Microbial preparation containing arbuscular mycorrhizal fungi and application thereof

Abstract

The present application relates to a kind of microbial preparation containing arbuscular mycorrhizal fungi and its application, belong to the field of plant cultivation technology.The present application finds that the young set of arbuscular mycorrhizal fungi, endogone intraradices, moses glomus, branch giant spore cystomycetes can promote the growth of plants under the condition of double pollution of molybdenum and microplastic, effectively improve the plant height and grain yield of plants.Moreover, it is found through further experiment that under the same conditions, the effect of applying mixed microbial agent of young set of glomus, endogone intraradices and moses glomus or young set of glomus, moses glomus and branch giant spore cystomycetes is better than applying single above-mentioned arbuscular mycorrhizal fungi agent.Further, when adding beneficial bacteria and auxiliary materials in mixed microbial agent, its promoting effect on the growth of plants under the condition of double pollution of molybdenum and microplastic is better.The microbial preparation provided by the present application can reduce the toxicity of microplastic and molybdenum to plants, so that plants can grow normally under double pollution.

Description

Technical Field

[0001] This invention relates to a microbial preparation containing arbuscular mycorrhizal fungi and its application, belonging to the field of plant cultivation technology. Background Technology

[0002] Molybdenum is a transition element, an essential trace element for the growth of organisms including plants. However, excessive molybdenum intake can lead to poisoning. Although plants are generally resistant to molybdenum, high levels within plants can still be toxic. High concentrations of molybdenum reduce crop quality, while excessive molybdenum inhibits photosynthesis and transpiration, further disrupting reactive oxygen species metabolism and causing membrane lipid peroxidation, ultimately affecting normal plant growth. Studies have shown that excessive molybdenum can cause tomatoes and cabbage leaves to turn purple, and soybean leaves to turn yellow; it can inhibit the growth of the roots and above-ground parts of chickpeas, altering the cross-sectional structure of their roots, leaves, and stems; and it can reduce the chlorophyll content of winter wheat, leading to a decrease in biological yield. Furthermore, increasing research demonstrates that higher concentrations of molybdenum pollution (3000–4000 mg / kg) can cause direct crop death.

[0003] Microplastics (MPs) refer to plastic particles or fibers with a diameter of less than 5 millimeters. They are widely used in various fields, such as agricultural microplastic mulch films and decomposition products of plastic waste. Microplastics can adsorb onto the surface of heavy metals, both of which disrupt the ecological balance of soil, inhibit soil microbial activity and diversity, affect soil nutrient cycling and organic matter decomposition, and negatively impact plant growth (such as reducing crop yield and quality), seriously threatening ecological and environmental security. Therefore, improving the environmental pollution caused by molybdenum (a heavy metal) and microplastics, and enhancing crop resistance to molybdenum and microplastics, are of great significance for promoting crop growth in polluted soils, mitigating the toxicity of microplastics and molybdenum, and improving crop indicators such as plant height and yield.

[0004] Mycorrhizae are highly evolved mutualistic symbiotic relationships formed between soil fungi and the roots of terrestrial vascular plants. Fungi capable of forming this symbiotic relationship with plant roots are called mycorrhizal fungi. They possess characteristics of both general plant roots and obligate fungi, and are widely distributed in nature. Currently, seven types of mycorrhizae have been identified: arbuscular mycorrhizae, ectomycorrhizae, endomycorrhizae, orchid mycorrhizae, heather or rhododendron mycorrhizae, monotypic mycorrhizae, and berry mycorrhizae. Among these types, arbuscular mycorrhizae (AM) are the most widely distributed and most closely related to plants in terrestrial ecosystems. After infecting the root system, arbuscular mycorrhizal fungi enter the root cortex cells and form a dichotomous branching structure, thus becoming arbuscular mycorrhizae; this type of fungus is called arbuscular mycorrhizal fungi.

[0005] Arbuscular mycorrhizal (AM) fungi not only improve plant nutrition but also significantly enhance plant stress resistance. Furthermore, AM fungi can improve soil physicochemical properties, promote the absorption and utilization of water and nutrients by plants, and enhance plant resistance to heavy metals, thereby reducing the damage caused by heavy metal stress. Studies have shown that AM fungi can mitigate the adverse effects of microplastics on soil nutrient availability and plant nutrient absorption. AM fungal hyphae can capture microplastics and further transport them to the roots, and the hyphal secretions of AM fungi can promote microplastic aggregation, thereby reducing their absorption.

[0006] Chinese invention patent CN108419605B, with an authorization announcement date of August 20, 2019, discloses an arbuscular mycorrhizal fungal agent and its preparation method and application. Specifically, it discloses a preparation method for the arbuscular mycorrhizal fungal agent, and the prepared arbuscular mycorrhizal fungal agent can promote the growth of corn in molybdenum-contaminated soil.

[0007] In his 2022 publication, "The Combined Toxicity-Reducing Effect of Arbuscular Mycorrhizal Fungi and Earthworms on Soil Microplastic Pollution," He Baiping documented that arbuscular mycorrhizal (AM) fungi and earthworms play important roles in improving plant nutrition and enhancing plant stress resistance within soil ecosystems. Specifically, the paper disclosed the adverse effects of microplastics on soil AM fungi and pepper growth, while clarifying the positive impact of AM fungi and earthworms on reducing the plant biotoxicity of soil microplastic pollution. The synergistic effect of earthworms and AM fungi shows great potential in promoting pepper growth.

[0008] Although the aforementioned articles or Chinese invention patents disclose that arbuscular mycorrhizal fungi can improve plant yield and quality by improving plant nutrition and enhance crop resistance to abiotic stresses such as heavy metals and microplastics, none of them involve the use of arbuscular mycorrhizal fungi under conditions of dual stress from microplastics and molybdenum. There are also no reports or patents on the use of microbial preparations containing arbuscular mycorrhizal fungi to promote crop growth under conditions of dual pollution from microplastics and molybdenum. Summary of the Invention

[0009] The first objective of this invention is to provide a microbial preparation containing arbuscular mycorrhizal fungi, which provides a microbial preparation that can effectively promote plant growth under conditions of dual pollution by microplastics and molybdenum, thus providing an alternative to the prior art.

[0010] The second objective of this invention is to provide an application of a microbial agent in promoting plant growth and development in soils contaminated with both microplastics and molybdenum, in order to address the current problem of poor plant growth in environments contaminated with molybdenum and microplastics.

[0011] To achieve the above objectives, the technical solution of the microbial preparation containing arbuscular mycorrhizal fungi in this invention is as follows:

[0012] A microbial preparation containing arbuscular mycorrhizal fungi, the microbial preparation comprising an arbuscular mycorrhizal fungal agent; the arbuscular mycorrhizal fungal agent is *Gyrocephalus jugatus*, *Rhizocystis radicans*, and *Gyrocephalus moses*, or *Gyrocephalus jugatus*, *Gyrocephalus moses*, and *Gyrocephalus cladoides*; the propagation ratio of the *Gyrocephalus jugatus*, *Rhizocystis radicans*, and *Gyrocephalus moses* is (1–1.1):(0.7–0.9):(1.3–1.4); the propagation ratio of the *Gyrocephalus jugatus*, *Gyrocephalus moses*, and *Gyrocephalus cladoides* is (0.9–1.1):(1.2–1.3):(0.8–1.0).

[0013] The beneficial effects of the above technical solution are as follows: This invention provides a combined microbial preparation containing arbuscular mycorrhizal fungi. Extensive preliminary field experiments have shown that under conditions of dual pollution by molybdenum and microplastics, plant growth is significantly inhibited, affecting plant height and grain yield. Experiments have revealed that *Claroideoglomus etunicatum*, *Rhizophagus intraradices*, *Glomus mosseae*, and *Gigasporara misporophora* among the arbuscular mycorrhizal fungi can promote plant growth under conditions of dual pollution by molybdenum and microplastics, effectively increasing plant height and grain yield. Furthermore, further experiments have shown that, under the same conditions, applying a mixed inoculum of *Claroideoglomus etunicatum*, *Rhizophagus intraradices*, and *Glomus mosseae*, or a mixture of *Claroideoglomus etunicatum*, *Glomus mosseae*, and *Gigasporara misporophora*, is more effective than applying a single inoculum of any of the aforementioned arbuscular mycorrhizal fungi. This indicates that the above-mentioned combined microbial agents have a synergistic effect, which can effectively promote plant growth under conditions of dual pollution of molybdenum and microplastics, and improve plant height, yield and other indicators. This provides an effective microbial agent for increasing plant yield under conditions of dual pollution of molybdenum and microplastics, and also lays the foundation for subsequent research on reducing the toxicity of microplastics and molybdenum to plants.

[0014] Specifically, the preparation process of the arbuscular mycorrhizal fungal inoculant is as follows: prepare a mixed substrate for cultivation, inoculate the arbuscular mycorrhizal fungal inoculant into the mixed substrate, and spread it in the field to coexist with the plants for 2 to 3 months. After harvesting the plant roots, mix them with the mixed substrate to obtain a mixture composed of spores, mycelia, root fragments and the mixed substrate, which is the arbuscular mycorrhizal fungal inoculant.

[0015] As a further improvement, the number of propagules in the arbuscular mycorrhizal fungal inoculum is not less than 110 per gram.

[0016] As a further improvement, the propagule consists of spores, extra-radical hyphae, and infected root segments.

[0017] Specifically, theoretically, any one of the arbuscular mycorrhizal fungi spores, arbuscular mycorrhizal hyphae, and infected root segments can function as an arbuscular mycorrhizal fungi inoculant. However, in practical applications, for ease of preparation, the arbuscular mycorrhizal fungi spores, arbuscular mycorrhizal hyphae, and infected root segments are often mixed together with a mixed substrate to form an arbuscular mycorrhizal fungi inoculant.

[0018] As a further improvement, the aforementioned *Gymnospermum julibrissin*, *Gymnospermum radicans*, *Gymnospermum moss*, and *Gymnospermum branchii* agents meet the following standards: spore count not less than 6 per gram, extraroot hyphae density not less than 18 m / g, and infected root segments not less than 20 cm / g.

[0019] As a further improvement, the microbial preparation includes Bacillus subtilis, Trichoderma longifolia, polyglutamic acid, humic acid, and ammonium molybdate.

[0020] The beneficial effects of the above technical solution are as follows: through further experiments, the present invention has found that when arbuscular mycorrhizal fungi inoculant is mixed with the above-mentioned components and applied to plants, the effect is better than that of a single arbuscular mycorrhizal fungi inoculant.

[0021] As a further improvement, the mass ratio of the arbuscular mycorrhizal fungal agent, Bacillus subtilis, Trichoderma longifolia, polyglutamic acid, humic acid and ammonium molybdate is 85:(4-5):(4-5):(2-3):(2-3):(0.04-0.05).

[0022] As a further improvement, the effective viable count of the Bacillus subtilis is not less than 4 × 10⁻⁶. 8 CFU / g, the effective viable count of *Trichoderma longifolia* is not less than 8 × 10⁻⁶. 8 CFU / g.

[0023] To achieve the above objectives, the technical solution of this invention for the application of a microbial agent in promoting plant growth and development in soils contaminated with both microplastics and molybdenum is as follows:

[0024] Application of a microbial agent in promoting plant growth and development in soils contaminated with both microplastics and molybdenum.

[0025] The beneficial effects of the above technical solution are as follows: This invention utilizes microbial agents containing arbuscular mycorrhizal fungi to cultivate crops in soils contaminated with microplastics and molybdenum, which can significantly improve plant height and yield, promote plant growth, reduce the toxicity of microplastics and molybdenum to plants, and enable crops to grow normally in soils under the dual stress of microplastics and molybdenum.

[0026] As a further improvement, the growth and development refers to plant height, yield, and / or chlorophyll content.

[0027] As a further improvement, the plants include wheat, mung beans, and corn.

[0028] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0029] (1) This invention utilizes the symbiotic relationship between arbuscular mycorrhizal fungi and host plants, and uses the hyphae of arbuscular mycorrhizal fungi to assist plants in obtaining nutrients, thereby improving the growth of crops in soils contaminated with microplastics and molybdenum.

[0030] (2) The mixed arbuscular mycorrhizal fungi agent used in this invention has high activity, which can effectively regulate soil structure, improve soil water and fertilizer retention capacity, promote crop growth in polluted soil, alleviate the toxicity caused by microplastics and molybdenum, improve plant height, yield and other indicators, promote plant growth, and achieve the beneficial effect of promoting crop growth.

[0031] (3) The present invention adds Bacillus subtilis, Trichoderma longifolia, polyglutamic acid, humic acid and ammonium molybdate to the microbial preparation. Bacillus subtilis and Trichoderma longifolia promote seed germination, improve seed disease resistance, improve fertilizer utilization, and enhance seed resistance to soil-borne diseases and soil harmful bacteria. Adding polyglutamic acid can effectively retain soil moisture, improve soil structure and aeration, and improve the soil's water and fertilizer retention capacity. Humic acid has the effect of improving soil structure, improving fertilizer efficiency, and improving crop stress resistance. Adding ammonium molybdate can not only promote the absorption of phosphorus by crops, but also improve the ability of arbuscular mycorrhizal fungi to cope with molybdenum stress from the source.

[0032] (4) The preparation process of the microbial preparation of the present invention is simple and easy to carry out, environmentally friendly and harmless, and has no side effects. Attached Figure Description

[0033] Figure 1 The graphs show the plant height, seed phosphorus content, and seed yield of mung beans under microplastic and molybdenum contamination conditions, with and without the application of a microbial preparation containing arbuscular mycorrhizal fungi (different letters indicate significant differences, while the same letter indicates no significant difference; AM1 is a microbial preparation containing a mixture of arbuscular mycorrhizal fungi including *Gyrodactylus juuta*, *Gyrodactylus radiata*, and *Gyrodactylus moses*; AM2 is a microbial preparation containing a mixture of arbuscular mycorrhizal fungi including *Gyrodactylus juuta*, *Gyrodactylus moses*, and *Gyrodactylus cladosporioides*).

[0034] Figure 2 The graphs show the maize plant height, kernel P content, and kernel yield under microplastic and molybdenum contamination conditions with and without the application of a microbial preparation containing arbuscular mycorrhizal fungi (AM1 is a microbial preparation containing a mixture of arbuscular mycorrhizal fungi, Rhizocystis radicans, and Rhizocystis moses; AM2 is a microbial preparation containing a mixture of arbuscular mycorrhizal fungi, Rhizocystis moses, and Rhizocystis radicans).

[0035] Figure 3The graphs show wheat plant height, grain phosphorus content, and grain yield under microplastic and molybdenum contamination conditions with and without the application of a microbial preparation containing arbuscular mycorrhizal fungi (AM1 is a microbial preparation containing a mixture of *Gyrodactylus juga*, *Gyrodactylus radicans*, and *Gyrodactylus moses*; AM2 is a microbial preparation containing a mixture of *Gyrodactylus juga*, *Gyrodactylus moses*, and *Gyrodactylus cladosporioides*).

[0036] Figure 4 The graph shows the maize yield in Example 7 of this invention under microplastic and molybdenum pollution conditions, with and without the application of a microbial preparation containing arbuscular mycorrhizal fungi (different letters indicate significant differences, and the same letter indicates no significant difference; AM1 is a microbial preparation containing a mixture of arbuscular mycorrhizal fungi including *Gyrodactylus juvenilee*, *Gyrodactylus radiata*, and *Gyrodactylus moses*; AM2 is a microbial preparation containing a mixture of arbuscular mycorrhizal fungi including *Gyrodactylus juvenilee*, *Gyrodactylus moses*, and *Gyrodactylus cladosporioides*). Detailed Implementation

[0037] Microplastics and molybdenum contamination in soil inhibit plant root development and nutrient absorption, thus affecting the overall growth of the plant. Furthermore, microplastics can adsorb onto the surface of heavy metals (molybdenum), further exacerbating the impact on soil quality, leading to reduced crop yields and quality, and threatening ecological security. Arbuscular mycorrhizal fungi, on the other hand, can form mutually beneficial symbiotic systems with most terrestrial plants, playing a vital role in improving plant nutrition, maintaining soil health, and ensuring ecosystem stability. Culture media containing arbuscular mycorrhizal fungi are highly permeable and water-retentive, meeting the crop's requirements for water, fertilizer, and oxygen.

[0038] This invention first provides a microbial preparation containing arbuscular mycorrhizal fungi. Utilizing the synergistic effects of *Gyrocephalus juutauriculatus*, *Rhizocarpus endocarpus*, and *Gyrocephalus moses*, or *Gyrocephalus juutauriculatus*, *Gyrocephalus moses*, and *Gyrocephalus branchi*, it can better improve crop growth in soils contaminated with microplastics and molybdenum, increasing plant height and grain yield. This microbial preparation provides a fertilizer for soils dually contaminated with microplastics and molybdenum that can improve crop yield, laying the foundation for subsequent fertilizer research and development.

[0039] Furthermore, this invention provides the application of microbial preparations containing arbuscular mycorrhizal fungi in promoting plant growth and development in soils contaminated with both microplastics and molybdenum. This invention demonstrates in advance that applying the microbial preparations of this invention to soils contaminated with both molybdenum and microplastics can effectively improve the nutritional status and yield of crops, while being environmentally friendly and enabling sustainable development.

[0040] The present invention will be further described in detail below with reference to specific embodiments. Unless otherwise specified, the equipment and reagents used in the embodiments, experimental examples and comparative examples are all commercially available.

[0041] In the following embodiments of the present invention, the characteristics of the fungal strains *Claroideoglomus etunicatum*, *Rhizophagus intraradices*, *Glomus mosseae*, and *Gigaspora ramisporophora* are utilized. The above-mentioned strains obtained through conventional methods that meet the requirements (e.g., number of propagules) in the embodiments can achieve the same or similar effects as those in the embodiments. Ammonium molybdate, microplastics, *Bacillus subtilis*, *Trichoderma longifolia*, polyglutamic acid, humic acid, and crop seeds were all commercially available.

[0042] I. Specific embodiments of a microbial preparation containing arbuscular mycorrhizal fungi according to the present invention:

[0043] Example 1: Preparation of Arbuscular Mycorrhizal Fungal Inoculant

[0044] The preparation process of the arbuscular mycorrhizal fungal inoculant in this embodiment is as follows:

[0045] Ceramic granules and bagasse were crushed and passed through a 2mm sieve. The granules and bagasse were then mixed evenly at a mass ratio of 3:1 to obtain a mixed substrate. The mixed substrate was sterilized at 121℃ for 2 hours, cooled, and air-dried for later use. A mixed soil sample containing *Gloydium juvenilee* fungal spores, mycelia, and infected root segments was used as an inoculum for *Gloydium juvenilee*. The inoculum was inoculated into the mixed substrate and mixed thoroughly. The substrate was spread to a thickness of 10–20 cm, and watered to 50–70% of field capacity. Corn seeds were sown, and the surface was covered with 1–2 cm of mixed substrate. The plants were cultured in an artificial climate chamber for 2–3 months, irrigated with Hoagland nutrient solution every 2 weeks during the growth process. The culture conditions in the artificial climate chamber were: 8000–12000 lux of light daily, 28–35℃ for 13–14 hours of temperature, and 18–25℃ for 10–11 hours of darkness daily. Then harvest and cut off the above-ground parts of the corn, and cut its roots into pieces less than 0.5cm. Mix them thoroughly with the mixed substrate to obtain a mixture (soil) consisting of spores, mycelium, root fragments and mixed substrate, which is the young Gloydius glomeratus fungicide.

[0046] Note: The mass of spores, mycelia, and root fragments is negligible compared to the mixed substrate.

[0047] The methods for preparing *Gymnospermum moses*, *Rhizocystis radicans*, and *Gymnospermum clavatum* are similar to those for preparing the aforementioned *Gymnospermum juvenileum*, except that *Gymnospermum juvenileum* is replaced with *Gymnospermum moses*, *Rhizocystis radicans*, or *Gymnospermum clavatum*.

[0048] Example 2: Screening of arbuscular mycorrhizal fungal inoculants

[0049] The screening process for arbuscular mycorrhizal fungal inoculants in this embodiment is as follows:

[0050] The *Arbuscular mycorrhizal* fungal agents prepared in Example 1 (a), (b), (c), and (d) were added to soil containing ammonium molybdate (100 mg / kg) and polyvinyl chloride (PVC) (1.2% w / w) and mixed thoroughly. The added fungal agents must meet the following two criteria simultaneously: (1) the total number of propagules of a single agent is not less than 60 per g of soil; (2) the number of spores of a single agent is not less than 6 per g of soil, the mycelial density is not less than 18 m / g of soil, and the number of infected root segments is not less than 20 cm / g of soil. At the same time, a treatment group was set up with a mixed fungal agent in which the three fungal agents were uniformly mixed at a mass ratio of 1:1:1, and a control group was set up without the addition of arbuscular mycorrhizal fungal agents.

[0051] Pot experiments were conducted using 1 kg of mixed soil inoculated with 10 wt% microbial agent (containing ammonium molybdate (100 mg / kg) and polyvinyl chloride (1.2% w / w)) in each pot, with 4 replicates. Six wheat seeds were sown in each pot, and after one week of germination, the seedlings were thinned to two plants per pot. The pots were then placed in a greenhouse for cultivation, with the following conditions: 14 hours of cultivation per day under 10,000 lux light and 30°C, followed by 10 hours of cultivation per day under 20°C darkness. Watering and other treatments were administered as needed during the growth process to ensure normal plant growth. After harvest, plant height, yield, and other indicators were measured. Specific experimental groups and results are shown in Table 1.

[0052] Table 1. Wheat plant height and grain yield (average values)

[0053]

[0054] Table 1 shows that the treatments with fungal agents all exhibited better plant growth than the control group without fungal agents. The effects of *Gyromycorrhiza juba*, *Gyromycorrhiza moses*, and *Rhizomycorrhiza radiata* agents were better than those of *Macromycorrhiza cladoides*. Furthermore, the effects of mixed fungal agents were superior to those of single fungal agents, and the effects of mixed fungal agents of *Gyromycorrhiza juba*, *Rhizomycorrhiza radiata*, and *Gyromycorrhiza moses*, as well as mixed fungal agents of *Gyromycorrhiza juba*, *Gyromycorrhiza moses*, and *Macromycorrhiza cladoides*, were significantly better than the effects of applying any of the four single fungal agents individually. Among these, the mixed fungal agent of *Gyromycorrhiza juba*, *Rhizomycorrhiza radiata*, and *Gyromycorrhiza moses* showed the best effect. Table 2 shows the ratio of the number of propagules of each arbuscular mycorrhizal fungus in the mixed fungal agents of experimental groups E–H. Based on previous experimental verification according to this invention, the infection capacity of 0.5 m mycelium is equivalent to one spore, and the infection capacity of 1 cm infected root segment is also equivalent to one spore.

[0055] The formula for calculating the total number of propagating bodies is: Total number of propagating bodies = Σ number of spores + 2 × mycelial density + infected root segments.

[0056] The number of spores, extracellular hyphae, infected root segments, and total number of propagules in each treatment group are shown in Table 3.

[0057] Table 2. Ratio of reproductive numbers

[0058]

[0059]

[0060] In other implementation scenarios, the ratio of propagules in *Gymnocarpus jujuba*, *Gymnocarpus endocarpus*, and *Gymnocarpus mosierifolius* agents was (1–1.1):(0.7–0.9):(1.3–1.4), achieving the same or similar experimental results as test group E; the ratio of propagules in *Gymnocarpus jujuba*, *Gymnocarpus mosierifolius*, and *Gymnocarpus cladoides* agents was (0.9–1.1):

[0061] (1.2~1.3):(0.8~1.0) can all achieve the same or similar experimental results as the experimental group G.

[0062] Table 3 Total number of reproductive bodies

[0063]

[0064] Example 3: Preparation of microbial preparations containing arbuscular mycorrhizal fungi

[0065] The preparation process of the microbial preparation containing arbuscular mycorrhizal fungi in this embodiment is as follows:

[0066] Based on the mixed arbuscular mycorrhizal fungal inoculant that showed better results in Example 2 (Experimental Group E and Experimental Group G), treatments were implemented with the addition of beneficial bacteria and synergists. The formulations for each experimental group are as follows (by mass ratio):

[0067] Experimental Group I: The mixed arbuscular mycorrhizal fungal inoculant of Experimental Group E contains 85% Bacillus subtilis, 5% Trichoderma longifolia, 2% polyglutamic acid, 2% humic acid, and 0.04% ammonium molybdate.

[0068] Experimental group J: The mixed arbuscular mycorrhizal fungal inoculant of experimental group E contains 85% Bacillus licheniformis, 5% Trichoderma longifolia, 2% potassium humate, 2% humic acid, and 0.04% ammonium molybdate.

[0069] Experimental group K: The mixed arbuscular mycorrhizal fungal inoculant of experimental group E contains 85% Bacillus licheniformis, 5% Bacillus megaterium, 2% potassium humate, 2% alginate, and 0.04% ammonium molybdate.

[0070] Experimental group L: The mixture of arbuscular mycorrhizal fungi inoculant of experimental group G contains 85% Bacillus subtilis, 5% Trichoderma longifolia, 2% polyglutamic acid, 2% humic acid, and 0.04% ammonium molybdate.

[0071] Experimental group M: The mixture of arbuscular mycorrhizal fungi inoculant of experimental group G contains 85% Bacillus licheniformis, 5% Trichoderma longifolia, 2% potassium humate, 2% humic acid, and 0.04% ammonium molybdate.

[0072] Experimental group N: The mixture of experimental group G and arbuscular mycorrhizal fungi inoculum consisted of 85% Bacillus licheniformis, 5% Bacillus megaterium, 2% potassium humate, 2% alginate, and 0.04% ammonium molybdate.

[0073] Note: Bacillus subtilis is a liquid inoculum, with an effective viable count concentration of 4 × 10⁻⁶. 8 ~4×10 9 CFU / g; *Trichoderma longifolia* is a solid powder inoculum agent with an effective viable count concentration of 3 × 10⁻⁶. 8 ~3×10 9 CFU / g; Bacillus licheniformis and Bacillus megaterium are solid powder inoculants, with an effective viable count concentration of 2×10⁻⁶ for Bacillus licheniformis. 9 ~2×10 10 CFU / g, the effective viable count concentration of Bacillus megater is 2×10⁻⁶. 8 ~5×10 8 CFU / g.

[0074] Pot experiments were conducted using a mixture of 1 kg of ammonium molybdate (100 mg / kg), polyvinyl chloride (1.2% w / w), and 10 wt% of the aforementioned microbial preparation, with four replicates. Six wheat seeds were sown in each pot, and after one week of germination, the seedlings were thinned to two plants per pot. The pots were then placed in a greenhouse for cultivation, with the following conditions: 14 hours of cultivation per day under 10,000 lux light and 30°C, followed by 10 hours of cultivation per day under 20°C darkness. Watering and other treatments were administered as needed during the growth process to ensure normal plant growth. After harvest, plant height, yield, and other indicators were measured. Specific experimental results are shown in Table 4.

[0075] Table 4 Wheat plant height and yield

[0076]

[0077] As shown in Table 4, the wheat growth effect of treatments with Bacillus subtilis, Trichoderma longifolia, polyglutamic acid, humic acid, and ammonium molybdate added to the arbuscular mycorrhizal fungi inoculant was better than that of treatments with only the arbuscular mycorrhizal fungi inoculant and treatments with other beneficial bacteria and synergists.

[0078] The correlation analysis results of yield are shown in Table 5. The results indicate that the effect of the mixed inoculant of *Gyrocephalus judoides*, *Gyrocephalus moses*, and *Rhizocystis radicans* is most strongly correlated with *Gyrocephalus judoides* and *Gyrocephalus moses*, and the treatment with the addition of beneficial bacteria and excipients shows the best correlation with this mixed inoculant. The effect of the mixed inoculant of *Gyrocephalus judoides*, *Gyrocephalus moses*, and *Rhizocystis radicans* is most strongly correlated with *Gyrocephalus moses*, and the treatment with the addition of beneficial bacteria and excipients shows the best correlation with this mixed inoculant.

[0079] Table 5 Correlation Analysis

[0080]

[0081] II. Specific Examples of the Application of a Microbial Agent in Promoting Plant Growth and Development in Soils Dually Contaminated with Microplastics and Molybdenum:

[0082] Example 4: Application of microbial agents in soil contaminated with microplastics and molybdenum 1

[0083] In this embodiment, the *Gyrocephalomyces juba*, *Gyrocephalomyces radicans*, and *Gyrocephalomyces moses* preparations from Example 1, as well as the *Gyrocephalomyces juba*, *Gyrocephalomyces moses*, and *Gyrocephalomyces cladomyces* preparations, were all mixed uniformly at a mass ratio of 1:1:1 to obtain their respective mixed arbuscular mycorrhizal fungal inoculants. The formulations containing 85% arbuscular mycorrhizal fungal inoculant, 5% *Bacillus subtilis*, 5% *Trichoderma longifolia*, 2% polyglutamic acid, 2% humic acid, and 0.04% ammonium molybdate were uniformly weighed and mixed to obtain microbial preparations containing arbuscular mycorrhizal fungi: AM1 (experimental group I) and AM2 (experimental group L). AM1 contained a total of 122 propagules, as shown in Table 3, Experimental Group E; AM2 contained a total of 121 propagules, as shown in Table 3, Experimental Group G.

[0084] Ammonium molybdate and polyvinyl chloride (PVC) were added to farmland soil that had passed through a 2mm sieve. Then, a microbial preparation containing arbuscular mycorrhizal fungi was added at an inoculum rate of 10 wt% to the soil mixed with ammonium molybdate (100 mg / kg) and PVC (1.2% w / w). A treatment without the arbuscular mycorrhizal fungi preparation was also included. Pot experiments were conducted with 1 kg of the mixed soil per pot, with four replicates. Six mung bean seeds were sown in each pot, and after one week of germination, the seedlings were thinned to two plants per pot. The plants were then placed in a greenhouse for cultivation, with the following conditions: 14 hours of light at 10,000 lux and 30°C, followed by 10 hours of darkness at 20°C. Watering and other treatments were administered as needed during the growth process to ensure normal mung bean growth. After harvest, plant height, yield, and chlorophyll content were measured.

[0085] Specific results are as follows Figure 1 As shown in the figure, under the conditions of mixed pollution of microplastics and molybdenum, the plant height of mung beans inoculated with arbuscular mycorrhizal fungi was 23.2 cm, while the plant height of uninoculated mung beans was 11.8 cm. Inoculation with arbuscular mycorrhizal fungi also increased the phosphorus content and grain yield of mung beans by 1.1 and 1.5 times, respectively.

[0086] Example 5: Application of microbial agents in microplastic and molybdenum contaminated soil 2

[0087] In this embodiment, the *Gyrocephalomycetes juvenile* fungal agents, *Rhizocarpus endophyticus* fungal agents, and *Gyrocephalomycetes moses* fungal agents prepared in Example 1, as well as the *Gyrocephalomycetes juvenile* fungal agents, *Gyrocephalomycetes moses* fungal agents, and *Gyrocephalomycetes cladomycetes* fungal agents, were all mixed at a mass ratio of 1:1:1 to obtain their respective mixed arbuscular mycorrhizal fungal agents. The mixed arbuscular mycorrhizal fungal agents, comprising 85% *Bacillus subtilis*, 5% *Trichoderma longifolia*, 2% polyglutamic acid, 2% humic acid, and 0.04% ammonium molybdate, were weighed and mixed evenly to obtain microbial preparations containing arbuscular mycorrhizal fungi, AM1 (experimental group I) and AM2 (experimental group L). AM1 contained a total of 122 propagules, as shown in Table 3, Experimental Group E; AM2 contained a total of 121 propagules, as shown in Table 3, Experimental Group G.

[0088] Polyvinyl chloride (PVC) was added to farmland soil that had passed through a 2mm sieve. Then, a microbial preparation containing arbuscular mycorrhizal fungi was added at a 10wt% inoculation rate to soil mixed with ammonium molybdate (100 mg / kg) and PVC (1.2% w / w). A control group without the arbuscular mycorrhizal fungi preparation was also included. Pot experiments were conducted with 1 kg of the mixed soil per pot, with four replicates. Six corn seeds were sown in each pot, and after one week of germination, the seedlings were thinned to two plants per pot. The pots were then placed in a greenhouse for cultivation, with the following conditions: 14 hours of light at 10,000 lux and 30°C, followed by 10 hours of darkness at 20°C. Water and other treatments were administered as needed during the growth process to ensure normal corn growth. After harvest, plant height, yield, and chlorophyll content were measured.

[0089] Specific results are as follows Figure 2 As shown in the figure, for potted maize, inoculation with microbial agents containing arbuscular mycorrhizal fungi increased maize plant height, chlorophyll content, and yield by 1.2, 1.5, and 2.2 times, respectively.

[0090] Example 6: Application of microbial agents in soil contaminated with microplastics and molybdenum 3

[0091] In this embodiment, the *Gyrocephalomycetes juvenile* fungal agents, *Rhizocarpus endophyticus* fungal agents, and *Gyrocephalomycetes moses* fungal agents prepared in Example 1, as well as the *Gyrocephalomycetes juvenile* fungal agents, *Gyrocephalomycetes moses* fungal agents, and *Gyrocephalomycetes cladomycetes* fungal agents, were all mixed at a mass ratio of 1:1:1 to obtain their respective mixed arbuscular mycorrhizal fungal agents. The mixed arbuscular mycorrhizal fungal agents, comprising 85% *Bacillus subtilis*, 5% *Trichoderma longifolia*, 2% polyglutamic acid, 2% humic acid, and 0.04% ammonium molybdate, were weighed and mixed evenly to obtain microbial preparations containing arbuscular mycorrhizal fungi, AM1 (experimental group I) and AM2 (experimental group L). AM1 contained a total of 122 propagules, as shown in Table 3, Experimental Group E; AM2 contained a total of 121 propagules, as shown in Table 3, Experimental Group G.

[0092] Ammonium molybdate was added to farmland soil that had passed through a 2mm sieve. Then, arbuscular mycorrhizal fungi (AMF) microbial preparation was added at a 10wt% inoculation rate to the soil mixed with ammonium molybdate (100 mg / kg) and polyvinyl chloride (1.2% w / w). A treatment without the AMF microbial preparation was also included. Pot experiments were conducted with 1 kg of the mixed soil per pot, with four replicates. Six wheat seeds were sown in each pot, and thinned to one seedling per week after emergence. The plants were then placed in a greenhouse for cultivation, maintaining the following conditions: 14 hours of light at 10,000 lux and 30°C daily, followed by 10 hours of darkness at 20°C. Water and other treatments were administered as needed during growth to ensure normal wheat growth. After harvest, plant height, yield, and chlorophyll content were measured.

[0093] Specific results are as follows Figure 3 As shown in the figure, for potted wheat, inoculation with microbial agents containing arbuscular mycorrhizal fungi increased wheat plant height, chlorophyll content, and grain yield by 1.4, 1.3, and 1.4 times, respectively.

[0094] Example 7 Field Experiment

[0095] In this embodiment, the *Gyrocephalomycetes juvenile* fungal agents, *Rhizocarpus endophyticus* fungal agents, and *Gyrocephalomycetes moses* fungal agents prepared in Example 1, as well as the *Gyrocephalomycetes juvenile* fungal agents, *Gyrocephalomycetes moses* fungal agents, and *Gyrocephalomycetes cladomycetes* fungal agents, were all mixed at a mass ratio of 1:1:1 to obtain their respective mixed arbuscular mycorrhizal fungal agents. The mixed arbuscular mycorrhizal fungal agents, comprising 85% *Bacillus subtilis*, 5% *Trichoderma longifolia*, 2% polyglutamic acid, 2% humic acid, and 0.04% ammonium molybdate, were weighed and mixed evenly to obtain microbial preparations containing arbuscular mycorrhizal fungi, AM1 (experimental group I) and AM2 (experimental group L). AM1 contained a total of 122 propagules, as shown in Table 3, Experimental Group E; AM2 contained a total of 121 propagules, as shown in Table 3, Experimental Group G.

[0096] Field experiments were conducted in farmland surrounding the molybdenum mining area in Luanchuan County, Luoyang City, where agricultural plastic film had been used for a long time. The molybdenum content was 360.84 mg / kg, and the microplastic abundance was 894 particles / kg. The experimental site was randomly divided into 6 experimental plots, each with an area of ​​40 m². 2(5m × 8m) Two treatments were set up with three replicates. Ten rows were planted per plot, with a row spacing of 0.5m and a row length of 8m. The plant spacing was 20cm, with 40 plants per row, resulting in a total density of 400 plants per plot. A 2m walkway was left between plots. Five corn seeds were sown per hole. For the inoculation treatment, 100g of a microbial preparation containing arbuscular mycorrhizal fungi was applied to each hole, covering the seeds and then covered with soil. For the non-inoculation treatment, soil was simply covered. Thinning was performed when the corn seedlings reached 10cm in height, leaving one seedling per hole. Field management measures such as weeding, pest control, and watering were implemented during the corn's growth period. Harvesting and performance indicators were measured at maturity.

[0097] Specific results are as follows Figure 4 As shown in the figure, inoculation with a microbial preparation containing arbuscular mycorrhizal fungi in field experiments increased maize yield by 1.1 times.

[0098] The above description is merely an illustrative embodiment of the present invention and is not intended to limit the scope of the invention. Any equivalent changes and modifications made by those skilled in the art without departing from the concept and principles of the present invention should fall within the scope of protection of the present invention.

Claims

1. A microbial preparation containing arbuscular mycorrhizal fungi, characterized in that: The microbial preparation includes an arbuscular mycorrhizal fungal agent; the arbuscular mycorrhizal fungal agent is *Gyrocephalus juga*, *Rhizocystis moses*, or *Gyrocephalus juga*, *Gyrocephalus moses*, and *Gyrocephalus cladoides*; the ratio of the number of propagules of *Gyrocephalus juga*, *Rhizocystis moses*, and *Gyrocephalus moses* is (1~1.1):(0.7~0.9):(1.3~1.4); the ratio of the number of propagules of *Gyrocephalus juga*, *Gyrocephalus moses*, and *Gyrocephalus cladoides* is (0.9~1.1):(1.2~1.3):(0.8~1.0); the propagules consist of spores, extraroot hyphae, and infected root segments.

2. The microbial preparation containing arbuscular mycorrhizal fungi according to claim 1, characterized in that: The number of vegetative cells in the arbuscular mycorrhizal fungal inoculum is not less than 110 per gram.

3. The microbial preparation containing arbuscular mycorrhizal fungi according to claim 1, characterized in that: The aforementioned *Gymnospermum julibrissin*, *Gymnospermum radicans*, *Gymnospermum moss*, and *Gymnospermum branchii* agents meet the following standards: spore count not less than 6 per gram, extraroot hyphae density not less than 18 m / g, and infected root segments not less than 20 cm / g.

4. The microbial preparation containing arbuscular mycorrhizal fungi according to claim 3, characterized in that: The microbial preparation includes Bacillus subtilis, Trichoderma longifolia, polyglutamic acid, humic acid, and ammonium molybdate.

5. The microbial preparation containing arbuscular mycorrhizal fungi according to claim 4, characterized in that: The mass ratio of the arbuscular mycorrhizal fungal agent, Bacillus subtilis, Trichoderma longifolia, polyglutamic acid, humic acid and ammonium molybdate is 85:(4~5):(4~5):(2~3):(2~3):(0.04~0.05).

6. The microbial preparation containing arbuscular mycorrhizal fungi according to claim 5, characterized in that: The effective viable count of the Bacillus subtilis is not less than 4 × 10⁻⁶. 8 CFU / g, the effective viable count of *Trichoderma longifolia* is not less than 8 × 10⁻⁶. 8 CFU / g.

7. The application of a microbial preparation as described in any one of claims 1 to 6 in promoting plant growth and development in soils contaminated with both microplastics and molybdenum, characterized in that: The plants mentioned are wheat, mung beans, and corn.

8. The application of the microbial agent according to claim 7 in promoting plant growth and development in soils contaminated with both microplastics and molybdenum, characterized in that: The growth and development are defined as plant height, yield, and / or chlorophyll content.

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