Effects of Pisha Sandstone Application Rate and Microbial Agents on Soil Nutrient Dynamics and Microbial Characteristics in Sandy Soil

Effects of Pisha Sandstone Application Rate and Microbial Agents on Soil Nutrient Dynamics and Microbial Characteristics in Sandy Soil | 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 Article Effects of Pisha Sandstone Application Rate and Microbial Agents on Soil Nutrient Dynamics and Microbial Characteristics in Sandy Soil Zhishui Liang, Xiuwen Fang, Haiying Gao, Bo Pan, Jishu Zhang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6408674/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 19 Nov, 2025 Read the published version in Scientific Reports → Version 1 posted 7 You are reading this latest preprint version Abstract Sandy soils are prone to water loss, low fertility, difficulties in vegetation growth, and severe wind erosion, and the employment of microorganisms and other techniques to improve sandy soils is an efficient way to realize their management. This study involved a field experiment in a demonstration garden located in the Hobq Desert region of China, aimed at examining the impacts on soil nutrients, microbial populations, enzyme activity, and licorice growth resulting from two experimental groups with varying volume ratios of Pisha sandstone (0%, 30%, 50%, 80%, and 100%) and sandy soils, categorized by the presence of microbial agents (M) or their absence (NM). The study's results indicated that the incorporation of Pisha sandstone markedly influenced soil nutrients, microbial populations, enzyme activity, and the height of licorice plants, in contrast to the 0% volume addition of Pisha sandstone without microbial agents (CK). The enhancement was notably greater following the introduction of microbial agents; the optimal overall treatment effect was observed with a 50% volume addition of Pisha sandstone, where soil nutrients, enzyme activity, and licorice growth metrics attained their peak values in NM and N. Correlation analysis indicates that the enhancement of soil organic carbon, available nitrogen, and available phosphorus can elevate the population of soil microorganisms and enzyme activity, with these parameters interacting and mutually reinforcing each other to facilitate the improvement of sandy soil. This offers an effective approach for enhancing sandy soil. Earth and environmental sciences/Biogeochemistry Earth and environmental sciences/Environmental social sciences Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Desertification, driven by anthropogenic activity, climate change, and disjointed environmental and developmental policies, results in environmental deterioration, and a significant reduction in agricultural productivity, and imposes substantial constraints on economic development while undermining social stability 1 – 3 . In 1992, the United Nations Environment Programme (UNEP) articulated a definition of desertification, characterizing it as "land degradation in arid, semi-arid, and dry sub-humid areas resulting from various factors, including climatic variations and human activities" 4 . China is among the nations with the most extensive desertification, the greatest impacted population, and the most severe wind and sand dangers. As per the fifth national desertification and monitoring report, by 2014, the total expanse of sandy terrain in China was 172,117,500 hectares, representing 17.93% of the overall land area 5 . Aeolian sandy soils possess a substandard soil structure, demonstrating inadequacies in organic matter and nitrogen levels. These soils have high permeability, resulting in substantial water infiltration and diminished capacity for nitrogen and fertilizer retention 6 . These properties represent a critical limiting factor in the ecological restoration of desertified areas. To solve these difficulties, enhancing sandy soils and alleviating environmental strain, while simultaneously increasing arable land and maintaining national food security, tackling desertification has emerged as a significant and widely recognized issue. Sandy soils exhibit markedly deficient water and nutrient retention capacities, rendering the augmentation of soil organic carbon, structural amelioration, enhancement of hydro-fertilizer retention capabilities, and improvement of crop survival rates pivotal for restoring self-sustaining edaphic ecosystems. Current enhancement strategies for sandy soils employ three principal approaches: engineering techniques, biological interventions, and soil amendments. Engineering methods, particularly sand barrier installation and sand compaction pile (SCP) implementation, present substantial economic costs and environmental burdens. Biological strategies primarily involve anti-desertification afforestation and cultivation of xerophytic vegetation, though their efficacy manifests gradually over extended temporal scales. Consequently, soil amendments have emerged as the predominant remediation approach, with Pisha sandstone demonstrating particular utility as an effective modifier 7 – 12 . In China's desertification-affected region, sandy soils and Pisha sandstone (a weakly lithified Mesozoic sedimentary formation) exhibit alternating stratigraphic distributions, with the latter constituting approximately 33% of the total substrate coverage 13 . Despite its low diagenetic grade and compromised structural integrity, Pisha sandstone contains substantial montmorillonite components, endowing it with exceptional physicochemical properties including elevated specific surface area, enhanced cation exchange capacity, and superior moisture retention characteristics 14 .Therefore, the mixture of the two substances in an optimal ratio contributes to the improvement of soil aggregate structure, enhances windbreak and sand-fixing capacity, improves water and nutrient retention properties, while also promoting the sustainable utilization and development of land resources 14 – 16 . Despite significant research, the current focus is mostly on the erosion resistance of soil following the amalgamation of Pisha sandstone and sandy soil, solute transport, and soil hydraulic properties 17 – 19 . The enhancement of Pisha sandstone and sand to soil fertility is restricted, and the basic improvement of desertified areas cannot be achieved. Soil microorganisms are essential constituents of soil ecosystems. Microbial agents, by the synergistic action of their internal microorganisms, effectively enhance soil quality, promote crop growth, mitigate soil disease transmission, and increase agricultural production. It is non-toxic, innocuous, environmentally benign, and economical 20 – 23 . This study investigates the impact of microbial agents on sandy soil and Pisha sandstone, focussing on soil fertility, the abundance of three primary bacterial families, and enzyme activity through field trials. It also examines the overall management solutions for sandy soil, which is crucial for mitigating soil erosion. It also looks at the comprehensive management strategies for sandy soil, which is extremely important for managing soil erosion. Materials and Methods Overview of the study area The study area is geographically positioned in Duguitala Township (40°19'N, 107°01'E), Hangjin Banner, located within the northwestern Ordos Plateau's geomorphological boundaries, specifically in the central Hobq Desert(Fig. 1 ). This region experiences a temperate continental monsoon climate (Köppen classification BWk), exhibiting substantial seasonal thermal and hygric variations. The average temperature is 6°C, with extreme minimum and maximum temperatures recorded at -19.7°C and 29.9°C, respectively. The maximum monthly average temperature reaches 25.5°C, while the lowest monthly average temperature is -10.2°C. Winters are characterized as cold and dry, whereas summers are typically warm and humid. The annual sunlight hours total 3166.3, the frost-free period spans 135 days, the plant growth period lasts 158 days, and the annual precipitation measures 250 mm. Sample Plot Layout and Sampling Methods The sample plots were established in the Amugulong Liquorice Industrial Demonstration Park of Yili Resource Group for field experiments. The research focused on enhancing windy sandy soil through the combination of Pisha sandstone and sandy soil, the cultivation of licorice, and the incorporation of organic fertilizers. The sample plots were organized into five blocks, each approximately 6666.67 m², characterized by windy sandy soils. The plots were leveled through both manual and mechanical means to ensure parameter consistency across each sample plot. The Pisha sandstone utilized in this study was weathered Pisha sandstone sourced from the Ertigou sub-basin (110°32′E ~ 111°6′E, 39°26′N ~ 39°56′N) of Ordos Jungar Banner, exhibiting a dry density ranging from 1.36 to 1.45 g/cm³ and a water content approximately 10.8%. Various volumes of Pisha sandstone (0%, 30%, 50%, 80%, and 100%) were initially applied to the surface of sandy soil. The soil was tilled using a machine to a depth of 0–30 cm, with the process repeated three times to maximize the mixing of Pisha sandstone with the sandy soil. The area of each sample plot was approximately 6666.67 m², where mixed tilling and planting of licorice occurred. Organic fertilizers were applied to the selected plots through furrow application, and watering and maintenance were conducted using sprinkler irrigation systems to ensure uniform water distribution across all plots during maintenance activities. In the subsequent year, three 10 × 10 m sample plots were randomly selected from each of the five fixed sample plots. Additional microbial agents were applied to the selected sample plots via furrow application, achieving a depth of 15–20 cm to manage the integrated measures effectively. The addition of microbial agents was recorded as M, and the absence of the addition of microbial agents was not recorded as NM. Soil sampling was conducted at the center of each 10×10 m sample plot, targeting a depth of 10–20 cm. One kilogram of samples was collected from each area. The nutrient contents of the samples, including available phosphorus and nitrogen concentrations as well as soil organic carbon, were determined after air drying, impurity removal, and screening. The sample was conducted at nine "S" shaped locations within each 10× 10 m survey plot, with the average value derived from nine repeated measurements. Composite soil samples were obtained by proportional mixing of equal-volume aliquots collected from three distinct soil depths (0–10, 10–20, 20–30 cm) at each georeferenced sampling node, with homogenization achieved. The samples were sealed in plastic ziplock bags, placed in a refrigerator with an ice box, and subsequently transported to the laboratory, where they were refrigerated at 4℃. Soil microbial counts and enzyme activities were assessed over a brief timeframe. Test material Organic fertilizers are cost-effective to prepare and, with prolonged application, can substantially enhance soil characteristics and its micro-ecological environment, hence fostering crop growth 24 – 26 , consequently, organic fertilizers were incorporated into the Pisha sandstone after being mixed with sandy soil. "The organic fertilizer formulation was independently developed by our research team through microbial fermentation coupled with aerobic composting technology(Fig. 2 ), demonstrating an optimal application dosage of 3 cubic meters per 666.67 square meters (equivalent to 1 Chinese mu) Organic fertilizer generated via aerobic composting offers benefits such as enhanced nutrient efficiency, elevated organic matter content, holistic nutrition, soil conditioning, stimulation of microbial activity in the soil, mitigation of soil crusting, improved soil aeration, and decreased water loss and evaporation, thereby facilitating crop growth and development. Southeast University developed a composite microbial agent comprising four strains: Strains Bacillus halotolerans P75, Sinorhizobium meliloti D10, Bacillus megaterium H3, and Bacillus subtilis HB01. P75 is a bacterial strain that facilitates plant-to-plant interaction, D10 belongs to the rhizobial group, which engages in symbiotic partnerships with legumes to improve nitrogen fixation, and H3 is a phosphate-solubilizing bacterium. Inter-root bacterium strain P75, rhizobium group strain D10, which symbiotically enhances nitrogen fixation in leguminous plants; phosphorus-solubilizing bacterium strain H3, which converts insoluble phosphorus into a soluble form for plant absorption; and HB01 27 , which suppresses pathogenic bacteria and bolsters plant resilience, are all administered at a dosage of 8 kg per 666.67 m². Licorice: In the arid region of northwest China, licorice, a perennial herb belonging to the genus Glycyrrhiza within the Leguminosae family, is cultivable due to its widespread agricultural use, medicinal efficacy, and resilience to extreme conditions such as cold, drought, and poor soil fertility 28 , 29 . The patented semi-wild biochemical cultivation technology, independently developed by Yili Resources Group Co. Ltd., was employed for planting. Licorice seedlings were sown in the experimental field during spring using a licorice planting machine, adhering to a row spacing of 33 cm, a plant spacing of 15 cm, and a depth of 25 cm. The quantity of licorice seedlings planted per mu was 60 kg, equating to approximately 9 plants/m². Methods for the determination of soil indicators and survey of licorice growth conditions Soil organic carbon content was determined by potassium dichromate oxidation spectrophotometry; available nitrogen was determined by the alkaline diffusion method; available phosphorus was extracted by sodium bicarbonate and determined by molybdenum antimony colourimetry. Soil urease levels were assessed through the indophenol blue colorimetric technique. Experimental protocol involved homogenizing 1.0 g soil with 50 mM urea solution in borate buffer (pH 10.0) under controlled incubation at 37°C for 2 hours. Post-reaction processing included quenching with 1 M KCl followed by membrane filtration. Ammonium ion quantification was performed using a sodium dichloroisocyanurate-salicylate/NaOH color development system, with absorbance readings recorded at 690 nm. Results were standardized as microgram ammonium nitrogen generated per gram desiccated soil per hour (µg NH₄⁺-N·g⁻¹·h⁻¹) 30 . The DNS colorimetric assay quantified invertase activity by measuring enzymatically liberated glucose. In brief, 5 g of air-dried soil was combined with 15 mL 8% (w/v) sucrose solution, 5 mL phosphate buffer (0.2 M, pH 5.5), and 5 drops toluene preservative, incubated at 37°C for 24 h. Post-incubation filtration yielded a clear supernatant, 1 mL of which underwent chromogenic reaction with 3 mL DNS reagent during 5-minute boiling water bath treatment. Spectrophotometric analysis at 550 nm determined glucose equivalence, with activity expressed as milligram glucose per gram soil per 24-hour cycle (mg glucose·g⁻¹·24 h⁻¹) 31 . Acid phosphatase measurements followed Tabatabai-Bremner's spectrophotometric protocol involving para-nitrophenyl phosphate hydrolysis, with enzymatic activity derived from p-nitrophenol calibration curves. Microbial enumeration was performed via serial dilution plating. Soil suspensions were prepared by homogenizing 10.0 g samples with 90 mL sterile water using orbital agitation (20 min, 25°C), followed by serial decimal dilutions through sequential 1 mL transfers to 9 mL sterile blanks. Target microorganisms received specific dilution gradients: fungi (10⁻⁴), actinomycetes (10⁻⁵), and bacteria (10⁻⁶). Aliquots (0.2 mL) from respective dilutions were plated on selective media - Gao's No.1 for fungi, Madin's medium for actinomycetes, and beef extract-peptone agar for bacteria. Inverted plates were parafilmed and incubated under optimal conditions. Concurrently, 5.0 g soil subsamples in 45 mL sterile water yielded 10⁻²-10⁻⁴ dilutions for differential cultivation: PDA medium with bacteriostatic antibiotics (penicillin-streptomycin, 50 mg·L⁻¹) for fungi (48h incubation), LB agar for bacteria (10⁻³-10⁻⁴), and dichromate-amended Gaoshi No.1 medium (K₂Cr₂O₇ 50 mg·L⁻¹) for actinomycetes (10⁻³-10⁻⁴). Actinomycete analysis required preliminary heat treatment (120°C, 60 min) of atrazine-containing samples before dilution 32 , 33 . Synchronized with the soil sampling time, within each 10×10 m survey sample plot, three 1×1 m licorice survey sample plots were selected along the diagonal, respectively, and the height of licorice plants in each sample plot was counted. Data processing Statistical analyses were conducted using SPSS 20.0. Two-way ANOVA was performed to evaluate main effects and interactions, followed by Duncan's multiple range test for post hoc comparisons at a significance threshold of p < 0.05. Pearson correlation analyses were subsequently implemented to examine relationships between soil nutrients (organic matter, available N/P/K), microbial populations (bacteria, fungi, actinomycetes), enzymatic activities (urease, invertase, acid phosphatase), and growth parameters of Glycyrrhiza uralensis (plant height, biomass, root morphology) under different experimental treatments. Results Effects of different Pisha sandstone addition volume ratios and microbial agents on soil organic carbon, available nitrogen, and available phosphorus changes The experimental data reveal significant variations in soil nutrient profiles with Pisha sandstone incorporation, as detailed in Fig. 3 . For organic carbon (Fig. 3 a), differential treatment responses emerged: NM demonstrated a unimodal pattern peaking at 50% amendment (2.07 g/kg), representing a 1.01-fold increase over CK (Pisha sandstone added at 0% by volume and no microbial agent applied). Conversely, M exhibited progressive carbon accumulation until stabilization above 50% amendment, achieving 1.65 times the CK level at optimal incorporation. At the same Pisha sandstone addition volume ratio, the soil organic carbon content of M was higher than that of NM, and there was no significant difference between M and NM. The available nitrogen content in both NM and M systems displayed a characteristic unimodal response to Pisha sandstone amendment ratios, as evidenced in Fig. 3 (b). Peak nitrogen accumulation occurred at 50% volumetric amendment, with NM and M attaining maximum concentrations of 7.41 mg/kg and 9.881 mg/kg, respectively, corresponding to 1.17-fold and 2.05-fold enhancements relative to CK controls. The nitrogen content of M was greater than that of NM at the same volume ratio of Pisha sandstone addition. Phosphorus dynamics (Fig. 3 c) mirrored these patterns with optimal values at 50% incorporation: 6.11 mg/kg (NM, 1.03 times CK) and 7.49 mg/kg (M, 1.49 times CK). Systematic performance advantages persisted in microbial-treated soils across all tested ratios, demonstrating 22.6% higher maximum phosphorus levels compared to non-microbial counterparts. Effect of different Pisha sandstone addition volume ratios and microbial agents on soil enzyme activities The experimental results demonstrate significant variations in soil enzyme activities under different Pisha sandstone incorporation ratios (Fig. 4 ). Regarding acid phosphatase activity (Fig. 4 a), both NM and M treatments exhibited a unimodal response pattern, achieving peak values of 13.52 and 38.47 µg /g soil respectively at 50% volumetric proportion. These maxima represented 56.12% and 344.23% enhancements compared to CK, with M treatment consistently demonstrating superior activity to NM across all tested ratios. Invertase dynamics (Fig. 4 b) revealed distinct behavioral patterns: M treatment showed a characteristic rise-decline trend peaking at 50% ratio (18.09 mg/g,94.21-fold increase vs CK), while NM displayed progressive enhancement until stabilization at 50% ratio. Notably, M maintained significantly higher invertase activity than NM at equivalent incorporation levels. Figure 4 (c) illustrates that following the compounding of Pisha sandstone and sandy soil, urease activity in M exhibited an initial increase followed by a decrease as the volume ratio of Pisha sandstone increased. The maximum urease activity, recorded at a 50% volume ratio of Pisha sandstone, was 330.91 µg NH3-N/g, representing a 19.52-fold increase compared to CK. Conversely, urease activity in NM consistently increased with the rising volume ratio of Pisha sandstone. The sucrase activity of M exceeded that of NM at volume ratios of Pisha sandstone addition of 0%, 30%, 50%, and 80%. However, at a 100% volume ratio of Pisha sandstone addition, the urease activity of NM surpassed that of M. Effects of different Pisha sandstone addition ratios and measures on the populations of the three major soil bacterial groups and the height of licorice plants Microbial population dynamics under Pisha sandstone amendment revealed distinct biphasic patterns in bacterial colonization (Fig. 5 a). Maximum bacterial densities were recorded at 80% volumetric incorporation, with NM and M systems achieving 21.37×10⁵ cfu/g and 78.03×10⁵ cfu/g respectively, corresponding to 17.91-fold and 68.05-fold enhancements relative to CK controls. Both systems demonstrated initial proliferation followed by population decline with increasing amendment ratios, while microbial augmentation (M) consistently maintained superior bacterial loads compared to non-microbial counterparts (NM) across equivalent treatment levels. Fungal community responses to Pisha sandstone amendment paralleled bacterial dynamics, as shown in Fig. 5 b. Optimal fungal proliferation occurred at 80% incorporation, with NM and M attaining peak values of 32.77×10⁵ cfu/g and 149.7×10⁵ cfu/g respectively, representing 58.58-fold and 271.18-fold increases over CK. The quantity of fungi in M exceeded that in NM at the identical Pisha sandstone addition volume ratio. Actinomycete colonization patterns diverged temporally from other microbial groups (Fig. 5 c). Maximum population densities emerged at 50% Pisha sandstone incorporation, with NM and M registering 27.37×10⁵ cfu/g and 95.2×10⁵ cfu/g respectively, equivalent to 0.073-fold and 2.73-fold CK increases. Notably, actinomycete communities demonstrated enhanced environmental stability compared to bacterial and fungal counterparts. The quantity of actinomycetes was greater in M compared to NM at the identical volume ratio of Pisha sandstone addition. Plant height dynamics under Pisha sandstone amendment exhibited biphasic responses in both treatment systems (Fig. 6 ). Peak growth metrics were recorded at 50% incorporation, with NM and M attaining 39.17 cm and 55.22 cm respectively, corresponding to 0.65-fold and 1.33-fold increases relative to CK. Significant differential responses emerged between systems: M maintained statistical significance (p < 0.05) across all amendment ratios, whereas NM showed significant divergence (p < 0.05) only below 30% incorporation. Both systems demonstrated initial height enhancement followed by progressive reduction with increasing Pisha sandstone proportions beyond optimal levels. Discussion Analysis of the correlation between soil nutrients, microbial populations, enzyme activity, and licorice growth. As can be seen in Figure 7, a markedly positive association existed between soil organic carbon and available nitrogen, as well as available phosphorus, suggesting that a rise in soil organic carbon is generally linked to increases in these parameters. This aligns with prior research regarding the significance of soil nutrient concentrations (e.g., available nitrogen, available phosphorus) on soil organic carbon accumulation in soil ecosystems 34,35 . Despite a low correlation between organic carbon and acid phosphatase, there exists a highly significant positive correlation with urease and sucrase, indicating that organic carbon substantially enhances the activity of these enzymes, potentially by supplying energy or other supportive substances. Moreover, soil organic carbon was also highly positively correlated with bacterial and fungal populations, possibly due to the improved correlation between bacterial and fungal availability of soil carbon. In conclusion, the increase in soil organic carbon favors the growth of soil microorganisms and the enhancement of soil readily available nutrients. A significantly substantial positive association existed between soil available nitrogen and available phosphorus, indicating coordinated variations in their concentrations. Soil acid phosphatase exhibited a highly significant positive correlation with available phosphorus and plant height, indicating that acid phosphatase activity facilitates the release and transformation of phosphorus, thereby enhancing plant phosphorus uptake and growth. This aligns with the understanding that phosphatase is a hydrolytic enzyme that aids in the decomposition and transformation of organic phosphorus, improving phosphorus availability for plant absorption in the soil 36 . Previous studies have shown that invertase is involved in converting and cycling soil carbon and nitrogen 37 , and urease can promote crop uptake of nitrogen. Moreover, substantial positive correlations were identified between invertase and available nitrogen, available phosphorus, and plant height, suggesting that invertase plays a crucial role in the transformation of nitrogen and phosphorus in the soil, thereby indirectly enhancing plant growth by influencing nutrient availability. A substantial positive association was identified between urease activity and both available nitrogen content and plant height, indicating that urease activity facilitates nitrogen release, hence enhancing the efficiency of nitrogen absorption by the plant. Purushotham et al. 38 and Hoyos et al. 39 demonstrated that actinomycetes can enhance the development of plant rhizomes and significantly increase plant height. A significant positive correlation was observed between actinomycete counts and licorice plant height, indicating that actinomycetes may promote plant growth. The direct impact of actinomycetes on plant growth may be less significant than that of phosphatase in relation to other factors. The correlation analysis results indicated a complex interrelationship among organic carbon, available nitrogen, available phosphorus, microorganisms, and enzyme activities in the soil. An increase in organic carbon, available nitrogen, and available phosphorus enhanced the number of microorganisms and their associated enzyme activities in the soil, positively influencing plant growth. Significant correlations among microorganisms suggest their complementary functions within the soil ecosystem, while the interactions of various enzymes additionally influence soil fertility and plant growth. The findings illustrate the intricate relationship between soil health and plant growth, highlighting the synergistic effects of various factors. The impact of varying volumes of Pisha sandstone and microbial agents on soil nutrient levels. Soil organic carbon is crucial for the formation and stabilization of soil aggregates, serving as a fundamental component of soil quality and significantly influencing soil fertility 40,41 . A reduction in soil organic carbon content correlates with diminished stability of soil structure 42,43 . Nitrogen and phosphorus deficiencies are essential indicators of soil fertility and have been recognized as key factors influencing plant growth 44,45 . The combination of Pisha sandstone with sandy soil resulted in a significant enhancement of soil organic carbon, available nitrogen, and available phosphorus compared to the control (CK), with no notable difference observed between the M and NM treatments (Figure 3). The organic carbon content in the soil remained stable after the addition of Pisha sandstone at a volume ratio of 50% (Figure 3 (a)). This stability may be attributed to the significant difference (p < 0.05) observed prior to reaching this volume ratio. At a volume ratio of 50% Pisha sandstone, the soil exhibited the highest levels of available nitrogen and available phosphorus (Figure 3 (b), 3 (c)), resulting in maximum nitrogen and phosphorus availability for licorice. The addition of Pisha sandstone at a volume ratio of 50% did not result in a significant difference in the available nitrogen content in the soil. However, the available phosphorus content in the soil showed a significant difference prior to the addition of Pisha sandstone at a volume ratio of 30% (p < 0.05). Consequently, based on the comprehensive soil organic carbon, available nitrogen, and available phosphorus content, the optimal improvement effect occurs when the volume ratio of Pisha sandstone reaches 50%. In comparison to CK, the soil organic carbon, available nitrogen, and available phosphorus content of NM increased by 1.01, 1.17, and 1.03 times, respectively. Meanwhile, the soil organic carbon, available nitrogen, and available phosphorus content of M increased by1.01, 1.17, and 1.03 times, respectively. The current soil structure exhibits greater stability compared to alternative measures, and soil fertility is at its peak. This indicates that the incorporation of microbial agents in the combination of Pisha sandstone with sandy soil is more effective than the compounding effect alone. Impact of varying Pisha sandstone addition volume ratios and microbial agents on enzyme activity, the abundance of three primary bacterial groups, and licorice growth. Soil enzymes are secreted into the soil by decomposing microorganisms associated with plant and animal residues. They are intricately connected to factors influencing soil fertility, serving as a critical indicator of soil fertility by reflecting microbial activity levels and the capacity for nutrient conversion, transport, and metabolism 46,47 . The ecological cycle of nature is significantly influenced by soil microbes. Their metabolism and associated processes directly influence the movement and circulation of materials and energy within the soil, making them essential indicators for assessing soil fertility 48,49 . The combination of Pisha sandstone with sandy soil resulted in a significant enhancement of acid phosphatase, invertase, and urease activities, as well as an increase in the populations of bacteria, fungi, and actinomycetes, in comparison to the control (Figures 4 and 5). Furthermore, at an equivalent volume ratio of Pisha sandstone, enzyme activity and the abundance of the three primary bacterial groups in M were significantly elevated compared to NM. This disparity may be attributed to the microbial agent's substantial content of microorganisms and organic residues from animals and plants. At a 50% volume ratio of Pisha sandstone, the activities of acid phosphatase, invertase, and urease in M peaked, exhibiting increases of 4.98 times, 81 times, and 18.37 times compared to CK, respectively. In NM, acid phosphatase activity also peaked at a 50% volume ratio of Pisha sandstone, though it was comparable to CK. The activities of invertase and urease rose with increasing Pisha sandstone volume, but after reaching a 50% volume ratio, the increase became more gradual, indicating that the addition of microbial agents enhances soil enzyme activity. With the increasing proportion of Pisha sandstone, the populations of bacteria and fungi rose markedly; however, the variation in actinomycete numbers was less pronounced (refer to Figure 5). No significant difference was observed between M and NM. Bacteria exhibited significant differences at a Pisha sandstone volume ratio of 30% (p<0.05), while no significant differences were noted for fungi and actinomycetes concerning the volume ratio of Pisha sandstone added. The quantity of actinomycetes exhibited minimal variation across different addition ratios, indicating a robust adaptation to the Pisha sandstone addition ratio. A high population of fungi can readily induce soil-borne illnesses and transform the soil from a fertile bacterial type to a less fertile fungal type 50-52 . Consequently, the fraction of fungi within the aggregate of the three predominant soil microorganisms should remain relatively low. At a Pisha sandstone volume ratio of 50%, fungi in NM constituted 34.45% of the total population of the three primary soil microorganisms (bacteria, fungi, and actinomycetes), while fungi in M represented40.24% of the total population of these microorganisms. When the volume ratio of Pisha sandstone was 80%, the fungal population in NM constituted 43.11% of the total of the three predominant soil floras, whereas the fungal population in M comprised 50.97% of the total of the three predominant soil floras. Furthermore, when the volume ratio of Pisha sandstone was 80%, both NM and M exerted a considerable detrimental effect on actinomycetes. When the volume ratio of Pisha sandstone was 50%, the height of the licorice plant for NM and M attained its maximum (Figure 5). It may be inferred that a 50% volume ratio of Pisha sandstone yields optimal enhancement of sandy soil, which is further increased by the addition of microbial agents. Conclusion This study thoroughly examined the impact of combining Pisha sandstone with sandy soil on soil enhancement, assessing the improvement effects at various addition volume ratios and the use of microbial agents. The findings indicated that the amalgamation of Pisha sandstone and sandy soil markedly enhanced the nutritional composition, microbial population, and enzymatic activity of the soil. In the experiment, irrespective of the addition of microbial agents, when the volume ratio of Pisha sandstone reached 50%, the soil exhibited peak levels of organic carbon, available nitrogen, and available phosphorus, alongside a significant increase in soil microorganisms and enzyme activity. The aforementioned indicates that incorporating Pisha sandstone at this volume ratio significantly enhances the physical and chemical qualities of the soil and fosters the growth of licorice. However, too high a volume ratio of Pisha sandstone may lead to negative changes in soil properties, thereby inhibiting plant growth. The findings indicate that a 50% volume ratio of Pisha sandstone and sandy soil, when supplemented with suitable fertilizers and microbial treatments, can significantly enhance sandy soil. This presents a viable approach for the management and amelioration of sandy soil, which is crucial for increasing licorice yield and offers a scientific foundation and practical guidance for sandy soil management. Nonetheless, there remains a deficiency in research regarding the optimal quantity of microbial agents to apply per square meter for improved sandy soil conditions involving Pisha sandstone and sandy soil. Future investigations will address this gap. Declarations Data availability The datasets generated, used, and/or analyzed during the current study are available from the corresponding author upon reasonable request. Acknowledgements (not compulsory) This research was funded by was supported by the National Natural Science Foundation of China (42207394), Guangxi Key Research and Development Program of China (AB21196048); Forestry and Grassland Science and Technology Promotion Demonstration Project of the Central Government ([2023] TG17) and Key Laboratory Autonomous Projects (2021-A-02-01). The authors are grateful to Guangxi Forest's new fertilizer research and development Center, and School of Civil Engineering, Southeast University, and National and Local Unified Engineering Research Center for Basalt Fiber Production and Application Technology, Southeast University. Special thanks go to the anonymous reviewers for their constrictive comments in improving this manuscript. Author contributions statement M Writing—original draft preparation, ZS. L. and XW. F.; validation B,P.; formal analysis LX. Z.; data curation and investigation, LX Z; resources, ZS. L.; writing—review and editing, ZS. 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Deep tillage combined with biofertilizer following soil fumigation improved chrysanthemum growth by regulating the soil microbiome. Microbiologyopen 9 , e1045. 10.1002/mbo3.1045 (2020). Deng, L., Wang, T., Luo, W., He, L. Y. & Liang, Z. S. Effects of a compound microbial agent and plants on soil properties, enzyme activities, and bacterial composition of Pisha sandstone. Environ. Sci. Pollut. Res. 28 , 53353–53364. 10.1007/s11356-021-14533-x (2021). Pan, Y., Wu, L. J. & Yu, Z. L. Effect of salt and drought stress on antioxidant enzymes activities and SOD isoenzymes of liquorice ( Glycyrrhiza uralensis Fisch ). Plant. Growth Regul. 49 , 157–165. 10.1007/s10725-006-9101-y (2006). Hayashi, H. & Sudo, H. Economic importance of licorice. Plant. Biotechnol. 26 , 101–104. 10.5511/plantbiotechnology.26.101 (2009). Wan, X. et al. Urban forest soil properties and microbial characteristics: seasonal and stand-specific variations. Appl. Soil. Ecol. 209 10.1016/j.apsoil.2025.105995 (2025). Zhang, C., Yu, X. W., Laipan, M., Wei, T. & Guo, J. K. Soil health improvement by inoculation of indigenous microalgae in saline soil. Environ. Geochem. Health . 46 10.1007/s10653-023-01790-7 (2024). Ji, X. H. et al. Responses of Soil Microbial Community to Herbicide Atrazine Contamination. Water Air Soil. Pollution . 234 10.1007/s11270-023-06284-x (2023). Chen, S., Dong, C. W., Gao, Y., Li, Y. J. & Shi, Y. Effects of nanocarbon and nano-calcium carbonate on soil enzyme activities and soil microbial community in wheat (Triticum aestivum L.) rhizosphere soil. J. Plant Nutr. Soil Sci. 187 , 639–652. 10.1002/jpln.202300146 (2024). Zhang, Y. et al. Change Characteristics of Soil Organic Carbon and Soil Available Nutrients and Their Relationship in the Subalpine Shrub Zone of Qilian Mountains in China. Sustainability 15 , 13028. 10.3390/su151713028 (2023). Liu, Z. et al. A simple assessment on spatial variability of rice yield and selected soil chemical properties of paddy fields in South China. Geoderma 235 , 39–47. 10.1016/j.geoderma.2014.06.027 (2014). Spiers, G. & McGill, W. Effects of phosphorus addition and energy supply on acid phosphatase production and activity in soils. Soil Biol. Biochem. 11 , 3–8 (1979). Jones, D. L., Nguyen, C. & Finlay, R. D. Carbon flow in the rhizosphere: carbon trading at the soil-root interface. Plant. Soil. 321 , 5–33. 10.1007/s11104-009-9925-0 (2009). Purushotham, N., Jones, E., Monk, J. & Ridgway, H. Community Structure of Endophytic Actinobacteria in a New Zealand Native Medicinal Plant Pseudowintera colorata (Horopito) and Their Influence on Plant Growth. Microb. Ecol. 76 , 729–740. 10.1007/s00248-018-1153-9 (2018). Vargas Hoyos, H. A. et al. An Actinobacterium Strain From Soil of Cerrado Promotes Phosphorus Solubilization and Plant Growth in Soybean Plants. Front. Bioeng. Biotechnol. 9 , 579906. 10.3389/fbioe.2021.579906 (2021). Johannes, A., Sauzet, O., Matter, A. & Boivin, P. Soil organic carbon content and soil structure quality of clayey cropland soils: A large-scale study in the Swiss Jura region. Soil Use Manag. 39 , 707–716. 10.1111/sum.12879 (2023). Reeves, D. W. The role of soil organic matter in maintaining soil quality in continuous cropping systems. Soil. Tillage Res. 43 , 131–167. 10.1016/s0167-1987(97)00038-x (1997). Rasool, R., Kukal, S. S. & Hira, G. S. Soil organic carbon and physical properties as affected by long-term application of FYM and inorganic fertilizers in maize-wheat system. Soil. Tillage Res. 101 , 31–36. 10.1016/j.still.2008.05.015 (2008). Filho, C. C., Lourenco, A., Guimaraes, M. D. F. & Fonseca, I. C. B. Aggregate stability under different soil management systems in a red latosol in the state of Parana, Brazil. Soil. Tillage Res. 65 , 45–51 (2002). Cheng, S., Ke, G., Li, Z., Cheng, Y. & Wu, H. Soil Available Phosphorus Investigated for Spatial Distribution and Effect Indicators Resulting from Ecological Construction on the Loess Plateau, China. Sustainability 13 , 12572. 10.3390/su132212572 (2021). Abaker, W. E., Berninger, F., Saiz, G., Pumpanen, J. & Starr, M. Linkages between soil carbon, soil fertility and nitrogen fixation in Acacia senegal plantations of varying age in Sudan. Peerj 6 , e5232. 10.7717/peerj.5232 (2018). Qiu, L., Jun, L., Wang, Yiquan, Huimin, S. & He, W. Research on relationship between soil enzyme activities and soil fertility. J. Plant. Nutr. Fertilizers , 277–280 (2004). Garcia-Ruiz, R., Ochoa, V., Belen Hinojosa, M. & Antonio Carreira, J. Suitability of enzyme activities for the monitoring of soil quality improvement in organic agricultural systems. Soil Biol. Biochem. 40 , 2137–2145. 10.1016/j.soilbio.2008.03.023 (2008). Lange, M. et al. Plant diversity increases soil microbial activity and soil carbon storage. Nat. Commun. 6 , 6707 (2015). Yaghoubi Khanghahi, M., Cucci, G., Lacolla, G., Lanzellotti, L. & Crecchio, C. Soil fertility and bacterial community composition in a semiarid Mediterranean agricultural soil under long-term tillage management. Soil Use Manag. 36 , 604–615. 10.1111/sum.12645 (2020). Wang, Y., Ren, J., Zhang, Y., Zheng, Y. & Tang, L. Effect of wheat and faba bean intercropping on improving rhizosphere microflora and reducing fusarium wilt of faba bean. Chin. J. Soil. Sci. 51 , 127–1133 (2020). Khanghahi, M. Y., Cucci, G., Lacolla, G., Lanzellotti, L. & Crecchio, C. Soil fertility and bacterial community composition in a semiarid Mediterranean agricultural soil under long-term tillage management. Soil Use Manag. 36 , 604–615. 10.1111/sum.12645 (2020). Ying, H., Xiaotong, X., Xiaori, H., Jun, L. & Jiaqi, L. Effects of different long-term fertilization patterns on soil microflora. J. South. China Agricultural Univ. 37 , 51–58 (2016). Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 19 Nov, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 28 Jul, 2025 Reviewers agreed at journal 17 Jun, 2025 Reviews received at journal 10 Jun, 2025 Reviewers agreed at journal 31 May, 2025 Reviewers invited by journal 09 May, 2025 Submission checks completed at journal 07 May, 2025 First submitted to journal 04 May, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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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-6408674","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":455214312,"identity":"0d55894e-f2d1-4202-8897-7bf9ce68e6bb","order_by":0,"name":"Zhishui Liang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA3ElEQVRIiWNgGAWjYHACxgMJQJIfwmEmTg9Yi2QDSVpAhMEBYrXI9x9gOPCg5o7d5vOHj0kwVFgnNrCfPYBXC+OMBKDDjj1L3nYjLU2C4Ux6YgNPXgJeLcwSIL+wHU42u8FjJsHYdjixQYLHAK8WNn6gwxL+HU427j//TYLxHxFaeBiADktsO2xnwJDDJsHYQIQWCQmQlr7DCRI30owtEo6lG7fx5ODXAgwxxoc/vh225+8//PDGhxpr2X72M/i1AKP9A4hMbACRCSDfEVAPB/bEKhwFo2AUjIIRCACL4EWkT7DNGwAAAABJRU5ErkJggg==","orcid":"","institution":"Southeast University","correspondingAuthor":true,"prefix":"","firstName":"Zhishui","middleName":"","lastName":"Liang","suffix":""},{"id":455214313,"identity":"992e5501-9597-4c08-8f4d-8d5a2b4d5ad0","order_by":1,"name":"Xiuwen Fang","email":"","orcid":"","institution":"Southeast University","correspondingAuthor":false,"prefix":"","firstName":"Xiuwen","middleName":"","lastName":"Fang","suffix":""},{"id":455214314,"identity":"34419260-2669-46cc-acc6-7d0318aa60f7","order_by":2,"name":"Haiying Gao","email":"","orcid":"","institution":"Southeast University","correspondingAuthor":false,"prefix":"","firstName":"Haiying","middleName":"","lastName":"Gao","suffix":""},{"id":455214315,"identity":"64848a11-252d-44a9-989e-344a32f424a7","order_by":3,"name":"Bo Pan","email":"","orcid":"","institution":"guangxi academy of forestry","correspondingAuthor":false,"prefix":"","firstName":"Bo","middleName":"","lastName":"Pan","suffix":""},{"id":455214316,"identity":"95905f6e-9285-4dc0-8b33-c0e623be68cf","order_by":4,"name":"Jishu Zhang","email":"","orcid":"","institution":"Inner Mongolia Kubuqi Desert Technology Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Jishu","middleName":"","lastName":"Zhang","suffix":""}],"badges":[],"createdAt":"2025-04-09 06:38:23","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6408674/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6408674/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-24467-w","type":"published","date":"2025-11-19T15:57:59+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":82694426,"identity":"2d5ffed2-57bd-4f9b-bf51-e86dee56db37","added_by":"auto","created_at":"2025-05-14 08:30:19","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":204738,"visible":true,"origin":"","legend":"\u003cp\u003eLocation of the study area.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-6408674/v1/498bce110c3b448c53d15b98.png"},{"id":82694429,"identity":"45219b19-6e18-45b8-9fc8-98aca6afae7b","added_by":"auto","created_at":"2025-05-14 08:30:19","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":140924,"visible":true,"origin":"","legend":"\u003cp\u003eOrganic fertilizer production process.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-6408674/v1/23bd0c305c5e2e461a30befc.png"},{"id":82694428,"identity":"c3763f9d-c99d-4d37-9261-de9497d2caf4","added_by":"auto","created_at":"2025-05-14 08:30:19","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":195325,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of different Pisha sandstone addition volume ratios and microbial agents on the content of soil organic carbon(a), available nitrogen(b), and available phosphorus(c)\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNote:\u003c/strong\u003e Different lowercase letters indicate significant differences among Pisha sandstone addition ratio treatments (p \u0026lt; 0.05). Significant main effects of microbial agent were observed for all measured parameters (soil organic matter: F = 149.784; available nitrogen: F = 167.628; available phosphorus: F = 95.969; p \u0026lt; 0.001), while no significant interaction effects between microbial agent and sandstone ratio were detected (p \u0026gt; 0.05). Data in the figure are presented as mean ± standard error (SE) (n = 3).\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-6408674/v1/6a047e2ab4675b6f76230daa.png"},{"id":82695867,"identity":"b65eb5b7-37b7-46f4-a504-7822a9e02ae7","added_by":"auto","created_at":"2025-05-14 08:46:19","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":177809,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of different Pisha sandstone addition volume ratios and microbial agents on the activities of soil acid phosphatase(a), invertase(b), and urease(c).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNote\u003c/strong\u003e: Different lowercase letters indicate significant differences among Pisha sandstone addition ratio treatments (p \u0026lt; 0.05). Significant main effects of microbial agent were observed for all measured parameters (acid phosphatase: F= 87.329, invertase: F =36.004, urease: F = 25.228; p \u0026lt; 0.001), while no significant interaction effects between microbial agent and sandstone ratio were detected (p \u0026gt; 0.05). Data in the figure are presented as mean ± standard error (SE) (n = 3).\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-6408674/v1/b4fba02e3a659d18c1429b66.png"},{"id":82694435,"identity":"66c11410-6e01-42ec-a330-78c7e1d08a8d","added_by":"auto","created_at":"2025-05-14 08:30:19","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":178273,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of different Pisha sandstone addition volume ratios and microbial agents on the populations of soil bacteria (a)、fungi(b), and actinobacteria(c).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNote\u003c/strong\u003e: Different lowercase letters indicate significant differences among Pisha sandstone addition ratio treatments (p \u0026lt; 0.05). Significant main effects of microbial agent were observed for all measured parameters (bacteria: F= 54.815, fungi: F = 41.238, actinobacteria: F = 133.734; p \u0026lt; 0.001), while no significant interaction effects between microbial agent and sandstone ratio were detected (p \u0026gt; 0.05). Data in the figure are presented as mean ± standard error (SE) (n = 3).\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-6408674/v1/810e87325b6faac4a5c6bafe.png"},{"id":82694434,"identity":"9c3681a9-6047-4351-8792-25da10707c11","added_by":"auto","created_at":"2025-05-14 08:30:19","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":92169,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of different Pisha sandstone addition ratios and microbial agents on licorice height.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNote\u003c/strong\u003e: Different lowercase letters indicate significant differences among Pisha sandstone addition ratio treatments (p \u0026lt; 0.05). Significant main effects of microbial agent were observed for all measured parameters (licorice height: F= 84.578; p \u0026lt; 0.001), while no significant interaction effects between microbial agent and sandstone ratio were detected (p \u0026gt; 0.05). Data in the figure are presented as mean ± standard error (SE) (n = 3).\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-6408674/v1/030bf4589d34d8162527bd76.png"},{"id":82695064,"identity":"549fd7db-a76c-4f4a-8a39-b97ef0814909","added_by":"auto","created_at":"2025-05-14 08:38:19","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":226025,"visible":true,"origin":"","legend":"\u003cp\u003eCorrelation between soil nutrients, microbial population, enzyme activities, and licorice plant height under different treatments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNote:\u003c/strong\u003e*P\u0026lt;0.05,**P\u0026lt;0.01.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAbbreviations:\u003c/strong\u003e SOC(soil organic carbon); AN(available nitrogen); AP(available phosphorus); BAC(bacterial count); FUN(fungi count); ACT(actinobacteria count); UA(urease activity); INA(invertase activity); APA(acid phosphatase activity); H(licorice height).\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-6408674/v1/3735b45a3ade217db815da80.png"},{"id":96650328,"identity":"c7b4a009-477b-463e-88a3-83a1c0870daf","added_by":"auto","created_at":"2025-11-24 16:11:11","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2186416,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6408674/v1/f2cc1724-8362-4e84-8401-b4ef41fddca5.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Effects of Pisha Sandstone Application Rate and Microbial Agents on Soil Nutrient Dynamics and Microbial Characteristics in Sandy Soil","fulltext":[{"header":"Introduction","content":"\u003cp\u003eDesertification, driven by anthropogenic activity, climate change, and disjointed environmental and developmental policies, results in environmental deterioration, and a significant reduction in agricultural productivity, and imposes substantial constraints on economic development while undermining social stability \u003csup\u003e\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. In 1992, the United Nations Environment Programme (UNEP) articulated a definition of desertification, characterizing it as \"land degradation in arid, semi-arid, and dry sub-humid areas resulting from various factors, including climatic variations and human activities\"\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. China is among the nations with the most extensive desertification, the greatest impacted population, and the most severe wind and sand dangers. As per the fifth national desertification and monitoring report, by 2014, the total expanse of sandy terrain in China was 172,117,500 hectares, representing 17.93% of the overall land area\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Aeolian sandy soils possess a substandard soil structure, demonstrating inadequacies in organic matter and nitrogen levels. These soils have high permeability, resulting in substantial water infiltration and diminished capacity for nitrogen and fertilizer retention\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. These properties represent a critical limiting factor in the ecological restoration of desertified areas.\u003c/p\u003e \u003cp\u003eTo solve these difficulties, enhancing sandy soils and alleviating environmental strain, while simultaneously increasing arable land and maintaining national food security, tackling desertification has emerged as a significant and widely recognized issue. Sandy soils exhibit markedly deficient water and nutrient retention capacities, rendering the augmentation of soil organic carbon, structural amelioration, enhancement of hydro-fertilizer retention capabilities, and improvement of crop survival rates pivotal for restoring self-sustaining edaphic ecosystems. Current enhancement strategies for sandy soils employ three principal approaches: engineering techniques, biological interventions, and soil amendments. Engineering methods, particularly sand barrier installation and sand compaction pile (SCP) implementation, present substantial economic costs and environmental burdens. Biological strategies primarily involve anti-desertification afforestation and cultivation of xerophytic vegetation, though their efficacy manifests gradually over extended temporal scales. Consequently, soil amendments have emerged as the predominant remediation approach, with Pisha sandstone demonstrating particular utility as an effective modifier\u003csup\u003e\u003cspan additionalcitationids=\"CR8 CR9 CR10 CR11\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. In China's desertification-affected region, sandy soils and Pisha sandstone (a weakly lithified Mesozoic sedimentary formation) exhibit alternating stratigraphic distributions, with the latter constituting approximately 33% of the total substrate coverage\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Despite its low diagenetic grade and compromised structural integrity, Pisha sandstone contains substantial montmorillonite components, endowing it with exceptional physicochemical properties including elevated specific surface area, enhanced cation exchange capacity, and superior moisture retention characteristics\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e.Therefore, the mixture of the two substances in an optimal ratio contributes to the improvement of soil aggregate structure, enhances windbreak and sand-fixing capacity, improves water and nutrient retention properties, while also promoting the sustainable utilization and development of land resources\u003csup\u003e\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Despite significant research, the current focus is mostly on the erosion resistance of soil following the amalgamation of Pisha sandstone and sandy soil, solute transport, and soil hydraulic properties\u003csup\u003e\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. The enhancement of Pisha sandstone and sand to soil fertility is restricted, and the basic improvement of desertified areas cannot be achieved.\u003c/p\u003e \u003cp\u003eSoil microorganisms are essential constituents of soil ecosystems. Microbial agents, by the synergistic action of their internal microorganisms, effectively enhance soil quality, promote crop growth, mitigate soil disease transmission, and increase agricultural production. It is non-toxic, innocuous, environmentally benign, and economical\u003csup\u003e\u003cspan additionalcitationids=\"CR21 CR22\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThis study investigates the impact of microbial agents on sandy soil and Pisha sandstone, focussing on soil fertility, the abundance of three primary bacterial families, and enzyme activity through field trials. It also examines the overall management solutions for sandy soil, which is crucial for mitigating soil erosion. It also looks at the comprehensive management strategies for sandy soil, which is extremely important for managing soil erosion.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eOverview of the study area\u003c/h2\u003e \u003cp\u003eThe study area is geographically positioned in Duguitala Township (40\u0026deg;19'N, 107\u0026deg;01'E), Hangjin Banner, located within the northwestern Ordos Plateau's geomorphological boundaries, specifically in the central Hobq Desert(Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). This region experiences a temperate continental monsoon climate (K\u0026ouml;ppen classification BWk), exhibiting substantial seasonal thermal and hygric variations. The average temperature is 6\u0026deg;C, with extreme minimum and maximum temperatures recorded at -19.7\u0026deg;C and 29.9\u0026deg;C, respectively. The maximum monthly average temperature reaches 25.5\u0026deg;C, while the lowest monthly average temperature is -10.2\u0026deg;C. Winters are characterized as cold and dry, whereas summers are typically warm and humid. The annual sunlight hours total 3166.3, the frost-free period spans 135 days, the plant growth period lasts 158 days, and the annual precipitation measures 250 mm.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eSample Plot Layout and Sampling Methods\u003c/h3\u003e\n\u003cp\u003eThe sample plots were established in the Amugulong Liquorice Industrial Demonstration Park of Yili Resource Group for field experiments. The research focused on enhancing windy sandy soil through the combination of Pisha sandstone and sandy soil, the cultivation of licorice, and the incorporation of organic fertilizers. The sample plots were organized into five blocks, each approximately 6666.67 m\u0026sup2;, characterized by windy sandy soils. The plots were leveled through both manual and mechanical means to ensure parameter consistency across each sample plot. The Pisha sandstone utilized in this study was weathered Pisha sandstone sourced from the Ertigou sub-basin (110\u0026deg;32\u0026prime;E\u0026thinsp;~\u0026thinsp;111\u0026deg;6\u0026prime;E, 39\u0026deg;26\u0026prime;N\u0026thinsp;~\u0026thinsp;39\u0026deg;56\u0026prime;N) of Ordos Jungar Banner, exhibiting a dry density ranging from 1.36 to 1.45 g/cm\u0026sup3; and a water content approximately 10.8%. Various volumes of Pisha sandstone (0%, 30%, 50%, 80%, and 100%) were initially applied to the surface of sandy soil. The soil was tilled using a machine to a depth of 0\u0026ndash;30 cm, with the process repeated three times to maximize the mixing of Pisha sandstone with the sandy soil. The area of each sample plot was approximately 6666.67 m\u0026sup2;, where mixed tilling and planting of licorice occurred. Organic fertilizers were applied to the selected plots through furrow application, and watering and maintenance were conducted using sprinkler irrigation systems to ensure uniform water distribution across all plots during maintenance activities. In the subsequent year, three 10 \u0026times; 10 m sample plots were randomly selected from each of the five fixed sample plots. Additional microbial agents were applied to the selected sample plots via furrow application, achieving a depth of 15\u0026ndash;20 cm to manage the integrated measures effectively. The addition of microbial agents was recorded as M, and the absence of the addition of microbial agents was not recorded as NM.\u003c/p\u003e \u003cp\u003eSoil sampling was conducted at the center of each 10\u0026times;10 m sample plot, targeting a depth of 10\u0026ndash;20 cm. One kilogram of samples was collected from each area. The nutrient contents of the samples, including available phosphorus and nitrogen concentrations as well as soil organic carbon, were determined after air drying, impurity removal, and screening. The sample was conducted at nine \"S\" shaped locations within each 10\u0026times; 10 m survey plot, with the average value derived from nine repeated measurements. Composite soil samples were obtained by proportional mixing of equal-volume aliquots collected from three distinct soil depths (0\u0026ndash;10, 10\u0026ndash;20, 20\u0026ndash;30 cm) at each georeferenced sampling node, with homogenization achieved. The samples were sealed in plastic ziplock bags, placed in a refrigerator with an ice box, and subsequently transported to the laboratory, where they were refrigerated at 4℃. Soil microbial counts and enzyme activities were assessed over a brief timeframe.\u003c/p\u003e\n\u003ch3\u003eTest material\u003c/h3\u003e\n\u003cp\u003eOrganic fertilizers are cost-effective to prepare and, with prolonged application, can substantially enhance soil characteristics and its micro-ecological environment, hence fostering crop growth\u003csup\u003e\u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003c/sup\u003e consequently, organic fertilizers were incorporated into the Pisha sandstone after being mixed with sandy soil. \"The organic fertilizer formulation was independently developed by our research team through microbial fermentation coupled with aerobic composting technology(Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), demonstrating an optimal application dosage of 3 cubic meters per 666.67 square meters (equivalent to 1 Chinese mu)\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eOrganic fertilizer generated via aerobic composting offers benefits such as enhanced nutrient efficiency, elevated organic matter content, holistic nutrition, soil conditioning, stimulation of microbial activity in the soil, mitigation of soil crusting, improved soil aeration, and decreased water loss and evaporation, thereby facilitating crop growth and development.\u003c/p\u003e \u003cp\u003eSoutheast University developed a composite microbial agent comprising four strains: Strains Bacillus halotolerans P75, Sinorhizobium meliloti D10, Bacillus megaterium H3, and Bacillus subtilis HB01. P75 is a bacterial strain that facilitates plant-to-plant interaction, D10 belongs to the rhizobial group, which engages in symbiotic partnerships with legumes to improve nitrogen fixation, and H3 is a phosphate-solubilizing bacterium. Inter-root bacterium strain P75, rhizobium group strain D10, which symbiotically enhances nitrogen fixation in leguminous plants; phosphorus-solubilizing bacterium strain H3, which converts insoluble phosphorus into a soluble form for plant absorption; and HB01\u003csup\u003e27\u003c/sup\u003e, which suppresses pathogenic bacteria and bolsters plant resilience, are all administered at a dosage of 8 kg per 666.67 m\u0026sup2;.\u003c/p\u003e \u003cp\u003eLicorice: In the arid region of northwest China, licorice, a perennial herb belonging to the genus Glycyrrhiza within the Leguminosae family, is cultivable due to its widespread agricultural use, medicinal efficacy, and resilience to extreme conditions such as cold, drought, and poor soil fertility\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. The patented semi-wild biochemical cultivation technology, independently developed by Yili Resources Group Co. Ltd., was employed for planting. Licorice seedlings were sown in the experimental field during spring using a licorice planting machine, adhering to a row spacing of 33 cm, a plant spacing of 15 cm, and a depth of 25 cm. The quantity of licorice seedlings planted per mu was 60 kg, equating to approximately 9 plants/m\u0026sup2;.\u003c/p\u003e\n\u003ch3\u003eMethods for the determination of soil indicators and survey of licorice growth conditions\u003c/h3\u003e\n\u003cp\u003eSoil organic carbon content was determined by potassium dichromate oxidation spectrophotometry; available nitrogen was determined by the alkaline diffusion method; available phosphorus was extracted by sodium bicarbonate and determined by molybdenum antimony colourimetry.\u003c/p\u003e \u003cp\u003eSoil urease levels were assessed through the indophenol blue colorimetric technique. Experimental protocol involved homogenizing 1.0 g soil with 50 mM urea solution in borate buffer (pH 10.0) under controlled incubation at 37\u0026deg;C for 2 hours. Post-reaction processing included quenching with 1 M KCl followed by membrane filtration. Ammonium ion quantification was performed using a sodium dichloroisocyanurate-salicylate/NaOH color development system, with absorbance readings recorded at 690 nm. Results were standardized as microgram ammonium nitrogen generated per gram desiccated soil per hour (\u0026micro;g NH₄⁺-N\u0026middot;g⁻\u0026sup1;\u0026middot;h⁻\u0026sup1;)\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. The DNS colorimetric assay quantified invertase activity by measuring enzymatically liberated glucose. In brief, 5 g of air-dried soil was combined with 15 mL 8% (w/v) sucrose solution, 5 mL phosphate buffer (0.2 M, pH 5.5), and 5 drops toluene preservative, incubated at 37\u0026deg;C for 24 h. Post-incubation filtration yielded a clear supernatant, 1 mL of which underwent chromogenic reaction with 3 mL DNS reagent during 5-minute boiling water bath treatment. Spectrophotometric analysis at 550 nm determined glucose equivalence, with activity expressed as milligram glucose per gram soil per 24-hour cycle (mg glucose\u0026middot;g⁻\u0026sup1;\u0026middot;24 h⁻\u0026sup1;)\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Acid phosphatase measurements followed Tabatabai-Bremner's spectrophotometric protocol involving para-nitrophenyl phosphate hydrolysis, with enzymatic activity derived from p-nitrophenol calibration curves.\u003c/p\u003e \u003cp\u003eMicrobial enumeration was performed via serial dilution plating. Soil suspensions were prepared by homogenizing 10.0 g samples with 90 mL sterile water using orbital agitation (20 min, 25\u0026deg;C), followed by serial decimal dilutions through sequential 1 mL transfers to 9 mL sterile blanks. Target microorganisms received specific dilution gradients: fungi (10⁻⁴), actinomycetes (10⁻⁵), and bacteria (10⁻⁶). Aliquots (0.2 mL) from respective dilutions were plated on selective media - Gao's No.1 for fungi, Madin's medium for actinomycetes, and beef extract-peptone agar for bacteria. Inverted plates were parafilmed and incubated under optimal conditions. Concurrently, 5.0 g soil subsamples in 45 mL sterile water yielded 10⁻\u0026sup2;-10⁻⁴ dilutions for differential cultivation: PDA medium with bacteriostatic antibiotics (penicillin-streptomycin, 50 mg\u0026middot;L⁻\u0026sup1;) for fungi (48h incubation), LB agar for bacteria (10⁻\u0026sup3;-10⁻⁴), and dichromate-amended Gaoshi No.1 medium (K₂Cr₂O₇ 50 mg\u0026middot;L⁻\u0026sup1;) for actinomycetes (10⁻\u0026sup3;-10⁻⁴). Actinomycete analysis required preliminary heat treatment (120\u0026deg;C, 60 min) of atrazine-containing samples before dilution\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e .\u003c/p\u003e \u003cp\u003eSynchronized with the soil sampling time, within each 10\u0026times;10 m survey sample plot, three 1\u0026times;1 m licorice survey sample plots were selected along the diagonal, respectively, and the height of licorice plants in each sample plot was counted.\u003c/p\u003e\n\u003ch3\u003eData processing\u003c/h3\u003e\n\u003cp\u003eStatistical analyses were conducted using SPSS 20.0. Two-way ANOVA was performed to evaluate main effects and interactions, followed by Duncan's multiple range test for post hoc comparisons at a significance threshold of p\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Pearson correlation analyses were subsequently implemented to examine relationships between soil nutrients (organic matter, available N/P/K), microbial populations (bacteria, fungi, actinomycetes), enzymatic activities (urease, invertase, acid phosphatase), and growth parameters of Glycyrrhiza uralensis (plant height, biomass, root morphology) under different experimental treatments.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eEffects of different Pisha sandstone addition volume ratios and microbial agents on soil organic carbon, available nitrogen, and available phosphorus changes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe experimental data reveal significant variations in soil nutrient profiles with Pisha sandstone incorporation, as detailed in Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e. For organic carbon (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ea), differential treatment responses emerged: NM demonstrated a unimodal pattern peaking at 50% amendment (2.07 g/kg), representing a 1.01-fold increase over CK (Pisha sandstone added at 0% by volume and no microbial agent applied). Conversely, M exhibited progressive carbon accumulation until stabilization above 50% amendment, achieving 1.65 times the CK level at optimal incorporation. At the same Pisha sandstone addition volume ratio, the soil organic carbon content of M was higher than that of NM, and there was no significant difference between M and NM.\u003c/p\u003e\n\u003cp\u003eThe available nitrogen content in both NM and M systems displayed a characteristic unimodal response to Pisha sandstone amendment ratios, as evidenced in Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e(b). Peak nitrogen accumulation occurred at 50% volumetric amendment, with NM and M attaining maximum concentrations of 7.41 mg/kg and 9.881 mg/kg, respectively, corresponding to 1.17-fold and 2.05-fold enhancements relative to CK controls. The nitrogen content of M was greater than that of NM at the same volume ratio of Pisha sandstone addition.\u003c/p\u003e\n\u003cp\u003ePhosphorus dynamics (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ec) mirrored these patterns with optimal values at 50% incorporation: 6.11 mg/kg (NM, 1.03 times CK) and 7.49 mg/kg (M, 1.49 times CK). Systematic performance advantages persisted in microbial-treated soils across all tested ratios, demonstrating 22.6% higher maximum phosphorus levels compared to non-microbial counterparts.\u003c/p\u003e\n\u003ch3\u003eEffect of different Pisha sandstone addition volume ratios and microbial agents on soil enzyme activities\u003c/h3\u003e\n\u003cp\u003eThe experimental results demonstrate significant variations in soil enzyme activities under different Pisha sandstone incorporation ratios (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e). Regarding acid phosphatase activity (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ea), both NM and M treatments exhibited a unimodal response pattern, achieving peak values of 13.52 and 38.47 \u0026micro;g /g soil respectively at 50% volumetric proportion. These maxima represented 56.12% and 344.23% enhancements compared to CK, with M treatment consistently demonstrating superior activity to NM across all tested ratios.\u003c/p\u003e\n\u003cp\u003eInvertase dynamics (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eb) revealed distinct behavioral patterns: M treatment showed a characteristic rise-decline trend peaking at 50% ratio (18.09 mg/g,94.21-fold increase vs CK), while NM displayed progressive enhancement until stabilization at 50% ratio. Notably, M maintained significantly higher invertase activity than NM at equivalent incorporation levels.\u003c/p\u003e\n\u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e(c) illustrates that following the compounding of Pisha sandstone and sandy soil, urease activity in M exhibited an initial increase followed by a decrease as the volume ratio of Pisha sandstone increased. The maximum urease activity, recorded at a 50% volume ratio of Pisha sandstone, was 330.91 \u0026micro;g NH3-N/g, representing a 19.52-fold increase compared to CK. Conversely, urease activity in NM consistently increased with the rising volume ratio of Pisha sandstone. The sucrase activity of M exceeded that of NM at volume ratios of Pisha sandstone addition of 0%, 30%, 50%, and 80%. However, at a 100% volume ratio of Pisha sandstone addition, the urease activity of NM surpassed that of M.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffects of different Pisha sandstone addition ratios and measures on the populations of the three major soil bacterial groups and the height of licorice plants\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMicrobial population dynamics under Pisha sandstone amendment revealed distinct biphasic patterns in bacterial colonization (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ea). Maximum bacterial densities were recorded at 80% volumetric incorporation, with NM and M systems achieving 21.37\u0026times;10⁵ cfu/g and 78.03\u0026times;10⁵ cfu/g respectively, corresponding to 17.91-fold and 68.05-fold enhancements relative to CK controls. Both systems demonstrated initial proliferation followed by population decline with increasing amendment ratios, while microbial augmentation (M) consistently maintained superior bacterial loads compared to non-microbial counterparts (NM) across equivalent treatment levels.\u003c/p\u003e\n\u003cp\u003eFungal community responses to Pisha sandstone amendment paralleled bacterial dynamics, as shown in Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eb. Optimal fungal proliferation occurred at 80% incorporation, with NM and M attaining peak values of 32.77\u0026times;10⁵ cfu/g and 149.7\u0026times;10⁵ cfu/g respectively, representing 58.58-fold and 271.18-fold increases over CK. The quantity of fungi in M exceeded that in NM at the identical Pisha sandstone addition volume ratio.\u003c/p\u003e\n\u003cp\u003eActinomycete colonization patterns diverged temporally from other microbial groups (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ec). Maximum population densities emerged at 50% Pisha sandstone incorporation, with NM and M registering 27.37\u0026times;10⁵ cfu/g and 95.2\u0026times;10⁵ cfu/g respectively, equivalent to 0.073-fold and 2.73-fold CK increases. Notably, actinomycete communities demonstrated enhanced environmental stability compared to bacterial and fungal counterparts. The quantity of actinomycetes was greater in M compared to NM at the identical volume ratio of Pisha sandstone addition.\u003c/p\u003e\n\u003cp\u003ePlant height dynamics under Pisha sandstone amendment exhibited biphasic responses in both treatment systems (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e). Peak growth metrics were recorded at 50% incorporation, with NM and M attaining 39.17 cm and 55.22 cm respectively, corresponding to 0.65-fold and 1.33-fold increases relative to CK. Significant differential responses emerged between systems: M maintained statistical significance (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) across all amendment ratios, whereas NM showed significant divergence (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) only below 30% incorporation. Both systems demonstrated initial height enhancement followed by progressive reduction with increasing Pisha sandstone proportions beyond optimal levels.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003e\u003cstrong\u003eAnalysis of the correlation between soil nutrients, microbial populations, enzyme activity, and licorice growth.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs can be seen in Figure 7, a markedly positive association existed between soil organic carbon and available nitrogen, as well as available phosphorus, suggesting that a rise in soil organic carbon is generally linked to increases in these parameters. This aligns with prior research regarding the significance of soil nutrient concentrations (e.g., available nitrogen, available phosphorus) on soil organic carbon accumulation in soil ecosystems\u003csup\u003e34,35\u003c/sup\u003e. Despite a low correlation between organic carbon and acid phosphatase, there exists a highly significant positive correlation with urease and sucrase, indicating that organic carbon substantially enhances the activity of these enzymes, potentially by supplying energy or other supportive substances. Moreover, soil organic carbon was also highly positively correlated with bacterial and fungal populations, possibly due to the improved correlation between bacterial and fungal availability of soil carbon. In conclusion, the increase in soil organic carbon favors the growth of soil microorganisms and the enhancement of soil readily available nutrients.\u003c/p\u003e\n\u003cp\u003eA significantly substantial positive association existed between soil available nitrogen and available phosphorus, indicating coordinated variations in their concentrations. Soil acid phosphatase exhibited a highly significant positive correlation with available phosphorus and plant height, indicating that acid phosphatase activity facilitates the release and transformation of phosphorus, thereby enhancing plant phosphorus uptake and growth. This aligns with the understanding that phosphatase is a hydrolytic enzyme that aids in the decomposition and transformation of organic phosphorus, improving phosphorus availability for plant absorption in the soil\u003csup\u003e36\u003c/sup\u003e. Previous studies have shown that invertase is involved in converting and cycling soil carbon and nitrogen\u003csup\u003e37\u003c/sup\u003e, and urease can promote crop uptake of nitrogen. Moreover, substantial positive correlations were identified between invertase and available nitrogen, available phosphorus, and plant height, suggesting that invertase plays a crucial role in the transformation of nitrogen and phosphorus in the soil, thereby indirectly enhancing plant growth by influencing nutrient availability. A substantial positive association was identified between urease activity and both available nitrogen content and plant height, indicating that urease activity facilitates nitrogen release, hence enhancing the efficiency of nitrogen absorption by the plant.\u003c/p\u003e\n\u003cp\u003ePurushotham et al. \u003csup\u003e38\u003c/sup\u003e and Hoyos et al. \u003csup\u003e39\u003c/sup\u003e demonstrated that actinomycetes can enhance the development of plant rhizomes and significantly increase plant height. A significant positive correlation was observed between actinomycete counts and licorice plant height, indicating that actinomycetes may promote plant growth. The direct impact of actinomycetes on plant growth may be less significant than that of phosphatase in relation to other factors.\u003c/p\u003e\n\u003cp\u003eThe correlation analysis results indicated a complex interrelationship among organic carbon, available nitrogen, available phosphorus, microorganisms, and enzyme activities in the soil. An increase in organic carbon, available nitrogen, and available phosphorus enhanced the number of microorganisms and their associated enzyme activities in the soil, positively influencing plant growth. Significant correlations among microorganisms suggest their complementary functions within the soil ecosystem, while the interactions of various enzymes additionally influence soil fertility and plant growth. The findings illustrate the intricate relationship between soil health and plant growth, highlighting the synergistic effects of various factors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe impact of varying volumes of Pisha sandstone and microbial agents on soil nutrient levels.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSoil organic carbon is crucial for the formation and stabilization of soil aggregates, serving as a fundamental component of soil quality and significantly influencing soil fertility\u003csup\u003e40,41\u003c/sup\u003e. A reduction in soil organic carbon content correlates with diminished stability of soil structure\u003csup\u003e42,43\u003c/sup\u003e. Nitrogen and phosphorus deficiencies are essential indicators of soil fertility and have been recognized as key factors influencing plant growth\u003csup\u003e44,45\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe combination of Pisha sandstone with sandy soil resulted in a significant enhancement of soil organic carbon, available nitrogen, and available phosphorus compared to the control (CK), with no notable difference observed between the M and NM treatments (Figure 3). The organic carbon content in the soil remained stable after the addition of Pisha sandstone at a volume ratio of 50% (Figure 3 (a)). This stability may be attributed to the significant difference (p \u0026lt; 0.05) observed prior to reaching this volume ratio. At a volume ratio of 50% Pisha sandstone, the soil exhibited the highest levels of available nitrogen and available phosphorus (Figure 3 (b), 3 (c)), resulting in maximum nitrogen and phosphorus availability for licorice. The addition of Pisha sandstone at a volume ratio of 50% did not result in a significant difference in the available nitrogen content in the soil. However, the available phosphorus content in the soil showed a significant difference prior to the addition of Pisha sandstone at a volume ratio of 30% (p \u0026lt; 0.05). Consequently, based on the comprehensive soil organic carbon, available nitrogen, and available phosphorus content, the optimal improvement effect occurs when the volume ratio of Pisha sandstone reaches 50%. In comparison to CK, the soil organic carbon, available nitrogen, and available phosphorus content of NM increased by 1.01, 1.17, and 1.03 times, respectively. Meanwhile, the soil organic carbon, available nitrogen, and available phosphorus content of M increased by1.01, 1.17, and 1.03 times, respectively. The current soil structure exhibits greater stability compared to alternative measures, and soil fertility is at its peak. This indicates that the incorporation of microbial agents in the combination of Pisha sandstone with sandy soil is more effective than the compounding effect alone.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImpact of varying Pisha sandstone addition volume ratios and microbial agents on enzyme activity, the abundance of three primary bacterial groups, and licorice growth.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSoil enzymes are secreted into the soil by decomposing microorganisms associated with plant and animal residues. They are intricately connected to factors influencing soil fertility, serving as a critical indicator of soil fertility by reflecting microbial activity levels and the capacity for nutrient conversion, transport, and metabolism\u003csup\u003e46,47\u003c/sup\u003e. The ecological cycle of nature is significantly influenced by soil microbes. Their metabolism and associated processes directly influence the movement and circulation of materials and energy within the soil, making them essential indicators for assessing soil fertility\u003csup\u003e48,49\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThe combination of Pisha sandstone with sandy soil resulted in a significant enhancement of acid phosphatase, invertase, and urease activities, as well as an increase in the populations of bacteria, fungi, and actinomycetes, in comparison to the control (Figures 4 and 5). Furthermore, at an equivalent volume ratio of Pisha sandstone, enzyme activity and the abundance of the three primary bacterial groups in M were significantly elevated compared to NM. This disparity may be attributed to the microbial agent\u0026apos;s substantial content of microorganisms and organic residues from animals and plants.\u003c/p\u003e\n\u003cp\u003eAt a 50% volume ratio of Pisha sandstone, the activities of acid phosphatase, invertase, and urease in M peaked, exhibiting increases of 4.98 times, 81 times, and 18.37 times compared to CK, respectively. In NM, acid phosphatase activity also peaked at a 50% volume ratio of Pisha sandstone, though it was comparable to CK. The activities of invertase and urease rose with increasing Pisha sandstone volume, but after reaching a 50% volume ratio, the increase became more gradual, indicating that the addition of microbial agents enhances soil enzyme activity.\u003c/p\u003e\n\u003cp\u003eWith the increasing proportion of Pisha sandstone, the populations of bacteria and fungi rose markedly; however, the variation in actinomycete numbers was less pronounced (refer to Figure 5). No significant difference was observed between M and NM. Bacteria exhibited significant differences at a Pisha sandstone volume ratio of 30% (p\u0026lt;0.05), while no significant differences were noted for fungi and actinomycetes concerning the volume ratio of Pisha sandstone added. The quantity of actinomycetes exhibited minimal variation across different addition ratios, indicating a robust adaptation to the Pisha sandstone addition ratio. A high population of fungi can readily induce soil-borne illnesses and transform the soil from a fertile bacterial type to a less fertile fungal type\u003csup\u003e50-52\u003c/sup\u003e. Consequently, the fraction of fungi within the aggregate of the three predominant soil microorganisms should remain relatively low. At a Pisha sandstone volume ratio of 50%, fungi in NM constituted 34.45% of the total population of the three primary soil microorganisms (bacteria, fungi, and actinomycetes), while fungi in M represented40.24% of the total population of these microorganisms. When the volume ratio of Pisha sandstone was 80%, the fungal population in NM constituted 43.11% of the total of the three predominant soil floras, whereas the fungal population in M comprised 50.97% of the total of the three predominant soil floras. Furthermore, when the volume ratio of Pisha sandstone was 80%, both NM and M exerted a considerable detrimental effect on actinomycetes.\u003c/p\u003e\n\u003cp\u003eWhen the volume ratio of Pisha sandstone was 50%, the height of the licorice plant for NM and M attained its maximum (Figure 5). It may be inferred that a 50% volume ratio of Pisha sandstone yields optimal enhancement of sandy soil, which is further increased by the addition of microbial agents.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study thoroughly examined the impact of combining Pisha sandstone with sandy soil on soil enhancement, assessing the improvement effects at various addition volume ratios and the use of microbial agents. The findings indicated that the amalgamation of Pisha sandstone and sandy soil markedly enhanced the nutritional composition, microbial population, and enzymatic activity of the soil. In the experiment, irrespective of the addition of microbial agents, when the volume ratio of Pisha sandstone reached 50%, the soil exhibited peak levels of organic carbon, available nitrogen, and available phosphorus, alongside a significant increase in soil microorganisms and enzyme activity. The aforementioned indicates that incorporating Pisha sandstone at this volume ratio significantly enhances the physical and chemical qualities of the soil and fosters the growth of licorice. However, too high a volume ratio of Pisha sandstone may lead to negative changes in soil properties, thereby inhibiting plant growth.\u003c/p\u003e\n\u003cp\u003eThe findings indicate that a 50% volume ratio of Pisha sandstone and sandy soil, when supplemented with suitable fertilizers and microbial treatments, can significantly enhance sandy soil. This presents a viable approach for the management and amelioration of sandy soil, which is crucial for increasing licorice yield and offers a scientific foundation and practical guidance for sandy soil management. Nonetheless, there remains a deficiency in research regarding the optimal quantity of microbial agents to apply per square meter for improved sandy soil conditions involving Pisha sandstone and sandy soil. Future investigations will address this gap.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated, used, and/or analyzed during the current study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements (not compulsory)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was funded by was supported by the National Natural Science Foundation of China (42207394), Guangxi Key Research and Development Program of China (AB21196048); Forestry and Grassland Science and Technology Promotion Demonstration Project of the Central\u0026nbsp;Government\u0026nbsp;([2023] TG17) and Key Laboratory Autonomous Projects (2021-A-02-01). The authors are grateful to Guangxi Forest\u0026apos;s new fertilizer research and development Center, and School of Civil Engineering, Southeast University, and National and Local Unified Engineering Research Center for Basalt Fiber Production and Application Technology, Southeast University. Special thanks go to the anonymous reviewers for their constrictive comments in improving this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eM Writing\u0026mdash;original draft preparation, ZS. L. and XW. F.; validation B,P.; formal analysis LX. Z.; data curation and investigation, LX Z; resources, ZS. L.; writing\u0026mdash;review and editing, ZS. L. and HY.G.; All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eKassas, M. Desertification -A general-Review. \u003cem\u003eJ. 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China Agricultural Univ.\u003c/em\u003e \u003cb\u003e37\u003c/b\u003e, 51\u0026ndash;58 (2016).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-6408674/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6408674/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSandy soils are prone to water loss, low fertility, difficulties in vegetation growth, and severe wind erosion, and the employment of microorganisms and other techniques to improve sandy soils is an efficient way to realize their management. This study involved a field experiment in a demonstration garden located in the Hobq Desert region of China, aimed at examining the impacts on soil nutrients, microbial populations, enzyme activity, and licorice growth resulting from two experimental groups with varying volume ratios of Pisha sandstone (0%, 30%, 50%, 80%, and 100%) and sandy soils, categorized by the presence of microbial agents (M) or their absence (NM). The study's results indicated that the incorporation of Pisha sandstone markedly influenced soil nutrients, microbial populations, enzyme activity, and the height of licorice plants, in contrast to the 0% volume addition of Pisha sandstone without microbial agents (CK). The enhancement was notably greater following the introduction of microbial agents; the optimal overall treatment effect was observed with a 50% volume addition of Pisha sandstone, where soil nutrients, enzyme activity, and licorice growth metrics attained their peak values in NM and N. Correlation analysis indicates that the enhancement of soil organic carbon, available nitrogen, and available phosphorus can elevate the population of soil microorganisms and enzyme activity, with these parameters interacting and mutually reinforcing each other to facilitate the improvement of sandy soil. This offers an effective approach for enhancing sandy soil.\u003c/p\u003e","manuscriptTitle":"Effects of Pisha Sandstone Application Rate and Microbial Agents on Soil Nutrient Dynamics and Microbial Characteristics in Sandy Soil","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-14 08:30:14","doi":"10.21203/rs.3.rs-6408674/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-07-28T08:05:19+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"8575385409220896403829404205867842574","date":"2025-06-17T23:51:32+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-10T12:21:06+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"113681923883980496619430023117187501050","date":"2025-05-31T06:41:42+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-05-09T11:04:59+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-05-07T09:41:36+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-05-04T16:06:57+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9bed42c0-105f-4110-b196-2b210d048dc2","owner":[],"postedDate":"May 14th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":48387446,"name":"Earth and environmental sciences/Biogeochemistry"},{"id":48387447,"name":"Earth and environmental sciences/Environmental social sciences"}],"tags":[],"updatedAt":"2025-11-24T16:06:03+00:00","versionOfRecord":{"articleIdentity":"rs-6408674","link":"https://doi.org/10.1038/s41598-025-24467-w","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-11-19 15:57:59","publishedOnDateReadable":"November 19th, 2025"},"versionCreatedAt":"2025-05-14 08:30:14","video":"","vorDoi":"10.1038/s41598-025-24467-w","vorDoiUrl":"https://doi.org/10.1038/s41598-025-24467-w","workflowStages":[]},"version":"v1","identity":"rs-6408674","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6408674","identity":"rs-6408674","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}