Mixed sowing improves soil fertility via enhancing plant facilitation in degraded grassland system

Mixed sowing improves soil fertility via enhancing plant facilitation in degraded grassland system | 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 Mixed sowing improves soil fertility via enhancing plant facilitation in degraded grassland system Shengjun Ji, Mohamed S. Sheteiwy, Wei Wang, Youcai Xiong, Fuying Niu, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8560681/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 7 You are reading this latest preprint version Abstract Background By enhancing plant complementarity and facilitation, mixed sowing may accelerate soil fertility recovery and improve ecosystem resilience. Nevertheless, the short-term impacts of mixed sowing on soil fertility improvement and the underlying plant-soil interaction mechanisms remain poorly understood. We hypothesize that interspecific interactions in mixed sowing systems may cause changes in rhizosphere soil properties, thereby affecting soil fertility. Results To test this hypothesis, monoculture and mixed sowing treatments were designed using Elymus breviaristatus , Medicago sativa , and moss to investigate their ecological adaptation. Our results showed that compared with monocropping, mixed sowing significantly increased above and belowground biomass, improved soil water content, and reduced bulk density, thereby creating a more favorable soil microenvironment. Compared with monocropping, mixed sowing enhanced microbial biomass C and N by 26% and 10%, respectively, and increased soil organic C by 30%. Fertility indices were positive in all mixed sowing systems, contrasting with negative values under monocropping and control. Across treatments, the facilitation index was positively correlated with the fertility index (R 2 = 0.76), demonstrating that interspecific facilitation was the primary driver of soil fertility gains. Conclusions These findings reveal that mixed sowing enhances soil fertility restoration in degraded grasslands through interspecific facilitation that promotes biomass accumulation, improves soil physicochemical conditions, and stimulates microbial-mediated nutrient cycling. These mechanisms highlight crop diversification as an effective strategy for accelerating soil fertility restoration. Plant-plant interaction Biodiversity Soil physical structure Soil fertility Mixed sowing Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Grasslands, as an important component of terrestrial ecosystems, play a pivotal role in global carbon (C) and nitrogen (N) cycling, soil and water conservation, biodiversity maintenance, and husbandry development [ 1 , 2 , 3 , 4 ]. Globally, they cover nearly 40% of the land surface (FAO, 2005) and are recognized as critical C sinks that can mitigate climate change while supporting multiple ecosystem services [ 5 ]. Despite this importance, grassland integrity is declining under converging pressures from climate change, overgrazing, and inappropriate land use [ 6 , 7 ]. These stressor drive widespread degradation characterized by reduced vegetation coverage, biodiversity loss, disrupted soil structure, and declining fertility, which in turn weaken the ecosystem service functions and ecological resilience [ 8 , 9 , 10 ]. Restoring degraded grasslands is therefore essential to stabilize and enhance ecosystem service while safeguarding co-benefits for biodiversity and livelihoods [ 11 ]. Qinghai-Tibet Plateau is one of the regions with the highest altitude and the most fragile ecosystems, dominated by alpine grasslands that play a critical role in regional climate regulation, water conservation, and pastoral production [ 12 , 13 , 14 ]. However, these ecosystems have been increasingly degraded due to the combined effects of climate warming, overgrazing, and unsustainable land use [ 16 ]. Therefore, restoring degraded alpine grasslands is of great ecological and socio-economic importance. Among various restoration strategies, mixed sowing (or interplanting) has emerged as an effective approach for accelerating ecosystem recovery in degraded grasslands. By combining plant species with niches complementary and plant facilitation, mixed communities can enhance nutrient acquisition, increase primary productivity, and improve soil physical and biochemical properties [ 17 ]. Plant facilitation, a key ecological process that regulates resource allocation within plant communities and drives their overproduction, typically refers to beneficial ecological interactions between adjacent plants, benefiting at least one of them [ 18 ]. For instance, when leguminous plants are mixed with gramineous plants, the former facilitate the growth of the latter through biological N fixation and cross-species N transfer, while the rapid growth of gramineous plants promotes the accumulation of soil C [ 19 , 20 ]. Although plant facilitation is common within plant communities, it remains unclear whether applying its fundamental principles can enhance ecosystem functions in mixed-species communities. Previous studies have shown that combinations of Elymus breviaristatus (a perennial grass with strong soil-holding capacity) and Medicago sativa (a legume capable of symbiotic N fixation) have been widely used to improve soil fertility and vegetation stability [ 21 , 22 , 23 ]. Biological soil crusts (BSCs) are complexes formed in degraded grassland ecosystems by cyanobacteria, lichens, mosses, and their secretions in conjunction with soil particles. They can cover the ground surface, effectively reducing soil water evaporation and soil erosion [ 24 ]. Introducing BSCs, such as moss, into mixed sowing systems may promote soil nutrient retention by secreting polysaccharides to facilitate soil aggregate formation, or enhance soil water content by covering the soil to slow down water evaporation [ 25 ]. These advantages could potentially facilitate the productivity of mixed planting systems and cascade to affect soil fertility. Despite these potential synergies, the interactive mechanisms by which plant mixtures and BSCs jointly influence soil fertility and ecosystem functions remain poorly understood. Most existing studies have focused on either mixed cropping or BSCs effects in isolation, while few have explored their combined impacts on plant–soil feedbacks and the short-term restoration of degraded alpine grasslands. Unraveling these mechanisms is therefore crucial for developing optimized mixed sowing models tailored to the unique ecological conditions of the Qinghai-Tibet Plateau and for advancing the theory and practice of grassland restoration in cold, arid regions [ 27 ]. Therefore, this study focuses on degraded grasslands on the Qinghai-Tibet Plateau, we established seven treatments: 1) blank control (S0); 2) monocropping of Elymus breviaristatus (S1); 3) monocropping of Medicago sativa (S2); 4) monocropping of moss (S3); 5) mixed sowing of Elymus breviaristatus and moss (S4); 6) mixed sowing of Medicago sativa and moss (S5); and 7) mixed sowing of Elymus breviaristatus and Medicago sativa (S6). To sum up: (1) the short-term impact of mixed sowing patterns on soil C, N, P pools, and aggregate stability; (2) whether legume-grass-moss mixed sowing can simultaneously enhance soil physical structure; and (3) whether soil structure improvement (MWD, bulk density) and microbial biomass (MBC, MBN) are the dominant driving factors for soil fertility restoration. The research results will provide theoretical support for the rapid restoration of alpine grasslands and fill the research gap in the synergistic enhancement of legume-grass-moss interactions. 2. Materials and Methods 2.1 Experimental location The research site is in Xihai Town, Haiyan County, Haibei Prefecture, Qinghai Province (36°44′N, 100°23′E), with an altitude of 3140 m. This region experiences relatively low precipitation and high evaporation, belonging to the plateau continental climate. Over the past 10 years, the annual average temperature has ranged from − 1.5 to 1.2 ℃, with annual precipitation of 400 mm, concentrated in June to September, and evaporation of 1538 mm (Fig. 1 ). In 2024, the total precipitation was 420 mm, slightly higher than the usual level, and the annual average temperature was approximately 1.0-1.5 ℃. According to the classification of the Food and Agriculture Organization of the United Nations (FAO), the local soil type is identified as chestnut soil. The initial physicochemical properties of the soil (0–20 cm soil layer) are: pH 8.22, SOC 16.18 g kg − 1 , TN 3.18 mg kg − 1 , AP 1.54 mg kg − 1 , and bulk density 1.45 g cm − ³. 2.2 Experimental design and sample plot management Elymus brevidentatus (Elymus breviaristatus cv. Tongde) and Medicago sativa (Qingda 1) seeds were sown in a field for the current experiment. The moss was collected from the topsoil within thestation nearby the study site during early spring. This study employed a randomized block experimental design, encompassing mixed sowing of Elymus brevidentatus - Medicago sativa , Elymus brevidentatus - moss , Medicago sativa - moss , as well as their respective monocropping treatments. Additionally, the experiment included a natural degraded grassland without planting as a control treatment. Consequently, this study involved a total of seven treatments, with each treatment replicated three times, resulting in a total of 21 plots. The area of each plot was 3 m × 2 m, with a spacing of 1 m between quadrats to prevent edge effects. Seeding was conducted using a strip sowing method. Elymus brevidentatus and Medicago sativa were planted in May during the growing season, adhering to the locally promoted planting density standards: 22.5 g m − 2 for Elymus brevidentatus and 15 g m − 2 for Medicago sativa . The sowing depth was 10 cm, with a row spacing of 20 cm. Two months prior to sowing, moss approximately 1 cm thick was collected and brought back to the laboratory. After being dried in the shade, it was crushed and sieved through a 2 mm sieve to remove weeds and root fragments. When the Elymus brevidentatus and Medicago sativa reached approximately 5 cm in height, 700 g m − 2 of crushed moss was uniformly spread onto the surface of the plots designated for mixed sowing and monocropping of moss according to the experimental design. During the plant growth period, weeds were removed and uniform field management was conducted. No fertilization treatment was applied to the plots. 2.3 Sampling and determination The sampling time was in late September. All above-ground plant parts were cut from a 0.5 m × 0.5 m quadrat in each plot. After being blanched at 105 ℃ for 2 hours, they were dried in an oven at 80℃ and weighed. The sampling depth for plant root biomass was at a soil depth of 0–20 cm. The collected roots were cleaned with water, dried, and weighed. Soil samples were collected using the root core method. Specifically, an 8 cm inner diameter root core was used to surround the plant roots and extract a soil core from 0–10 cm depth. After gently shaking to remove large soil clumps attached to the plant roots, the soil attached to the surface layer of the plant roots (0–3 mm) was collected. Meanwhile, the residual soil surrounding the roots from the above process was collected for aggregate stability and soil moisture storage measurements. Therefore, the collected soil samples were divided into two parts: one was stored at 4℃ as a rhizosphere soil sample for measuring microbial biomass C and N (MBC & MBN) and inorganic N (NH 4 + -N and NO 3 − −N), while the other was naturally air-dried for analyzing soil organic C (SOC), total nitrogen (TN), total P, available P, pH, and aggregate distribution. The contents of SOC and TN were measured using an elemental analyzer (Elementar, Germany). The contents of soil MBC and MBN were determined using the chloroform fumigation method [ 28 ]. Specifically, fresh soil samples equivalent to 10 g of oven-dried soil were divided into two equal parts. The first part was fumigated for 24 hours and then extracted with the 0.5 mol L − 1 K 2 SO 4 . The second one was non-fumigated as a control, followed by extraction with the same K 2 SO 4 . The MBC and MBN content were determined as the difference between C and N contents in fumigated and non-fumigated soil extract using Elemental vario TOC cube (Elementar, Germany) with corrections for conversion factor KC and KN of 0.45 and 0.54 [ 29 ]. The above unfumigated soil extracts were further used for the determination of soil NH 4 + -N and NO 3 − −N by using a FIAstar 5000 Analyser (Foss Tecator, Sweden). Soil total P was determined colorimetrically after perchloric acid digestion, while available P (AP) was determined using the Olsen method. Soil pH was determined in a soil: water suspension of 1:2.5. Soil moisture content (SWC %) is determined using the weighing method. The wet sieving method was used to separate soil aggregates according to Zhang et al [ 30 ]. These water-stable aggregates of different sizes were weighted to measure the mean weight diameter (MWD), which was used as an index of soil aggregate stability. 2.4 Data Processing In this study, we first employed one-way ANOVA and Tukey's HSD post-hoc test to compare the differences in all parameters between various monoculture treatments and mixed culture treatments at significance levels of 0.05, 0.01, and 0.001. Furthermore, we reclassified monoculture and mixed culture into two groups and utilized independent sample t-tests to analyze the differences between the two groups. In addition, we employed linear fitting to examine the relationship between the fertility index and the promotion index, and utilized heatmaps and Pearson's test to analyze the correlation between plant productivity and various soil properties. All statistical analyses were conducted using SPSS 22 (SPSS Inc., Chicago, USA), with a significance level set at p < 0.05. The charts were plotted using Origin 2021 software (Origin Lab, USA). 3. Results 3.1 The impact of unicast and mixed broadcast on the aboveground and underground biomass of plants In the monoculture treatment, the aboveground biomass of Elymus brevidentatus (S1) and Medicago sativa (S2) was 0.50 kg m − 2 and 0.47 kg m − 2 , respectively, with no significant difference between the two (both P < 0.05; Fig. 2 ). The biomass of moss was the lowest. In the mixture treatment, the Elymus breviden tatus - Medicago sativa mixture (S6) had the highest biomass of 0.75 kg m − 2 , followed by the Elymus brevidentatus - moss (S4) and Medicago sativa - moss mixtures (S5), with 0.6 kg m − 2 and 0.55 kg m − 2 , respectively. The root biomass exhibited different trends. In the monoculture treatment, the root biomass of S6 was the highest at 19.46 kg m − 2 , followed by S1 at 8.75 kg m − 2 , and moss was the lowest at 2.9 kg m − 2 . In the mixture treatment, there was no significant difference in root biomass between S5 and S6, while the S4 mixture had the lowest root biomass. Overall, the aboveground biomass in the mixed sowing treatment was higher than that in the monoculture treatment, and the root biomass also exhibited a similar trend (except for the Elymus brevidentatus - moss mixture). 3.2 The impact of monoculture and polyculture on soil fertility Overall, mix-planting treatments exhibited higher SOC, TN, and TP contents compared to monoculture treatments, although the specific differences varied across different indicators (both P < 0.05; Fig. 3 ). All mix-planting treatments had higher SOC contents, specifically 24.24 g kg − 1 , 28.02 g kg − 1 , and 26.47 g kg − 1 , respectively, compared to monoculture treatments (19.77 g kg − 1 , 22.77 g kg − 1 , 18.21 g kg − 1 ) and the control treatment (16.18 g kg − 1 ). Notably, S5 and S6 showed significant differences compared to monoculture and the control treatments. However, the differences in TN were not significant among the treatments, with only S5 (3.87 g kg − 1 ) showing significant differences compared to Control treatment (S0) (3.18 g kg − 1 ) and S2 (3.29 g kg − 1 ). TP exhibited a similar trend, with only S6 (1.37 g kg − 1 ) and S2 (1.13 g kg − 1 ) showing significant differences compared to S4 (1.21 g kg − 1 ) treatment, while the remaining planting treatments showed no significant differences. In terms of soil nutrient availability, there were no significant differences in ammonium N levels among all planting treatments, while nitrate N levels were higher in the mixed cropping treatment (4.82 ~ 6.33 mg kg − 1 ). Specifically, the nitrate nitrogen levels in all mixed cropping treatments were significantly different from those in the S0 (1.81 mg kg − 1 ), S1 (3.45 mg kg − 1 ), and S3 (3.31 mg kg − 1 ) treatments. Similarly, the available P levels in the soil were also higher in the mixed planting treatment, but there was no significant difference compared to the S2 (7.67 mg kg − 1 ) treatment. Regardless of the indicator, the level in the control treatment was the lowest, indicating a clear trend of soil degradation. 3.3 The impact of monoculture and mixed sowing on soil physicochemical properties Different treatment modes significantly influenced the levels of soil microbial C and N (both P < 0.05; Fig. 4 ). There were notable differences between mixed sowing and monoculture. The MBC of the S0 (137.80 mg kg − 1 ) was at the lowest level, while the MBC of the mixed sowing of S6 was the highest (217.81 mg kg − 1 ). In comparison, the MBC of the monoculture treatments (152.10 ~ 177.90 mg kg − 1 ) was lower than that of the mix-planting treatments (193.85 ~ 217.81 mg kg − 1 ). The trend of MBN variation was like that of MBC, with significant differences between the mixed sowing treatments (14.93 ~ 17.68 mg kg − 1 ) and the monoculture treatments (12.03 ~ 13.75 mg kg − 1 ). The S0 (10.28 mg kg − 1 ) had the lowest level. There were no significant differences in soil C/N among monoculture treatments and the control treatment. The soil C/N was highest in the mix-planting of S5 (8.17), which differed significantly from the other treatments. The pH differences among the treatments were not significant. The trends in soil moisture content (SWC%) and mean weight diameter (MWD) were the same, with the mixed sowing treatments generally being higher than the monoculture treatments and the control treatment, indicating that mixed sowing is beneficial for improving soil water retention capacity and structural stability. However, the trend reflected by soil bulk density was opposite, with S0 (1.45 g m − 3 ) being the highest, and the monoculture treatments (1.35 ~ 1.42 g m − 3 ) being higher than the mix-planting treatments (1.24 ~ 1.31 g m − 3 ). 3.4 The comparison of soil characteristics between monoculture and mix-planting By comparing the soil physical properties, plant biomass, soil C, N, and nutrients, as well as soil microbial characteristics between monoculture and mixed sowing, it was found that all indicators except soil bulk density and pH were significantly higher in the mixed sowing treatment compared to the monoculture treatment (both P < 0.05; Fig. 5 ). In the mixed sowing treatment, the aboveground biomass, belowground biomass, SOC, TN, NO 3 − -N, NH 4 + , AP, TP, C/N, MBC, MBN, SWC%, and MWD increased by an average of 25%, 16%, 30%, 3%, 41%, 8%, 19%, 9%, 38%, 26%, 10%, 19%, and 12% respectively compared to the monoculture treatment. Meanwhile, soil bulk density and pH decreased by 8% and 1% respectively. Therefore, compared to monoculture, the mixed sowing model significantly optimizes soil physical structure (reducing bulk density, enhancing aggregate stability, and increasing moisture content), promotes plant biomass accumulation, regulates soil C and N cycling and nutrient availability, activates microbial activity, and alters the soil chemical microenvironment. 3.5 The relationship between interspecific facilitation and soil fertility The facilitation index showed significant differences among different treatments, with the monoculture treatment exhibiting negative values (-1.43 to -0.75) and the mixed sowing treatment exhibiting positive values (0.72 to 1.01) (both P < 0.05; Fig. 6 a). The results indicate that the mixed sowing treatment is significantly different from the monoculture treatment and the control treatment. The fertility index of the S0 treatment is significantly lower than that of other treatments, exhibiting a negative value and reaching around − 6.7, indicating that this treatment has the worst fertility (both P < 0.05; Fig. 6 b). The fertility indices of the S1 and S3 treatments are also negative, at -1.5 and − 3.8, respectively, indicating that the soil fertility of these two treatments is also poor, but weaker than that of S0. However, the fertility indices of the S2, S4, S5, and S6 treatments are positive, at 3.3, 2.8, 3.8, and 2.2, respectively, indicating that these treatments can promote soil fertility. There are no significant differences in fertility index among these treatments, which are generally maintained between 2 and 4. The fertility index increased significantly with the facilitation index (R 2 = 0.757), indicating that stronger facilitation effectively enhanced soil fertility (Fig. 6 c). Correlation analysis revealed strong positive associations among most soil fertility indicators (Fig. 6 d). Aboveground and root biomass were significantly correlated with key soil nutrient and structural variables, including SOC, TN, MBC, MBN, SWS, MWD, NO₃⁻-N, NH₄⁺-N, AP, and TP. In contrast, bulk density, C/N ratio, and pH showed negative correlations with several fertility-related parameters. 4. Discussion 4.1 The Effects of Interactions of Plants in Mixed sowing Systems Effective ecological restoration in Qinghai-Tibet Plateau requires strategies that enhance plant establishment and promote sustainable soil fertility. While plant–plant interactions are recognized as a fundamental driver of community assembly and ecosystem functioning, their facilitative roles in restoring alpine grasslands remain insufficiently explored [ 31 ]. In this study, mixed sowing treatments significantly increased aboveground and root biomass (Fig. 2 ). This may be attributed to the interspecific interactions between different plant species. For instance, alfalfa, a leguminous plant, provides N for gramineous plants through N fixation, while gramineous plants, with their deep root systems, enhance the absorption of water and nutrients. In addition, the incorporation of moss may have improved the soil microenvironment, such as soil moisture (Fig. 4 ), thereby further promoting plant growth. Empirical studies have shown that the mixed sowing of gramineous plants and legumes exhibits a biomass advantage, which is because gramineous plants can not only achieve strong complementary utilization of water through belowground niche differentiation, but also acquire more water in the shared growth area due to their high competitiveness [ 32 ]. Leguminous crops can enhance P availability in the rhizosphere of neighboring cereal crops by regulating soil pH and activating insoluble P through acidic secretions [ 33 ]. The nodule N fixation of alfalfa provides a N advantage for Elymus breviaristatus , increasing its leaf N content by 15%-20% and improving its photosynthetic efficiency by 20%-30% [ 34 ]. In this study, S2, S5, and S6, which are related to the treatments of Medicago sativa , all increased soil P and nitrogen levels (Fig. 3 ). This result indicates that Medicago sativa can promote aboveground biomass accumulation by regulating the availability of soil nutrients. The core advantage of mixing moss with gramineous and leguminous plants lies in the role of moss as an “ecological engineer”. By physically covering the soil surface and regulating water and microclimatic conditions, moss creates a moister environment conducive to plant growth. This microhabitat modification not only reduces soil water evaporation and promotes soil water retention but also improves soil aggregate stability and stimulates microbial activity (Fig. 4 ). Consequently, all treatments involving moss exhibited higher belowground biomass than their respective monocultures, accompanied by an increased soil water content and reduced bulk density (Figs. 2 and 4 ). It is noted that all mixed-sowing treatments showed higher root biomass compared to their respective monocropping treatments (Fig. 2 ). All mixed sowing treatments showed greater root biomass compared with monoculture, suggesting that moss coverage facilitates the coordinated growth of Elymus breviaristatus and Medicago sativa by improving soil water availability. Moreover, when Elymus breviaristatus and Medicago sativa coexist, their simultaneous high water demand may lead to either competitive or complementary water use, which in turn promotes deeper root development and greater belowground investment. The above results indicate that mixed sowing communities can enhance overall biomass accumulation of plant communities through interspecific facilitation (S4-S6), primarily by improving soil nutrient availability and water content. In addition, a moderate level of resource competition (S6) may further stimulate root C investment, contributing to the development of a more balanced belowground system. Therefore, optimizing species composition, strengthening facilitative interactions, and minimizing excessive interspecific competition are essential strategies to improve plant community productivity and soil fertility in alpine grassland restoration. 4.2 The Effects of Mixed sowing on Soil Structure and Microorganisms The improvement of soil physical structure under mixed sowing treatment represents another important mechanism to enhanced soil fertility. The synergistic regulation of soil structure and microbial activity is central to maintaining grassland ecosystems health; particularly on the Qinghai–Tibet Plateau where fragile soils determine the stability and restoration potential of degraded grasslands ( Li et al., 2025). Under mixed-planting treatment, the average soil bulk density decreased to 1.27 g·cm⁻³, which was 8% lower than that of the monoculture treatment (1.38 g cm⁻³) and significantly lower than the control (1.45 g cm⁻³). Meanwhile, the MWD increased by 21.3% (Fig. 5 ). In contrast, the taproots of Medicago sativa extends into the 10–30 cm layer. The synergistic interaction between these root type enhances soil porosity and promotes microaggregate formation through root exudates (such as polysaccharides), thereby reducing soil bulk density. Such structural improvement alleviates the freeze–thaw–induced hardening commonly observed in Qinghai-Tibet Plateau [ 35 ]. Enhanced soil physical structure also creates a more suitable microenvironment for microbial activities by improving aeration and water retention. In the mixed-planting treatment, the MBC and MBN reached 217.81 mg kg⁻¹ and 32.65 mg kg⁻¹ respectively, increasing by 26% and 10% compared with the monoculture treatment. Among these treatments, the MBC value of the Elymus breviaristatus - M. sativa mixed sowing (S6) exhibited the highest MBC value, consistent with the positive feedback model of “soil structure-carbon input-microbial activity” [ 36 ]. In addition, the C/N ratio of the M. sativa - moss mixed sowing treatment is closer to the optimal range for microbial growth (C/N = 8–12), which may further optimize the efficiency of nutrient cycling by regulating the microbial community structure. Soil pH also differed significantly between monoculture and mixed sowing treatments. This is directly related to the process where alfalfa roots secrete organic acids (e.g., oxalic acid) to activate soil phosphorus [ 37 ], while the secretions of monocropped gramineous plants are relatively alkaline, resulting in a more stable soil pH. This pH difference further regulates the microbial community structure and accelerates nutrient transformation. Collectively, these findings suggest that mixed sowing improves nutrient availability and moderates constraints imposed by alkaline soils, thereby facilitating the restoration of degraded alpine grasslands. 4.3 Mixed sowing Enhancing Soil Fertility​ The average SOC content under mixed sowing reached 26.24 g kg⁻¹, which was 30% higher than that in the monoculture treatment. This finding aligns with previous studies showing that diversified planting systems enhance soil C accumulation [ 38 , 39 , 40 ]. The underlying mechanism can be attributed to two aspects. First, mixed sowing communities provide a broader and more continuous substrate input to the soil C pool due to greater diversity of root exudates and higher litter production. Second, functional complementarity between species extends the C input throughout the growing season, avoiding the seasonal fluctuation of C input in the monoculture system. Although the N-fixing function of M. sative did not significantly increase TN), it significantly increased the NO 3 − -N level (4.82 ~ 6.33 mg kg⁻¹). This indicates that interspecific facilitation accelerates N transformation by stimulating the nitrifying bacterial, consistent with microbial functions under N-limited conditions in alpine grasslands. A strong correlation (R 2 = 0.757) between the facilitation index and the fertility index indicates that interspecific facilitation is the primary driver of the mixed-planting effect. At the plant level, niche differentiation between deep- and shallow-rooted species (e.g., Elymus breviaristatus and M. sativa ) reduces direct resource competition. At the microbial level, synergistic interactions between leguminous-associated rhizobia and plant growth-promoting rhizobacteriaof gramineous plants enhances nutrient activation [ 41 , 42 ]. At the soil level, accelerated root turnover promotes aggregate formation, providing physical protection for C and N. Among all treatments, the Elymus breviaristatus - M. sative mixed sowing showed the the most pronounced positive effect, with the highest aboveground biomass, SOC, and MBC values, corresponding to its strong facilitation intensity. ​In contrast, the Elymus breviaristatus - moss mixed sowing showed the lowest root biomass and a limited SOC increase, likely due to resource competition between the shallow rhizoids of moss and the roots of Elymus breviaristatus . These contrasting results emphasize that the degree of functional trait complementarity determines the effectiveness of mixed-planting systems [ 43 , 44 ]. Selecting species with significant niche differentiation (e.g., deep- vs. shallow-rooted, N-fixing vs. non-N-fixing plants) is crucial for improving soil fertility and ecosystem functioning.​ Finally, the significant difference between the fertility index of the degraded control (S0: -6.7) and that of the optimal mixed-sowing treatments (S5, S6: 3.8) indicates that mixed sowing can effectively reverse soil degradation with a short timescale. While improving productivity, mixed sowing systematically improves soil fertility by optimizing soil structure and activating microbial activity, providing a promising ecological strategy for grassland restoration on the Qinghai-Tibet Plateau. 5. Conclusion​ Grassland degradation on the Qinghai-Tibet Plateau is becoming increasingly severe, and there is an urgent need to explore green solutions for soil restoration. As a natural restoration strategy, mixed sowing can effectively alleviate grassland degradation on the Qinghai-Tibet Plateau. Mixed sowing significantly promotes plant biomass accumulation through interspecific complementarity, providing continuous support for soil carbon and nitrogen input. Nutrients such as total nitrogen and available phosphorus are simultaneously increased, which confirms the driving effect of interspecific stratified resource utilization on carbon and nitrogen accumulation, as well as the role of interspecific niche differentiation in reducing interspecific competition. In addition, mixed sowing optimizes soil physical structure and activates microbial activity, providing effective nutrients for plant growth and forming a "plant-soil-microbe" synergistic feedback mechanism.. This has important practical value for grassland management on the Qinghai-Tibet Plateau: in the restoration of degraded grasslands, the adoption of mixed sowing of functional species (such as combinations of gramineous and leguminous plants) leverages their advantage in stratified resource utilization to reduce restoration costs in the short term and rapidly improve soil fertility. Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Data availability statement Date is provided within the manuscript. The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. Conflict of interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Funding This work was supported by the Natural Science Foundation of Qinghai Province (2025-ZJ-969T), the National Key R&D Program of China (2023YFF1304305), International (Regional) Cooperation and Exchange (ICE) Projects of the National Natural Science Foundation of China (NSFC) (W2412148), and 111 Project (D23029). Author contributions SJ: Writing­ - review & editing, Writing - original draft, Methodology, Supervision, Investigation, Software, Formal analysis, Data curation, Conceptualization. MS : Writing-review & editing. WW : Methodology, Formal analysis, Writing-review & editing. YC : Project administration, Methodology, Conceptualization. FY : Data curation. WY : Funding acquisition, Writing-review & editing. Acknowledgements Not applicable. References Chen J, Huang M, Zeng Y, Sui Y, Gao Y, Ochoa-Hueso R, et al. 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Agronomical and breeding approaches to improve the nutritional status of forage crops for better livestock productivity. Grass Forage Sci. 2022;77(1):11–32. Tahir M, Li C, Zeng T, Xin Y, Chen C, Javed HH et al. Mixture Composition Influenced the Biomass Yield and Nutritional Quality of Legume–Grass Pastures. Agronomy. 2022 June 17;12(6):1449. Wang W, Wang BZ, Zhou R, Ullah A, Zhao ZY, Wang PY, et al. Biocrusts as a nature-based strategy (NbS) improve soil carbon and nitrogen stocks and maize productivity in semiarid environment. Agric Water Manage. 2022;270:107742. Zhang Y, Liu B. Biological soil crusts and their potential applications in the sand land over Qinghai-Tibet Plateau. Res Cold Arid Reg. 2024;16(1):20–9. Wang W, Li MY, Zhang W, Khan A, Zhou R, Zhu SG, et al. Soil moisture drives the shift from selection to complementarity effect in the rainfed maize/faba bean intercropping system. Plant Soil. 2024 June;499(1–2):313–28. Lo Presti E, Kavamura VN, Abadie M, Romeo M, Reid TE, Heuer S, et al. Phosphorus availability drives the effect of legume-wheat intercropping on prokaryotic community interactions. Appl Soil Ecol. 2024 July;199:105414. Zhang T, Wang L, Liu W, Rihu S, Li J, Zhang D. Forage mixed planting can effectively improve soil enzyme activity and microbial community structure and diversity in agro-pastoral interlacing arid zone. Can J Soil Sci. 2022;102(3):697–706. Duan XY, Surigaoge S, Du YH, Fu DH, Yang H, Yang X, et al. Interspecific interactions increase soil aggregate stability through altered root traits in long-term legume/maize intercropping. Soil Tillage Res. 2026;255:106808. Vance ED, Brookes PC, Jenkinson DS. Microbial biomass measurements in forest soils: the use of the chloroform fumigation-incubation method in strongly acid soils. Soil Biol Biochem. 1987;19(87):697–702. https://doi.org/10.1016/0038-0717( . Brookes PC, Landman A, Pruden G, Jenkinson DS. Chloroform fumigation and the release of soil nitrogen: a rapid direct extraction method to measure microbial biomass nitrogen in soil. Soil Biol Biochem. 1985;17:837–42. Zhang T, Wang L, Liu W, Rihu S, Li J, Zhang D. Forage mixed planting can effectively improve soil enzyme activity and microbial community structure and diversity in agro-pastoral interlacing arid zone. Can J Soil Sci. 2022;102:697–706. 10.1139/cjss-2021-0106 . Li J, Wang X, Yuan M, Duan W, Xia J, Zhang X, et al. Effect of soil microbial community on ecosystem multifunctionality in an alpine grassland. CATENA. 2025;249:108714. Yang Z, Gu K, Wu G, Su J, Jiang Y, Hu B, et al. Mechanisms of tobacco yield enhancement in winter crop-tobacco rotations: Enhancing soil aggregate stability, organic carbon content, and microbial diversity. Appl Soil Ecol. 2025;214:106352. Six J, Bossuyt H, Degryze S, Denef K. A history of research on the link between (micro)aggregates, soil biota, and soil organic matter dynamics. Soil Tillage Res. 2004 Sept;79(1):7–31. Wei K, Sun Y, Cartmill AD, López IF, Ma C, Zhang Q. Long-term effects of nitrogen and phosphorus fertilizers on rhizosphere physicochemical characteristics and microbial composition in alfalfa. Ind Crops Prod. 2025;227:120776. Huang J, Wang S, Wu Y, Lu X, Bai Y, Wang B, et al. Monoculture-experiment evidence that plant species identity regulates soil biota attributes and soil functions. CATENA. 2025;258:109309. Wang W, Li MY, Wen QH, Ma Y, Zhang ZM, Rehman MMU, et al. Cereal-legume intercropping stimulates straw decomposition and promotes soil organic carbon stability. Sci China Life Sci. 2025;68(5):1498–508. Wang W, Li MY, Wang Y, Li JM, Zhang W, Wen QH, et al. Legume intercropping improves soil organic carbon stability in drylands: A 7-year experimental validation. Agric Ecosyst Environ. 2025;381:109456. Wu GL, Liu ZH, Zhang L, Hu TM, Chen JM. Effects of artificial grassland establishment on soil nutrients and carbon properties in a black-soil-type degraded grassland. Plant Soil. 2010;333(1–2):469–79. Wang W, Li MY, Zhou R, Zhu SG, Tao HY, Khan A, et al. Effects of interspecific interactions on soil carbon emission and efficiency in the semiarid intercropping systems. Soil Tillage Res. 2023;234:105857. Cong W, Hoffland E, Li L, Six J, Sun J, Bao X, et al. Intercropping enhances soil carbon and nitrogen. Glob Change Biol. 2015;21(4):1715–26. Kamran M, Zhang M, Jia Q, Usman M, Waris M, Chang S, et al. Legume-grass intercropping at low nitrogen input: Achieving the dual goals of lower greenhouse gas emissions and high quality forage productivity in the arid region. Agric Ecosyst Environ. 2025;393:109859. Wang X, Li Z, Li H, Shen T, Luo Y, Zhang F, et al. Maize straw application shows regional-scale improvements to soil fertility and crop yields in Chinese croplands: A meta-analysis. Field Crops Res. 2025;333:109908. Mętrak M, Wilk M, Jasser I, Khomutovska N, Korabiewski B, Niyatbekov T, et al. Morphology and distribution of biological soil crusts and their potential role in soil-forming processes under dry high-altitude periglacial conditions (Eastern Pamir, Tajikistan). Geoderma Reg. 2023 June;33:e00636. Wietrzyk-Pełka P, Rola K, Patchett A, Szymański W, Węgrzyn MH, Björk RG. Patterns and drivers of cryptogam and vascular plant diversity in glacier forelands. Sci Total Environ. 2021;770:144793. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 23 Feb, 2026 Reviewers agreed at journal 01 Feb, 2026 Reviewers invited by journal 30 Jan, 2026 Editor assigned by journal 19 Jan, 2026 Editor invited by journal 16 Jan, 2026 Submission checks completed at journal 14 Jan, 2026 First submitted to journal 14 Jan, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-8560681","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":583052557,"identity":"4dfdcf05-10db-4575-84c7-bdeaeae1ab25","order_by":0,"name":"Shengjun Ji","email":"","orcid":"","institution":"Qinghai Normal University","correspondingAuthor":false,"prefix":"","firstName":"Shengjun","middleName":"","lastName":"Ji","suffix":""},{"id":583052558,"identity":"3a3c3508-a67e-4db9-ae43-b081d07a0cea","order_by":1,"name":"Mohamed S. Sheteiwy","email":"","orcid":"","institution":"United Arab Emirates University","correspondingAuthor":false,"prefix":"","firstName":"Mohamed","middleName":"S.","lastName":"Sheteiwy","suffix":""},{"id":583052559,"identity":"ca4d3a09-451e-43d1-8a78-0390576f5e48","order_by":2,"name":"Wei Wang","email":"","orcid":"","institution":"Lanzhou University","correspondingAuthor":false,"prefix":"","firstName":"Wei","middleName":"","lastName":"Wang","suffix":""},{"id":583052560,"identity":"d084db70-5de9-455b-95e3-c67cea6b2a41","order_by":3,"name":"Youcai Xiong","email":"","orcid":"","institution":"Qinghai Normal University","correspondingAuthor":false,"prefix":"","firstName":"Youcai","middleName":"","lastName":"Xiong","suffix":""},{"id":583052561,"identity":"021775dd-0103-43e7-9770-3127e9af23cb","order_by":4,"name":"Fuying Niu","email":"","orcid":"","institution":"Qinghai Normal University","correspondingAuthor":false,"prefix":"","firstName":"Fuying","middleName":"","lastName":"Niu","suffix":""},{"id":583052562,"identity":"2e3e3798-2a5e-4760-8891-4d9a8aa4bdb0","order_by":5,"name":"Wenying Wang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA1ElEQVRIiWNgGAWjYPACCR429uYDBz78IF6LjQw/z7HEgzN7iNeSZiM5w8f4MAcbEWoNjp89/Jq37TCPwQ2eD4cZeBjk+cUOENByJi/NGqzldu+GwwUWDIYzZyfg12J2IMfMOBek5c7ZDYdn8DAkGNwmpOX8G6iWGzkPDvOwEaPlRo7x49y2NB7JGTkMxGmxv/HGjPnPORseYCAbAANZgrBfJPtzjD/OKJOwB0bl4w8fftjI80sT0AIEbBKMiOiQIKgcBJg/MPwhSuEoGAWjYBSMVAAAAGpJ9QjKBoMAAAAASUVORK5CYII=","orcid":"","institution":"Qinghai Normal University","correspondingAuthor":true,"prefix":"","firstName":"Wenying","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2026-01-09 11:53:46","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8560681/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8560681/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":101696562,"identity":"7025ccd0-62fa-49e0-9290-518c51822128","added_by":"auto","created_at":"2026-02-02 17:07:19","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1594696,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic diagrams of meteorological data and planting pattern in each plot. Figure (A) show the sampling field information location of the study area. Figure (B) show the temperature and precipitation of the growing seasons, 2024. Figure (C-I) show the the patterns of Elymus brevidentatus, Medicago sativa, and moss monoculture, Elymus brevidentatus-moss, Medicago sativa-moss, and Elymus brevidentatus-Medicago sativa mixture, and the field pictures respectively.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8560681/v1/3f818cea473218a844738082.jpg"},{"id":101753367,"identity":"d01ff579-fc8f-4e11-8525-894ca52f363f","added_by":"auto","created_at":"2026-02-03 10:39:52","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":824013,"visible":true,"origin":"","legend":"\u003cp\u003eAboveground biomass and root biomass of Elymus brevidentatus, Medicago sativa, and moss under the different treatments in 2024 growing seasons. S1, S2 and S3 refers to Elymus brevidentatus, Medicago sativa, and moss monoculture; S4, S5, and S6 refers to Elymus brevidentatus-moss, Medicago sativa-moss, and Elymus brevidentatus-Medicago sativa mixture. Different letters indicate significant differences among different treatments for the same year at the \u003cem\u003ep\u003c/em\u003e=0.05 level.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8560681/v1/1a7adb3d1c20543092c53029.jpg"},{"id":101696561,"identity":"a69fe016-ec31-4353-b3bb-a760b810914b","added_by":"auto","created_at":"2026-02-02 17:07:19","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2092758,"visible":true,"origin":"","legend":"\u003cp\u003eSoil organic carbon (SOC) (A), soil total nitrogen (TN) (B), soil total phosphorus (TP) (C), soil ammonium nitrogen (NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N) (D), soil nitrate nitrogen (NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e-N) (E), and soil available phosphorus (AP) (F) in Elymus brevidentatus, Medicago sativa, and moss fields under different treatments in 2024 growing seasons. S0 refers Control treatment. Different letters indicate significant differences among different treatments for the same year at the 0.05 level. Horizontal bars represent the LSD at \u003cem\u003ep\u003c/em\u003e=0.05 (n=3).\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8560681/v1/ee8d0a37d0c15d796792ff3c.jpg"},{"id":101696560,"identity":"9f63f991-d8e5-4b7a-9275-a7f3aa216608","added_by":"auto","created_at":"2026-02-02 17:07:19","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1487191,"visible":true,"origin":"","legend":"\u003cp\u003eDynamics of microbial biomass carbon (MBC) (A), microbial biomass nitrogen (MBN) (B), C/N ratio (C), pH (D), soil water storage (SWS) (E), mean weight diameter (MWD) (F), and bulk density (G) under the different treatments in 2024 growing seasons. The differences between monoculture and mixture were examined using Tukey test (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05). The t-test has been used to test the differences between monoculture and mixture, and all the data show \u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, showing a significant difference from.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8560681/v1/e24cdf25e672d8aba3ed7dd6.jpg"},{"id":101753408,"identity":"dcc92384-7419-47ee-9c7a-5b4825da56c4","added_by":"auto","created_at":"2026-02-03 10:40:00","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1578418,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of soil properties between monoculture and mixture systems.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8560681/v1/3b9e3dacda9992854a1f656f.jpg"},{"id":101696558,"identity":"4acd7d68-4238-4ea7-8a10-9bee01c9a3ca","added_by":"auto","created_at":"2026-02-02 17:07:19","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":640586,"visible":true,"origin":"","legend":"\u003cp\u003eFacilitation Index in different crop strips across the cropping patterns in growing seasons (2024) (A). Fertility index in different crop strips across the cropping patterns in growing seasons (2024) (B). The linear correlation between Facilitation index and Fertility index (C). Mantel test correlation analysis of soil physicochemical properties composition under different treatments (D). *, significant at \u003cem\u003ep≤\u003c/em\u003e0.05. **, significant at \u003cem\u003ep≤\u003c/em\u003e0.01. ***, significant at \u003cem\u003ep≤\u003c/em\u003e0.001.\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8560681/v1/8203e99aa7882d8eafc036e9.jpg"},{"id":101755610,"identity":"7e3fb4bf-0755-48dc-8877-7e34927ba320","added_by":"auto","created_at":"2026-02-03 10:53:18","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":9098554,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8560681/v1/72274595-c6d4-4334-8500-2eb3cd37bc38.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Mixed sowing improves soil fertility via enhancing plant facilitation in degraded grassland system","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eGrasslands, as an important component of terrestrial ecosystems, play a pivotal role in global carbon (C) and nitrogen (N) cycling, soil and water conservation, biodiversity maintenance, and husbandry development [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Globally, they cover nearly 40% of the land surface (FAO, 2005) and are recognized as critical C sinks that can mitigate climate change while supporting multiple ecosystem services [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Despite this importance, grassland integrity is declining under converging pressures from climate change, overgrazing, and inappropriate land use [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. These stressor drive widespread degradation characterized by reduced vegetation coverage, biodiversity loss, disrupted soil structure, and declining fertility, which in turn weaken the ecosystem service functions and ecological resilience [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Restoring degraded grasslands is therefore essential to stabilize and enhance ecosystem service while safeguarding co-benefits for biodiversity and livelihoods [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eQinghai-Tibet Plateau is one of the regions with the highest altitude and the most fragile ecosystems, dominated by alpine grasslands that play a critical role in regional climate regulation, water conservation, and pastoral production [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. However, these ecosystems have been increasingly degraded due to the combined effects of climate warming, overgrazing, and unsustainable land use [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Therefore, restoring degraded alpine grasslands is of great ecological and socio-economic importance. Among various restoration strategies, mixed sowing (or interplanting) has emerged as an effective approach for accelerating ecosystem recovery in degraded grasslands. By combining plant species with niches complementary and plant facilitation, mixed communities can enhance nutrient acquisition, increase primary productivity, and improve soil physical and biochemical properties [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Plant facilitation, a key ecological process that regulates resource allocation within plant communities and drives their overproduction, typically refers to beneficial ecological interactions between adjacent plants, benefiting at least one of them [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. For instance, when leguminous plants are mixed with gramineous plants, the former facilitate the growth of the latter through biological N fixation and cross-species N transfer, while the rapid growth of gramineous plants promotes the accumulation of soil C [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Although plant facilitation is common within plant communities, it remains unclear whether applying its fundamental principles can enhance ecosystem functions in mixed-species communities.\u003c/p\u003e \u003cp\u003ePrevious studies have shown that combinations of \u003cem\u003eElymus breviaristatus\u003c/em\u003e (a perennial grass with strong soil-holding capacity) and \u003cem\u003eMedicago sativa\u003c/em\u003e (a legume capable of symbiotic N fixation) have been widely used to improve soil fertility and vegetation stability [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Biological soil crusts (BSCs) are complexes formed in degraded grassland ecosystems by cyanobacteria, lichens, mosses, and their secretions in conjunction with soil particles. They can cover the ground surface, effectively reducing soil water evaporation and soil erosion [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Introducing BSCs, such as moss, into mixed sowing systems may promote soil nutrient retention by secreting polysaccharides to facilitate soil aggregate formation, or enhance soil water content by covering the soil to slow down water evaporation [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. These advantages could potentially facilitate the productivity of mixed planting systems and cascade to affect soil fertility. Despite these potential synergies, the interactive mechanisms by which plant mixtures and BSCs jointly influence soil fertility and ecosystem functions remain poorly understood. Most existing studies have focused on either mixed cropping or BSCs effects in isolation, while few have explored their combined impacts on plant\u0026ndash;soil feedbacks and the short-term restoration of degraded alpine grasslands. Unraveling these mechanisms is therefore crucial for developing optimized mixed sowing models tailored to the unique ecological conditions of the Qinghai-Tibet Plateau and for advancing the theory and practice of grassland restoration in cold, arid regions [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTherefore, this study focuses on degraded grasslands on the Qinghai-Tibet Plateau, we established seven treatments: 1) blank control (S0); 2) monocropping of \u003cem\u003eElymus breviaristatus\u003c/em\u003e (S1); 3) monocropping of \u003cem\u003eMedicago sativa\u003c/em\u003e (S2); 4) monocropping of moss (S3); 5) mixed sowing of \u003cem\u003eElymus breviaristatus\u003c/em\u003e and moss (S4); 6) mixed sowing of \u003cem\u003eMedicago sativa\u003c/em\u003e and moss (S5); and 7) mixed sowing of \u003cem\u003eElymus breviaristatus\u003c/em\u003e and \u003cem\u003eMedicago sativa\u003c/em\u003e (S6). To sum up: (1) the short-term impact of mixed sowing patterns on soil C, N, P pools, and aggregate stability; (2) whether legume-grass-moss mixed sowing can simultaneously enhance soil physical structure; and (3) whether soil structure improvement (MWD, bulk density) and microbial biomass (MBC, MBN) are the dominant driving factors for soil fertility restoration. The research results will provide theoretical support for the rapid restoration of alpine grasslands and fill the research gap in the synergistic enhancement of legume-grass-moss interactions.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Experimental location\u003c/h2\u003e \u003cp\u003eThe research site is in Xihai Town, Haiyan County, Haibei Prefecture, Qinghai Province (36\u0026deg;44\u0026prime;N, 100\u0026deg;23\u0026prime;E), with an altitude of 3140 m. This region experiences relatively low precipitation and high evaporation, belonging to the plateau continental climate. Over the past 10 years, the annual average temperature has ranged from \u0026minus;\u0026thinsp;1.5 to 1.2 ℃, with annual precipitation of 400 mm, concentrated in June to September, and evaporation of 1538 mm (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). In 2024, the total precipitation was 420 mm, slightly higher than the usual level, and the annual average temperature was approximately 1.0-1.5 ℃. According to the classification of the Food and Agriculture Organization of the United Nations (FAO), the local soil type is identified as chestnut soil. The initial physicochemical properties of the soil (0\u0026ndash;20 cm soil layer) are: pH 8.22, SOC 16.18 g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, TN 3.18 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, AP 1.54 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, and bulk density 1.45 g cm\u003csup\u003e\u0026minus;\u003c/sup\u003e\u0026sup3;.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Experimental design and sample plot management\u003c/h2\u003e \u003cp\u003e \u003cem\u003eElymus brevidentatus\u003c/em\u003e (Elymus breviaristatus cv. Tongde) \u003cem\u003eand Medicago sativa\u003c/em\u003e (Qingda 1) seeds were sown in a field for the current experiment. The moss was collected from the topsoil within thestation nearby the study site during early spring.\u003c/p\u003e \u003cp\u003eThis study employed a randomized block experimental design, encompassing mixed sowing of \u003cem\u003eElymus brevidentatus\u003c/em\u003e-\u003cem\u003eMedicago sativa\u003c/em\u003e, \u003cem\u003eElymus brevidentatus\u003c/em\u003e-\u003cem\u003emoss\u003c/em\u003e, \u003cem\u003eMedicago sativa\u003c/em\u003e-\u003cem\u003emoss\u003c/em\u003e, as well as their respective monocropping treatments. Additionally, the experiment included a natural degraded grassland without planting as a control treatment. Consequently, this study involved a total of seven treatments, with each treatment replicated three times, resulting in a total of 21 plots. The area of each plot was 3 m \u0026times; 2 m, with a spacing of 1 m between quadrats to prevent edge effects. Seeding was conducted using a strip sowing method. \u003cem\u003eElymus brevidentatus\u003c/em\u003e and \u003cem\u003eMedicago sativa\u003c/em\u003e were planted in May during the growing season, adhering to the locally promoted planting density standards: 22.5 g m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e for \u003cem\u003eElymus brevidentatus\u003c/em\u003e and 15 g m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e for \u003cem\u003eMedicago sativa\u003c/em\u003e. The sowing depth was 10 cm, with a row spacing of 20 cm. Two months prior to sowing, \u003cem\u003emoss\u003c/em\u003e approximately 1 cm thick was collected and brought back to the laboratory. After being dried in the shade, it was crushed and sieved through a 2 mm sieve to remove weeds and root fragments. When the \u003cem\u003eElymus brevidentatus\u003c/em\u003e and \u003cem\u003eMedicago sativa\u003c/em\u003e reached approximately 5 cm in height, 700 g m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e of crushed moss was uniformly spread onto the surface of the plots designated for mixed sowing and monocropping of moss according to the experimental design. During the plant growth period, weeds were removed and uniform field management was conducted. No fertilization treatment was applied to the plots.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Sampling and determination\u003c/h2\u003e \u003cp\u003eThe sampling time was in late September. All above-ground plant parts were cut from a 0.5 m \u0026times; 0.5 m quadrat in each plot. After being blanched at 105 ℃ for 2 hours, they were dried in an oven at 80℃ and weighed. The sampling depth for plant root biomass was at a soil depth of 0\u0026ndash;20 cm. The collected roots were cleaned with water, dried, and weighed. Soil samples were collected using the root core method. Specifically, an 8 cm inner diameter root core was used to surround the plant roots and extract a soil core from 0\u0026ndash;10 cm depth. After gently shaking to remove large soil clumps attached to the plant roots, the soil attached to the surface layer of the plant roots (0\u0026ndash;3 mm) was collected. Meanwhile, the residual soil surrounding the roots from the above process was collected for aggregate stability and soil moisture storage measurements. Therefore, the collected soil samples were divided into two parts: one was stored at 4℃ as a rhizosphere soil sample for measuring microbial biomass C and N (MBC \u0026amp; MBN) and inorganic N (NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N and NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e\u0026minus;N), while the other was naturally air-dried for analyzing soil organic C (SOC), total nitrogen (TN), total P, available P, pH, and aggregate distribution. The contents of SOC and TN were measured using an elemental analyzer (Elementar, Germany). The contents of soil MBC and MBN were determined using the chloroform fumigation method [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Specifically, fresh soil samples equivalent to 10 g of oven-dried soil were divided into two equal parts.\u003c/p\u003e \u003cp\u003eThe first part was fumigated for 24 hours and then extracted with the 0.5 mol L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e K\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e. The second one was non-fumigated as a control, followed by extraction with the same K\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e. The MBC and MBN content were determined as the difference between C and N contents in fumigated and non-fumigated soil extract using Elemental vario TOC cube (Elementar, Germany) with corrections for conversion factor KC and KN of 0.45 and 0.54 [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. The above unfumigated soil extracts were further used for the determination of soil NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N and NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e\u0026minus;N by using a FIAstar 5000 Analyser (Foss Tecator, Sweden). Soil total P was determined colorimetrically after perchloric acid digestion, while available P (AP) was determined using the Olsen method. Soil pH was determined in a soil: water suspension of 1:2.5. Soil moisture content (SWC %) is determined using the weighing method. The wet sieving method was used to separate soil aggregates according to Zhang et al [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. These water-stable aggregates of different sizes were weighted to measure the mean weight diameter (MWD), which was used as an index of soil aggregate stability.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Data Processing\u003c/h2\u003e \u003cp\u003eIn this study, we first employed one-way ANOVA and Tukey's HSD post-hoc test to compare the differences in all parameters between various monoculture treatments and mixed culture treatments at significance levels of 0.05, 0.01, and 0.001. Furthermore, we reclassified monoculture and mixed culture into two groups and utilized independent sample t-tests to analyze the differences between the two groups.\u003c/p\u003e \u003cp\u003eIn addition, we employed linear fitting to examine the relationship between the fertility index and the promotion index, and utilized heatmaps and Pearson's test to analyze the correlation between plant productivity and various soil properties. All statistical analyses were conducted using SPSS 22 (SPSS Inc., Chicago, USA), with a significance level set at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05. The charts were plotted using Origin 2021 software (Origin Lab, USA).\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e3.1 The impact of unicast and mixed broadcast on the aboveground and underground biomass of plants\u003c/h2\u003e \u003cp\u003eIn the monoculture treatment, the aboveground biomass of \u003cem\u003eElymus brevidentatus\u003c/em\u003e (S1) and \u003cem\u003eMedicago sativa\u003c/em\u003e (S2) was 0.50 kg m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e and 0.47 kg m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e, respectively, with no significant difference between the two (both \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The biomass of moss was the lowest. In the mixture treatment, the Elymus breviden\u003cem\u003etatus\u003c/em\u003e -\u003cem\u003eMedicago sativa\u003c/em\u003e mixture (S6) had the highest biomass of 0.75 kg m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e, followed by the \u003cem\u003eElymus brevidentatus\u003c/em\u003e-\u003cem\u003emoss\u003c/em\u003e (S4) and \u003cem\u003eMedicago sativa\u003c/em\u003e-\u003cem\u003emoss\u003c/em\u003e mixtures (S5), with 0.6 kg m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e and 0.55 kg m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e, respectively. The root biomass exhibited different trends. In the monoculture treatment, the root biomass of S6 was the highest at 19.46 kg m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e, followed by S1 at 8.75 kg m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e, and \u003cem\u003emoss\u003c/em\u003e was the lowest at 2.9 kg m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e. In the mixture treatment, there was no significant difference in root biomass between S5 and S6, while the S4 mixture had the lowest root biomass. Overall, the aboveground biomass in the mixed sowing treatment was higher than that in the monoculture treatment, and the root biomass also exhibited a similar trend (except for the \u003cem\u003eElymus brevidentatus\u003c/em\u003e-\u003cem\u003emoss\u003c/em\u003e mixture).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e3.2 The impact of monoculture and polyculture on soil fertility\u003c/h2\u003e \u003cp\u003eOverall, mix-planting treatments exhibited higher SOC, TN, and TP contents compared to monoculture treatments, although the specific differences varied across different indicators (both \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). All mix-planting treatments had higher SOC contents, specifically 24.24 g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 28.02 g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, and 26.47 g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively, compared to monoculture treatments (19.77 g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 22.77 g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 18.21 g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and the control treatment (16.18 g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). Notably, S5 and S6 showed significant differences compared to monoculture and the control treatments. However, the differences in TN were not significant among the treatments, with only S5 (3.87 g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) showing significant differences compared to Control treatment (S0) (3.18 g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and S2 (3.29 g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). TP exhibited a similar trend, with only S6 (1.37 g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and S2 (1.13 g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) showing significant differences compared to S4 (1.21 g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) treatment, while the remaining planting treatments showed no significant differences.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn terms of soil nutrient availability, there were no significant differences in ammonium N levels among all planting treatments, while nitrate N levels were higher in the mixed cropping treatment (4.82\u0026thinsp;~\u0026thinsp;6.33 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). Specifically, the nitrate nitrogen levels in all mixed cropping treatments were significantly different from those in the S0 (1.81 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), S1 (3.45 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), and S3 (3.31 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) treatments. Similarly, the available P levels in the soil were also higher in the mixed planting treatment, but there was no significant difference compared to the S2 (7.67 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) treatment. Regardless of the indicator, the level in the control treatment was the lowest, indicating a clear trend of soil degradation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.3 The impact of monoculture and mixed sowing on soil physicochemical properties\u003c/h2\u003e \u003cp\u003eDifferent treatment modes significantly influenced the levels of soil microbial C and N (both \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). There were notable differences between mixed sowing and monoculture. The MBC of the S0 (137.80 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) was at the lowest level, while the MBC of the mixed sowing of S6 was the highest (217.81 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). In comparison, the MBC of the monoculture treatments (152.10\u0026thinsp;~\u0026thinsp;177.90 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) was lower than that of the mix-planting treatments (193.85\u0026thinsp;~\u0026thinsp;217.81 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). The trend of MBN variation was like that of MBC, with significant differences between the mixed sowing treatments (14.93\u0026thinsp;~\u0026thinsp;17.68 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and the monoculture treatments (12.03\u0026thinsp;~\u0026thinsp;13.75 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). The S0 (10.28 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) had the lowest level. There were no significant differences in soil C/N among monoculture treatments and the control treatment. The soil C/N was highest in the mix-planting of S5 (8.17), which differed significantly from the other treatments. The pH differences among the treatments were not significant. The trends in soil moisture content (SWC%) and mean weight diameter (MWD) were the same, with the mixed sowing treatments generally being higher than the monoculture treatments and the control treatment, indicating that mixed sowing is beneficial for improving soil water retention capacity and structural stability. However, the trend reflected by soil bulk density was opposite, with S0 (1.45 g m\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e) being the highest, and the monoculture treatments (1.35\u0026thinsp;~\u0026thinsp;1.42 g m\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e) being higher than the mix-planting treatments (1.24\u0026thinsp;~\u0026thinsp;1.31 g m\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.4 The comparison of soil characteristics between monoculture and mix-planting\u003c/h2\u003e \u003cp\u003eBy comparing the soil physical properties, plant biomass, soil C, N, and nutrients, as well as soil microbial characteristics between monoculture and mixed sowing, it was found that all indicators except soil bulk density and pH were significantly higher in the mixed sowing treatment compared to the monoculture treatment (both \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). In the mixed sowing treatment, the aboveground biomass, belowground biomass, SOC, TN, NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N, NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e, AP, TP, C/N, MBC, MBN, SWC%, and MWD increased by an average of 25%, 16%, 30%, 3%, 41%, 8%, 19%, 9%, 38%, 26%, 10%, 19%, and 12% respectively compared to the monoculture treatment. Meanwhile, soil bulk density and pH decreased by 8% and 1% respectively. Therefore, compared to monoculture, the mixed sowing model significantly optimizes soil physical structure (reducing bulk density, enhancing aggregate stability, and increasing moisture content), promotes plant biomass accumulation, regulates soil C and N cycling and nutrient availability, activates microbial activity, and alters the soil chemical microenvironment.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.5 The relationship between interspecific facilitation and soil fertility\u003c/h2\u003e \u003cp\u003eThe facilitation index showed significant differences among different treatments, with the monoculture treatment exhibiting negative values (-1.43 to -0.75) and the mixed sowing treatment exhibiting positive values (0.72 to 1.01) (both \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). The results indicate that the mixed sowing treatment is significantly different from the monoculture treatment and the control treatment. The fertility index of the S0 treatment is significantly lower than that of other treatments, exhibiting a negative value and reaching around \u0026minus;\u0026thinsp;6.7, indicating that this treatment has the worst fertility (both \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb). The fertility indices of the S1 and S3 treatments are also negative, at -1.5 and \u0026minus;\u0026thinsp;3.8, respectively, indicating that the soil fertility of these two treatments is also poor, but weaker than that of S0. However, the fertility indices of the S2, S4, S5, and S6 treatments are positive, at 3.3, 2.8, 3.8, and 2.2, respectively, indicating that these treatments can promote soil fertility. There are no significant differences in fertility index among these treatments, which are generally maintained between 2 and 4.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe fertility index increased significantly with the facilitation index (R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.757), indicating that stronger facilitation effectively enhanced soil fertility (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec). Correlation analysis revealed strong positive associations among most soil fertility indicators (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed). Aboveground and root biomass were significantly correlated with key soil nutrient and structural variables, including SOC, TN, MBC, MBN, SWS, MWD, NO₃⁻-N, NH₄⁺-N, AP, and TP. In contrast, bulk density, C/N ratio, and pH showed negative correlations with several fertility-related parameters.\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e4.1 The Effects of Interactions of Plants in Mixed sowing Systems\u003c/h2\u003e \u003cp\u003eEffective ecological restoration in Qinghai-Tibet Plateau requires strategies that enhance plant establishment and promote sustainable soil fertility. While plant\u0026ndash;plant interactions are recognized as a fundamental driver of community assembly and ecosystem functioning, their facilitative roles in restoring alpine grasslands remain insufficiently explored [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. In this study, mixed sowing treatments significantly increased aboveground and root biomass (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). This may be attributed to the interspecific interactions between different plant species. For instance, alfalfa, a leguminous plant, provides N for gramineous plants through N fixation, while gramineous plants, with their deep root systems, enhance the absorption of water and nutrients. In addition, the incorporation of \u003cem\u003emoss\u003c/em\u003e may have improved the soil microenvironment, such as soil moisture (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e), thereby further promoting plant growth.\u003c/p\u003e \u003cp\u003eEmpirical studies have shown that the mixed sowing of gramineous plants and legumes exhibits a biomass advantage, which is because gramineous plants can not only achieve strong complementary utilization of water through belowground niche differentiation, but also acquire more water in the shared growth area due to their high competitiveness [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Leguminous crops can enhance P availability in the rhizosphere of neighboring cereal crops by regulating soil pH and activating insoluble P through acidic secretions [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. The nodule N fixation of alfalfa provides a N advantage for \u003cem\u003eElymus breviaristatus\u003c/em\u003e, increasing its leaf N content by 15%-20% and improving its photosynthetic efficiency by 20%-30% [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. In this study, S2, S5, and S6, which are related to the treatments of \u003cem\u003eMedicago sativa\u003c/em\u003e, all increased soil P and nitrogen levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). This result indicates that \u003cem\u003eMedicago sativa\u003c/em\u003e can promote aboveground biomass accumulation by regulating the availability of soil nutrients.\u003c/p\u003e \u003cp\u003eThe core advantage of mixing \u003cem\u003emoss\u003c/em\u003e with gramineous and leguminous plants lies in the role of moss as an \u0026ldquo;ecological engineer\u0026rdquo;. By physically covering the soil surface and regulating water and microclimatic conditions, moss creates a moister environment conducive to plant growth. This microhabitat modification not only reduces soil water evaporation and promotes soil water retention but also improves soil aggregate stability and stimulates microbial activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Consequently, all treatments involving \u003cem\u003emoss\u003c/em\u003e exhibited higher belowground biomass than their respective monocultures, accompanied by an increased soil water content and reduced bulk density (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). It is noted that all mixed-sowing treatments showed higher root biomass compared to their respective monocropping treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). All mixed sowing treatments showed greater root biomass compared with monoculture, suggesting that moss coverage facilitates the coordinated growth of \u003cem\u003eElymus breviaristatus\u003c/em\u003e and \u003cem\u003eMedicago sativa\u003c/em\u003e by improving soil water availability. Moreover, when \u003cem\u003eElymus breviaristatus\u003c/em\u003e and \u003cem\u003eMedicago sativa\u003c/em\u003e coexist, their simultaneous high water demand may lead to either competitive or complementary water use, which in turn promotes deeper root development and greater belowground investment.\u003c/p\u003e \u003cp\u003eThe above results indicate that mixed sowing communities can enhance overall biomass accumulation of plant communities through interspecific facilitation (S4-S6), primarily by improving soil nutrient availability and water content. In addition, a moderate level of resource competition (S6) may further stimulate root C investment, contributing to the development of a more balanced belowground system. Therefore, optimizing species composition, strengthening facilitative interactions, and minimizing excessive interspecific competition are essential strategies to improve plant community productivity and soil fertility in alpine grassland restoration.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e4.2 The Effects of Mixed sowing on Soil Structure and Microorganisms\u003c/h2\u003e \u003cp\u003eThe improvement of soil physical structure under mixed sowing treatment represents another important mechanism to enhanced soil fertility. The synergistic regulation of soil structure and microbial activity is central to maintaining grassland ecosystems health; particularly on the Qinghai\u0026ndash;Tibet Plateau where fragile soils determine the stability and restoration potential of degraded grasslands ( Li et al., 2025). Under mixed-planting treatment, the average soil bulk density decreased to 1.27 g\u0026middot;cm⁻\u0026sup3;, which was 8% lower than that of the monoculture treatment (1.38 g cm⁻\u0026sup3;) and significantly lower than the control (1.45 g cm⁻\u0026sup3;). Meanwhile, the MWD increased by 21.3% (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). In contrast, the taproots of \u003cem\u003eMedicago sativa\u003c/em\u003e extends into the 10\u0026ndash;30 cm layer. The synergistic interaction between these root type enhances soil porosity and promotes microaggregate formation through root exudates (such as polysaccharides), thereby reducing soil bulk density. Such structural improvement alleviates the freeze\u0026ndash;thaw\u0026ndash;induced hardening commonly observed in Qinghai-Tibet Plateau [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eEnhanced soil physical structure also creates a more suitable microenvironment for microbial activities by improving aeration and water retention. In the mixed-planting treatment, the MBC and MBN reached 217.81 mg kg⁻\u0026sup1; and 32.65 mg kg⁻\u0026sup1; respectively, increasing by 26% and 10% compared with the monoculture treatment. Among these treatments, the MBC value of the \u003cem\u003eElymus breviaristatus\u003c/em\u003e- \u003cem\u003eM. sativa\u003c/em\u003e mixed sowing (S6) exhibited the highest MBC value, consistent with the positive feedback model of \u0026ldquo;soil structure-carbon input-microbial activity\u0026rdquo; [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. In addition, the C/N ratio of the \u003cem\u003eM. sativa\u003c/em\u003e -\u003cem\u003emoss\u003c/em\u003e mixed sowing treatment is closer to the optimal range for microbial growth (C/N\u0026thinsp;=\u0026thinsp;8\u0026ndash;12), which may further optimize the efficiency of nutrient cycling by regulating the microbial community structure.\u003c/p\u003e \u003cp\u003eSoil pH also differed significantly between monoculture and mixed sowing treatments. This is directly related to the process where alfalfa roots secrete organic acids (e.g., oxalic acid) to activate soil phosphorus [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], while the secretions of monocropped gramineous plants are relatively alkaline, resulting in a more stable soil pH. This pH difference further regulates the microbial community structure and accelerates nutrient transformation. Collectively, these findings suggest that mixed sowing improves nutrient availability and moderates constraints imposed by alkaline soils, thereby facilitating the restoration of degraded alpine grasslands.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e4.3 Mixed sowing Enhancing Soil Fertility​\u003c/h2\u003e \u003cp\u003eThe average SOC content under mixed sowing reached 26.24 g kg⁻\u0026sup1;, which was 30% higher than that in the monoculture treatment. This finding aligns with previous studies showing that diversified planting systems enhance soil C accumulation [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. The underlying mechanism can be attributed to two aspects. First, mixed sowing communities provide a broader and more continuous substrate input to the soil C pool due to greater diversity of root exudates and higher litter production. Second, functional complementarity between species extends the C input throughout the growing season, avoiding the seasonal fluctuation of C input in the monoculture system. Although the N-fixing function of \u003cem\u003eM. sative\u003c/em\u003e did not significantly increase TN), it significantly increased the NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N level (4.82\u0026thinsp;~\u0026thinsp;6.33 mg kg⁻\u0026sup1;). This indicates that interspecific facilitation accelerates N transformation by stimulating the nitrifying bacterial, consistent with microbial functions under N-limited conditions in alpine grasslands.\u003c/p\u003e \u003cp\u003eA strong correlation (R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.757) between the facilitation index and the fertility index indicates that interspecific facilitation is the primary driver of the mixed-planting effect. At the plant level, niche differentiation between deep- and shallow-rooted species (e.g., \u003cem\u003eElymus breviaristatus\u003c/em\u003e and \u003cem\u003eM. sativa\u003c/em\u003e) reduces direct resource competition. At the microbial level, synergistic interactions between leguminous-associated rhizobia and plant growth-promoting rhizobacteriaof gramineous plants enhances nutrient activation [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. At the soil level, accelerated root turnover promotes aggregate formation, providing physical protection for C and N. Among all treatments, the \u003cem\u003eElymus breviaristatus\u003c/em\u003e-\u003cem\u003eM. sative\u003c/em\u003e mixed sowing showed the the most pronounced positive effect, with the highest aboveground biomass, SOC, and MBC values, corresponding to its strong facilitation intensity. ​In contrast, the \u003cem\u003eElymus breviaristatus\u003c/em\u003e-\u003cem\u003emoss\u003c/em\u003e mixed sowing showed the lowest root biomass and a limited SOC increase, likely due to resource competition between the shallow rhizoids of moss and the roots of \u003cem\u003eElymus breviaristatus\u003c/em\u003e. These contrasting results emphasize that the degree of functional trait complementarity determines the effectiveness of mixed-planting systems [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Selecting species with significant niche differentiation (e.g., deep- vs. shallow-rooted, N-fixing vs. non-N-fixing plants) is crucial for improving soil fertility and ecosystem functioning.​\u003c/p\u003e \u003cp\u003eFinally, the significant difference between the fertility index of the degraded control (S0: -6.7) and that of the optimal mixed-sowing treatments (S5, S6: 3.8) indicates that mixed sowing can effectively reverse soil degradation with a short timescale. While improving productivity, mixed sowing systematically improves soil fertility by optimizing soil structure and activating microbial activity, providing a promising ecological strategy for grassland restoration on the Qinghai-Tibet Plateau.\u003c/p\u003e \u003c/div\u003e"},{"header":"5. Conclusion​","content":"\u003cp\u003eGrassland degradation on the Qinghai-Tibet Plateau is becoming increasingly severe, and there is an urgent need to explore green solutions for soil restoration. As a natural restoration strategy, mixed sowing can effectively alleviate grassland degradation on the Qinghai-Tibet Plateau. Mixed sowing significantly promotes plant biomass accumulation through interspecific complementarity, providing continuous support for soil carbon and nitrogen input. Nutrients such as total nitrogen and available phosphorus are simultaneously increased, which confirms the driving effect of interspecific stratified resource utilization on carbon and nitrogen accumulation, as well as the role of interspecific niche differentiation in reducing interspecific competition. In addition, mixed sowing optimizes soil physical structure and activates microbial activity, providing effective nutrients for plant growth and forming a \"plant-soil-microbe\" synergistic feedback mechanism.. This has important practical value for grassland management on the Qinghai-Tibet Plateau: in the restoration of degraded grasslands, the adoption of mixed sowing of functional species (such as combinations of gramineous and leguminous plants) leverages their advantage in stratified resource utilization to reduce restoration costs in the short term and rapidly improve soil fertility.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDate is provided within the manuscript. The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Natural Science Foundation of Qinghai Province (2025-ZJ-969T), the National Key R\u0026amp;D Program of China (2023YFF1304305), International (Regional) Cooperation and Exchange (ICE) Projects of the National Natural Science Foundation of China (NSFC) (W2412148), and 111 Project (D23029).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSJ: Writing\u0026shy; - review \u0026amp; editing, Writing - original draft, Methodology, Supervision, Investigation, Software, Formal analysis, Data curation, Conceptualization. MS : Writing-review \u0026amp; editing. WW : Methodology, Formal analysis, Writing-review \u0026amp; editing. YC : Project administration, Methodology, Conceptualization. FY : Data curation. WY : Funding acquisition, Writing-review \u0026amp; editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eChen J, Huang M, Zeng Y, Sui Y, Gao Y, Ochoa-Hueso R, et al. Grassland degradation status modulates the relative contribution of soil nematode diversity and community structure to ecosystem functioning under nitrogen fertilization and irrigation management. Agric Ecosyst Environ. 2026;396:109954.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNiu B, Fu G. 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Sci Total Environ. 2021;770:144793.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Plant-plant interaction, Biodiversity, Soil physical structure, Soil fertility, Mixed sowing","lastPublishedDoi":"10.21203/rs.3.rs-8560681/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8560681/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eBy enhancing plant complementarity and facilitation, mixed sowing may accelerate soil fertility recovery and improve ecosystem resilience. Nevertheless, the short-term impacts of mixed sowing on soil fertility improvement and the underlying plant-soil interaction mechanisms remain poorly understood. We hypothesize that interspecific interactions in mixed sowing systems may cause changes in rhizosphere soil properties, thereby affecting soil fertility.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eTo test this hypothesis, monoculture and mixed sowing treatments were designed using \u003cem\u003eElymus breviaristatus\u003c/em\u003e, \u003cem\u003eMedicago sativa\u003c/em\u003e, and moss to investigate their ecological adaptation. Our results showed that compared with monocropping, mixed sowing significantly increased above and belowground biomass, improved soil water content, and reduced bulk density, thereby creating a more favorable soil microenvironment. Compared with monocropping, mixed sowing enhanced microbial biomass C and N by 26% and 10%, respectively, and increased soil organic C by 30%. Fertility indices were positive in all mixed sowing systems, contrasting with negative values under monocropping and control. Across treatments, the facilitation index was positively correlated with the fertility index (R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.76), demonstrating that interspecific facilitation was the primary driver of soil fertility gains.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThese findings reveal that mixed sowing enhances soil fertility restoration in degraded grasslands through interspecific facilitation that promotes biomass accumulation, improves soil physicochemical conditions, and stimulates microbial-mediated nutrient cycling. These mechanisms highlight crop diversification as an effective strategy for accelerating soil fertility restoration.\u003c/p\u003e","manuscriptTitle":"Mixed sowing improves soil fertility via enhancing plant facilitation in degraded grassland system","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-02 17:07:08","doi":"10.21203/rs.3.rs-8560681/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-02-23T15:56:34+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"303679250751028466451764543006219452800","date":"2026-02-01T16:49:15+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-01-30T06:55:42+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-01-19T06:17:18+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-01-16T21:19:51+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-01-14T07:15:13+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Plant Biology","date":"2026-01-14T07:06:18+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d2fa00fb-8608-47e7-a2cc-d8ddd2bc8148","owner":[],"postedDate":"February 2nd, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-02-02T17:07:08+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-02 17:07:08","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8560681","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8560681","identity":"rs-8560681","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}