Preparation method and application of bacillus velezensis compound microbial fertilizer

Abstract

The invention relates to the technical field of preparation of compound microbial fertilizers, in particular to a preparation method and application of a bacillus velezensis compound microbial fertilizer, and the preparation method comprises the following steps: uniformly mixing livestock and poultry manure and plant raw materials, adding a primary fermentation microbial agent, fermenting, stirring and mixing through trough type turning compost, and adjusting the water content, the fermentation temperature and the fermentation time to obtain the bacillus velezensis compound microbial fertilizer. Stirring once every 2-3 days to obtain a primarily fermented compost material; after the water content of the compost material after the primary fermentation is reduced to 45%-55% and the temperature is less than or equal to 45 DEG C, adding functional bacteria and a functional auxiliary agent in the secondary fermentation, stirring and mixing, continuously fermenting, controlling the temperature of the compost body in the secondary fermentation to be not more than 60 DEG C, stirring once every day, naturally airing after the fermentation is finished, and adding a nutrient element auxiliary agent for mixing to obtain the compound microbial fertilizer. According to the invention, a single disease prevention and control function is converted into comprehensive effects of nutrition supply, prevention and control of multiple diseases, soil remediation and ecological balance maintenance, so that plant growth is promoted.

Description

Technical Field

[0001] This invention relates to the field of compound microbial fertilizer preparation technology, and in particular to a method for preparing and applying Bacillus vesiculosus compound microbial fertilizer. Background Technology

[0002] With the adjustment of planting structure and the vigorous development of agricultural industry, the cultivation of Chinese medicinal herbs and vegetables is becoming increasingly intensive, large-scale, and monoculture-oriented, leading to widespread continuous cropping of some Chinese medicinal herbs and greenhouse vegetables. Long-term continuous cropping deteriorates soil physical properties, leads to deficiencies in certain micronutrients, strong allelopathic effects, or homogenization of plant residues, selectively selecting rhizosphere microorganisms, altering the soil microbial community, and significantly reducing microbial functional diversity. This is mainly manifested in a significant increase in the number of pathogenic fungi, resulting in an imbalance in the soil microbial community structure, which is a major factor in the occurrence of continuous cropping obstacles. To alleviate the problem of continuous cropping obstacles, compound microbial fertilizers have received widespread attention as an environmentally friendly solution. Compound microbial fertilizers are composed of three main components: organic fertilizer, microbial bacteria, and inorganic fertilizer. They not only provide nutrients to crops but also inhibit soil pathogens through the antagonistic effect of biocontrol bacteria, improving the soil microbial community structure and playing an increasingly important role in agricultural production. Currently, the nutrient content of solid compound microbial fertilizers on the market is generally between 8% and 25%, with products containing 10% to 18% nutrient content being the most common. This is because excessively high nutrient content may have a fumigation effect on the microorganisms caused by chemical fertilizers, leading to a decrease in the survival rate of the microorganisms and affecting the biological efficacy of the microbial fertilizer.

[0003] Existing patent CN202111551155.0 discloses *Bacillus belyssiensis* and its application in the control of crop diseases. The provided *Bacillus belyssiensis* can be prepared into microbial agents, compound microbial agents, and microbial fertilizers, exhibiting good inhibitory effects against agricultural fungal pathogens such as early blight of potato, bakanae disease of rice, Fusarium head blight of wheat, sheath blight of rice, black scurf of potato, and large leaf spot of corn. It can also be applied to the control of early blight of potato. However, the microbial fertilizers prepared using the above-mentioned technical solutions have relatively limited soil remediation functions, mainly focusing on disease control. They lack systematic solutions for the comprehensive problems commonly found in continuously cropped soils, such as heavy metal accumulation and organic matter loss, thus affecting plant growth. Summary of the Invention

[0004] In view of this, the present invention proposes a method for preparing and applying Bacillus vesiculosus compound microbial fertilizer to solve the problem that existing microbial fertilizers have a single function in soil remediation and are difficult to effectively promote plant growth.

[0005] The technical solution of this invention is achieved as follows: This invention provides a method for preparing Bacillus belyssus compound microbial fertilizer, comprising the following steps: (1) Mix livestock and poultry manure and plant materials in a ratio of (6-7): (3-4) evenly, add primary fermentation microbial agent, and ferment and mix them by turning and turning in a trough, adjust the moisture content to 50%-55%, the fermentation temperature is 55-65℃, the fermentation time is 20-30 days, and stir once every 2-3 days to obtain the compost material after primary fermentation; (2) After the first fermentation, the moisture content of the compost material drops to 45%-55% and the temperature drops to below 45℃. Add functional bacteria and functional additives, stir and mix, and continue fermentation. Control the temperature of the compost pile during the second fermentation to not exceed 60℃. Stir once a day to promote the rapid growth and reproduction of functional microorganisms. After fermentation is completed, let it air dry naturally, add nutrient element additives to compound it, and obtain compound microbial fertilizer.

[0006] This invention employs a staged fermentation process, which enables the orderly inoculation and synergistic effect of different functional microorganisms. The resulting compound microbial fertilizer not only has the nutrient supply function of traditional organic fertilizer, but also has excellent disease control effects, effectively improving the soil microbial community structure and promoting plant growth.

[0007] Based on the above technical solutions, preferably, the plant raw materials, by weight, include 30-50 parts rapeseed cake and 25-35 parts tea oil powder; the nutrient element adjuvants include three or more of urea, potassium sulfate, monoammonium phosphate, magnesium sulfate, zinc sulfate, humic acid and potassium fulvate, and the amount of nutrient element adjuvants added is 15%-40% of the mass of the compost material after secondary fermentation.

[0008] Specifically, rapeseed cake is rich in high-quality protein, amino acids, and phospholipids, making it an excellent source of organic nitrogen and phosphorus. Tea oil powder contains residual oil, protein, and cellulose, providing a carbon source and organic matter to improve soil structure. It also contains some tea saponins, which have a good insect-repelling effect, reducing the occurrence of underground pests in crops. During fermentation, both are decomposed by microorganisms into amino acids, small-molecule nitrogen compounds, and soluble sugars, forming a nutrient matrix that is easily utilized by microorganisms. Urea, potassium sulfate, monoammonium phosphate, and magnesium sulfate provide readily available nitrogen, phosphorus, potassium, and magnesium nutrients, quickly supplementing the nutritional needs of crops and promoting the growth and reproduction of microorganisms. Humic acid and potassium fulvate, as bioactive organic substances, combine with calcium and magnesium ions in the soil to form a water-stable aggregate structure. This synergistic effect with organic matter increases soil porosity, creating habitat space for microorganisms. At the same time, the short-chain active molecules it contains continuously release the carbon and nitrogen sources needed by microorganisms, which can be directly utilized by microorganisms, promoting their growth and colonization. This achieves a triple synergistic effect of soil improvement, nutrient supply, and microbial protection and growth promotion.

[0009] Based on the above technical solutions, preferably, the amount of the primary fermentation microbial agent added is 0.02%-0.2% of the total mass of the compost raw materials, and the effective viable bacteria count is (1-10)×10⁻¹⁰. 10 CFU / g, wherein the primary fermentation microbial agent comprises sparsely cottony thermophilic filamentous fungi, thermophilic actinomycetes and Bacillus licheniformis in a mass ratio of 4-5:3-4:2-3.

[0010] Based on the above technical solutions, preferably, in step (2), the mass ratio of the compost material, functional bacteria and functional additives after primary fermentation is 100:0.8-5:0.5-5.

[0011] Based on the above technical solutions, preferably, the functional bacteria are Bacillales velezensis and Bacillales amyloliquefaciens, with a mass ratio of 3-4:1-2. The Bacillales velezensis is Bacillales velezensis YFB3-1, with the accession number CCTCC NO:M20221140.

[0012] The functional bacteria are composed of Bacillus bellis YFB3-1 and Bacillus amyloliquefaciens in a mass ratio of 3-4:1-2, forming a synergistic biocontrol system with complementary functions. Bacillus bellis YFB3-1, as a highly efficient and broad-spectrum pathogen-resistant fungal strain isolated and screened from earthworm castings, has unique physiological characteristics such as rapid growth and reproduction, strong resistance, high temperature tolerance, and preference for amino acid nutrition. It is perfectly matched with the secondary fermentation process of this invention: when the primary high-temperature fermentation is completed and the temperature of the compost pile drops to a mesophilic environment below 45°C, the rich amino acid nutrition in the compost provides a high-quality nutrient source for the YFB3-1 strain, promoting its rapid reproduction and colonization; Bacillus amyloliquefaciens, with its strong enzymatic activity, decomposes complex organic matter, creating a more suitable growth environment for Bacillus bellis. The two strains are deeply integrated with the organic fertilizer through the secondary fermentation process to form a stable functional microbiome. After the microbial agent is applied to the soil, Bacillus belye can form a mutually beneficial symbiotic relationship with earthworms in the soil. Through multiple mechanisms such as the production of antibacterial substances, competitive repulsion, and nutrient competition, it synergistically enhances the antagonistic ability against soil-borne pathogenic fungi such as Fusarium oxysporum. At the same time, Bacillus amyloliquefaciens continuously secretes extracellular enzymes to improve the availability of soil nutrients. The synergistic effect of the two fundamentally improves the structure of the soil microbial community, significantly enhances soil biological activity, and effectively promotes plant growth.

[0013] Based on the above technical solutions, preferably, the preparation method of the functional additive includes: S1. Air-dry the earthworm castings until loose, then dry them completely. Mix the dried earthworm castings with an activator and impregnate them. Then place them in an activation furnace and heat them to 300-350℃ for 1-1.2 hours for pretreatment. Then, purge them with ammonia gas and heat them to 400-500℃ under an ammonia atmosphere for 1-1.5 hours to obtain ammoniated earthworm castings. S2. Mix guanidinoacetic acid solution and zinc sulfate solution, adjust the pH of the solution to 7.5-10, and react at 50-60℃ for 30-60 min to obtain guanidinoacetic acid-zinc complex; S3. Mix aminated earthworm castings, guanidinoacetic acid-zinc complex and ethylene glycol glycidyl ether, add triethylamine, react at 35-45℃ for 2-4 hours, and after the reaction is completed, separate and dry to obtain the functional additive.

[0014] In step S1, earthworm castings are impregnated with phosphoric acid and aluminum chloride activator and then activated at high temperature in stages. Phosphoric acid can corrode the internal pore structure of earthworm castings, increasing the specific surface area and porosity. At the same time, aluminum ions loaded on earthworm castings play a complexing role against heavy metals. Subsequently, high-temperature amination treatment under an ammonia atmosphere introduces a large number of amino functional groups on the surface of the carrier, significantly improving the chemical activity and cross-linking ability of the carrier. In step S2, a complex is formed by the complexation reaction of guanidinoacetic acid and zinc sulfate under alkaline conditions. This complex retains the slow-release nitrogen source characteristics of guanidinoacetic acid and provides the essential trace element zinc for plants. In step S3, the aminated earthworm castings react with the aldehyde group of glutaraldehyde to form a stable cross-linked structure, firmly binding the guanidinoacetic acid-zinc complex to the surface of the carrier, forming a composite carrier material with excellent slow-release function and heavy metal adsorption capacity.

[0015] This functional adjuvant forms a highly efficient synergistic system with functional bacteria, exerting multiple functions such as carrier protection, nutrient supply, and soil improvement. Earthworm castings, as a carrier substrate, possess excellent biocompatibility and a microporous structure, providing an ideal habitat and reproduction site for Bacillus belyssus YFB3-1 and Bacillus amyloliquefaciens. In particular, the YFB3-1 strain, originating from the earthworm casting environment, exhibits natural affinity and adaptability to the earthworm casting carrier. The aminated earthworm casting carrier has well-developed pores and a large specific surface area, enabling it to adsorb heavy metal ions in the soil through ion exchange and chelation, thus facilitating soil remediation. Guanidoacetic acid-zinc complex, as a functional slow-release nutrient source, has an excellent nitrogen retention effect due to its stable guanidino structure, significantly reducing nitrogen volatilization and leaching losses, improving nitrogen fertilizer utilization, and continuously providing Bacillus belyssus with its preferred amino acid nutrition, satisfying the strain's physiological characteristics of preferring amino acid nutrition. The slow-release supply of zinc enhances plant disease resistance. The stable carrier structure formed by the cross-linking of epoxy groups on ethylene glycol glycidyl ether gradually degrades in the soil environment, releasing nutrients and functional strains, achieving a triple synergistic effect of nutrient supply, microbial protection, and soil improvement, significantly enhancing the comprehensive efficacy and sustainability of the compound microbial fertilizer.

[0016] Based on the above technical solutions, preferably, in step S1, the solid-liquid ratio of earthworm castings and activator is 1g:3-5ml, the activator is phosphoric acid and aluminum chloride, the immersion treatment temperature is 85-95℃, and the immersion treatment time is 8-10h.

[0017] Based on the above technical solutions, preferably, in step S2, the mass ratio of guanidinoacetic acid and zinc sulfate is 2.5-3.5:1; in step S3, the mass ratio of aminated earthworm castings, guanidinoacetic acid-zinc complex, ethylene glycol glycidyl ether and triethylamine is 10:2.5-3.5:0.8-1.2:0.1-0.2.

[0018] The present invention also provides a Bacillus vesiculosus compound microbial fertilizer, which is prepared by the preparation method described above.

[0019] This invention also provides the application of the Bacillus vesiculosus compound microbial fertilizer described above in promoting plant growth.

[0020] The preparation method and application of the Bacillus vesiculosus compound microbial fertilizer of the present invention have the following advantages over the prior art: (1) This invention constructs a compound microbial fertilizer system by integrating plant raw material biocontrol system, specific functional strains and functional adjuvants. Among them, rapeseed cake in plant raw materials provides nitrogen and phosphorus sources, creating a favorable colonization environment for biocontrol strains such as Bacillus belyssus YFB3-1; the specific Bacillus belyssus YFB3-1 strain is derived from earthworm excrement environment, and can form a mutually beneficial symbiotic relationship with soil earthworms after being applied to the soil. Through the biological-soil-earthworm ternary synergistic system, the control effect of soil-borne diseases is continuously enhanced. At the same time, the tea oil powder in plant raw materials can further reduce the occurrence of underground pests of crops through synergistic effects; the functional adjuvants ensure the long-term activity of biocontrol strains and improve soil environmental quality through multiple functions such as carrier protection, slow release of nutrients and heavy metal passivation. The entire technical system realizes the transformation from a single disease prevention function to a comprehensive function of nutrient supply, disease control, soil remediation and ecological balance maintenance, thereby promoting plant growth. (2) The functional adjuvant of the present invention achieves the dual effects of microbial protection and nutrient supply through the activation modification of earthworm castings carrier and the slow-release function of guanidinoacetic acid-zinc complex. The activation treatment increases the specific surface area and adsorption active sites of earthworm castings carrier, and can also adsorb soil heavy metal ions through ion exchange and chelation, and introduce amino functional groups to provide binding sites for the ethylene glycol glycidyl ether reaction; the stable structure of guanidinoacetic acid-zinc complex has excellent nitrogen retention effect, significantly reducing nitrogen loss, while providing continuous amino acid nutrition for Bacillus belyssus, ensuring the long-term activity and biocontrol effect of functional strains in the soil. Detailed Implementation

[0021] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0022] It should be noted that the Bacillus belyssin inoculum YFB3-1 has the accession number CCTCC M 20221140 at the China Center for Type Culture Collection (CCTCC), with the accession date of July 21, 2022. The accession address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province. *Thermophilic filamentosa* was purchased from Mingzhou Biotechnology (catalog number BMZ148400); *Thermophilic actinomycetes* was purchased from Mingzhou Biotechnology (catalog number BMZ135187); and *Bacillus licheniformis* was purchased from Mingzhou Biotechnology (catalog number BMZ135440).

[0023] Example 1 This embodiment provides a Bacillus vesiculosus compound microbial fertilizer and its preparation method, including the following steps: (1) Mix 65 kg of livestock and poultry manure and 35 kg of plant materials evenly. The plant materials consist of 40 parts rapeseed cake and 30 parts tea oil powder. Add 0.1 kg of primary fermentation microbial agent, with a mass ratio of 4.5:3.5:2.5 of *Thermophilic Mycorrhizal* (microbial concentration 5 × 10⁻⁶). 10 CFU / g), thermophilic actinomycetes (bacterial concentration 5×10⁻⁶) 10 CFU / g) and Bacillus licheniformis (bacterial concentration 5×10⁻⁶) 10 Composed of CFU / g, the compost was fermented and mixed in a trough-type turning and turning composting process, with the moisture content adjusted to 52%, the fermentation temperature set at 60℃, and the fermentation time set at 25 days. The compost was stirred for 15 minutes every 2.5 days to obtain the compost material after one fermentation. (2) After the moisture content of the compost material drops to 50% and the temperature drops to below 45℃ after the first fermentation, take 100kg of the compost material after the first fermentation, add 3kg of functional bacteria and 3kg of functional adjuvant, and the mass ratio of the functional bacteria is 3.5:1.5 with Bacillus belyssus (bacterial concentration 1×10⁻⁶). 9 CFU / g) and Bacillus amyloliquefaciens (bacterial concentration 8×10⁻⁶) 8Mix (CFU / g), stir and continue fermentation for 18 days, controlling the temperature of the secondary fermentation pile to not exceed 60℃, stirring once a day for 12 minutes each time. After fermentation, let it air dry naturally until the moisture content is ≤30%, then add 27kg of nutrient element adjuvants, which are urea, potassium sulfate, monoammonium phosphate, magnesium sulfate and zinc sulfate in a mass ratio of 1:1:1:1:1. Pass through a 6-mesh sieve (approximately 3.8mm aperture) to obtain compound microbial fertilizer.

[0024] The preparation methods of functional additives include: S1. Air-dry earthworm castings until loose, then dry at 100℃ for 2 hours. Mix 100g of dried earthworm castings with 400ml of activator, which is a mixture of 15% phosphoric acid solution and 10% aluminum chloride solution at a volume ratio of 2:1. Impregnate at 90℃ for 9 hours, stirring every 2 hours. After impregnation, filter out excess liquid, place the impregnated earthworm castings in an activation furnace and heat to 330℃. Pre-treat under an inert atmosphere for 1.1 hours, then purge with ammonia for 3 minutes. Heat to 450℃ under an ammonia atmosphere and react for 1.2 hours. Cool to room temperature to obtain activated earthworm castings. S2. Dissolve 30g of guanidinoacetic acid in water to prepare a 15% guanidinoacetic acid solution, and dissolve 10g of zinc sulfate in water to prepare a 10% zinc sulfate solution. Mix the guanidinoacetic acid solution and the zinc sulfate solution, adjust the pH to 8.5 with 2mol / L sodium hydroxide solution, and react at 55℃ for 45 min with continuous stirring. After the reaction is complete, centrifuge to obtain the guanidinoacetic acid-zinc complex precipitate, wash and dry to obtain the guanidinoacetic acid-zinc complex. S3. Mix 100g of amination-treated earthworm castings, 30g of guanidinoacetic acid-zinc complex, and 10g of ethylene glycol glycidyl ether. Add an appropriate amount of distilled water to adjust the consistency to a paste. Add 1.5g of triethylamine and react at 40℃ for 3 hours, stirring every 30 minutes. After the reaction is complete, separate the solid product by centrifugation, wash, dry, and pulverize through an 80-mesh sieve to obtain the functional additive.

[0025] Example 2 This embodiment provides a Bacillus vesiculosus compound microbial fertilizer and its preparation method, including the following steps: (1) Mix 60 kg of livestock and poultry manure and 40 kg of plant materials evenly. The plant materials consist of 30 parts rapeseed cake and 25 parts tea oil powder. Add 0.02 kg of primary fermentation microbial agent, which is 4:3:2 of the mass ratio of *Thermophilic Mycorrhizal* (1×10⁻⁶ bacteria concentration). 10 CFU / g), thermophilic actinomycetes (bacterial concentration 1×10⁻⁶) 10 CFU / g) and Bacillus licheniformis (bacterial concentration 1×10⁻⁶) 10Composed of CFU / g, the compost is fermented and mixed in a trough-type turning and turning composting process, with the moisture content adjusted to 50%, the fermentation temperature set at 55℃, and the fermentation time set at 30 days. The compost is stirred for 15 minutes every 2-3 days to obtain the compost material after one fermentation. (2) After the moisture content of the compost material drops to 45% and the temperature drops below 45℃ after the first fermentation, take 100kg of the compost material after the first fermentation and add 0.8kg of functional bacteria and 0.5kg of functional adjuvant. The functional bacteria have a mass ratio of 3:1 and contain Bacillus belyssus (bacterial concentration 1×10⁻⁶). 9 CFU / g) and Bacillus amyloliquefaciens (bacterial concentration 8×10⁻⁶) 8 Mix the ingredients (CFU / g) and continue fermentation for 15 days. Control the temperature of the fermentation pile to not exceed 60℃. Stir once a day for 10 minutes each time. After fermentation, let it air dry until the moisture content is ≤30%. Add 15kg of nutrient element adjuvants and mix. The nutrient element adjuvants are urea, potassium sulfate, monoammonium phosphate, magnesium sulfate and zinc sulfate in a mass ratio of 1:1:1:1:1. Pass the mixture through a 6-mesh sieve (about 3.8mm aperture) to obtain compound microbial fertilizer.

[0026] The preparation methods of functional additives include: S1. Air-dry the earthworm castings until loose, then dry at 100℃ for 2 hours. Mix 100g of the dried earthworm castings with 300ml of activator, which is a mixture of 15% phosphoric acid solution and 10% aluminum chloride solution at a volume ratio of 2:1. Impregnate at 85℃ for 10 hours, stirring every 2 hours. After impregnation, filter out excess liquid, place the impregnated earthworm castings in an activation furnace and heat to 300℃. Pre-treat under an inert atmosphere for 1.2 hours, then purge with ammonia for 3 minutes. Heat to 400℃ under an ammonia atmosphere and react for 1.5 hours. Cool to room temperature to obtain activated earthworm castings. S2. Dissolve 25g of guanidinoacetic acid in water to prepare a 15% guanidinoacetic acid solution, and dissolve 10g of zinc sulfate in water to prepare a 10% zinc sulfate solution. Mix the guanidinoacetic acid solution and the zinc sulfate solution, adjust the pH to 7.5-10 with 2mol / L sodium hydroxide solution, and react at 50℃ for 60 min with continuous stirring. After the reaction is complete, centrifuge to obtain the guanidinoacetic acid-zinc complex precipitate, wash and dry to obtain the guanidinoacetic acid-zinc complex. S3. Mix 100g of amination-treated earthworm castings, 25g of guanidinoacetic acid-zinc complex, and 8g of ethylene glycol glycidyl ether. Add an appropriate amount of distilled water to adjust the consistency to a paste. Add 1g of triethylamine and react at 35℃ for 4 hours, stirring every 30 minutes. After the reaction is complete, separate the solid product by centrifugation, wash, dry, and pulverize through an 80-mesh sieve to obtain the functional additive.

[0027] Example 3 This embodiment provides a Bacillus vesiculosus compound microbial fertilizer and its preparation method, including the following steps: (1) Mix 70 kg of livestock and poultry manure and 30 kg of plant materials evenly. The plant materials consist of 50 parts rapeseed cake and 35 parts tea oil powder. Add 0.2 kg of primary fermentation microbial agent, which is 5:4:3 of the mass ratio of *Thermophilic Mycorrhizalis* (10×10⁻⁶ bacteria concentration). 10 CFU / g), thermophilic actinomycetes (bacterial concentration 10×10⁻⁶) 10 CFU / g) and Bacillus licheniformis (bacterial concentration 10×10⁻⁶) 10 Composed of CFU / g, the compost is fermented and mixed in a trough-type turning and turning composting process, with the moisture content adjusted to 50-55%, the fermentation temperature set at 65℃, and the fermentation time set at 20 days. The compost is stirred for 15 minutes every 2-3 days to obtain the compost material after one fermentation. (2) After the moisture content of the compost material drops to 55% and the temperature drops to below 45℃ after the first fermentation, take 100kg of the compost material after the first fermentation, add 5kg of functional bacteria and 5kg of functional adjuvant, and the functional bacteria are Bacillus belyssus with a mass ratio of 4:2 (bacterial concentration 1×10⁻⁶). 9 CFU / g) and Bacillus amyloliquefaciens (bacterial concentration 8×10⁻⁶) 8 Mix the ingredients (CFU / g) and continue fermentation for 20 days. Control the temperature of the fermentation pile to not exceed 60℃. Stir once a day for 15 minutes each time. After fermentation, let it air dry until the moisture content is ≤30%. Add 40kg of nutrient element adjuvants and mix them. The nutrient element adjuvants are urea, potassium sulfate, monoammonium phosphate, magnesium sulfate and zinc sulfate in a mass ratio of 1:1:1:1:1. Pass the mixture through a 6-mesh sieve (about 3.8mm aperture) to obtain compound microbial fertilizer.

[0028] The preparation methods of functional additives include: S1. Air-dry earthworm castings until loose, then dry at 100℃ for 2 hours. Mix 100g of dried earthworm castings with 500ml of activator, which is a mixture of 15% phosphoric acid solution and 10% aluminum chloride solution at a volume ratio of 2:1. Impregnate at 95℃ for 8 hours, stirring every 2 hours. After impregnation, filter out excess liquid, place the impregnated earthworm castings in an activation furnace and heat to 350℃. Pre-treat under an inert atmosphere for 1 hour, then purge with ammonia for 3 minutes. Heat to 500℃ under an ammonia atmosphere and react for 1 hour. Cool to room temperature to obtain ammoniated earthworm castings. S2. Dissolve 35g of guanidinoacetic acid in water to prepare a 15% guanidinoacetic acid solution, and dissolve 10g of zinc sulfate in water to prepare a 10% zinc sulfate solution. Mix the guanidinoacetic acid solution and the zinc sulfate solution, adjust the pH to 10 with 2mol / L sodium hydroxide solution, and react at 60℃ for 30 min with continuous stirring. After the reaction is complete, centrifuge to obtain the guanidinoacetic acid-zinc complex precipitate, wash and dry to obtain the guanidinoacetic acid-zinc complex. S3. Mix 100g of amination-treated earthworm castings, 35g of guanidinoacetic acid-zinc complex, and 12g of ethylene glycol glycidyl ether. Add an appropriate amount of distilled water to adjust the consistency to a paste. Add 2g of triethylamine and react at 45℃ for 2 hours, stirring every 30 minutes. After the reaction is complete, separate the solid product by centrifugation, wash, dry, and pulverize through an 80-mesh sieve to obtain the functional additive.

[0029] Comparative Example 1 This comparative example provides a Bacillus belyssus compound microbial fertilizer and its preparation method. The preparation method is the same as in Example 1, except that: in step (2), Bacillus belyssus was purchased from the China General Microbiological Culture Collection Center, with the number S10B1, and the bacterial concentration was 1×10⁻⁶. 9 CFU / g.

[0030] Comparative Example 4 This comparative example provides a Bacillus vesiculosus compound microbial fertilizer and its preparation method. The preparation method is the same as in Example 1, except that the functional bacteria are Bacillus vesiculosus (bacterial concentration 1.8 × 10⁻⁶). 9 CFU / g).

[0031] Comparative Example 3 This comparative example provides a Bacillus vesiculosus compound microbial fertilizer and its preparation method. The preparation method is the same as in Example 1, except that: S1. Dry biomass activated carbon at 100℃ for 2 hours. Mix 100g of dried activated carbon with 400ml of activating agent, which is a mixture of 15% phosphoric acid solution and 10% aluminum chloride solution at a volume ratio of 2:1. Impregnate at 90℃ for 9 hours, stirring every 2 hours. After impregnation, filter out excess liquid. Place the impregnated activated carbon in an activation furnace and heat to 330℃. Pre-treat under an inert atmosphere for 1.1 hours, then purge with ammonia for 3 minutes. Heat to 450℃ under an ammonia atmosphere and react for 1.2 hours. Cool to room temperature to obtain activated carbon. S2. Dissolve 30g of guanidinoacetic acid in water to prepare a 15% guanidinoacetic acid solution, and dissolve 10g of zinc sulfate in water to prepare a 10% zinc sulfate solution. Mix the guanidinoacetic acid solution and the zinc sulfate solution, adjust the pH to 8.5 with 2mol / L sodium hydroxide solution, and react at 55℃ for 45 min with continuous stirring. After the reaction is complete, centrifuge to obtain the guanidinoacetic acid-zinc complex precipitate, wash and dry to obtain the guanidinoacetic acid-zinc complex. S3. Mix 100g of amination-treated earthworm castings, 30g of guanidinoacetic acid-zinc complex, and 10g of ethylene glycol glycidyl ether. Add an appropriate amount of distilled water to adjust the consistency to a paste. Add 1.5g of triethylamine and react at 40℃ for 3 hours, stirring every 30 minutes. After the reaction is complete, separate the solid product by centrifugation, wash, dry, and pulverize through an 80-mesh sieve to obtain the functional additive.

[0032] Comparative Example 4 This comparative example provides a Bacillus vesiculosus compound microbial fertilizer and its preparation method. The preparation method is the same as in Example 1, except that: The preparation methods of functional additives include: S1. Air-dry earthworm castings until loose, then dry at 100℃ for 2 hours. Mix 100g of dried earthworm castings with 400ml of activator, which is a mixture of 15% phosphoric acid solution and 10% aluminum chloride solution at a volume ratio of 2:1. Impregnate at 90℃ for 9 hours, stirring every 2 hours. After impregnation, filter out excess liquid, place the impregnated earthworm castings in an activation furnace and heat to 330℃. Pre-treat under an inert atmosphere for 1.1 hours, then purge with ammonia for 3 minutes. Heat to 450℃ under an ammonia atmosphere and react for 1.2 hours. Cool to room temperature to obtain activated earthworm castings. S2. Dissolve 30g of guanidinoacetic acid in water to prepare a 15% guanidinoacetic acid solution, and dissolve 10g of zinc sulfate in water to prepare a 10% zinc sulfate solution. Mix the guanidinoacetic acid solution and the zinc sulfate solution, adjust the pH to 8.5 with 2mol / L sodium hydroxide solution, and react at 55℃ for 45 min with continuous stirring. After the reaction is complete, centrifuge to obtain the guanidinoacetic acid-zinc complex precipitate, wash and dry to obtain the guanidinoacetic acid-zinc complex. S3. Mix 100g of activated earthworm castings and 30g of guanidinoacetic acid-zinc complex, add an appropriate amount of distilled water to adjust to a paste, mix at 35℃ for 3h, stirring once every 30min during the process. After mixing, separate the solid product by centrifugation, wash, dry, and pulverize through an 80-mesh sieve to obtain the functional additive.

[0033] Performance testing 1. Disease prevention and control effectiveness Select tomato seedlings with uniform growth at the 5-leaf stage, and inoculate them with Fusarium oxysporum (the causal agent of tomato wilt) using the root soaking method. Rinse off any soil particles from the roots with sterile water, then cut the main root 0.2 cm from the base using sterile scissors. Soak the roots in a prepared suspension of Fusarium oxysporum spores (10...). 6 After soaking in water (CFU / mL) for 20 minutes, transfer to a flowerpot for planting.

[0034] Experimental Design: A total of eight groups were established: Example 1, Example 2, Example 3, Comparative Example 1, Comparative Example 2, Comparative Example 3, Comparative Example 4, and a control group. Each group was treated with the corresponding compound microbial fertilizer. The control group received no compound microbial fertilizer. Each group had six flowerpots, with two flowerpots in the same group forming a parallel group, for a total of 48 flowerpots. The flowerpots used in the experiment had an inner diameter of 0.40 m × 0.30 m and a height of 0.16 m, with small holes at the bottom. Each flowerpot contained 21 kg of soil to a depth of approximately 0.15 m, and six tomato plants were planted in each pot. The row spacing for the tomato plants was 0.15 m, the row spacing was 0.12 m, and the distance from the edge of the pot was approximately 0.075 m. The compound microbial fertilizer prepared in the examples and comparative examples was applied to each flowerpot at a rate of 210 g / pot. The plants were cultivated in a greenhouse at 25-30℃ under natural light, with regular watering to keep the soil moist. Ten days after the tomato seedlings developed two true leaves, they were inoculated with fungicide. Thirty days after inoculation, the severity of tomato wilt was assessed. Disease incidence was recorded according to disease severity criteria, and the disease index and control effectiveness were calculated. Throughout the growing season, morphological differences in tomato seedlings, including plant height, stem diameter, and total fresh weight, were monitored.

[0035] Grading criteria for tomato wilt disease (Wang Jing et al., 2018): Grade 0: No symptoms; Grade 1: One or two cotyledons turn yellow; Grade 2: Three or four true leaves turn yellow and wilt and droop; Grade 3: Five or six true leaves turn yellow or wilt and droop; Grade 4: The whole plant wilts severely and dies.

[0036] Incidence rate = (Number of infected plants / Total number of plants) × 100% Disease index = (Number of diseased plants at each level × Corresponding disease level) / (Total number of plants × Highest disease level) × 100; Prevention and control effect = (control disease index - treatment disease index) / control disease index × 100%. The test results are shown in Table 1 and Table 2.

[0037] Table 1 Disease control effectiveness

[0038] As shown in Table 1, the data on incidence rate, disease index, and control effect indicate that using Bacillus vesiculosus to produce compound microbial fertilizer products can reduce the incidence rate and disease index of tomato seedling wilt, and improve the control effect of the compound microbial fertilizer products prepared by the technical solution of this invention on tomato seedling wilt.

[0039] Table 2. Growth-promoting effects

[0040] As shown in Table 2, the plant height, stem diameter and whole plant fresh weight of the compound microbial fertilizer prepared by the technical solution of the present invention are significantly higher than those of the example group, and it has a better growth-promoting effect.

[0041] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. 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 method for preparing a Bacillus vesiculosus compound microbial fertilizer, characterized in that: Includes the following steps: (1) Mix livestock and poultry manure and plant materials in a ratio of (6-7): (3-4) evenly, add primary fermentation microbial agent, and ferment and mix them by turning and turning in a trough, adjust the moisture content to 50%-55%, the fermentation temperature is 55-65℃, the fermentation time is 20-30 days, and stir once every 2-3 days to obtain the compost material after primary fermentation; (2) After the first fermentation, the moisture content of the compost material drops to 45%-55% and the temperature drops to below 45℃. Add functional bacteria and functional additives, stir and mix, and continue fermentation. Control the temperature of the compost pile during the second fermentation to not exceed 60℃. Stir once a day. After the fermentation is completed, let it air dry naturally, add nutrient element additives and mix to obtain compound microbial fertilizer.

2. The method for preparing a Bacillus vesiculosus compound microbial fertilizer as described in claim 1, characterized in that: The plant raw materials, by weight, include 30-50 parts rapeseed cake and 25-35 parts tea oil powder; the nutrient element adjuvants are three or more of urea, potassium sulfate, monoammonium phosphate, magnesium sulfate, zinc sulfate, humic acid and potassium fulvate, and the amount of nutrient element adjuvants added is 15%-40% of the mass of the compost material after secondary fermentation.

3. The method for preparing a Bacillus vesiculosus compound microbial fertilizer as described in claim 1, characterized in that: The amount of the primary fermentation microbial agent added is 0.02%-0.2% of the total mass of the compost raw materials, and the effective viable bacteria count is (1-10)×10⁻⁶. 10 CFU / g, wherein the primary fermentation microbial agent comprises sparsely cottony thermophilic filamentous fungi, thermophilic actinomycetes and Bacillus licheniformis in a mass ratio of 4-5:3-4:2-3.

4. The method for preparing a Bacillus vesiculosus compound microbial fertilizer as described in claim 1, characterized in that: In step (2), the mass ratio of the compost material, functional bacteria and functional additives after primary fermentation is 100:0.8-5:0.5-5.

5. The method for preparing a Bacillus vesiculosus compound microbial fertilizer as described in claim 4, characterized in that: The functional bacteria are Bacillales velezensis and Bacillales amyloliquefaciens, with a mass ratio of 3-4:1-2. The Bacillales velezensis is Bacillales velezensis YFB3-1, with accession number CCTCCNO:M20221140.

6. The method for preparing a Bacillus vesiculosus compound microbial fertilizer as described in claim 1, characterized in that: The preparation method of the functional additive includes: S1. Air-dry the earthworm castings until loose, then dry them completely. Mix the dried earthworm castings with an activator and impregnate them. Then place them in an activation furnace and heat them to 300-350℃ for 1-1.2 hours for pretreatment. Then, purge them with ammonia gas and heat them to 400-500℃ under an ammonia atmosphere for 1-1.5 hours to obtain ammoniated earthworm castings. S2. Mix guanidinoacetic acid solution and zinc sulfate solution, adjust the pH of the solution to 7.5-10, and react at 50-60℃ for 30-60 min to obtain guanidinoacetic acid-zinc complex; S3. Mix aminated earthworm castings, guanidinoacetic acid-zinc complex and ethylene glycol glycidyl ether, and react at 30-40℃ for 2-4 hours. After the reaction is completed, separate and dry to obtain the functional additive.

7. The method for preparing a Bacillus vesiculosus compound microbial fertilizer as described in claim 5, characterized in that: In step S1, the solid-liquid ratio of earthworm castings and activator is 1g:3-5ml, the activator is phosphoric acid and aluminum chloride, the impregnation temperature is 85-95℃, and the impregnation time is 8-10h.

8. The method for preparing a Bacillus vesiculosus compound microbial fertilizer as described in claim 1, characterized in that: In step S2, the mass ratio of guanidinoacetic acid to zinc sulfate is 2.5-3.5:1; in step S3, the mass ratio of aminated earthworm castings, guanidinoacetic acid-zinc complex, ethylene glycol glycidyl ether, and triethylamine is 10:2.5-3.5:0.8-1.2:0.1-0.

2.

9. A Bacillus belesii compound microbial fertilizer, characterized in that: The compound microbial fertilizer is prepared using the preparation method described in any one of claims 1-8.

10. The application of the Bacillus vesiculosus compound microbial fertilizer as described in claim 9 in promoting plant growth.

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