Graphene and arbuscular mycorrhizal fungus composite microbial agent, preparation method thereof and application in gangue dump reclamation

Abstract

The application discloses a kind of graphene and arbuscular mycorrhizal fungi composite microbial inoculant and its preparation method and application technology in gangue hill reclamation, belong to the cross field of microbial technology and ecological restoration.The preparation method directly uses the purified graphene sol, through arbuscular mycorrhizal fungi propagation, two-step core process of graphene sol-fungus composite system construction, obtain the composite microbial inoculant with good dispersibility, high stability and strong stress resistance;When the composite microbial inoculant is applied to gangue hill reclamation, through the supporting process of soil pretreatment-microbial inoculant directional application-later monitoring, the survival rate of plants in gangue hill reclamation is as high as 90% or more.The application simplifies the preparation process, solves the technical problems of low survival rate of microbial inoculant and poor soil improvement effect in traditional gangue hill reclamation, and provides an efficient and low-cost technical solution for ecological restoration in coal mine area.

Description

Technical Field

[0001] This invention relates to the field of microbial preparation and ecological restoration technology, specifically to a composite microbial agent of graphene and arbuscular mycorrhizal fungi and its preparation method, as well as the application of the composite microbial agent in the reclamation of coal mine gangue hills. Background Technology

[0002] Coal mine gangue piles are solid waste accumulations generated during coal mining. Their surface soil suffers from problems such as lack of organic matter, high content of heavy metals (such as Pb, Cd, and Cu), and pH imbalance (mostly strongly acidic or strongly alkaline), resulting in low microbial activity and difficulty for plants to survive, making them a key and difficult point for ecological restoration in mining areas.

[0003] Microbial treatment technology is considered a green and low-cost activation pathway. Mycorrhizal fungi, especially arbuscular mycorrhizal fungi (AMF), are a type of microorganism that can form a symbiotic relationship with plant roots. They can effectively weather minerals and release elements such as K, P, Si, and Ca through the physical penetration of hyphae and the secretion of metabolic products such as organic acids (e.g., oxalic acid, citric acid), chelating agents, and extracellular polymers. This bioweathering ability provides a possibility for the dissociation and activation of coal gangue. However, traditional AMF preparations have drawbacks in the harsh environment of gangue piles, such as low survival rate (less than 30%), short action period (only 1-2 months), and limited synergistic effect, which restricts their large-scale application in land reclamation. Summary of the Invention

[0004] The inventors discovered that graphene sol, as a carbon-based material with excellent dispersibility, possesses an ultra-large specific surface area (≥500 m² / g), good biocompatibility, and strong adsorption capacity. It can serve as a microbial carrier to protect fungi from harsh environmental stresses and can also adsorb heavy metals in the soil and regulate the soil microenvironment through surface functional groups (hydroxyl and carboxyl groups), further enhancing the metabolic activity of fungi. Currently, mature graphene sol purification and preparation technologies exist, directly yielding high-purity and highly stable products. However, there are no reports on its direct application for enhancing the efficacy of arbuscular mycorrhizal fungi, nor on the development of specialized formulations and supporting application technologies for waste rock reclamation.

[0005] To address the technical problems of low survival rate and poor soil improvement effect of existing microbial agents in coal gangue reclamation, as well as the cumbersome preparation process of traditional synergistic compound agents, this invention directly utilizes purified graphene sol to provide a simplified method for preparing a compound agent of graphene sol and arbuscular mycorrhizal fungi. Considering that arbuscular mycorrhizal fungi cannot be cultured in pure culture media, this method utilizes the synergistic effect of graphene sol during the propagation stage of arbuscular mycorrhizal fungi to create a suitable microenvironment for fungal survival and colonization. Simultaneously, it provides the application technology of this agent in coal gangue reclamation, ultimately achieving the goals of improving fungal survival rate, shortening the preparation cycle, improving soil quality in coal gangue piles, and increasing plant survival rate.

[0006] To achieve the above objectives, the following technical solution is adopted: This invention provides a composite microbial agent, which is obtained by mixing graphene sol and AMF crude microbial agent; The concentration of graphene sol added to the compound microbial agent is 20 ~ 200 mg / kg; The mass ratio of the graphene sol to the AMF crude bacterial agent is (5~15):100.

[0007] According to an embodiment of the present invention, in the graphene sol, the graphene interlayer spacing is 1.0 nm to 1.8 nm, and the number of layers is less than 5.

[0008] According to an embodiment of the present invention, the AMF crude agent comprises plant root powder infected with AMF (Gymnocytophyte molasses) and AMF (Gymnocytophyte molasses) propagation substrate; The AMF (Gymnocytogenes moses) propagation substrate is a substrate containing Gymnocytogenes moses spores and hyphae, with a Gymnocytogenes moses spore density ≥ 50 spores / cm³.

[0009] According to an embodiment of the present invention, the substrate comprises sterilized soil and river sand in a mass ratio of 1:1.

[0010] According to an embodiment of the present invention, the plant is a crop, preferably a crop with a well-developed root system and a high AMF infection rate, such as corn.

[0011] The present invention also provides a method for preparing the above-mentioned compound microbial agent, comprising the following steps: 1. Preparation of cultivation substrate: Sterilized soil and river sand are mixed at a mass ratio of 1:(0.8~1.2) (e.g., 1:1) to obtain the cultivation substrate; 2. Pretreatment of plant (e.g., corn) seeds: Select seeds of plants with well-developed root systems and high AMF infection rate (e.g., corn) for germination, and select germinated seeds with uniform sprout length for later use; 3. Pot propagation: Fill the cultivation substrate into a sterilized pot, inoculate the original strain of *Glomus mosy* into the cultivation substrate at an inoculation amount of 3-6% (e.g., 5%) of the cultivation substrate mass, and mix well; sow the germinated seeds described in step 2 into the cultivation substrate, cover with the sterilized cultivation substrate, and cover with a thin film to retain moisture. 4. Cultivation and Management: Place the pots from step 3 in a sterilized greenhouse. After the seedlings emerge, remove the film. When the density of *Gymnocytosporum moses* spores in the substrate is ≥50 spores / cm³, harvest the AMF propagation substrate (containing mycelium, spores, and infected roots). 5. Preparation of crude AMF inoculant: Select the AMF propagation substrate harvested in step 4, remove the above-ground parts of the corn, separate the roots from the AMF propagation substrate, dry the roots and crush them into powder; mix the root powder with the dried AMF propagation substrate evenly to obtain crude AMF inoculant. 6. Preparation of the composite microbial agent of graphene sol and AMF: The graphene sol is added at a rate of 5-15% of the mass of crude AMF. The two are mixed under sterile conditions and placed in a cool, ventilated place to stand (e.g., stand for 24 hours) to obtain the composite microbial agent.

[0012] According to an embodiment of the present invention, the sterilized soil in step 1 is obtained by the following method: select uncontaminated loam, remove impurities and large particles, pass through a 2mm sieve, and treat it by intermittent sterilization, that is, dry at 160℃ for 2 hours, let it cool, dry it again at 160℃ for 2 hours, and then cool it for later use.

[0013] According to an embodiment of the present invention, step 2, the germination process includes: selecting seeds of plants with well-developed root systems and high AMF infection rates (e.g., corn), rinsing the surface coating with water, disinfecting with hydrogen peroxide (e.g., disinfecting with a 10% hydrogen peroxide solution for 10 minutes), rinsing with distilled water, and soaking in a saturated calcium sulfate solution for 3-6 hours; subsequently, placing the seeds in a petri dish lined with moistened filter paper and germinating them in a dark incubator at a temperature of 25-30°C and a humidity of 60-70% for 24-36 hours.

[0014] According to an embodiment of the present invention, step 3, the propagation of potted plants, includes: filling a flowerpot sterilized by wiping with 75% alcohol into a cultivation substrate, with the substrate filling volume being 2 / 3 of the flowerpot volume; inoculating the substrate with the original strain of *Glomus mosyensis*, with an inoculation amount of 5% of the cultivation substrate mass, and gently mixing; sowing pretreated germinated corn seeds into the substrate, with 3 seeds per pot, covering with a layer of sterilized cultivation substrate with a thickness of 1 to 2 cm, and covering with a thin film to retain moisture.

[0015] According to an embodiment of the present invention, step 4, cultivation and management, includes: placing the flowerpots in a glass greenhouse that has been disinfected with alcohol, controlling the temperature at 15-35℃, and removing the film after emergence; watering with distilled water every 2-3 days to keep the substrate moist; watering with nutrient solution every 10-15 days; cultivating for 3-4 months, detecting the density of *Gloydium moss* spores in the substrate, and harvesting the AMF propagation substrate (containing mycelium, spores, and infected roots) when the spore density is ≥50 spores / cm³.

[0016] According to an embodiment of the present invention, the solid content of the graphene sol in step 6 is 4-6‰, for example 5‰.

[0017] According to an embodiment of the present invention, the concentration of the graphene sol in step 6 is 10~200 mg / kg, preferably 40~60 mg / kg, and more preferably 50 mg / kg.

[0018] According to an embodiment of the present invention, the preparation method of the compound microbial agent includes the following steps: 1. Propagation of *Gymnospermum mosesense* in potted corn: Preparation of cultivation substrate: Select uncontaminated loam, remove impurities and large particles, pass through a 2mm sieve, and treat with intermittent sterilization method, that is, dry at 160℃ for 2 hours, cool and dry again at 160℃ for 2 hours, and then cool for use; mix sterilized loam with river sand at a mass ratio of 1:1 to obtain cultivation substrate. 2. Pretreatment of corn seeds: Select corn seeds with well-developed root systems and high AMF infection rate. Rinse the surface coating with water to remove the coating, disinfect with 10% hydrogen peroxide solution for 10 minutes, rinse quickly with distilled water, and soak in saturated calcium sulfate solution for 5 hours. Then place the seeds in a petri dish lined with moist filter paper and germinate in a dark incubator at 25-30℃ and 60-70% humidity for 24-36 hours. Select germinated seeds with uniform sprout length for later use. 3. Pot propagation: Fill flower pots sterilized by wiping with 75% alcohol with the cultivation substrate, filling the pots to 2 / 3 of their volume; inoculate the substrate with the original *Gastromyxomorpha moses* strain at 5% of the substrate mass, and mix gently; sow the pretreated germinated corn seeds into the substrate, 3 seeds per pot, cover with a 1-2 cm thick layer of sterilized cultivation substrate, and cover with a thin film to retain moisture. 4. Cultivation and Management: Place the flowerpots in a glass greenhouse that has been sterilized with alcohol, and control the temperature at 15-35℃. Remove the film after the seedlings emerge. Water with distilled water every 2-3 days to keep the substrate moist. Water with nutrient solution every 10-15 days. Cultivate for 3-4 months, and test the density of *Gloydium moss* spores in the substrate. When the spore density is ≥50 spores / cm³, harvest the AMF propagation substrate (containing mycelium, spores, and infected roots). 5. Preparation of AMF crude inoculum: Select the AMF propagation substrate harvested in step 1, remove the above-ground parts of the corn, separate the roots from the substrate, dry the roots and crush them into powder; mix the root powder with the dried AMF propagation substrate evenly to obtain crude AMF inoculum. 6. Preparation of the composite microbial agent of graphene sol and AMF: The graphene sol (concentration of graphene sol is 50 mg / kg) is added at an amount of 5-15% of the mass of crude AMF. The two are mixed under sterile conditions and placed in a cool and ventilated place to stand (e.g., stand for 24 hours) to obtain the composite microbial agent.

[0019] The present invention also provides the application of the above-mentioned compound microbial agent in the reclamation of gangue hills, the method comprising the following steps: 1. Pre-treatment of the waste rock reclamation area: The waste rock pile is leveled, and large pieces of waste rock (diameter > 10cm) and sharp debris are removed; a layer of topsoil with a thickness of 40-50cm is laid on the surface to improve the surface soil structure and water retention capacity; 2. Vegetation establishment: Select reclaimed plants that are tolerant of poor soil and have strong stress resistance (such as European plum, forsythia, etc.), and the planting density of reclaimed plants is 400-500 plants / acre; 3. Application of microbial agent: When planting, apply the compound microbial agent around the roots of the reclaimed plants using the hole application method. The application depth is 8-12cm, and the dosage of microbial agent is 8-10 g / hole. After application, cover with soil and compact to ensure that the microbial agent comes into contact with the plant roots. 4. Post-planting management: Water thoroughly after sowing to keep the topsoil moist; water thoroughly within 24 hours after applying compound microbial agent to bring the soil moisture content to 60%~70% of field capacity; water every 15 days thereafter to keep the soil moist; apply additional microbial agent every 30 days (8~10 g / hole) for 2~3 times to extend the synergistic effect period; regularly monitor soil nutrients and plant growth indicators (plant height, fresh weight, survival rate).

[0020] The beneficial effects of this invention are as follows: The addition of graphene sol in this invention can significantly enhance the activity of AMF (e.g., *Glomus mosyensis*). Its ultra-large specific surface area can adsorb nutrient ions in the soil, increase the nutrient migration rate, and promote the absorption of nutrients and water by plant roots. The synergistic effect of graphene and AMF can enhance plant stress resistance, improve the micro-ecological environment of coal gangue soil, and enhance the soil's water and fertilizer retention capacity. Simultaneously, by utilizing graphene to enhance the growth of *Glomus mosyensis*, the mycelium directly contacts coal gangue particles, causing mechanical damage. At the same time, it secretes low-molecular-weight organic acids (such as oxalic acid and citric acid) to undergo complexation reactions with aluminosilicate minerals and secretes extracellular polymers to encapsulate particles, altering their interfacial properties. These combined effects lead to the decomposition of coal gangue particles and the development of pore structures.

[0021] After applying this technology, the survival rate of plants (such as European plum and forsythia) in the coal gangue hill reclamation can reach 80% to 90%, and the crop height can be increased by 10% to 20%, achieving a dual improvement in soil quality and vegetation cover.

[0022] The preparation process of this invention is simple and low-cost, and the application process is easy to operate, making it suitable for large-scale promotion. It provides an efficient and feasible technical solution for the reclamation of gangue hills in coal mining areas. Attached Figure Description

[0023] Figure 1Example 1: Effects of different concentrations of graphene combined with AMF on the total dry weight and root-to-shoot ratio of maize plants; Note: Different lowercase letters indicate significant differences between different treatments for the same indicator. P <0.05).

[0024] Figure 2 Example 2: Prunus cerasifera plant height and survival rate in a waste rock reclamation area under different treatments; Note: Different lowercase letters indicate significant differences between different treatments for the same indicator. P <0.05).

[0025] Figure 3 Example 2: Soil fertility indicators in the gangue hill reclamation area under different treatments; Note: Different lowercase letters indicate significant differences between different treatments for the same indicator. P <0.05). Detailed Implementation

[0026] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention, and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0027] Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available products or can be prepared by known methods.

[0028] Arbuscular mycorrhizal fungi (AMF) were selected from *Glomus mosseae*, a strain provided by the mycorrhizal research team of the College of Resources and Environment, China Agricultural University.

[0029] The graphene sol was provided by the Ministry of Education Engineering Research Center for Coal-based Ecological Carbon Sequestration Technology of Shanxi Datong University; the graphene interlayer spacing is between 1.0nm and 1.8nm, the number of layers is less than 5, the specific surface area is ≥500m² / g, and the solid content is 5‰.

[0030] AMF crude inoculum (Glomus mosysum spore density ≥ 50 spores / cm³) and its preparation: 1. Propagation of *Gymnospermum mosesense* in potted corn: Preparation of cultivation substrate: Select uncontaminated loam, remove impurities and large particles, and pass through a 2mm sieve. Use intermittent sterilization method, that is, dry at 160℃ for 2 hours, let cool, and dry again at 160℃ for 2 hours. After cooling, use for later use. Mix sterilized loam with river sand at a mass ratio of 1:1 to obtain cultivation substrate.

[0031] 2. Pretreatment of corn seeds: Select corn seeds with well-developed root systems and high AMF infection rate. Rinse the surface coating with water to remove the seeds, disinfect them with 10% hydrogen peroxide solution for 10 minutes, rinse them quickly with distilled water, and soak them in saturated calcium sulfate solution for 5 hours. Then place the seeds in a petri dish lined with moist filter paper and germinate them in a dark incubator at a temperature of 25-30℃ and a humidity of 60-70% for 24-36 hours. Select germinated seeds with uniform sprout length for later use.

[0032] 3. Pot propagation: Fill flower pots sterilized by wiping with 75% alcohol with the cultivation substrate, filling the pots to 2 / 3 of their volume; inoculate the substrate with the original *Gastromyxomorpha moses* strain at 5% of the substrate mass, and mix gently; sow the pretreated germinated corn seeds into the substrate, 3 seeds per pot, cover with a 1-2 cm thick layer of sterilized cultivation substrate, and cover with a thin film to retain moisture.

[0033] 4. Cultivation and Management: Place the flowerpots in a glass greenhouse that has been disinfected with alcohol, and control the temperature at 15-35℃. Remove the film after the seedlings emerge. Water with distilled water every 2-3 days to keep the substrate moist. Water with nutrient solution every 10-15 days. Cultivate for 3-4 months and test the density of Gastrodia moss spores in the substrate. When the spore density is ≥50 spores / cm³, harvest the AMF propagation substrate (containing mycelium, spores, and infected roots).

[0034] 5. Preparation of AMF crude inoculum Select the AMF propagation substrate harvested in step 4, remove the above-ground parts of the corn, separate the roots from the substrate, dry the roots and crush them into powder; mix the root powder with the dried AMF propagation substrate evenly to obtain crude AMF inoculum.

[0035] Example 1: Evaluation of the synergistic effect of graphene (using corn as an example) 1. Experimental materials: Corn seedlings: Select "Zhengdan 958" corn seedlings that have germinated well and are growing uniformly (about 10cm tall).

[0036] AMF inoculum: AMF crude inoculum prepared by the above method (M. mossy spore density ≥ 50 spores / cm³).

[0037] Graphene sol: provided by the Ministry of Education Engineering Research Center for Coal-based Ecological Carbon Sequestration, Shanxi Datong University, with a solid content of 5‰.

[0038] Cultivation substrate and flower pots: Sterilized loam-river sand (1:1) mixed substrate, 50cm×16cm×16cm sterilized flower pots.

[0039] 2. Experimental Design: There were 6 treatments in total, with 4 replicates for each treatment, and 24 potted plants. ① Control group (CK): no AMF inoculation and no graphene application; ② AMF group: AMF inoculation only, no graphene application; ③ Graphene group: no AMF inoculation, different concentrations of graphene applied; ④ Graphene sol preparation synergistic agent group (AMF + graphene): AMF inoculation and different concentrations of graphene applied.

[0040] 3. Preparation of graphene sol and AMF composite bacterial agent: Graphene sol was added to the crude AMF bacterial agent and the concentration was adjusted to 0 mg / kg, 50 mg / kg and 100 mg / kg. The mixture was stirred thoroughly under sterile conditions and placed in a cool and ventilated place for 24 hours to obtain graphene sol and AMF composite bacterial agent.

[0041] 4. Treatment Methods: After transplanting, corn seedlings were allowed to acclimatize for 3 days, and then each group received a corresponding treatment. The graphene sol-based synergistic microbial agent group and the AMF group received a single application of the corresponding microbial agent (30g / pot) per planting hole, followed by covering with soil, compacting, and immediately watering thoroughly. The graphene group received root irrigation with graphene sol after transplanting (0mg / kg, 50mg / kg, and 100mg / kg). Environmental conditions were maintained consistently for all groups (temperature 15-35℃, moisture content maintained at 60-70% of field capacity).

[0042] 5. Data monitoring: Two months later, the above-ground and underground parts of the corn were harvested, and indicators such as corn biomass, root-to-shoot ratio, and root morphology were measured.

[0043] 6. Effectiveness Evaluation: The addition of graphene sol and arbuscular mycorrhizal fungi has a promoting effect on the total dry weight of corn. Figure 1 Furthermore, the best results were observed at a graphene concentration of 50 mg / kg. When graphene sol and AMF compound microbial agent were added, and the graphene concentration was 50 mg / kg, the root-to-shoot ratio of corn was closer to 1, indicating that the root system of corn was more developed at a graphene concentration of 50 mg / kg. Figure 1 Meanwhile, in the graphene sol and AMF compound bacterial agent group, when the graphene concentration was 50 mg / kg, the number of root tips, number of branches, root projected surface area, root surface area, and root diameter growth of maize roots were all significantly higher than those of the control (Table 1). This indicates that adding an appropriate concentration of graphene sol (50 mg / kg) can promote the symbiosis between arbuscular mycorrhizal fungi and maize roots, promote nutrient absorption, and thus benefit the growth of the entire maize plant.

[0044] Table 1. Effects of different graphene-AMF combinations on maize root morphology indices

[0045] Note: Different lowercase letters indicate significant differences between different treatments for the same indicator. P<0.05).

[0046] Example 2: Application of graphene sol-gel synergistic microbial agent in the reclamation of gangue hills 1. Overview of the Experimental Area: In a coal mine waste rock pile, after removing large pieces of waste rock with a diameter >10cm from the surface, the surface was leveled using a bulldozer, and a 40-50cm thick layer of topsoil was laid on top to improve the surface soil structure and water retention capacity. In late March, European plum seedlings with identical initial conditions were transplanted at a planting density of 5-6 seedlings / hole, resulting in a planting density of 400-500 seedlings / acre, covering a total area of ​​15 acres. At planting time, holes 8-12cm deep were dug around the plant roots (for applying microbial agents).

[0047] 2. Experimental materials: European plum seedlings: Select European plum, a reclaimed plant that is tolerant of poor soil, has strong stress resistance, and grows evenly.

[0048] AMF inoculum: AMF crude inoculum prepared by the above method (M. mossy spore density ≥ 50 spores / cm³).

[0049] Graphene sol: provided by the Ministry of Education Engineering Research Center for Coal-based Ecological Carbon Sequestration, Shanxi Datong University, with a solid content of 5‰.

[0050] Preparation of graphene sol and AMF composite inoculant: Graphene sol was added to the crude AMF inoculant and the concentration was adjusted to 50 mg / kg of graphene. The mixture was stirred thoroughly under sterile conditions and placed in a cool and ventilated place to stand for 24 hours to obtain the graphene sol-enhanced arbuscular mycorrhizal fungal inoculant.

[0051] 3. Experimental Design: Three treatment groups and one control group were set up. The crude AMF inoculant was applied in the holes at a dosage of 10 g / plant and was designated as group J; the AMF inoculant with enhanced effect was prepared by applying graphene sol in the holes at a dosage of 10 g / plant and was designated as group JS; the graphene sol was applied in the holes at a dosage of 10 g / plant and was designated as group S; and the control group was treated with an equal amount of water and was designated as group CK.

[0052] After each group of reagents is applied, immediately cover with soil and compact. Irrigate thoroughly within 24 hours of applying the inoculant, so that the soil moisture content reaches 60%~70% of field capacity. Thereafter, irrigate once every 15 days, and apply each group of reagents again every 30 days, using the same amount as the first time.

[0053] 3. Application effect evaluation: like Figure 2As shown, after 90 days, the survival rate of *Prunus cerasifera* in the JS group was the highest at 98%, significantly higher than the control group (CK) by 22%. Regarding plant height, compared to the CK group, the treatment with graphene sol and AMF compound inoculant increased plant height by 18.07%, while the treatment with AMF alone and graphene alone increased plant height by 12.82% and 12.84%, respectively. Figure 3 As shown, compared with the control group, single AMF, single graphene, or a combination of both treatments significantly increased soil organic matter content. The JS treatment showed the highest organic matter content, increasing by 59.94% compared to the CK group, while there was no significant difference between the J and S groups. The JS group had the highest available nitrogen content, increasing by 40.43% compared to the CK group, while the single AMF and single graphene groups showed no significant difference compared to the control group. The JS treatment group had the highest available phosphorus content, showing a significant difference compared to the other treatment groups; the available phosphorus content in the control and single AMF treatment groups was at a moderate level, with no significant difference between the groups.

[0054] The above results fully demonstrate that the present invention, through the synergistic effect of graphene and arbuscular mycorrhizal fungi, can effectively improve the soil quality of coal gangue hills, significantly increase organic matter and nitrogen and phosphorus indicators, improve soil fertility, and significantly promote the growth of Prunus armeniaca by optimizing the soil micro-ecological environment. Moreover, the graphene-enhanced mycorrhizal fungi treatment is more effective than the single AMF or single graphene treatment, providing efficient and sustainable technical support for the ecological restoration of coal gangue hills and the subsequent high-value-added resource utilization of vegetation.

[0055] The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiments. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A compound microbial agent, characterized in that, The composite microbial agent is obtained by mixing graphene sol and AMF crude microbial agent; The concentration of graphene sol added to the compound microbial agent is 20 ~ 200 mg / kg; The mass ratio of the graphene sol to the AMF crude bacterial agent is (5~15):100; The AMF crude agent includes plant root powder infected with AMF (Aureobasidium mosesense) and AMF (Aureobasidium mosesense) propagation substrate; The AMF (Gymnocytogenes moses) propagation substrate is a substrate containing Gymnocytogenes moses spores and hyphae, with a Gymnocytogenes moses spore density ≥ 50 spores / cm³.

2. The compound microbial agent according to claim 1, characterized in that, In the graphene sol, the interlayer spacing of the graphene is 1.0 nm to 1.8 nm, and the number of layers is less than 5. And / or, the plant is a crop, preferably a crop with a well-developed root system and a high AMF infection rate, such as maize.

3. The method for preparing the compound microbial agent according to claim 1 or 2, characterized in that, The preparation method includes the following steps: (1) Preparation of cultivation substrate: Sterilized soil and river sand are mixed at a mass ratio of 1:(0.8~1.2) to obtain the cultivation substrate; (2) Pretreatment of plant (e.g., corn) seeds: Select plant seeds with well-developed root systems and high AMF infection rate for germination, and select germinated seeds with uniform bud length for later use; (3) Pot propagation: The cultivation substrate is placed into a sterilized pot, and the original strain of *Glomus mosy* is inoculated into the cultivation substrate at a rate of 3-6% of the mass of the cultivation substrate. Mix well; the germinated seeds described in step (2) are sown into the cultivation substrate, covered with the sterilized cultivation substrate, and covered with a film to keep it moist. (4) Cultivation and management: Place the pots from step (3) in a sterilized greenhouse and remove the film after the seedlings emerge; when the density of Moses spores in the substrate is ≥50 spores / cm³, harvest the AMF propagation substrate. (5) Preparation of AMF crude inoculant: Select the AMF propagation substrate harvested in step (4), remove the aboveground parts of the corn, separate the roots from the AMF propagation substrate, dry the roots and crush them into powder; mix the root powder with the dried AMF propagation substrate evenly to obtain AMF crude inoculant; (6) Preparation of the composite microbial agent of graphene sol and AMF: The amount of graphene sol added is 5-15% of the mass of crude AMF microbial agent. The two are mixed under sterile conditions and placed in a cool and ventilated place to stand to obtain the composite microbial agent.

4. The preparation method according to claim 3, characterized in that, The sterilized soil in step (1) is obtained by the following method: select uncontaminated loam, remove impurities and large particles, pass through a 2mm sieve, dry at 160℃ for 2 hours, let cool, dry again at 160℃ for 2 hours, and then cool for later use.

5. The preparation method according to claim 3, characterized in that, Step (2) involves germination as follows: selecting plant seeds with well-developed root systems and high AMF infection rates, rinsing the surface coating with water, disinfecting with hydrogen peroxide, rinsing with distilled water, and soaking in a saturated calcium sulfate solution for 3-6 hours; then placing the seeds in a petri dish lined with moist filter paper and germinating them in a dark incubator at a temperature of 25-30℃ and a humidity of 60-70% for 24-36 hours.

6. The preparation method according to claim 3, characterized in that, Step (3) of potted plant propagation includes: filling the cultivation substrate into flower pots that have been sterilized by wiping with 75% alcohol, with the substrate filling amount being 2 / 3 of the flower pot volume; inoculating the substrate with the original strain of *Glomus mossicae*, with an inoculation amount of 5% of the cultivation substrate mass, and gently mixing; sowing the pretreated germinated corn seeds into the substrate, with 3 seeds per pot, covering with a layer of sterilized cultivation substrate with a thickness of 1 to 2 cm, and covering with a thin film to retain moisture.

7. The preparation method according to claim 3, characterized in that, Step (4) Cultivation management includes: placing the flowerpots in a glass greenhouse that has been disinfected with alcohol, controlling the temperature at 15-35℃, and removing the film after the seedlings emerge; watering with distilled water every 2-3 days to keep the substrate moist; watering with nutrient solution every 10-15 days; cultivating for 3-4 months, testing the density of Moses spores in the substrate, and harvesting the AMF propagation substrate when the spore density is ≥50 spores / cm³.

8. The preparation method according to claim 3, characterized in that, The solid content of the graphene sol in step (6) is 4-6‰; In step (6), the concentration of graphene sol added to the composite bacterial agent is 40~60 mg / kg.

9. A method for reclamation of waste rock hills, characterized in that, The method includes applying the compound microbial agent as described in claim 1 or 2 to the planting of vegetation in the reclamation of gangue hills; Preferably, the method includes the following steps: (S1) Pretreatment of the gangue mountain reclamation area: The gangue mountain accumulation is leveled, and gangue with a diameter >10cm and sharp debris are removed; a layer of topsoil with a thickness of 40~50cm is laid on the surface to improve the surface soil structure and water retention capacity; (S2) Vegetation establishment: Select reclaiming plants that are tolerant of poor soil and have strong resistance (such as European plum, forsythia, etc.), and the planting density of reclaiming plants is 400-500 plants / mu; (S3) Application of microbial agent: When planting, apply the compound microbial agent around the roots of the reclaimed plants using the hole application method. The application depth is 8-12cm, and the amount of microbial agent is 8-10 g / hole. After application, cover with soil and compact to avoid the microbial agent failing to come into contact with the plant roots. (S4) Post-planting management: Water thoroughly after sowing to keep the topsoil moist; water thoroughly within 24 hours after applying compound microbial agent to make the soil moisture content reach 60%~70% of field capacity; water once every 15 days thereafter to keep the soil moist; apply microbial agent once every 30 days for 2~3 times.

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