Bacillus YWF-075, microbial inoculum and preparation method of microbial inoculum

Abstract

The invention discloses a bacillus velezensis microbial agent for preventing and treating fusarium wilt and a preparation method thereof, relates to the technical field of fusarium wilt prevention and treatment, and solves the technical problems of pesticide residues and environmental pollution caused by adopting chemical pesticides to prevent and control fusarium wilt in the prior art. The preparation method comprises the following steps: taking bacillus YWF-075 as a strain, and activating the strain; the method comprises the following steps: culturing a bacillus YWF-075 strain to obtain a bacillus YWF-075 primary seed solution; the bacillus YWF-075 primary seed solution is cultured, and a bacillus YWF-075 secondary seed solution is obtained; the bacillus YWF-075 secondary seed solution is cultured, a bacillus YWF-075 fermentation solution is obtained, and the bacillus YWF-075 fermentation solution is the biological preparation for preventing and treating the wilt disease. The biological preparation for preventing and controlling the fusarium wilt can effectively prevent and control the tomato soil-borne disease fusarium wilt, and can replace the traditional method for preventing and controlling pesticide residues generated by plant diseases only depending on chemical pesticides.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial control of wilt disease, and in particular relates to a Bacillus YWF-075, a bacterial agent and a preparation method thereof. Background Art

[0002] In recent years, as the planting area of ​​greenhouse tomatoes continues to increase, soil-borne tomato wilt has also become serious, becoming a bottleneck problem in tomato greenhouse production. Tomato wilt is caused by Fusarium oxysporum tomato-specific type ( Fusarium oxysporum f.sp. Lycopersici, Fol ) infection, belonging to the subphylum Ascomycota. Studies have shown that the incidence of tomato wilt is as high as 20% to 40% in fields with continuous cropping for many years, and there is a trend of increasing severity year by year. At present, there are no completely disease-resistant varieties and effective chemical agents, and the widespread use and increased dosage of chemical pesticides have brought a series of problems, including increased resistance of pathogenic bacteria and environmental pollution. The development of eco-friendly biofungicides has become a top priority for agricultural production.

[0003] Bacillus has been widely studied and applied in biological control of plant diseases due to its rapid reproduction, strong stress resistance, good bactericidal effect, ability to induce plant disease resistance and promote growth and production, ability to reduce the interference of chemical agents on the environment, and green and safe. It is considered to be one of the most promising control methods. Therefore, it is urgent to research and develop new biological control technologies for the prevention and control of Fusarium wilt. Summary of the invention

[0004] The purpose of the present invention is to overcome the deficiencies in the prior art and provide a Bacillus YWF-075 and a bacterial agent thereof.

[0005] The first aspect of the present invention is to provide a Bacillus YWF-075, named: Bacillus Velezii ( Bacillus velezensis )YWF-075, registered and preserved in China Center for Type Culture Collection, with the registration number CCTCC NO: M 20242105.

[0006] The second aspect of the present invention is to provide a fermentation broth of Bacillus YWF-075 as described in the first aspect of the present invention.

[0007] The third aspect of the present invention is to provide a preparation containing the Bacillus YWF-075 described in the first aspect of the present invention or the fermentation broth described in the second aspect of the present invention.

[0008] The beneficial effects of the present invention are as follows: the fermentation liquid of Bacillus YWF-075 obtained after seed activation and at least three-stage culture using Bacillus YWF-075 as the strain can effectively prevent and control the soil-borne disease Fusarium wilt of tomatoes, can greatly reduce the application amount of chemical pesticides, reduce environmental pollution, and improve crop safety; on the other hand, the biological agent for preventing and controlling Fusarium wilt of the present invention can also promote plant growth; thirdly, the preparation method of the biological agent for preventing and controlling Fusarium wilt of the present invention is simple and easy, and the production cost is low.

[0009] The bacillus microbial agent of the present invention has various dosage forms and can be prepared into fermentation liquid, powder, granule or suspension. The method of use is diverse. It can be applied to holes as powder or granule, can be mixed and diluted with water for root irrigation and watering, and can also be added to substrates and fertilizers as beneficial microorganisms for prevention and control in the seedling stage and fertilization stage. It has good application and promotion value. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 The graph is a test result of the antibacterial activity of the strains screened in Example 1 against the pathogenic bacteria of Fusarium wilt; Figure 2 This is the phylogenetic tree of Bacillus YWF-075 constructed using MEGA 7.0 software. DETAILED DESCRIPTION

[0011] To make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described in detail below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other implementation methods obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention.

[0012] The preparation method of the biological agent for preventing and treating wilt disease by Bacillus YWF-075 comprises the following steps: S1. Take out the Bacillus preserved by glycerol and streak culture for 24 hours at -80℃, pick out 3-5 single colonies with better growth, inoculate them into LB liquid medium, and culture them at 35-37℃ for 24 hours to obtain activated Bacillus YWF-075 seed liquid; S2. Inoculate the activated seed liquid into the primary seed culture medium at a rate of 5-10%, and culture it on a shaking table at a temperature of 35-37°C and a speed of 200 rpm / min for 16-20 hours. Then transfer the primary seed liquid to the secondary seed culture medium in the seed tank at a rate of 5-10%, and culture it at 35-37°C, a speed of 200 rpm / min, a ventilation volume of 0.5-1vvm, and a tank pressure of 0.04 MPa-0.06 MPa for 16-18 hours to obtain the secondary seed liquid of Bacillus YWF-075; S3. Inoculate the secondary seed liquid into the fermentation medium in the fermentation tank at a rate of 5-8%, and culture it at 35-37 °C, a speed of 200 rpm / min, a ventilation volume of 0.5-1vvm, and a tank pressure of 0.05MPa for 20-24 h. The effective viable count of Bacillus YWF-075 in the fermentation liquid after culture is not less than 5×10 9 cfu / ml, the fermentation was stopped, and the obtained fermentation liquid of Bacillus YWF-075 was the biological agent for preventing and treating Fusarium wilt; S4. The fermentation liquid of Bacillus YWF-075 is packaged as a microbial agent to form a product. The microbial agent dosage form of the fermentation liquid is a fermentation liquid liquid, a powder, a granule or a suspension.

[0013] The primary seed culture medium is a liquid LB culture medium, and its components by mass fraction are 10.0‰ bacteriological peptone, 5.0‰ yeast extract powder, 5‰ sodium chloride, and the balance is water, and the pH is natural.

[0014] The secondary seed culture medium comprises the following components in mass fraction: 5-8 ‰ soybean meal powder, 10-15 ‰ corn steep liquor dry powder, 5-8 ‰ glucose, 2-3 ‰ yeast extract, 2-3 ‰ peptone, 0.5-1 ‰ magnesium sulfate, 0.3-0.5 ‰ sodium chloride, 3-5 ‰ calcium carbonate, 1-2 ‰ dipotassium hydrogen phosphate, 0.5-1 ‰ defoaming agent, and the balance is water, and the pH value is 7.0-7.3.

[0015] The fermentation medium comprises the following components in mass fraction: 5-8 ‰ soybean meal powder, 10-20 ‰ corn steep liquor powder, 10-15 ‰ molasses, 5-8 ‰ glucose, 2-3 ‰ yeast extract, 2-3 ‰ peptone, 0.5-1 ‰ magnesium sulfate, 0.3-0.5 ‰ sodium chloride, 3-5 ‰ calcium carbonate, 1-2 ‰ dipotassium hydrogen phosphate, 0.5-1 ‰ defoaming agent, and the balance is water, and the pH value is 7.0-7.3.

[0016] The classification name of the Bacillus Velez YWF-075 of the present invention is: Bacillus Velez ( Bacillus velezensis)YWF-075, the deposit registration number is CCTCC NO: M 20242105, the deposit date is September 27, 2024, the status is: alive, the depository unit is China Center for Type Culture Collection, and the deposit address is Wuhan University, Wuhan, China.

[0017] The present invention is described in detail below in conjunction with Examples 1 to 4.

[0018] Example 1

[0019] This example illustrates the acquisition and identification of Bacillus YWF-075 of the present invention.

[0020] 1. Method for obtaining Bacillus sp. YWF-075 of the present invention 1.1 Materials Rhizosphere soil of healthy tomato plants in the tomato planting area all year round.

[0021] Melt the LB solid medium and PDA medium, pour them into sterilized culture dishes, cool them, and make them into plates. The LB solid medium is used to separate and preserve endophytes, and the PDA medium is used to culture wilt pathogens and antagonistic bacteria to detect the activity of the pathogens.

[0022] 1.2. Isolation and culture The bacteria in the soil were separated by the serial gradient dilution method and the plate spreading method, and colonies with different morphologies were picked for purification, culture and preservation.

[0023] 1.3. Screening of antagonistic endophytic fermentation broth The hole method was used: the wilt pathogen was inoculated in the center of the PDA culture dish, and a 0.5 cm hole was punched at 3 cm from the center to remove the culture medium. 40 μL of YWF-075 fermentation liquid was pipetted into the hole with a pipette, and only the pathogen was inoculated as the control group. After the plate was placed in a 28 ℃ incubator for 5 days, the fermentation liquid of the strain was observed to see whether it had an inhibitory effect on the wilt pathogen. The Bacillus YWF-075 preparation had a strong inhibitory effect on the growth of the wilt pathogen. The test results are as follows: Figure 1 shown.

[0024] 2. Identification of Bacillus YWF-075 The bacterium YWF-075 was identified by molecular biology. The genomic DNA of the strain YWF-075 was used as a template and the bacterial universal primers 27F / 1492R were used to amplify YWF-075 by PCR. After sequencing, the 16S rDNA gene sequence of the strain YWF-075 was input into the NCBI database for Blast similarity comparison analysis. The phylogenetic tree of the strain was constructed using MEGA 7.0 software. Figure 2As shown, the 16S rDNA gene sequence of strain YWF-075 is B. velezensis H168 (MW737443.1) is in a branch, and strain YWF-075 is Bacillus velezinoffii ( Bacillus velezensis ).

[0025] Example 2

[0026] This example illustrates the biological agent for preventing and treating Fusarium wilt of the present invention.

[0027] The method for preparing the biological agent for preventing and treating wilt disease in this embodiment comprises the following steps: S1. Using Bacillus YWF-075 as the strain, the strain was activated. Specifically, 3-5 single colonies with better growth were selected from the Bacillus preserved by glycerol and cultured for 24 hours at -80°C, and inoculated into LB liquid culture medium, and cultured at 37°C for 24 hours to obtain activated Bacillus YWF-075 seed liquid.

[0028] S2. Cultivate the Bacillus subtilis YWF-075 strain to obtain a primary seed solution of Bacillus subtilis YWF-075. Specifically, inoculate the activated Bacillus subtilis seed solution into a primary seed culture medium at a 5% inoculation amount, and culture on a shaking table for 16 hours at a culture temperature of 35°C and a rotation speed of 200 rpm / min to obtain a primary seed solution of Bacillus subtilis YWF-075.

[0029] Preferably, the primary seed culture medium comprises the following components in mass fraction: bacteriological peptone 10.0‰, yeast extract powder 5.0‰, sodium chloride 5.0‰, and natural pH.

[0030] S3. Transfer the primary seed liquid to the secondary seed culture medium in the seed tank according to the inoculation amount of 8%, and culture it at 37°C, 200 rpm / min, 0.5 vvm of ventilation, and 0.05 MPa of tank pressure for 16 h to obtain the secondary seed liquid of Bacillus YWF-075.

[0031] Preferably, the secondary seed culture medium comprises the following components in mass fraction: 6‰ soybean meal powder, 14‰ corn steep liquor powder, 8‰ glucose, 2.5‰ yeast extract, 3‰ peptone, 0.5‰ magnesium sulfate, 0.3‰ sodium chloride, 3‰ calcium carbonate, 1‰ dipotassium hydrogen phosphate, 0.8‰ defoaming agent, and the remainder is water, and the pH is 7.0~7.3.

[0032] S4. Cultivate the secondary seed liquid of Bacillus YWF-075 to obtain Bacillus YWF-075 fermentation liquid, which is the biological agent for preventing and treating Fusarium wilt.

[0033] Specifically, the secondary seed liquid of Bacillus YWF-075 was inoculated into the fermentation medium in the fermentation tank at an inoculation rate of 8%, and cultured for 22 hours at 37°C, a speed of 200 rpm / min, a ventilation volume of 0.5vvm, and a tank pressure of 0.05MPa. The effective viable count of Bacillus YWF-075 in the fermentation liquid after culture was not less than 5×10 9 cfu / ml, the fermentation is stopped, and the obtained fermentation liquid is the fermentation liquid of Bacillus YWF-075. The fermentation liquid is packaged as a bacterial agent to form a product.

[0034] Preferably, the fermentation medium comprises the following components in mass fraction: 5‰ soybean meal powder, 12‰ corn steep liquor, 12‰ molasses liquid, 5‰ glucose, 2.5‰ yeast extract, 2.5‰ peptone, 0.5‰ magnesium sulfate, 0.3‰ sodium chloride, 3‰ calcium carbonate, 1‰ dipotassium hydrogen phosphate, 1‰ defoaming agent, and the balance is water, and the pH is 7.0-7.3.

[0035] S5, Bacillus YWF-075 fermentation liquid can be packaged in bottles. The microbial agent can be applied to crops by one or more methods such as root irrigation, pouring, and hole application.

[0036] Example 3

[0037] The method for preparing the biological agent for preventing and treating wilt disease in this embodiment comprises the following steps: S1. Use Bacillus YWF-075 as the strain and activate the strain. Specifically, take out 3-5 single colonies with better growth from the Bacillus preserved by glycerol and cultured for 24 hours at -80°C, pick them and inoculate them into LB liquid culture medium, and culture them at 35-37°C for 24 hours to obtain the activated Bacillus YWF-075 seed liquid.

[0038] S2. Cultivate the Bacillus subtilis YWF-075 strain to obtain a primary seed solution of Bacillus subtilis YWF-075. Specifically, inoculate the activated Bacillus subtilis seed solution into a primary seed culture medium at an inoculation rate of 8%, and culture on a shaking table for 18 hours at a culture temperature of 37° C. and a rotation speed of 200 rpm / min to obtain a primary seed solution of Bacillus subtilis YWF-075.

[0039] Preferably, the primary seed culture medium comprises the following components in mass fraction: bacteriological peptone 10.0‰, yeast extract powder 5.0‰, sodium chloride 5‰, and the balance is water, with a natural pH.

[0040] S3. Transfer the primary seed liquid to the secondary seed culture medium in the seed tank at an inoculation rate of 8%, and culture for 16 h at 37°C, a rotation speed of 200 rpm / min, a ventilation volume of 0.5 vvm, and a tank pressure of 0.05 MPa to obtain the secondary seed liquid of Bacillus YWF-075.

[0041] Preferably, the secondary seed culture medium comprises the following components in mass fraction: 5‰ soybean meal powder, 10‰ corn steep liquor powder, 10‰ glucose, 2‰ yeast extract, 3‰ peptone, 1‰ magnesium sulfate, 0.3‰ sodium chloride, 3‰ calcium carbonate, 1.5‰ dipotassium hydrogen phosphate, 0.5‰ defoaming agent, and the remainder is water, and the pH is 7.0~7.3.

[0042] S4. Cultivate the secondary seed liquid of Bacillus YWF-075 to obtain the fermentation liquid of Bacillus YWF-075, which is the biological agent for preventing and treating Fusarium wilt. Specifically, the secondary seed liquid of Bacillus YWF-075 is inoculated into the fermentation medium in the fermentation tank at an inoculation amount of 8%, and cultured for 22 hours at 37°C, a speed of 200 rpm / min, a ventilation volume of 0.8vvm, and a tank pressure of 0.05MPa. The effective viable count of Bacillus YWF-075 in the fermentation liquid after culture is not less than 5×10 9 cfu / ml, the fermentation is stopped, and the obtained fermentation liquid is the fermentation liquid of Bacillus YWF-075. The fermentation liquid is packaged as a bacterial agent to form a product.

[0043] Preferably, the fermentation medium comprises the following components in mass fraction: 5‰ soybean meal powder, 15‰ corn steep liquor powder, 15‰ molasses liquid, 5‰ glucose, 2.5‰ yeast extract, 2.5‰ peptone, 0.5‰ magnesium sulfate, 0.3‰ sodium chloride, 3‰ calcium carbonate, 1‰ dipotassium hydrogen phosphate, 1‰ defoaming agent, and the balance is water, and the pH is 7.0-7.3.

[0044] The bacillus microbial agent of the present invention can be combined with a matrix to prepare a functional seedling matrix for use, and disease prevention and control can be carried out in the seedling stage, which can effectively reduce the outbreak of diseases after crop transplanting.

[0045] Example 4

[0046] The method for preparing the biological agent for preventing and treating wilt disease in this embodiment comprises the following steps: S1. Use Bacillus YWF-075 as the strain and activate the strain. Specifically, take out 3-5 single colonies with better growth from the Bacillus preserved by glycerol and cultured for 24 hours at -80°C, pick them and inoculate them into LB liquid culture medium, and culture them at 35-37°C for 24 hours to obtain the activated Bacillus YWF-075 seed liquid.

[0047] S2. Cultivate the Bacillus subtilis YWF-075 strain to obtain a primary seed solution of Bacillus subtilis YWF-075. Specifically, inoculate the activated Bacillus subtilis seed solution into a primary seed culture medium at a 10% inoculation amount, and culture on a shaking table for 16 hours at a culture temperature of 36° C. and a rotation speed of 200 rpm / min to obtain a primary seed solution of Bacillus subtilis YWF-075.

[0048] Preferably, the primary seed culture medium comprises the following components in mass fraction: bacteriological peptone 10.0‰, yeast extract powder 5.0‰, sodium chloride 5‰, and the balance is water, with a natural pH.

[0049] S3. Transfer the primary seed liquid to the secondary seed culture medium in the seed tank at an inoculation rate of 8%, and culture for 18 hours at 36°C, a rotation speed of 180 rpm / min, a ventilation volume of 0.8vvm, and a tank pressure of 0.05MPa to obtain the secondary seed liquid of Bacillus YWF-075.

[0050] Preferably, the secondary seed culture medium comprises the following components in mass fraction: 5‰ soybean meal powder, 10‰ corn steep liquor powder, 8‰ glucose, 3‰ yeast extract, 3‰ peptone, 0.5‰ magnesium sulfate, 0.3‰ sodium chloride, 3‰ calcium carbonate, 1‰ dipotassium hydrogen phosphate, 1‰ defoaming agent, and the remainder is water, and the pH is 7.0~7.3.

[0051] S4. Cultivate the secondary seed liquid of Bacillus YWF-075 to obtain the fermentation liquid of Bacillus YWF-075, which is the biological agent for preventing and treating Fusarium wilt. Specifically, the secondary seed liquid of Bacillus YWF-075 is inoculated into the fermentation medium in the fermentation tank at a 10% inoculation amount, and cultured for 22 hours at 36°C, a speed of 200 rpm / min, a ventilation volume of 0.8vvm, and a tank pressure of 0.06MPa. The effective viable count of Bacillus YWF-075 in the fermentation liquid after culture is not less than 5×10 9 cfu / ml, the fermentation is stopped, and the obtained fermentation liquid is the fermentation liquid of Bacillus YWF-075. The fermentation liquid is packaged as a bacterial agent to form a product.

[0052] Preferably, the fermentation medium comprises the following components in mass fraction: 8‰ soybean meal powder, 10‰ corn steep liquor powder, 10‰ molasses liquid, 8‰ glucose, 2.5‰ yeast extract, 3‰ peptone, 1‰ magnesium sulfate, 0.3‰ sodium chloride, 3‰ calcium carbonate, 1‰ dipotassium hydrogen phosphate, 1‰ defoaming agent, and the balance is water, and the pH is 7.0-7.3.

[0053] The bacillus microbial agent of the present invention can be combined with fertilizers to prepare functional biological fertilizers, which can enter the soil when applying base fertilizers and prevent and control diseases during crop transplanting, thereby effectively reducing the outbreak of diseases after crop transplanting.

Claims

1. A Bacillus YWF-075, characterized in that: Bacillus YWF-075 is named as: Bacillus Velezii ( Bacillus velezensis )YWF-075, registered and deposited in the China Center for Type Culture Collection, the deposit registration number is CCTCC NO: M 20242105, and the deposit date is September 27, 2024.

2. A Bacillus YWF-075 fermentation broth, characterized in that: The fermentation liquid is prepared by using the Bacillus YWF-075 described in claim 1.

3. The Bacillus YWF-075 fermentation broth according to claim 2, characterized in that: The effective viable count of the fermentation broth of Bacillus subtilis YWF-075 is not less than 5×10 9 cfu / ml.

4. A Bacillus YWF-075 preparation, characterized in that: Contains the Bacillus Velez subtilis according to claim 1 or the fermentation liquid according to claim 2.

5. The Bacillus YWF-075 preparation according to claim 4, characterized in that: The Bacillus YWF-075 preparation is a fermentation liquid, powder, granule or suspension.

Images