A complex microbial agent containing bacillus thuringiensis and a preparation method and application thereof

Abstract

This application relates to the field of biological control, specifically disclosing a compound bacterial agent containing Bacillus thuringiensis, its preparation method, and its application. A method involves fermenting Bacillus thuringiensis HP-SYJ2 to a concentration of (3-8) × 10⁻⁶. 9 A Bacillus thuringiensis HP-SYJ2 bacterial suspension was obtained by mixing cfu / ml with hydroxyapatite and biochar, then mixing the mixture with the Bacillus thuringiensis HP-SYJ2 bacterial suspension, soaking, and air-drying until the moisture content is 30-40%, followed by maturation for 1-2 weeks to obtain the loading material. Fenugreek seed oil, emulsifier, soil penetrant, and water were mixed and sheared to obtain an emulsion with a fenugreek seed oil concentration of 0.1-0.5 wt%. The loading material and emulsion were packaged separately and mixed before application. This compound microbial agent can be used for the prevention and treatment of root rot in medicinal herbs or to promote their growth. It has the advantages of both preventing and treating root rot in medicinal herbs and promoting their growth.

Description

Technical Field

[0001] This application relates to the field of biological control technology, and more specifically, to a compound bacterial agent containing Bacillus thuringiensis, its preparation method, and its application. Background Technology

[0002] Root rot, often called "plant cancer," is a typical soil-borne disease caused by the combined infection of multiple pathogenic fungi, including Fusarium and Alternaria. It is characterized by its insidious onset, rapid spread, and severe damage, leading to root rot, plant wilting, and even large-scale death of medicinal herbs, resulting in significant economic losses. For a long time, the control of this disease has primarily relied on chemical agents such as propiconazole and difenoconazole. While these agents can quickly and effectively control plant diseases, long-term improper use can lead to increased pathogen resistance, reduced soil microbial diversity, and excessive pesticide residues, ultimately impacting human health, animal safety, and the balance of the ecological environment.

[0003] In recent years, research on the biological control of traditional Chinese medicinal herbs using biocontrol strains has been reported. For example, Bacillus subtilis, arbuscular mycorrhizal fungi, and Trichoderma harzianum can effectively control root rot in traditional Chinese medicinal herbs. However, in actual large-scale cultivation and application of traditional Chinese medicinal herbs, existing biological control methods still have significant limitations and challenges, resulting in poor overall treatment efficacy for root rot, insufficient stability and broad spectrum. Specific problems include: single and unstable control efficacy, weak field colonization and competitive ability, and lack of synergistic and enhanced composite systems.

[0004] Bacillus thuringiensis has long been known for its excellent insecticidal activity. Recent studies have found that certain strains also produce abundant antimicrobial metabolites, exhibiting good inhibitory activity against various plant pathogenic fungi, and possessing good soil adaptability and rhizosphere colonization potential. However, its systematic application in the prevention and control of root rot in traditional Chinese medicinal herbs, particularly as a core component scientifically formulated with other complementary beneficial components to construct a multi-mechanism synergistic compound microbial agent that prevents disease and promotes growth, in order to systematically address the problems of poor efficacy and instability in existing technologies, has yet to be fully researched and successfully reported. Summary of the Invention

[0005] In order to achieve a more efficient, stable and lasting effect in preventing and controlling root rot of Chinese medicinal herbs, while promoting plant growth, this application provides a compound bacterial agent containing Bacillus thuringiensis, its preparation method and application.

[0006] In a first aspect, this application provides a method for preparing a compound bacterial agent containing Bacillus thuringiensis, using the following technical solution: A method for preparing a compound bacterial agent containing Bacillus thuringiensis includes the following steps: Bacillus thuringiensis HP-SYJ2 was fermented to a concentration of (3-8)×10. 9 CFU / ml was used to obtain Bacillus thuringiensis HP-SYJ2 bacterial suspension; Hydroxyapatite and biochar were mixed evenly, then mixed with Bacillus thuringiensis HP-SYJ2 bacterial solution, soaked, and air-dried until the moisture content was 30-40%. The mixture was then matured for 1-2 weeks to obtain the loading material. Fenugreek seed oil, emulsifier, soil permeable agent, and water are mixed and sheared to emulsify, thus obtaining an emulsion with a fenugreek seed oil concentration of 0.1-0.5 wt%. The loading material and emulsion are packaged separately and mixed before application.

[0007] By employing the above-mentioned technical solutions, fenugreek seed oil contains active ingredients such as diosgenin and alkaloids. When it comes into contact with pathogens causing root rot (such as Fusarium and Pythium), it can directly destroy the mycelium or spore cell membrane, playing a local bactericidal or bacteriostatic role. Moreover, carrot seed oil is rich in unsaturated fatty acids, which are important signaling molecules in plants. When absorbed or sensed by the roots, they can activate the plant's systemic resistance, causing it to produce more defensive compounds, thereby enhancing the plant's overall resistance to root rot. Through the low concentration of carrot seed oil, combined with the action of emulsifiers, it forms an extremely thin protective film on the root surface and in the soil around the roots, changing the root surface microenvironment and blocking the colonization of pathogens. The low concentration of fenugreek seed oil also avoids its inactivation effect on Bacillus thuringiensis HP-SYJ2.

[0008] Bacillus thuringiensis (Bt) exhibits excellent inhibitory activity against various plant pathogenic fungi. It secretes a variety of antibacterial metabolites, including lipopeptides (such as surfactants and fenbacin), chitinases, proteases, and β-glucanases. These substances can directly destroy or dissolve the cell walls and cell membranes of pathogenic fungi (such as Fusarium and Alternaria), inhibiting spore germination and mycelial growth. As a beneficial rhizosphere bacterium, it can rapidly seize living space and nutrient resources in the roots, displacing pathogens and forming a "biological protective barrier." It can activate the plant's natural defense system, enabling the plant to produce its own defensive substances, becoming stronger and more vigilant, thus resisting pathogen invasion. It has good soil adaptability and rhizosphere colonization potential, promoting root development, healthy plant growth, and increased disease resistance.

[0009] Biochar has a huge specific surface area and abundant pore structure, and hydroxyapatite is also a porous material. It can adsorb the spores of Bacillus thuringiensis HP-SYJ2 inside the pores or on the surface, playing a physical barrier role. This partially prevents the active ingredients in the oil from directly contacting and damaging the Bacillus thuringiensis HP-SYJ2 cells. Moreover, the survival time, colonization ability and slow release effect of Bacillus thuringiensis HP-SYJ2 loaded on biochar and hydroxyapatite are improved in the soil.

[0010] In addition, biochar and hydroxyapatite can improve soil permeability, thus acting as a framework and channel in the soil, breaking down the continuous barrier formed by fenugreek seed oil, creating pathways for air and water, fundamentally avoiding the serious air closure problem caused by simply drenching fenugreek seed oil, and preventing soil compaction after drenching.

[0011] During preparation, zinc-loaded hydroxyapatite and iron-modified biochar are first used to load Bacillus thuringiensis, which acts as a physical carrier to protect the bacteria and reduce the risk of fenugreek seed oil killing the bacteria. The pre-activated wetting carrier is simply mixed with the pre-emulsified fenugreek seed oil emulsion at a very low concentration before application and the roots are immediately irrigated. At this time, Bacillus thuringiensis is relatively stable on the iron-modified biochar and zinc-loaded hydroxyapatite, reducing the contact time between fenugreek seed oil and Bacillus thuringiensis.

[0012] Preferably, the compound microbial agent contains the following parts by weight of each raw material: 40-60 parts of Bacillus thuringiensis HP-SYJ2 bacterial solution, 1-5 parts of fenugreek seed oil, 10-20 parts of hydroxyapatite, 30-50 parts of biochar, 0.1-1 parts of emulsifier, and 1-3 parts of soil permeable agent.

[0013] By adopting the above technical solution, the combination of the components in the above dosage can not only achieve multiple antibacterial effects, but also strongly induce plant systemic resistance, regulate the root environment, and improve soil structure. Through the synergistic effects of biological, chemical and physical processes, it can achieve the functions of promoting growth, soil improvement and disease control.

[0014] Preferably, the biochar undergoes the following pretreatment: A 0.2-0.5% chitosan solution and a 0.5-1% seaweed extract solution were mixed and the pH was adjusted to 5.5-6.5 to obtain the treatment solution. Add biochar to the treatment solution, soak for 12-24 hours, drain, and air dry.

[0015] By adopting the above technical solution, the biochar is pretreated before loading Bacillus thuringiensis by soaking it in chitosan solution and seaweed extract solution. The amino groups of chitosan and the various functional groups of seaweed extract modify the surface of biochar, making it hydrophilic instead of hydrophobic, which is more conducive to the adhesion and colonization of negatively charged bacterial cells. Moreover, seaweed extract and chitosan, as slow-release organic carbon and nitrogen sources, are stored in the pores of biochar to provide nutrients for the subsequently loaded microorganisms. In addition, chitosan strongly adsorbs negatively charged Bacillus thuringiensis cells through electrostatic interaction, improving loading efficiency. Furthermore, chitosan and seaweed extract can significantly accelerate the germination rate of Bacillus thuringiensis after entering the soil and support its continuous reproduction, extending its shelf life.

[0016] When the prepared compound microbial agent is applied to the rhizosphere, it can continuously release chitosan and seaweed extract. As a plant immune inducer, chitosan can continuously and gently activate the systemic resistance of Chinese medicinal materials, making them more resistant to root rot, leaf diseases, etc. Chitosan itself also has certain antibacterial activity, and works synergistically with Bacillus thuringiensis to form a dual mechanism of immunity and biocontrol. Seaweed extract is rich in natural auxins, cytokinins, etc., which directly stimulate root meristems, increase the number and length of fibrous roots, and build a strong root system. The betaine, proline and other osmotic regulators it contains can improve tolerance and improve the content of active ingredients in Chinese medicinal materials.

[0017] Therefore, pretreated biochar can not only serve as a carrier for Bacillus thuringiensis, but also provide it with nutrients, ensuring that it can quickly occupy its ecological niche, reproduce in large quantities, and remain effective for a long time after entering the soil. Moreover, in the soil, biochar provides beneficial bacteria while continuously releasing biostimulants and immune signals, promoting root development and plant health from multiple dimensions.

[0018] Preferably, the mass ratio of chitosan, seaweed extract and biochar is 0.4-0.5:0.1-0.2:100.

[0019] By adopting the above technical solutions, at the above dosage ratio, chitosan can form a monomolecular membrane in biochar film, effectively changing the surface charge, while seaweed extract provides sufficient natural hormones and trace elements, optimizing the microbial colonization environment, plant root development, and rhizosphere soil microecology.

[0020] Preferably, potassium humate is also added to the treatment solution, and the mass ratio of potassium humate to biochar is 1-1.5:100.

[0021] By adopting the above technical solution, potassium humate can increase the loading strength of chitosan and seaweed extract on biochar and slow down their release rate to achieve long-term release. At the same time, its strong ion exchange capacity can provide potassium nutrition for the entire system, strongly promote root growth, improve soil structure, chelate nutrients, buffer pH, and provide a slow-release carbon source.

[0022] Preferably, the biochar is iron-modified sesame stalk biochar, which is prepared by pyrolyzing sesame stalks, washing, drying, soaking in ferric nitrate solution, and heat-treating under a nitrogen atmosphere.

[0023] By adopting the above technical solution and using iron-modified sesame stalk biochar, essential micronutrients are provided to the plants, participating in chlorophyll synthesis, cellular respiration, and enzymatic reactions, improving photosynthetic efficiency, plant height, and stem diameter. Iron ions can also provide nutrients for beneficial bacteria in the soil, promote their reproduction, inhibit the growth of soil-borne pathogens, optimize the rhizosphere microenvironment, improve soil fertility and fertilizer retention, and enhance the plants' resistance to salinity and disease.

[0024] Preferably, the soil penetrant is selected from at least one of polyether-modified siloxane, ethoxylated polytrisiloxane, fatty alcohol polyoxyethylene ether, sodium di-2-octyl sulfonate, and nonylphenol polyoxyethylene ether.

[0025] Preferably, the fermentation method of Bacillus thuringiensis HP-SYJ2 is as follows: Bacillus thuringiensis HP-SYJ2 is inoculated into liquid culture medium at an inoculation amount of 1-2%, activated at 30-35℃ for 20-24h, then centrifuged, washed 2-3 times with sterile physiological saline, and sterile physiological saline is added to obtain Bacillus thuringiensis HP-SYJ2 bacterial solution.

[0026] Secondly, this application provides a compound bacterial agent containing Bacillus thuringiensis, employing the following technical solution: A compound bacterial agent containing Bacillus thuringiensis is prepared using the aforementioned method for preparing a compound bacterial agent containing Bacillus thuringiensis.

[0027] Thirdly, this application provides a compound microbial agent containing Bacillus thuringiensis for the application of root rot in Chinese medicinal materials or for promoting the growth of Chinese medicinal materials.

[0028] Preferred Chinese medicinal materials include, but are not limited to, Danshen, Panax notoginseng, and Astragalus membranaceus.

[0029] In summary, this application has the following beneficial effects: 1. Because this application uses hydroxyapatite and biochar as physical carriers, it protects Bacillus thuringiensis HP-SYJ, reducing the risk of it killing fenugreek seed oil. It also greatly alleviates the aeration of fenugreek seed oil root irrigation, achieving the effects of insect control, soil improvement, antibacterial activity and induction of resistance, reducing the incidence of plant diseases, promoting root development, plant health, improving soil productivity, enriching the population of superior microorganisms, and enhancing the sustainable growth capacity of the soil, thereby achieving the goal of efficient and long-lasting prevention and control of root rot.

[0030] 2. In this application, chitosan and seaweed extract are preferred for pretreatment of biochar, so that biochar becomes a nutrient-functional material, providing nutrients for Bacillus thuringiensis and achieving high load, high survival, high activity and durability. At the same time, it delivers immune stimulation signals and growth stimuli to the roots of Chinese medicinal materials, thereby improving the health of plant roots. Detailed Implementation

[0031] The present application will be further described in detail below with reference to the embodiments. Example

[0032] In Example 1, Bacillus thuringiensis HP-SYJ2 (Bt) was deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, 430072, China, on March 3, 2025, with accession number CCTCC NO: M2025343. Hydroxyapatite was selected from Beijing Deco Island, model HAP0040. The rice husk biochar had a pH of 9.76, ammonium nitrogen content of 1.95 mg / kg, nitrate nitrogen content of 35.71 mg / kg, available phosphorus content of 854.59 mg / kg, available potassium content of 1.1 mg / kg, and total nitrogen content of 0.12%. The specific surface area of ​​the sesame stalk biochar was 351.79 m². 2 / g, total pore volume is 0.176cm³ 3 / g, with an average pore size of 1nm and a micropore volume of 0.149cm³. 3 / g, ash content is 4.71; chitosan is selected from Zhengzhou Ruoxiang Trading Co., Ltd., product number 0.9, and seaweed extract is selected from Xi'an Ruilin Biotechnology Co., Ltd., product number RLHZ.

[0033] Example 1: A method for preparing a compound bacterial agent containing Bacillus thuringiensis, comprising the following steps: S1. Inoculate Bacillus thuringiensis HP-SYJ2 at a rate of 2% into liquid culture medium, activate by fermentation at 30℃ for 20 hours, centrifuge, wash twice with sterile physiological saline, and then dilute with sterile physiological saline to a concentration of 8×10⁻⁶. 9Bacillus thuringiensis HP-SYJ2 bacterial suspension was prepared using cfu / ml. The liquid culture medium formula is as follows: soluble corn starch 20 g / L, sodium chloride 0.5 g / L, potassium nitrate 1 g / L, potassium phosphate 0.5 g / L, magnesium sulfate 0.5 g / L, ferric sulfate 0.01 g / L, agar 15 g / L, pH 7.5; S2. Mix 20g hydroxyapatite and 40g biochar evenly, mix with 60g Bacillus thuringiensis HP-SYJ2 bacterial solution, soak for 24h, air dry until the moisture content is 30%, store in a breathable bag, and mature for 1 week to obtain the loading material. The biochar is rice husk char. S3. Mix 5g fenugreek seed oil with 1g emulsifier, 3g soil penetrant and water, and shear emulsify at 500r / min for 6h to obtain an emulsion with fenugreek seed oil concentration of 0.5wt%. The penetrant is nonylphenol polyoxyethylene ether. S4. Seal the loading material and emulsion separately; mix the loading material and emulsion thoroughly before application.

[0034] Example 2: A method for preparing a compound bacterial agent containing Bacillus thuringiensis, comprising the following steps: S1. Inoculate Bacillus thuringiensis HP-SYJ2 at a rate of 1% into liquid culture medium, activate by fermentation at 35°C for 24 hours, centrifuge, wash three times with sterile physiological saline, and then dilute with sterile physiological saline to a concentration of 8×10⁻⁶. 9 Bacillus thuringiensis HP-SYJ2 bacterial suspension was prepared using cfu / ml. The liquid culture medium formula is as follows: soluble corn starch 20 g / L, sodium chloride 0.5 g / L, potassium nitrate 1 g / L, potassium phosphate 0.5 g / L, magnesium sulfate 0.5 g / L, ferric sulfate 0.01 g / L, agar 15 g / L, pH 7.5; S2. Mix 10g of hydroxyapatite and 50g of biochar evenly, mix with 40g of Bacillus thuringiensis HP-SYJ2 bacterial solution, soak for 24h, air dry until the moisture content is 40%, store in a breathable bag, and mature for 1 week to obtain the loading material. The biochar is rice husk char. S3. Mix 3g fenugreek seed oil with 0.5g emulsifier, 2g soil penetrant and water, and shear emulsify at 500r / min for 6h to obtain an emulsion with fenugreek seed oil concentration of 0.3wt%. The penetrant is nonylphenol polyoxyethylene ether. S4. Seal the loading material and emulsion separately; mix the loading material and emulsion thoroughly before application.

[0035] Example 3: A method for preparing a compound bacterial agent containing Bacillus thuringiensis, comprising the following steps: S1. Inoculate Bacillus thuringiensis HP-SYJ2 at a rate of 2% into liquid culture medium, activate by fermentation at 30℃ for 20 hours, centrifuge, wash twice with sterile physiological saline, and then dilute with sterile physiological saline to a concentration of 8×10⁻⁶. 9 Bacillus thuringiensis HP-SYJ2 bacterial suspension was prepared using cfu / ml. The liquid culture medium formula is as follows: soluble corn starch 20 g / L, sodium chloride 0.5 g / L, potassium nitrate 1 g / L, potassium phosphate 0.5 g / L, magnesium sulfate 0.5 g / L, ferric sulfate 0.01 g / L, agar 15 g / L, pH 7.5; S2. Mix 20g hydroxyapatite and 30g biochar evenly, mix with 50g Bacillus thuringiensis HP-SYJ2 bacterial solution, soak for 24h, air dry until the moisture content is 35%, store in a breathable bag, and mature for 1 week to obtain the loading material. The biochar is rice husk char. S3. Mix 1g fenugreek seed oil with 0.1g emulsifier, 1g soil penetrant and water, and shear emulsify at 500r / min for 6h to obtain an emulsion with fenugreek seed oil concentration of 0.1wt%. The penetrant is nonylphenol polyoxyethylene ether. S4. Seal the loading material and emulsion separately; mix the loading material and emulsion thoroughly before application.

[0036] Example 4: A method for preparing a compound bacterial agent containing Bacillus thuringiensis, differing from Example 1 in that the biochar is iron-modified sesame stalk biochar. The method for preparing iron-modified sesame stalk biochar is as follows: Naturally dried sesame stalks are washed four times with distilled water, dried at 80°C for 8 hours, pulverized, and placed in a muffle furnace for pyrolysis at 600°C for 5 hours to obtain biochar. After washing with pure water until neutral, the biochar is dried at 105°C for 24 hours to obtain sesame stalk biochar. Ferric hydroxide is placed in pure water, and sesame stalk biochar is added. The mixture is stirred and soaked at 25°C for 2.5 hours, then the water is evaporated at 90°C. Finally, the mixture is heat-treated at 550°C and a nitrogen atmosphere at 150 mL / min for 0.5 hours to obtain iron-modified sesame stalk biochar. The mass ratio of sesame stalk biochar to ferric hydroxide is 10:1.

[0037] Example 5: A method for preparing a compound bacterial agent containing Bacillus thuringiensis, which differs from Example 4 in that the biochar is sesame stalk biochar. Naturally dried sesame stalks are washed four times with distilled water, dried at 80°C for 8 hours, pulverized, and placed in a muffle furnace for pyrolysis at 600°C for 5 hours to obtain biochar. After washing with pure water until neutral, it is dried at 105°C for 24 hours to obtain sesame stalk biochar.

[0038] Example 6: A method for preparing a compound bacterial agent containing Bacillus thuringiensis, differing from Example 1 in that the biochar undergoes the following pretreatment: Dissolve 0.5g of chitosan in water to prepare a chitosan solution with a concentration of 0.5wt%. Add 1g of seaweed extract to water to prepare a seaweed extract solution with a concentration of 1wt%. Mix the chitosan solution and the seaweed extract solution evenly and adjust the pH to 6 to obtain the treatment solution. The seaweed extract is small-leaved seaweed extract. Add 100g of biochar to the treatment solution, soak for 24 hours, drain, and air dry until the moisture content is 30%.

[0039] Example 7: A method for preparing a compound bacterial agent containing Bacillus thuringiensis, differing from Example 1 in that the biochar undergoes the following pretreatment: Dissolve 0.2g of chitosan in water to prepare a chitosan solution with a concentration of 0.2wt%. Add 0.5g of seaweed extract to water to prepare a seaweed extract solution with a concentration of 0.5wt%. Mix the chitosan solution and the seaweed extract solution evenly and adjust the pH to 6.5 to obtain the treatment solution. Add 100g of biochar to the treatment solution, soak for 12 hours, drain, and air dry until the moisture content is 40%.

[0040] Example 8: A method for preparing a compound bacterial agent containing Bacillus thuringiensis, differing from Example 6 in that the biochar undergoes the following pretreatment: Dissolve 0.2g of chitosan in water to prepare a chitosan solution with a concentration of 0.2wt%. Add 0.5g of seaweed extract to water to prepare a seaweed extract solution with a concentration of 0.5wt%. Mix the chitosan solution and the seaweed extract solution evenly and adjust the pH to 6.5 to obtain the treatment solution. Add potassium humate to the treatment solution, add 100g of biochar to the treatment solution, soak for 12 hours, drain, and air dry until the moisture content is 40%. The mass ratio of potassium humate to biochar is 1.5:100.

[0041] Example 9: A method for preparing a compound bacterial agent containing Bacillus thuringiensis, differing from Example 6 in that the biochar undergoes the following pretreatment: Dissolve 0.2g of chitosan in water to prepare a chitosan solution with a concentration of 0.2wt%. Adjust the pH to 6.5 to obtain the treatment solution. Add 100g of biochar to the treatment solution, soak for 12 hours, drain, and air dry until the moisture content is 40%.

[0042] Example 10: A method for preparing a compound bacterial agent containing Bacillus thuringiensis, differing from Example 1 in that the biochar is iron-modified sesame stalk biochar. The method for preparing the iron-modified sesame stalk biochar is as follows: Naturally dried sesame stalks are washed four times with distilled water, dried at 80°C for 8 hours, pulverized, and placed in a muffle furnace for pyrolysis at 600°C for 5 hours to obtain biochar. After washing with pure water until neutral, the biochar is dried at 105°C for 24 hours to obtain sesame stalk biochar. Ferric hydroxide is placed in pure water, and the sesame stalk biochar is added. The mixture is stirred and soaked at 25°C for 2.5 hours, then the water is evaporated at 90°C. Finally, the mixture is heat-treated at 550°C and under a nitrogen atmosphere of 150 mL / min for 0.5 hours to obtain iron-modified sesame stalk biochar. The mass ratio of sesame stalk biochar to ferric hydroxide is 10:1. The iron-modified sesame stalk biochar undergoes the following pretreatment: Dissolve 0.5g of chitosan in water to prepare a chitosan solution with a concentration of 0.5wt%. Add 1g of seaweed extract to water to prepare a seaweed extract solution with a concentration of 1wt%. Mix the chitosan solution and the seaweed extract solution evenly and adjust the pH to 6 to obtain the treatment solution. Add potassium humate to the treatment solution, add 100g of iron-modified sesame stalk biochar to the treatment solution, soak for 24 hours, drain, and air dry until the moisture content is 30%. The mass ratio of potassium humate to biochar is 1.5:100.

[0043] Comparative Example

[0044] Comparative Example 1: A method for preparing a compound bacterial agent containing Bacillus thuringiensis, which differs from Example 1 in that an equal amount of hydroxyapatite is used instead of biochar.

[0045] Comparative Example 2: A method for preparing a compound microbial agent containing Bacillus thuringiensis, which differs from Example 1 in that no fenugreek seed oil, emulsifier, or penetrant is added, and the loading material is the compound microbial agent.

[0046] Performance testing

[0047] The compound microbial agent was prepared according to the methods in the examples and comparative examples. The compound microbial agent was used to test the growth-promoting effect and root rot prevention effect on Chinese medicinal materials (taking Danshen). The specific test methods are as follows: I. Growth-promoting effect: Healthy Salvia miltiorrhiza seedlings after transplanting were selected and inoculated with a compound microbial agent using the root irrigation method. Before root irrigation, the soil was moistened with 10 mL of sterile water. 10 mL of the treatment solution (prepared from the compound microbial agent and water at a mass ratio of 1:100) was then applied to the root zone of the Salvia miltiorrhiza plants. This was repeated every 5 days for 3 consecutive treatments. Water was used as a blank control. Each treatment group consisted of 6 plants, with 3 replicates. 28 days after the first root irrigation, the plant height, leaf width, leaf length, number of leaves, and fresh weight of the above-ground and underground parts of each group of Salvia miltiorrhiza plants were measured. The measurement methods are as follows: plant height (height from the base of the stem to the tip of the new leaf), leaf length and width (length and width of the first fully unfolded new leaf), and fresh weight of the above-ground and underground parts (the above-ground and underground parts were cut off, and the root zone soil was removed before weighing).

[0048] Table 1. Growth-promoting effect of compound microbial agents on Salvia miltiorrhiza.

[0049] Combining the data from Examples 1-3 and Table 1, it can be seen that when Codonopsis pilosula was treated with the compound microbial agent prepared in Examples 1-3 for root irrigation, compared with the blank control group, the plant height, leaf width, leaf length, number of leaves, and fresh bacterial weight of the above-ground and underground parts of the Codonopsis pilosula plants treated with the compound microbial agent increased to varying degrees. This indicates that the use of the compound microbial agent has a significant promoting effect on the growth of Codonopsis pilosula plants.

[0050] In Example 4, iron-modified sesame stalk biochar was used. Compared with Example 1, Table 1 shows that after root irrigation treatment of Salvia miltiorrhiza plants with the compound microbial agent prepared in Example 4, various growth indicators of the Salvia miltiorrhiza plants treated in Example 4 were improved, indicating that iron-modified sesame stalk biochar can improve the growth-promoting effect of the compound microbial agent on Salvia miltiorrhiza plants.

[0051] Compared with Example 1, Example 5 only used sesame stalk biochar. It can be seen that after the compound microbial agent prepared in Example 5 was used to drench the roots of Salvia miltiorrhiza, the growth trend of Salvia miltiorrhiza was similar to that in Example 1, and the growth-promoting effect was not as good as that in Example 4 which used iron-modified sesame stalk biochar.

[0052] Compared with Example 1, Examples 6 and 7 used chitosan and seaweed extract to pretreat biochar. As can be seen from the data comparison in Table 1, the compound bacterial agent prepared in Examples 6 and 7 improved the growth parameters of Salvia miltiorrhiza plants, such as leaf size, plant height, and fresh weight of above-ground and underground parts, after being applied to the rhizosphere of the plants through root irrigation.

[0053] Compared with Example 6, Example 8 added potassium humate to the treatment solution. It can be seen that after the compound bacterial agent was used to drench the roots of Salvia miltiorrhiza, the growth trend of Salvia miltiorrhiza was further improved compared with Example 6.

[0054] Compared with Example 6, Example 9 only used chitosan solution to soak biochar for pretreatment. It can be seen that the fresh weight of the underground part of Salvia miltiorrhiza decreased, and other growth parameters also changed.

[0055] Compared with Example 1, Example 10 not only uses iron-modified sesame stalk biochar, but also pre-treats the biochar with a treatment solution containing chitosan, seaweed extract and potassium humate. It can be seen that the prepared compound microbial agent further enhances the growth-promoting effect of Salvia miltiorrhiza.

[0056] In Comparative Example 1, hydroxyapatite was used instead of biochar, and the growth-promoting effect of the prepared compound microbial agent was not as good as that of Example 1. In Comparative Example 2, no ingredients such as fenugreek seed oil were added, and hydroxyapatite and biochar loaded with Bacillus thuringiensis HP-SYJ2 bacterial solution were used as the compound microbial agent. Compared with Example 1, the growth-promoting effect of the compound microbial agent prepared in Comparative Example 2 on Salvia miltiorrhiza was also reduced.

[0057] II. Control Effect of Root Rot: Healthy 1-year-old Salvia miltiorrhiza seedlings with uniform growth were selected and transplanted into plastic pots 24cm high and 17cm wide, filled with a mixed nutrient soil (nutrient soil: vermiculite: perlite = 3:1:1). One seedling was placed in each pot. Seven days after transplanting, each seedling was inoculated with 10mL of a treatment solution (prepared from compound bacterial agent and water at a mass ratio of 1:10). Three days later, each seedling was inoculated with a Salvia miltiorrhiza root rot pathogen spore suspension (1×10⁻⁶) using the root dredging method. 7 10 mL of sterile water (CFU / mL) was used to inoculate Fusarium solani and Fusarium latifolium separately. The control group was replaced with an equal volume of sterile water. Each group consisted of 12 pots, and the treatment was repeated 3 times.

[0058] The above-mentioned treatment groups were transferred to greenhouses for further cultivation for 28-35 days. Disease incidence was observed and statistically analyzed, and the disease index and control effect were calculated. The severity of *Salvia miltiorrhiza* root rot was classified into five levels: Level 0 indicates healthy roots with no disease; Level 1 indicates rot only on the surface of the tuber, with a rotten root area of ​​less than 25%, and normal leaves; Level 2 indicates yellowing leaves, rot extending to the interior, and a rotten root area of ​​25-35%; Level 3 indicates wilting leaves, rot extending to the interior, all fibrous roots rot, and a rotten root area of ​​50-75%, with wilting leaves; Level 4 indicates completely dried leaves, complete rot of the entire tuber, and a rotten root area of ​​over 75%. The incidence rate, disease index, and control effect of *Salvia miltiorrhiza* root rot were calculated using the following formulas.

[0059] Incidence rate (%) = Number of diseased plants in the treatment group / Total number of plants in the treatment group × 100; Disease index (%) = ∑ (number of diseased plants at each level × corresponding disease level) / (total number of plants surveyed × highest disease level) × 100; Prevention and control effect (%) = (disease index of control group - disease index of treatment group) / disease index of control group × 100.

[0060] Table 2. Control effect of compound microbial agents on root rot of Salvia miltiorrhiza.

[0061] Based on the test data in Examples 1-3 and Table 2, it can be seen that the compound microbial agent prepared in Examples 1-3 can inhibit the growth of various pathogens after root irrigation treatment of Salvia miltiorrhiza. It can be added to the soil near the roots of the plant during transplanting and released slowly, which helps to reduce the occurrence of diseases. It can also be used for root irrigation treatment of growing plants to prevent root rot.

[0062] In Example 4, iron-modified sesame stalk biochar was used. Compared with Example 1, the compound microbial agent prepared in Example 4 reduced the incidence of salvia miltiorrhiza and increased the prevention and control effect after root irrigation treatment.

[0063] In Example 5, sesame stalk biochar was used, and the compound microbial agent prepared in Example 5 had similar effects to that in Example 1.

[0064] Compared with Example 1, Examples 6 and 7 used chitosan and seaweed extract to treat biochar, and the compound bacterial agents prepared in Examples 6 and 7 showed improved control effects on root rot.

[0065] Compared with Example 6, Example 8 also added potassium humate to the treatment solution. The compound microbial agent prepared in Example 8 had a similar effect on the prevention and control of root rot as in Example 6.

[0066] In Example 9, only chitosan solution was used for biochar pretreatment. Compared with Example 6, the compound bacterial agent prepared in Example 9 showed a decreased effect on the prevention and control of root rot in Salvia miltiorrhiza.

[0067] In Example 10, iron-modified sesame stalk biochar was used and pretreated. As shown in Table 2, the compound microbial agent prepared in Example 10 has the best control effect on root rot of Salvia miltiorrhiza.

[0068] In Comparative Example 1, hydroxyapatite was used instead of biochar, while in Comparative Example 2, fenugreek seed oil was not added. Compared with Example 1, the compound microbial agents prepared in Comparative Examples 1 and 2 showed a decrease in the control effect on root rot.

[0069] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. A method for preparing a complex microbial agent containing Bacillus thuringiensis, characterized by, Includes the following steps: Ferment Bacillus thuringiensis HP-SYJ2 to a concentration of (3-8) x 10 9 cfu / ml to obtain Bacillus thuringiensis HP-SYJ2 bacterial liquid; Hydroxyapatite and biochar were mixed evenly, then mixed with Bacillus thuringiensis HP-SYJ2 bacterial solution, soaked, and air-dried until the moisture content was 30-40%. The mixture was then matured for 1-2 weeks to obtain the loading material. Fenugreek seed oil, emulsifier, soil permeable agent, and water are mixed and sheared to emulsify, thus obtaining an emulsion with a fenugreek seed oil concentration of 0.1-0.5 wt%. The loading material and emulsion are packaged separately and mixed before application.

2. The preparation method of the Bacillus thuringiensis-containing complex microbial agent according to claim 1, characterized by: The compound microbial agent contains the following raw materials in parts by weight: 40-60 parts of Bacillus thuringiensis HP-SYJ2 bacterial solution, 1-5 parts of fenugreek seed oil, 10-20 parts of hydroxyapatite, 30-50 parts of biochar, 0.1-1 parts of emulsifier, and 1-3 parts of soil permeable agent.

3. The Bacillus thuringiensis-comprising complex microbial agent according to claim 1, characterized by: The biochar undergoes the following pretreatment: A 0.2-0.5% chitosan solution and a 0.5-1% seaweed extract solution were mixed and the pH was adjusted to 5.5-6.5 to obtain the treatment solution. Add biochar to the treatment solution, soak for 12-24 hours, drain, and air dry.

4. The method for preparing the compound bacterial agent containing Bacillus thuringiensis according to claim 3, characterized in that: The mass ratio of chitosan, seaweed extract and biochar is 0.4-0.5:0.1-0.2:

100.

5. The method of claim 3, wherein the Bacillus thuringiensis -containing complex microbial agent is prepared by the steps of: (a) mixing Bacillus thuringiensis with a carrier; (b) adding a surfactant to the mixture; (c) adding a stabilizer to the mixture; and (d) adding a dispersant to the mixture. The treatment solution also contains potassium humate, and the mass ratio of potassium humate to biochar is 1-1.5:

100.

6. The preparation method of the Bacillus thuringiensis-containing complex microbial agent according to claim 1, characterized by: The biochar is iron-modified sesame stalk biochar, which is prepared by pyrolyzing sesame stalks, washing, drying, soaking in ferric nitrate solution, and heat-treating under a nitrogen atmosphere.

7. The method for preparing the compound bacterial agent containing Bacillus thuringiensis according to claim 1, characterized in that: The fermentation method of Bacillus thuringiensis HP-SYJ2 is as follows: Bacillus thuringiensis HP-SYJ2 is inoculated into liquid culture medium at an inoculation rate of 1-2%, activated at 30-35℃ for 20-24 hours, then centrifuged, washed 2-3 times with sterile physiological saline, and sterile physiological saline is added to obtain Bacillus thuringiensis HP-SYJ2 bacterial solution.

8. The preparation method of the Bacillus thuringiensis-containing complex microbial agent according to claim 1, characterized by: The soil penetrant is selected from at least one of polyether-modified siloxane, ethoxylated polytrisiloxane, fatty alcohol polyoxyethylene ether, sodium di-2-octyl sulfonate, and nonylphenol polyoxyethylene ether.

9. A complex microbial agent containing Bacillus thuringiensis, characterized by, It is prepared using the method for preparing the compound bacterial agent containing Bacillus thuringiensis as described in any one of claims 1-8.

10. The application of a compound bacterial agent containing Bacillus thuringiensis prepared by the method according to any one of claims 1-8 in the treatment of root rot of Chinese medicinal materials or in promoting the growth of Chinese medicinal materials.

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