Bacillus velezensis microbial inoculant, preparation method and application thereof

Abstract

The application provides a bacillus velezensis microbial agent, a preparation method and application thereof, and belongs to the technical field of microorganisms; the preparation method comprises the steps of primary fermentation seed culture, secondary fermentation seed culture, post-treatment and final mixing; the final mixing comprises bentonite pretreatment and mixing; in the mixing step, solid fermentation air-dried products, pretreated bentonite and malt dextrin are mixed to obtain a solid-phase base; liquid microbial preparation of bacillus velezensis, tea saponin, polyethylene glycol 400 and potassium humate are mixed to obtain a liquid-phase composite material; the liquid-phase composite material is sprayed into the solid-phase base to obtain the bacillus velezensis microbial agent; the product prepared by the application has a high number of effective living bacteria, and when used in tomato planting, can effectively reduce the incidence of tomato fusarium wilt, and improve the yield and quality of tomatoes.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a Bacillus belye inoculum agent, its preparation method, and its application. Background Technology

[0002] Fusarium wilt of tomato is a systemic soil-borne disease caused by Fusarium oxysporum f. sp. lycopersici. The pathogen infects the roots, blocks the vascular bundles and secretes fusarin toxins, causing yellowing, wilting and even death of tomato plants. In continuous cropping, the incidence of this disease often reaches more than 30%, and in severely affected areas, it can lead to total crop failure. Currently, production mainly relies on chemical pesticides such as hymexazol and carbendazim for root irrigation control. However, long-term chemical control has led to a series of problems, including increased pathogen resistance, soil microecological imbalance, excessive pesticide residues in agricultural products, and environmental pollution. Utilizing beneficial microorganisms for biological control is an inevitable trend for achieving green and sustainable agricultural development.

[0003] Bacillus spp. has become the mainstream species for biological control agents due to its rapid reproduction rate, strong resistance (spore production), and ability to produce a variety of secondary metabolites. However, existing Bacillus products used to control tomato wilt generally have the following drawbacks: unstable field efficacy; many strains show significant antagonistic effects under laboratory conditions, but their efficacy is greatly reduced in complex field soil environments due to poor colonization and weak competitiveness; narrow spectrum of inhibition; existing products are mostly targeted at single diseases and cannot address soil-borne diseases such as root rot and bacterial wilt that occur simultaneously, increasing farmers' pesticide costs and management difficulties; and insignificant growth-promoting effects; most strains have single functions and lack the ability to produce growth-promoting substances such as indoleacetic acid (IAA) and siderophores, making it difficult to achieve integrated "prevention-treatment-cultivation" treatment.

[0004] Therefore, there is an urgent need for a biocontrol strain with strong colonization ability, broad antibacterial spectrum, and growth-promoting function, and to establish a set of efficient and stable methods for the preparation and application of the bacterial agent. Summary of the Invention

[0005] This invention aims to overcome the shortcomings of existing technologies and provide a new strain of Bacillus vesiculus with stable genetic traits, excellent control efficacy against tomato wilt, and broad-spectrum antagonistic activity. It also provides liquid fermentation and solid-state fermentation processes for this strain, as well as its application in controlling tomato wilt and improving tomato yield and quality.

[0006] To address the aforementioned technical problems, the present invention adopts the following technical solution: A method for preparing Bacillus vesiculosus inoculum includes primary fermentation seed culture, secondary fermentation seed culture, post-treatment, and final mixing steps, as detailed below: 1. Primary fermentation seed culture The slant culture of Bacillus belye was inoculated into liquid NB medium at an inoculation rate of one loop of inoculation needle, and cultured with shaking at 26-28℃ and 150-160 rpm for 24-25 h to obtain liquid primary fermentation seed of Bacillus belye. The Bacillus belyssus was deposited on May 24, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 30756.

[0007] 2. Secondary fermentation seed culture Inoculate the liquid primary fermentation seed of Bacillus belye into the secondary culture medium at an inoculation rate of 6-10% based on the volume of the liquid fermentation medium, and culture with shaking at 26-28℃ and 150-160 rpm for 24-25 h to obtain the liquid secondary fermentation seed of Bacillus belye. The secondary culture medium consists of 10-20 g / L glycerol, 5-10 g / L corn steep liquor powder, 2-5 g / L yeast powder, 0.5-1.0 g / L ferric ammonium citrate, 1-3 g / L KH2PO4, 0.2-0.5 g / L MgSO4·7H2O, and 1-2 g / L sodium L-glutamate, with a pH of 7.0-7.2.

[0008] 3. Post-processing (1) Preparation of liquid microbial preparations Inoculate the liquid secondary fermentation seed of Bacillus belye into the tertiary culture medium at an inoculation rate of 5-10% of the liquid fermentation medium volume. Ferment for 46-48 hours at 26-28℃, 150-160 rpm and an aeration rate of 2-3% of the liquid fermentation medium volume per minute to obtain a liquid microbial preparation containing Bacillus belye. The tertiary culture medium consists of 12-18 g / L sucrose, 7-12 g / L peptone, 2.0-3.0 g / L ammonium dihydrogen phosphate, 0.10-0.12 g / L zinc sulfate heptahydrate, and 0.4-0.8 g / L proline, with a pH of 7.0-7.2. (2) Preparation of solid-state fermentation products Inoculate the liquid secondary fermentation seed of Bacillus belye into the solid fermentation medium at an inoculation rate of 18-20% based on the volume of the liquid fermentation medium. Incubate at 27-28℃ for 70-72 hours, and then dry in a cold air dryer for 8-12 hours at a temperature of 10-20℃ and a relative humidity of 20-30% to obtain the solid fermentation air-dried product. The solid fermentation medium is composed of fermentation substrate and deionized water at a ratio of 1:0.8-1.1; The fermentation substrate, based on 100g dry weight, consists of 50-55g wheat bran, 20-25g soybean meal powder, 3-5g glycerol, 2-3g calcium carbonate, 5-6g diatomaceous earth, 1.0-1.5g sodium alginate, with the remainder being corn stalk powder.

[0009] 4. Final Mixing (1) Bentonite pretreatment Sodium-based bentonite was dried at 105-110℃ for 1.8-2.2h, and then autoclaved at 120-125℃ for 30-35min to obtain sterile bentonite. Deionized water was added to the sterile bentonite, and the mixture was stirred at 58-62℃ and 280-310rpm for 27-35min. Polyaluminum chloride aqueous solution was added, and the addition time was controlled at 40-45min. After the addition was completed, the mixture was stirred at 64-66℃ for 2.0-2.3h, and then the temperature was lowered to 44-46℃. Chitosan solution was added, and the mixture was stirred for 45-50min. Disodium EDTA was added, and the mixture was stirred for 20-25min. The pH of the system was controlled at 6.8-7.2 throughout the process. The mixture was allowed to stand at room temperature for 10-12h, and then dried after centrifugation to obtain pretreated bentonite. The sodium-based bentonite has a particle size of 180-200 mesh and a CEC ≥ 85 mmol / 100g; The polyaluminum chloride aqueous solution is a mixture of polyaluminum chloride and deionized water, with a mass-to-volume ratio of 5-6 g:100 mL. The chitosan solution has a pH of 5.4-5.5; the mass ratio of chitosan to acetic acid solution in the chitosan solution is 1-2:100, and the mass concentration of acetic acid solution is 2-3%. The mass-to-volume ratio of the sterile bentonite, deionized water, polyaluminum chloride aqueous solution, chitosan solution, and disodium EDTA is 100g:250-300g:50-60mL:110-120g:0.4-0.7g.

[0010] (2) Mixing Solid fermentation dried product, pretreated bentonite, and maltodextrin are mixed and stirred at 12-15℃ and 25-30 rpm for 8-10 min to obtain a solid phase substrate; liquid microbial preparation of Bacillus vesiculus, tea saponin, polyethylene glycol 400, and potassium humate are mixed and placed in a homogenizer and homogenized at 20-25℃ and 6000-6300 rpm for 5-8 min to obtain a liquid phase composite material; All the above liquid phase composite materials are sprayed into all the above solid phase substrates. While spraying, the stirring speed of the solid phase substrate is controlled at 25-30 rpm and the spraying time is controlled at 20-30 min. After spraying, stir for 15-20 min and then let it stand and mature at 20-23℃ for 22-24 h to obtain Bacillus vesiculosus inoculum. The mass ratio of the solid fermentation dried product, pretreated bentonite, maltodextrin, liquid microbial preparation of Bacillus vesiculus, tea saponin, polyethylene glycol 400, and potassium humate is 44-47:12-15:6-8:20-23:0.5-1.0:2.0-2.5:4-6.

[0011] A Bacillus bellis inoculum agent was prepared using the aforementioned preparation method.

[0012] Application of a Bacillus vesiculosus inoculant, specifically the application of Bacillus vesiculosus in tomato cultivation.

[0013] This invention employs a three-stage culture process for Bacillus belyssus. First, primary activation restores the physiological activity of the bacteria. Then, a secondary culture medium rich in organic nitrogen, trace elements, and amino acids rapidly increases the bacterial count, allowing the strain to adapt to the fermentation environment. In the tertiary liquid fermentation stage, zinc ions and proline are added to regulate the strain's metabolism, significantly improving its storage tolerance and environmental stress resistance. In the solid-state fermentation stage, calcium carbonate and diatomaceous earth provide loose and breathable material, while sodium alginate helps retain moisture, achieving high-density accumulation of the bacteria. In the final mixing step, the solid-state fermentation components and... The liquid fermentation inoculant is mixed with pretreated bentonite, specifically sodium-based bentonite, which is sterilized at high temperature and then cross-linked with polyaluminum chloride and pretreated with chitosan. The layered structure is expanded to form a porous adsorption carrier, increasing the specific surface area of ​​the carrier, firmly adsorbing and encapsulating the spore-forming inoculant, and isolating it from external stimuli. Tea saponin and polyethylene glycol enhance wettability and permeability, while potassium humate serves as a nutrient component. This results in a compound inoculant with strong stability and high viable bacteria retention rate, effectively preventing tomato wilt, improving nutrient utilization, thereby increasing yield and enhancing the flavor and quality of tomatoes.

[0014] Compared with the prior art, the present invention has achieved the following beneficial effects: 1. The Bacillus belysinian agent prepared by this invention has an effective viable count of 15.1-16.4 billion CFU / g. After being placed at room temperature for 3 months, the effective viable count is 14.9-16.3 billion CFU / g; after being placed at room temperature for 9 months, the effective viable count is 12.1-13.5 billion CFU / g; and after being placed at room temperature for 12 months, the effective viable count is 8.4-9.6 billion CFU / g. 2. The Bacillus vesiculosus inoculant prepared by this invention, when used in tomato cultivation, can reduce the incidence of tomato wilt disease to 6-10%, reduce the disease index to 11.2-14.4, and achieve a control efficacy of 82-86%. 3. When the Bacillus berberis inoculant prepared by this invention is used in tomato cultivation, the tomato plant height is 241.8-259.8cm and the root length is 34.8-37.4cm; 4. When the Bacillus berberis inoculant prepared by this invention is used in tomato cultivation, the tomato yield per mu is 4823.4-4981.7 kg, the soluble solids content is 5.21-5.56%, and the vitamin C content is 19.28-20.96 mg / 100g. Detailed Implementation

[0015] To provide a clearer understanding of the technical features, objectives, and effects of the present invention, specific embodiments of the present invention are now described.

[0016] Example 1 1. Primary fermentation seed culture The slant culture of Bacillus belye was inoculated into liquid NB medium at an inoculation rate of one loop of inoculation needle, and cultured with shaking at 26°C and 150 rpm for 24 h to obtain liquid primary fermentation seed of Bacillus belye. The Bacillus belyssus was deposited on May 24, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 30756.

[0017] 2. Secondary fermentation seed culture The liquid primary fermentation seed of Bacillus belye was inoculated into the secondary culture medium at an inoculation rate of 5% based on the volume of the liquid fermentation medium. The culture was shaken at 26°C and 150 rpm for 24 h to obtain the liquid secondary fermentation seed of Bacillus belye. The secondary culture medium consisted of 10 g / L glycerol, 5 g / L corn steep liquor powder, 2 g / L yeast powder, 0.5 g / L ferric ammonium citrate, 1 g / L KH2PO4, 0.2 g / L MgSO4·7H2O, and 1 g / L sodium L-glutamate, with a pH of 7.0.

[0018] 3. Post-processing (1) Preparation of liquid microbial preparations The liquid secondary fermentation seed of Bacillus belye was inoculated into the tertiary culture medium at an inoculation rate of 5% based on the volume of the liquid fermentation medium. Fermentation was carried out at 26°C, 150 rpm and an aeration rate of 2% based on the volume of the liquid fermentation medium per minute for 46 hours to obtain a liquid microbial preparation containing Bacillus belye. The tertiary culture medium consisted of 12 g / L sucrose, 7 g / L peptone, 2.0 g / L ammonium dihydrogen phosphate, 0.10 g / L zinc sulfate heptahydrate, and 0.4 g / L proline, with a pH of 7.0. (2) Preparation of solid-state fermentation products At an inoculation rate of 18% based on the volume of the liquid fermentation medium, the liquid secondary fermentation seed of Bacillus belye was inoculated into the solid fermentation medium and cultured at 27°C for 70 hours. Then, it was placed in a cold air dryer and dried for 8 hours at a temperature of 10°C and a relative humidity of 20% to obtain the solid fermentation air-dried product. The solid fermentation medium is composed of fermentation substrate and deionized water in a ratio of 1:0.8; The fermentation substrate, based on 100g dry weight, consists of 50g wheat bran, 20g soybean meal powder, 3g glycerol, 2g calcium carbonate, 5g diatomaceous earth, 1.0g sodium alginate, with the remainder being corn stalk powder.

[0019] 4. Final Mixing (1) Bentonite pretreatment Sodium-based bentonite was dried at 105℃ for 1.8 h, and then autoclaved at 120℃ for 30 min to obtain sterile bentonite. 250 g of deionized water was added to 100 g of sterile bentonite, and the mixture was stirred at 58℃ and 280 rpm for 27 min. 50 mL of polyaluminum chloride aqueous solution was added, and the addition time was controlled at 40 min. After the addition was completed, the mixture was stirred at 64℃ for 2.0 h, and then the temperature was lowered to 44℃. 110 g of chitosan solution was added, and the mixture was stirred for another 45 min. 0.4 g of disodium ethylenediaminetetraacetate was added, and the mixture was stirred for 20 min. The pH of the system was controlled at 6.8 throughout the process. The mixture was allowed to stand at room temperature for 10 h, and then dried after centrifugation to obtain pretreated bentonite. The sodium-based bentonite has a particle size of 180 mesh and a CEC ≥ 85 mmol / 100g. The polyaluminum chloride aqueous solution is a mixture of polyaluminum chloride and deionized water, with a mass-to-volume ratio of 5g:100mL. The chitosan solution has a pH of 5.4; the chitosan solution contains chitosan and acetic acid in a mass ratio of 1:100, and the acetic acid solution has a mass concentration of 2%. (2) Mixing 44g of solid fermentation dried product, 12g of pretreated bentonite, and 6g of maltodextrin were mixed and stirred at 12℃ and 25rpm for 8min to obtain a solid phase substrate; 20g of liquid microbial preparation of Bacillus belye, 0.5g of tea saponin, 2.0g of polyethylene glycol 400, and 4g of potassium humate were mixed and placed in a homogenizer and homogenized at 20℃ and 6000rpm for 5min to obtain a liquid phase composite material; All the above liquid phase composite materials were sprayed onto all the above solid phase substrates. During spraying, the stirring speed of the solid phase substrate was controlled at 25 rpm and the spraying time was controlled at 20 min. After spraying, the mixture was stirred for 15 min and then allowed to stand and mature at 20℃ for 22 h to obtain Bacillus vesiculosus inoculum.

[0020] Example 2 1. Primary fermentation seed culture The slant culture of Bacillus belye was inoculated into liquid NB medium at an inoculation rate of one loop of inoculation needle, and cultured with shaking at 27°C and 160 rpm for 25 h to obtain liquid primary fermentation seed of Bacillus belye. The Bacillus belyssus was deposited on May 24, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 30756.

[0021] 2. Secondary fermentation seed culture The liquid primary fermentation seed of Bacillus belye was inoculated into the secondary culture medium at an inoculation rate of 8% based on the volume of the liquid fermentation medium. The culture was shaken and cultured at 27°C and 155 rpm for 25 h to obtain the liquid secondary fermentation seed of Bacillus belye. The secondary culture medium consisted of 15 g / L glycerol, 7 g / L corn steep liquor powder, 3 g / L yeast powder, 1.0 g / L ferric ammonium citrate, 2 g / L KH2PO4, 0.3 g / L MgSO4·7H2O, and 2 g / L sodium L-glutamate, with a pH of 7.0.

[0022] 3. Post-processing (1) Preparation of liquid microbial preparations The liquid secondary fermentation seed of Bacillus belye was inoculated into the tertiary culture medium at an inoculation rate of 8% based on the volume of the liquid fermentation medium. Fermentation was carried out at 27°C, 155 rpm and an aeration rate of 3% based on the volume of the liquid fermentation medium per minute for 47 hours to obtain a liquid microbial preparation containing Bacillus belye. The tertiary culture medium consisted of 15 g / L sucrose, 10 g / L peptone, 2.5 g / L ammonium dihydrogen phosphate, 0.12 g / L zinc sulfate heptahydrate, and 0.6 g / L proline, with a pH of 7.2. (2) Preparation of solid-state fermentation products At an inoculation rate of 20% based on the volume of the liquid fermentation medium, the liquid secondary fermentation seed of Bacillus belye was inoculated into the solid fermentation medium and cultured at 28°C for 72 hours. Then, it was placed in a cold air dryer and dried for 10 hours at a temperature of 15°C and a relative humidity of 25% to obtain the solid fermentation air-dried product. The solid fermentation medium is composed of fermentation substrate and deionized water in a ratio of 1:1.0; The fermentation substrate, based on 100g dry weight, consists of 52g wheat bran, 23g soybean meal powder, 4g glycerol, 3g calcium carbonate, 6g diatomaceous earth, 1.3g sodium alginate, with the remainder being corn stalk powder.

[0023] 4. Final Mixing (1) Bentonite pretreatment Sodium-based bentonite was dried at 108℃ for 2.0 h, and then autoclaved at 123℃ for 32 min to obtain sterile bentonite. 280 g of deionized water was added to 100 g of sterile bentonite, and the mixture was stirred at 60℃ and 300 rpm for 30 min. 55 mL of polyaluminum chloride aqueous solution was added, and the addition time was controlled at 43 min. After the addition was completed, the mixture was stirred at 65℃ for 2.2 h, and then the temperature was lowered to 45℃. 115 g of chitosan solution was added, and the mixture was stirred for 48 min. 0.6 g of disodium EDTA was added, and the mixture was stirred for 23 min. The pH of the system was controlled at 7.0 throughout the process. The mixture was allowed to stand at room temperature for 11 h, and then dried after centrifugation to obtain pretreated bentonite. The sodium-based bentonite has a particle size of 200 mesh and a CEC ≥ 85 mmol / 100g; The polyaluminum chloride aqueous solution is a mixture of polyaluminum chloride and deionized water, with a mass-to-volume ratio of 6g:100mL. The chitosan solution has a pH of 5.5; the chitosan solution contains chitosan and acetic acid in a mass ratio of 2:100, and the acetic acid solution has a mass concentration of 3%. (2) Mixing 46g of solid fermentation dried product, 13g of pretreated bentonite, and 7g of maltodextrin were mixed and stirred at 13℃ and 28rpm for 10min to obtain a solid-phase substrate; 22g of liquid microbial preparation of Bacillus belye, 0.7g of tea saponin, 2.3g of polyethylene glycol 400, and 5g of potassium humate were mixed and placed in a homogenizer and homogenized at 23℃ and 6200rpm for 7min to obtain a liquid-phase composite material. All the above liquid phase composite materials were sprayed onto all the above solid phase substrates. During spraying, the stirring speed of the solid phase substrate was controlled at 28 rpm and the spraying time was controlled at 25 min. After spraying, the mixture was stirred for 18 min and then allowed to stand and mature at 22℃ for 23 h to obtain Bacillus belye inoculum.

[0024] Example 3 1. Primary fermentation seed culture The slant culture of Bacillus belye was inoculated into liquid NB medium at an inoculation rate of one loop of inoculation needle, and cultured with shaking at 28°C and 160 rpm for 25 h to obtain liquid primary fermentation seed of Bacillus belye. The Bacillus belyssus was deposited on May 24, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 30756.

[0025] 2. Secondary fermentation seed culture The liquid primary fermentation seed of Bacillus belye was inoculated into the secondary culture medium at an inoculation rate of 10% based on the volume of the liquid fermentation medium. The culture was shaken and cultured at 28°C and 160 rpm for 25 h to obtain the liquid secondary fermentation seed of Bacillus belye. The secondary culture medium consisted of 20 g / L glycerol, 10 g / L corn steep liquor powder, 5 g / L yeast powder, 1.5 g / L ferric ammonium citrate, 3 g / L KH2PO4, 0.5 g / L MgSO4·7H2O, and 2 g / L sodium L-glutamate, with a pH of 7.2.

[0026] 3. Post-processing (1) Preparation of liquid microbial preparations The liquid secondary fermentation seed of Bacillus belye was inoculated into the tertiary culture medium at an inoculation rate of 10% of the liquid fermentation medium volume. Fermentation was carried out at 28°C, 160 rpm and an aeration rate of 3% of the liquid fermentation medium volume per minute for 48 hours to obtain a liquid microbial preparation containing Bacillus belye. The tertiary culture medium consisted of 18 g / L sucrose, 12 g / L peptone, 3.0 g / L ammonium dihydrogen phosphate, 0.12 g / L zinc sulfate heptahydrate, and 0.8 g / L proline, with a pH of 7.2. (2) Preparation of solid-state fermentation products At an inoculation rate of 20% based on the volume of the liquid fermentation medium, the liquid secondary fermentation seed of Bacillus belye was inoculated into the solid fermentation medium and cultured at 28°C for 72 hours. Then, it was placed in a cold air dryer and dried for 12 hours at a temperature of 20°C and a relative humidity of 30% to obtain the solid fermentation air-dried product. The solid fermentation medium is composed of fermentation substrate and deionized water in a ratio of 1:1.1; The fermentation substrate, based on 100g dry weight, consists of 55g wheat bran, 25g soybean meal powder, 5g glycerol, 3g calcium carbonate, 6g diatomaceous earth, 1.5g sodium alginate, with the remainder being corn stalk powder.

[0027] 4. Final Mixing (1) Bentonite pretreatment Sodium-based bentonite was dried at 110℃ for 2.2 h, and then autoclaved at 125℃ for 35 min to obtain sterile bentonite. 300 g of deionized water was added to 100 g of sterile bentonite, and the mixture was stirred at 62℃ and 310 rpm for 35 min. 60 mL of polyaluminum chloride aqueous solution was added, and the addition time was controlled at 45 min. After the addition was completed, the mixture was stirred at 66℃ for 2.3 h, and then the temperature was lowered to 46℃. 120 g of chitosan solution was added, and the mixture was stirred for another 50 min. 0.7 g of disodium ethylenediaminetetraacetate was added, and the mixture was stirred for 25 min. The pH of the system was controlled at 7.28 throughout the process. The mixture was allowed to stand at room temperature for 12 h, and then dried after centrifugation to obtain pretreated bentonite. The sodium-based bentonite has a particle size of 200 mesh and a CEC ≥ 85 mmol / 100g; The polyaluminum chloride aqueous solution is a mixture of polyaluminum chloride and deionized water, with a mass-to-volume ratio of 6g:100mL. The chitosan solution has a pH of 5.5; the chitosan solution contains chitosan and acetic acid in a mass ratio of 2:100, and the acetic acid solution has a mass concentration of 3%. (2) Mixing 47g of solid fermentation dried product, 15g of pretreated bentonite, and 8g of maltodextrin were mixed and stirred at 15℃ and 30rpm for 10min to obtain a solid-phase substrate; 23g of liquid microbial preparation of Bacillus belye, 1.0g of tea saponin, 2.5g of polyethylene glycol 400, and 6g of potassium humate were mixed and placed in a homogenizer and homogenized at 25℃ and 6300rpm for 8min to obtain a liquid-phase composite material. All the above liquid phase composite materials were sprayed onto all the above solid phase substrates. During spraying, the stirring speed of the solid phase substrate was controlled at 30 rpm and the spraying time was controlled at 30 min. After spraying, the mixture was stirred for 20 min and then allowed to stand and mature at 23℃ for 24 h to obtain Bacillus belye inoculum.

[0028] Comparative Example 1 The changes made in Example 2 are as follows: In the post-processing step, the liquid microbial preparation containing Bacillus belye was replaced in equal amounts with the liquid secondary fermentation seed of Bacillus belye obtained in the secondary fermentation seed culture step. The step of preparing solid fermentation product is as follows: place the liquid secondary fermentation seed of Bacillus belye in a cold air dryer and dry it for 10 hours at a temperature of 15°C and a relative humidity of 25% to obtain the solid fermentation air-dried product. The rest of the operations are exactly the same.

[0029] Comparative Example 2 The changes made in Example 2 are as follows: In the secondary fermentation seed culture step, ferric ammonium citrate in the culture medium is replaced with an equal amount of glycerol; In the post-processing step for preparing solid fermentation products, calcium carbonate and sodium alginate in the fermentation substrate are replaced with wheat bran in equal amounts. In the final mixing step, the bentonite pretreatment step is omitted; the pretreated bentonite in the mixing step is replaced with an equal amount of untreated sodium-based bentonite; the sodium-based bentonite has a particle size of 200 mesh and a CEC ≥ 85 mmol / 100g. The rest of the operations are exactly the same.

[0030] Performance testing The viability of Bacillus belychnophorus inoculants prepared in Examples 1-5 was periodically tested at room temperature, and the number of viable bacteria was determined according to GB 20287-2006 standard for agricultural microbial inoculants; the results are as follows:

[0031] The above experimental results clearly show that the Bacillus belye prepared in Examples 1-3 have strong viability and the rate of bacterial count decline is significantly lower than that of the comparative examples. After 12 months of storage at room temperature, the bacterial survival rates of Examples 1-3 reached 55.6%, 58.5%, and 57.7%, respectively, all of which are stably higher than the 50% threshold. In contrast, the bacterial agents prepared in Comparative Examples 1 and 2 had a survival rate of only 32.6% and 48.3% after 12 months, respectively, which are significantly lower. The high viable bacterial count during the shelf life can ensure the effective colonization during field application and avoid the problem of unstable efficacy caused by a significant decrease in bacterial count during storage.

[0032] Application performance The Bacillus berberis inoculant prepared in Examples 1-3 and Comparative Examples 1-2 was used to conduct field control trials of tomato wilt disease. The trial sites were selected in greenhouses in the main tomato-growing area of ​​Shouguang City, Shandong Province, where tomato wilt disease was more severe. The inoculant was applied at a rate of 5 kg / mu in the planting furrow at the tomato transplanting stage. The tomato variety was Provence and the application method was basal application. The negative control was no treatment, and the positive control was the existing chemical control agent 30% hymexazol aqueous solution purchased from Tianjin Hanbang Plant Protection Agent Co., Ltd., diluted 600 times, applied to the roots, 200-300 mL per plant, once every 7 days, for 2 consecutive applications. Throughout the entire growth period of tomatoes, observe the growth of tomatoes and the occurrence of tomato wilt disease, count plant height and root length, and after the tomatoes are harvested, count the yield per acre, and test the soluble solids content and vitamin C content of tomatoes. The severity of tomato wilt disease is divided into 5 levels. Grade 0: Disease-free, plant growing normally; Grade 1: Less than 1 / 4 of the leaves are yellow and slightly wilted; Grade 2: 1 / 4-1 / 2 of the leaves are wilted and yellow, and the base of the stem is slightly browned; Grade 3: 1 / 2-3 / 4 of the leaves are wilted, and the vascular bundles are noticeably browned; Level 4: The entire plant wilts and dies; The formulas for calculating the disease index and prevention efficacy are as follows: Disease index = [∑(number of disease-grade plants × representative value) / (total number of plants × highest disease grade representative value)] × 100; Efficacy (%) = (Disease index in control area - Disease index in treatment area) / Disease index in control area × 100; Incidence rate (%) = Number of infected plants / Total number of plants surveyed × 100.

[0033] The test results are as follows:

[0034] The experimental results of each treatment group are as follows: The incidence of tomato wilt in the negative control group was 36%, and the plant growth, yield and fruit quality indicators were all at a low level; the incidence of tomato wilt in the positive control group was 9%, which had a certain disease prevention effect, but the effect on improving tomato growth, quality and yield was limited; the incidence of tomato wilt in comparative 1 and comparative 2 was 23% and 16% respectively, with poor disease prevention effect and no obvious effect on improving plant growth and fruit quality.

[0035] Compared with the control groups, the Bacillus berberis inoculant prepared in Examples 1-3 of this invention showed excellent comprehensive application effects, significantly inhibiting the occurrence of tomato wilt disease and effectively controlling the incidence of tomato wilt disease to 6-10%. The disease prevention effect was significantly better than the control group and almost equivalent to the control effect of chemical pesticides. At the same time, the inoculant of this invention can significantly promote the growth of tomato plants. The tomato plant height in each example was 241.8-259.8cm, the root length was 34.8-37.4cm, the root system was well developed, the plants were vigorous, and the growth indicators were significantly better than the control treatment and the pesticide treatment.

[0036] In terms of yield and fruit quality, the tomato yield per mu (667 square meters) in Examples 1-3 of this invention can reach 4823.4-4981.7 kg, which is a significant increase compared with the negative control, positive control and comparative ratio; the soluble solids content of the fruit is 5.21-5.56%, and the vitamin C content is 19.28-20.96 mg / 100g, which is significantly improved compared with the control group, effectively improving the nutritional quality and marketability of tomato fruit.

[0037] In summary, the Bacillus vesiculosus inoculant prepared in this invention can effectively control tomato wilt, reduce the probability of plant disease, and significantly promote tomato growth and development, increase tomato yield, and improve fruit quality. It possesses multiple functions of disease prevention, seedling strengthening, yield increase, and quality improvement, demonstrating excellent application results and suitability for large-scale promotion and application. Unless otherwise stated, all percentages used in this invention are mass percentages.

[0038] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. 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 belye inoculum, characterized in that, This includes primary fermentation seed culture, secondary fermentation seed culture, post-processing, and final mixing steps; The final mixing includes bentonite pretreatment and mixing steps; The bentonite pretreatment steps are as follows: add deionized water to sterile bentonite, stir at 58-62℃ for 27-35 min, add polyaluminum chloride aqueous solution, control the addition time to 40-45 min, after the addition is completed, stir at 64-66℃ for 2.0-2.3 h, cool to 44-46℃, add chitosan solution, continue stirring for 45-50 min, add disodium ethylenediaminetetraacetate, stir for 20-25 min, control the pH value of the system to 6.8-7.2 throughout the process, and after standing at room temperature, the pretreated bentonite is obtained. The mixing steps are as follows: Solid fermentation dried product, pretreated bentonite, and maltodextrin are mixed and stirred at 12-15℃ and 25-30 rpm for 8-10 minutes to obtain a solid substrate; liquid microbial preparation of Bacillus belesii, tea saponin, polyethylene glycol 400, and potassium humate are mixed and placed in a homogenizer, homogenized at 20-25℃ and 6000-6300 rpm for 5-8 minutes to obtain a liquid composite material; the liquid composite material is sprayed onto the solid substrate, while controlling the stirring speed of the solid substrate at 25-30 rpm and the spraying time at 20-30 minutes; after spraying, stirring is performed for 15-20 minutes, and then the mixture is allowed to stand and mature at 20-23℃ for 22-24 hours to obtain Bacillus belesii inoculum.

2. The method for preparing a Bacillus belye inoculum agent according to claim 1, characterized in that, The primary fermentation seed culture step is as follows: the slant culture of Bacillus belye is inoculated into liquid NB medium at an inoculation amount of one loop of inoculation needle, and cultured with shaking at 26-28℃ and 150-160 rpm for 24-25 h to obtain liquid primary fermentation seed of Bacillus belye.

3. The method for preparing a Bacillus belye inoculum agent according to claim 1, characterized in that, The secondary fermentation seed culture step is as follows: at an inoculation rate of 6-10% of the volume of the liquid fermentation medium, the liquid primary fermentation seed of Bacillus belye is inoculated into the secondary culture medium, and cultured with shaking at 26-28℃ and 150-160 rpm for 24-25 hours to obtain the liquid secondary fermentation seed of Bacillus belye. The secondary culture medium consists of 10-20 g / L glycerol, 5-10 g / L corn steep liquor powder, 2-5 g / L yeast powder, 0.5-1.0 g / L ferric ammonium citrate, 1-3 g / L KH2PO4, 0.2-0.5 g / L MgSO4·7H2O, and 1-2 g / L sodium L-glutamate, with a pH of 7.0-7.

2.

4. The method for preparing a Bacillus belye inoculum agent according to claim 1, characterized in that, The post-processing includes the steps of preparing liquid microbial preparations and preparing solid fermentation products; Inoculate the liquid secondary fermentation seed of Bacillus belye into the tertiary culture medium at an inoculation rate of 5-10% of the liquid fermentation medium volume. Ferment for 46-48 hours at 26-28℃, 150-160 rpm and an aeration rate of 2-3% of the liquid fermentation medium volume per minute to obtain a liquid microbial preparation containing Bacillus belye. The tertiary culture medium consists of 12-18 g / L sucrose, 7-12 g / L peptone, 2.0-3.0 g / L ammonium dihydrogen phosphate, 0.10-0.12 g / L zinc sulfate heptahydrate, and 0.4-0.8 g / L proline, with a pH of 7.0-7.

2.

5. The method for preparing a Bacillus belye inoculum agent according to claim 4, characterized in that, Inoculate the liquid secondary fermentation seed of Bacillus belye into the solid fermentation medium at an inoculation rate of 18-20% based on the volume of the liquid fermentation medium. Incubate at 27-28℃ for 70-72 hours, and then dry in a cold air dryer for 8-12 hours at a temperature of 10-20℃ and a relative humidity of 20-30% to obtain the solid fermentation air-dried product. The solid fermentation medium is composed of fermentation substrate and deionized water at a ratio of 1:0.8-1.1; The fermentation substrate, based on 100g dry weight, consists of 50-55g wheat bran, 20-25g soybean meal powder, 3-5g glycerol, 2-3g calcium carbonate, 5-6g diatomaceous earth, 1.0-1.5g sodium alginate, with the remainder being corn stalk powder.

6. The method for preparing a Bacillus belye inoculum agent according to claim 1, characterized in that, In the bentonite pretreatment step, the sodium-based bentonite has a particle size of 180-200 mesh and a CEC ≥ 85 mmol / 100g; The polyaluminum chloride aqueous solution is a mixture of polyaluminum chloride and deionized water, with a mass-to-volume ratio of 5-6 g:100 mL. The chitosan solution has a pH of 5.4-5.5; the mass ratio of chitosan to acetic acid solution in the chitosan solution is 1-2:100, and the mass concentration of acetic acid solution is 2-3%. The mass-to-volume ratio of the sterile bentonite, deionized water, polyaluminum chloride aqueous solution, chitosan solution, and disodium EDTA is 100g:250-300g:50-60mL:110-120g:0.4-0.7g.

7. The method for preparing a Bacillus belye inoculum agent according to claim 1, characterized in that, In the mixing step, the mass ratio of the solid fermentation dried product, pretreated bentonite, maltodextrin, liquid microbial preparation of Bacillus vesiculosus, tea saponin, polyethylene glycol 400, and potassium humate is 44-47:12-15:6-8:20-23:0.5-1.0:2.0-2.5:4-6.

8. A Bacillus belesiensis inoculant, characterized in that, It is prepared by the preparation method according to any one of claims 1-7.

9. The application of a Bacillus belysin inoculant, characterized in that, Application of Bacillus berberis inoculant prepared by any one of claims 1-7 in tomato cultivation.

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