Phosphorus Improves Stoichiometric Characteristics of Saline-Sodic Soil and Alfalfa

Phosphorus Improves Stoichiometric Characteristics of Saline-Sodic Soil and Alfalfa | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Phosphorus Improves Stoichiometric Characteristics of Saline-Sodic Soil and Alfalfa Anni Bai, MingCong Zhang, Chen Wang, HanShuo Zhang, Mingfen Shan, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8299360/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract Background: To optimize saline-sodic soil amelioration strategies, enhance soil fertility and forage productivity, and elucidate aluminum sulfate's regulatory mechanisms on C-N-P stoichiometric characteristics in both saline-sodic soils and alfalfa, we conducted this study at Yinlang Ranch of Daqing City, Heilongjiang Province in China. Results: This study investigated the effects of aluminum sulfate application at different doses (0, 24, 48, and 72 kg·hm -2 ) on the distribution of carbon (C), nitrogen (N), and phosphorus (P), as well as stoichiometric ratios and environmental factors of alfalfa and soil layers. The results indicated significant differences in the C-N-P stoichiometric characteristics of soil and alfalfa under different aluminum sulfate applications. An application rate of 48 kg·hm -2 significantly increased the C, N, and P contents in both soil and alfalfa while maintaining a relatively stable soil N/P ratio. Soil C and P exhibited significant positive correlations with alfalfa P. During the growth process, alfalfa growth was primarily limited by P. The plants enhanced their growth and development by adjusting the balance between elemental requirements and nutrient absorption, as well as modifying nutrient utilization strategies to adapt to the saline-sodic soil environment. Furthermore, alfalfa P showed significant negative correlations with soil pH and electrical conductivity (EC). Conclusions: The study concludes that aluminum sulfate significantly influences the C-N-P stoichiometric characteristics of the soil-alfalfa system. The optimal application rate of 48 kg·hm ⁻2 enhanced nutrient content in soil and alfalfa, stabilized the soil N/P ratio, and promoted P uptake of the alfalfa by improving soil environmental conditions. These findings establish a theoretical foundation for precision nutrient management. Ecological stoichiometry Saline-sodic soil Alfalfa Improver Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 1. Introduction Saline-sodic soil is one of the most widely distributed soil types globally, with over 100 countries and regions currently facing salinization issues [1] ,Among global saline-sodic distribution areas, the Songnen Plain was one of the highest rates of ecological degradation, leading to declining land productivity, biodiversity loss, groundwater pollution, and other ecological problems. These issues have become major obstacles to sustainable agricultural development [2] . The high alkalinity and sodicity in saline-sodic soils cause severe structural degradation characterized by enhanced colloidal dispersion, soil compaction, reduced porosity (<30%), and impaired hydraulic conductivity [3] 。Concurrently, saline-sodic soils exhibit low nutrient availability [4] . Under salt stress, plant roots experience osmotic imbalance and ion toxicity, leading to stomatal closure, impaired photosynthesis, and leaf oxidative stress [5 - 6] , ultimately causing crop yield reductions [7 - 8] .Therefore, enhancing nutrient availability and the productive capacity of saline-sodic soil through amelioration measures has become a central issue for achieving the sustainable utilization of saline-sodic soil resources [9] . Biological and chemical amelioration are two prominent technologies for managing saline-sodic soil. Biological amelioration facilitates soil desalination by planting salt-tolerant plants or through microbial inoculation, offering advantages such as low cost and environmental sustainability [10] . For instance, the introduction of salt-tolerant forage crops in Xinjiang's saline regions has resulted in a 20% to 30% reduction in surface soil salinity and a 15% increase in organic matter content [11] . However, biological amelioration is characterized by a lengthy process, limited effectiveness in restoring severely saline-sodic soils [12] . In contrast, chemical amelioration quickly modifies soil physical and chemical properties by adding acidic substances making it an effective strategy for enhancing productivity in the short term [13] . Aluminum sulfate (Al 2 (SO 4 ) 3 ) functions as a typical acidic ameliorant through several mechanisms: (1) it releases H + ions to neutralize soil alkalinity and lower pH; (2) it replaces Na + ions on soil colloids, thereby enhancing aggregate structure; and (3) sulfate ions (SO 4 2- ) react with carbonates to form soluble salts, which promotes salt leaching [14 -15] . Studies have demonstrated that a single application of aluminum sulfate can reduce soil pH from 9.2 to 8.0 and decrease the sodium adsorption ratio (SAR) by 40% [16 -17] . However, the use of single ameliorants can easily lead to elemental imbalances, and excessive application of aluminum sulfate may inhibit P availability [18 -19] . Existing research has primarily concentrated on the effects of aluminum sulfate on the physical properties of soil, while the regulatory mechanisms affecting C-N-P stoichiometric characteristics remain unclear [20] . Traditional soil research has primarily focused on the isolated analysis of individual elements, such as N and P. However, the cycling of C, N, and P in saline-sodic soils is highly interconnected, and their stoichiometric ratios (C:N:P) can systematically reveal mechanisms of nutrient limitation and utilization efficiency [21] . Ecological stoichiometry, which originated from the Redfield ratio (C:N:P = 106:16:1) in marine ecosystems, has been adapted to terrestrial ecosystems and has become a fundamental tool for studying elemental balance and energy flow [22] . Its key advantages include: N/P ratios that can determine the threshold for plant N or P limitation (N/P 16 indicates P limitation) [23] ; increasing C/N or C/P ratios that suggest slow organic matter decomposition and enhanced nutrient retention [24] ; and the ability to evaluate the long-term effects of amelioration measures through correlation models between elemental ratios and environmental factors, such as pH and electrical conductivity [25] . In saline-sodic soil amelioration, ecological stoichiometry demonstrates distinct advantages. P is often fixed by calcium and iron ions, leading to P limitation for its growth [26] . Zhao and Niu et al. found that gypsum application significantly decreased soil C/P ratios while enhancing P availability and moderately increasing crop yield [27 -29] However, existing studies primarily focus on the effects of single ameliorants on certain elements, lacking systematic analysis of C-N-P synergistic changes.Moreover, the interaction effects between salt-tolerant plants (such as alfalfa) and chemical ameliorants are rarely reported, yet this interaction is crucial for optimizing improvement strategies [30] 。 Alfalfa ( Medicago sativa ), a leguminous perennial forage, is widely utilized for the restoration of saline-sodic land due to its remarkable salt tolerance (withstanding electrical conductivity levels of up to 8 dS/m) and high biomass production (annual hay yield of 10-15 tons per hectare) [32 -33] . The mechanisms underlying its salt tolerance include: (1) the root secretion of organic acids (such as malic acid) that chelate sodium ions (Na + ) and mitigate ion toxicity; (2) the synthesis of osmoregulatory substances (such as proline) to maintain cellular water potential; and (3) symbiotic N fixation with rhizobia to alleviate soil N deficiency [34 -35] . Planting alfalfa in moderately saline-sodic soil (pH 8.5-9.0) in the Songnen Plain has resulted in a 20% increase in SOC content and a 15% reduction in surface salinity [36] . However, alfalfa has high P requirements, with tissue P content needing to reach 0.2%-0.4%. This study focuses on saline-sodic soils in the Songnen Plain, employing ecological stoichiometry to investigate aluminum sulfate's effects on C-N-P stoichiometric ratios in both soil and alfalfa. The research objectives are: Quantify the roles of C/P and N/P ratios in nutrient limitation and use efficiency . Analyze synergistic effects among multiple elements to elucidate how modulating C-N-P balance enhances soil structure and alfalfa nutrient uptake when combined with soil amendments . (iii) Determine optimal application rates to refine remediation strategies, providing a scientific basis for precision amendment and ecological restoration of saline-sodic lands. 2. Materials and Methods 2.1 Materials The experimental site was located at Yinlang Ranch, Daqing City, Heilongjiang Province, China (46°30′0.2″N, 124°50′4.7″E), which is part of the Songnen Plain and has an altitude of 149 meters.The alfalfa ( Medicago sativa L .) used in this study was the salt-tolerant cultivar “Gongnong 5”. The plants were grown under field conditions and sampled during the mid-growth stage in mid-August 2024. The soil type was classified as saline-sodic soil. The soil was classified as saline-sodic soil. A randomized complete block design was implemented with 10 plots (7 m × 3 m each). In mid-August 2024, soil and plant samples were collected during the mid-growth stage, avoiding fertilizer application zones. Three 1 m × 1 m subplots were randomly designated within each experimental area, and five sampling points were selected using an S-shaped sampling pattern in each subplot to collect soil samples at three depths: 0–10 cm, 10–20 cm, and 20–30 cm. Following pretreatment and air-drying, soil samples were ground through a 2 mm sieve and stored. Alfalfa stems were processed by grinding with a mechanical mill, sieving, and packaging in self-sealing bags for subsequent analysis. The basic physical and chemical properties of the soil samples are presented in Table 1 Table 1 Basic physical and chemical properties of soil before planting alfalfa 2.1.1 Soil Parameter Analysis The experimental treatment scheme is presented in Table 2. To ensure representativeness and accuracy during sampling, soil samples were collected at various depths using a soil auger after removing surface litter. Soil samples from three quadrants were combined by layer, with roots and large stones removed. The samples were thoroughly mixed and quartered to obtain composite samples, which were labeled and transported to the laboratory. The samples were then ground and sieved to determine the contents of soil C, N, and P. Nutrient content analysis was conducted using standard methods: Soil organic carbon (SOC) was determined using the external-heat potassium dichromate titration method. Total nitrogen (TN) was quantified by the Kjeldahl method (hd-kn10 analyzer, HORDE ELECTRIC). Total phosphorus (TP) was measured via the molybdenum blue spectrophotometric method (721 spectrophotometer, Shanghai Jinghua). Nitrate N was analyzed by phenol disulfonic acid colorimetry. Available P was extracted with sodium bicarbonate and determined by molybdenum blue spectrophotometry. Exchangeable potassium was extracted with ammonium acetate and quantified using flame photometry (FP6400, Bona Technology). Soil pH was measured potentiometrically (Sartorius PB-10 pH meter, Germany) at a 1:2.5 soil:water ratio [36] . Table 2 Test treatment scheme 2.1.2 Alfalfa Parameter Analysis Plant total organic carbon (TOC) was determined using the potassium dichromate (K₂Cr₂O₇) wet oxidation method. Approximately 0.200 g of oven-dried plant sample was digested at 180°C for 5 minutes in an oil bath, followed by titration with ferrous sulfate (FeSO₄). TN and TP contents were analyzed using a continuous flow analyzer (QC8500, HACH). Samples (~0.200 g) were digested in 100 mL tubes with 5 mL concentrated H₂SO 4 at 240°C for 1 hour, then at 380°C with intermittent additions of 30% H₂O₂ until the digest became colorless/clear. Digests were diluted to 50 mL with deionized water and analyzed for TN and TP content via continuous flow analysis using appropriate calibration standards and reagent blanks [37] . 2.2 Statistical analysis Data calculations and variance analyses ( P = 0.05) were performed using Excel 2019 and SPSS version 24.0. Post-hoc tests were conducted using Duncan's multiple comparison method. Origin 2021 was utilized for plotting. Redundancy analysis (RDA) was carried out using the vegan package in R, with visualizations created using the ggplot2 package. Additionally, structural equation modeling (SEM) was constructed using the plspm package in R, employing the least squares method. Model fit was evaluated using fitness indices (CFI/TLI > 0.9, RMSEA < 0.08). 3. Results 3.1 Ecological chemical metering of alfalfa 3.1.1 Effects of aluminum sulfate application on C, N and P content in alfalfa stems Different applications of aluminum sulfate had specific effects on N and P levels in alfalfa (Figure 1). The N and P concentrations in the CL2 treatment were significantly higher than those in the other treatments ( P < 0.05, Figures 1A and 1B). Compared to the CK, the N content increased by 41.81%, while the P content rose by 15.27%. However, varying doses of aluminum sulfate did not have significant effects on the C content of alfalfa (Figure 1C). The CL3 treatment exhibited the highest C content at 314.13 g·kg -1 , representing an increase of 14.36% compared to the control. Figure 1 Effects of aluminum sulfate doses on TOC content (Figure 1A), TN content (Figure 1B), and TP content (Figure 1C) in alfalfa stems. CK: 0 kg·hm -2 (control); CL1: 24 kg·hm -2 ; CL2: 48 kg·hm -2 ; CL3: 72 kg·hm -2 . Lowercase letters indicate significant differences at ( P < 0.05), and uppercase letters indicate significant differences at ( P < 0.01). 3.1.2 Effects of aluminum sulfate application on C-N-P chemical characteristics of alfalfa rhizomes Alfalfa's C, N, and P contents varied under different applications, resulting in significant differences in their ecological stoichiometric characteristics (Figure 2 ). The C/N ratio in the CK was significantly higher than in the treatments CL1, CL2, and CL3 ( P < 0.05, Figure 2A). Specifically, the C/N ratio in CK was 34.04% higher compared to CL2. An excessively high C/N ratio may negatively impact the rate of organic matter decomposition in plants. In terms of the C/P ratio, CL2 exhibited a significantly lower value than the other treatments ( P < 0.01, Figure 2B), decreasing by 6.39% compared to CK, which is contrary to the results observed for the N/P ratio (Figure 2C). Figure 2 Effects of aluminum sulfate doses on C/N ratio (Figure 2A), C/P ratio (Figure 2B), and N/P ratio (Figure 2C) in alfalfa stems. Treatments as in Figure 1. Lowercase letters indicate significant differences at ( P < 0.05), and uppercase letters indicate significant differences at ( P < 0.01). 3.2 Ecological chemical metering of soil 3.2.1 Effects of aluminum sulfate application on soil C, N and P content Different applications of aluminum sulfate influenced the concentrations of C, N, and P in the 0-30 cm soil layer (Figure 3). In the 0-10 cm layer, the C content in the CL3 treatment was significantly higher than in the other treatments ( P < 0.05, Figure 3A), showing an increase of 64% compared to the CK At an application rate of 48 kg·hm -2 , the C content in the 10-20 cm layer was significantly greater by 6.9% compared to the 20-30 cm layer ( P < 0.05). Different applications of aluminum sulfate also influenced soil N content. At various depths, the N content in CL3 was significantly higher than in CL2, CL1, and CK ( P < 0.05, Figure 3B). Soil N was concentrated in the 10-30 cm layer, with lower concentrations in the surface layer, which is contrary to the distribution of P. In the soil layer of 20 to 30 cm, the P content of CL1 reached 2.93 g·kg -1 , significantly higher than that of CK. In addition, the P content of CL1 was 7.33% and 9.12% higher than that of CL2 and CL3 respectively ( P < 0.05, see Figure 3C). Figure 3 Effects of aluminum sulfate doses on soil C content (Figure 3A), TN content (Figure 3B), and TP content (Figure 3C) at 0–10 cm, 10–20 cm, and 20–30 cm depths. Treatments as in Figure 1. Different lowercase letters indicate significant differences between different soil layers of the same treatment ( P < 0.05) and different uppercase letters indicate significant differences between different treatments of the same soil layer ( P < 0.05) 3.2.2 Effects of aluminum sulfate application on C-N-P chemical characteristics of soil As illustrated in Figure 4, the ratios of C/N, C/P, and N/P generally decreased with increasing soil depth. The C/N ratio in CL1 and CL2 was significantly higher than that in CK and CL3 ( P < 0.05, Figure 4A); however, no significant difference was observed between CL1 and CL2. In the 10-20 cm soil layer, the C/N ratio in CL3 was lower, showing a decrease of 12.87% compared to CK at the same depth. After the application of aluminum sulfate, the C/P ratio in the soil was generally lower than that of the CK, with significant differences observed in the 0-10 cm layer ( P < 0.05, Figure 4B). In the 10-20 cm layer, the C/P ratio in the CK was 66.73% higher than that in CL1, 64.18% higher than in CL2, and 65.91% higher than in CL3. Significant differences were noted among the depths within the same treatment ( P < 0.05). Different doses resulted in varying N/P ratios due to differing inputs of N and P. Compared to the CK, treatments CL1 and CL2 reduced the N/P ratio by 36.81% and 49.71%, respectively, in the surface layer, with more pronounced differences observed in the 10-30 cm layer. No significant differences were found among depths under the same treatment (Figure 4C). Figure 4 Effects of aluminum sulfate doses on soil C/N ratio (Figure 4A), C/P ratio (Figure 4B), and N/P ratio (Figure 4C) at 0–10 cm, 10–20 cm, and 20–30 cm depths. Treatments as in Figure 1. Different lowercase letters indicate significant differences between different soil layers of the same treatment ( P < 0.05) and different uppercase letters indicate significant differences between different treatments of the same soil layer ( P < 0.05) 3.3 Regression analysis of C 、 N 、 P Different applications of aluminum sulfate demonstrated strong linear relationships with average soil nutrients in the 0-30 cm layer (Figure 5). Soil C exhibited the highest correlation with P. As C levels increased, both N and P also rose, indicating positive correlations. Similar trends were observed in the linear regression analysis of alfalfa nutrients. Figure 5 The linear regression relationship between soil C and soil N (Figure 5A), soil C and soil P (Figure 5B), alfalfa C and alfalfa N (Figure 5C) and alfalfa C and alfalfa P (Figure 5D) in the 0-30cm soil layer treated with aluminum sulfate. As illustrated in Figure 6, C exhibited a positive correlation with the C/N ratio and a negative correlation with the C/P ratio. N demonstrated a negative correlation with both the C/N and N/P ratios, although these correlations were weaker in terms of ecological stoichiometry. In contrast, P displayed stronger correlations with the C/P and N/P ratios (R 2 = 0.8993 and 0.8375, respectively), indicating negative trends. Figure 6 The linear regression relationship between soil C and C/N (Figure 6A), N and C/N (Figure 6B), soil P and C/P (Figure 6C), soil C and C/P Figure 6D), soil N and N/P (Figure 6E) and soil P and N/P (Figure 6F) in the 0-30cm soil layer after aluminum sulfate treatment. 3.4 Correlations with the C:N:P stoichiometry As illustrated in Figure 7, the alfalfa-soil system demonstrated a highly significant positive correlation between soil C and P ( P < 0.01). This finding suggests a synergistic accumulation or mutual enhancement of these two components within the soil. Additionally, both soil C and P exhibited significant positive correlations with alfalfa P content ( P < 0.05), indicating that improved soil P availability directly facilitates alfalfa's P absorption. The soil C/P and N/P were significantly negatively correlated with soil pH, BD, and EC ( P < 0.05). Furthermore, pH showed a highly significant negative correlation with alfalfa P ( P < 0.01), suggesting that elevated alkalinity inhibits alfalfa's P absorption. The N and P in alfalfa were significantly positively correlated ( P < 0.05), indicating a synergistic absorption or functional coupling between these nutrients. The alfalfa N/P ratio positively correlated with both N and P, suggesting that the N/P ratio reflects the plant's balanced demand for N and P nutrients. Figure 7 Correlation network of C-N-P stoichiometric characteristics in the alfalfa-soil system. Red/blue lines indicate positive/negative correlations. The thickness of the line indicates the strength of the correlation, and the asterisk indicates statistical significance ( * P < 0.05, ** P < 0.01, *** P < 0.001). S.C: soil carbon; S.N: soil nitrogen; S.P: soil phosphorus; S.CN: soil C:N; S.CP: soil C:P; S.NP: soil N:P; A.C: alfalfa carbon; A.N: alfalfa nitrogen; A.P: alfalfa phosphorus; A.CN: alfalfa C:N; A.CP: alfalfa C:P; A.NP: alfalfa N:P; EC: electrical conductivity; BD: bulk density. 3.5 Redundancy Analysis of C:N:P stoichiometry Changes in the soil environment and the application of soil amendments significantly influenced the stoichiometric characteristics of C, N, and P in both the soil and alfalfa (Figure 8). Redundancy analysis (RDA) revealed that amendments and soil properties collectively accounted for 82.03% of the total variation in the data, with RDA1 (79.31%) and RDA2 (2.72%) serving as the principal ordination axes. Soil C/N exhibited the strongest positive correlation with RDA1, identifying it as the primary positive factor driving the effects of soil amendments. Conversely, alfalfa C/P demonstrated a significant negative correlation with RDA1, indicating its role as a secondary negative factor reflecting the plant's adaptive adjustments in C/P balance. Additionally, soil N showed a negative correlation with RDA2, suggesting interactive effects between soil N and alfalfa C/P. Alfalfa P displayed a positive correlation with RDA1 but negative correlations with pH and EC ( P <0.001), confirming P as a key driver. Soil C and P exhibited significant positive correlations along the first axis of the redundancy analysis (RDA1) ( P < 0.001), supporting the "C-P co-accumulation" hypothesis. Conversely, soil N/P ratios correlated negatively with RDA1, indicating that amendments alleviated N limitation by regulating the N/P balance. The CL2 treatment was positioned in the positive direction of RDA1, demonstrating significant positive correlations with soil C/N ratios and alfalfa P, which suggests its effectiveness in enhancing the soil C-N balance and P uptake through reductions in pH and EC. Soil C/P ratios exhibited negative correlations with the CL2 treatment, further supporting its role in enhancing P bioavailability. The CK group clustered along the negative axis of RDA1, which is associated with high C/N ratios, elevated pH, and EC, all indicative of soil degradation. Although CL3 showed a positive correlation with RDA1, it deviated from the main trend, potentially due to aluminum toxicity resulting from high amendment doses that inhibit root growth. Alfalfa N/P ratios correlated positively with the first axis of redundancy analysis (RDA1), supporting its role as an indicator of N limitation. Additionally, alfalfa C and N exhibited positive correlations along the second axis of redundancy analysis (RDA2), suggesting coordinated uptake mechanisms. Both pH and EC correlated negatively with RDA1 and demonstrated significant negative relationships with alfalfa P and soil C/P ratios ( P < 0.001). This indicates that high salinity inhibits nutrient absorption and that reducing pH is critical for alleviating P limitation. Figure 8 Redundancy analysis (RDA) of C-N-P stoichiometric characteristics and environmental factors. S.C: soil carbon; S.N: soil nitrogen; S.P: soil phosphorus; S.CN: soil C:N; S.CP: soil C:P; S.NP: soil N:P; A.C: alfalfa carbon; A.N: alfalfa nitrogen; A.P: alfalfa phosphorus; A.CN: alfalfa C:N; A.CP: alfalfa C:P; A.NP: alfalfa N:P; EC: electrical conductivity; BD: bulk density. Arrows indicate direction and strength of variable contributions. Treatments: CK (black), CL1 (blue), CL2 (red), CL3 (green). RDA1 and RDA2 explain 79.31% and 2.72% of variance, respectively. ( ** P < 0.001). 3.6 Structural equation modeling of multiple elements in different soil layers Based on structural equation modeling (SEM) analysis (Figure 9), aluminum sulfate regulated C-N-P coupling in the soil-plant system differently across soil depths. In the 0-10 cm layer (Figure 9A), amendments directly lowered soil pH and EC, promoting the release of available nutrients. These nutrients drove both soil and alfalfa nutrient dynamics, establishing a synergistic nutrient interaction between soil and alfalfa. In this layer, available nutrients (primarily P) acted as key mediators in nutrient cycling. Deeper down, while amendment effects on pH and EC lessened, their influence on soil nutrients grew stronger. Soil nutrients directly governed alfalfa nutrient uptake, and the coupling between soil and alfalfa stoichiometric traits strengthened significantly. In the 10-20 cm soil layer (Figure 9B), alfalfa's nutrient acquisition primarily depended on direct soil supply, exhibiting the highest sensitivity to nutrient balance. In the 20-30 cm soil layer (Figure 9C), the effectiveness of amendments was limited, and EC remained the primary factor inhibiting nutrient availability. The contribution of soil nutrients to alfalfa uptake decreased significantly, and stoichiometric coupling was at its weakest. In deeper soil layers, alfalfa's nutrient absorption was constrained, prompting adaptive adjustments in the plant's elemental demand in response to environmental conditions. Collectively, the results demonstrate that the regulatory effects of amendments on pH and EC gradually diminished from the surface to deeper soil layers. Available P emerged as the key driver primary soil of nutrient accumulation in both alfalfa nutrient accumulation layers, validating the hypothesis that "P is the critical driver for improving stoichiometric characteristics in saline-sodic soils". This highlights soils. pivotal underscores of essential availability in mediating soil-plant C-N-P interactions under C-N-P application. following the application of amendments. Note: Structural equation model for 0-10 cm layer (GFI=0.9001) Note: Structural equation model for 10-20 cm layer (GFI=0.8276) Note: Structural equation model for 20-30 cm layer (GFI=0.8505) Figure 9 Structural equation models (SEM) illustrating aluminum sulfate regulation on C-N-P coupling in soil-alfalfa systems at 0–10 cm (A), 10–20 cm (B), and 20–30 cm (C) depths. Solid/dashed arrows indicate positive/negative pathways ( P < 0.05). Arrow width reflects standardized path coefficients. GFI: Goodness-of-Fit Index. AP: available P; EC: electrical conductivity 4. Discussion 4.1 The effect of aluminum sulfate on nutrient uptake in alfalfa and its stoichiometric control The use of aluminum sulfate significantly changed how alfalfa absorbed nutrients. As shown in Figure. 1, under the CL2 treatment condition, the N and P contents in alfalfa were significantly increased compared with the CK, while the C content showed a slight increase. This is consistent with the findings of Mu et al [38] , indicating that aluminum sulfate promotes alfalfa growth by regulating soil pH and ion forms, thereby improving the bioavailability of N and P. [39 -41] . Notably, Figure 2 demonstrates that following aluminum sulfate application, the CL2 treatment exhibited the lowest C/N and C/P ratios, indicating optimal nutrient utilization efficiency at this dosage. This finding aligns with the observations of Yu et al. [41] , where a low C/N ratio typically corresponds to high decomposition rates and nutrient turnover. Alfalfa N/P ratios (12.69-13.78) observed in this study were lower than those reported by Luo et al . [42] on the Qinghai-Tibet Plateau (N/P = 14.5-16.2) and Pan et al. [43] in temperate grasslands (N/P = 15.1-17.3). which can be attributed to the unique edaphic conditions of the Songnen Plain. Specifically, the study site exhibited a baseline soil pH of 8.2 (Table 1), significantly higher than the mildly acidic to neutral pH ranges (6.0–7.5) typically reported in the Qinghai-Tibet Plateau and temperate grassland ecosystems [44 -45] . This elevated alkalinity promotes ammonium-N conversion to gaseous ammonia (NH 3 ) via volatilization, while high sodium ion (Na + ) saturation (over 80% in soil colloids [2] ) disrupts soil aggregate structure, enhancing N leaching through increased permeability [43] .. Additionally, no P limitation was observed, which contrasts with findings by Harpole et al. [24] in global grasslands, likely due to significantly improved P availability resulting from the ameliorant. Compared to the study by Jiaping et al. [46] in Xinjiang cotton fields, alfalfa in this research contributed more to C fixation (14.36% increase in C), possibly due to the N-fixing capabilities of legumes [33] . However, alfalfa had limited effects on deep soil improvement, consistent with the findings of Díaz et al . [29] in arid regions, indicating the limitations of perennial forages in enhancing deep soil quality. Furthermore, the co-accumulation of soil C and P supports the "C-P co-accumulation" hypothesis [43] . However, this effect diminishes in deeper soil layers, likely due to reduced microbial activity with increasing depth [44] . 4.2 Vertical Differentiation and Environmental Drivers of Soil-Plant C-N-P Coupling The vertical distribution of soil C, N, and P exhibited significant dose-dependent responses to amendments. In the 0-10 cm layer, the C content under the treatment increased by 64% compared to CK, which is with with the findings of Elser et al. [45] regarding cycle-enhanced C sequestration. However, in the 10-20 cm layer, the treatment showed demonstrated highest C content, suggesting that doses optimize mid-layer accumulation in mid-layers. The content was significantly higher in in the cm layers than in surface in the aligning with prior studies previous N leaching in saline-sodic soils [46] . Notably, the yielded the highest P content in the 20-30 cm layer, likely due to low-dose amendments activating insoluble phosphates in deeper soils [47] . Vertical differentiation of soil C/P ratios revealed spatial variability in P availability. The C/P ratio in surface soil was significantly lower than that in deeper layers, corroborating the findings of Ivanova et al . [39] regarding organic acid-mediated P activation. Furthermore, a significant negative correlation ( P < 0.01) was observed between soil C/P and alfalfa C/P, indicating that plants adapt to P limitation by enhancing their utilization efficiency [17] . This adaptive strategy is particularly critical in the Songnen Plain, where high pH levels exacerbate P fixation. Alfalfa mitigates this issue by secreting malic and oxalic acids to chelate Ca 2+ and Fe 3+ ions [32] , thereby maintaining cellular P homeostasis. 4.3 Nutrient Limitation Mechanisms from an Ecological Stoichiometry Perspective As shown in Figure 4, the soil C/N ratio (21.5) under CL2 treatment approached the ideal 25:1 [49] indicating an effective C-N balance. A higher dosage of aluminum sulfate increased the C/N ratio to 25.3, which not only reduced C sequestration efficiency [50] but also induced aluminum toxicity, inhibiting root growth [50 -51] , consistent with the findings of Batjes et al [52] . Reduced C/P ratios reflected improved P bioavailability, which is directly linked to an increase in available soil P. Notably, CL2 reduced the surface soil N/P by 49.71%, likely due to pH-driven N mineralization and P release. The stability of the N/P ratios, with no significant differences observed across layers within treatments, suggests that the amendment induced vertical homogenization of nutrient distribution. As shown in Figures 5 and 7, N and P in alfalfa exhibited a significant positive correlation ( P < 0.01), indicating synergistic uptake between the two nutrients. This phenomenon may result from N-enhanced photosynthetic product synthesis driving P demand [53] . Reduced C/P under CL2 further highlighted elevated P utilization efficiency, directly related to soil available P. Such synergy is consistent with the findings of Wang et al. [19] , highlighting the universal plant co-demand for N and P. 4.4 Environmental Drivers of Soil-Plant C-N-P Coupling As indicated by the RDA analysis (Figure 8), soil pH exhibited a significant negative correlation with alfalfa P conten ( P <0.01), and positive correlations between pH and EC ( P <0.05). In high-pH soils, Na⁺ competes with PO 4 3- for root adsorption sites, thereby impeding P transmembrane transport [4] . This ion antagonism is particularly pronounced in the Songnen Plain, where Na⁺ saturation in soil colloids exceeds 80% [5] . Unlike the halophytes studied by Flowers et al. [30] , alfalfa has adapted to salinity by modulating N/P ratios rather than accumulating osmolytes, possibly due to its leguminous characteristics [33] . Co-accumulation of soil C and P further supports the "C-P synergy" hypothesis [54] . CL2 increased soil C by 64% and P by 15.27%, aligning with the findings of Cai et al. [48] regarding sulfur-driven C fixation. The enhanced availability of P likely stimulates the decomposition and stabilization of microbial organic C [54] . However, this synergy appears to weaken in deeper soils, potentially due to a decline in microbial activity with increasing depth [49] . 4.5 Aluminum sulfate's distinct regulation of the C-N-P coupling process in soil-plant systems. Structural equation modeling (SEM) elucidated amendment-driven regulatory networks across various soil layers(Figure 9). In the 0-10 cm layer, amendments improved enhanced cycling through two primary (1) reducing pH/EC alleviate ion toxicity toxicity, (2) enhancing promoting the release of nutrient, with the findings of Yu et al. [41] in North China Plain. Acidic amendments neutralize alkalinity via by releasing H⁺ ions, which liberate P that is fixed in carbonate forms. the availability of directly drove influenced uptake more strongly significantly soil reserves, likely due to root-secreted organic acids chelating that chelate Fe 3+ and Al 3+ ions, thereby solubilizing phosphates [32] . In the 10-20 cm layer, SEM revealed diminished pH/EC regulation but intensified nutrient-driven soil-plant interactions intensified. An increased organic matter content may promote the binding of sulfate (SO 4 2- ) and calcium ions (Ca 2+) to form gypsum, thereby restoring the aggregate structure [18] . The direct uptake of nutrients by alfalfa surpassed the effects observed in the surface layer, aligning with the findings of Wu et al. [6] regarding the root density of alfalfa in the mid-layer. Enhanced stoichiometric coupling indicates a heightened sensitivity of the mid-layer to nutrient fluctuations, which may be associated with vertical gradients in microbial activity. In the 20-30 cm layer, SEM showed limited penetration of amendments, with EC remaining the primary inhibitor of nutrient availability. This finding aligns with the research conducted by Zhao et al. [26] on gypsum amendments. Contributions from deeper soil layers to alfalfa uptake were significantly lower, potentially due to suppressed proton pump activity under alkaline conditions [55 -56] . Weak stoichiometric coupling suggests that plants adapt to nutrient scarcity by regulating elemental demand [57 -58] . 5. Conclusion Aluminum sulfate has a notable impact on the C-N-P chemical stoichiometry of the soil-alfalfa system. When applied at a rate of 48 kg·hm -2 , it significantly boosts the levels of C, N, and P in both the soil and alfalfa, while keeping the N/P ratio in the soil relatively stable. It also improves P uptake in alfalfa by reducing soil pH and EC. Structural equation modeling (SEM) indicates that P plays a crucial role in nutrient cycling in surface soils, showing a significant negative relationship between alfalfa P and soil C/P. This research identifies 48 kg·hm -2 as the ideal application rate, which greatly enhances soil fertility and alfalfa yield by addressing P deficiencies and optimizing the C-N-P relationship. Declarations Ethical approval and field permissions All field studies, including plant material collection, were conducted in accordance with local legislation and with permission from the management of Yinlang Ranch, Daqing City. The research protocol complies with the guidelines for ethical conduct in plant studies issued by the Heilongjiang Bayi Agricultural University. Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Availability of data and materials All data generated or analysed during this study are included in this published article and its supplementary information files. Competing interests The authors declare that they have no competing interests. Funding National Key R&D Program of China (No. 2023YFD2300102), Postdoctoral Scientific Research Startup Fund Project of Heilongjiang Provincial (No. LBH-Q21162), Natural Science Foundation Project of Heilongjiang Provincial (No. LH2022D019), and Horizontal research project of Heilongjiang Bayi Agricultural University (No. 2041200084). Authors' contributions A.B. conducted the investigation, developed the methodology, wrote the original draft, and created visualizations. M.Z. conceptualized the study, developed the methodology, supervised the work, and reviewed and edited the writing. All authors reviewed the manuscript.C.W. reviewed and edited the writing. H.Z. worked on the methodology, created visualizations, and supervised the study. M.S. Methodology, Supervision. W.Z. and M.Q. Supervision. All authors read and approved the final manuscript. Acknowledgements Not applicable. References Centofanti T, Banuelos G. Evaluation of the halophyte Salsola soda as an alternative crop for saline soils high in selenium and boron. J Environ Manage. 2015;157:96–102. 2015;157:96–102. doi: 10.1016/j.jenvman.2015.04.005. Qi H, Ma R, Shi C, et al. Novel low-cost carboxymethyl cellulose microspheres with excellent fertilizer absorbency and release behavior for saline-alkali soil. Int J Biol Macromol. 2019;131:412-419. doi:10.1016/j.ijbiomac.2019.03.047. Munns R, Tester M. Mechanisms of salinity tolerance. Annu Rev Plant Biol. 2008;59:651-681. doi:10.1146/annurev.arplant.59.032607.092911. Shabala S, Cuin TA. Potassium transport and plant salt tolerance. Physiol Plant. 2008;133(4):651-669. doi:10.1111/j.1399-3054.2007.01008.x. Hasanuzzaman M, et al. Potential use of halophytes to remediate saline soils. Biomed Res Int. 2014;2014:589341. doi:10.1155/2014/589341. Wu J, et al. Alfalfa cultivation in saline soils: Physiological responses and soil reclamation. Agron J. 2014;106(2):685-694. doi:10.2134/agronj2013.0355. Rengasamy P. Soil processes affecting crop production in salt-affected soils. Funct Plant Biol. 2010;37(7):613-620. doi:10.1071/FP09249. Rayment GE, Higginson FR. Australian Laboratory Handbook of Soil and Water Chemical Methods. Inkata Press; 1992. Elser JJ, et al. Global analysis of nitrogen and phosphorus limitation of primary producers in freshwater, marine, and terrestrial ecosystems. Ecol Lett. 2007;10(12):1135-1142. doi:10.1111/j.1461-0248.2007.01113.x. Sterner RW, Elser JJ. Ecological Stoichiometry: The Biology of Elements from Molecules to the Biosphere. Princeton University Press; 2002. doi:10.1515/9781400885695. Koerselman W, Meuleman AFM. The vegetation N:P ratio: A new tool to detect the nature of nutrient limitation. J Appl Ecol. 1996;33(6):1441-1450. doi:10.2307/2404783. Güsewell S. N:P ratios in terrestrial plants: Variation and functional significance. New Phytol. 2004;164(2):243-266. doi:10.1111/j.1469-8137.2004.01192.x. Li X, et al. Effects of combined amendments on soil salinity and nutrient availability in coastal saline soil. Soil Tillage Res. 2019;194:104318. doi:10.1016/j.still.2019.104318. Clark GJ, et al. Sulfate sorption and release in acid sulfate soils: Effects of pH and redox potential. Geoderma. 2007;138(3-4):357-366. doi:10.1016/j.geoderma.2006.11.017. Daliakopoulos IN, Tsanis IK, Koutroulis AG. Soil salinity assessment, monitoring and mitigation: An overview. Sci Total Environ. 2016;542:727-739. doi:10.1016/j.scitotenv.2015.11.074. Jones DL, Darrah PR. Role of root derived organic acids in the mobilization of nutrients from the rhizosphere. Plant Soil. 1994;166(2):247-257. doi:10.1007/BF00008338. Bolan NS, et al. Influence of low-molecular-weight organic acids on the solubilization of phosphates. Biol Fertil Soils. 1994;18(4):311-319. doi:10.1007/BF00570634. Lindsay WL. Chemical Equilibria in Soils. Wiley; 1979. doi:10.2136/sssaj1979.03615995004300050021x. Wang Y, et al. Synergistic effects of aluminum sulfate and ferrous sulfate on saline-alkali soil amelioration. J Soils Sediments. 2021;21(5):2103-2115. doi:10.1007/s11368-021-02922-1. Du E, et al. Global patterns of nitrogen and phosphorus limitation in terrestrial ecosystems. Nat Geosci. 2022;15(11):867-873. doi:10.1038/s41561-022-01034-3. Andersen T, Elser JJ, Hessen DO. Stoichiometry and population dynamics. Ecol Lett. 2004;7(10):884-900. doi:10.1111/j.1461-0248.2004.00646.x. Weber TS, Deutsch C. Ocean nutrient ratios governed by plankton biogeography. Nature. 2010;467(7315):550-554. doi:10.1038/nature09403. LeBauer DS, Treseder KK. Nitrogen limitation of net primary productivity in terrestrial ecosystems is globally distributed. Ecology. 2008;89(2):371-379. doi:10.1890/07-0527.1. Harpole WS, Tilman D. Nitrogen and phosphorus limitation of productivity in worldwide herbaceous communities. Ecology. 2007;88(10):2391-2397. doi:10.1890/06-1892.1. Hessen DO, et al. Ecological stoichiometry: An elementary approach using basic principles. Limnol Oceanogr. 2013;58(6):2219-2236. doi:10.4319/lo.2013.58.6.2219. Marra LM, et al. Initial pH of medium affects organic acids production but do not affect phosphate solubilization. Braz J Microbiol. 2015;46(2):367-375. doi:10.1590/S1517-838246246220131102. Zhao S, et al. Gypsum amendment improves soil properties and crop productivity in saline-alkali soils of North China. Agric Water Manag. 2019;222:28-36. doi:10.1016/j.agwat.2019.05.037. Niu R L, et al. Carbon, nitrogen, and phosphorus stoichiometric characteristics of soil and leaves from young and middle aged Larix principis-rupprechtii growing in a Qinling Mountain plantation. Acta Ecologica Sinica. 2016,36(22):7384-7392. doi: 10.5846/stxb201601080057 . Cox, K.H., Jacinthe, PA. Phosphorus Mobility in Gypsum-Amended Soils in Relation to Soil Type and Timing of P Fertilizer Application. Water Air Soil Pollut 234, 368 (2023). doi:10.1007/s11270-023-06388-4. Díaz FJ, et al. Using saline soil and marginal quality water to produce alfalfa in arid climates. Agric Water Manag. 2018;199:11-21. doi:10.1016/j.agwat.2017.12.003. Flowers TJ, Colmer TD. Salinity tolerance in halophytes. New Phytol. 2008;179(4):945-963. doi:10.1111/j.1469-8137.2008.02480.x. Shannon MC, Grieve CM. Tolerance of vegetable crops to salinity. Sci Hortic. 1999;78(1-4):5-38. doi:10.1016/S0304-4238(98)00189-7. Ashraf M, Harris PJC. Potential biochemical indicators of salinity tolerance in plants. Plant Sci. 2004;166(1):3-16. doi:10.1016/j.plantsci.2003.10.024. Zahran HH. Rhizobium-legume symbiosis and nitrogen fixation under severe conditions and in an arid climate. Microbiol Mol Biol Rev. 1999;63(4):968-989. doi:10.1128/MMBR.63.4.968-989.1999. Shams MK, Khadivi A. Mechanisms of salinity tolerance and their possible application in the breeding of vegetables. BMC Plant Biol. 2023;23(1):139. doi:10.1186/s12870-023-04152-8. Bremner JM. Nitrogen—Total. In: Sparks DL, editor. Methods of Soil Analysis: Part 3 Chemical Methods. Soil Science Society of America; 1996:1085-1121. Liu Y S. Effects of forage reseeding on plant-soil ecological stoichiometric characteristics in degraded grasslands [PhD Dissertation]. Northwest A&F University, 2023. doi:10.27409/d.cnki.gxbnu.2023.000752. Mu C, et al. Response of Extracellular Enzyme Stoichiometric Properties and Microbial Metabolic Limitations to the Ecosystem Transition Mode Employed in Red Jujube Economic Forests on the Loess Plateau. Microorganisms. 2025,13(4):729. doi: 10.3390/microorganisms13040729. Ivanova RP, et al. The Solubilization of Rock Phosphate by Organic Acids. Crit Rev Environ Sci Technol. 2006;36(4):2541-2554. doi:10.1080/10426500600758399. Li R, et al. Amelioration of saline-alkali soil using aluminum sulfate: Effects on soil properties and crop growth. J Environ Manage. 2018;215:1-8. doi:10.1016/j.jenvman.2018.03.042. Yu YF, et al. Stoichiometric characteristics of plant and soil C, N and P in different forest types in depressions between karst hills, southwest China. Acta Ecol Sin. 2014;34(4):947-954. doi:10.1007/s12665-014-3553-6. Luo X, et al. Nitrogen:Phosphorus Supply Ratio and Allometry in Five Alpine Plant Species. Ecol Evol. 2017;7(21):8905-8915. doi:10.1002/ece3.2587. Pan Y, et al. Global patterns of nitrogen and phosphorus resorption efficiency in terrestrial ecosystems. Ecol Lett. 2015;18(12):1395-1404. doi:10.1111/ele.12515. Larrainzar E, et al. Hemoglobins in the legume–Rhizobium symbiosis. New Phytol. 2020;228(2):472-484. doi:10.1111/nph.16673. Elser JJ, et al. Organism size, life history, and N:P stoichiometry: Toward a unified view of cellular and ecosystem processes. Bioscience. 1996;46(9):674-684. doi:10.2307/1312897. Jiaping L, Wenjuan S. Cotton/halophytes intercropping decreases salt accumulation and improves soil physicochemical properties and crop productivity in saline-alkali soils under mulched drip irrigation: A three-year field experiment. Field Crops Res. 2021;262:108027. doi: 10.1016/j.fcr.2020.108027. Wang X, et al. Nitrogen and phosphorus addition alter leaf nutrient concentrations of dominant grass species and regulate primary productivity in Inner Mongolia meadow steppe. Grassland Res. 2023;6(1):100126. doi:10.1016/j.grs.2023.100126. Cai Z, et al. Sulfur cycling in acid sulfate soils: A review. Geoderma. 2020;375:114574. doi:10.1016/j.geoderma.2020.114574. Jēkabsone A, et al. Dependence on Nitrogen Availability and Rhizobial Symbiosis of Different Accessions of Trifolium fragiferum, a Crop Wild Relative Legume Species, as Related to Physiological Traits. Plants. 2022;11(9):1141. doi:10.3390/plants11091141. Hu Y, et al. Storage of C, N, and P affected by afforestation with Salix cupularis in an alpine semiarid desert ecosystem. Land Degrad Dev. 2018;29(1):188-198. doi:10.1002/ldr.2862. Wang X, et al. Nitrogen and phosphorus addition alter leaf nutrient concentrations of dominant grass species and regulate primary productivity in Inner Mongolia meadow steppe. Grassland Res. 2023;6(1):100126. doi:10.1016/j.grs.2023.100126. Batjes NH. Total carbon and nitrogen in the soils of the world. Eur J Soil Sci. 1996;47(2):151-163. doi:10.1111/j.1365-2389.1996.tb01386.x. Wang Y, et al. Influence of Alfalfa Planting Years on Soil Carbon Sequestration and Enzyme Activity in Saline-Alkali Soils of the Songnen Plain. Sustainability. 2023;15(14):10772. doi:10.3390/su151410772. McGroddy ME, et al. Scaling of C:N:P stoichiometry in forests worldwide: Implications of terrestrial Redfield-type ratios. Ecology. 2004;85(9):2390-2401. doi:10.1890/03-0240. Mooshammer M, et al. Adjustment of microbial nitrogen use efficiency to carbon:nitrogen imbalances regulates soil nitrogen cycling. Nat Commun. 2014;5:3694. doi:10.1038/ncomms4694. Jēkabsone A, et al. Dependence on Nitrogen Availability and Rhizobial Symbiosis of Different Accessions of Trifolium fragiferum, a Crop Wild Relative Legume Species, as Related to Physiological Traits. Plants. 2022;11(9):1141. doi:10.3390/plants11091141. Bohn HL, McNeal BL, O'Connor GA. Soil Chemistry. Wiley; 2001. doi:10.1002/0471224437. Zhu JK. Salt Tolerance. Curr Biol. 2016;26(16):R706-R710. doi:10.1016/j.cub.2016.06.047. Tables Table 1 and 2 are available in the Supplementary Files section. Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8299360","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":560325285,"identity":"bc3d0e6a-b24f-4614-acaf-33d22c7da97a","order_by":0,"name":"Anni Bai","email":"","orcid":"","institution":"Heilongjiang Bayi Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Anni","middleName":"","lastName":"Bai","suffix":""},{"id":560325286,"identity":"d3800247-ceb1-4edd-919a-5ef2d4ef2e7e","order_by":1,"name":"MingCong Zhang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5ElEQVRIiWNgGAWjYDACCSSSgaFCQk6eRC1nLIwNG4jTAgWMbRWJDAcI6JCf3fzs4dccizx598PHHn6dJ5HA2MD88NENPFoY5xwzN5bdJlFseCYtHcTIY2dgMzbOwaOFWSLBTFpym0Tixhk8YEYxYwMPmzQ+LWwS6d+QtMyRSGw4QEALj0SOmeRHoJb5EjxARgMRWiQkcsqkGYFaNvCkpUkzHJMwNmwm4Bf5GenbJH9uq0uc3374mOSPmjo5efbmh4/xaQEBZh4gYXAAymBgJqAcBBh/gKxrgDJGwSgYBaNgFKADAMlKRasne1UQAAAAAElFTkSuQmCC","orcid":"","institution":"Heilongjiang Bayi Agricultural University","correspondingAuthor":true,"prefix":"","firstName":"MingCong","middleName":"","lastName":"Zhang","suffix":""},{"id":560325287,"identity":"f65329cc-c17d-412e-a669-d58b49f296aa","order_by":2,"name":"Chen Wang","email":"","orcid":"","institution":"Heilongjiang Bayi Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Chen","middleName":"","lastName":"Wang","suffix":""},{"id":560325288,"identity":"d1ed4650-b040-40a4-88ba-ac59129a1e0c","order_by":3,"name":"HanShuo Zhang","email":"","orcid":"","institution":"Heilongjiang Bayi Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"HanShuo","middleName":"","lastName":"Zhang","suffix":""},{"id":560325293,"identity":"dd3b9951-1da2-4124-848d-e44737f072e6","order_by":4,"name":"Mingfen Shan","email":"","orcid":"","institution":"Heilongjiang Bayi Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Mingfen","middleName":"","lastName":"Shan","suffix":""},{"id":560325297,"identity":"cbc50c77-1c69-42b7-96f9-8a9221e6888b","order_by":5,"name":"Wei Zhou","email":"","orcid":"","institution":"Daqing Qilong Agricultural Science and Technology Limited Company","correspondingAuthor":false,"prefix":"","firstName":"Wei","middleName":"","lastName":"Zhou","suffix":""},{"id":560325298,"identity":"ade8d5f6-d411-43c2-9ec3-7ec5a71f7440","order_by":6,"name":"Shanmin Qu","email":"","orcid":"","institution":"Heilongjiang Bayi Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Shanmin","middleName":"","lastName":"Qu","suffix":""}],"badges":[],"createdAt":"2025-12-07 11:08:21","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8299360/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8299360/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":98344964,"identity":"218fb0bf-285d-46c4-ae90-2e10d396637f","added_by":"auto","created_at":"2025-12-16 18:39:18","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2660522,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of aluminum sulfate doses on TOC content (Figure 1A), TN content (Figure 1B), and TP content (Figure 1C) in alfalfa stems. CK: 0 kg·hm\u003csup\u003e-2\u003c/sup\u003e (control); CL1: 24 kg·hm\u003csup\u003e-2\u003c/sup\u003e; CL2: 48 kg·hm\u003csup\u003e-2\u003c/sup\u003e; CL3: 72 kg·hm\u003csup\u003e-2\u003c/sup\u003e. Lowercase letters indicate significant differences at (\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05), and uppercase letters indicate significant differences at (\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.01).\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-8299360/v1/c294493adf77ed8eca9b7086.png"},{"id":98438046,"identity":"1f0ec107-41e2-4161-8fd0-518b18d2f193","added_by":"auto","created_at":"2025-12-17 16:58:33","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2450727,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of aluminum sulfate doses on C/N ratio (Figure 2A), C/P ratio (Figure 2B), and N/P ratio (Figure 2C) in alfalfa stems. Treatments as in Figure 1. Lowercase letters indicate significant differences at (\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05), and uppercase letters indicate significant differences at (\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.01).\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-8299360/v1/169b29060f318ada48a89faa.png"},{"id":98344970,"identity":"d60ad6c5-bdf8-47ef-8fca-9387ab436baf","added_by":"auto","created_at":"2025-12-16 18:39:18","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":4182039,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of aluminum sulfate doses on soil C content (Figure 3A), TN content (Figure 3B), and TP content (Figure 3C) at 0–10 cm, 10–20 cm, and 20–30 cm depths. Treatments as in Figure 1. Different lowercase letters indicate significant differences between different soil layers of the same treatment (\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05) and different uppercase letters indicate significant differences between different treatments of the same soil layer (\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05)\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-8299360/v1/8579fd450f1141090b13a43c.png"},{"id":98344968,"identity":"6057bdc3-4735-433d-8ba7-460a4f6ef743","added_by":"auto","created_at":"2025-12-16 18:39:18","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":4062993,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of aluminum sulfate doses on soil C/N ratio (Figure 4A), C/P ratio (Figure 4B), and N/P ratio (Figure 4C) at 0–10 cm, 10–20 cm, and 20–30 cm depths. Treatments as in Figure 1. Different lowercase letters indicate significant differences between different soil layers of the same treatment (\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05) and different uppercase letters indicate significant differences between different treatments of the same soil layer (\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05)\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-8299360/v1/cdd31cc63ce1615745a82e7f.png"},{"id":98438750,"identity":"203bcd34-bc40-42ca-a5de-4d0f3ab8d4e7","added_by":"auto","created_at":"2025-12-17 16:59:59","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2307659,"visible":true,"origin":"","legend":"\u003cp\u003eThe linear regression relationship between soil C and soil N (Figure 5A), soil C and soil P (Figure 5B), alfalfa C and alfalfa N (Figure 5C) and alfalfa C and alfalfa P (Figure 5D) in the 0-30cm soil layer treated with aluminum sulfate.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-8299360/v1/c26318c604d3044feeb039df.png"},{"id":98344973,"identity":"b9f67a72-45a3-4f5f-bf60-7f5f3ce1f746","added_by":"auto","created_at":"2025-12-16 18:39:18","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":5170473,"visible":true,"origin":"","legend":"\u003cp\u003eThe linear regression relationship between soil C and C/N (Figure 6A), N and C/N (Figure 6B), soil P and C/P (Figure 6C), soil C and C/P Figure 6D), soil N and N/P (Figure 6E) and soil P and N/P (Figure 6F) in the 0-30cm soil layer after aluminum sulfate treatment.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-8299360/v1/cd1a0fa48182309d0fae1409.png"},{"id":98344972,"identity":"e84e33e0-992b-4fe1-a147-e4d186ee061a","added_by":"auto","created_at":"2025-12-16 18:39:18","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":3870797,"visible":true,"origin":"","legend":"\u003cp\u003eCorrelation network of C-N-P stoichiometric characteristics in the alfalfa-soil system. Red/blue lines indicate positive/negative correlations. The thickness of the line indicates the strength of the correlation, and the asterisk indicates statistical significance (\u003csup\u003e*\u003c/sup\u003e \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e**\u003c/sup\u003e \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, \u003csup\u003e***\u003c/sup\u003e \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001). S.C: soil carbon; S.N: soil nitrogen; S.P: soil phosphorus; S.CN: soil C:N; S.CP: soil C:P; S.NP: soil N:P; A.C: alfalfa carbon; A.N: alfalfa nitrogen; A.P: alfalfa phosphorus; A.CN: alfalfa C:N; A.CP: alfalfa C:P; A.NP: alfalfa N:P; EC: electrical conductivity; BD: bulk density.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-8299360/v1/9c3bd335144213204b6da193.png"},{"id":98438078,"identity":"25ae799f-6b7d-4af0-82eb-2e2a8934e807","added_by":"auto","created_at":"2025-12-17 16:58:37","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1454247,"visible":true,"origin":"","legend":"\u003cp\u003eRedundancy analysis (RDA) of C-N-P stoichiometric characteristics and environmental factors. S.C: soil carbon; S.N: soil nitrogen; S.P: soil phosphorus; S.CN: soil C:N; S.CP: soil C:P; S.NP: soil N:P; A.C: alfalfa carbon; A.N: alfalfa nitrogen; A.P: alfalfa phosphorus; A.CN: alfalfa C:N; A.CP: alfalfa C:P; A.NP: alfalfa N:P; EC: electrical conductivity; BD: bulk density. Arrows indicate direction and strength of variable contributions. Treatments: CK (black), CL1 (blue), CL2 (red), CL3 (green). RDA1 and RDA2 explain 79.31% and 2.72% of variance, respectively. (\u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"Figure8.png","url":"https://assets-eu.researchsquare.com/files/rs-8299360/v1/62bf98243c36018f5e9a8674.png"},{"id":98439719,"identity":"b6837184-090c-48f4-8bbe-9f4898fcab04","added_by":"auto","created_at":"2025-12-17 17:02:49","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":1892582,"visible":true,"origin":"","legend":"\u003cp\u003eStructural equation models (SEM) illustrating aluminum sulfate regulation on C-N-P coupling in soil-alfalfa systems at 0–10 cm (A), 10–20 cm (B), and 20–30 cm (C) depths. Solid/dashed arrows indicate positive/negative pathways (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05). Arrow width reflects standardized path coefficients. GFI: Goodness-of-Fit Index. AP: available P; EC: electrical conductivity\u003c/p\u003e","description":"","filename":"Figure9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8299360/v1/b3d6b7b2fc1ee16181e5e31b.jpg"},{"id":98774643,"identity":"526e35d8-5531-48be-869b-b89cce0d6684","added_by":"auto","created_at":"2025-12-22 12:07:36","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":25031531,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8299360/v1/37838626-22ad-4db3-bbf8-4722c15819fa.pdf"},{"id":98344962,"identity":"9c2cdb82-8d41-48e3-b627-1d6ea1e7e4ed","added_by":"auto","created_at":"2025-12-16 18:39:18","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":12597,"visible":true,"origin":"","legend":"","description":"","filename":"Table2.docx","url":"https://assets-eu.researchsquare.com/files/rs-8299360/v1/2e78b8d3de9f6bfe54a1b454.docx"},{"id":98437688,"identity":"6f813df7-5fd5-4aa4-b27d-fda7c5284e73","added_by":"auto","created_at":"2025-12-17 16:57:34","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":14729,"visible":true,"origin":"","legend":"","description":"","filename":"Table1.docx","url":"https://assets-eu.researchsquare.com/files/rs-8299360/v1/daae8d1275d4a1c34478f02d.docx"},{"id":98344967,"identity":"d04c81db-8772-4f1a-a124-db1a3b364177","added_by":"auto","created_at":"2025-12-16 18:39:18","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":15664,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformation.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8299360/v1/2052e8de0579d04187c9f075.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Phosphorus Improves Stoichiometric Characteristics of Saline-Sodic Soil and Alfalfa","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eSaline-sodic soil is one of the most widely distributed soil types globally, with over 100 countries and regions currently facing salinization issues\u003csup\u003e[1]\u003c/sup\u003e,Among global saline-sodic distribution areas, the Songnen Plain was one of the highest rates of ecological degradation, leading to declining land productivity, biodiversity loss, groundwater pollution, and other ecological problems. These issues have become major obstacles to sustainable agricultural development\u003csup\u003e[2]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThe high alkalinity and sodicity in saline-sodic soils cause severe structural degradation characterized by enhanced colloidal dispersion, soil compaction, reduced porosity (\u0026lt;30%), and impaired hydraulic conductivity\u003csup\u003e[3]\u003c/sup\u003e。Concurrently, saline-sodic soils exhibit low nutrient availability\u003csup\u003e[4]\u003c/sup\u003e. Under salt stress, plant roots experience osmotic imbalance and ion toxicity, leading to stomatal closure, impaired photosynthesis, and leaf oxidative stress\u003csup\u003e[5\u003c/sup\u003e\u003csup\u003e-\u003c/sup\u003e\u003csup\u003e6]\u003c/sup\u003e, ultimately causing crop yield reductions\u003csup\u003e[7\u003c/sup\u003e\u003csup\u003e-\u003c/sup\u003e\u003csup\u003e8]\u003c/sup\u003e.Therefore, enhancing nutrient availability and the productive capacity of saline-sodic soil through amelioration measures has become a central issue for achieving the sustainable utilization of saline-sodic soil resources\u003csup\u003e[9]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eBiological and chemical amelioration are two prominent technologies for managing saline-sodic soil. Biological amelioration facilitates soil desalination by planting salt-tolerant plants or through microbial inoculation, offering advantages such as low cost and environmental sustainability\u003csup\u003e[10]\u003c/sup\u003e. For instance, the introduction of salt-tolerant forage crops in Xinjiang's saline regions has resulted in a 20% to 30% reduction in surface soil salinity and a 15% increase in organic matter content\u003csup\u003e[11]\u003c/sup\u003e. However, biological amelioration is characterized by a lengthy process, limited effectiveness in restoring severely saline-sodic soils\u003csup\u003e[12]\u003c/sup\u003e. In contrast, chemical amelioration quickly modifies soil physical and chemical properties by adding acidic substances making it an effective strategy for enhancing productivity in the short term\u003csup\u003e[13]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eAluminum sulfate (Al\u003csub\u003e2\u003c/sub\u003e(SO\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e3\u003c/sub\u003e) functions as a typical acidic ameliorant through several mechanisms: (1) it releases H\u003csup\u003e+\u003c/sup\u003e ions to neutralize soil alkalinity and lower pH; (2) it replaces Na\u003csup\u003e+\u003c/sup\u003e ions on soil colloids, thereby enhancing aggregate structure; and (3) sulfate ions (SO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e2-\u003c/sup\u003e) react with carbonates to form soluble salts, which promotes salt leaching\u003csup\u003e[14\u003c/sup\u003e\u003csup\u003e-15]\u003c/sup\u003e. Studies have demonstrated that a single application of aluminum sulfate can reduce soil pH from 9.2 to 8.0 and decrease the sodium adsorption ratio (SAR) by 40%\u003csup\u003e[16\u003c/sup\u003e\u003csup\u003e-17]\u003c/sup\u003e. However, the use of single ameliorants can easily lead to elemental imbalances, and excessive application of aluminum sulfate may inhibit P availability\u003csup\u003e[18\u003c/sup\u003e\u003csup\u003e-19]\u003c/sup\u003e. Existing research has primarily concentrated on the effects of aluminum sulfate on the physical properties of soil, while the regulatory mechanisms affecting C-N-P stoichiometric characteristics remain unclear\u003csup\u003e[20]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eTraditional soil research has primarily focused on the isolated analysis of individual elements, such as N and P. However, the cycling of C, N, and P in saline-sodic soils is highly interconnected, and their stoichiometric ratios (C:N:P) can systematically reveal mechanisms of nutrient limitation and utilization efficiency\u003csup\u003e[21]\u003c/sup\u003e. Ecological stoichiometry, which originated from the Redfield ratio (C:N:P = 106:16:1) in marine ecosystems, has been adapted to terrestrial ecosystems and has become a fundamental tool for studying elemental balance and energy flow\u003csup\u003e[22]\u003c/sup\u003e. Its key advantages include: N/P ratios that can determine the threshold for plant N or P limitation (N/P \u0026lt; 14 indicates N limitation, while N/P \u0026gt; 16 indicates P limitation)\u003csup\u003e[23]\u003c/sup\u003e; increasing C/N or C/P ratios that suggest slow organic matter decomposition and enhanced nutrient retention\u003csup\u003e[24]\u003c/sup\u003e; and the ability to evaluate the long-term effects of amelioration measures through correlation models between elemental ratios and environmental factors, such as pH and electrical conductivity\u003csup\u003e[25]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eIn saline-sodic soil amelioration, ecological stoichiometry demonstrates distinct advantages. P is often fixed by calcium and iron ions, leading to P limitation for its growth\u003csup\u003e[26]\u003c/sup\u003e.\u0026nbsp;Zhao and Niu et al. found that gypsum application significantly decreased soil C/P ratios while enhancing\u0026nbsp;P\u0026nbsp;availability and moderately increasing crop yield\u003csup\u003e[27\u003c/sup\u003e\u003csup\u003e-29]\u003c/sup\u003eHowever, existing studies primarily focus on the effects of single ameliorants on certain elements, lacking systematic analysis of C-N-P synergistic changes.Moreover, the interaction effects between salt-tolerant plants (such as alfalfa) and chemical ameliorants are rarely reported, yet this interaction is crucial for optimizing improvement strategies\u003csup\u003e[30]\u003c/sup\u003e。\u003c/p\u003e\n\u003cp\u003eAlfalfa (\u003cem\u003eMedicago sativa\u003c/em\u003e), a leguminous perennial forage, is widely utilized for the restoration of saline-sodic land due to its remarkable salt tolerance (withstanding electrical conductivity levels of up to 8 dS/m) and high biomass production (annual hay yield of 10-15 tons per hectare)\u003csup\u003e[32\u003c/sup\u003e\u003csup\u003e-33]\u003c/sup\u003e. The mechanisms underlying its salt tolerance include: (1) the root secretion of organic acids (such as malic acid) that chelate sodium ions (Na\u003csup\u003e+\u003c/sup\u003e) and mitigate ion toxicity; (2) the synthesis of osmoregulatory substances (such as proline) to maintain cellular water potential; and (3) symbiotic N fixation with rhizobia to alleviate soil N deficiency\u003csup\u003e[34\u003c/sup\u003e\u003csup\u003e-35]\u003c/sup\u003e. Planting alfalfa in moderately saline-sodic soil (pH 8.5-9.0) in the Songnen Plain has resulted in a 20% increase in SOC content and a 15% reduction in surface salinity\u003csup\u003e[36]\u003c/sup\u003e. However, alfalfa has high P requirements, with tissue P content needing to reach 0.2%-0.4%.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThis study focuses on saline-sodic soils in the Songnen Plain, employing ecological stoichiometry to investigate aluminum sulfate's effects on C-N-P stoichiometric ratios in both soil and alfalfa. The research objectives are:\u003c/strong\u003e\u003c/p\u003e\n\u003col\u003e\n \u003cli\u003e\u003cstrong\u003eQuantify the roles of C/P and N/P ratios in nutrient limitation and use efficiency\u003c/strong\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eAnalyze synergistic effects among multiple elements to elucidate how modulating C-N-P balance enhances soil structure and alfalfa nutrient uptake when combined with soil amendments\u003c/strong\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003e\u003cstrong\u003e(iii) Determine optimal application rates to refine remediation strategies, providing a scientific basis for precision amendment and ecological restoration of\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003esaline-sodic\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;lands.\u003c/strong\u003e\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003e2.1 Materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe experimental site was located at Yinlang Ranch, Daqing City, Heilongjiang Province, China (46°30′0.2″N, 124°50′4.7″E), which is part of the Songnen Plain and has an altitude of 149 meters.The alfalfa (\u003cem\u003eMedicago sativa\u0026nbsp;L\u003c/em\u003e.) used in this study was the salt-tolerant cultivar “Gongnong 5”. The plants were grown under field conditions and sampled during the mid-growth stage in mid-August 2024. The soil type was classified as saline-sodic soil.\u0026nbsp;\u003cstrong\u003eThe soil was classified as\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003esaline-sodic\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;soil. A randomized complete block design was implemented with 10 plots (7 m × 3 m each). In mid-August 2024, soil and plant samples were collected during the mid-growth stage, avoiding fertilizer application zones.\u003c/strong\u003e Three 1 m × 1 m subplots were randomly designated within each experimental area, and five sampling points were selected using an S-shaped sampling pattern in each subplot to collect soil samples at three depths: 0–10 cm, 10–20 cm, and 20–30 cm. Following pretreatment and air-drying, soil samples were ground through a 2 mm sieve and stored. Alfalfa stems were processed by grinding with a mechanical mill, sieving, and packaging in self-sealing bags for subsequent analysis.\u003c/p\u003e\n\u003cp\u003eThe basic physical and chemical properties of the soil samples are presented in\u0026nbsp;Table 1\u003c/p\u003e\n\u003cp\u003eTable\u0026nbsp;1\u0026nbsp;Basic physical and chemical properties of soil before planting alfalfa\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.1.1 Soil Parameter Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe experimental treatment scheme is presented in\u0026nbsp;Table 2. To ensure representativeness and accuracy during sampling, soil samples were collected at various depths using a soil auger after removing surface litter. Soil samples from three quadrants were combined by layer, with roots and large stones removed. The samples were thoroughly mixed and quartered to obtain composite samples, which were labeled and transported to the laboratory. The samples were then ground and sieved to determine the contents of soil C, N, and P. Nutrient content analysis was conducted using standard methods: Soil organic carbon (SOC) was determined using the external-heat potassium dichromate titration method. Total nitrogen (TN) was quantified by the Kjeldahl method (hd-kn10 analyzer, HORDE ELECTRIC). Total phosphorus (TP) was measured via the molybdenum blue spectrophotometric method (721 spectrophotometer, Shanghai Jinghua). Nitrate N was analyzed by phenol disulfonic acid colorimetry. Available P was extracted with sodium bicarbonate and determined by molybdenum blue spectrophotometry. Exchangeable potassium was extracted with ammonium acetate and quantified using flame photometry (FP6400, Bona Technology). Soil pH was measured potentiometrically (Sartorius PB-10 pH meter, Germany) at a 1:2.5 soil:water ratio\u003csup\u003e[36]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eTable\u0026nbsp;2\u0026nbsp;Test treatment scheme\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.1.2 Alfalfa Parameter Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePlant total organic carbon (TOC) was determined using the potassium dichromate (K₂Cr₂O₇) wet oxidation method. Approximately 0.200 g of oven-dried plant sample was digested at 180°C for 5 minutes in an oil bath, followed by titration with ferrous sulfate (FeSO₄). TN and TP contents were analyzed using a continuous flow analyzer (QC8500, HACH). Samples (~0.200 g) were digested in 100 mL tubes with 5 mL concentrated H₂SO\u003csub\u003e4\u003c/sub\u003e at 240°C for 1 hour, then at 380°C with intermittent additions of 30% H₂O₂ until the digest became colorless/clear. Digests were diluted to 50 mL with deionized water and analyzed for TN and TP content via continuous flow analysis using appropriate calibration standards and reagent blanks\u003csup\u003e[37]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2 Statistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData calculations and variance analyses (\u003cem\u003eP\u003c/em\u003e = 0.05) were performed using Excel 2019 and SPSS version 24.0. Post-hoc tests were conducted using Duncan's multiple comparison method. Origin 2021 was utilized for plotting. Redundancy analysis (RDA) was carried out using the vegan package in R, with visualizations created using the ggplot2 package. Additionally, structural equation modeling (SEM) was constructed using the plspm package in R, employing the least squares method. Model fit was evaluated using fitness indices (CFI/TLI \u0026gt; 0.9, RMSEA \u0026lt; 0.08).\u003c/p\u003e"},{"header":"3. Results","content":"\u003cp\u003e\u003cstrong\u003e3.1 Ecological chemical metering of alfalfa\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.1.1 Effects of aluminum sulfate application on C, N and P content in alfalfa stems\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDifferent applications of aluminum sulfate had specific effects on N and P levels in alfalfa (Figure 1). The N and P concentrations in the CL2 treatment were significantly higher than those in the other treatments (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, Figures 1A and 1B). Compared to the CK, the N content increased by 41.81%, while the P content rose by 15.27%. However, varying doses of aluminum sulfate did not have significant effects on the C content of alfalfa (Figure 1C). The CL3 treatment exhibited the highest C content at 314.13 g·kg\u003csup\u003e-1\u003c/sup\u003e, representing an increase of 14.36% compared to the control.\u003c/p\u003e\n\u003cp\u003eFigure\u0026nbsp;1\u0026nbsp;Effects of aluminum sulfate doses on TOC content (Figure 1A), TN content (Figure 1B), and TP content (Figure 1C) in alfalfa stems. CK: 0 kg·hm\u003csup\u003e-2\u003c/sup\u003e (control); CL1: 24 kg·hm\u003csup\u003e-2\u003c/sup\u003e; CL2: 48 kg·hm\u003csup\u003e-2\u003c/sup\u003e; CL3: 72 kg·hm\u003csup\u003e-2\u003c/sup\u003e. Lowercase letters indicate significant differences at (\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05), and uppercase letters indicate significant differences at (\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.01).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.1.2 Effects of aluminum sulfate application on C-N-P chemical characteristics of alfalfa rhizomes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAlfalfa's C, N, and P contents varied under different applications, resulting in significant differences in their ecological stoichiometric characteristics (Figure 2\u0026nbsp;). The C/N ratio in the CK was significantly higher than in the treatments CL1, CL2, and CL3 (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, Figure 2A). Specifically, the C/N ratio in CK was 34.04% higher compared to CL2. An excessively high C/N ratio may negatively impact the rate of organic matter decomposition in plants. In terms of the C/P ratio, CL2 exhibited a significantly lower value than the other treatments (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, Figure 2B), decreasing by 6.39% compared to CK, which is contrary to the results observed for the N/P ratio (Figure 2C).\u003c/p\u003e\n\u003cp\u003eFigure\u0026nbsp;2 Effects of aluminum sulfate doses on C/N ratio (Figure 2A), C/P ratio (Figure 2B), and N/P ratio (Figure 2C) in alfalfa stems. Treatments as in Figure 1. Lowercase letters indicate significant differences at (\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05), and uppercase letters indicate significant differences at (\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.01).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2 Ecological chemical metering of soil\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2.1 Effects of aluminum sulfate application on soil C, N and P content\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDifferent applications of aluminum sulfate influenced the concentrations of C, N, and P in the 0-30 cm soil layer (Figure 3). In the 0-10 cm layer, the C content in the CL3 treatment was significantly higher than in the other treatments (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, Figure 3A), showing an increase of 64% compared to the CK At an application rate of 48 kg·hm\u003csup\u003e-2\u003c/sup\u003e, the C content in the 10-20 cm layer was significantly greater by 6.9% compared to the 20-30 cm layer (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e\n\u003cp\u003eDifferent applications of aluminum sulfate also influenced soil N content. At various depths, the N content in CL3 was significantly higher than in CL2, CL1, and CK (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, Figure 3B). Soil N was concentrated in the 10-30 cm layer, with lower concentrations in the surface layer, which is contrary to the distribution of P.\u003c/p\u003e\n\u003cp\u003eIn the soil layer of 20 to 30 cm, the P content of CL1 reached 2.93 g·kg\u003csup\u003e-1\u003c/sup\u003e, significantly higher than that of CK. In addition, the P content of CL1 was 7.33% and 9.12% higher than that of CL2 and CL3 respectively (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, see Figure 3C).\u003c/p\u003e\n\u003cp\u003eFigure\u0026nbsp;3\u0026nbsp;Effects of aluminum sulfate doses on soil C content (Figure 3A), TN content (Figure 3B), and TP content (Figure 3C) at 0–10 cm, 10–20 cm, and 20–30 cm depths. Treatments as in Figure 1. Different lowercase letters indicate significant differences between different soil layers of the same treatment (\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05) and different uppercase letters indicate significant differences between different treatments of the same soil layer (\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2.2 Effects of aluminum sulfate application on C-N-P chemical characteristics of soil\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs illustrated in Figure 4, the ratios of C/N, C/P, and N/P generally decreased with increasing soil depth. The C/N ratio in CL1 and CL2 was significantly higher than that in CK and CL3 (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, Figure 4A); however, no significant difference was observed between CL1 and CL2. In the 10-20 cm soil layer, the C/N ratio in CL3 was lower, showing a decrease of 12.87% compared to CK at the same depth.\u003c/p\u003e\n\u003cp\u003eAfter the application of aluminum sulfate, the C/P ratio in the soil was generally lower than that of the CK, with significant differences observed in the 0-10 cm layer (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, Figure 4B). In the 10-20 cm layer, the C/P ratio in the CK was 66.73% higher than that in CL1, 64.18% higher than in CL2, and 65.91% higher than in CL3. Significant differences were noted among the depths within the same treatment (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e\n\u003cp\u003eDifferent doses resulted in varying N/P ratios due to differing inputs of N and P. Compared to the CK, treatments CL1 and CL2 reduced the N/P ratio by 36.81% and 49.71%, respectively, in the surface layer, with more pronounced differences observed in the 10-30 cm layer. No significant differences were found among depths under the same treatment (Figure 4C).\u003c/p\u003e\n\u003cp\u003eFigure\u0026nbsp;4\u0026nbsp;Effects of aluminum sulfate doses on soil C/N ratio (Figure 4A), C/P ratio (Figure 4B), and N/P ratio (Figure 4C) at 0–10 cm, 10–20 cm, and 20–30 cm depths. Treatments as in Figure 1. Different lowercase letters indicate significant differences between different soil layers of the same treatment (\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05) and different uppercase letters indicate significant differences between different treatments of the same soil layer (\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3 Regression analysis of C\u003c/strong\u003e\u003cstrong\u003e、\u003c/strong\u003e\u003cstrong\u003eN\u003c/strong\u003e\u003cstrong\u003e、\u003c/strong\u003e\u003cstrong\u003eP\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDifferent applications of aluminum sulfate demonstrated strong linear relationships with average soil nutrients in the 0-30 cm layer (Figure 5). Soil C exhibited the highest correlation with P. As C levels increased, both N and P also rose, indicating positive correlations. Similar trends were observed in the linear regression analysis of alfalfa nutrients.\u003c/p\u003e\n\u003cp\u003eFigure\u0026nbsp;5\u0026nbsp;The linear regression relationship between soil C and soil N (Figure 5A), soil C and soil P (Figure 5B), alfalfa C and alfalfa N (Figure 5C) and alfalfa C and alfalfa P (Figure 5D) in the 0-30cm soil layer treated with aluminum sulfate.\u003c/p\u003e\n\u003cp\u003eAs illustrated in Figure 6, C exhibited a positive correlation with the C/N ratio and a negative correlation with the C/P ratio. N demonstrated a negative correlation with both the C/N and N/P ratios, although these correlations were weaker in terms of ecological stoichiometry. In contrast, P displayed stronger correlations with the C/P and N/P ratios (R\u003csup\u003e2\u003c/sup\u003e = 0.8993 and 0.8375, respectively), indicating negative trends.\u003c/p\u003e\n\u003cp\u003eFigure\u0026nbsp;6\u0026nbsp;The linear regression relationship between soil C and C/N (Figure 6A), N and C/N (Figure 6B), soil P and C/P (Figure 6C), soil C and C/P Figure 6D), soil N and N/P (Figure 6E) and soil P and N/P (Figure 6F) in the 0-30cm soil layer after aluminum sulfate treatment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4 Correlations with the C:N:P stoichiometry\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs illustrated in Figure 7, the alfalfa-soil system demonstrated a highly significant positive correlation between soil C and P (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01). This finding suggests a synergistic accumulation or mutual enhancement of these two components within the soil. Additionally, both soil C and P exhibited significant positive correlations with alfalfa P content (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05), indicating that improved soil P availability directly facilitates alfalfa's P absorption. The soil C/P and N/P were significantly negatively correlated with soil pH, BD, and EC (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05). Furthermore, pH showed a highly significant negative correlation with alfalfa P (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01), suggesting that elevated alkalinity inhibits alfalfa's P absorption. The N and P in alfalfa were significantly positively correlated (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05), indicating a synergistic absorption or functional coupling between these nutrients. The alfalfa N/P ratio positively correlated with both N and P, suggesting that the N/P ratio reflects the plant's balanced demand for N and P nutrients.\u003c/p\u003e\n\u003cp\u003eFigure\u0026nbsp;7\u0026nbsp;Correlation network of C-N-P stoichiometric characteristics in the alfalfa-soil system. Red/blue lines indicate positive/negative correlations. The thickness of the line indicates the strength of the correlation, and the asterisk indicates statistical significance (\u003csup\u003e*\u003c/sup\u003e \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e**\u003c/sup\u003e \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, \u003csup\u003e***\u003c/sup\u003e \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001). S.C: soil carbon; S.N: soil nitrogen; S.P: soil phosphorus; S.CN: soil C:N; S.CP: soil C:P; S.NP: soil N:P; A.C: alfalfa carbon; A.N: alfalfa nitrogen; A.P: alfalfa phosphorus; A.CN: alfalfa C:N; A.CP: alfalfa C:P; A.NP: alfalfa N:P; EC: electrical conductivity; BD: bulk density.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.5 Redundancy Analysis of C:N:P stoichiometry\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eChanges in the soil environment and the application of soil amendments significantly influenced the stoichiometric characteristics of C, N, and P in both the soil and alfalfa (Figure 8). Redundancy analysis (RDA) revealed that amendments and soil properties collectively accounted for 82.03% of the total variation in the data, with RDA1 (79.31%) and RDA2 (2.72%) serving as the principal ordination axes. Soil C/N exhibited the strongest positive correlation with RDA1, identifying it as the primary positive factor driving the effects of soil amendments. Conversely, alfalfa C/P demonstrated a significant negative correlation with RDA1, indicating its role as a secondary negative factor reflecting the plant's adaptive adjustments in C/P balance. Additionally, soil N showed a negative correlation with RDA2, suggesting interactive effects between soil N and alfalfa C/P. Alfalfa P displayed a positive correlation with RDA1 but negative correlations with pH and EC (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001), confirming P as a key driver.\u003c/p\u003e\n\u003cp\u003eSoil C and P exhibited significant positive correlations along the first axis of the redundancy analysis (RDA1) (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001), supporting the \"C-P co-accumulation\" hypothesis. Conversely, soil N/P ratios correlated negatively with RDA1, indicating that amendments alleviated N limitation by regulating the N/P balance. The CL2 treatment was positioned in the positive direction of RDA1, demonstrating significant positive correlations with soil C/N ratios and alfalfa P, which suggests its effectiveness in enhancing the soil C-N balance and P uptake through reductions in pH and EC.\u003c/p\u003e\n\u003cp\u003eSoil C/P ratios exhibited negative correlations with the CL2 treatment, further supporting its role in enhancing P bioavailability. The CK group clustered along the negative axis of RDA1, which is associated with high C/N ratios, elevated pH, and EC, all indicative of soil degradation. Although CL3 showed a positive correlation with RDA1, it deviated from the main trend, potentially due to aluminum toxicity resulting from high amendment doses that inhibit root growth.\u003c/p\u003e\n\u003cp\u003eAlfalfa N/P ratios correlated positively with the first axis of redundancy analysis (RDA1), supporting its role as an indicator of N limitation. Additionally, alfalfa C and N exhibited positive correlations along the second axis of redundancy analysis (RDA2), suggesting coordinated uptake mechanisms. Both pH and EC correlated negatively with RDA1 and demonstrated significant negative relationships with alfalfa P and soil C/P ratios (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001). This indicates that high salinity inhibits nutrient absorption and that reducing pH is critical for alleviating P limitation.\u003c/p\u003e\n\u003cp\u003eFigure\u0026nbsp;8\u0026nbsp;Redundancy analysis (RDA) of C-N-P stoichiometric characteristics and environmental factors. S.C: soil carbon; S.N: soil nitrogen; S.P: soil phosphorus; S.CN: soil C:N; S.CP: soil C:P; S.NP: soil N:P; A.C: alfalfa carbon; A.N: alfalfa nitrogen; A.P: alfalfa phosphorus; A.CN: alfalfa C:N; A.CP: alfalfa C:P; A.NP: alfalfa N:P; EC: electrical conductivity; BD: bulk density. Arrows indicate direction and strength of variable contributions. Treatments: CK (black), CL1 (blue), CL2 (red), CL3 (green). RDA1 and RDA2 explain 79.31% and 2.72% of variance, respectively. (\u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.6 Structural equation modeling of multiple elements in different soil layers\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBased on structural equation modeling (SEM) analysis (Figure 9), aluminum sulfate regulated C-N-P coupling in the soil-plant system differently across soil depths. In the 0-10 cm layer (Figure 9A), amendments directly lowered soil pH and EC, promoting the release of available nutrients. These nutrients drove both soil and alfalfa nutrient dynamics, establishing a synergistic nutrient interaction between soil and alfalfa. In this layer, available nutrients (primarily P) acted as key mediators in nutrient cycling. Deeper down, while amendment effects on pH and EC lessened, their influence on soil nutrients grew stronger. Soil nutrients directly governed alfalfa nutrient uptake, and the coupling between soil and alfalfa stoichiometric traits strengthened significantly.\u003c/p\u003e\n\u003cp\u003eIn the 10-20 cm soil layer (Figure 9B), alfalfa's nutrient acquisition primarily depended on direct soil supply, exhibiting the highest sensitivity to nutrient balance. In the 20-30 cm soil layer (Figure 9C), the effectiveness of amendments was limited, and EC remained the primary factor inhibiting nutrient availability. The contribution of soil nutrients to alfalfa uptake decreased significantly, and stoichiometric coupling was at its weakest. In deeper soil layers, alfalfa's nutrient absorption was constrained, prompting adaptive adjustments in the plant's elemental demand in response to environmental conditions.\u003c/p\u003e\n\u003cp\u003eCollectively, the results demonstrate that the regulatory effects of amendments on pH and EC gradually diminished from the surface to deeper soil layers. Available P emerged as the key driver primary soil of nutrient accumulation in both alfalfa nutrient accumulation layers, validating the hypothesis that \"P is the critical driver for improving stoichiometric characteristics in saline-sodic soils\". This highlights soils. pivotal underscores of essential availability in mediating soil-plant C-N-P interactions under C-N-P application. following the application of amendments.\u003c/p\u003e\n\u003cp\u003eNote: Structural equation model for 0-10 cm layer (GFI=0.9001)\u003c/p\u003e\n\u003cp\u003eNote: Structural equation model for 10-20 cm layer (GFI=0.8276)\u003c/p\u003e\n\u003cp\u003eNote: Structural equation model for 20-30 cm layer (GFI=0.8505)\u003c/p\u003e\n\u003cp\u003eFigure\u0026nbsp;9\u0026nbsp;Structural equation models (SEM) illustrating aluminum sulfate regulation on C-N-P coupling in soil-alfalfa systems at 0–10 cm (A), 10–20 cm (B), and 20–30 cm (C) depths. Solid/dashed arrows indicate positive/negative pathways (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05). Arrow width reflects standardized path coefficients. GFI: Goodness-of-Fit Index. AP: available P; EC: electrical conductivity\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003e\u003cstrong\u003e4.1\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eThe effect of aluminum sulfate on nutrient uptake in alfalfa and its stoichiometric control\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe use of aluminum sulfate significantly changed how alfalfa absorbed nutrients. As shown in Figure. 1, under the CL2 treatment condition, the N and P contents in alfalfa were significantly increased compared with the CK, while the C content showed a slight increase. This is consistent with the findings of Mu et al\u003csup\u003e[38]\u003c/sup\u003e, indicating that aluminum sulfate promotes alfalfa growth by regulating soil pH and ion forms, thereby improving the bioavailability of\u0026nbsp;N\u0026nbsp;and\u0026nbsp;P.\u003csup\u003e[39\u003c/sup\u003e\u003csup\u003e-41]\u003c/sup\u003e. Notably, Figure 2 demonstrates that following aluminum sulfate application, the CL2 treatment exhibited the lowest C/N and C/P ratios, indicating optimal nutrient utilization efficiency at this dosage. This finding aligns with the observations of Yu et al.\u003csup\u003e[41]\u003c/sup\u003e, where a low C/N ratio typically corresponds to high decomposition rates and nutrient turnover.\u003c/p\u003e\n\u003cp\u003eAlfalfa N/P ratios (12.69-13.78) observed in this study were lower than those reported by Luo et al\u003csup\u003e.\u003c/sup\u003e\u003csup\u003e[42]\u003c/sup\u003e on the Qinghai-Tibet Plateau (N/P = 14.5-16.2) and Pan et al.\u003csup\u003e[43]\u003c/sup\u003e in temperate grasslands (N/P = 15.1-17.3). which can be attributed to the unique edaphic conditions of the Songnen Plain. Specifically, the study site exhibited a baseline soil pH of 8.2 (Table 1), significantly higher than the mildly acidic to neutral pH ranges (6.0–7.5) typically reported in the Qinghai-Tibet Plateau and temperate grassland ecosystems\u003csup\u003e[44\u003c/sup\u003e\u003csup\u003e-45]\u003c/sup\u003e. This elevated alkalinity promotes ammonium-N conversion to gaseous ammonia (NH\u003csub\u003e3\u003c/sub\u003e) via volatilization, while high sodium ion (Na\u003csup\u003e+\u003c/sup\u003e) saturation (over 80% in soil colloids\u003csup\u003e[2]\u003c/sup\u003e) disrupts soil aggregate structure, enhancing N leaching through increased permeability\u003csup\u003e[43]\u003c/sup\u003e.. Additionally, no P limitation was observed, which contrasts with findings by Harpole et al.\u003csup\u003e[24]\u003c/sup\u003e in global grasslands, likely due to significantly improved P availability resulting from the ameliorant. Compared to the study by Jiaping et al.\u003csup\u003e[46]\u003c/sup\u003e in Xinjiang cotton fields, alfalfa in this research contributed more to C fixation (14.36% increase in C), possibly due to the N-fixing capabilities of legumes\u003csup\u003e[33]\u003c/sup\u003e. However, alfalfa had limited effects on deep soil improvement, consistent with the findings of Díaz et al\u003csup\u003e.\u003c/sup\u003e\u003csup\u003e[29]\u003c/sup\u003e in arid regions, indicating the limitations of perennial forages in enhancing deep soil quality. Furthermore, the co-accumulation of soil C and P supports the \"C-P co-accumulation\" hypothesis\u003csup\u003e[43]\u003c/sup\u003e. However, this effect diminishes in deeper soil layers, likely due to reduced microbial activity with increasing depth\u003csup\u003e[44]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.2 Vertical Differentiation and Environmental Drivers of Soil-Plant C-N-P Coupling\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe vertical distribution of soil C, N, and P exhibited significant dose-dependent responses to amendments. In the 0-10 cm layer, the C content under the treatment increased by 64% compared to CK, which is with with the findings of Elser et al.\u003csup\u003e[45]\u003c/sup\u003e regarding cycle-enhanced C sequestration. However, in the 10-20 cm layer, the treatment showed demonstrated highest C content, suggesting that doses optimize mid-layer accumulation in mid-layers. The content was significantly higher in in the cm layers than in surface in the aligning with prior studies previous N leaching in saline-sodic soils\u003csup\u003e[46]\u003c/sup\u003e. Notably, the yielded the highest P content in the 20-30 cm layer, likely due to low-dose amendments activating insoluble phosphates in deeper soils\u003csup\u003e[47]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eVertical differentiation of soil C/P ratios revealed spatial variability in P availability. The C/P ratio in surface soil was significantly lower than that in deeper layers, corroborating the findings of Ivanova et al\u003csup\u003e.\u003c/sup\u003e\u003csup\u003e[39]\u003c/sup\u003e regarding organic acid-mediated P activation. Furthermore, a significant negative correlation (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01) was observed between soil C/P and alfalfa C/P, indicating that plants adapt to P limitation by enhancing their utilization efficiency\u003csup\u003e[17]\u003c/sup\u003e. This adaptive strategy is particularly critical in the Songnen Plain, where high pH levels exacerbate P fixation. Alfalfa mitigates this issue by secreting malic and oxalic acids to chelate Ca\u003csup\u003e2+\u003c/sup\u003e and Fe\u003csup\u003e3+\u003c/sup\u003e ions\u003csup\u003e[32]\u003c/sup\u003e, thereby maintaining cellular P homeostasis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.3 Nutrient Limitation Mechanisms from an Ecological Stoichiometry Perspective\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs shown in Figure 4, the soil C/N ratio (21.5) under CL2 treatment approached the ideal 25:1\u003csup\u003e[49]\u003c/sup\u003e indicating an effective C-N balance. A higher dosage of aluminum sulfate increased the C/N ratio to 25.3, which not only reduced C sequestration efficiency\u003csup\u003e[50]\u003c/sup\u003e but also induced aluminum toxicity, inhibiting root growth\u003csup\u003e[50\u003c/sup\u003e\u003csup\u003e-51]\u003c/sup\u003e, consistent with the findings of Batjes et al\u003csup\u003e[52]\u003c/sup\u003e. Reduced C/P ratios reflected improved P bioavailability, which is directly linked to an increase in available soil P. Notably, CL2 reduced the surface soil N/P by 49.71%, likely due to pH-driven N mineralization and P release. The stability of the N/P ratios, with no significant differences observed across layers within treatments, suggests that the amendment induced vertical homogenization of nutrient distribution.\u003c/p\u003e\n\u003cp\u003eAs shown in Figures 5 and 7, N and P in alfalfa exhibited a significant positive correlation (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01), indicating synergistic uptake between the two nutrients.\u0026nbsp;This phenomenon may result from N-enhanced photosynthetic product synthesis driving P demand\u003csup\u003e[53]\u003c/sup\u003e. Reduced C/P under CL2 further highlighted elevated P utilization efficiency, directly related to soil available P. Such synergy is consistent with the findings of Wang et al.\u003csup\u003e[19]\u003c/sup\u003e, highlighting the universal plant co-demand for N and P.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.4 Environmental Drivers of Soil-Plant C-N-P Coupling\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs indicated by the RDA analysis (Figure 8), soil pH exhibited a significant negative correlation with alfalfa P conten (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01), and positive correlations between pH and EC (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05). In high-pH soils, Na⁺ competes with PO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e3-\u003c/sup\u003e for root adsorption sites, thereby impeding P transmembrane transport\u003csup\u003e[4]\u003c/sup\u003e. This ion antagonism is particularly pronounced in the Songnen Plain, where Na⁺ saturation in soil colloids exceeds 80%\u003csup\u003e[5]\u003c/sup\u003e. Unlike the halophytes studied by Flowers et al.\u003csup\u003e[30]\u003c/sup\u003e, alfalfa has adapted to salinity by modulating N/P ratios rather than accumulating osmolytes, possibly due to its leguminous characteristics\u003csup\u003e[33]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eCo-accumulation of soil C and P further supports the \"C-P synergy\" hypothesis\u003csup\u003e[54]\u003c/sup\u003e. CL2 increased soil C by 64% and P by 15.27%, aligning with the findings of Cai et al.\u003csup\u003e[48]\u003c/sup\u003e regarding sulfur-driven C fixation. The enhanced availability of P likely stimulates the decomposition and stabilization of microbial organic C\u003csup\u003e[54]\u003c/sup\u003e. However, this synergy appears to weaken in deeper soils, potentially due to a decline in microbial activity with increasing depth\u003csup\u003e[49]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.5 Aluminum sulfate's distinct regulation of the C-N-P coupling process in soil-plant systems.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStructural equation modeling (SEM) elucidated amendment-driven regulatory networks across various soil layers(Figure 9). In the 0-10 cm layer, amendments improved enhanced cycling through two primary (1) reducing pH/EC alleviate ion toxicity toxicity, (2) enhancing promoting the release of nutrient, with the findings of Yu et al.\u003csup\u003e[41]\u003c/sup\u003e in North China Plain. Acidic amendments neutralize alkalinity via by releasing H⁺ ions, which liberate P that is fixed in carbonate forms. the availability of directly drove influenced uptake more strongly significantly soil reserves, likely due to root-secreted organic acids chelating that chelate Fe\u003csup\u003e3+\u003c/sup\u003e and Al\u003csup\u003e3+\u003c/sup\u003e ions, thereby solubilizing phosphates\u003csup\u003e[32]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eIn the 10-20 cm layer, SEM revealed diminished pH/EC regulation but intensified nutrient-driven soil-plant interactions intensified. An increased organic matter content may promote the binding of sulfate (SO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e2-\u003c/sup\u003e) and calcium ions (Ca\u003csup\u003e2+)\u003c/sup\u003e to form gypsum, thereby restoring the aggregate structure\u003csup\u003e[18]\u003c/sup\u003e. The direct uptake of nutrients by alfalfa surpassed the effects observed in the surface layer, aligning with the findings of Wu et al.\u003csup\u003e[6]\u003c/sup\u003e regarding the root density of alfalfa in the mid-layer. Enhanced stoichiometric coupling indicates a heightened sensitivity of the mid-layer to nutrient fluctuations, which may be associated with vertical gradients in microbial activity.\u003c/p\u003e\n\u003cp\u003eIn the 20-30 cm layer, SEM showed limited penetration of amendments, with EC remaining the primary inhibitor of nutrient availability. This finding aligns with the research conducted by Zhao et al.\u003csup\u003e[26]\u003c/sup\u003e on gypsum amendments. Contributions from deeper soil layers to alfalfa uptake were significantly lower, potentially due to suppressed proton pump activity under alkaline conditions\u003csup\u003e[55\u003c/sup\u003e\u003csup\u003e-56]\u003c/sup\u003e. Weak stoichiometric coupling suggests that plants adapt to nutrient scarcity by regulating elemental demand\u003csup\u003e[57\u003c/sup\u003e\u003csup\u003e-58]\u003c/sup\u003e.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eAluminum sulfate has a notable impact on the C-N-P chemical stoichiometry of the soil-alfalfa system. When applied at a rate of 48 kg\u0026middot;hm\u003csup\u003e-2\u003c/sup\u003e, it significantly boosts the levels of C, N, and P in both the soil and alfalfa, while keeping the N/P ratio in the soil relatively stable. It also improves P uptake in alfalfa by reducing soil pH and EC. Structural equation modeling (SEM) indicates that P plays a crucial role in nutrient cycling in surface soils, showing a significant negative relationship between alfalfa P and soil C/P. This research identifies 48 kg\u0026middot;hm\u003csup\u003e-2\u003c/sup\u003e as the ideal application rate, which greatly enhances soil fertility and alfalfa yield by addressing P deficiencies and optimizing the C-N-P relationship.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthical approval and field permissions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll field studies, including plant material collection, were conducted in accordance with local legislation and with permission from the management of Yinlang Ranch, Daqing City. The research protocol complies with the guidelines for ethical conduct in plant studies issued by the Heilongjiang Bayi Agricultural University.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analysed during this study are included in this published article and its supplementary information files.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNational Key R\u0026amp;D Program of China (No. 2023YFD2300102), Postdoctoral Scientific Research Startup Fund Project of Heilongjiang Provincial (No. LBH-Q21162), Natural Science Foundation Project of Heilongjiang Provincial (No. LH2022D019), and Horizontal research project of Heilongjiang Bayi Agricultural University (No. 2041200084).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors' contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA.B. conducted the investigation, developed the methodology, wrote the original draft, and created visualizations. M.Z. conceptualized the study, developed the methodology, supervised the work, and reviewed and edited the writing. All authors reviewed the manuscript.C.W. reviewed and edited the writing. H.Z. worked on the methodology, created visualizations, and supervised the study. M.S. Methodology, Supervision. W.Z. and M.Q. Supervision. All authors read and approved the final manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eCentofanti T, Banuelos G. Evaluation of the halophyte Salsola soda as an alternative crop for saline soils high in selenium and boron. J Environ Manage. 2015;157:96\u0026ndash;102. 2015;157:96\u0026ndash;102. doi: 10.1016/j.jenvman.2015.04.005. \u0026nbsp;\u003c/li\u003e\n \u003cli\u003eQi H, Ma R, Shi C, et al. Novel low-cost carboxymethyl cellulose microspheres with excellent fertilizer absorbency and release behavior for saline-alkali soil. Int J Biol Macromol. 2019;131:412-419. doi:10.1016/j.ijbiomac.2019.03.047.\u003c/li\u003e\n \u003cli\u003eMunns R, Tester M. Mechanisms of salinity tolerance. Annu Rev Plant Biol. 2008;59:651-681. doi:10.1146/annurev.arplant.59.032607.092911.\u003c/li\u003e\n \u003cli\u003eShabala S, Cuin TA. Potassium transport and plant salt tolerance. Physiol Plant. 2008;133(4):651-669. doi:10.1111/j.1399-3054.2007.01008.x.\u003c/li\u003e\n \u003cli\u003eHasanuzzaman M, et al. Potential use of halophytes to remediate saline soils. Biomed Res Int. 2014;2014:589341. doi:10.1155/2014/589341.\u003c/li\u003e\n \u003cli\u003eWu J, et al. Alfalfa cultivation in saline soils: Physiological responses and soil reclamation. Agron J. 2014;106(2):685-694. doi:10.2134/agronj2013.0355.\u003c/li\u003e\n \u003cli\u003eRengasamy P. Soil processes affecting crop production in salt-affected soils. Funct Plant Biol. 2010;37(7):613-620. doi:10.1071/FP09249.\u003c/li\u003e\n \u003cli\u003eRayment GE, Higginson FR. Australian Laboratory Handbook of Soil and Water Chemical Methods. Inkata Press; 1992.\u003c/li\u003e\n \u003cli\u003eElser JJ, et al. Global analysis of nitrogen and phosphorus limitation of primary producers in freshwater, marine, and terrestrial ecosystems. Ecol Lett. 2007;10(12):1135-1142. doi:10.1111/j.1461-0248.2007.01113.x.\u003c/li\u003e\n \u003cli\u003eSterner RW, Elser JJ. Ecological Stoichiometry: The Biology of Elements from Molecules to the Biosphere. Princeton University Press; 2002. doi:10.1515/9781400885695.\u003c/li\u003e\n \u003cli\u003eKoerselman W, Meuleman AFM. The vegetation N:P ratio: A new tool to detect the nature of nutrient limitation. J Appl Ecol. 1996;33(6):1441-1450. doi:10.2307/2404783.\u003c/li\u003e\n \u003cli\u003eG\u0026uuml;sewell S. N:P ratios in terrestrial plants: Variation and functional significance. New Phytol. 2004;164(2):243-266. doi:10.1111/j.1469-8137.2004.01192.x.\u003c/li\u003e\n \u003cli\u003eLi X, et al. Effects of combined amendments on soil salinity and nutrient availability in coastal saline soil. Soil Tillage Res. 2019;194:104318. doi:10.1016/j.still.2019.104318.\u003c/li\u003e\n \u003cli\u003eClark GJ, et al. Sulfate sorption and release in acid sulfate soils: Effects of pH and redox potential. Geoderma. 2007;138(3-4):357-366. doi:10.1016/j.geoderma.2006.11.017.\u003c/li\u003e\n \u003cli\u003eDaliakopoulos IN, Tsanis IK, Koutroulis AG. Soil salinity assessment, monitoring and mitigation: An overview. Sci Total Environ. 2016;542:727-739. doi:10.1016/j.scitotenv.2015.11.074.\u003c/li\u003e\n \u003cli\u003eJones DL, Darrah PR. Role of root derived organic acids in the mobilization of nutrients from the rhizosphere. Plant Soil. 1994;166(2):247-257. doi:10.1007/BF00008338.\u003c/li\u003e\n \u003cli\u003eBolan NS, et al. Influence of low-molecular-weight organic acids on the solubilization of phosphates. Biol Fertil Soils. 1994;18(4):311-319. doi:10.1007/BF00570634.\u003c/li\u003e\n \u003cli\u003eLindsay WL. Chemical Equilibria in Soils. Wiley; 1979. doi:10.2136/sssaj1979.03615995004300050021x.\u003c/li\u003e\n \u003cli\u003eWang Y, et al. Synergistic effects of aluminum sulfate and ferrous sulfate on saline-alkali soil amelioration. J Soils Sediments. 2021;21(5):2103-2115. doi:10.1007/s11368-021-02922-1.\u003c/li\u003e\n \u003cli\u003eDu E, et al. Global patterns of nitrogen and phosphorus limitation in terrestrial ecosystems. Nat Geosci. 2022;15(11):867-873. doi:10.1038/s41561-022-01034-3.\u003c/li\u003e\n \u003cli\u003eAndersen T, Elser JJ, Hessen DO. Stoichiometry and population dynamics. Ecol Lett. 2004;7(10):884-900. doi:10.1111/j.1461-0248.2004.00646.x.\u003c/li\u003e\n \u003cli\u003eWeber TS, Deutsch C. Ocean nutrient ratios governed by plankton biogeography. Nature. 2010;467(7315):550-554. doi:10.1038/nature09403.\u003c/li\u003e\n \u003cli\u003eLeBauer DS, Treseder KK. Nitrogen limitation of net primary productivity in terrestrial ecosystems is globally distributed. Ecology. 2008;89(2):371-379. doi:10.1890/07-0527.1.\u003c/li\u003e\n \u003cli\u003eHarpole WS, Tilman D. Nitrogen and phosphorus limitation of productivity in worldwide herbaceous communities. Ecology. 2007;88(10):2391-2397. doi:10.1890/06-1892.1.\u003c/li\u003e\n \u003cli\u003eHessen DO, et al. Ecological stoichiometry: An elementary approach using basic principles. Limnol Oceanogr. 2013;58(6):2219-2236. doi:10.4319/lo.2013.58.6.2219.\u003c/li\u003e\n \u003cli\u003eMarra LM, et al. Initial pH of medium affects organic acids production but do not affect phosphate solubilization. Braz J Microbiol. 2015;46(2):367-375. doi:10.1590/S1517-838246246220131102.\u003c/li\u003e\n \u003cli\u003eZhao S, et al. Gypsum amendment improves soil properties and crop productivity in saline-alkali soils of North China. Agric Water Manag. 2019;222:28-36. doi:10.1016/j.agwat.2019.05.037.\u003c/li\u003e\n \u003cli\u003eNiu R L, et al. Carbon, nitrogen, and phosphorus stoichiometric characteristics of soil and leaves from young and middle aged Larix principis-rupprechtii\u0026nbsp;growing in a Qinling Mountain plantation. Acta Ecologica Sinica. 2016,36(22):7384-7392. doi:\u003ca href=\"http://dx.doi.org/10.5846/stxb201601080057\"\u003e10.5846/stxb201601080057\u003c/a\u003e.\u003c/li\u003e\n \u003cli\u003eCox, K.H., Jacinthe, PA. Phosphorus Mobility in Gypsum-Amended Soils in Relation to Soil Type and Timing of P Fertilizer Application.\u0026nbsp;Water Air Soil Pollut\u0026nbsp;234, 368 (2023). doi:10.1007/s11270-023-06388-4.\u003c/li\u003e\n \u003cli\u003eD\u0026iacute;az FJ, et al. Using saline soil and marginal quality water to produce alfalfa in arid climates. Agric Water Manag. 2018;199:11-21. doi:10.1016/j.agwat.2017.12.003.\u003c/li\u003e\n \u003cli\u003eFlowers TJ, Colmer TD. Salinity tolerance in halophytes. New Phytol. 2008;179(4):945-963. doi:10.1111/j.1469-8137.2008.02480.x.\u003c/li\u003e\n \u003cli\u003eShannon MC, Grieve CM. Tolerance of vegetable crops to salinity. Sci Hortic. 1999;78(1-4):5-38. doi:10.1016/S0304-4238(98)00189-7.\u003c/li\u003e\n \u003cli\u003eAshraf M, Harris PJC. Potential biochemical indicators of salinity tolerance in plants. Plant Sci. 2004;166(1):3-16. doi:10.1016/j.plantsci.2003.10.024.\u003c/li\u003e\n \u003cli\u003eZahran HH. Rhizobium-legume symbiosis and nitrogen fixation under severe conditions and in an arid climate. Microbiol Mol Biol Rev. 1999;63(4):968-989. doi:10.1128/MMBR.63.4.968-989.1999.\u003c/li\u003e\n \u003cli\u003eShams MK, Khadivi A. Mechanisms of salinity tolerance and their possible application in the breeding of vegetables. BMC Plant Biol. 2023;23(1):139. doi:10.1186/s12870-023-04152-8.\u003c/li\u003e\n \u003cli\u003eBremner JM. Nitrogen\u0026mdash;Total. In: Sparks DL, editor. Methods of Soil Analysis: Part 3 Chemical Methods. Soil Science Society of America; 1996:1085-1121.\u003c/li\u003e\n \u003cli\u003eLiu Y S. Effects of forage reseeding on plant-soil ecological stoichiometric characteristics in degraded grasslands [PhD Dissertation]. Northwest A\u0026amp;F University, 2023. doi:10.27409/d.cnki.gxbnu.2023.000752.\u003c/li\u003e\n \u003cli\u003eMu C, et al. Response of Extracellular Enzyme Stoichiometric Properties and Microbial Metabolic Limitations to the Ecosystem Transition Mode Employed in Red Jujube Economic Forests on the Loess Plateau. Microorganisms. 2025,13(4):729. doi: 10.3390/microorganisms13040729.\u003c/li\u003e\n \u003cli\u003eIvanova RP, et al. The Solubilization of Rock Phosphate by Organic Acids. Crit Rev Environ Sci Technol. 2006;36(4):2541-2554. doi:10.1080/10426500600758399.\u003c/li\u003e\n \u003cli\u003eLi R, et al. Amelioration of saline-alkali soil using aluminum sulfate: Effects on soil properties and crop growth. J Environ Manage. 2018;215:1-8. doi:10.1016/j.jenvman.2018.03.042.\u003c/li\u003e\n \u003cli\u003eYu YF, et al. Stoichiometric characteristics of plant and soil C, N and P in different forest types in depressions between karst hills, southwest China. Acta Ecol Sin. 2014;34(4):947-954. doi:10.1007/s12665-014-3553-6.\u003c/li\u003e\n \u003cli\u003eLuo X, et al. Nitrogen:Phosphorus Supply Ratio and Allometry in Five Alpine Plant Species. Ecol Evol. 2017;7(21):8905-8915. doi:10.1002/ece3.2587.\u003c/li\u003e\n \u003cli\u003ePan Y, et al. Global patterns of nitrogen and phosphorus resorption efficiency in terrestrial ecosystems. Ecol Lett. 2015;18(12):1395-1404. doi:10.1111/ele.12515.\u003c/li\u003e\n \u003cli\u003eLarrainzar E, et al. Hemoglobins in the legume\u0026ndash;Rhizobium symbiosis. New Phytol. 2020;228(2):472-484. doi:10.1111/nph.16673.\u003c/li\u003e\n \u003cli\u003eElser JJ, et al. Organism size, life history, and N:P stoichiometry: Toward a unified view of cellular and ecosystem processes. Bioscience. 1996;46(9):674-684. doi:10.2307/1312897.\u003c/li\u003e\n \u003cli\u003eJiaping L, Wenjuan S. Cotton/halophytes intercropping decreases salt accumulation and improves soil physicochemical properties and crop productivity in saline-alkali soils under mulched drip irrigation: A three-year field experiment. Field Crops Res. 2021;262:108027. doi: 10.1016/j.fcr.2020.108027. \u0026nbsp;\u003c/li\u003e\n \u003cli\u003eWang X, et al. Nitrogen and phosphorus addition alter leaf nutrient concentrations of dominant grass species and regulate primary productivity in Inner Mongolia meadow steppe. Grassland Res. 2023;6(1):100126. doi:10.1016/j.grs.2023.100126.\u003c/li\u003e\n \u003cli\u003eCai Z, et al. Sulfur cycling in acid sulfate soils: A review. Geoderma. 2020;375:114574. doi:10.1016/j.geoderma.2020.114574.\u003c/li\u003e\n \u003cli\u003eJēkabsone A, et al. Dependence on Nitrogen Availability and Rhizobial Symbiosis of Different Accessions of Trifolium fragiferum, a Crop Wild Relative Legume Species, as Related to Physiological Traits. Plants. 2022;11(9):1141. doi:10.3390/plants11091141.\u003c/li\u003e\n \u003cli\u003eHu Y, et al. Storage of C, N, and P affected by afforestation with Salix cupularis in an alpine semiarid desert ecosystem. Land Degrad Dev. 2018;29(1):188-198. doi:10.1002/ldr.2862.\u003c/li\u003e\n \u003cli\u003eWang X, et al. Nitrogen and phosphorus addition alter leaf nutrient concentrations of dominant grass species and regulate primary productivity in Inner Mongolia meadow steppe. Grassland Res. 2023;6(1):100126. doi:10.1016/j.grs.2023.100126.\u003c/li\u003e\n \u003cli\u003eBatjes NH. Total carbon and nitrogen in the soils of the world. Eur J Soil Sci. 1996;47(2):151-163. doi:10.1111/j.1365-2389.1996.tb01386.x.\u003c/li\u003e\n \u003cli\u003eWang Y, et al. Influence of Alfalfa Planting Years on Soil Carbon Sequestration and Enzyme Activity in Saline-Alkali Soils of the Songnen Plain. Sustainability. 2023;15(14):10772. doi:10.3390/su151410772.\u003c/li\u003e\n \u003cli\u003eMcGroddy ME, et al. Scaling of C:N:P stoichiometry in forests worldwide: Implications of terrestrial Redfield-type ratios. Ecology. 2004;85(9):2390-2401. doi:10.1890/03-0240.\u003c/li\u003e\n \u003cli\u003eMooshammer M, et al. Adjustment of microbial nitrogen use efficiency to carbon:nitrogen imbalances regulates soil nitrogen cycling. Nat Commun. 2014;5:3694. doi:10.1038/ncomms4694.\u003c/li\u003e\n \u003cli\u003eJēkabsone A, et al. Dependence on Nitrogen Availability and Rhizobial Symbiosis of Different Accessions of Trifolium fragiferum, a Crop Wild Relative Legume Species, as Related to Physiological Traits. Plants. 2022;11(9):1141. doi:10.3390/plants11091141.\u003c/li\u003e\n \u003cli\u003eBohn HL, McNeal BL, O\u0026apos;Connor GA. Soil Chemistry. Wiley; 2001. doi:10.1002/0471224437.\u003c/li\u003e\n \u003cli\u003eZhu JK. Salt Tolerance. Curr Biol. 2016;26(16):R706-R710. doi:10.1016/j.cub.2016.06.047.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1 and 2 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Ecological stoichiometry, Saline-sodic soil, Alfalfa, Improver","lastPublishedDoi":"10.21203/rs.3.rs-8299360/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8299360/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground: \u003c/strong\u003eTo optimize saline-sodic soil amelioration strategies, enhance soil fertility and forage productivity, and elucidate aluminum sulfate's regulatory mechanisms on C-N-P stoichiometric characteristics in both saline-sodic soils and alfalfa, we conducted this study at Yinlang Ranch of Daqing City, Heilongjiang Province in China.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eThis study investigated the effects of aluminum sulfate application at different doses (0, 24, 48, and 72 kg·hm\u003csup\u003e-2\u003c/sup\u003e) on the distribution of carbon (C), nitrogen (N), and phosphorus (P), as well as stoichiometric ratios and environmental factors of alfalfa and soil layers.\u003cstrong\u003e \u003c/strong\u003eThe results indicated significant differences in the C-N-P stoichiometric characteristics of soil and alfalfa under different aluminum sulfate applications. An application rate of 48 kg·hm\u003csup\u003e-2\u003c/sup\u003e significantly increased the C, N, and P contents in both soil and alfalfa while maintaining a relatively stable soil N/P ratio. Soil C and P exhibited significant positive correlations with alfalfa P. During the growth process, alfalfa growth was primarily limited by P. The plants enhanced their growth and development by adjusting the balance between elemental requirements and nutrient absorption, as well as modifying nutrient utilization strategies to adapt to the saline-sodic soil environment. Furthermore, alfalfa P showed significant negative correlations with soil pH and electrical conductivity (EC).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions:\u003c/strong\u003eThe study concludes that aluminum sulfate significantly influences the C-N-P stoichiometric characteristics of the soil-alfalfa system. The optimal application rate of 48 kg·hm\u003csup\u003e⁻2\u003c/sup\u003e enhanced nutrient content in soil and alfalfa, stabilized the soil N/P ratio, and promoted P uptake of the alfalfa by improving soil environmental conditions. These findings establish a theoretical foundation for precision nutrient management.\u003c/p\u003e","manuscriptTitle":"Phosphorus Improves Stoichiometric Characteristics of Saline-Sodic Soil and Alfalfa","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-16 18:39:13","doi":"10.21203/rs.3.rs-8299360/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2025-12-29T23:37:38+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"131565069223899384968550004137435907563","date":"2025-12-17T22:59:28+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-15T00:39:54+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"206783558064805202814697004374530436772","date":"2025-12-11T02:11:54+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-12-10T13:32:30+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-12-09T13:12:44+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-12-09T13:08:47+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-12-09T09:08:02+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Plant Biology","date":"2025-12-09T08:51:10+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"71851f0a-63e5-4546-9b89-b40dc20b0049","owner":[],"postedDate":"December 16th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-12-16T18:39:13+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-16 18:39:13","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8299360","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8299360","identity":"rs-8299360","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}