Mechanisms of Bio-Based Paper Mulch and Calcium Sulfonate Water-Soluble Fertilizer in Ameliorating Saline-Alkali Soils

Mechanisms of Bio-Based Paper Mulch and Calcium Sulfonate Water-Soluble Fertilizer in Ameliorating Saline-Alkali Soils | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Mechanisms of Bio-Based Paper Mulch and Calcium Sulfonate Water-Soluble Fertilizer in Ameliorating Saline-Alkali Soils Huanjun Liu, Xiao Mou, Yuwen Wang, Diyi Qian This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9457087/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 4 You are reading this latest preprint version Abstract Background and Aims Soil salinization threatens agricultural productivity and ecosystem health. Bio-based paper film and bio-based calcium sulfonate have the potential to reduce salinity and enhance ecosystem multifunctionality (EMF). However, their mechanisms in heavily saline-alkali soils remain unclear. Methods A pot experiment was conducted with five treatments: CK (water only), M2 (17.00 g·pot − 1 bio-based paper film + 25.00 g·pot − 1 bio-based calcium sulfonate), M4 (27.20 g·pot − 1 bio-based paper film + 25.00 g·pot − 1 bio-based calcium sulfonate), M55 (37.40 g·pot − 1 bio-based paper film + 25.00 g·pot − 1 bio-based calcium sulfonate), and JF (microbial fertilizer + PVC film). Soil properties, microbial communities, and EMF were analyzed. Results M4 showed the best performance. Compared with CK, it promoted alfalfa (Medicago sativa) germination and growth and increased rhizosphere organic acids. It reduced soil pH and electrical conductivity (EC) and increased soil temperature, moisture, nutrients, and enzyme activities. Although microbial alpha diversity decreased, bacterial and fungal community structures were optimized, forming a more cooperative and complex network. Beneficial taxa involved in nitrogen fixation and phosphate solubilization (e.g., Pseudomonadota, Alphaproteobacteria, and Anaerolineae) were enriched. The higher abundance of Basidiomycota enhanced cellulose and lignin decomposition. Conclusions The combined application of bio-based paper film and calcium sulfonate improved soil quality and reshaped microbial communities, thereby enhancing ecosystem functioning in saline-alkali soils. Bio-based paper film Soil microbial community Soil properties Ecosystem multifunctionality Saline-alkali soil Alfalfa (Medicago sativa) Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 1. Introduction Soil salinization poses a serious threat to agricultural production and soil ecology. Under salt stress, plant growth is inhibited, and microbial activity and survival are suppressed. These effects disrupt soil material cycling and lead to the degradation of ecological functions (Ahlawi et al. 2018 ; Lu et al. 2020 ). In recent years, soil salinization has intensified due to human-induced soil degradation and climate change. According to the Global Status of Salt-Affected Soils report released by the FAO in 2024, salt-affected soils cover more than 1.381 billion hectares worldwide, accounting for 10.7% of the global land area (FAO. 2024). In China, saline-alkali soils cover approximately 100 million hectares. Xinjiang is the most severely affected region, accounting for about one-third of the national total. Within Xinjiang, 274,000 hectares are classified as heavily saline-alkali soil. Despite this, most soils in this region have deep profiles and represent a potential resource for arable land (Jiang et al. 2019 ). Therefore, rehabilitating saline-alkali soils in Xinjiang is a critical strategy for improving soil ecology and enhancing agricultural productivity. High evaporation is a primary driver of soil salinization in Xinjiang. Mulching can reduce water loss from the soil surface. As an important agricultural practice, it improves soil warming, moisture retention, weed control, and salt inhibition (Liu et al. 2014 ; Li et al. 2014 ; Zhang et al. 2024 ). However, plastic mulch is difficult to fully recover. Residual plastic fragments impede water and nutrient movement and disrupt soil structure. In addition, leached plasticizers can inhibit root development and harm soil biota (Koskei et al. 2021 ). Therefore, replacing conventional plastic mulch with biodegradable alternatives is an effective way to reduce plastic pollution in agricultural soils (Li et al. 2024b ). Biodegradable mulches can be divided into two categories: those derived from natural macromolecules and those synthesized from biodegradable polymers. European countries began developing biodegradable mulch films in the early 1990s. In 2018, the European Union introduced the EN 17033 standard for biodegradable mulch films used in agriculture and horticulture. This established a clear technical framework for the industry (Žaimis et al. 2023 ; Ajao et al. 2018 ; Sadeghifar et al. 2020). In recent years, China has made rapid progress in biodegradable mulch technology. This includes films derived from biomass resources such as lignin and cellulose (Chen et al. 2023 ; Li et al. 2022 ; Wang et al. 2021 ). After degradation, these materials are converted into soil organic matter and support sustainable agriculture. The bio-based products used in this study were derived from crop straw. A sulfonation process produced water-soluble biomacromolecules as bio-based calcium sulfonate fertilizer. The remaining insoluble fraction was used to produce biodegradable bio-based paper film (Li et al. 2018 ). Bio-based calcium sulfonate exchanges cations with sodium ions, driving sodium downward from the plow layer. Calcium ions then react with carbonate to form calcium carbonate, reducing soil salinity and pH (Cao. 2020).The insoluble fraction mainly contains lignin, cellulose, and hemicellulose. These components can be directly utilized by microorganisms. As a result, the bio-based paper film acts as a nutrient-rich substrate and enriches microbes adapted to high nutrient availability. When applied to the soil surface, it also retains heat and moisture and suppresses salt resurgence. Conventional methods for ameliorating saline-alkali soils often require high water input, incur high costs, and have poor scalability. They also carry a high risk of salt reaccumulation (Han et al. 2025 ). In contrast, bio-based calcium sulfonate is fully water-soluble. Combined with the widespread use of integrated water–fertilizer systems in Xinjiang and the potential for spray application of bio-based paper film, this approach shows strong potential for saline-alkali soil remediation. A single approach is insufficient for the sustainable amelioration of saline-alkali soils. Therefore, integrated chemical, physical, and biological measures are required (Li et al. 2025 ). Alfalfa (Medicago sativa) is a moderately salt- and alkali-tolerant leguminous forage crop with both economic and ecological value for soil improvement (Chen et al. 2022 ). Alfalfa develops a strong root system and high aboveground biomass. Symbiotic rhizobia supply nitrogen to the plant. They also enhance soil fertility, improve soil structure, and contribute to water and soil conservation (Xu et al. 2024 ). Therefore, the combined application of bio-based paper film, bio-based calcium sulfonate, and alfalfa is an effective strategy for the long-term remediation of saline-alkali soils. Ecosystem multifunctionality (EMF) refers to the capacity of an ecosystem to provide multiple functions simultaneously. It emphasizes the combined performance and synergistic effects of different functions and enables a comprehensive evaluation of complex ecosystems (Hu et al. 2021 ). Previous studies have shown that EMF is influenced by plant communities, soil physicochemical properties, enzyme activities, and microbial diversity. Among these factors, plant and microbial diversity are the main drivers of EMF. They jointly regulate EMF through aboveground-belowground feedback mechanisms (Antiqueira et al. 2018 ). EMF also reflects ecosystem resilience and sustainability. High EMF indicates greater functional redundancy. Even if some species or processes are impaired, other components can maintain overall ecosystem functioning. This enhances the system's resistance to external disturbances (Ma et al. 2025 ). Therefore, EMF was used in this study to evaluate the effectiveness of bio-based paper film in improving saline-alkali soil ecosystems. In this study, a combined strategy was employed to ameliorate saline-alkali soil. Bio-based calcium sulfonate was applied to leach salts downward. Bio-based paper film was used to suppress salt resurgence, and alfalfa (Medicago sativa) was planted as a salt-tolerant crop. The effects of different application rates of bio-based paper film on alfalfa growth, rhizosphere soil properties, microbial community structure, and ecosystem multifunctionality (EMF) were investigated. The goal was to clarify how bio-based paper film improves saline-alkali soil and regulates the microbial community. This will provide a theoretical basis for its application in the remediation of saline-alkali soils in Xinjiang. 2. Materials and methods 2.1. Soil Collection and Experimental Site The saline-alkali soil was collected from Que'erpan Village, Cha County, Ili Prefecture, Xinjiang (43.92°N, 81.33°E), located on the south bank of the Ili River. The site has a temperate continental semi-arid climate, with an annual mean temperature of 7.9°C, mean annual precipitation of 222 mm, and evaporation of 1630 mm. Before soil collection, the field had been planted with oil sunflower and alfalfa, but both crops failed completely due to severe soil salinization. The pot experiment was conducted in a greenhouse at the Production-Education-Research Base of Yili Normal University. 2.2. Experimental Materials and Design The alfalfa (Medicago sativa) seeds used in this study were purchased from a local agricultural supply market, with the variety being Omekanog (Eurekanog). The bio-based paper film, mainly composed of lignin, cellulose, and hemicellulose, and bio-based calcium sulfonate (containing Organic Matter (OM): 44.5%; Ca: 4250 mg·L⁻¹; N: 61.51 mg·L⁻¹; K: 125.79 mg·L⁻¹; S: 847.58 mg·L⁻¹; Si: 25.05 mg·L⁻¹) were all provided by Beijing Unisplendour Yingli Company. The microbial inoculant was purchased from Xinjiang Tiankai Ecological Technology Co., Ltd. Plastic pots (24 cm in diameter, 20 cm in height) and perlite were obtained from a local flower market. Saline-alkali soil was randomly collected from a depth of 0–20 cm, and the collected soil was thoroughly mixed to prepare a homogeneous saline-alkali soil with a salinity of 0.6%. On June 15, 2025, a pot experiment was conducted to cultivate alfalfa (Medicago sativa) in a greenhouse. Each pot was filled with 5 kg of saline-alkali soil and 100 g of perlite, which were mixed thoroughly. Five treatments were established, each with five replicates: CK (irrigation with water only), M2 (17.00 g·pot − 1 bio-based paper film + 25.00 g·pot − 1 bio-based calcium sulfonate), M4 (27.20 g·pot − 1 bio-based paper film + 25.00 g·pot − 1 bio-based calcium sulfonate), M55 (37.40 g·pot − 1 bio-based paper film + 25.00 g·pot − 1 bio-based calcium sulfonate), and JF (microbial fertilizer + PVC plastic film). The dosage of bio-based calcium sulfonate was determined based on our previous experiments(Liu et al. 2025 ). For the M2, M4, and M55 treatments, after laying and forming the bio-based paper film, 50 seeds were uniformly sown per pot, followed by the application of bio-based calcium sulfonate (diluted and applied in multiple increments). For the CK and JF treatments, 50 seeds were sown per pot, followed by normal watering. Except for CK, all other treatments received a topdressing one month later, consisting of 0.93 g of diammonium phosphate and 0.71 g of potassium sulfate per pot. The experiment was terminated on October 3, 2025. 2.3. Sample Collection Emergence counts were recorded 10 days after sowing. At the end of the experiment, the number of surviving plants in each pot was counted to calculate the survival rate. Three alfalfa plants with uniform growth were selected from each pot to measure fresh weight, dry weight, and plant height. The entire alfalfa plant was removed from each pot, and large soil aggregates attached to the roots were gently detached. Rhizosphere soil (0–5 mm from the root surface) was collected using sterile brushes and other tools, placed into 10 mL sterile, nuclease-free centrifuge tubes, and stored at -80°C for subsequent high-throughput sequencing of the rhizosphere microbial community and determination of rhizosphere organic acids. The remaining soil was passed through a 2 mm sieve to remove debris and air-dried for subsequent measurements of soil physicochemical properties and enzyme activities (Hu et al. 2023 ). 2.4. Soil Physicochemical Properties Soil pH was measured using a pH meter at a soil-to-water ratio of 1:2.5 (Wu et al. 2024 ). Electrical conductivity (EC) was determined with a conductivity meter at a soil-to-water ratio of 1:5 (Hu et al. 2024 ). Soil temperature and moisture were measured using a portable soil temperature-moisture rapid meter (Wufuer et al. 2023 ). Soil organic matter (SOM) content was determined using the potassium dichromate volumetric method (Schecter et al. 2004 ). Available nitrogen (AN) content was measured with a Kjeldahl nitrogen analyzer (Fulford and Roberts 2022 ). Available phosphorus (AP) content was determined using the 0.5 mol·L⁻¹ NaHCO₃ extraction method (Veloso et al. 2023 ). 2.5. Soil Enzyme Activities and Organic Acids Commercial kits purchased from Beijing Solarbio Science & Technology Co., Ltd. were used to quantify the activities of alkaline phosphatase (AKP), neutral phosphatase (NP), cellulase (CL), β-glucosidase (β-GC), and urease (UE) in the soil, following the manufacturer's instructions. Organic acids, including oxalic acid, citric acid, and malic acid, were measured using LC-ESI-MS/MS (UHPLC-Qtrap) (Bueno-Muiño et al. 2023 ). 2.6. High-Throughput Sequencing Analysis Total microbial genomic DNA was extracted using the E.Z.N.A.® Soil DNA Kit (Omega Bio-tek, Norcross, GA, USA). DNA integrity was assessed by 1% agarose gel electrophoresis. DNA concentration and purity were determined using a NanoDrop 2000 spectrophotometer (Thermo Scientific, USA). The extracted DNA was used as a template to amplify the V3-V4 hypervariable region of the 16S rRNA gene with primers 338F and 806R carrying barcode sequences. The fungal internal transcribed spacer (ITS) region was amplified using primers ITS1F and ITS2R (Sinha et al. 2022 ). The PCR program for both bacterial and fungal DNA was as follows: initial denaturation at 95°C for 3 min; 30 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 45 s; and a final extension at 72°C for 10 min, followed by holding at 10°C. PCR products from each sample were pooled and examined by 2% agarose gel electrophoresis. Target fragments were excised and purified using the AxyPrep DNA Gel Extraction Kit (AXYGEN) and eluted with Tris-HCl. The purified products were re-examined by 2% agarose gel electrophoresis. Quantification was performed using the QuantiFluor™-ST fluorometric system (Promega). Sequencing libraries were constructed from the purified PCR products using the NEXTFLEX Rapid DNA-Seq Kit. Sequencing was conducted on the Illumina NextSeq 2000 platform (Shanghai Majorbio Bio-Pharm Technology Co., Ltd.). The raw sequence data were deposited in the NCBI Sequence Read Archive (SRA) database. 2.7. Statistical analysis Raw paired-end sequencing reads were quality-controlled using fastp (v0.23.4) and assembled with FLASH (v1.2.11). The quality-filtered and assembled sequences were then denoised using the DADA2 plugin within the QIIME2 pipeline under default parameters. The resulting sequences were defined as amplicon sequence variants (ASVs). Taxonomic assignment was conducted using the classify-sklearn (Naive Bayes) algorithm in QIIME2 against the Silva138.2/16S bacterial database, with a confidence threshold of 70% (Bokulich et al. 2018 ). For both bacteria and fungi, sequence counts were rarefied to 50,887 and 51,487 per sample, respectively. In total, 9,915 bacterial ASVs and 2,515 fungal ASVs were obtained and used for subsequent analyses of microbial diversity and community structure. Statistical analyses of plant agronomic traits, soil physicochemical properties, enzyme activities, and organic acids were performed using SPSS 26. One-way analysis of variance (ANOVA) followed by Duncan's multiple range test was used to determine significant differences. Box plots were generated using Origin 2021. Microbial data were analyzed and visualized using R software (v4.4.3). Alpha diversity indices of bacteria and fungi were calculated using the otuSummary package, and box plots were generated with the ggplot2 package (Liu et al. 2023). Chord diagrams of community abundance at the phylum level were generated using the circlize package. Principal coordinate analysis (PCoA) based on Bray-Curtis distances was performed, followed by PERMANOVA tests (Yang et al. 2024 ). Linear discriminant analysis effect size (LEfSe) was conducted using the microeco package, with an LDA score > 4 and P 0.8, P < 0.001) (Jacomy et al. 2014 ). Redundancy analysis (RDA) and Mantel tests were conducted based on Spearman's correlation using the vegan and linkET packages, respectively (Kim and Cui 2023 ; Cromer et al. 2023 ). A total of 27 indicators were selected to calculate ecosystem multifunctionality (EMF). The averaging approach was applied after Z-score normalization (Hooper and Vitousek 1998 ). These indicators included plant dry and fresh weight. Soil indicators included temperature, moisture, electrical conductivity (EC), pH, soil organic matter (SOM), available phosphorus (AP), available nitrogen (AN), oxalic acid (CAS), citric acid (CA), malic acid (MAL), neutral phosphatase (NP), alkaline phosphatase (AKP), urease (UE), β-glucosidase (β-GC), and cellulase (CL). Microbial indicators included the Shannon, Chao1, Simpson, ACE, and coverage indices for both bacteria and fungi. EMF was calculated as follows: $$\:\text{EMF}=\frac{1}{n}\sum\:_{i=1}^{n}{Z}_{i}$$ Where \(\:{Z}_{i}\) is the Z-score normalized value of indicator \(\:i\) , and \(\:n\) is the total number of indicators. 3. Results 3.1 Plant Growth The application of bio-based paper film improved the growth of alfalfa under saline-alkali stress. The results are presented in Fig. 1 . Compared with the control (CK), the application of bio-based paper film significantly increased alfalfa dry weight, fresh weight, plant height, emergence rate, and survival rate ( P < 0.05). Among all treatments, M4 showed the best performance. Relative to CK, M4 increased dry weight by 400.78%, fresh weight by 323.93%, plant height by 109.05%, emergence rate by 131.91%, and survival rate by 127.72% ( P < 0.001). 3.2 Soil Physicochemical Properties and Enzyme Activities The application of bio-based paper film improved the physicochemical properties of saline-alkali soil, with M4 showing the best performance (Fig. 2 ). The bio-based paper film significantly increased soil organic matter, available phosphorus, and available nitrogen. Compared with CK, M4 increased these parameters by 138.66%, 57.19%, and 30.16%, respectively ( P < 0.001). The bio-based paper film also reduced soil electrical conductivity (EC) and pH. Relative to CK, M4 decreased EC by 48.23% ( P < 0.001) and pH by 0.60 units ( P < 0.05). In addition, the bio-based paper film helped maintain soil temperature and moisture. Compared with CK, M4 increased soil temperature by 2.53% and soil moisture by 95.86% ( P < 0.001). The application of bio-based paper film increased the content of organic acids in rhizosphere soil, with M4 showing the best performance (Fig. 3 ). The bio-based paper film significantly increased malic acid (MAL), oxalic acid (CAS), and citric acid (CA) in the rhizosphere soil of alfalfa. Compared with CK, M4 increased these organic acids by 318.92%, 321.35%, and 197.48%, respectively ( P < 0.001). The application of bio-based paper film enhanced soil enzyme activities, with M4 showing the best performance (Fig. 4 ). The activities of cellulase, alkaline phosphatase, β-glucosidase, neutral phosphatase, and urease were significantly increased under the bio-based paper film treatments. Compared with CK, M4 increased the activities of these enzymes by 139.46% ( P < 0.001), 78.64% ( P < 0.001), 48.24% ( P < 0.01), 56.49% ( P < 0.001), and 37.10% (P < 0.001), respectively. 3.3 Effect of Bio-Based Paper Film on Rhizosphere Soil Microbial Community The application of bio-based paper film had a minor effect on the alpha diversity of soil bacteria but a greater impact on that of soil fungi (Fig. 5 ). Compared with CK, the M4 treatment decreased the bacterial ACE and Chao1 indices by 3.21% and the Shannon index by 1.03%, while the Simpson index increased by 50.00%; however, none of these differences were statistically significant. In contrast, for soil fungi, M4 significantly decreased the ACE and Chao1 indices by 29.87% and the Shannon index by 23.18% ( P < 0.05), while the Simpson index increased by 42.31%. Analysis of microbial β-diversity showed that the first two principal coordinates (PCo1 and PCo2) explained 32.526% of the variation in bacterial community structure (R² = 0.39, P = 0.001), indicating significant differences among treatment groups (Fig. 6 a). The M4 and CK samples were located in different quadrants, suggesting a marked difference between these two treatments. For fungal communities, PCo1 and PCo2 explained 35.00% of the structural variation (R² = 0.53, P = 0.001), also indicating significant differences among groups (Fig. 6 b). The M4 and CK samples were again distributed in distinct quadrants, demonstrating a clear difference between them. At the phylum level, bacterial and fungal taxa with a relative abundance greater than 1.0% were defined as dominant. The dominant bacterial phyla included Pseudomonadota (28.7%), Bacillota (15.5%), Actinomycetota (11.1%), Chloroflexota (10.8%), Bacteroidota (6.5%), Gemmatimonadota (4.7%), Acidobacteriota (4.3%), Myxococcota (4.2%), Cyanobacteriota (2.7%), Patescibacteria (2.2%), Rhodothermota (1.5%), Planctomycetota (1.4%), and Verrucomicrobiota (1.1%). These phyla together accounted for more than 94% of the total bacterial ASVs. Among all treatments, M4 showed the highest proportion of Pseudomonadota, while the relative abundances of other bacterial phyla varied little among treatments. The dominant fungal phyla included Ascomycota (47.6%), Basidiomycota (38.9%), unclassified_k_Fungi (5.8%), Fungi_Phy_Incertae_sedis (3.3%), Aphelidiomycota (2.0%), and Rozellomycota (1.9%). These phyla together accounted for more than 99% of the total fungal ASVs. Ascomycota dominated in the CK and JF treatments, whereas Basidiomycota dominated in the M2, M4, and M55 treatments. LEfSe analysis (LDA > 2, P < 0.05) was performed to identify taxa with significantly different abundances among treatment groups. The top 20 taxa with the highest LDA scores are shown. Among bacterial taxa, four significantly enriched groups were identified in CK: P__Actinomycetota, P__Bacteroidota, c__Bacteroidia, and c__Actinobacteria. In contrast, M4 showed seven significantly enriched taxa, including P__Pseudomonadota, c__Alphaproteobacteria, o__Hyphomicrobiales, c__Polyangiia, c__Anaerolineae, o__Aggregatilineales, and P__Myxococcota. Among fungal taxa, six significantly enriched groups were detected in CK: P__Ascomycota, c__Sordariomycetes, o__Hypocreales, g__unclassified_f__Nectriaceae, s__unclassified_f__Nectriaceae, and g__Enterocarpus. No significantly enriched fungal taxa were detected in the M4 treatment. 3.4. Co-occurrence Network of Rhizosphere Soil Microorganisms of Alfalfa A co-occurrence network of soil bacterial and fungal communities was constructed based on ASV-level data (r > 0.8, P < 0.001) (Fig. 9 ). Compared with CK, the bio-based paper film treatments increased the number of edges, the proportion of positive correlations, and the average degree in the bacterial-fungal network. These changes indicate strengthened mutualistic relationships between bacteria and fungi. In the M4 treatment, the network comprised 259 nodes and 3,015 edges. Positive correlations accounted for 57.55% (1,735 edges), and the average degree was 23.282. Compared with CK, M4 increased the number of edges by 176.61%, the proportion of positive correlations by 8.52%, the number of positively correlated edges by 200.17%, and the average degree by 185.14%, while the number of nodes decreased by 3.00%. 3.5. Correlations Among Microbial Communities, Soil Properties, and Alfalfa Growth Redundancy analysis (RDA) revealed the relationships between soil microbial communities and soil properties. For bacteria (Fig. 10 a), RDA1 and RDA2 explained 46.92% of the community variation. The CK treatment was mainly associated with the pH and EC axes and showed significant positive correlations with these variables ( P < 0.05). In contrast, the M4 treatment was distributed along the directions of soil nutrients, enzyme activities, temperature, moisture, and organic acids, and showed significant positive correlations with these factors ( P < 0.05). Key bacterial taxa, including Hyphomicrobiales, Polyangiales, Beijerinckiaceae, and Beduibacterium, were positively correlated with NP, ST, β-GC, AN, AKP, SOM, AP, CA, MAL, UE, CL, and CAS ( P < 0.05). Soil pH and EC were negatively correlated with soil nutrients, enzyme activities, temperature, moisture, and organic acids (P < 0.05). For fungi (Fig. 10 b), RDA1 and RDA2 explained 58.99% of the variation. The CK treatment was again associated with pH and EC and showed significant positive correlations with these variables ( P < 0.05). The M4 treatment was positively correlated with soil nutrients, enzyme activities, temperature, moisture, and organic acids ( P < 0.05). Key fungal taxa, including Agaricomycetes, Coprinellus, Coprinellus alkalinus, Basidiomycota, Agaricales, and Psathyrellaceae, were positively correlated with NP, AN, MAL, SOM, β-GC, ST, AP, CL, AKP, CAS, SM, CA, and UE ( P < 0.05). Soil pH and EC were negatively correlated with these environmental factors ( P < 0.05). Mantel tests were conducted to assess the relationships among key microbial taxa, soil properties, and alfalfa growth (Fig. 10 c). Key bacterial taxa were significantly correlated with AP, CAS, NP, AKP, UE, CL, and fresh weight (FW). Among these, AP showed the strongest correlation (Mantel's r = 0.44). Key fungal taxa were significantly correlated with SM, EC, SOM, AN, CAS, CA, UE, CL, FW, and dry weight (DW). Among these, SOM showed the strongest correlation (Mantel's r = 0.535).Ecosystem multifunctionality (EMF) differed significantly among treatments (Fig. 10 d). M4 showed the highest EMF, whereas CK exhibited the lowest level. 4. Discussion 4.1. Effects of Bio-Based Products on Alfalfa Growth and Soil Properties Plant growth is closely linked to the soil environment. In this study, compared with CK, the bio-based paper film treatments promoted alfalfa seed germination and growth. Plant dry weight, fresh weight, plant height, emergence rate, and survival rate were all significantly increased ( P < 0.05), with M4 showing the best performance. These positive effects may be attributed to multiple factors. The water-soluble fertilizer reduced salt accumulation in the surface soil layer. Meanwhile, the bio-based paper film maintained soil temperature and moisture and inhibited salt resurgence. Together, these changes created favorable conditions for seed germination and seedling growth. This finding is consistent with previous studies showing that mulching promotes the growth of maize, alfalfa, and taro (Sheng et al. 2019; Gu et al. 2018 ; Anikwe et al. 2007 ). Soil physicochemical properties, enzyme activities, and organic acids are key indicators of soil quality. Soil quality is closely linked to agricultural management practices, plant (root) activity, and microbial communities, which interact to regulate these indicators. In this study, compared with CK, all bio-based paper film treatments significantly increased soil organic matter, available phosphorus, and available nitrogen ( P < 0.05), with M4 showing the best performance. This improvement may be attributed to the application of water-soluble fertilizer and the input of alfalfa root exudates, which increased soil organic matter content. Increased microbial activity, together with higher urease and phosphatase activities, contributed to the accumulation of available phosphorus and nitrogen. The M4 treatment showed the lowest EC and pH values. This was related to the salt-leaching effect of the water-soluble fertilizer and the inhibition of salt resurgence by the bio-based paper film. The reduction in EC and pH restored root metabolic activity. This led to increased secretion of organic acids and promoted microbial proliferation in the rhizosphere soil. Microorganisms are the primary source of soil enzymes, and their increased abundance is generally associated with higher enzyme activities. The M4 treatment enriched Basidiomycota and its lower taxonomic units, as well as Anaerolineae. This significantly increased β-glucosidase and cellulase activities ( P < 0.05). In addition, the enrichment of Alphaproteobacteria enhanced nitrogen fixation in the alfalfa rhizosphere. Rapid plant growth increased nutrient demand, which further stimulated urease and phosphatase activities. These findings are consistent with previous studies showing that mulching increases soil nutrients and enzyme activities while reducing pH and electrical conductivity (Liu et al. 2024 ; Mao et al. 2023 ; Jacquiod et al. 2024 ). 4.2. Bio-Based Products Reshape the Microbial Interaction Network in the Alfalfa Rhizosphere Rhizosphere soil microorganisms originate from bulk soil and undergo self-regulation to adapt to new environmental conditions. This process generally reduces microbial diversity (Essel et al. 2019 ). In this study, the alpha diversity of both bacteria and fungi was lower in the bio-based paper film treatments than in CK, with bacterial diversity being less affected. The difference between bacterial and fungal diversity may be attributed to variations in rhizosphere soil physicochemical properties and their distinct nutrient acquisition strategies (Philippot et al. 2024 ). The application of bio-based paper film and bio-based calcium sulfonate reduced soil EC and inhibited salt resurgence. This created a favorable environment and provided sufficient nutrients for microbial growth. Due to their shorter generation times and stronger substrate utilization capacities, bacteria had a competitive advantage over fungi in nutrient and spatial competition. This resulted in a suppressive effect on fungal abundance (Dămătîrcă et al. 2023 ). Differences in nutrient substrates can selectively enrich microbial taxa with distinct resource utilization preferences (Rinnan and Baath. 2009). The bio-based paper film and bio-based calcium sulfonate provided abundant soluble organic matter, as well as cellulose and lignin that can be directly decomposed by microorganisms. This created clear substrate differences compared with CK. As a result, the bio-based treatments caused significant changes in rhizosphere microbial β-diversity. These changes indicate the formation of new microbial assemblages adapted to the altered environment and shifts in community structure. LEfSe analysis further showed that, among bacteria, the key taxa in the M4 treatment were Pseudomonadota and its lower taxonomic units, as well as Anaerolineae, which are associated with high nutrient availability. Among fungi, CK was dominated by pathogenic taxa belonging to Ascomycota and its lower taxonomic units. In contrast, the bio-based paper film treatments were dominated by saprotrophic fungi of Basidiomycota and its lower taxonomic units, which have strong capacities to decompose cellulose and lignin. These findings are consistent with previous studies showing that organic fertilizer application regulates microbial community structure and composition (Liu et al. 2023b ). Soil microbial network analysis reveals potential interspecific interactions and the organizational principles of microbial communities (Huo et al. 2023 ; Yue et al. 2023 ). In this study, network analysis showed that the application of bio-based products increased the complexity of the alfalfa rhizosphere microbial network compared with CK. Among all treatments, M4 showed more nodes and edges, as well as the highest proportion of positive correlations. This indicates a more “cooperative, complex, and efficient” network with greater resistance to environmental disturbances. These effects may be attributed to multiple factors. Bio-based products leached salts from the soil surface and inhibited salt resurgence. They also supplied water-soluble organic matter, calcium, and decomposable substrates such as lignin and cellulose. These changes created favorable conditions for both alfalfa and microorganisms. As alfalfa resumed normal metabolism, root exudates were released into the soil in large quantities. This increased nutrient availability and promoted microbial growth, thereby enhancing network complexity (Wang et al. 2022a ; Wang et al. 2022b ; Shi et al. 2016 ). 4.3. Correlations Between Key Microbial Taxa and Rhizosphere Soil Properties Microbial community structure is shaped by environmental filtering, biological interactions, host regulation, and spatiotemporal dynamics. Among these, environmental filtering is the primary driver of microbial community assembly (Banerjee et al. 2018 ; Xun et al. 2024 ). RDA results showed that the CK treatment was mainly influenced by pH and EC. Bacterial key taxa were associated with CAS, whereas fungal key taxa were associated with SOM. Mantel tests further showed that bacterial key taxa were significantly correlated with AP, CAS, NP, AKP, UE, CL, and fresh weight (FW). Fungal key taxa were significantly correlated with SM, EC, SOM, AN, CAS, CA, UE, CL, FW, and dry weight (DW). These patterns may be explained by changes in soil conditions. The application of bio-based paper film and bio-based calcium sulfonate reduced soil pH and EC. This shifted the M4 treatment from a stress-dominated environment to a substrate-dominated environment. Under these conditions, alfalfa roots released large amounts of CAS into the soil. This enriched Pseudomonadota and its lower taxonomic units, as well as Anaerolineae. Meanwhile, SOM derived from roots, exudates, microbial residues, and bio-based materials enriched Basidiomycota and its lower taxonomic units (Ling et al. 2022 ). 4.4. Bio-based Paper Film Enhances the EMF of Saline-Alkali Soils The performance of individual functions does not reflect overall ecosystem health. Ecosystem multifunctionality (EMF) provides a comprehensive evaluation of ecosystem functions (Manning et al. 2018 ; Wang et al. 2022c ). In this study, M4 showed the highest EMF. This is because the alfalfa root system, soil, and microorganisms formed an integrated system. Root exudates promoted microbial recruitment and increased community complexity. This enhanced enzyme activities and nutrient cycling. These interactions collectively improved EMF (Li et al. 2024a ; Liu et al. 2023a ). 5. Conclusion This study investigated the effects of bio-based paper film on the alfalfa rhizosphere and evaluated its role in improving soil properties, microbial communities, and ecosystem multifunctionality (EMF) in heavily saline-alkali soil. The results showed that the application of 27.20 g·pot⁻¹ (M4) achieved the best amelioration effect. The M4 treatment reduced soil EC and pH and maintained soil temperature and moisture. It also promoted alfalfa germination and growth. As a result, alfalfa roots released large amounts of organic acids. Soil nutrient contents and enzyme activities were significantly increased. Although the alpha diversity of bacteria and fungi decreased, their community structures were optimized. A “cooperative, complex, and efficient” microbial interaction network was formed. Bacterial taxa involved in nitrogen fixation and phosphate solubilization, such as Pseudomonadota, Alphaproteobacteria, and Anaerolineae, were enriched. The higher abundance of Basidiomycota and its lower taxonomic units enhanced the decomposition of cellulose and lignin. Overall, the M4 treatment showed the highest EMF. This study provides a theoretical basis and practical reference for the remediation of saline-alkali soils in Xinjiang. Statements and Declarations Acknowledgements This study was supported bythe Xinjiang Talent Development Fund Research Innovation Platform Talent Team Project(XJRCSWZTD2025) , the Innovation and Entrepreneurship Training Program of Yili Normal University(202410764012), and the Key R&D Program of Xinjiang Uygur Autonomous Region (2022B02021). Competing Interests The authors have no competing interests to declare that are relevant to the content of this article. Author Contributions Huanjun Liu: Writing – review & editing, Writing – original draft, Visualization, Methodology, Data curation, Conceptualization. Dayi Qian: Writing – review & editing, Visualization, Validation, Supervision, Project administration, Methodology, Funding acquisition, Data curation, Conceptualization. Xiao Mou: Collected plant and soil samples and conducted sample analyses. Yuwen Wang: Writing – review & editing. Data availability Data will be made available on request. 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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-9457087","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":630416644,"identity":"e2cc5e79-a45f-4d02-9fea-e8586e207da4","order_by":0,"name":"Huanjun Liu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA0klEQVRIie3RIQvCUBDA8RsPznKYn2xsn0A4GAgWP8sb1glGw8DJQIsfYEH0QwjmB8JMdqvFNHAWMRhc1LRnE3z//uPuOACb7QfDVnZ4KE58DPbajLSpULIaF2FbojIjvoy5k1ciWktiw8VAcUiMztxd3k4lDPxu2ki0GhJ7Ar3jtr+CYdjTTcRJ9b6eggijnUugo10jEc4sIxaEEF8MCQrh5CwkyhgNCdUrVVwwUhH2V2xwS7C53kE9k+lmkZ1P5WTgN5KPjF/zTr4VNpvN9he9ALsuOkwyJDknAAAAAElFTkSuQmCC","orcid":"","institution":"Yili Normal University","correspondingAuthor":true,"prefix":"","firstName":"Huanjun","middleName":"","lastName":"Liu","suffix":""},{"id":630416645,"identity":"b56e0ff6-654e-45c0-bbb1-93a5e7d1b4fd","order_by":1,"name":"Xiao Mou","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Xiao","middleName":"","lastName":"Mou","suffix":""},{"id":630416647,"identity":"619f70d0-2faf-494d-9898-26c0181e07b1","order_by":2,"name":"Yuwen Wang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Yuwen","middleName":"","lastName":"Wang","suffix":""},{"id":630416648,"identity":"76e60134-d9fe-40d6-ad61-f5038bb81a44","order_by":3,"name":"Diyi Qian","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Diyi","middleName":"","lastName":"Qian","suffix":""}],"badges":[],"createdAt":"2026-04-18 17:08:06","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9457087/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9457087/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":108635249,"identity":"e0709451-ae51-426b-9d08-e3522dc787d1","added_by":"auto","created_at":"2026-05-06 17:40:18","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":22473,"visible":true,"origin":"","legend":"\u003cp\u003ePlant dry weight, fresh weight, plant height, emergence rate, and survival rate. “*”indicates significant differences among treatments at \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05; “**” indicates \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01; “***” indicates \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001. CK: irrigation with water only; M2: 17.00 g·pot\u003csup\u003e-1\u003c/sup\u003e bio-based paper film + 25.00 g·pot\u003csup\u003e-1\u003c/sup\u003e bio-based calcium sulfonate; M4: 27.20 g·pot\u003csup\u003e-1\u003c/sup\u003e bio-based paper film + 25.00 g·pot\u003csup\u003e-1\u003c/sup\u003e bio-based calcium sulfonate; M55: 37.40 g·pot\u003csup\u003e-1\u003c/sup\u003e bio-based paper film + 25.00 g·pot\u003csup\u003e-1\u003c/sup\u003e bio-based calcium sulfonate; JF: microbial fertilizer + PVC plastic film. The same notations apply to the figures below\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-9457087/v1/85dcab4bab910abe0e3eca72.png"},{"id":108635246,"identity":"80c4a02b-5bcd-4b0d-9470-b962e7e25fdb","added_by":"auto","created_at":"2026-05-06 17:40:18","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":31588,"visible":true,"origin":"","legend":"\u003cp\u003eSoil physicochemical properties. \u003cstrong\u003eSOM:\u003c/strong\u003e Soil organic matter; \u003cstrong\u003eAP:\u003c/strong\u003eAvailable phosphorus; \u003cstrong\u003eAN:\u003c/strong\u003e Available nitrogen; \u003cstrong\u003eEC:\u003c/strong\u003e Electrical conductivity; \u003cstrong\u003eST:\u003c/strong\u003e Soil temperature; \u003cstrong\u003eSM:\u003c/strong\u003e Soil moisture\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-9457087/v1/51c671cb267b45dddd9dbc1c.png"},{"id":108635253,"identity":"a294f83a-1ea1-4585-8529-3edc8e7ef23d","added_by":"auto","created_at":"2026-05-06 17:40:18","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":11599,"visible":true,"origin":"","legend":"\u003cp\u003eOrganic acids in rhizosphere soil. \u003cstrong\u003eMAL\u003c/strong\u003e: Malic acid; CAS: Oxalic acid; \u003cstrong\u003eCA\u003c/strong\u003e: Citric acid\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-9457087/v1/b2c374da9e2601d41cedac1d.png"},{"id":108635247,"identity":"d2594b07-8e97-49ee-8cfb-b40fa575846e","added_by":"auto","created_at":"2026-05-06 17:40:18","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":24849,"visible":true,"origin":"","legend":"\u003cp\u003eSoil enzyme activities.\u003cstrong\u003e CL\u003c/strong\u003e: Cellulase; \u003cstrong\u003eAKP\u003c/strong\u003e: Alkaline phosphatase; \u003cstrong\u003eβ-GC\u003c/strong\u003e: β-glucosidase; \u003cstrong\u003eNP\u003c/strong\u003e: Neutral phosphatase; \u003cstrong\u003eUE\u003c/strong\u003e: Urease\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-9457087/v1/736e02b6cbee8270e93276d5.png"},{"id":108805458,"identity":"354da818-1ac0-4cf2-a25e-165c6479f7df","added_by":"auto","created_at":"2026-05-08 15:26:02","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":155526,"visible":true,"origin":"","legend":"\u003cp\u003eAlpha diversity indices of bacteria and fungi. (a) Bacterial ACE index; (b) Bacterial Chao1 index; (c) Bacterial Shannon index; (d) Bacterial Simpson index; (e) Bacterial Coverage index; (f) Fungal ACE index; (g) Fungal Chao1 index; (h) Fungal Shannon index; (i) Fungal Simpson index; (j) Fungal Coverage index\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-9457087/v1/9910c0cab318070eabb6768c.png"},{"id":108635248,"identity":"25783b6d-3ea5-4a17-b2a5-dba0719c91f7","added_by":"auto","created_at":"2026-05-06 17:40:18","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":94724,"visible":true,"origin":"","legend":"\u003cp\u003ePCoA analysis of β-diversity of bacteria and fungi. (a) PCoA analysis of bacterial communities; (b) PCoA analysis of fungal communities\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-9457087/v1/a7e1899ae40d1ab5866bca3f.png"},{"id":108635251,"identity":"32296595-f3b0-414d-98e2-3885916c6fbc","added_by":"auto","created_at":"2026-05-06 17:40:18","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":225061,"visible":true,"origin":"","legend":"\u003cp\u003eBacterial (a) and fungal (b) taxa with relative abundance greater than 1% at the phylum level\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-9457087/v1/b7866813ce254e215238b32e.png"},{"id":108805930,"identity":"6db79409-31eb-4f1f-9982-21561ca4546d","added_by":"auto","created_at":"2026-05-08 15:27:13","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":361396,"visible":true,"origin":"","legend":"\u003cp\u003e(a) LEfSe multi-level taxonomic hierarchy diagram and LDA score bar chart for bacteria; (b) LEfSe multi-level taxonomic hierarchy diagram and LDA score bar chart for fungi\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-9457087/v1/10f4002fcae62bbc8fdb8926.png"},{"id":108635284,"identity":"fdc3d88a-8396-4d87-97bf-9c0c4a8f9d34","added_by":"auto","created_at":"2026-05-06 17:40:26","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":551081,"visible":true,"origin":"","legend":"\u003cp\u003eCo-occurrence networks of soil bacteria and fungi under CK (a), M2 (b), M4 (c), M55 (d), and JF (e). The topological parameters shown are total number of edges, total number of nodes, number of positive edges, average degree, and proportion of positive edges\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-9457087/v1/a46f2675a4c107d80b75775f.png"},{"id":108635257,"identity":"d8436af4-4837-455a-92f5-2fd14f44c0c7","added_by":"auto","created_at":"2026-05-06 17:40:20","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":257814,"visible":true,"origin":"","legend":"\u003cp\u003e(a) RDA analysis of key bacterial taxa with soil properties, soil extracellular enzyme activities, and rhizosphere soil organic acids; (b) RDA analysis of key fungal taxa with soil properties, soil extracellular enzyme activities, and rhizosphere soil organic acids; (c) Mantel test analysis of key bacterial and fungal taxa with soil properties, soil extracellular enzyme activities, and rhizosphere soil organic acids; (d) Ecosystem multifunctionality in the rhizosphere of alfalfa under different treatments\u003c/p\u003e","description":"","filename":"10.png","url":"https://assets-eu.researchsquare.com/files/rs-9457087/v1/805f92e93a5c1e138a43f907.png"},{"id":108809668,"identity":"11c9e6d0-bb60-4644-9311-f4aa8f29f13e","added_by":"auto","created_at":"2026-05-08 15:54:48","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1797552,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9457087/v1/eea68f31-7fb0-49b6-b589-be100d643a43.pdf"}],"financialInterests":"","formattedTitle":"Mechanisms of Bio-Based Paper Mulch and Calcium Sulfonate Water-Soluble Fertilizer in Ameliorating Saline-Alkali Soils","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eSoil salinization poses a serious threat to agricultural production and soil ecology. Under salt stress, plant growth is inhibited, and microbial activity and survival are suppressed. These effects disrupt soil material cycling and lead to the degradation of ecological functions (Ahlawi et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Lu et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In recent years, soil salinization has intensified due to human-induced soil degradation and climate change. According to the Global Status of Salt-Affected Soils report released by the FAO in 2024, salt-affected soils cover more than 1.381\u0026nbsp;billion hectares worldwide, accounting for 10.7% of the global land area (FAO. 2024). In China, saline-alkali soils cover approximately 100\u0026nbsp;million hectares. Xinjiang is the most severely affected region, accounting for about one-third of the national total. Within Xinjiang, 274,000 hectares are classified as heavily saline-alkali soil. Despite this, most soils in this region have deep profiles and represent a potential resource for arable land (Jiang et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Therefore, rehabilitating saline-alkali soils in Xinjiang is a critical strategy for improving soil ecology and enhancing agricultural productivity.\u003c/p\u003e \u003cp\u003eHigh evaporation is a primary driver of soil salinization in Xinjiang. Mulching can reduce water loss from the soil surface. As an important agricultural practice, it improves soil warming, moisture retention, weed control, and salt inhibition (Liu et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Li et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Zhang et al. \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). However, plastic mulch is difficult to fully recover. Residual plastic fragments impede water and nutrient movement and disrupt soil structure. In addition, leached plasticizers can inhibit root development and harm soil biota (Koskei et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Therefore, replacing conventional plastic mulch with biodegradable alternatives is an effective way to reduce plastic pollution in agricultural soils (Li et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2024b\u003c/span\u003e). Biodegradable mulches can be divided into two categories: those derived from natural macromolecules and those synthesized from biodegradable polymers. European countries began developing biodegradable mulch films in the early 1990s. In 2018, the European Union introduced the EN 17033 standard for biodegradable mulch films used in agriculture and horticulture. This established a clear technical framework for the industry (Žaimis et al. \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Ajao et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Sadeghifar et al. 2020). In recent years, China has made rapid progress in biodegradable mulch technology. This includes films derived from biomass resources such as lignin and cellulose (Chen et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Li et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Wang et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). After degradation, these materials are converted into soil organic matter and support sustainable agriculture. The bio-based products used in this study were derived from crop straw. A sulfonation process produced water-soluble biomacromolecules as bio-based calcium sulfonate fertilizer. The remaining insoluble fraction was used to produce biodegradable bio-based paper film (Li et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Bio-based calcium sulfonate exchanges cations with sodium ions, driving sodium downward from the plow layer. Calcium ions then react with carbonate to form calcium carbonate, reducing soil salinity and pH (Cao. 2020).The insoluble fraction mainly contains lignin, cellulose, and hemicellulose. These components can be directly utilized by microorganisms. As a result, the bio-based paper film acts as a nutrient-rich substrate and enriches microbes adapted to high nutrient availability. When applied to the soil surface, it also retains heat and moisture and suppresses salt resurgence. Conventional methods for ameliorating saline-alkali soils often require high water input, incur high costs, and have poor scalability. They also carry a high risk of salt reaccumulation (Han et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). In contrast, bio-based calcium sulfonate is fully water-soluble. Combined with the widespread use of integrated water\u0026ndash;fertilizer systems in Xinjiang and the potential for spray application of bio-based paper film, this approach shows strong potential for saline-alkali soil remediation.\u003c/p\u003e \u003cp\u003eA single approach is insufficient for the sustainable amelioration of saline-alkali soils. Therefore, integrated chemical, physical, and biological measures are required (Li et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Alfalfa (Medicago sativa) is a moderately salt- and alkali-tolerant leguminous forage crop with both economic and ecological value for soil improvement (Chen et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Alfalfa develops a strong root system and high aboveground biomass. Symbiotic rhizobia supply nitrogen to the plant. They also enhance soil fertility, improve soil structure, and contribute to water and soil conservation (Xu et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Therefore, the combined application of bio-based paper film, bio-based calcium sulfonate, and alfalfa is an effective strategy for the long-term remediation of saline-alkali soils.\u003c/p\u003e \u003cp\u003eEcosystem multifunctionality (EMF) refers to the capacity of an ecosystem to provide multiple functions simultaneously. It emphasizes the combined performance and synergistic effects of different functions and enables a comprehensive evaluation of complex ecosystems (Hu et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Previous studies have shown that EMF is influenced by plant communities, soil physicochemical properties, enzyme activities, and microbial diversity. Among these factors, plant and microbial diversity are the main drivers of EMF. They jointly regulate EMF through aboveground-belowground feedback mechanisms (Antiqueira et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). EMF also reflects ecosystem resilience and sustainability. High EMF indicates greater functional redundancy. Even if some species or processes are impaired, other components can maintain overall ecosystem functioning. This enhances the system's resistance to external disturbances (Ma et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Therefore, EMF was used in this study to evaluate the effectiveness of bio-based paper film in improving saline-alkali soil ecosystems.\u003c/p\u003e \u003cp\u003eIn this study, a combined strategy was employed to ameliorate saline-alkali soil. Bio-based calcium sulfonate was applied to leach salts downward. Bio-based paper film was used to suppress salt resurgence, and alfalfa (Medicago sativa) was planted as a salt-tolerant crop. The effects of different application rates of bio-based paper film on alfalfa growth, rhizosphere soil properties, microbial community structure, and ecosystem multifunctionality (EMF) were investigated. The goal was to clarify how bio-based paper film improves saline-alkali soil and regulates the microbial community. This will provide a theoretical basis for its application in the remediation of saline-alkali soils in Xinjiang.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Soil Collection and Experimental Site\u003c/h2\u003e \u003cp\u003eThe saline-alkali soil was collected from Que'erpan Village, Cha County, Ili Prefecture, Xinjiang (43.92\u0026deg;N, 81.33\u0026deg;E), located on the south bank of the Ili River. The site has a temperate continental semi-arid climate, with an annual mean temperature of 7.9\u0026deg;C, mean annual precipitation of 222 mm, and evaporation of 1630 mm. Before soil collection, the field had been planted with oil sunflower and alfalfa, but both crops failed completely due to severe soil salinization. The pot experiment was conducted in a greenhouse at the Production-Education-Research Base of Yili Normal University.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Experimental Materials and Design\u003c/h2\u003e \u003cp\u003eThe alfalfa (Medicago sativa) seeds used in this study were purchased from a local agricultural supply market, with the variety being Omekanog (Eurekanog). The bio-based paper film, mainly composed of lignin, cellulose, and hemicellulose, and bio-based calcium sulfonate (containing Organic Matter (OM): 44.5%; Ca: 4250 mg\u0026middot;L⁻\u0026sup1;; N: 61.51 mg\u0026middot;L⁻\u0026sup1;; K: 125.79 mg\u0026middot;L⁻\u0026sup1;; S: 847.58 mg\u0026middot;L⁻\u0026sup1;; Si: 25.05 mg\u0026middot;L⁻\u0026sup1;) were all provided by Beijing Unisplendour Yingli Company. The microbial inoculant was purchased from Xinjiang Tiankai Ecological Technology Co., Ltd. Plastic pots (24 cm in diameter, 20 cm in height) and perlite were obtained from a local flower market. Saline-alkali soil was randomly collected from a depth of 0\u0026ndash;20 cm, and the collected soil was thoroughly mixed to prepare a homogeneous saline-alkali soil with a salinity of 0.6%.\u003c/p\u003e \u003cp\u003eOn June 15, 2025, a pot experiment was conducted to cultivate alfalfa (Medicago sativa) in a greenhouse. Each pot was filled with 5 kg of saline-alkali soil and 100 g of perlite, which were mixed thoroughly. Five treatments were established, each with five replicates: CK (irrigation with water only), M2 (17.00 g\u0026middot;pot\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e bio-based paper film\u0026thinsp;+\u0026thinsp;25.00 g\u0026middot;pot\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e bio-based calcium sulfonate), M4 (27.20 g\u0026middot;pot\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e bio-based paper film\u0026thinsp;+\u0026thinsp;25.00 g\u0026middot;pot\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e bio-based calcium sulfonate), M55 (37.40 g\u0026middot;pot\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e bio-based paper film\u0026thinsp;+\u0026thinsp;25.00 g\u0026middot;pot\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e bio-based calcium sulfonate), and JF (microbial fertilizer\u0026thinsp;+\u0026thinsp;PVC plastic film). The dosage of bio-based calcium sulfonate was determined based on our previous experiments(Liu et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). For the M2, M4, and M55 treatments, after laying and forming the bio-based paper film, 50 seeds were uniformly sown per pot, followed by the application of bio-based calcium sulfonate (diluted and applied in multiple increments). For the CK and JF treatments, 50 seeds were sown per pot, followed by normal watering. Except for CK, all other treatments received a topdressing one month later, consisting of 0.93 g of diammonium phosphate and 0.71 g of potassium sulfate per pot. The experiment was terminated on October 3, 2025.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Sample Collection\u003c/h2\u003e \u003cp\u003eEmergence counts were recorded 10 days after sowing. At the end of the experiment, the number of surviving plants in each pot was counted to calculate the survival rate. Three alfalfa plants with uniform growth were selected from each pot to measure fresh weight, dry weight, and plant height. The entire alfalfa plant was removed from each pot, and large soil aggregates attached to the roots were gently detached. Rhizosphere soil (0\u0026ndash;5 mm from the root surface) was collected using sterile brushes and other tools, placed into 10 mL sterile, nuclease-free centrifuge tubes, and stored at -80\u0026deg;C for subsequent high-throughput sequencing of the rhizosphere microbial community and determination of rhizosphere organic acids. The remaining soil was passed through a 2 mm sieve to remove debris and air-dried for subsequent measurements of soil physicochemical properties and enzyme activities (Hu et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Soil Physicochemical Properties\u003c/h2\u003e \u003cp\u003eSoil pH was measured using a pH meter at a soil-to-water ratio of 1:2.5 (Wu et al. \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Electrical conductivity (EC) was determined with a conductivity meter at a soil-to-water ratio of 1:5 (Hu et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Soil temperature and moisture were measured using a portable soil temperature-moisture rapid meter (Wufuer et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Soil organic matter (SOM) content was determined using the potassium dichromate volumetric method (Schecter et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). Available nitrogen (AN) content was measured with a Kjeldahl nitrogen analyzer (Fulford and Roberts \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Available phosphorus (AP) content was determined using the 0.5 mol\u0026middot;L⁻\u0026sup1; NaHCO₃ extraction method (Veloso et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Soil Enzyme Activities and Organic Acids\u003c/h2\u003e \u003cp\u003eCommercial kits purchased from Beijing Solarbio Science \u0026amp; Technology Co., Ltd. were used to quantify the activities of alkaline phosphatase (AKP), neutral phosphatase (NP), cellulase (CL), β-glucosidase (β-GC), and urease (UE) in the soil, following the manufacturer's instructions. Organic acids, including oxalic acid, citric acid, and malic acid, were measured using LC-ESI-MS/MS (UHPLC-Qtrap) (Bueno-Mui\u0026ntilde;o et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. High-Throughput Sequencing Analysis\u003c/h2\u003e \u003cp\u003eTotal microbial genomic DNA was extracted using the E.Z.N.A.\u0026reg; Soil DNA Kit (Omega Bio-tek, Norcross, GA, USA). DNA integrity was assessed by 1% agarose gel electrophoresis. DNA concentration and purity were determined using a NanoDrop 2000 spectrophotometer (Thermo Scientific, USA). The extracted DNA was used as a template to amplify the V3-V4 hypervariable region of the 16S rRNA gene with primers 338F and 806R carrying barcode sequences. The fungal internal transcribed spacer (ITS) region was amplified using primers ITS1F and ITS2R (Sinha et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The PCR program for both bacterial and fungal DNA was as follows: initial denaturation at 95\u0026deg;C for 3 min; 30 cycles of 95\u0026deg;C for 30 s, 55\u0026deg;C for 30 s, and 72\u0026deg;C for 45 s; and a final extension at 72\u0026deg;C for 10 min, followed by holding at 10\u0026deg;C. PCR products from each sample were pooled and examined by 2% agarose gel electrophoresis. Target fragments were excised and purified using the AxyPrep DNA Gel Extraction Kit (AXYGEN) and eluted with Tris-HCl. The purified products were re-examined by 2% agarose gel electrophoresis. Quantification was performed using the QuantiFluor\u0026trade;-ST fluorometric system (Promega). Sequencing libraries were constructed from the purified PCR products using the NEXTFLEX Rapid DNA-Seq Kit. Sequencing was conducted on the Illumina NextSeq 2000 platform (Shanghai Majorbio Bio-Pharm Technology Co., Ltd.). The raw sequence data were deposited in the NCBI Sequence Read Archive (SRA) database.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Statistical analysis\u003c/h2\u003e \u003cp\u003eRaw paired-end sequencing reads were quality-controlled using fastp (v0.23.4) and assembled with FLASH (v1.2.11). The quality-filtered and assembled sequences were then denoised using the DADA2 plugin within the QIIME2 pipeline under default parameters. The resulting sequences were defined as amplicon sequence variants (ASVs). Taxonomic assignment was conducted using the classify-sklearn (Naive Bayes) algorithm in QIIME2 against the Silva138.2/16S bacterial database, with a confidence threshold of 70% (Bokulich et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). For both bacteria and fungi, sequence counts were rarefied to 50,887 and 51,487 per sample, respectively. In total, 9,915 bacterial ASVs and 2,515 fungal ASVs were obtained and used for subsequent analyses of microbial diversity and community structure.\u003c/p\u003e \u003cp\u003eStatistical analyses of plant agronomic traits, soil physicochemical properties, enzyme activities, and organic acids were performed using SPSS 26. One-way analysis of variance (ANOVA) followed by Duncan's multiple range test was used to determine significant differences. Box plots were generated using Origin 2021. Microbial data were analyzed and visualized using R software (v4.4.3). Alpha diversity indices of bacteria and fungi were calculated using the otuSummary package, and box plots were generated with the ggplot2 package (Liu et al. 2023). Chord diagrams of community abundance at the phylum level were generated using the circlize package. Principal coordinate analysis (PCoA) based on Bray-Curtis distances was performed, followed by PERMANOVA tests (Yang et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Linear discriminant analysis effect size (LEfSe) was conducted using the microeco package, with an LDA score\u0026thinsp;\u0026gt;\u0026thinsp;4 and P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 indicating significant enrichment (Bian et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Network analysis was performed using the igraph package based on Spearman's correlation (r\u0026thinsp;\u0026gt;\u0026thinsp;0.8, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Jacomy et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Redundancy analysis (RDA) and Mantel tests were conducted based on Spearman's correlation using the vegan and linkET packages, respectively (Kim and Cui \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Cromer et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eA total of 27 indicators were selected to calculate ecosystem multifunctionality (EMF). The averaging approach was applied after Z-score normalization (Hooper and Vitousek \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). These indicators included plant dry and fresh weight. Soil indicators included temperature, moisture, electrical conductivity (EC), pH, soil organic matter (SOM), available phosphorus (AP), available nitrogen (AN), oxalic acid (CAS), citric acid (CA), malic acid (MAL), neutral phosphatase (NP), alkaline phosphatase (AKP), urease (UE), β-glucosidase (β-GC), and cellulase (CL). Microbial indicators included the Shannon, Chao1, Simpson, ACE, and coverage indices for both bacteria and fungi. EMF was calculated as follows:\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:\\text{EMF}=\\frac{1}{n}\\sum\\:_{i=1}^{n}{Z}_{i}$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eWhere \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{Z}_{i}\\)\u003c/span\u003e\u003c/span\u003e is the Z-score normalized value of indicator \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:i\\)\u003c/span\u003e\u003c/span\u003e, and \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:n\\)\u003c/span\u003e\u003c/span\u003e is the total number of indicators.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Plant Growth\u003c/h2\u003e \u003cp\u003eThe application of bio-based paper film improved the growth of alfalfa under saline-alkali stress. The results are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCompared with the control (CK), the application of bio-based paper film significantly increased alfalfa dry weight, fresh weight, plant height, emergence rate, and survival rate (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Among all treatments, M4 showed the best performance. Relative to CK, M4 increased dry weight by 400.78%, fresh weight by 323.93%, plant height by 109.05%, emergence rate by 131.91%, and survival rate by 127.72% (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Soil Physicochemical Properties and Enzyme Activities\u003c/h2\u003e \u003cp\u003eThe application of bio-based paper film improved the physicochemical properties of saline-alkali soil, with M4 showing the best performance (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The bio-based paper film significantly increased soil organic matter, available phosphorus, and available nitrogen. Compared with CK, M4 increased these parameters by 138.66%, 57.19%, and 30.16%, respectively (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). The bio-based paper film also reduced soil electrical conductivity (EC) and pH. Relative to CK, M4 decreased EC by 48.23% (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and pH by 0.60 units (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). In addition, the bio-based paper film helped maintain soil temperature and moisture. Compared with CK, M4 increased soil temperature by 2.53% and soil moisture by 95.86% (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe application of bio-based paper film increased the content of organic acids in rhizosphere soil, with M4 showing the best performance (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The bio-based paper film significantly increased malic acid (MAL), oxalic acid (CAS), and citric acid (CA) in the rhizosphere soil of alfalfa. Compared with CK, M4 increased these organic acids by 318.92%, 321.35%, and 197.48%, respectively (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe application of bio-based paper film enhanced soil enzyme activities, with M4 showing the best performance (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The activities of cellulase, alkaline phosphatase, β-glucosidase, neutral phosphatase, and urease were significantly increased under the bio-based paper film treatments. Compared with CK, M4 increased the activities of these enzymes by 139.46% (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), 78.64% (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), 48.24% (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), 56.49% (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), and 37.10% (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001), respectively.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Effect of Bio-Based Paper Film on Rhizosphere Soil Microbial Community\u003c/h2\u003e \u003cp\u003eThe application of bio-based paper film had a minor effect on the alpha diversity of soil bacteria but a greater impact on that of soil fungi (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Compared with CK, the M4 treatment decreased the bacterial ACE and Chao1 indices by 3.21% and the Shannon index by 1.03%, while the Simpson index increased by 50.00%; however, none of these differences were statistically significant. In contrast, for soil fungi, M4 significantly decreased the ACE and Chao1 indices by 29.87% and the Shannon index by 23.18% (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), while the Simpson index increased by 42.31%.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAnalysis of microbial β-diversity showed that the first two principal coordinates (PCo1 and PCo2) explained 32.526% of the variation in bacterial community structure (R\u0026sup2; = 0.39, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.001), indicating significant differences among treatment groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). The M4 and CK samples were located in different quadrants, suggesting a marked difference between these two treatments. For fungal communities, PCo1 and PCo2 explained 35.00% of the structural variation (R\u0026sup2; = 0.53, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.001), also indicating significant differences among groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb). The M4 and CK samples were again distributed in distinct quadrants, demonstrating a clear difference between them.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eAt the phylum level, bacterial and fungal taxa with a relative abundance greater than 1.0% were defined as dominant. The dominant bacterial phyla included Pseudomonadota (28.7%), Bacillota (15.5%), Actinomycetota (11.1%), Chloroflexota (10.8%), Bacteroidota (6.5%), Gemmatimonadota (4.7%), Acidobacteriota (4.3%), Myxococcota (4.2%), Cyanobacteriota (2.7%), Patescibacteria (2.2%), Rhodothermota (1.5%), Planctomycetota (1.4%), and Verrucomicrobiota (1.1%). These phyla together accounted for more than 94% of the total bacterial ASVs. Among all treatments, M4 showed the highest proportion of Pseudomonadota, while the relative abundances of other bacterial phyla varied little among treatments. The dominant fungal phyla included Ascomycota (47.6%), Basidiomycota (38.9%), unclassified_k_Fungi (5.8%), Fungi_Phy_Incertae_sedis (3.3%), Aphelidiomycota (2.0%), and Rozellomycota (1.9%). These phyla together accounted for more than 99% of the total fungal ASVs. Ascomycota dominated in the CK and JF treatments, whereas Basidiomycota dominated in the M2, M4, and M55 treatments.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eLEfSe analysis (LDA\u0026thinsp;\u0026gt;\u0026thinsp;2, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) was performed to identify taxa with significantly different abundances among treatment groups. The top 20 taxa with the highest LDA scores are shown. Among bacterial taxa, four significantly enriched groups were identified in CK: P__Actinomycetota, P__Bacteroidota, c__Bacteroidia, and c__Actinobacteria. In contrast, M4 showed seven significantly enriched taxa, including P__Pseudomonadota, c__Alphaproteobacteria, o__Hyphomicrobiales, c__Polyangiia, c__Anaerolineae, o__Aggregatilineales, and P__Myxococcota. Among fungal taxa, six significantly enriched groups were detected in CK: P__Ascomycota, c__Sordariomycetes, o__Hypocreales, g__unclassified_f__Nectriaceae, s__unclassified_f__Nectriaceae, and g__Enterocarpus. No significantly enriched fungal taxa were detected in the M4 treatment.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Co-occurrence Network of Rhizosphere Soil Microorganisms of Alfalfa\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eA co-occurrence network of soil bacterial and fungal communities was constructed based on ASV-level data (r\u0026thinsp;\u0026gt;\u0026thinsp;0.8, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e). Compared with CK, the bio-based paper film treatments increased the number of edges, the proportion of positive correlations, and the average degree in the bacterial-fungal network. These changes indicate strengthened mutualistic relationships between bacteria and fungi. In the M4 treatment, the network comprised 259 nodes and 3,015 edges. Positive correlations accounted for 57.55% (1,735 edges), and the average degree was 23.282. Compared with CK, M4 increased the number of edges by 176.61%, the proportion of positive correlations by 8.52%, the number of positively correlated edges by 200.17%, and the average degree by 185.14%, while the number of nodes decreased by 3.00%.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.5. Correlations Among Microbial Communities, Soil Properties, and Alfalfa Growth\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eRedundancy analysis (RDA) revealed the relationships between soil microbial communities and soil properties. For bacteria (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003ea), RDA1 and RDA2 explained 46.92% of the community variation. The CK treatment was mainly associated with the pH and EC axes and showed significant positive correlations with these variables (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). In contrast, the M4 treatment was distributed along the directions of soil nutrients, enzyme activities, temperature, moisture, and organic acids, and showed significant positive correlations with these factors (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Key bacterial taxa, including Hyphomicrobiales, Polyangiales, Beijerinckiaceae, and Beduibacterium, were positively correlated with NP, ST, β-GC, AN, AKP, SOM, AP, CA, MAL, UE, CL, and CAS (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Soil pH and EC were negatively correlated with soil nutrients, enzyme activities, temperature, moisture, and organic acids (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). For fungi (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eb), RDA1 and RDA2 explained 58.99% of the variation. The CK treatment was again associated with pH and EC and showed significant positive correlations with these variables (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The M4 treatment was positively correlated with soil nutrients, enzyme activities, temperature, moisture, and organic acids (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Key fungal taxa, including Agaricomycetes, Coprinellus, Coprinellus alkalinus, Basidiomycota, Agaricales, and Psathyrellaceae, were positively correlated with NP, AN, MAL, SOM, β-GC, ST, AP, CL, AKP, CAS, SM, CA, and UE (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Soil pH and EC were negatively correlated with these environmental factors (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Mantel tests were conducted to assess the relationships among key microbial taxa, soil properties, and alfalfa growth (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003ec). Key bacterial taxa were significantly correlated with AP, CAS, NP, AKP, UE, CL, and fresh weight (FW). Among these, AP showed the strongest correlation (Mantel's r\u0026thinsp;=\u0026thinsp;0.44). Key fungal taxa were significantly correlated with SM, EC, SOM, AN, CAS, CA, UE, CL, FW, and dry weight (DW). Among these, SOM showed the strongest correlation (Mantel's r\u0026thinsp;=\u0026thinsp;0.535).Ecosystem multifunctionality (EMF) differed significantly among treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003ed). M4 showed the highest EMF, whereas CK exhibited the lowest level.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e4.1. Effects of Bio-Based Products on Alfalfa Growth and Soil Properties\u003c/h2\u003e \u003cp\u003ePlant growth is closely linked to the soil environment. In this study, compared with CK, the bio-based paper film treatments promoted alfalfa seed germination and growth. Plant dry weight, fresh weight, plant height, emergence rate, and survival rate were all significantly increased (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), with M4 showing the best performance. These positive effects may be attributed to multiple factors. The water-soluble fertilizer reduced salt accumulation in the surface soil layer. Meanwhile, the bio-based paper film maintained soil temperature and moisture and inhibited salt resurgence. Together, these changes created favorable conditions for seed germination and seedling growth. This finding is consistent with previous studies showing that mulching promotes the growth of maize, alfalfa, and taro (Sheng et al. 2019; Gu et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Anikwe et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2007\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSoil physicochemical properties, enzyme activities, and organic acids are key indicators of soil quality. Soil quality is closely linked to agricultural management practices, plant (root) activity, and microbial communities, which interact to regulate these indicators. In this study, compared with CK, all bio-based paper film treatments significantly increased soil organic matter, available phosphorus, and available nitrogen (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), with M4 showing the best performance. This improvement may be attributed to the application of water-soluble fertilizer and the input of alfalfa root exudates, which increased soil organic matter content. Increased microbial activity, together with higher urease and phosphatase activities, contributed to the accumulation of available phosphorus and nitrogen. The M4 treatment showed the lowest EC and pH values. This was related to the salt-leaching effect of the water-soluble fertilizer and the inhibition of salt resurgence by the bio-based paper film. The reduction in EC and pH restored root metabolic activity. This led to increased secretion of organic acids and promoted microbial proliferation in the rhizosphere soil. Microorganisms are the primary source of soil enzymes, and their increased abundance is generally associated with higher enzyme activities. The M4 treatment enriched Basidiomycota and its lower taxonomic units, as well as Anaerolineae. This significantly increased β-glucosidase and cellulase activities (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). In addition, the enrichment of Alphaproteobacteria enhanced nitrogen fixation in the alfalfa rhizosphere. Rapid plant growth increased nutrient demand, which further stimulated urease and phosphatase activities. These findings are consistent with previous studies showing that mulching increases soil nutrients and enzyme activities while reducing pH and electrical conductivity (Liu et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Mao et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Jacquiod et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e4.2. Bio-Based Products Reshape the Microbial Interaction Network in the Alfalfa Rhizosphere\u003c/h2\u003e \u003cp\u003eRhizosphere soil microorganisms originate from bulk soil and undergo self-regulation to adapt to new environmental conditions. This process generally reduces microbial diversity (Essel et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In this study, the alpha diversity of both bacteria and fungi was lower in the bio-based paper film treatments than in CK, with bacterial diversity being less affected. The difference between bacterial and fungal diversity may be attributed to variations in rhizosphere soil physicochemical properties and their distinct nutrient acquisition strategies (Philippot et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The application of bio-based paper film and bio-based calcium sulfonate reduced soil EC and inhibited salt resurgence. This created a favorable environment and provided sufficient nutrients for microbial growth. Due to their shorter generation times and stronger substrate utilization capacities, bacteria had a competitive advantage over fungi in nutrient and spatial competition. This resulted in a suppressive effect on fungal abundance (Dămăt\u0026icirc;rcă et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDifferences in nutrient substrates can selectively enrich microbial taxa with distinct resource utilization preferences (Rinnan and Baath. 2009). The bio-based paper film and bio-based calcium sulfonate provided abundant soluble organic matter, as well as cellulose and lignin that can be directly decomposed by microorganisms. This created clear substrate differences compared with CK. As a result, the bio-based treatments caused significant changes in rhizosphere microbial β-diversity. These changes indicate the formation of new microbial assemblages adapted to the altered environment and shifts in community structure. LEfSe analysis further showed that, among bacteria, the key taxa in the M4 treatment were Pseudomonadota and its lower taxonomic units, as well as Anaerolineae, which are associated with high nutrient availability. Among fungi, CK was dominated by pathogenic taxa belonging to Ascomycota and its lower taxonomic units. In contrast, the bio-based paper film treatments were dominated by saprotrophic fungi of Basidiomycota and its lower taxonomic units, which have strong capacities to decompose cellulose and lignin. These findings are consistent with previous studies showing that organic fertilizer application regulates microbial community structure and composition (Liu et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2023b\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSoil microbial network analysis reveals potential interspecific interactions and the organizational principles of microbial communities (Huo et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Yue et al. \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In this study, network analysis showed that the application of bio-based products increased the complexity of the alfalfa rhizosphere microbial network compared with CK. Among all treatments, M4 showed more nodes and edges, as well as the highest proportion of positive correlations. This indicates a more \u0026ldquo;cooperative, complex, and efficient\u0026rdquo; network with greater resistance to environmental disturbances. These effects may be attributed to multiple factors. Bio-based products leached salts from the soil surface and inhibited salt resurgence. They also supplied water-soluble organic matter, calcium, and decomposable substrates such as lignin and cellulose. These changes created favorable conditions for both alfalfa and microorganisms. As alfalfa resumed normal metabolism, root exudates were released into the soil in large quantities. This increased nutrient availability and promoted microbial growth, thereby enhancing network complexity (Wang et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2022a\u003c/span\u003e; Wang et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2022b\u003c/span\u003e; Shi et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e4.3. Correlations Between Key Microbial Taxa and Rhizosphere Soil Properties\u003c/h2\u003e \u003cp\u003eMicrobial community structure is shaped by environmental filtering, biological interactions, host regulation, and spatiotemporal dynamics. Among these, environmental filtering is the primary driver of microbial community assembly (Banerjee et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Xun et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). RDA results showed that the CK treatment was mainly influenced by pH and EC. Bacterial key taxa were associated with CAS, whereas fungal key taxa were associated with SOM. Mantel tests further showed that bacterial key taxa were significantly correlated with AP, CAS, NP, AKP, UE, CL, and fresh weight (FW). Fungal key taxa were significantly correlated with SM, EC, SOM, AN, CAS, CA, UE, CL, FW, and dry weight (DW). These patterns may be explained by changes in soil conditions. The application of bio-based paper film and bio-based calcium sulfonate reduced soil pH and EC. This shifted the M4 treatment from a stress-dominated environment to a substrate-dominated environment. Under these conditions, alfalfa roots released large amounts of CAS into the soil. This enriched Pseudomonadota and its lower taxonomic units, as well as Anaerolineae. Meanwhile, SOM derived from roots, exudates, microbial residues, and bio-based materials enriched Basidiomycota and its lower taxonomic units (Ling et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e4.4. Bio-based Paper Film Enhances the EMF of Saline-Alkali Soils\u003c/h2\u003e \u003cp\u003eThe performance of individual functions does not reflect overall ecosystem health. Ecosystem multifunctionality (EMF) provides a comprehensive evaluation of ecosystem functions (Manning et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Wang et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2022c\u003c/span\u003e). In this study, M4 showed the highest EMF. This is because the alfalfa root system, soil, and microorganisms formed an integrated system. Root exudates promoted microbial recruitment and increased community complexity. This enhanced enzyme activities and nutrient cycling. These interactions collectively improved EMF (Li et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2024a\u003c/span\u003e; Liu et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2023a\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eThis study investigated the effects of bio-based paper film on the alfalfa rhizosphere and evaluated its role in improving soil properties, microbial communities, and ecosystem multifunctionality (EMF) in heavily saline-alkali soil. The results showed that the application of 27.20 g\u0026middot;pot⁻\u0026sup1; (M4) achieved the best amelioration effect. The M4 treatment reduced soil EC and pH and maintained soil temperature and moisture. It also promoted alfalfa germination and growth. As a result, alfalfa roots released large amounts of organic acids. Soil nutrient contents and enzyme activities were significantly increased. Although the alpha diversity of bacteria and fungi decreased, their community structures were optimized. A \u0026ldquo;cooperative, complex, and efficient\u0026rdquo; microbial interaction network was formed. Bacterial taxa involved in nitrogen fixation and phosphate solubilization, such as Pseudomonadota, Alphaproteobacteria, and Anaerolineae, were enriched. The higher abundance of Basidiomycota and its lower taxonomic units enhanced the decomposition of cellulose and lignin. Overall, the M4 treatment showed the highest EMF. This study provides a theoretical basis and practical reference for the remediation of saline-alkali soils in Xinjiang.\u003c/p\u003e"},{"header":"Statements and Declarations","content":"\u003cp\u003eAcknowledgements\u003c/p\u003e\n\u003cp\u003eThis study was supported bythe Xinjiang Talent Development Fund Research Innovation Platform Talent Team Project(XJRCSWZTD2025) , the Innovation and Entrepreneurship Training Program of Yili Normal University(202410764012), and the Key R\u0026amp;D Program of Xinjiang Uygur Autonomous Region (2022B02021).\u003c/p\u003e\n\u003cp\u003eCompeting Interests\u003c/p\u003e\n\u003cp\u003eThe authors have no competing interests to declare that are relevant to the content of this article.\u003c/p\u003e\n\u003cp\u003eAuthor Contributions\u003c/p\u003e\n\u003cp\u003eHuanjun Liu: Writing \u0026ndash; review \u0026amp; editing, Writing \u0026ndash; original draft, Visualization, Methodology, Data curation, Conceptualization. Dayi Qian: Writing \u0026ndash; review \u0026amp; editing, Visualization, Validation, Supervision, Project administration, Methodology, Funding acquisition, Data curation, Conceptualization. Xiao Mou: Collected plant and soil samples and conducted sample analyses. Yuwen Wang: Writing \u0026ndash; review \u0026amp; editing.\u003c/p\u003e\n\u003cp\u003eData availability\u003c/p\u003e\n\u003cp\u003eData will be made available on request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAhlawi S, Naeem A, Rengel Z, et al (2018) Biochar application for the remediation of salt-affected soils: Challenges and opportunities. Science of the Total Environment 625: 35-320. https://doi.org/10.1016/j.scitotenv.2017.12.257\u003c/li\u003e\n\u003cli\u003eAjao O, Jeaidi J, Benali M, et al (2018) Quantification and variability analysis of lignin optical properties for colour-dependent industrial applications. 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Industrial Crops and Products 215: 118634. https://doi.org/10.1016/j.indcrop.2024.118634\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"plant-and-soil","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"plso","sideBox":"Learn more about [Plant and Soil](https://www.springer.com/journal/11104)","snPcode":"11104","submissionUrl":"https://submission.nature.com/new-submission/11104/3","title":"Plant and Soil","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Bio-based paper film, Soil microbial community, Soil properties, Ecosystem multifunctionality, Saline-alkali soil, Alfalfa (Medicago sativa)","lastPublishedDoi":"10.21203/rs.3.rs-9457087/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9457087/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground and Aims\u003c/h2\u003e \u003cp\u003eSoil salinization threatens agricultural productivity and ecosystem health. Bio-based paper film and bio-based calcium sulfonate have the potential to reduce salinity and enhance ecosystem multifunctionality (EMF). However, their mechanisms in heavily saline-alkali soils remain unclear.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eA pot experiment was conducted with five treatments: CK (water only), M2 (17.00 g\u0026middot;pot\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e bio-based paper film\u0026thinsp;+\u0026thinsp;25.00 g\u0026middot;pot\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e bio-based calcium sulfonate), M4 (27.20 g\u0026middot;pot\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e bio-based paper film\u0026thinsp;+\u0026thinsp;25.00 g\u0026middot;pot\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e bio-based calcium sulfonate), M55 (37.40 g\u0026middot;pot\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e bio-based paper film\u0026thinsp;+\u0026thinsp;25.00 g\u0026middot;pot\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e bio-based calcium sulfonate), and JF (microbial fertilizer\u0026thinsp;+\u0026thinsp;PVC film). Soil properties, microbial communities, and EMF were analyzed.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eM4 showed the best performance. Compared with CK, it promoted alfalfa (Medicago sativa) germination and growth and increased rhizosphere organic acids. It reduced soil pH and electrical conductivity (EC) and increased soil temperature, moisture, nutrients, and enzyme activities. Although microbial alpha diversity decreased, bacterial and fungal community structures were optimized, forming a more cooperative and complex network. Beneficial taxa involved in nitrogen fixation and phosphate solubilization (e.g., Pseudomonadota, Alphaproteobacteria, and Anaerolineae) were enriched. The higher abundance of Basidiomycota enhanced cellulose and lignin decomposition.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThe combined application of bio-based paper film and calcium sulfonate improved soil quality and reshaped microbial communities, thereby enhancing ecosystem functioning in saline-alkali soils.\u003c/p\u003e","manuscriptTitle":"Mechanisms of Bio-Based Paper Mulch and Calcium Sulfonate Water-Soluble Fertilizer in Ameliorating Saline-Alkali Soils","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-06 17:40:13","doi":"10.21203/rs.3.rs-9457087/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewersInvited","content":"","date":"2026-04-27T14:23:33+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Plant and Soil","date":"2026-04-21T22:33:42+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-21T04:45:31+00:00","index":"","fulltext":""},{"type":"submitted","content":"Plant and Soil","date":"2026-04-19T22:41:18+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"plant-and-soil","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"plso","sideBox":"Learn more about [Plant and Soil](https://www.springer.com/journal/11104)","snPcode":"11104","submissionUrl":"https://submission.nature.com/new-submission/11104/3","title":"Plant and Soil","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"36a430dd-3237-4b2a-94ac-ad69fe3675cd","owner":[],"postedDate":"May 6th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-06T17:40:14+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-06 17:40:13","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9457087","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9457087","identity":"rs-9457087","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}