A method for improving soil substrate in alpine regions based on nitrogen-fixing plants

Abstract

The application relates to the technical field of high-cold region soil remediation, in particular to a high-cold region soil substrate improvement method based on nitrogen-fixing plants. The high-cold region soil substrate improvement method comprises the following steps: sowing nitrogen-fixing plants and companion plants in the high-cold region soil to be remediated in a mixed sowing mode; when the nitrogen-fixing plants are in the true leaf stage, nitrogen-fixing rhizobium suspension is applied, and inoculation material containing active AMF spores is applied synchronously or after an interval of 1-2 days; organic substrate is applied at an interval of 20-30 days after 7-15 days and after the sowing, and the soil water content is maintained at 50%-70% of the field water holding capacity; after the nitrogen-fixing plants and the companion plants experience one or two complete growth seasons, the soil substrate improvement is completed. The application realizes rapid vegetation establishment and soil function recovery in the high-cold environment by constructing a three-way symbiotic system of the nitrogen-fixing plants, the rhizobium and the arbuscular mycorrhizal fungi.

Description

Technical Field

[0001] This invention relates to the field of soil remediation technology in high-altitude and cold regions, specifically to a method for improving soil matrix in high-altitude and cold regions based on nitrogen-fixing plants. Background Technology

[0002] High-altitude, cold-region disaster surfaces (such as debris flow deposits and landslides) typically exhibit characteristics such as weak soil formation, extreme nutrient deficiency, poor water retention capacity, and intense environmental stress, leading to difficulties in vegetation establishment and constituting a key factor limiting ecological restoration. Therefore, in selecting plant species, priority should be given to functional species that can overcome nutrient limitations and rapidly establish community structure.

[0003] While existing technologies include research on the ecological restoration of stony slopes and degraded surfaces using nitrogen-fixing plants or plant combinations, most rely solely on the plants' own mechanisms or simple species combinations. The restoration process primarily depends on the gradual establishment of microorganisms under natural conditions, resulting in long restoration cycles, poor stability, and susceptibility to extreme environmental disturbances. Furthermore, in high-altitude and cold regions, limiting factors such as low temperatures, drought, and nutrient scarcity lead to low soil microbial activity, hindering the rapid formation of symbiotic systems and resulting in slow plant growth. This makes vegetation degradation or secondary disturbances more likely during the restoration process. Summary of the Invention

[0004] To address the problems of existing technologies, such as long recovery cycles in high-altitude and cold environments, slow and unstable formation of symbiotic systems, and difficulty in forming stable vegetation communities, the present invention aims to provide a method for improving soil matrix in high-altitude and cold regions based on nitrogen-fixing plants, thereby enabling rapid vegetation establishment and soil function restoration in high-altitude and barren environments.

[0005] The technical solution adopted by this invention to solve the technical problem is:

[0006] A method for improving soil matrix in high-altitude and cold regions based on nitrogen-fixing plants includes the following steps:

[0007] Step 1: From March to May, nitrogen-fixing plants and associated plants are sown in the soil of the high-altitude cold region to be restored using a mixed sowing method.

[0008] Step 2: When the nitrogen-fixing plant grows to the seedling stage with 2-4 true leaves, apply nitrogen-fixing rhizobium suspension to the rhizosphere of the nitrogen-fixing plant. Simultaneously or at intervals of 1-2 days, apply inoculation material containing active AMF spores to the rhizosphere of the nitrogen-fixing plant.

[0009] Step 3: Apply organic substrate for the first time 7-15 days after sowing, and then apply organic substrate again every 20-30 days. Maintain soil moisture content at 50%-70% of field capacity throughout the planting period.

[0010] Step 4: After the nitrogen-fixing plants and associated plants have gone through one or two complete growing seasons, the soil substrate improvement is completed.

[0011] In the above-mentioned method for improving soil matrix in high-altitude cold regions based on nitrogen-fixing plants, in the mixed sowing method, the nitrogen-fixing plants and the associated plants are mixed at a seed mass ratio of 1:1 to 2.

[0012] In the above-mentioned method for improving soil matrix in high-altitude cold regions based on nitrogen-fixing plants, the plant spacing between the nitrogen-fixing plants and the companion plants is 5-15 cm.

[0013] In the above-mentioned method for improving soil substrate in high-altitude and cold regions based on nitrogen-fixing plants, the sowing depth of the mixed sowing method is 1-3 cm.

[0014] In the above-mentioned method for improving soil matrix in high-altitude cold regions based on nitrogen-fixing plants, the nitrogen-fixing plant is Astragalus membranaceus;

[0015] In the above-mentioned method for improving soil matrix in high-altitude and cold regions based on nitrogen-fixing plants, the associated plant is cogongrass.

[0016] In the above-mentioned method for improving soil matrix in high-altitude and cold regions based on nitrogen-fixing plants, the method for preparing the nitrogen-fixing rhizobium suspension is as follows: healthy root nodules are collected from the root system of the target nitrogen-fixing plant, and after isolation, purification and culture, a nitrogen-fixing rhizobium suspension of a set concentration is prepared.

[0017] In the above-mentioned method for improving soil substrate in high-altitude cold regions based on nitrogen-fixing plants, the concentration of the nitrogen-fixing rhizobium suspension at the set concentration is 10. 6 -10 8 CFU·mL -1 ;

[0018] In the above-mentioned method for improving soil matrix in high-altitude cold regions based on nitrogen-fixing plants, 5-10 mL of nitrogen-fixing rhizobium suspension of the set concentration is applied to the rhizosphere of each nitrogen-fixing plant.

[0019] In the above-mentioned method for improving soil substrate in high-altitude and cold regions based on nitrogen-fixing plants, AMF spores are extracted from soil that has formed a mycorrhizal symbiotic system using a combination of wet sieving and centrifugation. After washing, inoculation material containing active AMF spores is obtained.

[0020] In the above-mentioned method for improving soil substrate in high-altitude and cold regions based on nitrogen-fixing plants, the inoculation material containing active AMF spores is uniformly applied to the rhizosphere of the nitrogen-fixing plants. The inoculation material includes soil inoculum containing AMF spores and their mycelium, spore suspension or enrichment thereof.

[0021] In the above-mentioned method for improving soil substrate in high-altitude and cold regions based on nitrogen-fixing plants, the organic substrate is a bacterial residue suspension or humus.

[0022] In the above-mentioned method for improving soil substrate in high-altitude and cold regions based on nitrogen-fixing plants, the fungal residue suspension is prepared by mixing fungal residue and water at a mass ratio of 1:5 to 1:15. The fungal residue is obtained by natural decomposition and composting of waste culture medium from edible fungi cultivation.

[0023] In the above-mentioned method for improving soil matrix in high-altitude and cold regions based on nitrogen-fixing plants, the organic humus source of the nitrogen-fixing plant litter can be obtained from the aboveground or underground litter of nitrogen-fixing plants after natural decomposition or microbial degradation.

[0024] In the above-mentioned method for improving soil substrate in high-altitude and cold regions based on nitrogen-fixing plants, when the organic substrate is a bacterial residue suspension, 50-150 mL is applied to each plant.

[0025] In the above-mentioned method for improving soil substrate in high-altitude and cold regions based on nitrogen-fixing plants, when the organic substrate is humus, 10-50 g is applied per plant.

[0026] In the above-mentioned method for improving soil substrate in high-altitude and cold regions based on nitrogen-fixing plants, the organic substrate is applied by uniformly spreading it around the rhizosphere or by shallow mixing it.

[0027] By employing the above technical solution, the present invention has at least the following advantages:

[0028] 1) This invention constructs a three-way symbiotic system of "nitrogen-fixing plants-rhizobium-arbuscular mycorrhizal fungi" and uses associated grasses to expand the mycorrhizal network, which can form a multi-species synergistic nutrient acquisition and distribution network, thereby achieving rapid vegetation establishment and soil function restoration in high-altitude and barren environments.

[0029] 2) This invention utilizes Astragalus membranaceus as a nitrogen-fixing plant. Astragalus membranaceus can form a symbiotic relationship with rhizobia to achieve biological fixation of atmospheric nitrogen, thereby providing a continuous nitrogen source in nitrogen-limited environments. Astragalus membranaceus can also form a symbiotic system with arbuscular mycorrhizal fungi, significantly enhancing its ability to acquire difficult-to-migrate nutrients such as phosphorus, thereby forming a nitrogen-phosphorus synergistic acquisition mechanism.

[0030] 3) This invention uses cogongrass as a companion plant to enhance the colonization and network expansion of arbuscular mycorrhizal fungi in the roots of Astragalus membranaceus. Cogongrass has strong stress resistance and a well-developed root structure, which can quickly increase the ground cover and enhance soil stability in the early stage of recovery, thereby promoting the synergistic effect between Astragalus membranaceus and mycorrhizal fungi, thereby improving the availability of nitrogen and phosphorus in the soil, achieving synergistic enhancement of nutrients and rapid establishment of vegetation communities.

[0031] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it in accordance with the contents of the specification, the preferred embodiments of the present invention are described in detail below. Detailed Implementation

[0032] To further illustrate the technical means and effects adopted by the present invention to achieve the intended purpose, the following detailed description of the specific implementation methods, structures, features and effects of the present invention, in conjunction with preferred embodiments, is provided below.

[0033] This embodiment discloses a method for improving soil substrate in high-altitude cold regions based on nitrogen-fixing plants, comprising the following steps: Step 1, from March to May, nitrogen-fixing plants and associated plants are sown in the high-altitude cold region soil to be remediated using a mixed sowing method; Step 2, when the nitrogen-fixing plants grow to the seedling stage of 2-4 true leaves, a nitrogen-fixing rhizobium suspension is applied to the rhizosphere of the nitrogen-fixing plants, and simultaneously or at intervals of 1-2 days, an inoculation material containing active AMF spores is applied to the rhizosphere of the nitrogen-fixing plants. The inoculation material includes soil inoculum containing AMF spores and their mycelium, spore... The inoculation material includes AMF spores, mycelium, and / or infected root segments, which can be obtained by collecting the rhizosphere soil, infected roots, and culture medium of the host plant that has formed a mycorrhizal symbiotic system; Step 3: Apply organic substrate for the first time 7-15 days after sowing, and then apply organic substrate again every 20-30 days, maintaining the soil moisture content at 50%-70% of field capacity throughout the planting period; Step 4: After the nitrogen-fixing plants and companion plants have experienced one complete growing season, the soil substrate improvement is completed.

[0034] In this embodiment, the nitrogen-fixing plant is Astragalus membranaceus; the associated plant is Imperata cylindrica. Specifically, the nitrogen-fixing plant is preferably an Astragalus species (Astragalus mahoschanicus), and the associated plant is preferably an Imperata cylindrica. The combination of Astragalus membranaceus and Imperata cylindrica has significant advantages in promoting soil nutrient enhancement and vegetation community establishment.

[0035] From a functional perspective, Astragalus membranaceus can form a symbiotic relationship with rhizobia, achieving biofixation of atmospheric nitrogen and thus providing a continuous nitrogen source in nitrogen-limited environments. Simultaneously, Astragalus membranaceus can also form a symbiotic system with arbuscular mycorrhizal fungi, significantly enhancing its ability to acquire recalcitrant nutrients such as phosphorus, thus forming a nitrogen-phosphorus synergistic acquisition mechanism. This three-way symbiotic system is key for plants to overcome the dual limitations of nitrogen and phosphorus in exposed substrates in high-altitude and cold regions. As an associated grass, Imperata cylindrica possesses strong stress resistance and a well-developed root system, enabling it to rapidly increase surface cover and enhance soil stability in the early stages of recovery. Furthermore, Imperata cylindrica exhibits high mycorrhizal dependence, forming a symbiotic relationship with arbuscular mycorrhizal fungi with a high infection rate, thereby forming a relatively developed mycorrhizal network in the soil. Through this network, it establishes indirect connections with nitrogen-fixing plants, participating in underground nutrient cycling processes and promoting the stability and sustainability of the overall system function.

[0036] This invention is designed to improve soil substrates in high-altitude and cold regions. Since plants typically have only one growing season per calendar year, soil improvement can only be carried out once per calendar year. In this embodiment, soil improvement is completed after the plants have completed one full growing season, meaning that planting in March to May is followed by soil improvement in July and August.

[0037] If the improvement effect is not ideal after one attempt, a second improvement can be carried out. Since Astragalus is a perennial nitrogen-fixing herb, the seeds can be mixed and sown in the soil to be improved at the beginning of the growing season (around March to May, depending on regional climate conditions). A stable vegetation community can be formed after one growing season. If the seedling survival rate is low, re-sowing can be carried out in the second year. Since the seedlings go through the flowering and pod-setting process after sowing, the mature pods will release a large number of seeds in the soil, so there is no need to repeat sowing every year.

[0038] Because low temperatures, drought, and nutrient scarcity in high-altitude and cold regions can significantly inhibit the activity of rhizobia and mycorrhizal fungi, the establishment of symbiotic relationships is slow and the stability is poor. In addition, both nitrogen-fixing symbiosis and mycorrhizal symbiosis require plants to provide carbon sources. In barren environments, the two symbiotic processes may compete for resources, thereby affecting their synergistic effect. This invention solves the carbon source problem by adding an organic matrix in step 3.

[0039] In degraded, bare soil surfaces, low vegetation cover and poor soil development hinder the effective expansion of mycorrhizal networks, limiting nutrient transfer and sharing among different plants. When nitrogen-fixing plants grow alongside other associated plants, enhancing mycorrhizal network connectivity to further improve soil nitrogen and phosphorus availability and promote community building is crucial for soil texture improvement. The Astragalus and Imperata cylindrica symbiotic system of this invention maintains a high mycorrhizal infection rate and significantly increases soil available nitrogen and phosphorus content, indicating that associated plants may enhance the synergistic effect of the three-way symbiotic system by promoting mycorrhizal network expansion, thereby achieving rapid restoration of degraded soils in high-altitude and cold regions.

[0040] The proposed solution not only utilizes pioneer nitrogen-fixing plants to restore aboveground vegetation communities, but also further utilizes functional microorganisms, namely rhizobia and mycorrhizal fungi, in combination with associated plants to expand the mycorrhizal network, ultimately achieving significant and rapid improvement of the soil matrix by aboveground and underground biological communities.

[0041] Specifically, this invention proposes to construct a three-way symbiotic system of "nitrogen-fixing plants-rhizobium-arbuscular mycorrhizal fungi" and use associated grasses to expand the mycorrhizal network, thereby forming a multi-species synergistic nutrient acquisition and distribution network, thus enabling rapid vegetation establishment and soil function restoration in high-altitude and barren environments.

[0042] In the mixed-sowing method, the nitrogen-fixing plants and the associated plants are mixed at a seed mass ratio of 1:1 to 2. This mass ratio is chosen to balance the competition and synergy between the nitrogen-fixing plants and the associated plants in the early stages of recovery. The nitrogen-fixing plants act as pioneer plants, responsible for providing nitrogen input and promoting the establishment of a symbiotic system of rhizobia and mycorrhizal fungi; the associated plants mainly enhance the mycorrhizal network, promote nutrient flow, and accelerate soil formation. Too low or too high a proportion may affect their functional performance.

[0043] Furthermore, the distance between the nitrogen-fixing plants and the companion plants is 5-15 cm, thereby ensuring that the rhizosphere space of the two types of plants has an overlapping area (promoting mycorrhizal network connection).

[0044] Furthermore, the sowing depth of the mixed-sowing method is 1-3 cm. This not only maintains the nitrogen-fixing function of the nitrogen-fixing plant Astragalus membranaceus, but also promotes the expansion of the mycorrhizal network through Imperata cylindrica, achieving a stable tripartite symbiotic relationship and associated system. In this method, Astragalus membranaceus provides nitrogen to Imperata cylindrica, and Imperata cylindrica provides phosphorus to Astragalus membranaceus.

[0045] Specifically, the method for preparing the nitrogen-fixing rhizobium suspension is as follows: healthy root nodules are collected from the roots of the target nitrogen-fixing plant (i.e., Astragalus membranaceus of this invention), isolated, purified, and cultured to prepare a nitrogen-fixing rhizobium suspension of a set concentration. The specific steps are as follows: 1) First, root nodules are isolated (removed) from the Astragalus membranaceus roots and broken down to obtain root nodule tissue fluid; 2) The root nodule tissue fluid is inoculated onto yeast extract mannitol agar (YMA) medium for rhizobium isolation and purification, and single-cell amplification culture is performed; 3) A nitrogen-fixing rhizobium suspension of a set concentration is prepared. The amplified rhizobium cells are resuspended in sterile water or buffer solution, and the bacterial concentration is adjusted to prepare a nitrogen-fixing rhizobium suspension of a set concentration.

[0046] Specifically, in this embodiment, the concentration of the nitrogen-fixing rhizobium suspension at the set concentration is 10. 6 -10 8 CFU·mL -1 Apply 5-10 mL of the specified concentration of nitrogen-fixing rhizobium suspension to the rhizosphere of each nitrogen-fixing plant. This method ensures, firstly, that the inoculated suspension maintains a sufficient number of viable rhizobia in cold environments, thereby improving the rhizobium's colonization efficiency and nodule formation capacity, and promoting biological nitrogen fixation. Simultaneously, this concentration range avoids problems such as intensified competition among rhizosphere microorganisms and localized oxygen deficiency caused by excessively high concentrations. Secondly, the application amount ensures the formation of an effective microbial community around the roots while avoiding excessive application that could lead to suspension loss or resource waste.

[0047] Specifically, the preparation method of the inoculation material containing active AMF spores is as follows: AMF spores are extracted from soil in which a mycorrhizal symbiotic system has been formed (the rhizosphere soil of the host plant that has formed a stable mycorrhizal symbiotic system is selected, which is the type of soil that is not to be remediated under natural conditions; the host plant of this invention is thatch grass), using a wet sieving method combined with centrifugation separation method, and the inoculation material containing active AMF spores is obtained after washing.

[0048] The inoculation material containing active AMF spores is uniformly applied to the rhizosphere of the nitrogen-fixing plant, with approximately 50-200 spores applied per plant. The number of spores can be determined using a microscope. The inoculation material contains AMF spores, mycelium, and / or infected root segments. It can be obtained by collecting the roots of a host plant that has formed a mycorrhizal symbiotic system and the culture medium. After the mycorrhizal-containing roots and culture medium are naturally air-dried, pulverized, and mixed, a solid inoculation material or suspension is prepared.

[0049] Specifically, the organic substrate is a fungal residue suspension, with 50-150 mL of fungal residue suspension applied to each plant; this invention utilizes edible fungal residue suspension as the organic substrate. Here, the application of fungal residue provides a carbon source for the rhizosphere microorganisms of Astragalus membranaceus.

[0050] The mushroom residue suspension is prepared by mixing mushroom residue and water at a mass ratio of 1:5 to 1:15. The mushroom residue is obtained by natural decomposition and composting of waste culture medium from edible mushroom cultivation.

[0051] The organic substrate here is mainly used to enhance the activity of rhizosphere microorganisms. The organic substrate is one that promotes microbial activity and rhizosphere nutrient cycling. When the selected organic substrate is a microbial residue suspension, 50-150 mL is applied per plant; when the selected organic substrate is humus, 10-50 g is applied per plant. (Nitrogen-fixing plant litter forms organic humus through natural decomposition or microbial degradation.)

[0052] The mushroom residue suspension is prepared by mixing mushroom residue and water at a mass ratio of 1:5 to 1:15. The mushroom residue is obtained by natural decomposition and composting of waste culture medium from edible mushroom cultivation, and is measured by wet weight.

[0053] As an alternative implementation, the organic matrix can also be humus, and the organic humus source of the nitrogen-fixing plant litter can be obtained from the aboveground or underground litter of nitrogen-fixing plants after natural decomposition or microbial degradation.

[0054] The organic substrate can be applied by uniformly spreading it around the rhizosphere or by shallow mixing. During this period, water should be replenished according to the soil moisture content and rainfall, preferably maintaining the soil moisture content at 50%-70% of field capacity. Under conditions of insufficient natural rainfall, water can be replenished every 5-10 days to keep the rhizosphere moist but avoid waterlogging, thereby promoting the colonization and spread of rhizobia and arbuscular mycorrhizal fungi.

[0055] The target nitrogen-fixing plant was identified based on the pioneer nitrogen-fixing plants in the study area and belongs to the genus Astragalus. It can not only be stably established in the target high-altitude and cold regions, but also form both root nodule symbiosis and AMF symbiosis.

[0056] To characterize the resource-limiting features of typical exposed soils in high-altitude cold regions, the physicochemical properties of the soils in the high-altitude cold regions to be remediated were measured. The results are as follows:

[0057]

[0058] The above results indicate that exposed soils in high-altitude and cold regions are characterized by significant nitrogen deficiency and extremely low phosphorus availability (available phosphorus is only 0.71 mg·kg⁻¹). -1 Meanwhile, the low water content and high pH further limit nutrient availability, which is a major limiting factor for vegetation restoration.

[0059] To achieve rapid ecological restoration of degraded land surfaces in high-altitude and cold regions, this embodiment constructs a symbiotic system based on nitrogen-fixing plants, rhizobia, and arbuscular mycorrhizal fungi, combined with an associated plant synergistic restoration method. The specific implementation process is as follows:

[0060] (1) Seed collection and processing

[0061] a. Select robust, disease- and pest-free nitrogen-fixing plants (preferably Astragalus mahoschanicus) and associated plants (preferably Imperata cylindrica) mother plants in the target high-altitude and cold regions, and collect mature seeds;

[0062] b. Screen the seeds to remove shriveled grains and impurities;

[0063] c. Perform surface disinfection on the seeds (treat with 75% ethanol for 30-60 seconds, then rinse with sterile water 3-5 times);

[0064] d. Perform a soaking treatment (soak in clean water for 12-24 hours) to improve the germination rate.

[0065] (2) Sowing and community building

[0066] Three treatment methods were set up to construct different plant combination systems: monoculture of nitrogen-fixing plants, monoculture of companion plants, and mixed sowing of nitrogen-fixing plants and companion plants. In the mixed sowing, nitrogen-fixing plants and companion plants were sown at a seed weight ratio of 1:1-2. This ratio was chosen to balance the competition and synergy between nitrogen-fixing plants and companion plants in the early stages of recovery. Nitrogen-fixing plants act as pioneer plants, responsible for providing nitrogen input and promoting the establishment of a symbiotic system of rhizobia and mycorrhizal fungi. Companion plants mainly enhance the mycorrhizal network, promote nutrient flow, and accelerate soil formation; too low or too high a ratio may affect their function. Plant spacing was controlled within the range of 5-15 cm; sowing depth was 1-3 cm; and overlapping areas in the rhizosphere of the two types of plants were ensured (to promote mycorrhizal network connection). This overlapping area facilitates the expansion of arbuscular mycorrhizal fungi (AMF) hyphae between different plant rhizospheres and the formation of a continuous mycorrhizal network, thereby enhancing the underground nutrient and water transport capacity and promoting the activation of nitrogen, phosphorus, and other nutrients in the soil. Meanwhile, the formation of mycorrhizal networks can also improve soil microbial activity and rhizosphere organic matter accumulation, enhance soil aggregate stability and water retention capacity, thereby improving the soil structure of bare substrate in cold regions and promoting soil formation and rapid establishment of vegetation communities.

[0067] (3) Preparation and inoculation of rhizobium suspension

[0068] Healthy root nodules were collected from the roots of the target nitrogen-fixing plant, surface-sterilized, and then crushed. Rhizobium bacteria were isolated under aseptic conditions, cultured, and prepared into a bacterial suspension of a predetermined concentration (10 in this example). 6 -10 8 CFU·mL -1 ).

[0069] Inoculation method: Apply bacterial suspension to the rhizosphere when the seedlings have 2-4 true leaves; apply 5-10 mL of bacterial suspension to each plant; keep the soil moist (but not waterlogged) after inoculation to promote the establishment of symbiosis.

[0070] (4) AMF spore extraction and inoculation

[0071] Samples were collected from soils that had formed a mycorrhizal symbiotic system. AMF spores were extracted using a combination of wet sieving and centrifugation to obtain inoculation material containing live spores (which can be appropriately enriched).

[0072] Inoculation method: Apply evenly to the plant rhizosphere (0-10 cm soil layer), applying about 50-200 spores per plant. This should be done simultaneously with or 1-2 days after rhizobium inoculation.

[0073] (5) Construction of symbiotic system and seedling management

[0074] a. Avoid applying high concentrations of chemical fertilizers (to prevent inhibition of symbiosis);

[0075] b. Low-concentration organic substrates (such as microbial residue) can be applied in appropriate amounts to promote microbial activity;

[0076] c. Regularly observe the plant's growth status and root development.

[0077] (6) Vegetation maintenance and management

[0078] a. Avoid human trampling and mechanical disturbance;

[0079] b. Avoid weeding during the initial recovery period (1-2 months) to promote community establishment;

[0080] c. In the mid-to-late stages, adjustments can be made based on coverage levels.

[0081] (7) Measurement indicators

[0082] Soil and plant parameters were measured after different combinations of plant improvements were tested over a growing season.

[0083] The soil parameters include moisture, pH, organic carbon, total nitrogen, total phosphorus, dissolved carbon, dissolved nitrogen, and available phosphorus; the plant parameters include biomass and mycorrhizal infection rate.

[0084] (1) Improve soil nitrogen and phosphorus availability, improve soil microenvironment, and promote soil formation.

[0085]

[0086] Overall Results: Compared with single-plant treatments, the Astragalus and Imperata cylindrica mixture showed significant improvements in both dissolved nitrogen and available phosphorus, reaching 86.4 mg·kg⁻¹. -1 and 3.67 mg·kg -1 The mycorrhizal biomass was the highest among all treatments; simultaneously, both aboveground and belowground biomass increased significantly, indicating that the combination effectively promoted vegetation growth. Furthermore, *Imperata cylindrica* exhibited a high mycorrhizal infection rate (89.18%), indicating it is a strongly mycorrhizally dependent plant. In the mixed planting system, the overall mycorrhizal infection rate remained at a high level (75.22%), demonstrating that the associated system could maintain and expand the colonization of mycorrhizal fungi.

[0087] The above results indicate that associated plants may enhance the colonization and network expansion of arbuscular mycorrhizal fungi, thereby promoting the synergistic effect between nitrogen-fixing plants and mycorrhizal fungi, thus improving soil nitrogen and phosphorus availability, achieving synergistic nutrient enhancement and rapid establishment of vegetation communities.

[0088] (2) Promote plant establishment and community building, and rapidly form vegetation communities.

[0089] By constructing a pioneer plant system centered on Astragalus membranaceus, rapid vegetation reconstruction and community succession were achieved in high-altitude, barren areas. The Astragalus membranaceus cushion structure provides a warming and shading microenvironment, allowing other herbaceous plants to germinate and establish themselves within its gaps. Simultaneously, Astragalus membranaceus' nitrogen fixation and rhizosphere carbon input enhance soil microbial activity and nitrogen availability, creating nutrient hotspots that favor the establishment of adjacent non-nitrogen-fixing plants. With enhanced interplant interactions, overall community productivity and diversity increase, leading to an early stable community structure.

[0090] Overall Results: In high-altitude and cold regions, bare land can form a vegetation community dominated by Astragalus membranaceus and accompanied by grasses within one year, achieving rapid and stable surface greening. Furthermore, to further increase the dissolved C, N, available phosphorus, and above-ground and below-ground biomass in the soil, the soil in high-altitude and cold regions can be remediated once more, that is, through two years (two growing seasons) of remediation, the soil texture and surface greening can be further stabilized.

[0091] To evaluate the role of microorganisms in ecological restoration, the abundance of rhizobia and arbuscular mycorrhizal fungi (AMF) in different plant combinations was quantitatively analyzed. The results are as follows:

[0092]

[0093] In the mixed system of nitrogen-fixing plants and associated plants, the abundance of microorganisms was significantly increased. Among them, the soil rhizobium marker gene rpoB increased by 2.24 × 10⁻⁶. 8 copies·g -1 Increased to 5.03×10 8 copies·g -1 This represents an increase of approximately 125%, with the root system containing 4.28 × 10⁻⁶ cells / day. 8 Increased to 6.08×10 8 copies·g -1 This represents an increase of approximately 42%; simultaneously, the abundance of AMF in the soil increased from 3.64 × 10⁻⁶. 6 Increased to 5.15×10 6 copies·g -1 The colonization rate of rhizobia and arbuscular mycorrhizal fungi in the soil and root system was increased by approximately 41%. This result indicates that by optimizing plant combinations and constructing symbiotic systems, the colonization rate of rhizobia and arbuscular mycorrhizal fungi in the soil and root system can be significantly improved, thereby enhancing biological nitrogen fixation and phosphorus acquisition functions, promoting synergistic nutrient enhancement, and providing microbial support for rapid vegetation establishment and soil function restoration.

[0094] This embodiment utilizes the nitrogen-fixing plant Astragalus membranaceus as a pioneer plant. Within the constructed plant-microbe synergistic system, it significantly improves the physicochemical properties of degraded soils in high-altitude, cold regions, thus creating favorable conditions for the establishment of other cold-resistant plants. Specifically, Astragalus membranaceus achieves nitrogen input through a symbiotic relationship with rhizobia, while simultaneously promoting the activation and absorption of nutrients such as phosphorus through synergistic interactions with arbuscular mycorrhizal fungi. With the participation of associated plants, it further enhances soil nutrient cycling and structural stability.

[0095] The experimental results of this embodiment show that the system can significantly increase the content of available nitrogen and available phosphorus in the soil, and promote soil moisture retention and microbial activity, thereby improving the originally nutrient-poor, loosely structured, and unfavorable alpine degraded soil environment. Under these conditions, in addition to pioneer plants, other cold-resistant plants (such as grasses and subsequent community species) can achieve effective establishment and normal growth.

[0096] In contrast, under natural conditions without human intervention, vegetation recovery in high-altitude and cold regions is extremely slow due to nutrient limitations and environmental stress. It typically takes about 20-30 years for an initial herbaceous community to gradually evolve into a more stable community structure, and the establishment of woody plants is even more difficult. Therefore, by constructing a nitrogen-fixing plant-microbe synergistic system, the ecological restoration cycle can be significantly shortened, the vegetation succession process accelerated, and a rapid transition from pioneer species to stable communities achieved.

[0097] After screening, it was determined that the selected Astragalus mahoschanicus could grow and complete its life cycle in the target high-altitude and cold regions, thus demonstrating a certain degree of adaptability as a pioneer plant. However, under solitary planting conditions, its growth rate and its effect on improving the soil environment were relatively limited due to soil nutrient deficiency and the incomplete establishment of a microbial symbiotic system.

[0098] Comparative experimental results showed that compared with Astragalus membranaceus planted alone, the introduction of associated plants and a microbial symbiotic system significantly enhanced plant growth and soil improvement. For example, in the Astragalus membranaceus monoculture system, the aboveground biomass was 37.84 g, while in the Astragalus membranaceus and Imperata cylindrica mixed system, it increased to 51.67 g, an increase of approximately 36%; the underground biomass increased from 18.20 g to 33.59 g, an increase of approximately 84%. Simultaneously, soil nutrient levels were significantly improved, with dissolved nitrogen increasing from 69.2 mg·kg⁻¹. -1 Increased to 86.4 mg·kg -1 Available phosphorus is 2.46 mg·kg -1 Increased to 3.67 mg·kg -1 Furthermore, the construction of the symbiotic system significantly enhanced microbial function, such as increasing the abundance and colonization capacity of rhizobia and arbuscular mycorrhizal fungi, thereby further promoting nitrogen input and phosphorus acquisition.

[0099] This invention constructs a synergistic system of "nitrogen-fixing plants-microorganisms-associated plants," which can significantly enhance their growth performance and soil improvement capabilities, thereby accelerating the ecological restoration process and achieving a rapid transition from pioneer species establishment to stable community succession.

[0100] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention shall still fall within the scope of the technical solution of the present invention.

Claims

1. A method for improving soil matrix in high-altitude and cold regions based on nitrogen-fixing plants, characterized in that, Includes the following steps: Step 1: From March to May, nitrogen-fixing plants and associated plants are sown in the soil of the high-altitude cold region to be restored using a mixed sowing method. Step 2: When the nitrogen-fixing plant grows to the seedling stage with 2-4 true leaves, apply nitrogen-fixing rhizobium suspension to the rhizosphere of the nitrogen-fixing plant. Simultaneously or at intervals of 1-2 days, apply inoculation material containing active AMF spores to the rhizosphere of the nitrogen-fixing plant. Step 3: Apply organic substrate for the first time 7-15 days after sowing, and then apply organic substrate again every 20-30 days. Maintain soil moisture content at 50%-70% of field capacity throughout the planting period. Step 4: After the nitrogen-fixing plants and associated plants have gone through one or two complete growing seasons, the soil substrate improvement is completed.

2. The method for improving soil matrix in high-altitude cold regions based on nitrogen-fixing plants according to claim 1, characterized in that, In the mixed sowing method, the nitrogen-fixing plant and the companion plant are mixed at a seed mass ratio of 1:1 to 2; The plant spacing between the nitrogen-fixing plants and the associated plants is 5-15 cm; The sowing depth for the mixed sowing method is 1-3 cm.

3. The method for improving soil matrix in high-altitude cold regions based on nitrogen-fixing plants according to claim 1, characterized in that, The nitrogen-fixing plant is Astragalus membranaceus; The associated plant is thatch grass.

4. The method for improving soil matrix in high-altitude cold regions based on nitrogen-fixing plants according to claim 1, characterized in that, The method for preparing the nitrogen-fixing rhizobium suspension is as follows: healthy root nodules are collected from the roots of the target nitrogen-fixing plant, and after isolation, purification, and culture, a nitrogen-fixing rhizobium suspension of a set concentration is prepared.

5. The method for improving soil matrix in high-altitude cold regions based on nitrogen-fixing plants according to claim 4, characterized in that, The concentration of the nitrogen-fixing rhizobium suspension at the set concentration is 10. 6 -10 8 CFU·mL -1 ; Apply 5-10 mL of the specified concentration of nitrogen-fixing rhizobium suspension to the rhizosphere of each nitrogen-fixing plant.

6. The method for improving soil matrix in high-altitude cold regions based on nitrogen-fixing plants according to claim 1, characterized in that, AMF spores were extracted from soil that had formed a mycorrhizal symbiotic system using a combination of wet sieving and centrifugation. After washing, inoculation material containing active AMF spores was obtained.

7. The method for improving soil matrix in high-altitude cold regions based on nitrogen-fixing plants according to claim 1, characterized in that, The inoculation material containing active AMF spores is uniformly applied to the rhizosphere of the nitrogen-fixing plant. The inoculation material includes soil inoculum containing AMF spores and their mycelium, spore suspension or enrichment thereof.

8. The method for improving soil matrix in high-altitude cold regions based on nitrogen-fixing plants according to claim 1, characterized in that, The organic matrix is ​​a bacterial residue suspension or humus; The mushroom residue suspension is prepared by mixing mushroom residue and water at a mass ratio of 1:5 to 1:

15. The mushroom residue is obtained by natural decomposition and composting fermentation of waste culture medium from edible mushroom cultivation. The organic humus source of the nitrogen-fixing plant litter can be obtained from the aboveground or underground litter of nitrogen-fixing plants through natural decomposition or microbial degradation.

9. The method for improving soil matrix in high-altitude cold regions based on nitrogen-fixing plants according to claim 8, characterized in that, When the organic matrix is ​​a fungal residue suspension, apply 50-150 mL to each plant. When the organic substrate is humus, apply 10-50g per plant.

10. The method for improving soil matrix in high-altitude cold regions based on nitrogen-fixing plants according to claim 1, characterized in that, The organic substrate is applied by uniformly spreading it around the rhizosphere or by shallow mixing it.

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