⁺ uptake releases H⁺ and acidifies the rhizosphere^[43]. To maintain pH balance in the rhizosphere, wheat may increase NH₄⁺ uptake after an initial phase of elevated NO₃⁻ uptake, thereby optimizing nutrient use efficiency. This mechanism may partly explain the N uptake pattern observed in this study and could represent a competitive advantage under high nitrification conditions that limit NH₄⁺ availability.

In contrast, wheat in acid soil showed a preference for ¹⁵NO₃⁻ at 48 h (Fig. 2a). Because NH₄⁺ is less mobile and may form depletion zones in the rhizosphere, NO₃⁻ becomes a more accessible N source for plants in later stages^[39,44]. Moreover, NH₄⁺ can exert toxic effects on plants^[45], and NO₃⁻ application has been shown to alleviate NH₄⁺ toxicity^[45,46], leading plants to increase NO₃⁻ uptake under such conditions^[28]. Overall, the uptake and metabolism of inorganic N by plants are influenced by multiple interacting factors, including N supply dynamics, soil chemical conditions, rhizosphere microbial communities, growth stage, and intrinsic physiological regulatory mechanisms. Plants may adjust the relative uptake of NO₃⁻ and NH₄⁺ to adapt to the spatiotemporal heterogeneity of soil N availability, while simultaneously regulating rhizosphere pH and internal N metabolism to optimize nutrient use efficiency and growth adaptability.

There is indeed uncertainty regarding the estimation of certain ¹⁵N forms uptake by plants in this study^[47]. In the ¹⁵NH₄⁺ labeled treatment, without the constraint of nitrification, it is impossible to fully distinguish whether plant ¹⁵N uptake originated directly from ¹⁵NH₄⁺ or indirectly from ¹⁵NO₃⁻ produced by nitrification. This may lead to overestimation of ¹⁵NH₄⁺ uptake even under short isotope exposure times. To address the limitation, the methodology should be improved, such as using nitrification inhibitors to decrease the nitrification rate or modelling approaches^[48,49].

Microbial-plant N competition under different soil pH

It was found that plant N uptake rates and ¹⁵N recovery increased continuously, and exceeded microbial uptake after 48 h (Fig. 3, Table 3), regardless of soil pH. Several reasons may explain this pattern. First, incubation conditions in small pots could constrain root growth, increasing root density and enhancing plant competitive ability^[5,50]. Microbes, however, can rapidly assimilate large amounts of inorganic N in the short term due to faster growth and higher surface-area-to-volume ratios^[10,51]. Second, plants generally outcompete microorganisms for NO₃⁻ more effectively than for NH₄^+[50,52], especially wheat, which prefers NO₃⁻. Because NO₃⁻ availability remained high in both soils (Table 1), microbes may reduce competition for N uptake, leading to temporary microbial N retention followed by rapid turnover^[9], and decreasing constraints on plant inorganic N uptake^[3,53].

In contrast, soil pH appeared to influence microbial ¹⁵N recovery during the early stages. Microbial ¹⁵N recovery was higher in the acid soil than in the calcareous soil after 48 h (Table 3), indicating a greater sensitivity to pH fluctuations^[54,55]. The ratio of microbial N immobilization to plant N uptake was substantially higher in the acid soil than in the calcareous soil (95.23% vs 41.3%), indicating that microorganisms can compete with wheat for inorganic N under acid conditions. This result was inconsistent with the previous study, which found that low pH has a stronger inhibitory effect on microbes than on plants, thereby weakening microbial competition for inorganic N and enhancing the competitive advantage of plants in the rhizosphere environment^[3]. A likely explanation is that higher nitrification rates in the calcareous soil-wheat system produced lower NH₄⁺ concentration (Table 2), limiting microbial NH₄⁺ assimilation relative to the acid soil. Besides, as soil organic carbon in calcareous soil was lower than that in acid soil (Table 1), carbon availability might differ between calcareous and acid soils, which can affect microbial growth strategies and their ability to assimilate N^[9].

Currently, studies have shown that plants and rhizosphere microbes reduce direct competition through niche differentiation, thereby optimizing N resource utilization^[5,49]. However, no significant plant-microbe niche differentiation was observed under NH₄NO₃ treatment in this study. This may be attributed to the high inorganic N availability in agricultural soils (Table 1) and the low ratio of microbial N assimilation to plant N uptake (Table 3), which reduces competition pressure compared to N-limited natural ecosystems^[52]. Moreover, plants can also absorb organic N, including low molecular weight amino acids and peptides from the soil solution^[56], which can influence N competition and has not been investigated in this study. Previous studies have suggested that microbes act as N reservoirs in soil systems, with a greater N retention potential than plants^[7]. Due to rapid turnover rates^[9], microbes exhibit strong N retention potential within specific timeframes. Notably, despite similar NO₃⁻ concentrations in calcareous and acid soils (Table 2), quicker mineralization and nitrification processes in calcareous soils led to faster N turnover. Thus, combined with continuous N uptake by both plants and microbes, the calcareous soil-wheat system could result in a higher actual N supply compared to acid soils.

Total wheat ¹⁵N recovery (¹⁵NH₄⁺ + ¹⁵NO₃⁻) was higher in calcareous soil than in acid soil, whereas microbial ¹⁵N recovery was higher in acid soil at 24 and 48 h after labelling. Specifically, ¹⁵N uptake by wheat continuously increased in both soil-wheat systems, varying from 1.0% to 30.2% in calcareous soil and 2.3% to 28.0% in acid soil. ¹⁵N recovery of microbes in acid soil was stable, while microbial ¹⁵N recovery in calcareous soil decreased after 48 h. In addition, microbial ¹⁵NH₄⁺ recovery increased first and then reduced, except in the calcareous soil-wheat system, which decreased continuously after labelling. Both wheat ¹⁵NH₄⁺ and ¹⁵NO₃⁻ recovery were higher in acid soil than in calcareous soil after 48 h. Overall, the results indicated that wheat (preferring NO₃⁻uptake) dominated N uptake over microorganisms in the calcareous soil, rather than in the acid soil, after labelling for 48 h. These findings provide new insights for understanding the regulatory role of soil pH in short-term plant-microbe N competition in agricultural soils.