Exogenous phosphorus compounds interact with nitrogen availability to regulate dynamics of soil inorganic phosphorus fractions in a meadow steppe

Here we investigated the effects of P compounds (KH₂PO₄ and Ca(H₂PO₄)₂) with different addition rates of 0, 20, 40, 60, 80, and 100 kg P ha⁻¹ yr⁻¹ and NH₄NO₃ addition (0 and 100 kg N ha⁻¹ yr⁻¹) on soil labile inorganic phosphorus (IP) (dicalcium phosphate, Ca₂-P), moderate-cycling IP, and recalcitrant IP fractions in a calcareous grassland of northeastern China. Soil moderate-cycling IP fractions, not readily available to plants but transforming into soil-available P quickly, include variscite (Al-P), strengite (Fe-P) and octacalcium phosphate (Ca₈-P); recalcitrant IP fractions include hydroxylapatite (Ca₁₀-P) and occluded P (O-P). Soil labile and moderate-cycling IP fractions and total P significantly increased with increasing P addition rates, with higher concentrations detected for KH₂PO₄ than for Ca(H₂PO₄)₂ addition. Combined N and P treatments showed lower soil labile IP and moderate-cycling IP fractions compared to ambient N conditions, due to enhanced plant productivity. Moderate-cycling IP was mainly regulated by P addition and plant P uptake to further enhance labile IP and total P concentrations with KH₂PO₄ and Ca(H₂PO₄)₂ addition. Soil labile IP was also directly and negatively affected by soil pH and plant P uptake with Ca(H₂PO₄)₂ addition. Ca(H₂PO₄)₂ addition significantly increased the soil recalcitrant IP (Ca₁₀-P) fraction, while KH₂PO₄ addition showed no impact on it. A significant positive correlation was detected between soil labile IP, moderate-cycling IP fractions and soil Olsen-P which illustrated that labile IP and moderate-cycling IP fractions were important sources for soil-available P. Our results suggest that moderate-cycling IP fractions are essential for grassland P biogeochemical cycling and the chemical form of P fertilizer should be considered during fertilization management for maintaining soil-available P.

1 Introduction

Phosphorus (P) is an essential nutrient affecting terrestrial plant productivity and ecosystem functions (Luo et al., 2015). Soil inorganic P (IP) could occupy 50 %–90 % of soil total P (Jiang and Gu, 1989; Feng et al., 2016) and mainly includes adsorbed and mineral forms of phosphate (Hinsinger, 2001). Soil mineral-bound phosphate falls into two groups: those containing calcium (Ca-P) and those containing iron and aluminum (Chen et al., 2002), which are the leading sources of plant-available P (Meena et al., 2018). The detailed classification of these two groups includes soil variscite (Al-P), strengite (Fe-P), dicalcium phosphate (Ca₂-P), octacalcium phosphate (Ca₈-P), hydroxylapatite (Ca₁₀-P) and occluded P (O-P) (Zhao et al., 2019). Soil IP is dominated by Al-P and Fe-P in acidic soils and by Ca-P fractions in calcareous soils (Baumann et al., 2017). The solubility of soil Ca-P fractions decreases in the order Ca₂-P > Ca₈-P > Ca₁₀-P (Jiang and Gu, 1989). Although these element-bound P fractions are not readily available to plants, soil Al-P, Fe-P, Ca₂-P and Ca₈-P can be converted to free phosphates, serving as important buffering pools for available P (Herlihy and McGrath, 2006; Zhao et al., 2019). Based on P transforming dynamics, soil IP could be fractionated into soil labile (Ca₂-P) or available P (Olsen-P), moderate-cycling P fractions (Al-P, Fe-P and Ca₈-P) and recalcitrant P fractions (Ca₁₀-P and O-P). But the category is not universal and mainly depends on the sensitivity of soil IP fractions in different studies (Schrijver et al., 2012 vs. Zhao et al., 2019). Soil Olsen-P could be directly absorbed and utilized by plants, which includes all water-soluble P, some of the adsorbed and soluble IP, and mineralizable organic P (Tang et al., 2009; Cao et al., 2012). Ca₂-P, chemically similar to Olsen-P, includes water-soluble P, citrate-soluble P and partially surface-adsorbed P (Shen et al., 2004; Zhao et al., 2019). Soil recalcitrant IP is relatively stable and unavailable for plants, which is mainly converted from the fixation of labile and moderate-cycling IP (Shen et al., 2004; Zhao et al., 2019). Therefore, soil IP transformation is crucial for nutrient cycling in terrestrial ecosystems.

Adding P to soil is an effective way to enhance soil-available P and sustain plant productivity (Marklein and Houlton, 2012). However, P addition changes ecosystem P cycling (Herlihy and McGrath, 2006) and the reactions between free phosphate and cations in soil, thereby influencing the concentration and transformation of soil IP fractions (Walker and Syers, 1976). As such, chronic P input can result in insoluble phosphate accumulation and decrease P use efficiency without providing additional benefits for plant growth (Maistry et al., 2015; Halajnia et al., 2009; Maistry, 2015). This could then increase the risk of soil P leaching losses (Shen et al., 2004). Therefore, exploring the effect of P addition on soil IP fractions is important for understanding soil-available P supply and P dynamics in terrestrial ecosystems (Sigua et al., 2017).

Biogeochemical P dynamics can also be influenced by the chemical form of applied P (Margenot et al., 2017). Frequently used P compounds include Ca(H₂PO₄)₂ (slow-release P fertilizer) and KH₂PO₄ (fast-release P fertilizer) (Mortvedt et al., 1999), which have played critical roles in agricultural ecosystems (Nguyen et al., 2012). Both P fertilizers can convert into various inorganic P fractions including soil Fe-P (especially in paddy soils with high Fe²⁺) (Sah et al., 1989) and soil Al-P, which can account for 33 % of the total soil P in weathered soils (Margenot et al., 2017). Soil Ca₂-P and Ca₈-P could be also formed with P addition, especially in calcareous soils with pH higher than 7 (Al-Khateeb et al., 1986). In a previous study it was found that long-term application of Ca(H₂PO₄)₂ increased soil IP fractions (Al-P, Fe-P, Ca₂-P, Ca₈-P and O-P) and total P (TP) concentrations in a calcareous soil (Wang et al., 2010), and another study showed that long-term Ca(H₂PO₄)₂ addition decreased soil P availability due to fixation of P to the soil constituents (Xiong et al., 2018). KH₂PO₄ addition was suggested to enhance the reaction of phosphate with Fe and Al oxides and with hydroxyl ions under lower soil pH conditions (Shuman, 1988). While these studies provided insight into the relationships between P addition and IP fractions (Condron and Goh, 1989), little is known about the effects of different P compound additions on soil IP fractions and P availability.

Nitrogen enrichment can influence soil chemical properties, thereby affecting soil P transformations and consequently aboveground biomass and foliar P concentrations (Crowley et al., 2012). For instance, N addition often decreases soil pH and promotes the release of P from Fe and Al oxides (Gustafsson et al., 2012; Yang et al., 2014) and can result in redistribution of soil IP fractions (Zhao et al., 2019). Nitrogen addition could also promote the mineralization of soil organic P by increasing phosphatase activities (Olander and Vitousek, 2000). The effect of combined N and P addition on ecosystem P dynamics may further depend on their relative amounts added to the soil. For instance, inorganic P solubility and availability were particularly large when P was applied with N (Ross et al., 1995), while N and P applied at higher N:P ratios increased plant P use efficiency and leaf phosphatase activity (Hogan et al., 2010). It is well known that variation in N:P input ratios can also alter plant litter N:P ratios (Vitousek et al., 2010; Güsewell, 2004; Sun et al., 2018), thereby further affecting N and P availability through litter decomposition. However, it remains unclear how applications of N and P added at different ratios will affect soil IP fractions.

The meadow steppe in northern China is an important part of the Eurasian grassland ecosystem (Kang et al., 2007). With the intensive environmental stresses of overgrazing and enhanced outputs of animal products from a sharply rising population, Inner Mongolian grassland ecosystems have potentially become more nutrient-limited, predominantly in regards to N and P (Kang et al., 2007; Gong et al., 2011). Hence, N and P additions are necessary to enhance ecosystem productivity in the meadow steppe. The purpose of our study was to monitor the effects of various levels of KH₂PO₄ and Ca(H₂PO₄)₂ with and without N addition on concentrations of soil IP fractions, available P and TP. We hypothesized that (1) soil labile IP (Ca₂-P) and moderate-cycling IP fractions (Al-P, Fe-P and Ca₈-P) would significantly increase with KH₂PO₄ and Ca(H₂PO₄)₂ addition, while soil recalcitrant IP fractions (Ca₁₀-P and O-P) would not be affected because of different solubility in IP fractions; (2) addition of soluble KH₂PO₄ would be more efficient in increasing soil labile IP, moderate-cycling IP fractions, recalcitrant P fractions, and total P (TP) than less soluble Ca(H₂PO₄)₂ because of faster conversion of KH₂PO₄ into labile and moderate-cycling IP fractions, consequentially promoting the formation of recalcitrant fractions; and (3) the concentrations of soil IP fractions would be lower under combined P and N additions than those under P addition alone due to facilitation of plant P uptake with N addition.

2 Materials and methods

2.1 Study sites and experimental design

The study site (50^∘10^′ N, 119^∘22^′ E, elevation 523 m a.s.l.) is located at the Erguna Forest-Steppe Ecotone Research Station of Inner Mongolia, China. This area belongs to a temperate continental monsoon climate. The mean annual precipitation and mean annual temperature are 375 mm and −3 ^∘C, respectively. Rainfall at the site is mainly concentrated during the period from June to August and the average growing season is about 150 d. The soil is a chernozem according to the Food and Agricultural Organization of the United Nations classification (IUSS Working Group WRB, 2014), and the soil chemical characteristics of the site are reported in Table 1. The dominant plant species include Stipa baicalensis, Leymus chinensis and Carex duriuscula. The relative biomass proportion of the three dominant species combined to the total aboveground biomass were 50.29 %–74.75 % and 72.43 %–91.92 % in the plots without and with N addition, respectively.

Table 1 General characteristics of the soil from the experimental site before nitrogen and phosphorus manipulation. Selected parameters include sand, silt, and clay fractions; soil pH; soil organic matter (SOM); total nitrogen (TN); total phosphorus (TP); total inorganic phosphorus (TIP); organic P; exchangeable Ca and Al; and available Fe and Mn. Data are mean ± standard error (n=5).

The experiment, established in 2014, was arranged in a randomized block design with 24 treatments and five replicates. Phosphorus addition included two compounds, i.e., KH₂PO₄ and Ca(H₂PO₄)₂, and were applied at six levels: 0, 20, 40, 60, 80 and 100 kg P ha⁻¹ yr⁻¹. Half of the plots were applied with 100 kg N ha⁻¹ yr⁻¹ in the form of NH₄NO₃. P and N were added in the middle of May. All treatment plots were balanced for K using potassium chloride (KCl) to maintain the same amount of K input as in the treatment with 100 kg P ha⁻¹ yr⁻¹ of KH₂PO₄ addition (132 kg K ha⁻¹). To balance for Cl along with KCl addition, CaCl₂ was applied to maintain equal Cl inputs for all plots (121 kg Cl ha⁻¹). Calcium was not balanced in this calcareous soil, where Ca is already abundant. In this study we chose the same control plots for the KH₂PO₄ and Ca(H₂PO₄)₂ treatment, so that, in total, there were 110 plots of 8 m × 8 m, separated by 1 m wide buffer zones. The initial pH of KH₂PO₄ and Ca(H₂PO₄)₂ solutions were 4.91 and 4.50, respectively.

2.2 Sample collection

In August 2016, aboveground biomass was harvested by clipping all living tissues using a 1 m × 1 m quadrat placed randomly within each plot. The plants were sorted to species and oven-dried at 65 ^∘C for 48 h, then weighed and ground. Soil samples were taken from the surface (10 cm depth) using a 5 cm diameter soil auger. Five soil cores were collected from each plot and mixed into one composite sample. Each sample was air-dried and passed through a 2 mm sieve to remove litter and detritus. A subsample of the air-dried soil was ground using a ball mill to pass through a 0.15 mm sieve for further analyses of IP fractions and TP.

2.3 Soil chemical analyses

Soil pH was measured in a 1:5 soil-to-water slurry with a pH meter (S210 SevenCompact^™, Mettler, Germany). Soil IP fractions were extracted according to the sequential fractionation scheme proposed by Chang and Jackson (1958) which is modified to suit for calcareous soils (Jiang and Gu, 1989). Briefly, soil Ca₂-P was determined by shaking 0.5 g soil with 25 mL 0.25 M NaHCO₃ (pH 7.5) and then centrifuged at 3500 rpm for 8 min to measure soil Ca₂-P. The remaining soil was washed two times with 25 mL 95 % C₂H₅OH and extracted with 25 mL 0.5 M NH₄Ac (pH = 4.2) to determine soil Ca₈-P (Jiang and Gu, 1989). After this, the soil was shaken with 25 mL 1 M NH₄Cl and centrifuged at 3500 rpm to discard the supernatant. The remaining soil was then shaken with 25 mL 0.5 M NH₄F (pH 8.2) and centrifuged at 3500 rpm for 8 min to analyze the soil Al-P fraction in the supernatant. The remaining soil was washed two times with 25 mL saturated NaCl and then sequentially shaken with 25 mL mixture of 0.1 M NaOH and Na₂CO₃ for 2 h at 25 ^∘C and then centrifuged at 4500 rpm for 10 min to measure soil Fe-P in the supernatant. The remaining samples were washed as above and extracted with 25 mL mixture of 0.3 M Na₃ (citrate)-Na₂S₂O₄ and 0.5 M NaOH to measure soil O-P. Finally, the remaining samples were shaken with 0.25 M H₂SO₄ for 1 h at 25 ^∘C and centrifuged at 3800 rpm for 10 min to determine soil Ca₁₀-P. The P concentration in all the extractants was determined by the molybdenum blue colorimetric method at a wavelength of 700 nm with a UV-VIS spectrometer (UV-1700, Shimadzu) (Murphy and Riley, 1962). Soil total IP (TIP) concentration was defined as the sum of soil labile IP (Ca₂-P), moderate-cycling IP (Al-P, Fe-P and Ca₈-P) and recalcitrant IP (Ca₁₀-P and O-P). Total organic P (TOP) was calculated as the difference between TP and TIP concentrations. The potential limitation of the extraction methods is that they may not be very specific in separating the different forms of P minerals, as a small amount of other phosphates dissolved in the extractants (Jiang and Gu, 1989).

Soil TP concentration was determined after digestion with 8 mL 85 % HNO₃ + 4 mL 72 % HClO₄ + 1 mL 40 % HF (Sommers and Nelson, 1972), and Olsen-P was extracted from air-dried soil with 0.5 M NaHCO₃ (pH 8.5) (Olsen et al., 1954). Plant TP concentration was determined by acid digestion using H₂SO₄-H₂O₂ (Thomas et al., 1967). Soil TP, Olsen-P and plant TP were analyzed by the molybdenum blue colorimetric method at 700 nm.

2.4 Statistical analyses

Plant P uptake of three dominant species was calculated using the following equation:

$$\mathrm{P}\mathrm{uptake}=\sum _i^n{P}_i\times B_i,$$

where P_i is TP concentration of species i and B_i is the biomass of species i. All the data were shown as mean ± standard error. The Kolmogorov–Smirnov test was performed to determine whether the data had a normal distribution. Three-way ANOVAs were conducted to determine the effects of N addition (N), P addition rate (P_r), P compounds (P_t) and their interactions on soil IP fractions and Olsen-P concentration. For each P compound and N treatment, the effect of P addition rates on moderate-cycling IP fractions and plant P were determined using polynomial contrasts, the effect of P addition rates on plant biomass, soil pH, soil recalcitrant IP fractions, Olsen-P and TP were analyzed using Duncan's multiple-range tests. A Student's t test was used to determine the difference between two P compounds within each P addition rate and N treatment (without and with N) and between N treatments within each P compound and addition rate. For moderate-cycling IP fractions, one-way analysis of covariance (ANCOVA) was employed to distinguish the slopes between the two N treatments (without N vs. with N) for each P compound and between the two P compounds (KH₂PO₄ vs. Ca(H₂PO₄)₂) for each N treatment. Pearson correlations were used to test the relationships between soil variables. All of the above statistics were carried out using SPSS 16.0 (SPSS Inc., Chicago, USA).

Structural equation models (SEMs) were built to clarify direct and indirect N and P addition effects on soil IP fractions through the changes in plant P uptake and soil pH. A chi-square test, Akaike information criteria (AIC) and root-mean-square error of approximation (RMSEA) were used to evaluate the fit of the model. The SEM analyses were performed using AMOS 7.0 (Amos Development Co., Greene, Maine, USA). Statistical significance was accepted at P<0.05.

3 Results

3.1 Aboveground plant biomass and soil pH

Nitrogen addition increased aboveground biomass production (Fig. 1a, b). Aboveground biomass production did not show a clear trend in response to different levels of P addition, either as KH₂PO₄ or as Ca(H₂PO₄)₂ (Fig. 1a, b). Nitrogen addition significantly increased plant P uptake of the three dominant species Stipa baicalensis, Leymus chinensis and Carex duriuscula for both P compounds (Fig. 1c, d). Plant P uptake increased with increasing P addition rates with significantly higher overall KH₂PO₄ effect than Ca(H₂PO₄)₂. KH₂PO₄ addition showed no impact on soil pH (Fig. 1e), while Ca(H₂PO₄)₂ tended to decrease soil pH at 80 and 100 kg P ha⁻¹ yr⁻¹ without N addition and at 60, 80 and 100 kg P ha⁻¹ yr⁻¹ with N addition (Fig. 1f).

Figure 1 Aboveground plant biomass (a, b), biomass-weighted phosphorus (P) uptake of three dominant plant species (c, d) and soil pH (e, f) as affected by KH₂PO₄ and Ca(H₂PO₄)₂ additions with and without N inputs. Data are represented as mean ± standard error for panels (a), (b), (e) and (f). Letters indicate significant differences between P rates of KH₂PO₄ or Ca(H₂PO₄)₂ addition without N (lowercase letters) and with N addition (capital letters). Asterisks represent significance between N treatments within each P type and rate (^* and ${}^{**}$ for P<0.05 and 0.01, respectively).

3.2 Soil labile and moderate-cycling inorganic phosphorus fractions

For both KH₂PO₄ and Ca(H₂PO₄)₂ additions, soil Al-P and Fe-P concentrations significantly increased with increased P addition rates (Fig. 2a, b, c, d). Soil Al-P and Fe-P concentrations were higher with KH₂PO₄ than with Ca(H₂PO₄)₂ addition, especially at higher P rates (Fig. 2; Table S1 in the Supplement). This resulted in significant interactive P_t×P_r effects on soil Al-P and Fe-P concentrations (Table 2). Significant interactive P_r×N and $P_{\mathrm{t}}\times P_{\mathrm{r}}\times N$ effects were also found on soil Al-P. For instance, Al-P concentration was higher with KH₂PO₄ addition than Ca(H₂PO₄)₂ at P addition level of 60 kg P ha⁻¹ yr⁻¹ when N was not added, but it was higher for P addition levels of 60, 80 and 100 kg P ha⁻¹ yr⁻¹ when N was added. Nitrogen addition decreased both soil Al-P and Fe-P concentrations, particularly at higher levels of P addition (Fig. 2; Tables 2 and S1).

Figure 2 Effects of phosphorus (P) and nitrogen (N) additions on soil variscite (Al-P) and strengite (Fe-P). Data are represented as mean ± standard error. Phosphorus types include KH₂PO₄ and Ca(H₂PO₄)₂ at rates of 0, 20, 40, 60, 80 and 100 kg P ha⁻¹ yr⁻¹. Fitted lines are based on linear regression models. The black and red lines represent without N and N addition, respectively. Significance is labeled for slopes of the black and red lines.

Addition of both P compounds significantly increased soil Ca₂-P and Ca₈-P concentrations with and without N addition (Fig. 3a, b, c, d). With KH₂PO₄ addition, soil Ca₂-P and Ca₈-P concentrations were higher than with Ca(H₂PO₄)₂ addition (Fig. 3; Table S1). Nitrogen addition significantly decreased soil Ca₂-P and Ca₈-P concentrations for both P compounds in some P addition levels (Fig. 3; Table S1). Therefore, significant P_t×P_r interactive effects were detected on soil Ca₂-P and Ca₈-P and P_r×N interactive effect on soil Ca₂-P (Table 2).

Table 2 Results (F values) of three-way ANOVAs on the effect of phosphorus (P) fertilizer type (P_t), P addition rate (P_r), and nitrogen (N) addition and their interactions on soil inorganic phosphorus fractions of variscite (Al-P), strengite (Fe-P), dicalcium phosphate (Ca₂-P), octacalcium phosphate (Ca₈-P), hydroxylapatite (Ca₁₀-P), occluded phosphate (O-P), Olsen-P, total inorganic phosphorus (TIP), and soil total P (TP) with KH₂PO₄ and Ca(H₂PO₄)₂ addition.

^∗ Significance level at 0.05. ${}^{\ast \ast }$ Significance level at 0.01.

Figure 3 Effects of phosphorus (P) and nitrogen (N) additions on soil dicalcium phosphate (Ca₂-P) and octacalcium phosphate (Ca₈-P). Phosphorus types include KH₂PO₄ and Ca(H₂PO₄)₂ at rates of 0, 20, 40, 60, 80 and 100 kg P ha⁻¹ yr⁻¹. Data are represented as mean ± standard error. Fitted lines are based on linear regression models. The black and red lines represent without N and N addition, respectively. Significance was labeled for slopes of the black and red lines.

3.3 Soil recalcitrant inorganic phosphorus fractions

Ca(H₂PO₄)₂ addition significantly increased soil Ca₁₀-P concentration, while KH₂PO₄ addition showed no impact (Fig. 4a, b). Soil Ca₁₀-P concentration was higher with Ca(H₂PO₄)₂ addition than with KH₂PO₄ addition for all levels except for 60 kg P ha⁻¹ yr⁻¹ without N addition and for 20, 60 and 80 kg P ha⁻¹ yr⁻¹ with N addition (Fig. 4a, b; Table S2). There was no main N addition and N×P_r interactive effect on soil Ca₁₀-P for both P compounds (Table 2). Nitrogen addition also had no significant effect on soil O-P with KH₂PO₄ addition but significantly decreased it with Ca(H₂PO₄)₂ addition at 60 and 80 kg P ha⁻¹ yr⁻¹ (Fig. 4c, d). Soil O-P showed a hump-shaped relationship along the P addition gradient with KH₂PO₄ when added with N and with Ca(H₂PO₄)₂ independent of N addition. Soil O-P concentration was lower with Ca(H₂PO₄)₂ addition than with KH₂PO₄ addition for 60 and 80 kg P ha⁻¹ yr⁻¹ with N addition (Fig. 4c, d; Table S2). The relative proportions of soil O-P and Ca₁₀–P to soil TIP decreased while the proportions of soil Al-P, Ca₂-P, and Ca₈-P increased with increasing P addition rates for both P compounds (Fig. S1 in the Supplement).

Figure 4 Effects of phosphorus (P) and nitrogen (N) additions on soil hydroxylapatite (Ca₁₀-P) and occluded P (O-P). Phosphorus types include KH₂PO₄ and Ca(H₂PO₄)₂ at rates of 0, 20, 40, 60, 80 and 100 kg P ha⁻¹ yr⁻¹. Data are represented as mean ± standard error. Letters indicate significant differences between P rates of KH₂PO₄ or Ca(H₂PO₄)₂ addition without N (lowercase letters) and with N addition (capital letters). Asterisks represent significance between N treatments within each P type and rate (^* and ${}^{**}$ for P<0.05 and 0.01, respectively).

3.4 Soil Olsen-P and total P

For both P compounds, P addition significantly increased soil Olsen-P concentration regardless of N addition (Fig. 5a, b). Soil Olsen-P concentration increased more strongly with increased levels of KH₂PO₄ addition than with Ca(H₂PO₄)₂ addition, resulting in significantly higher soil Olsen-P with KH₂PO₄ addition in the 100 kg P ha⁻¹ yr⁻¹ treatment with and without N addition (Fig. 5a, b). KH₂PO₄ addition increased soil TP irrespective of N addition, while Ca(H₂PO₄)₂ addition only increased soil TP without N addition (Fig. 5c, d).

Figure 5 Effects of phosphorus (P) and nitrogen (N) additions on soil Olsen-P and total P (TP) in the meadow steppe. Phosphorus types include KH₂PO₄ and Ca(H₂PO₄)₂ at rates of 0, 20, 40, 60, 80 and 100 kg P ha⁻¹ yr⁻¹. Data are represented as mean ± standard error. Letters indicate significant differences between P rates of KH₂PO₄ or Ca(H₂PO₄)₂ addition without N (lowercase letters) and with N addition (capital letters). Asterisks represent significance between N treatments within each P type and rate (^* and ${}^{**}$ for P<0.05 and 0.01, respectively).

3.5 Correlation between soil inorganic fractions with soil characteristics

For both P compounds, soil Al-P, Fe-P, Ca₂-P and Ca₈-P had significantly positive correlations with each other. In addition, soil TP and Olsen-P were all positively correlated with soil Al-P, Fe-P, Ca₂-P and Ca₈-P (P<0.01) (Table 3). Soil Ca₁₀-P had significantly positive correlations with soil Fe-P and Ca₈-P for both P compounds (Table 3). Soil TOP had significantly positive correlations with soil TP (Table 3). Furthermore, soil TIP concentration was positively correlated with the level of P addition for both P compounds (Fig. S2).

Table 3 Correlation analyses (R values) among soil inorganic fractions, Olsen-P, total phosphorus (TP) and organic P (TOP) with Ca(H₂PO₄)₂ and KH₂PO₄ addition in the meadow steppe.

^∗ Significance level at 0.05. ${}^{\ast \ast }$ Significance level at 0.01.

The SEM suggested that P addition had a positive impact on plant P uptake and soil moderate-cycling IP both with KH₂PO₄ (Fig. 6a) and with Ca(H₂PO₄)₂ addition (Fig. 6b). N addition had a positive impact on plant P uptake under both P compound additions (Fig. 6a, b). Plant P uptake had a negative impact on soil moderate-cycling IP both with KH₂PO₄ (Fig. 6a) and with Ca(H₂PO₄)₂ addition (Fig. 6b), while soil pH also negatively influenced soil labile IP with Ca(H₂PO₄)₂. Soil moderate-cycling IP fractions drove the increase in soil labile IP concentration for both P compounds, with a higher contribution for KH₂PO₄ addition (Fig. 6a, b). Soil moderate-cycling IP contributed to an increase in soil TP under both P compounds (Fig. 6a, b).

Figure 6 Structural equation model of the relationships between soil pH, plant P uptake, soil moderate-cycling inorganic phosphorus (IP), labile P, and TP under KH₂PO₄ (a, χ²=23.44, P=0.38, RMSEA = 0.03, AIC = 67.44) and Ca(H₂PO₄)₂ additions (b, χ²=27.01, P=0.08, RMSEA = 0.09, AIC = 79.01) across N treatments. Arrows indicate positive (black), negative (red) and neutral (dotted grey) effects. Arrow width is proportional to the strength of the relationship. The number adjacent to each arrow is the standardized path coefficient with corresponding significance (^*, ${}^{**}$ for P<0.05 and 0.01, respectively).

4 Discussion

4.1 Effect of P additions rates on soil IP fractions

With increasing P addition levels, the increase in soil labile IP and moderate-cycling IP fractions was consistent with our first hypothesis and the findings from previous studies (Wang et al., 2010; Zhao et al., 2019). Soil IP fractions can be affected by nutrient addition, soil type and soil chemical properties (Daly et al., 2001; Stroia et al., 2011). The positive linear correlation of soil TIP concentration and P addition rates (Fig. S2) indicates that applied P was fixed mainly into inorganic forms (Chauhan et al., 1981) and converted to various soil P fractions (Piegholdt et al., 2013). Most of the applied P transformed into soil moderate-cycling IP fractions as seen from the decrease in the relative proportion of soil recalcitrant IP fractions (Fig. S1). Indeed, applied P fertilizer can be quickly bound by P-fixing constituents, e.g., Fe and Al oxides and clay minerals (Devau et al., 2011). In calcareous soils, precipitation is the main process retaining applied P in soils, especially precipitation with Ca at relatively high soil pH (Wang et al., 2010). Likely, the amount of P from input processes (exogenous P and weathering) was higher than the amount from output processes (plant uptake, erosion and leaching losses), which can eventually cause P accumulation in the soil as insoluble soil P fractions (Song et al., 2017). Therefore, both the monopotassium phosphate and monocalcium phosphate fertilizers transformed into soil labile IP and moderate-cycling IP fractions and contributed to the increase in soil Al-P, Fe-P, Ca₂-P and Ca₈-P concentrations (Fig. 7).

Figure 7 Schematic diagram shows the effects of phosphorus (P) compound addition on soil inorganic P transformations in the calcareous grassland of Inner Mongolia. The percentages represent increase (“↑” in the diagram) of soil inorganic P fractions, plant P uptake and plant biomass as affected by fertilization across P addition rates. KH₂PO₄ addition had a larger impact on moderate-cycling IP (sum of Al-P, Fe-P and Ca₈-P) as compared to Ca(H₂PO₄)₂ (109 %–814 % vs. 75 %–676 %). However, recalcitrant P (sum of Ca₁₀-P and O-P) increased more with Ca(H₂PO₄)₂ addition relative to KH₂PO₄ (6 %–49 % vs. 0 %–31 %). Nitrogen addition decreased moderate-cycling P by enhancing plant biomass and plant P uptake for both P compound additions across P addition rates. Therefore, moderate-cycling P showed a higher contribution to soil labile P than recalcitrant P, as represented by “$+++$” and “+”, respectively.

Inconsistent with our first hypothesis, we found significant changes in soil recalcitrant P fractions (Ca₁₀-P and O-P), which accounted for 21 %–73 % of soil TIP (Fig. S1) and potentially played an important role in supplying soil-available P in this meadow steppe (Fig. 7). The significant increase in soil Ca₁₀-P with Ca(H₂PO₄)₂ addition was not expected; and it was inconsistent with a previous study from a calcareous soil showing unchanged soil Ca₁₀-P after 21 years of superphosphate application (Wang et al., 2010). In this calcareous soil, Ca(H₂PO₄)₂ addition enhanced the transformation of soil Ca₂-P and Ca₈-P into more stable soil Ca₁₀-P fractions for reasons that are not clear. In return, soil Ca₁₀-P can be a potential P sink of soil moderate-cycling IP pools because of its significant correlation with soil Fe-P under both chemical P forms (Table 3). Soil O-P showed a hump-shaped relationship with P addition for both P compounds when N was also supplied, while no relationship was found for KH₂PO₄ without N addition (Fig. 4c, d). This is in contrast to a 21 year long study where increased levels of Ca(H₂PO₄)₂ significantly increased soil O-P (Wang et al., 2010). The discrepancy might be due to (1) differences in soil type affecting soil P dynamics differently (a Calcarid Regosol in the Wang et al., 2010, study and a Chernozem in this study); (2) different P compounds having different effects on soil O-P cycling; (3) N addition interacting with P to affect plant P uptake and soil O-P transformations (Marklein and Houlton, 2012; Zhang et al., 2004). Thus, P addition effects on soil O-P and other soil recalcitrant P fractions were complex, particularly when N was also added.

The significant increase in soil Olsen-P with P addition levels was consistent with previous studies (Karaca et al., 2002; Zhou et al., 2018). Significant increase in soil Olsen-P most likely was a consequence of inputs of P going into this pool surpassing output processes of mineral fixation, leaching and plant uptake. Dissolution of exogenous P compounds could be the primary input process, which then directly contributed to the increase in soil Olsen-P concentration. Moreover, the soil IP pools, especially soil Fe-P, Al-P, Ca₂-P and Ca₈-P, can also be an important source of bioavailable P (Zhang et al., 2012). Significantly positive correlations between soil moderate-cycling IP fractions and soil labile IP (Table 3; Fig. 6) suggest that soil moderate-cycling IP fractions contributed strongly to enhance soil P availability (Fig. 7). Indeed, soil moderate-cycling IP fractions could release soil-available P more easily than recalcitrant IP fractions (Zhang et al., 2012).

4.2 Effects of compound-specific P additions

Consistent with our second hypothesis, soil labile IP and moderate-cycling IP fractions of Al-P, Fe-P and Ca₈-P tended to be higher with soluble KH₂PO₄ addition than with less soluble Ca(H₂PO₄)₂ addition with significant increases in some P addition levels (Figs. 2 and 3). Compared to Ca(H₂PO₄)₂, KH₂PO₄ is more effective in elevating soil phosphate levels to form soil labile IP and moderate-cycling IP fractions by rapidly interacting with Fe- and Al-oxides and CaCO₃ (Havlin et al., 2005). The decrease in soil pH contributed to the increase in labile IP with Ca(H₂PO₄)₂ addition (Fig. 6b). Lower soil pH could accelerate soil recalcitrant IP fractions converting into soil labile IP, moderate-cycling IP and Olsen-P (Alt et al., 2013). Under the Ca(H₂PO₄)₂ treatment, significant negative relationships of soil O-P and Olsen-P further support this argument (Table 3). On the other hand, plant P uptake instead of soil pH was more responsible for variations in soil moderate-cycling IP fractions with KH₂PO₄ addition (Fig. 6a). Under both P compounds, soil moderate-cycling IP fractions contributed to an increase in soil labile IP across N treatments (Fig. 6), which suggested that higher plant growth and P demand (Fig. 1c, d) could enhance the conversion of soil moderate-cycling IP fraction into labile or available P.

In contrast to our expectation, soil recalcitrant IP fractions of O-P and Ca₁₀-P were higher with Ca(H₂PO₄)₂ than with KH₂PO₄ addition (Figs. 4 and 7). This suggests that a higher proportion of Ca(H₂PO₄)₂ was converted into stable inorganic P forms than the more soluble KH₂PO₄. Additionally, lower soil O-P and Ca₁₀-P concentrations could be caused by enhanced plant P uptake intensity under KH₂PO₄ addition, which facilitated more soil recalcitrant IP fractions transforming into moderate-cycling IP and labile IP. Thus, Ca(H₂PO₄)₂ appears to be better than the KH₂PO₄ at maintaining soil recalcitrant IP fractions in the grassland soil. Although soil recalcitrant IP fractions (O-P and Ca₁₀–P) are relatively stable, they play essential roles in buffering the depletion of soil moderate-cycling P and maintaining soil-available P levels (Seeling and Jungk, 1996; Vu et al., 2008).

As expected, more soluble KH₂PO₄ could result in higher labile IP (Fig. 7) in soil solution through dissolution. It is generally observed that this more soluble P fertilizer type dissolves in soil water over a shorter time period and generates more free ${\mathrm{PO}}_{\mathrm{4}}^{\mathrm{3}-}$ (Chien et al., 2011). Soil moderate-cycling IP fractions also contributed to elevated soil Olsen-P concentrations with stronger correlations under KH₂PO₄ addition (Table 3). Soil moderate-cycling IP factions showed a stronger direct and positive effect on soil labile IP with KH₂PO₄ addition than Ca(H₂PO₄)₂ addition, while plant P uptake showed both indirect negative (by negatively affecting soil moderate-cycling IP) and direct negative effects on labile IP with Ca(H₂PO₄)₂ addition (Fig. 6). Based on the correlation analyses between soil IP fractions and Olsen-P, we found that soil Ca₂-P and Al-P were the dominant soil IP fractions to improve soil P availability in the calcareous soil. Our results suggest that KH₂PO₄ was better than Ca(H₂PO₄)₂ at alleviating P limitations of soil microorganisms and plants by promoting the formation of soil labile IP and moderate-cycling IP fractions in the meadow steppe.

4.3 Nitrogen-addition-regulated P effects on soil inorganic P fractions

We predicted that the concentrations of soil IP fractions would be lower under combined P and N additions than under P addition alone because of increased plant P uptake with N addition (third hypothesis). We found some support for this, where combined P and N additions decreased most soil IP fractions compared to P addition alone, except for the soil Ca₁₀-P fraction. We also found a significant increase in plant biomass with combined P and N addition (Fig. 1a, b) and a negative correlation between plant P uptake and moderate-cycling IP fractions (Fig. 6a, b). Therefore, the decrease in soil moderate-cycling IP fractions with N addition could have been due to enhanced plant P uptake (Fig. 1c, d) as a result of increased plant biomass (Figs. 1a, b and 7). Under N addition, simultaneous increases in soil Olsen-P output (plant uptake) and input pathways (transformation from soil moderate-cycling IP fractions) may have resulted in mostly nonsignificant difference in soil Olsen-P concentrations between combined P and N addition and P addition alone (Fig. 5a, b). Nitrogen addition can potentially increase soil P availability by promoting solubilization of soil IP fractions in the short term (Wang et al., 2016). However, long-term N deposition resulted in soil IP-exhaustion, thereby constraining the growth of plants (Olander and Vitousek, 2000; Yang et al., 2014). Previous research has also found that decades of N addition could accelerate ${\mathrm{PO}}_{\mathrm{4}}^{\mathrm{3}-}$ release (Malik et al., 2012; Stroia et al., 2011) and enhance conversion of soil recalcitrant IP fractions to soil labile IP and moderate-cycling IP fractions as a result of soil acidification (Alt et al., 2013). Additionally, N addition was found to suppress acid and alkaline phosphatase enzymes, resulting in a decrease in soil organic P mineralization in a similar semiarid grassland (Tian et al., 2016). Therefore, combined N and P addition might decrease soil IP fractions by reducing the conversion of soil organic P to IP as compared to P addition alone. Our results clearly illustrate that N effects on soil IP fractions depended on P inputs, where combined N and P additions could accelerate conversion of soil moderate-cycling IP fractions into soil-available P and enhance plant P uptake and biomass (Fig. 7).

5 Conclusions

Addition of P compounds significantly increased soil moderate-cycling IP fractions of Al-P, Fe-P, Ca₂-P and Ca₈-P, which may have contributed to higher available P (Olsen-P) in the soil. Soil moderate-cycling IP fractions were higher with soluble KH₂PO₄ addition, but, in contrast, soil recalcitrant fractions of Ca₁₀-P and O-P were higher with Ca(H₂PO₄)₂ addition. Combined N and P addition decreased soil IP fractions due to enhanced plant P uptake compared to P addition alone for both P compounds. Thus, N addition promoted the transformation of soil moderate and recalcitrant IP fractions into available forms. Soil moderate-cycling IP fractions had a greater contribution to soil P availability than soil recalcitrant P fractions. Our findings elucidated the interactive effects of N and P addition on soil IP dynamics and presented the first evidence for the relative roles of exogenous P compounds in regulating P availability in the meadow steppe grassland. Overall, P fertilization is necessary for promoting productivity and sustainable management of grasslands by maintaining soil P availability and pools under scenarios of ecosystem N enrichment.