Preparation method and application of bio-organic fertilizer based on secondary fermentation propagation of bacillus velezensis

Abstract

The invention relates to the technical field of bio-organic fertilizers, in particular to a preparation method and application of a bio-organic fertilizer based on secondary fermentation propagation of bacillus velezensis, and the preparation method comprises the following steps: uniformly mixing livestock and poultry manure and straw powder, adding a high-efficiency decay-promoting microbial inoculum and a compound additive, and carrying out trough type turning, stirring and mixing to obtain the bio-organic fertilizer. A compost material after primary fermentation is obtained; and after the water content of the compost material after primary fermentation is reduced to 45-55% and the temperature is reduced to 45 DEG C or below, adding an amino acid raw material and a bacillus velezensis inoculant, stirring and mixing, continuing fermentation, controlling the temperature of the compost body for secondary fermentation to be less than or equal to 60 DEG C, stirring once a day, and naturally airing after fermentation is completed, thereby obtaining the biological organic fertilizer. According to the technical scheme, the effective viable count in the biological organic fertilizer and the soil colonization effect of the biological organic fertilizer are improved, so that the prevention and control effect on continuous cropping obstacle related pathogenic fungal diseases is improved, and an efficient and reliable technical scheme is provided for biological prevention and control of continuous cropping obstacles of traditional Chinese medicinal materials and greenhouse vegetables.

Description

Technical Field

[0001] The present invention relates to the technical field of bio-organic fertilizers, and in particular to a preparation method and application of bio-organic fertilizers based on secondary fermentation and propagation of Bacillus velez. Background Art

[0002] With the adjustment of planting structures and the vigorous development of the agricultural industry, the cultivation of Chinese medicinal herbs and vegetables has become increasingly intensive, large-scale, and monocultured. This has led to the widespread practice of continuous cropping of some Chinese medicinal herbs and greenhouse vegetables. Long-term continuous cropping leads to deterioration of soil physical properties, deficiencies in certain trace elements, strong allelopathic effects, or a homogenous decomposition of residues. This selectively selects for rhizosphere microorganisms, altering soil microbial communities and significantly reducing microbial functional diversity, primarily manifested by a significant increase in the number of pathogenic fungi. This leads to an imbalance in the soil microbial community structure and is a major factor in the development of continuous cropping problems.

[0003] To alleviate the problem of continuous cropping, bio-organic fertilizer has received widespread attention as an environmentally friendly solution. Bio-organic fertilizer is made by inoculating beneficial microorganisms into organic fertilizer. It not only provides nutrients for crops, but also inhibits soil pathogens through the antagonistic effects of biocontrol bacteria, thereby improving the structure of soil microbial communities. However, most organic fertilizer plants currently produce bio-organic fertilizer by directly mixing biocontrol microbial strains with organic fertilizer. This method has obvious limitations: on the one hand, due to the large number of microorganisms in organic fertilizer and the complex environment, the added biocontrol bacteria have difficulty surviving and maintaining activity; on the other hand, biocontrol bacteria have difficulty colonizing in the soil, making it difficult to achieve a sustained biocontrol effect.

[0004] Existing patent CN202411411538.1 discloses a multi-effect bio-organic fertilizer prepared from landscaping waste. This technology combines biochar prepared from landscaping waste with peanut shell nanocellulose to provide a habitat and breeding ground for microorganisms. However, this technology still has shortcomings: first, biocontrol bacteria have difficulty surviving during high-temperature fermentation and need to be re-inoculated after fermentation is completed. In addition, the inoculated biocontrol bacteria have a low survival rate in complex fertilizer environments; second, the carrier material used lacks specific protection and nutrient supply functions for functional microorganisms, and cannot ensure that the biocontrol bacteria maintain stable activity during storage and application; third, the bio-organic fertilizer prepared by this technical solution lacks effective long-term control measures for the pathogenic fungi that are the main problem of continuous cropping problems. Summary of the Invention

[0005] In view of this, the present invention proposes a preparation method and application of a bio-organic fertilizer based on secondary fermentation and propagation of Bacillus velezensis, so as to solve the problems of low survival rate of biocontrol bacteria in existing bio-organic fertilizers and difficulty in soil colonization, resulting in poor effect in preventing and controlling continuous cropping disorders.

[0006] The technical solution of the present invention is achieved as follows: The present invention provides a method for preparing a bio-organic fertilizer based on secondary fermentation and propagation of Bacillus velezensis, comprising the following steps:

[0007] (1) livestock and poultry manure and straw powder are mixed evenly, and a high-efficiency decay-promoting agent and a composite auxiliary agent are added, wherein the composite auxiliary agent includes biochar loaded with ergosterol, and the mixture is stirred and mixed by a trough-type composting fermentation method, and the moisture content is adjusted to 50-55%, the fermentation temperature is 55-65°C, the fermentation time is 20-30 days, and the mixture is stirred every 2-3 days to obtain a compost material after fermentation;

[0008] (2) After the water content of the compost material after the primary fermentation drops to 45-55%, the temperature is reduced to below 45° C., amino acid raw materials and Velez Bacillus inoculum are added, stirred and mixed, and fermentation is continued. The temperature of the heap body of the secondary fermentation is controlled to be no more than 60° C. by the technical processes such as the height of the heap and timely turning of the heap, which is conducive to the rapid growth and reproduction of Velez Bacillus and obtains a bio-organic fertilizer product with a high bacterial cell concentration. The compost material is stirred once a day. After the fermentation is completed, the compost material is naturally dried to obtain a bio-organic fertilizer.

[0009] In the present invention, during the first fermentation stage, high-temperature fermentation at 55-65°C ensures the full decomposition of livestock and poultry manure and straw, and the thorough inactivation of pathogens. Simultaneously, the ergosterol-loaded biochar composite adjuvant not only promotes the efficient decomposition of organic materials during this stage but also creates an ideal carrier environment for the subsequent inoculation of biocontrol bacteria. When the temperature drops below 45°C and enters the second fermentation, Bacillus velezensis achieves efficient propagation under suitable temperature conditions and amino acid nutritional support. The biochar carrier in the composite adjuvant provides a protective microenvironment for the strain, significantly improving the survival rate and activity retention of the biocontrol bacteria. The sustained release of ergosterol further enhances the strain's colonization ability and biocontrol effectiveness.

[0010] Based on the above technical solution, preferably, the Bacillus velezensis is Bacillales velezensis YFB3-1, and its deposit number is CCTCC NO: M20221140.

[0011] On the basis of the above technical solution, preferably, the amino acid raw material includes one or more of glycine, leucine, cystine, serine, tyrosine, γ-polyglutamic acid and aspartic acid.

[0012] The Bacillales velezensis YFB3-1 strain is a highly efficient and broad-spectrum anti-pathogenic fungal strain isolated and screened from earthworm feces, and has a strong biocontrol effect. The strain has unique physiological characteristics of rapid growth and reproduction, strong stress resistance, high temperature resistance and preference for amino acid nutrition. These characteristics are perfectly matched with the secondary fermentation process of the present invention: after the first high-temperature fermentation is completed, when the pile temperature drops below 45°C, the added amino acid raw materials provide a high-quality nutrient source for the YFB3-1 strain, promoting its rapid reproduction and colonization under medium temperature conditions. Through the secondary fermentation process, the functional bacteria and organic fertilizer are deeply integrated to form a stable microbial group, which not only significantly improves the activity and biocontrol function of Bacillales velezensis, but more importantly, after the strain is applied to the soil, it can form a mutually beneficial symbiotic relationship with the earthworms in the soil, synergistically enhance the antagonistic ability to soil-borne pathogenic fungi such as Fusarium oxysporum, fundamentally improve the soil microbial community structure, and effectively alleviate the problem of continuous cropping obstacles.

[0013] On the basis of the above technical solution, preferably, the preparation method of the composite auxiliary agent includes:

[0014] S1, ball-milling the mushroom dregs and shell powder to obtain a mixture, and subjecting the mixture to high-temperature pyrolysis and carbonization to obtain composite biochar;

[0015] S2. Dispersing the composite biochar in an ethanol aqueous solution, adding a mercaptosilane coupling agent under an inert environment, and ball milling at 30-40° C. for 8-10 hours to obtain thiol biochar;

[0016] S3. Disperse the thiol-modified biochar in ethanol, add ergosterol and benzophenone, and react at 25-40° C. for 8-10 hours under ultraviolet light to obtain a composite additive.

[0017] Mushroom husks are waste materials after edible fungus cultivation. They are rich in biomass components such as lignin, cellulose and hemicellulose. After high-temperature pyrolysis, they can form a developed porous structure and provide a good carbon skeleton foundation. The main component of shell powder is calcium carbonate, which decomposes to produce calcium oxide and carbon dioxide during high-temperature pyrolysis. Calcium oxide, as a pore-forming agent, can significantly increase the specific surface area and porosity of biochar. At the same time, it adjusts the pH value of biochar, making it more suitable for microbial attachment, which is conducive to improving the survival rate of biocontrol bacteria. Then, a sulfhydryl group is introduced by a silane coupling reaction between a mercaptosilane coupling agent and the hydroxyl groups on the surface of the composite biochar. The sulfhydryl group is grafted with ergosterol to achieve a firm fixation of ergosterol on the biochar carrier, avoiding the problem of ergosterol being easily decomposed by light or heat in the conventional blending method. The composite adjuvant prepared by the present invention has both the porous carrier function of biochar and the biological activity promotion function of ergosterol, which can provide a protective microenvironment and a nutrient-promoting factor for Bacillus velezini, significantly improving the survival rate, activity and soil colonization ability of biocontrol bacteria.

[0018] On the basis of the above technical solution, preferably, in step S1, the mass ratio of mushroom bran and shell powder is 6-8:1; high-temperature pyrolysis specifically includes: heating the mixture to 450-550℃ at a rate of 15-20℃ / min and keeping it warm for 2-3h, and then continuing to heat it to 700-800℃ and keeping it warm for 1-1.5h.

[0019] On the basis of the above technical solution, preferably, in step S2, the mass ratio of the composite biochar to the mercaptosilane coupling agent is 100:3-8, and the mercaptosilane coupling agent includes 3-mercaptopropyltrimethoxysilane or 3-mercaptopropyltriethoxysilane.

[0020] Based on the above technical solution, preferably, in step S3, the mass ratio of thiolated biochar, ergosterol and benzophenone is 100:5-15:0.5-2.

[0021] On the basis of the above technical solution, preferably, in step (1), the mass ratio of livestock and poultry manure to straw powder is 7-8:2-3; the amount of the high-efficiency decay-promoting bacterium added is 0.3-0.8% of the total weight of the mixture of livestock and poultry manure and straw powder, and the amount of the composite auxiliary agent added is 0.5-1.5% of the total weight of the mixture of livestock and poultry manure and straw powder; the amount of the amino acid raw material added is 0.3-0.6% of the total weight of the compost material after the first fermentation, and the amount of the Bacillus Velez bacteria agent added is 0.1-0.3% of the total weight of the compost material after the first fermentation.

[0022] On the basis of the above technical solution, preferably, the high-efficiency decay-promoting bacteria agent comprises Thermomyces lanuginosa, Thermoactinomycetes and Bacillus licheniformis in a mass ratio of 4-5:3-4:2-3.

[0023] Thermomyces lanuginosus primarily secretes cellulases and hemicellulases, specifically degrading cellulose and hemicellulose in straw. Thermoactinomycetes produce lignin-degrading enzymes such as lignin peroxidase and laccase. Bacillus licheniformis secretes powerful proteases and lipases, effectively breaking down proteins and lipids in livestock and poultry manure. These three bacteria form a comprehensive enzyme network that comprehensively degrades the main components of organic waste, including cellulose, hemicellulose, lignin, protein, and fat.

[0024] The present invention provides a bio-organic fertilizer, which is prepared by the above-mentioned preparation method.

[0025] The invention provides an application of a bio-organic fertilizer, which is used for planting Chinese medicinal materials and vegetables.

[0026] The method for preparing a bio-organic fertilizer based on secondary fermentation and propagation of Bacillus velezensis and its application have the following beneficial effects compared with the prior art:

[0027] (1) The present invention adopts a secondary fermentation process to solve the key technical problems of low survival rate of biocontrol bacteria and difficulty in soil colonization in the preparation of traditional bio-organic fertilizers. The first stage is a high-temperature fermentation of 55-65°C to achieve full decomposition of organic materials and inactivation of pathogens. The second stage is a medium-temperature fermentation below 45°C to provide a suitable environment for the expansion and propagation of Bacillus velezinoff. Combined with the protection of the composite biochar carrier loaded with ergosterol and the nutritional support of amino acids, the number of viable bacteria in the bio-organic fertilizer and the soil colonization rate are increased, thereby further improving the control effect of pathogenic fungi related to continuous cropping obstacles, and providing an efficient and reliable technical solution for the biological control of continuous cropping obstacles of Chinese medicinal materials and facility vegetables;

[0028] (2) Bacillales velezensis YFB3-1 was used as the functional strain. This strain was isolated from earthworm feces and has high-efficiency and broad-spectrum anti-pathogenic fungal activity. Its physiological characteristics are highly compatible with the secondary fermentation process and amino acid nutrient supply system. After application to the soil, it can form a mutually beneficial symbiotic relationship with earthworms, synergistically enhancing the antagonistic effect against soil-borne pathogenic fungi and fundamentally improving the soil microbial community structure.

[0029] (3) The composite biochar carrier prepared by pyrolysis of mushroom bran and shell powder provides an ideal habitat for biocontrol bacteria; the covalent grafting and fixation of ergosterol are achieved by surface modification with mercaptosilane coupling agent and mercapto-olefin addition reaction, forming a stable sustained-release system. Compared with the conventional physical blending method, it avoids the rapid loss of active ingredients and environmental interference, continuously provides membrane stabilizing factors for Bacillus velezensis, and significantly improves the survival rate, activity retention ability and soil colonization efficiency of the strain;

[0030] (4) A combination of thermophilic hyphomycetes, thermophilic actinomycetes and Bacillus licheniformis is used to form a complete cellulase, ligninase and protease system to achieve comprehensive and efficient degradation of the main components of organic waste. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0032] Figure 1 It is the effective viable bacterial count of the biocontrol bacteria in the biological organic fertilizer prepared in Example 1 of the present invention. DETAILED DESCRIPTION

[0033] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0034] It should be noted that the Bacillus velezensis agent YFB3-1 was deposited at the China Center for Type Culture Collection with the number CCTCC M 20221140, the deposit date was July 21, 2022, and the deposit address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province. Thermomyces lanuginosus was purchased from Mingzhou Biotechnology with the catalog number BMZ148400; thermophilic actinomycetes were purchased from Mingzhou Biotechnology with the catalog number BMZ135187; and Bacillus licheniformis was purchased from Mingzhou Biotechnology with the catalog number BMZ135440.

[0035] Example 1

[0036] This embodiment provides a method for preparing a bio-organic fertilizer based on secondary fermentation and propagation of Bacillus velezensis, comprising the following steps:

[0037] (1) 75 kg of livestock and poultry manure and 25 kg of straw powder were mixed evenly, and 550 g of high-efficiency decay-promoting bacteria and 1000 g of composite additives were added. The high-efficiency decay-promoting bacteria were Thermomyces lanuginosa (with a mass ratio of 4.5:3.5:2.5) (bacterial concentration 1×10 8 CFU / g), thermophilic actinomycetes (bacterial concentration 8×10 7 CFU / g) and Bacillus licheniformis (bacterial concentration 5×10 7 CFU / g), stirred and mixed for 18 minutes by trough composting fermentation, etc., adjusted the moisture content to 53%, the fermentation temperature to 60°C, the fermentation time to 25 days, and stirred for 15 minutes every 3 days to obtain a compost material after primary fermentation;

[0038] (2) After the water content of the compost material after the primary fermentation dropped to 50% and the temperature dropped to below 45°C, 50 kg of the compost material after the primary fermentation was taken, 225 g of amino acid raw materials (82 g of glycine, 75 g of cystine, 45 g of γ-polyglutamic acid and 23 g of aspartic acid) and 100 g of Bacillus velezensis YFB3-1 (with a bacterial concentration of 5 × 10 9CFU / g), fully stirred and mixed in a blender for 20 minutes, and continued to ferment for 15 days. The temperature of the pile during the secondary fermentation was controlled to not exceed 60°C by means of technical processes such as the height of the pile and timely turning of the pile. The pile was stirred for 10 minutes every day to maintain the temperature of the pile at 30-40°C. After the fermentation was completed, it was naturally dried to a moisture content of ≤30%, and passed through a 2mm sieve to obtain a biological organic fertilizer.

[0039] The preparation method of the composite auxiliary agent includes:

[0040] S1. 70 g of edible fungus husk (water content ≤ 10%) and 10 g of shell powder (200 mesh) were ball-milled in a planetary ball mill at a speed of 300 r / min for 2 h to obtain a mixture, the mixture was placed in a tubular furnace quartz boat, and under nitrogen protection (flow rate 200 ml / min), the temperature was raised to 450-550° C. at a rate of 18° C. / min and kept warm for 2.5 h, then continued to rise to 750° C. and kept warm for 1.2 h, and then naturally cooled to room temperature after high-temperature pyrolysis and carbonization to obtain composite biochar;

[0041] S2. Disperse 100 g of composite biochar in 1000 ml of ethanol-water solution, add 5.5 g of 3-mercaptopropyltrimethoxysilane under an inert environment, and ball-mill at 35 °C for 9 h. After the reaction is completed, filter and wash with anhydrous ethanol three times, and vacuum dry to obtain thiolated biochar;

[0042] S3. Disperse 100 g of thiolated biochar in 1000 ml of ethanol, add 10 g of ergosterol and 1.0 g of benzophenone, and react at 35 ° C for 9 h under ultraviolet light irradiation (wavelength 365 nm, power 250 W, distance 15 cm). After the reaction is completed, centrifuge (8000 r / min, 10 min), wash with ethanol and acetone in turn, and vacuum dry to obtain a composite auxiliary agent.

[0043] Example 2

[0044] This embodiment provides a method for preparing a bio-organic fertilizer based on secondary fermentation and propagation of Bacillus velezensis, comprising the following steps:

[0045] (1) 70 kg of livestock and poultry manure and 30 kg of straw powder were mixed evenly, and 300 g of high-efficiency decay-promoting bacteria and 500 g of composite additives were added. The high-efficiency decay-promoting bacteria were Thermomyces lanuginosus (bacterial concentration 1×10 8 CFU / g), thermophilic actinomycetes (bacterial concentration 8×10 7 CFU / g) and Bacillus licheniformis (bacterial concentration 5×10 7CFU / g), stirred and mixed for 15 minutes by trough composting fermentation, etc., adjusted the moisture content to 50%, the fermentation temperature to 55°C, the fermentation time to 30 days, and stirred for 10 minutes every 2 days to obtain a compost material after primary fermentation;

[0046] (2) After the water content of the compost material after the primary fermentation dropped to 45% and the temperature dropped to below 45°C, 50 kg of the compost material after the primary fermentation was taken, 150 g of amino acid raw materials (55 g of glycine, 50 g of cystine, 30 g of γ-polyglutamic acid and 15 g of aspartic acid) and 50 g of Bacillus velezensis YFB3-1 (with a bacterial concentration of 5 × 10 9 CFU / g), fully stirred and mixed in a blender for 20 minutes, and continued to ferment for 15 days. The temperature of the pile during the secondary fermentation was controlled to not exceed 60°C by means of technical processes such as the height of the pile and timely turning of the pile. The pile was stirred for 10 minutes every day to maintain the temperature of the pile at 30-40°C. After the fermentation was completed, it was naturally dried to a moisture content of ≤30%, and passed through a 2mm sieve to obtain a biological organic fertilizer.

[0047] The preparation method of the composite auxiliary agent includes:

[0048] S1. 60 g of edible fungus husk (water content ≤ 10%) and 10 g of shell powder (200 mesh) were ball-milled in a planetary ball mill at a speed of 300 r / min for 2 h to obtain a mixture, and the mixture was placed in a tubular furnace quartz boat. Under nitrogen protection (flow rate 200 ml / min), the temperature was raised to 450° C. at a rate of 15° C. / min and kept warm for 3 h, and then continued to rise to 700° C. and kept warm for 1.5 h. After high-temperature pyrolysis and carbonization, it was naturally cooled to room temperature to obtain composite biochar;

[0049] S2. Disperse 100 g of composite biochar in 1000 ml of ethanol-water solution, add 3 g of 3-mercaptopropyltrimethoxysilane under an inert environment, and ball-mill for 10 h at 30 °C. After the reaction is complete, filter, wash with anhydrous ethanol three times, and vacuum dry to obtain thiolated biochar;

[0050] S3. Disperse 100 g of thiolated biochar in 1000 ml of ethanol, add 5 g of ergosterol and 0.5 g of benzophenone, and react at 25 ° C for 10 h under ultraviolet light irradiation (wavelength 365 nm, power 250 W, distance 15 cm). After the reaction is completed, centrifuge (8000 r / min, 10 min), wash with ethanol and acetone in turn, and vacuum dry to obtain a composite auxiliary agent.

[0051] Example 3

[0052] This embodiment provides a method for preparing a bio-organic fertilizer based on secondary fermentation and propagation of Bacillus velezensis, comprising the following steps:

[0053] (1) 70 kg of livestock and poultry manure and 30 kg of straw powder were mixed evenly, and 800 g of high-efficiency pro-rotting agent and 1500 g of composite additive were added, wherein the high-efficiency pro-rotting agent was Thermomyces lanuginosus (bacterial concentration 1×10 8 CFU / g), thermophilic actinomycetes (bacterial concentration 8×10 7 CFU / g) and Bacillus licheniformis (bacterial concentration 5×10 7 CFU / g), stirred and mixed for 20 minutes by trough composting fermentation, etc., adjusted the moisture content to 55%, the fermentation temperature to 65°C, the fermentation time to 20 days, and stirred for 15 minutes every 3 days to obtain a compost material after primary fermentation;

[0054] (2) After the water content of the compost material after the primary fermentation dropped to 55% and the temperature dropped to below 45°C, 50 kg of the compost material after the primary fermentation was taken, 300 g of amino acid raw materials (108 g of glycine, 102 g of cystine, 60 g of γ-polyglutamic acid and 30 g of aspartic acid) and 150 g of Bacillus velezensis YFB3-1 (with a bacterial concentration of 5 × 10 9 CFU / g), fully stirred and mixed in a blender for 20 minutes, and continued to ferment for 15 days. The temperature of the pile during the secondary fermentation was controlled to not exceed 60°C by means of technical processes such as the height of the pile and timely turning of the pile. The pile was stirred for 10 minutes every day to maintain the temperature of the pile at 30-40°C. After the fermentation was completed, it was naturally dried to a moisture content of ≤30%, and passed through a 2mm sieve to obtain a biological organic fertilizer.

[0055] The preparation method of the composite auxiliary agent includes:

[0056] S1. 80 g of edible fungus husk (water content ≤ 10%) and 10 g of shell powder (200 mesh) were ball-milled in a planetary ball mill at a speed of 300 r / min for 2 h to obtain a mixture, the mixture was placed in a tubular furnace quartz boat, and the temperature was raised to 550° C. at a rate of 20° C. / min under nitrogen protection (flow rate 200 ml / min) and kept warm for 2 h, then continued to rise to 800° C. and kept warm for 1 h, and high-temperature pyrolysis and carbonization were performed to obtain composite biochar;

[0057] S2. Disperse 100 g of composite biochar in 1000 ml of ethanol-water solution, add 8 g of 3-mercaptopropyltrimethoxysilane under an inert environment, and ball-mill at 40° C. for 8 h to obtain thiolated biochar;

[0058] S3. Disperse 100 g of thiolated biochar in 1000 ml of ethanol, add 15 g of ergosterol and 2 g of benzophenone, and react at 40 ° C for 8 h under ultraviolet light irradiation (wavelength 365 nm, power 250 W, distance 15 cm). After the reaction is completed, centrifuge (8000 r / min, 10 min), wash with ethanol and acetone in turn, and vacuum dry to obtain a composite auxiliary agent.

[0059] Comparative Example 1

[0060] This comparative example provides a method for preparing a bio-organic fertilizer based on secondary fermentation and propagation of Bacillus velezensis. The preparation method is the same as that of Example 1, except that: in step (1), the composite adjuvant is not grafted with ergosterol. The specific preparation method is as follows:

[0061] S1. 70 g of edible fungus husk (water content ≤ 10%) and 10 g of shell powder (200 mesh) were ball-milled in a planetary ball mill at a speed of 300 r / min for 2 h to obtain a mixture, the mixture was placed in a tubular furnace quartz boat, and under nitrogen protection (flow rate 200 ml / min), the temperature was raised to 450-550° C. at a rate of 18° C. / min and kept warm for 2.5 h, then continued to rise to 750° C. and kept warm for 1.2 h, and then naturally cooled to room temperature after high-temperature pyrolysis and carbonization to obtain composite biochar;

[0062] S2. Disperse 100 g of composite biochar in 1000 ml of ethanol, add 10 g of ergosterol, and blend at 35° C. for 9 h. After mixing, centrifuge (8000 rpm, 10 min) and vacuum dry to obtain a composite additive.

[0063] Comparative Example 2

[0064] This comparative example provides a method for preparing a bio-organic fertilizer based on secondary fermentation and propagation of Bacillus velezensis. The preparation method is the same as that in Example 1, except that the biochar raw material of the composite adjuvant is straw. The specific preparation method is as follows:

[0065] S1. 80 g of straw powder (200 mesh) was ball-milled in a planetary ball mill at a speed of 300 r / min for 2 h to obtain a mixture. The mixture was placed in a tubular furnace quartz boat, and heated to 450-550° C. at a rate of 18° C. / min under nitrogen protection (flow rate 200 ml / min) and kept warm for 2.5 h. The temperature was then continued to be raised to 750° C. and kept warm for 1.2 h. After high-temperature pyrolysis and carbonization, the mixture was naturally cooled to room temperature to obtain composite biochar;

[0066] S2-S3 are the same as in Example 1.

[0067] Comparative Example 3

[0068] This comparative example provides a method for preparing a bio-organic fertilizer based on secondary fermentation and propagation of Bacillus velez. The preparation method is the same as that in Example 1, except that: in step (2), Bacillus velez was purchased from the China General Microbial Culture Collection Center and is numbered S10B1. The bacterial concentration is 5×10 9 CFU / g.

[0069] Comparative Example 4

[0070] This comparative example provides a method for preparing a bio-organic fertilizer based on secondary fermentation and propagation of Bacillus velezensis. The preparation method is the same as that in Example 1, except that:

[0071] In step (1), the high-efficiency decay-promoting agent is the thermophilic stearobacillus (bacterial concentration 1×10 8 CFU / g), thermophilic actinomycetes (bacterial concentration 8×10 7 CFU / g) and Bacillus licheniformis (bacterial concentration 5×10 7 CFU / g).

[0072] Performance testing

[0073] 1. Effective live bacteria rate

[0074] The effective viable bacteria count of the biological organic fertilizer prepared in the embodiment and the comparative example was tested, wherein the effective viable bacteria count was tested according to the national standard NY / T798-2004, and the detection object was Bacillus velezensis, and the initial effective viable bacteria count and the effective viable bacteria rate during the 4-month storage period were tested respectively, wherein the effective viable bacteria rate during the 4-month storage period = the effective viable bacteria count in the organic fertilizer after 3 months of storage / the effective viable bacteria count in the organic fertilizer after fermentation was completed × 100%. The test results are shown in Tables 1 and Figure 1 .in, Figure 1 The graph shows the change in the effective viable bacterial rate of the biocontrol bacteria (i.e., Bacillus Velezii) in the biological organic fertilizer prepared in Example 1 as the storage time increases.

[0075] Table 1 Effective viable bacteria rate

[0076]

[0077]

[0078] 2. Disease prevention and control effects

[0079] Select tomato seedlings at the 5-leaf stage with uniform growth, inoculate tomato wilt pathogen (i.e., Fusarium oxysporum) by root dipping method, flush the soil particles on the roots with sterile water, and then cut the main root 0.2 cm from the base with sterile scissors. 6CFU / mL) for 20 min and then transferred to pots for planting.

[0080] Experimental design: A total of eight groups: divided into Example 1 group, Example 2 group, Example 3 group, Comparative Example 1 group, Comparative Example 2 group, Comparative Example 3 group, Comparative Example 4 group and blank group, each group applied corresponding organic fertilizer, wherein the blank control group did not apply organic fertilizer, each group was provided with 6 flower pots, and the two flower pots in the same group were 1 parallel, for a total of 48 pots. The experimental pots were 0.40m × 0.30m in inner diameter at the upper and lower bottoms, 0.16m high, with small holes at the bottom, 21kg of soil per pot, and a soil depth of about 0.15m. Six tomato plants were planted in each pot. The row spacing of the tomatoes planted in the pots was 0.15m, the spacing was 0.12m, and the distance from the pot edge was about 0.075m. The organic fertilizers prepared by the embodiments and comparative examples were applied to each flower pot respectively, and the amount of organic fertilizer applied was 210g / pot. The plants were cultured in a greenhouse at 25-30°C, with natural light and regular watering to keep the soil moist. Inoculation was performed 10 days after the tomato seedlings grew two true leaves. Thirty days after inoculation, the disease status of tomato Fusarium wilt was investigated. The disease severity was recorded according to the disease grade standard, and the disease index and control efficacy were calculated.

[0081] Tomato wilt disease grading standard (Wang Jing et al., 2018): Level 0: no symptoms; Level 1: 1 or 2 cotyledons obviously turn yellow; Level 2: 3 or 4 true leaves turn yellow, and the leaves wilt and droop; Level 3: 5 or 6 true leaves turn yellow or wilt and droop; Level 4: The whole plant wilts severely and even dies.

[0082] Incidence rate = (number of diseased plants / total number of plants) × 100%

[0083] Disease index = (number of diseased plants at each level × corresponding disease level) / (total number of plants × highest disease level) × 100%;

[0084] Control effect = (control disease index - treatment disease index) / control disease index × 100%. The test results are shown in Table 2.

[0085] Table 2 Disease prevention and control effects

[0086] Incidence (%) Disease index (%) Control effect (%) Example 1 27.78 13.19 76.25 Example 2 33.33 15.28 72.5 Example 3 36.11 14.58 73.75 Comparative Example 1 66.67 36.11 35 Comparative Example 2 52.78 24.31 56.25 Comparative Example 3 83.33 47.22 15 Comparative Example 4 44.44 22.92 58.75 Blank group 91.67 55.56 -

[0087] As shown in Table 2, the data on incidence rate, disease index and control effect show that the use of Bacillus Velezii to produce bio-organic fertilizer products can reduce the incidence rate and disease index of tomato seedling wilt disease, and improve the control effect of the bio-organic fertilizer product prepared by the technical solution of the embodiment of the present invention on tomato seedling wilt disease.

[0088] 3. Soil properties

[0089] Soil samples from the potted plants were collected before and after the planting experiment, primarily for basic physical and chemical properties and soil microbial analysis. Soil sampling methods: Soil was drilled to a depth of 20 cm. Three replicates were collected from each treatment group. Three replicates from each treatment group were combined into a single sample, and visible debris, such as gravel and plant roots, were removed. The samples were then brought back to the laboratory for analysis. The collected soil samples were thoroughly mixed and divided into two aliquots. One aliquot was air-dried in a well-ventilated area and used to measure soil enzyme activity and organic matter; the other aliquot was refrigerated at 4°C and used for soil microbial analysis. Soil organic matter was measured using the potassium dichromate method; soil urease activity was determined using the phenol-sodium hypochlorite colorimetric method; soil bulk density was determined using the ring knife method; and pH was measured using the potentiometric method (water:soil = 5:1). Soil microbial counts, including Bacillus and Fusarium oxysporum counts, were determined using a dilution plate count method using a specialized culture medium three months after planting. Results are shown in Table 3.

[0090] Table 3 Soil properties

[0091]

[0092] As can be seen from Tables 2 and 3, the bio-organic fertilizer prepared using Bacillus Velez subtilis in the present invention improves the effective colonization effect of Bacillus in the bio-organic fertilizer in the soil, reduces the number of Fusarium oxysporum, thereby reducing the incidence and disease index of tomato wilt at the seedling stage, enhancing the wilt control effect of tomatoes, and further improving the control effect of tomatoes on other related pathogenic fungal diseases caused by continuous cropping disorders.

[0093] 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 in the scope of protection of the present invention.

Claims

1. A method for preparing a bio-organic fertilizer based on secondary fermentation and propagation of Bacillus velezinii, characterized in that: The following steps are involved: (1) livestock and poultry manure and straw powder are mixed evenly, and a high-efficiency decay-promoting agent and a composite auxiliary agent are added, wherein the composite auxiliary agent includes biochar loaded with ergosterol, and the mixture is stirred and mixed by trough-type composting fermentation, and the moisture content is adjusted to 50-55%, the fermentation temperature is 55-65° C., the fermentation time is 20-30 days, and the mixture is stirred every 2-3 days to obtain a fermented compost material; (2) After the primary fermentation, the moisture content of the compost material drops to 45-55%, the temperature drops to below 45° C., the amino acid raw materials and the Velezac bacteria agent are added, stirred and mixed, and the fermentation is continued. The temperature of the compost body during the secondary fermentation is controlled to be no more than 60° C., stirred once a day, and after the fermentation is completed, naturally dried to obtain a biological organic fertilizer.

2. a kind of bio-organic fertilizer preparation method based on secondary fermentation and propagation of Bacillus Velez subtilis as claimed in claim 1, is characterized in that: The Bacillus velezensis is Bacillales velezensis YFB3-1, and its deposit number is CCTCC NO: M20221140.

3. A method for preparing a bio-organic fertilizer based on secondary fermentation and propagation of Bacillus Velez subtilis as claimed in claim 1, characterized in that: The preparation method of the composite auxiliary agent comprises: S1, ball-milling the mushroom dregs and shell powder to obtain a mixture, and subjecting the mixture to high-temperature pyrolysis and carbonization to obtain composite biochar; S2. Dispersing the composite biochar in an ethanol aqueous solution, adding a mercaptosilane coupling agent under an inert environment, and ball milling at 30-40° C. for 8-10 hours to obtain thiol biochar; S3. Disperse the thiol-modified biochar in ethanol, add ergosterol and benzophenone, and react at 25-40° C. for 8-10 hours under ultraviolet light to obtain a composite additive.

4. a kind of bio-organic fertilizer preparation method based on secondary fermentation and propagation of Bacillus Velez subtilis as claimed in claim 3, is characterized in that: In step S1, the mass ratio of mushroom bran to shell powder is 6-8:1; high-temperature pyrolysis specifically includes: heating the mixture to 450-550°C at a rate of 15-20°C / min and keeping it warm for 2-3 hours, and then continuing to heat it to 700-800°C and keeping it warm for 1-1.5 hours.

5. A method for preparing a bio-organic fertilizer based on secondary fermentation and propagation of Bacillus Velez subtilis as claimed in claim 3, characterized in that: In step S2, the mass ratio of the composite biochar to the mercaptosilane coupling agent is 100:3-8, and the mercaptosilane coupling agent includes 3-mercaptopropyltrimethoxysilane or 3-mercaptopropyltriethoxysilane.

6. A method for preparing a bio-organic fertilizer based on secondary fermentation and propagation of Bacillus Velez subtilis as claimed in claim 3, characterized in that: In step S3, the mass ratio of thiolated biochar, ergosterol and benzophenone is 100:5-15:0.5-2.

7. A method for preparing a bio-organic fertilizer based on secondary fermentation and propagation of Bacillus Velez subtilis as claimed in claim 1, characterized in that: In step (1), the amount of the high-efficiency pro-rot bacteria agent added is 0.3-0.8% of the total weight of the mixture of livestock and poultry manure and straw powder, and the amount of the composite auxiliary agent added is 0.5-1.5% of the total weight of the mixture of livestock and poultry manure and straw powder; in step (2), the amount of the amino acid raw material added is 0.3-0.6% of the total weight of the compost material after the first fermentation, and the amount of the Bacillus Velez bacteria agent added is 0.1-0.3% of the total weight of the compost material after the first fermentation.

8. A method for preparing a bio-organic fertilizer based on secondary fermentation and propagation of Bacillus Velez subtilis as claimed in claim 1, characterized in that: The high-efficiency decay-promoting bacteria agent includes Thermomyces lanuginosa, Thermoactinomycetes and Bacillus licheniformis in a mass ratio of 4-5:3-4:2-3, and the amino acid raw materials include one or more of glycine, leucine, cystine, serine, tyrosine, γ-polyglutamic acid and aspartic acid.

9. A bio-organic fertilizer, characterized in that: The bio-organic fertilizer is prepared by the preparation method according to any one of claims 1 to 8.

10. The use of a bio-organic fertilizer as claimed in claim 9, wherein: The bio-organic fertilizer is used for planting Chinese medicinal materials and vegetables.

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