Shrub and grass combined planting method suitable for soil of strip mine dump and application of shrub and grass combined planting method

Abstract

The invention provides a shrub and grass combined planting method suitable for soil of a strip mine dump and application of the shrub and grass combined planting method, and relates to the technical field of ecological remediation, the shrub and grass combined planting method comprises the steps of planting amorpha fruticosa in a to-be-improved soil area, and sowing alfalfa in gaps of the amorpha fruticosa; the arbuscular mycorrhizal fungi inoculant and the dark-color separated endophytic fungi inoculant are applied to the root of each amorpha fruticosa plant in a hole application mode, and the arbuscular mycorrhizal fungi inoculant and the dark-color separated endophytic fungi inoculant are applied in planting gaps of the amorpha fruticosa plant by digging shallow ditches. The long-term stable symbiosis of the two fungi at the rhizosphere is realized. The photosynthetic rate and the CO2 quantum efficiency of the amorpha fruticosa and the alfalfa are remarkably improved, and the obvious synergistic interaction effect is achieved in the aspects of improving the soil nutrient components and the activity of soil enzymes such as sucrase and phosphatase. The technical problems that in the prior art, the AMF and the DSE have ecological niche competition, the coexistence stability is poor and inoculation balance is difficult to control in the process of improving the soil of the strip mine dumping site, and consequently soil remediation is difficult are solved.

Description

Technical Field

[0001] This invention relates to the field of ecological restoration technology, and in particular to a method for planting shrubs and grasses in combination with soil in open-pit mine spoil heaps, and its application. Background Technology

[0002] Coal mining places immense environmental pressure on land, ecology, and human settlements. Coal mining primarily employs two methods: open-pit mining and underground mining. Open-pit mining damages the original surface, topography, and landforms, and the excavated waste creates large areas of wasteland, with spoil heaps being the most significant source of environmental disturbance. Under natural conditions, vegetation succession in mining areas is extremely slow. Natural vegetation restoration is only possible with sufficient seed sources or propagules, and it takes much longer than artificial restoration. Therefore, in natural succession, using artificial support and guidance measures to introduce vegetation, such as planting artificial forests, is highly beneficial in accelerating this process, shortening the succession cycle and increasing the rate of vegetation succession to some extent. Generally, after 15 years of vegetation restoration, the soil nutrient content in spoil heaps shows a significant increase, with vegetation restoration showing a particularly noticeable improvement in topsoil nutrients.

[0003] Shrub-grass combinations are a common vegetation pattern in artificial reclamation measures in mining areas. In arid and semi-arid mining areas in western China, trees are not often used as the main tree species due to their high water requirements, and shrub-grass configurations are more common. Studies have found that under three remediation methods for coal mine waste sites, the soil organic carbon, N, and P levels after vegetation remediation are in the order of cypress groves > sumac forests > natural restoration. Hu Qili et al. believe that shrub-grass combinations can significantly promote the accumulation of soil nutrients and have a good water and soil conservation effect. Considering the maximum economic benefits of land reclamation, economic shrubs are given priority. Hu Yigang et al. believe that after nearly 20 years of remediation, the pure herbaceous configuration of mixed leguminous forage grasses in the Heidaigou open-pit coal mine spoil heap showed the best restoration effect and is the preferred vegetation configuration for initial soil remediation.

[0004] Arbuscular mycorrhizal fungi (AMF) are an important component of almost all terrestrial ecosystems, playing a particularly crucial role in enhancing plant nutrient and water uptake under arid conditions. AMF also enhances plant competitiveness and determines community structure and ecosystem stability. Dark septate endophytes (DSE) are small endophytic fungi primarily distributed within or between plant root cells. These fungi exhibit high colonization rates in plant roots under various extreme environments. DSE effectively decomposes soil organic phosphorus and nitrogen compounds, improves plant potassium and calcium translocation, and promotes plant growth and tolerance to stress environments. A significant advantage of DSE over AMF is that it can be obtained in large quantities through pure culture.

[0005] In existing technologies, although AMF and DSE are applied to vegetation restoration in mining areas, under extreme mining soil conditions, the two have problems such as niche competition, poor coexistence stability, and difficulty in controlling inoculation balance, which limit their synergistic application effect.

[0006] In view of this, the present invention is hereby proposed. Summary of the Invention

[0007] One of the objectives of this invention is to provide a method for combined planting of shrubs and grasses suitable for open-pit mine spoil heaps, in order to solve the technical problems in the prior art where AMF and DSE have niche competition, poor coexistence stability, and difficulty in controlling inoculation balance, leading to difficulties in soil remediation.

[0008] The second objective of this invention is to provide the application of the above-mentioned shrub-grass combination planting method in soil improvement of open-pit mine spoil heaps.

[0009] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted: In a first aspect, the present invention provides a method for combined shrub and grass planting suitable for soil in open-pit mine spoil heaps, comprising the following steps: A. Plant Amorpha fruticosa in the area where the soil needs to be improved, and sow alfalfa between the Amorpha fruticosa plants; B. Apply arbuscular mycorrhizal fungi inoculant and dark-colored septate endophytic fungi inoculant to the base of each Amorpha fruticosa plant by hole application, and apply arbuscular mycorrhizal fungi inoculant and dark-colored septate endophytic fungi inoculant by opening shallow trenches in the gaps between Amorpha fruticosa plants; The application rate of the arbuscular mycorrhizal fungal agent in holes is 40-50g / hole, and the application rate of the arbuscular mycorrhizal fungal agent in shallow trenches is 200-300 kg / ha. Each gram of arbuscular mycorrhizal fungal agent contains 66 spores. The application rate of the dark-colored septate endophytic fungal agent is 40-50 ml / hole, the application rate of the dark-colored septate endophytic fungal agent in shallow trenches is 1000-1500 L / ha, and the inoculation potential of the dark-colored septate endophytic fungus is 8 × 10⁻⁶. 4 CFU / ml.

[0010] Furthermore, the row spacing of the Amorpha fruticosa is 3m; the plant spacing of the Amorpha fruticosa is 2m.

[0011] Furthermore, the alfalfa is sown in full. Preferably, the density of the full-sowing is 8 g / m³. 3 .

[0012] Furthermore, the aforementioned Amorpha fruticosa is an annual seedling.

[0013] Furthermore, the diameter of the seedling is 4-7 mm; Preferably, the height of the seedling is 30cm.

[0014] Furthermore, the alfalfa mentioned is a drought-resistant variety.

[0015] Furthermore, the preparation method of the arbuscular mycorrhizal fungal agent includes using sterilized sandy soil substrate, with corn as the host plant, and inoculating with *Morchella mosierifolia* for propagation. Preferably, the method for preparing the dark-colored septate endophytic fungal inoculum includes inoculating a dark-colored septate endophytic fungal cake that has been cultured in the dark into a liquid culture medium and shaking it. Preferably, the shaking culture time is at least 15 days.

[0016] Furthermore, the shallow trench is 15cm deep and 30cm wide, and the edge of the trench is 50-100cm away from the Amorpha fruticosa plant.

[0017] Secondly, the present invention provides the application of the above-mentioned shrub-grass combination planting method in soil improvement of open-pit mine spoil heaps; Furthermore, the soil remediation of the open-pit mine spoil heap includes any of the following: C1. Increase the organic carbon content of soil in open-pit mine spoil heaps; C2. Increase the carbon-nitrogen ratio of soil in open-pit mine spoil heaps; C3. Increase the total nitrogen content in soil at open-pit mine spoil heaps; C4. Increase soil enzyme activity in open-pit mine spoil heaps; Preferably, the enzyme includes at least one of catalase, sucrase, and phosphatase.

[0018] This invention provides a shrub-grass combination planting method suitable for open-pit mine spoil heaps. By using a combination of Amorpha fruticosa and Alfalfa, the nitrogen-fixing effect of leguminous plants is utilized to continuously improve the nutrient status of the spoil heap. Simultaneously, arbuscular mycorrhizal fungi (AMF) and dark-colored septate endophytic fungi (DSE) are applied, introducing AMF and DSE into the shrub-grass combination via hole application and shallow trench application, respectively. This creates a cross-spatial distribution between AMF and Alfalfa. Furthermore, by controlling the application amount at different locations, the symbiosis of AMF and DSE is promoted, fostering positive interactions between plants, microorganisms, and soil, accelerating soil nutrient enrichment. Field trial results show that this dual-inoculation shrub-grass combination not only significantly improves the photosynthetic rate and CO2 quantum efficiency of AMF and Alfalfa, but also exhibits superior combined enhancement effects compared to single-inoculation and existing technologies in key indicators such as soil organic carbon, available phosphorus, and carbon-nitrogen ratio. In particular, it exhibits a significant synergistic effect on the activity of soil enzymes such as sucrase and phosphatase. It demonstrates that the appropriate spatial distribution of AMF and DSE application rates in holes and trenches, along with their respective locations, can exert synergistic effects such as niche complementarity, superimposed stress resistance mechanisms, and accelerated nutrient cycling. This not only overcomes the poor colonization stability of single-strain inoculation in the extreme environment of mining areas but also significantly improves the soil carbon-nitrogen ratio, enzyme activity, and long-term carbon sequestration potential. Therefore, this model has technical advantages in improving soil fertility, promoting rapid vegetation recovery, and enhancing carbon sequestration capacity, providing a new technical pathway for ecological restoration in mining areas. It solves the technical problems in existing technologies where AMF and DSE face challenges in improving open-pit mine spoil heap soils due to niche competition, poor coexistence stability, and difficulty in controlling inoculation balance, leading to difficulties in soil remediation. Attached Figure Description

[0019] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0020] Figure 1 This is a layout diagram of a demonstration area for combined shrub and grass planting provided by the present invention; Figure 2 The photosynthetic parameters of alfalfa in a demonstration area of ​​shrub-grass combination planting with different treatments provided by the present invention; Figure 3 The photosynthetic parameters of Amorpha fruticosa in a demonstration area of ​​shrub-grass combination planting with different treatments provided by this invention; Figure 4 Comparison of soil nutrient elements in June and September in demonstration areas of shrub-grass combination planting with different treatments; Figure 5A comparison of soil factors in June and September in demonstration areas of shrub-grass combination planting with different treatments; Figure 6 Comparison of soil factors in June and September in demonstration areas of shrub-grass combination planting with different treatments. Detailed Implementation

[0021] Unless otherwise defined herein, the scientific and technical terms used in conjunction with this invention shall have the meanings commonly understood by one of ordinary skill in the art. The meaning and scope of terms shall be clear; however, in any case of potential ambiguity, the definitions provided herein shall prevail over any dictionary or foreign definitions. In this application, unless otherwise stated, the use of "or" means "and / or". Furthermore, the use of the term "comprising" and other forms is non-limiting.

[0022] Unless otherwise stated, the methods and techniques of the present invention are generally carried out according to conventional methods well known in the art and as described in various general and more specific references, which are cited and discussed throughout this specification.

[0023] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0024] This invention provides a method for combined shrub and grass planting suitable for soil in open-pit mine spoil heaps, comprising the following steps: A. Plant Amorpha fruticosa in the area where the soil needs to be improved, and sow alfalfa between the Amorpha fruticosa plants; B. Apply arbuscular mycorrhizal fungi inoculant and dark-colored septate endophytic fungi inoculant to the base of each Amorpha fruticosa plant by hole application, and apply arbuscular mycorrhizal fungi inoculant and dark-colored septate endophytic fungi inoculant by opening shallow trenches in the gaps between Amorpha fruticosa plants; The application rate of the arbuscular mycorrhizal fungal agent in holes is 40-50g / hole, and the application rate of the arbuscular mycorrhizal fungal agent in shallow trenches is 200-300 kg / ha. Each gram of arbuscular mycorrhizal fungal agent contains 66 spores. The application rate of the dark-colored septate endophytic fungal agent is 40-50 ml / hole, the application rate of the dark-colored septate endophytic fungal agent in shallow trenches is 1000-1500 L / ha, and the inoculation potential of the dark-colored septate endophytic fungus is 8 × 10⁻⁶. 4 CFU / ml.

[0025] By using a combination of Amorpha fruticosa and Alfalfa as a shrub-grass mixture, the nitrogen-fixing effect of leguminous plants was utilized to continuously improve the nutrient status of the spoil heap. Simultaneously, arbuscular mycorrhizal fungi (AMF) and dark-colored septate endophytic fungi (DSE) were applied, introducing AMF and DSE into the shrub-grass mixture via hole application and shallow trench application, respectively. This created a cross-spatial distribution between AMF and Alfalfa. Furthermore, by controlling the application rate at different locations, the symbiosis of AMF and DSE was promoted, fostering positive interactions between plants, microorganisms, and soil, and accelerating soil nutrient enrichment. Field trial results showed that this dual-inoculation shrub-grass mixture not only significantly improved the photosynthetic rate and CO2 quantum efficiency of AMF and Alfalfa, but also demonstrated a superior combined enhancement effect compared to single-inoculation and existing technologies in key indicators such as soil organic carbon, available phosphorus, and carbon-nitrogen ratio. In particular, it exhibits a significant synergistic effect on the activity of soil enzymes such as sucrase and phosphatase. It demonstrates that the appropriate spatial distribution of AMF and DSE application rates in holes and trenches, along with their respective locations, can exert synergistic effects such as niche complementarity, superimposed stress resistance mechanisms, and accelerated nutrient cycling. This not only overcomes the poor colonization stability of single-strain inoculation in the extreme environment of mining areas but also significantly improves the soil carbon-nitrogen ratio, enzyme activity, and long-term carbon sequestration potential. Therefore, this model has technical advantages in improving soil fertility, promoting rapid vegetation recovery, and enhancing carbon sequestration capacity, providing a new technical pathway for ecological restoration in mining areas. It solves the technical problems in existing technologies where AMF and DSE face challenges in improving open-pit mine spoil heap soils due to niche competition, poor coexistence stability, and difficulty in controlling inoculation balance, leading to difficulties in soil remediation.

[0026] The application rate of the arbuscular mycorrhizal fungal agent per hole can be, but is not limited to, 40g / hole, 41g / hole, 42g / hole, 43g / hole, 44g / hole, 45g / hole, 46g / hole, 47g / hole, 48g / hole, 49g / hole or 50g / hole, or any value between 40 and 50g / hole.

[0027] The shallow trench application rate of the arbuscular mycorrhizal fungal agent can be, but is not limited to, 200 kg / ha, 210 kg / ha, 220 kg / ha, 230 kg / ha, 240 kg / ha, 250 kg / ha, 260 kg / ha, 270 kg / ha, 280 kg / ha, 290 kg / ha or 300 kg / ha, or any value between 200 and 300 kg / ha.

[0028] The dosage of the dark-colored septate endophytic fungal agent applied per acupoint can be, but is not limited to, 40 ml / acupoint, 41 ml / acupoint, 42 ml / acupoint, 43 ml / acupoint, 44 ml / acupoint, 45 ml / acupoint, 46 ml / acupoint, 47 ml / acupoint, 48 ml / acupoint, 49 ml / acupoint, or 50 ml / acupoint, or any value between 40 and 50 ml / acupoint.

[0029] The shallow trench application rate of the dark-colored septate endophytic fungal agent can be, but is not limited to, 1000 L / ha, 1050 L / ha, 1100 L / ha, 1150 L / ha, 1200 L / ha, 1250 L / ha, 1300 L / ha, 1350 L / ha, 1400 L / ha, 1450 L / ha, or 1500 L / ha, or any value between 1000 and 1500 L / ha.

[0030] In some specific implementations, the row spacing of the Amorpha fruticosa is 3m; the plant spacing is 2m. Controlling the ratio of shrubs to grasses accelerates the remediation of soil by AMF, DSE, and plants.

[0031] In some specific embodiments, the shallow trench is 15 cm deep and 30 cm wide, with the edge of the trench 50-100 cm away from the Amorpha fruticosa plant. Controlling the depth of the trench and its distance from the Amorpha fruticosa ensures the symbiotic relationship between the inoculated AMF and DSE microorganisms and the alfalfa sown among the Amorpha fruticosa.

[0032] In some specific embodiments, the alfalfa is sown in full sowing; in some specific embodiments, the density of full sowing is 8 g / m². 3 .

[0033] In some specific embodiments, the Amorpha fruticosa is an annual seedling. In some specific embodiments, the diameter of the seedling is 4-7 mm; in some specific embodiments, the height of the seedling is 30 cm.

[0034] In some specific implementations, the alfalfa is a drought-resistant variety, such as WL168HQ; before sowing, damaged seeds need to be screened to remove them, and seeds of uniform size should be selected.

[0035] In some specific embodiments, the preparation method of the arbuscular mycorrhizal fungal agent includes using sterilized sandy soil substrate, with corn as the host plant, and inoculating with *Morchella mosierifolia* for propagation.

[0036] In some specific embodiments, the method for preparing the dark-colored septate endophytic fungal inoculum includes inoculating a dark-colored septate endophytic fungal cake that has been cultured in the dark into a liquid culture medium and shaking it; in some specific embodiments, the shaking culture time is at least 15 days.

[0037] Secondly, the present invention provides the application of the above-mentioned shrub-grass combination planting method in soil improvement of open-pit mine spoil heaps; In some specific embodiments, the soil improvement of the open-pit mine spoil heap includes any of the following: C1. Increase the organic carbon content of soil in open-pit mine spoil heaps; C2. Increase the carbon-nitrogen ratio of soil in open-pit mine spoil heaps; C3. Increase the total nitrogen content in soil at open-pit mine spoil heaps; C4. Increase soil enzyme activity in open-pit mine spoil heaps; In some specific embodiments, the enzyme includes at least one of catalase, sucrase, and phosphatase.

[0038] The present invention will be further illustrated by the following examples. Unless otherwise specified, the materials in the examples are prepared according to existing methods or purchased directly from the market.

[0039] In the following examples, the arbuscular mycorrhizal fungus (AMF) used was Funneliformis mosseae (Fm), which was provided by the Western Mining Ecological Environment Restoration Research Institute of Xi'an University of Science and Technology.

[0040] In the following examples, the dark-colored septate endophytic fungus (DSE) used was Alternaria sp., which is deposited at the China General Microbiological Culture Collection Center with accession number CGMCC No. 17463.

[0041] For Amorpha fruticosa, select 1-year-old seedlings with a diameter of 4-7 mm and a height of 30±5 cm.

[0042] Alfalfa, specifically the drought-resistant variety WL168HQ, should have its damaged seeds removed, and only seeds of uniform size selected for use.

[0043] Example 1 This embodiment provides a method for combined shrub and grass planting suitable for open-pit mine spoil heaps. It utilizes the AMF inoculant *Funneliformis mosseae* (Fm) and liquid-cultured DSE inoculant to establish a plant-fungus symbiosis with *Amorpha fruticosa* and *Alfalfa*, promoting plant growth and continuously improving soil physicochemical properties. The main steps include: 1. For example Figure 1 As shown, the planting pattern for Amorpha fruticosa is 3m × 2m (row spacing × plant spacing), while alfalfa was sown in full within the demonstration area using drone seeding at a density of 8g / m². 3 .

[0044] 2. Inoculate pure cultured DSE strains into PDA medium and incubate in the dark at 28°C for 14 days. Prepare MMN liquid culture medium and autoclave at 121°C for 30 minutes. Remove the cultured DSE strains and take mycelial discs of the same size along the edge of the colony, placing them in sterilized MMN liquid culture medium. Shake and incubate a portion for 15 days as DSE inoculum. The AMF inoculum is Funneliformis mosseae (Fm), with 66 spores per gram of arbuscular mycorrhizal fungal inoculum.

[0045] 3. Inoculation treatment should be carried out simultaneously with planting. The specific method is as follows: After placing the Amorpha fruticosa plants in the planting pit, apply microbial inoculum to the roots of the plants. The application rate of AMF fungal inoculum is 50g per plant per pit, and the application rate of DSE inoculum is 50ml per plant per pit (inoculation potential is 8×10). 4 (CFU / ml). For alfalfa planting areas, the application method is as follows: dig shallow trenches (15cm deep, 30cm wide, with the edge of the trench 75cm away from the alfalfa plant) in the gaps between alfalfa plants. Apply the microbial inoculum and then cover it. The application rate is 300kg / ha of AMF inoculum and 1500 L / ha of DSE inoculum. After application, re-cover with the removed topsoil.

[0046] 4. After each treatment is completed, water to the maximum saturation water holding capacity of the soil, then water once every two weeks. After one month, watering is no longer necessary, and the plants can be managed naturally. Subsequent watering will be done as needed during periods of extreme drought, depending on the weather. After the first year of planting, water the plants with winter water between the Minor Snow and Major Snow solar terms to ensure their survival through the winter.

[0047] Comparative Example 1 Unlike Example 1, no AMF inoculum or DSE inoculum was applied, serving as a control (CK).

[0048] Comparative Example 2 Unlike Example 1, only AMF inoculum was applied.

[0049] Comparative Example 3 Unlike Example 1, only DSE inoculum was applied.

[0050] During the vigorous growth period of plants, photosynthetic parameters of plants are monitored using a photosynthesis meter. At the same time, soil samples are collected from the experimental area during different growing seasons to monitor changes in available nutrients and soil enzymes.

[0051] Experiment 1: Measurement of photosynthetic parameters of plants in a demonstration area of ​​shrub-grass hybrid planting 1. Soil samples were taken from different treatment areas (Comparative Example 1: CK; Comparative Example 2: AMF; Comparative Example 3: DSE; Example 1: AMF+DSE). Total nitrogen, available phosphorus, and available potassium were measured. During the peak growing season, using a Li-6400XT portable CO2 / H2O analysis system, on a clear, windless morning from 9:00 to 11:00, photosynthetic rate (Pn), stomatal conductance (Gs), transpiration rate (Tr), CO2 quantum efficiency (Qe), and intercellular CO2 concentration (Ci) were measured on the third fully expanded functional leaf from the main stem of a well-grown plant. The effective radiation source was 1000 μmol / m². 2 The gas flow rate was 500 mmol / s. Fifteen Amorpha fruticosa and Alfalfa plants were randomly selected from each plot for testing. Water use efficiency (WUE) was the ratio of net photosynthetic rate to transpiration rate.

[0052] 2. Test Results: like Figure 2 As shown, the photosynthetic indices of alfalfa differed significantly between the control and inoculated treatments (P<0.05). Net photosynthetic rate, stomatal conductance, intercellular carbon dioxide concentration, transpiration rate, and CO2 quantum efficiency were all lowest in the control treatment. Intercellular carbon dioxide concentration was highest in the AMF+DSE treatment, while net photosynthetic rate, transpiration rate, stomatal conductance, and CO2 quantum efficiency were highest in the DSE treatment. Water use efficiency was highest in the control (CK) treatment and lowest in the AMF+DSE treatment.

[0053] like Figure 3 As shown, the net photosynthetic rate, stomatal conductance, transpiration rate, and CO2 quantum efficiency of *Amorpha fruticosa* were all minimized in the control treatment. There was no significant difference in intercellular carbon dioxide concentration among the treatments (P>0.05). The net photosynthetic rate and stomatal conductance were maximized in the DSE treatment, and the inoculated treatment was significantly higher than the control treatment, but the differences between inoculated treatments were not significant. The transpiration rate and CO2 quantum efficiency were maximized in the AMF+DSE treatment, with the order being double-inoculated treatment > single-inoculated treatment > control. The maximum water use efficiency was maximized in the AMF treatment, but the differences among treatments were not significant.

[0054] Experiment 2: Determination of soil organic carbon (SOC), total nitrogen (TN), and available phosphorus in a demonstration area of ​​shrub-grass intercropping Soil samples were collected from different treatment areas in June and August 2023 (Comparative Example 1 was CK, Comparative Example 2 was AMF, Comparative Example 3 was DSE, and Example 1 was AMF+DSE). The organic carbon (SOC), total nitrogen (TN), and available phosphorus of the soil in the shrub-grass combination planting demonstration area were tested, and the soil carbon-nitrogen ratio was calculated.

[0055] The results are as follows Figure 4As shown, soil nutrient elements showed significant differences between the control and inoculated treatments in June and August (P<0.05). Overall, with the growth of plants in the reclaimed area, the soil organic carbon content, available phosphorus content, and soil carbon-nitrogen ratio were higher in August than in June.

[0056] Soil organic carbon reached its maximum value in the DSE treatment in June and its minimum value in the DSE treatment in August. The maximum value was found in the double-inoculation treatment. Both the AMF inoculation treatment and the double-inoculation treatment showed a significant increase compared to June, while the DSE treatment and the control showed a smaller increase. The double-inoculation treatment significantly increased soil organic carbon over time.

[0057] In June, the AMF treatment showed the highest value, which was significantly higher than that of the DSE and dual-inoculation treatments. In August, the AMF treatment also showed the highest value, which was significantly higher than that of other treatments. However, the dual-inoculation treatment did not show significant changes between June and August. The dual-inoculation treatment can keep the available phosphorus in a moderate state and maintain its stability.

[0058] Soil carbon-nitrogen ratios reached their maximum values ​​in both June and August with the double-inoculation treatment. In June, the DSE and double-inoculation treatments were significantly higher than the AMF treatment, while in August, the double-inoculation treatment was significantly higher than the other treatments.

[0059] Soil total nitrogen showed a maximum value in the control treatment in June, which was significantly higher than that of the inoculated treatment, while the minimum value was found in the double-inoculated treatment. In August, the double-inoculated treatment showed a maximum value, which was significantly higher than that of other treatments.

[0060] The soil carbon-nitrogen ratio had the highest value in the double-inoculation treatment and was significantly higher than in other treatments.

[0061] Experiment 3: Measurement of soil factors including pH, electrical conductivity, catalase, phosphatase, and sucrase in a demonstration area of ​​shrub-grass hybrid planting. Soil samples were collected in June and August 2023 from different treatment areas (Comparative Example 1 was CK, Comparative Example 2 was AMF, Comparative Example 3 was DSE, and Example 1 was AMF+DSE). The soil pH, electrical conductivity, catalase, phosphatase and sucrase of the shrub-grass combination planting demonstration area were tested.

[0062] The results are as follows Figure 5 and Figure 6 As shown, soil factors differed significantly between the control and inoculated treatments in June and August (P<0.05). Overall trends indicated that soil pH decreased in August compared to June, while soil electrical conductivity and catalase levels increased, although phosphatase levels showed no significant change.

[0063] Soil pH showed a minimum in the control treatment and a maximum in the DSE treatment in June. The inoculation treatment was significantly higher than the control treatment, which may be related to the release of ions after the introduction of microbial agents. In August, the DSE treatment showed a minimum and the control treatment had a maximum. The inoculation treatment showed a significant decrease compared to June, while the control treatment showed a slight increase.

[0064] In June, the conductivity was highest in the DSE treatment and lowest in the double-inoculation treatment, which was significantly lower than other treatments. In August, the conductivity was highest in the double-inoculation treatment and significantly higher than other treatments, while the control treatment had the lowest conductivity. Overall, the conductivity was double-inoculation > single-inoculation > control, and all treatments showed improvement compared to June.

[0065] Soil catalase showed a maximum value in the DSE treatment and a minimum value in the control treatment in June, with the overall trend being double-inoculated bacteria > single-inoculated bacteria > control; in August, the control treatment showed a maximum value, which was significantly higher than other treatments.

[0066] Soil phosphatase levels were highest in the AMF treatment in June, significantly higher than in other treatments, while the control treatment showed the lowest values.

[0067] Soil sucrase levels reached their maximum in the AMF treatment in August, and were significantly higher than in other treatments.

[0068] In summary, this invention establishes a method for promoting carbon sequestration and growth of the *Amorpha fruticosa* + *Alfalfa* shrub-shrub combination using AMF and DSE double inoculation. On the one hand, the AMF and DSE double inoculation agents improve relevant photosynthetic parameters of the plants and enhance their physiological adaptability, thereby promoting the accumulation of organic carbon in the soil. On the other hand, the AMF+DSE inoculation treatment significantly increases soil carbon, nitrogen, and soil enzyme activity within the same time frame. For plant physiological indicators, the AMF+DSE treatment improves indicators such as net photosynthetic rate and quantum efficiency, demonstrating good performance. The AMF+DSE combination with *Alfalfa* + *Amorpha fruticosa* shrub-shrub combination is suitable for open-field soils.

[0069] The combined microbial agent, when applied to the soil, creates a favorable microbial environment for plants. By improving photosynthetic efficiency and leaf characteristics, it promotes plant growth and environmental adaptability, playing a significant role in increasing crop yield and protecting the sustainable development of the ecological environment, thus improving soil in open fields. In this experiment, the combined AM fungus and DSE microbial agent improved the relevant photosynthetic parameters of Amorpha fruticosa and Alfalfa to a certain extent. The application of the combined microbial agent in this invention has certain potential for promotion and application in land reclamation, vegetation restoration, and ecological protection, and also provides microbial technical support for ecological reconstruction.

[0070] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for combined shrub and grass planting suitable for soil in open-pit mine spoil heaps, characterized in that, Includes the following steps: A. Plant Amorpha fruticosa in the area where the soil needs to be improved, and sow alfalfa between the Amorpha fruticosa plants; B. Apply arbuscular mycorrhizal fungi inoculant and dark-colored septate endophytic fungi inoculant to the base of each Amorpha fruticosa plant by hole application, and apply arbuscular mycorrhizal fungi inoculant and dark-colored septate endophytic fungi inoculant by opening shallow trenches in the gaps between Amorpha fruticosa plants; The application rate of the arbuscular mycorrhizal fungal agent in holes is 40-50g / hole, and the application rate of the arbuscular mycorrhizal fungal agent in shallow trenches is 200-300 kg / ha. Each gram of arbuscular mycorrhizal fungal agent contains 66 spores. The hole application amount of the dark-septate endophytic fungus agent is 40-50 ml / hole, the shallow trench application amount of the dark-septate endophytic fungus agent is 1000-1500 L / ha, and the inoculation potential of the dark-septate endophytic fungus is 8×10 4 CFU / ml.

2. The method for combined planting of shrubs and grasses according to claim 1, characterized in that, The row spacing of the Amorpha fruticosa is 3m; the plant spacing of the Amorpha fruticosa is 2m.

3. The method for combined planting of shrubs and grasses according to claim 1, characterized in that, The alfalfa was sown in full. Preferably, the density of the full-sowing is 8 g / m³. 3 .

4. The method for combined planting of shrubs and grasses according to claim 1, characterized in that, The Amorpha fruticosa mentioned is an annual seedling.

5. The method for combined planting of shrubs and grasses according to claim 4, characterized in that, The diameter of the seedlings is 4-7 mm; Preferably, the height of the seedling is 30cm.

6. The method for combined planting of shrubs and grasses according to claim 1, characterized in that, The alfalfa mentioned is a drought-resistant variety.

7. The method for combined planting of shrubs and grasses according to claim 1, characterized in that, The preparation method of the arbuscular mycorrhizal fungal agent includes using sterilized sandy soil substrate, with corn as the host plant, and inoculating with *Mammillaria mosie* for propagation. Preferably, the method for preparing the dark-colored septate endophytic fungal inoculum includes inoculating a dark-colored septate endophytic fungal cake that has been cultured in the dark into a liquid culture medium and shaking it. Preferably, the shaking culture time is at least 15 days.

8. The method for combined planting of shrubs and grasses according to claim 1, characterized in that, The shallow trench is 15cm deep and 30cm wide, and the edge of the trench is 50-100cm away from the Amorpha fruticosa plant.

9. The application of the shrub-grass combination planting method according to any one of claims 1-8 in soil improvement of open-pit mine spoil heaps.

10. The application according to claim 9, characterized in that, Soil remediation for open-pit mine spoil heaps includes any of the following: C1. Increase the organic carbon content of soil in open-pit mine spoil heaps; C2. Increase the carbon-nitrogen ratio of soil in open-pit mine spoil heaps; C3. Increase the total nitrogen content in soil at open-pit mine spoil heaps; C4. Increase soil enzyme activity in open-pit mine spoil heaps; Preferably, the enzyme includes at least one of catalase, sucrase, and phosphatase.

Images