A Highly Effective Antagonistic Agent for Verticillium Wilt and Its Application

Abstract

This invention belongs to the field of agricultural microbiology and provides a highly effective antagonistic agent against Verticillium wilt and its application. The highly effective antagonistic agent against Verticillium wilt involved in this invention is a compound of Bacillus subtilis and Bacillus belye. Bacillus subtilis is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 20013, and Bacillus belye is deposited at the same center with accession number CGMCC No. 25095. The highly effective antagonistic agent against Verticillium dahliae, the pathogen of Verticillium wilt, shows significant antagonistic effects. When diluted 200 times and applied to tomato Verticillium wilt, the control efficacy reached 98.53%. When applied to strawberry cultivation, this agent effectively reduced strawberry mortality by more than 95%. The antagonistic agent against Verticillium wilt involved in this invention has a significant control effect on Verticillium wilt and improves crop resistance, thus safeguarding crop yield and income. Therefore, it has broad application prospects in agricultural disease control and yield enhancement.

Description

Technical Field

[0001] This invention relates to the field of agricultural microbiology, and in particular to a highly effective antagonistic agent against Verticillium wilt and its application. Background Technology

[0002] Verticillium wilt is a relatively serious fungal vascular system disease caused by Verticillium dahliae. This fungus has a wide host range, infecting plants including tomatoes, eggplants, cotton, bell peppers, radishes, and roses. After infection, plants often exhibit leaf wilting and death, with a prolonged disease period leading to the gradual withering and death of the plant, affecting crop quality and yield. In recent years, the disease has shown a trend of increasing severity and has become one of the major obstacles to high-efficiency crop production in my country.

[0003] Currently, the main methods for controlling Verticillium wilt include breeding resistant varieties, chemical pesticides, and biological control. Chemical pesticides cause soil and environmental pollution, harm human health, and easily lead to pesticide resistance in pests, hindering sustainable agricultural development. Against the backdrop of vigorously developing green and ecological agriculture, biological control is receiving increasing attention. Biological control is safe for humans, livestock, and plants, causes no environmental pollution, and is less likely to cause resistance in pathogens. Utilizing antagonistic microorganisms to control pests and diseases is a major method of biological control. Currently, biocontrol bacteria in production and application include Pseudomonas, Bacillus, and certain actinomycetes. Among them, Bacillus can produce heat-stable endospores, exhibits strong resistance, is non-toxic, and leaves no residue, making it an increasingly important biocontrol material. Therefore, researching a novel antagonistic microbial agent for the control of Verticillium wilt is an inevitable direction for sustainable development. Summary of the Invention

[0004] To address the problems of existing technologies, this invention provides a highly effective antagonistic agent against Verticillium wilt and its application. The highly effective antagonistic agent is a compound of Bacillus subtilis and Bacillus belye. This agent exhibits significant antagonistic effects against the Verticillium dahliae pathogen, achieving a control efficacy of 98.53% against tomato Verticillium wilt. Applying this agent to strawberry cultivation effectively reduces strawberry mortality by over 95%. The Verticillium wilt antagonistic agent of this invention has significant control effects against Verticillium wilt and enhances crop resistance, thus safeguarding crop yield and income. Therefore, it has broad application prospects in agricultural disease control and yield enhancement.

[0005] To achieve the above objectives, the specific technical solution of the present invention is as follows:

[0006] First, this invention proposes a highly effective antagonistic agent against Verticillium wilt, which includes Bacillus subtilis and Bacillus velezensis. Both strains are deposited at the China General Microbiological Culture Collection Center (CGMCC), with Bacillus subtilis having accession number CGMCC No. 20013 and Bacillus velezensis having accession number CGMCC No. 25095.

[0007] The Bacillus subtilis strain was screened from a laboratory strain bank; the Bacillus berberis strain was screened from cotton rhizosphere soil.

[0008] The *Bacillus velezensis* colonies described in this invention are round, with a rough, opaque surface, milky white color, and neat edges. Microscopic observation reveals short rod-shaped cells, 0.5–1.2 μm in diameter and 1.5–2.5 μm in length. BLAST alignment of the strain's 16S rDNA sequence showed greater than 99% homology with the nucleotide sequences of different *Bacillus* strains, and 100% homology with strains specifically identified as *Bacillus velezensis*.

[0009] The aforementioned Bacillus subtilis is a Gram-positive bacterium, without a capsule, short rod-shaped, and its colonies on ordinary nutrient agar are round protrusions with a smooth and opaque surface.

[0010] Preferably, the effective viable count ratio of Bacillus subtilis and Bacillus belye is 1:1 to 1.5.

[0011] Furthermore, the antagonistic bacterial agent can be a liquid bacterial agent or a solid bacterial agent.

[0012] The present invention also provides a method for preparing the above-mentioned antagonistic bacterial agent, the steps of which include:

[0013] (1) Activation of bacterial strains: Take out the Bacillus subtilis and Bacillus belye strains stored at 4℃ and activate them at room temperature for 3 hours. Since the bacterial strains are stored on test tube slant nutrient agar medium, no additional nutrients are needed during the activation process.

[0014] (2) Liquid seed preparation: In a sterile operating table, use a pipette to add 10 mL of sterile water into the bacterial culture tube, and use a pipette to repeatedly blow and agitate to prepare a bacterial suspension. Then, inoculate the bacterial suspension into 200 mL of liquid culture medium and shake and culture for 20 h. The culture conditions are: temperature 35℃, shaking speed 170 r / min.

[0015] (3) High-density fermentation: Bacillus subtilis and Bacillus belye liquid seeds were inoculated into the corresponding fermentation medium at an inoculation rate of 1.5% by volume for liquid deep fermentation;

[0016] The formula for high-density culture medium for Bacillus subtilis is as follows: soybean meal 2%-3.5%, corn flour 3%-4%, maltodextrin 1.8%-3.2%, urea 0.1%-0.16%, calcium carbonate 0.1%-0.3%, dimethyl hydrogen phosphate 0.2%-0.3%, magnesium sulfate 0.05%-0.15%, manganese sulfate 0.01%-0.02%, polyether defoamer 0.1%, and the balance being water.

[0017] High-density fermentation conditions for Bacillus subtilis: tank pressure 0.02-0.05 MPa, temperature 30-37℃, rotation speed 180-400 r / min, dissolved oxygen 30%, and culture time 15-36 h.

[0018] The formula for high-density culture medium for Bacillus vesiculosus is as follows: 2%-4% soybean meal, 1.5%-2% corn starch, 1%-2% glucose, 0.02%-0.04% manganese sulfate, 0.1%-0.2% ammonium sulfate, 0.2%-0.4% magnesium sulfate, 0.01%-0.03% sodium chloride, 0.1% polyether defoamer, and the balance being water.

[0019] High-density fermentation conditions for Bacillus vesiculosus: tank pressure 0.02-0.05MPa, temperature 30-37℃, rotation speed 180-400r / min, dissolved oxygen 30%, and culture time 15-36h;

[0020] (4) The liquid or solid inoculum of the highly effective antagonistic fungal agent against Verticillium wilt is further prepared according to the following method:

[0021] Liquid bacterial agent: Bacillus subtilis and Bacillus belye bacterial solutions are mixed according to the ratio of effective viable bacteria;

[0022] Solid bacterial agent: The bacterial solutions of the two strains obtained in step (3) are spray-dried separately with an outlet air temperature of 85°C and an inlet air temperature of 180°C. The carrier is Jia Yi powder, and the amount is 5-10%. The bacterial powder is prepared and then mixed according to the effective live bacteria ratio.

[0023] This invention further discloses the application of the above-mentioned highly effective antagonistic fungicide against Verticillium wilt in crop cultivation. The application method includes: applying the highly effective antagonistic fungicide against Verticillium wilt of this invention to the cultivation of crops such as tomatoes or strawberries via root irrigation.

[0024] Preferably, the microbial agent is applied by diluting it 200-300 times and then applying it to the roots.

[0025] More preferably, when applied, the initial effective viable count of the microbial agent is ≥10. 8 cfu / mL (or cfu / g).

[0026] In summary, the highly effective antagonistic fungicide and application method for Verticillium wilt provided by this invention can effectively prevent and control Verticillium wilt in crops, improve crop resistance, and safeguard increased crop yields and income. Therefore, it has broad application prospects in agricultural disease control and yield enhancement.

[0027] Preservation Information

[0028] Collection date: June 3, 2020;

[0029] Name of the depository: China General Microbiological Culture Collection Center, China Committee on the Preservation and Management of Microbial Cultures;

[0030] Accession number: CGMCC No.20013;

[0031] Address of the depository: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing;

[0032] Classification and nomenclature: Bacillus subtilis.

[0033] Preservation date: June 16, 2022;

[0034] Name of the depository: China General Microbiological Culture Collection Center, China Committee on the Preservation and Management of Microbial Cultures;

[0035] Accession number: CGMCC No. 25095;

[0036] Address of the depository: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing;

[0037] Classification and nomenclature: Bacillus velezensis. Attached Figure Description

[0038] Figure 1 This is a diagram showing the inhibition zone effect of each strain in the secondary screening of Verticillium wilt antagonistic strains in Example 1 of the present invention;

[0039] Figure 2 The diagram shows the application effect of the antagonistic bacteria agent of the present invention in strawberry production, where a represents the strawberry before treatment with the antagonistic bacteria agent of the present invention, and b represents the strawberry after treatment with the antagonistic bacteria agent of the present invention. Detailed Implementation

[0040] To better explain this invention, the technical solution is explained below with reference to specific embodiments; however, the scope of this invention is not limited to these embodiments. Unless otherwise specified, all technologies and materials described in this invention are conventional products and are commercially available.

[0041] Example 1: Screening of Verticillium Wilt Antagonistic Strains

[0042] 1. Strains Isolation

[0043] In areas severely affected by Verticillium wilt, healthy cotton plants were selected, and rhizosphere soil samples were collected and brought back to the laboratory for isolation. The plate dilution method was used for isolation. 10g of soil sample was added to an Erlenmeyer flask containing 90mL of sterile water, and the mixture was shaken at 30℃ and 180rpm for 30min. 1mL of the soil suspension was then serially diluted, and 10g of the diluted sample was taken. -4 10 -5 10 -6 0.1 ml of each culture was spread onto LB medium and incubated at 30°C for 1-3 days. Colonies with different morphologies were picked and further purified to obtain single colonies. After 2-3 purifications, the single colonies were stored at 4°C. A total of 18 bacterial strains were isolated.

[0044] 2. Initial screening of Verticillium wilt antagonistic strains

[0045] Verticillium dahliae stored at 4℃ was transferred to PDA plates and activated for 7-10 days. Mycelial discs were taken from the edge of the colony and inoculated into the center of the PDA plate. After incubation at 25℃ for 2-3 days, 18 different bacterial strains isolated from the rhizosphere of the soil were inoculated 20 mm away from the mycelial discs. The plates were then incubated at 25℃ for another 7 days, after which the width of the antagonistic zone was measured.

[0046] The plate confrontation method was used for the initial screening of antagonistic strains. A total of 18 strains were screened, and the results are shown in Table 1. A total of 12 strains formed antagonistic bands with Verticillium dahliae, and 5 strains had antagonistic band widths exceeding 6 mm. The strain numbers were 12, 13, 14, 15, and 16, respectively. These 5 strains were used for secondary screening.

[0047] Table 1 Initial screening results of antagonistic strains

[0048] strain number Antagonistic band width (mm) strain number Antagonistic band width (mm) 1 4 10 4 2 3 11 0 3 4 12 6 4 5 13 10 5 1 14 7 6 3.5 15 7 7 0 16 7 8 0 17 0 9 0 18 0

[0049] 3. Rescreening of Verticillium wilt antagonistic strains

[0050] (1) Preparation of Verticillium dahliae spore suspension

[0051] A small amount of activated cotton wilt mycelium from a PDA plate was inoculated into PDA liquid medium and cultured at 25°C with shaking at 120 rpm for 6 days. The mycelium was then filtered out from the culture medium using sterile gauze, and the spore concentration was adjusted to 1×10⁻⁶ spores with sterile water. 7 CFU / mL available for use.

[0052] (2) Re-screening of inhibition zones

[0053] Pour 1.5% agar into agar plates and allow it to solidify. Prepare a mixed culture medium (take 1 mL of the prepared spore suspension and add it to melted and cooled PDA medium (0.7% agar content) to approximately 45°C, then mix well). Take 10 mL of the mixed culture medium and add it to the solidified agar, spreading it evenly. After the medium cools and solidifies, punch wells in the plate. Seal each well with an appropriate amount of heated sterile agar, and add 20 μL of the test bacterial solution to each well. Incubate the plates at 28°C for 48 hours and observe whether inhibition zones are formed. Use LB liquid medium as a blank control. Set up three replicates for each treatment and measure the diameter of the inhibition zones.

[0054] Five strains initially screened were used as research subjects for secondary screening. The inhibition zone method was used for antagonistic screening. The results are shown in Table 2. The results of the secondary screening showed that except for strain 14, which did not produce an inhibition zone, the other four strains all produced inhibition zones. Among them, the inhibition zone diameter of strain 15 exceeded 20 mm, reaching 23 cm, while the inhibition zones of the other strains were all less than 20 mm.

[0055] Table 2 Results of strain rescreening

[0056] strain number blank 12 13 14 15 16 Transparent ring (mm) 0 17 19 0 23 16

[0057] Example 2: Identification of 16S rDNA from strain 15

[0058] Strain 15 colonies are round, with a rough, opaque surface, milky white color, and neat edges. Under a microscope, the cells appear as short rods, with a diameter of 0.5–1.2 μm and a length of 1.5–2.5 μm.

[0059] The genome of this strain was extracted. Using the genome as a template, the upstream primer 27F (5'-AGAGTTTGATCCTGGCTC-3') and the downstream primer 1492R (5'-AGAGTTTGATCCTGGCTC-3') of the 16S rDNA universal primers were used.

[0060] PCR amplification was performed using the -GGTTACCTTGTTACGACTT-3' gene. The PCR amplification program was as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 55℃ annealing for 45 s, 72℃ extension for 60 s, 30 cycles; 72℃ extension for 5 min. The target DNA fragment was purified and recovered using a gel purification kit (AXYGEN) and sequenced. The sequencing results are shown in SEQ ID NO.1, and BLAST comparison on NCBI confirmed that the 16S rDNA gene sequence similarity between this strain and Bacillus belyssus was 100%.

[0061] Based on the analysis of colony morphology and 16S rDNA sequencing results, the strain was identified as Bacillus velezensis, and it was deposited at the China General Microbiological Culture Collection Center on June 16, 2022, with the accession number CGMCC No. 25095.

[0062] Example 3: Compound formulation of Verticillium wilt antagonistic bacteria

[0063] First, using the same method as in Example 1, the bacterial strains preserved in our laboratory were screened. It was ultimately found that *Bacillus subtilis* with accession number CGMCC No. 20013 had a good antagonistic effect against *Verticillium wilt*. After testing using the same re-screening method as in Example 1, the diameter of its inhibition zone reached 21 cm, exceeding 20 cm. Therefore, we believe this strain may have a good antagonistic effect against *Verticillium wilt*. Therefore, the inventors further combined this *Bacillus subtilis* with the aforementioned *Bacillus bereaves* to test its antagonistic effect.

[0064] The inhibition zone method was used to optimize the bacterial agent compound. The inhibition zone test method was the same as the strain screening in Example 1. The results are shown in Table 3. When Bacillus subtilis and Bacillus belye were compounded in equal proportions, the antagonistic effect against Verticillium dahliae was the best, and the diameter of the inhibition zone reached 25 mm.

[0065] Table 3. Compound formulations of antagonistic fungal agents against Verticillium wilt

[0066]

[0067]

[0068] Example 4: Preparation of Verticillium Wilt Antagonistic Agent

[0069] (1) Activation of bacterial strains: Take out the Bacillus subtilis and Bacillus belye strains stored at 4℃ and activate them at room temperature for 3 hours. Since the bacterial strains are stored on test tube slant nutrient agar medium, no additional nutrients are needed during the activation process.

[0070] (2) Liquid seed preparation: In a sterile operating table, use a pipette to add 10 mL of sterile water into the bacterial culture tube, and use a pipette to repeatedly blow and agitate to prepare a bacterial suspension. Then, inoculate the bacterial suspension into 200 mL of liquid culture medium and shake and culture for 20 h. The culture conditions are: temperature 35℃, shaking speed 170 r / min.

[0071] (3) High-density fermentation: Bacillus subtilis and Bacillus belye liquid seeds were inoculated into the corresponding fermentation medium at an inoculation amount of 1.5% by volume for liquid deep fermentation.

[0072] The formula for high-density culture medium for Bacillus subtilis is as follows: soybean meal 2%-3.5%, corn flour 3%-4%, maltodextrin 1.8%-3.2%, urea 0.1%-0.16%, calcium carbonate 0.1%-0.3%, dimethyl hydrogen phosphate 0.2%-0.3%, magnesium sulfate 0.05%-0.15%, manganese sulfate 0.01%-0.02%, polyether defoamer 0.1%, and the balance being water.

[0073] High-density fermentation conditions for Bacillus subtilis: tank pressure 0.02 MPa, temperature 35℃, rotation speed 200 r / min, dissolved oxygen 30%, and culture time 36 h;

[0074] The formula for high-density culture medium for Bacillus vesiculosus is as follows: soybean meal 2%-4%, corn starch 1.5%-2%, glucose 1%-2%, manganese sulfate 0.02%-0.04%, ammonium sulfate 0.1%-0.2%, magnesium sulfate 0.2%-0.4%, sodium chloride 0.01%-0.03%, polyether defoamer 0.1%, and the balance being water.

[0075] High-density fermentation conditions for Bacillus vesiculosus: tank pressure 0.02 MPa, temperature 35℃, rotation speed 200 r / min, dissolved oxygen 30%, and culture time 36 h;

[0076] (4) Spray drying

[0077] The bacterial cells of the liquid bacterial agent obtained in step (3) are spray-dried at an outlet temperature of 85°C and an inlet temperature of 180°C. The carrier is Jia Yi powder, and the dosage is 5-10%. The bacterial powder is prepared and then mixed with the effective live bacteria in a ratio of 1:1 to obtain a solid antagonistic bacterial agent.

[0078] Take 1g of the prepared antagonistic bacterial agent, dilute it according to the serial dilution method, spread it, incubate it at 30℃ for 24h, and count it. The effective viable count of the antagonistic bacterial agent was found to be 10. 10 cfu / g.

[0079] Example 5: Colonization ability of microbial agents in tomato rhizosphere soil

[0080] The following bacterial preparations were used: a 1:1 mixture of Bacillus subtilis (CGMCC NO.17357) and Bacillus vesiliflorus of the present invention (control group 1); a 1:1 mixture of Bacillus subtilis and Bacillus vesiliflorus of the present invention (CGMCC NO.12744) of the present invention (control group 2); Bacillus subtilis powder from Example 4 (control group 3); Bacillus vesiliflorus powder (control group 4); and a compound antagonistic bacterial preparation. The effective viable count in each group was 10. 10The concentration of cfu / g was approximately 100 cfu / g. Each group was diluted 200 times and sprayed onto the roots of tomatoes at a rate of 20 mL / plant. Ten tomatoes were treated in each group. Rhizosphere soil samples were collected by shaking at 0, 30, and 90 days. The samples were then diluted using a serial dilution method, spread on LB plates, and incubated at 37°C for 24 hours before counting. The colonization capacity of Bacillus subtilis, Bacillus belyssus, the compound antagonistic agent, and the other two compound agents in the rhizosphere soil of tomatoes was determined. (The Bacillus subtilis (CGMCC NO. 17357) and Bacillus belyssus (CGMCC NO. 12744) agents were prepared by fermentation and spray drying according to conventional methods to obtain the corresponding single agents.)

[0081] The test results are as follows:

[0082] Table 4 Comparison of colonization ability of compound antagonistic bacteria agent and control group in tomato rhizosphere soil

[0083]

[0084] Experiments show that compound microbial agents have a stronger and more stable colonization ability in the rhizosphere soil of tomatoes, which is significantly better than single microbial agents and other similar compound microbial agents.

[0085] Example 6: Comparison of the effects of Verticillium wilt antagonist agents and commonly used agents

[0086] The antagonistic fungal agent prepared in Example 4 was compared with the control effects of two pesticides (commercial pesticide 1 is hymexazol, and commercial pesticide 2 is cymoxanil) and two compound fungal agents (compound fungal agent 1 is the same as control group 1 in Example 5, and compound fungal agent 2 is the same as control group 2 in Example 5).

[0087] Tomato seedlings with 2-4 leaves were soaked in pathogen spore solution for 20 minutes, then transplanted into pots containing vermiculite, 5 seedlings per pot. The roots were then drenched with 20 mL of pathogen spore solution. After 6 hours, the seedlings were treated with the aforementioned fungal agents and pesticides. The antagonistic fungal agent of this invention was diluted 200 times and then drenched with 20 mL of solution. Commercially available pesticides 1 and 2 were diluted 400 times and 1400 times respectively, and then drenched with 20 mL of solution. The control group was treated with water. Each group consisted of 20 tomato plants managed under standard conditions. Disease incidence was assessed after 30 days of growth, and the control efficacy was calculated.

[0088] The disease severity index standards are as follows: Level 0: No symptoms; Level 1: One or two cotyledons turn yellow and fall off; Level 2: Three or four true leaves turn yellow and wilt and droop; Level 3: Five or six true leaves turn yellow or wilt and droop; Level 4: The entire plant wilts severely and dies.

[0089] Incidence rate = (Number of diseased plants treated / Total number of plants treated) × 100%;

[0090] Disease index = ∑(number of diseased plants at each level × representative level value) / (total number of plants surveyed × highest representative level value) × 100%;

[0091] Prevention and control effect = (control disease index - treatment disease index) / control disease index × 100%.

[0092] Table 5 Comparison of the control effects of microbial agents and pesticides on tomato Verticillium wilt.

[0093] Incidence rate (%) Disease severity index (%) Prevention and control efficacy (%) control group 100 85 Antagonistic bacteria 5 1.25 98.53 Compound bacterial agent 1 65 41.25 51.47 Compound bacterial agent 2 75 48.75 42.65 Commercially available pesticides 1 70 41.25 51.47 Commercially available pesticides 2 75 37.5 55.88

[0094] The above results indicate that the antagonistic microbial agent of this invention has a good control effect on tomato Verticillium wilt, with a control efficacy of 98.53%, which is significantly better than other microbial agents and pesticides.

[0095] Example 7: Application of microbial agents in strawberry production

[0096] The antagonistic bacterial agent prepared in Example 4 was applied to strawberry cultivation. In a diseased strawberry field, the strawberry mortality rate was 40% before treatment, and strawberries still died even after replanting to the original number of plants. 240 diseased strawberry plants were selected, and four treatments were set up, with 60 strawberry plants in each treatment group. The water control group received 50ml of water per plant. When using the Bacillus subtilis powder, Bacillus vesiculosus powder, and antagonistic bacterial agent prepared in Example 4, the corresponding agents were diluted 200 times and 50ml was applied to the roots of each plant. The same routine management was carried out under greenhouse conditions at 26℃. After 10 days, the strawberry growth was observed, and the strawberry mortality rate was recorded as shown in Table 6.

[0097] Table 6. Effects of each group of inoculants on diseased strawberries.

[0098] Number of plants that died after treatment (plants) mortality rate(%) control group 22 36.7 Bacillus subtilis powder 15 25 Bacillus vesiculosus powder 13 21.67 Antagonistic bacteria 1 1.67

[0099] Experimental results show that the antagonistic fungicide of this invention can effectively improve strawberry root systems and increase strawberry survival rates. Strawberry plants treated with this antagonistic fungicide showed significant root improvement, with new fine hairs growing from the roots, and strawberry mortality rates decreased by more than 95%. The growth status of strawberries before and after treatment with this antagonistic fungicide is as follows: Figure 2 As shown, left a is before treatment with the antagonistic bacteria agent, and right b is after treatment with the antagonistic bacteria agent. It is very obvious that before the antagonistic bacteria agent was used, the tomato seedlings were wilted and in a state of death and stunting. After the antagonistic bacteria agent was used, the tomato seedlings grew vigorously and had large and thick leaves.

[0100] In summary, the antagonistic fungicide of this invention can effectively reduce root rot in strawberries, significantly reduce strawberry mortality, and is environmentally friendly and pollution-free, making it a novel environmentally friendly fungicide with broad application prospects. Furthermore, the above experimental demonstrations show that the combination of Bacillus subtilis and Bacillus vesalis in this invention is specific and unique to each other; not any arbitrary or conventional combination of Bacillus subtilis or Bacillus vesalis with related functions can achieve the above-mentioned technical effects!

[0101] The above description is only a preferred embodiment of the present invention. The application of the present invention is not limited thereto. For any person skilled in the art, there may be various variations or substitutions of this product. Any changes made within the principles and technical scope of the present invention are included within the protection scope of the present invention.

Claims

1. A highly effective antagonistic agent against Verticillium wilt, characterized in that, This inoculant is made from Bacillus subtilis ( Bacillus subtilis ) and Bacillus belesi ( Bacillus velezensis The composition is as follows: the effective viable count ratio of Bacillus subtilis and Bacillus belyssus is 1:1 to 1.

5. Both strains are deposited at the China General Microbiological Culture Collection Center, with Bacillus subtilis having the accession number CGMCC No.20013 and Bacillus belyssus having the accession number CGMCC No.25095.

2. The highly effective antagonistic agent against Verticillium wilt according to claim 1, characterized in that, The antagonistic bacterial agent is a liquid bacterial agent or a solid bacterial agent.

3. The highly effective antagonistic agent against Verticillium wilt according to claim 2, characterized in that, The effective viable count of the bacterial agent is ≥10 8 cfu / mL or cfu / g.

4. The application of the highly effective antagonistic fungicide for Verticillium wilt according to claim 1 in the control of Verticillium wilt, characterized in that, Application methods include applying highly effective antagonistic bacteria for Verticillium wilt to the cultivation of tomatoes or strawberries by root irrigation.

5. The application of the highly effective antagonistic fungal agent for Verticillium wilt according to claim 4 in the control of Verticillium wilt, characterized in that, The application method of the microbial agent is as follows: dilute the microbial agent 200-300 times and then apply it to the roots.

6. The method for preparing the antagonistic bacterial agent according to claim 1, characterized in that, The steps include: (1) Activation of bacterial strains: Take out the Bacillus subtilis and Bacillus belye strains stored at 4℃ and activate them at room temperature for 3 h. Since the bacterial strains are stored on test tube slant nutrient agar medium, no additional nutrients are needed during the activation process. (2) Liquid seed preparation: In a sterile operating table, use a pipette to add 10 mL of sterile water into the bacterial culture tube, and use a pipette to repeatedly blow and agitate to prepare a bacterial suspension. Then, inoculate the bacterial suspension into 200 mL of liquid culture medium and shake and culture for 20 h. The culture conditions are: temperature 35℃, shaking speed 170 r / min. (3) High-density fermentation: Bacillus subtilis and Bacillus belye liquid seeds were inoculated into the corresponding fermentation medium at a volume ratio of 1.5% for liquid deep fermentation; The formula for high-density culture medium for Bacillus subtilis is as follows: soybean meal 2%-3.5%, corn flour 3%-4%, maltodextrin 1.8%-3.2%, urea 0.1%-0.16%, calcium carbonate 0.1%-0.3%, dimethyl hydrogen phosphate 0.2%-0.3%, magnesium sulfate 0.05%-0.15%, manganese sulfate 0.01%-0.02%, polyether defoamer 0.1%, and the balance being water; High-density fermentation conditions for Bacillus subtilis: tank pressure 0.02-0.05 MPa, temperature 30-37℃, rotation speed 180-400 r / min, dissolved oxygen 30%, and culture time 15-36 h; The formula for high-density culture medium for Bacillus vesiculosus is as follows: soybean meal 2%-4%, corn starch 1.5%-2%, glucose 1%-2%, manganese sulfate 0.02%-0.04%, ammonium sulfate 0.1%-0.2%, magnesium sulfate 0.2%-0.4%, sodium chloride 0.01%-0.03%, polyether defoamer 0.1%, and the balance being water; High-density fermentation conditions for Bacillus vesiculosus: tank pressure 0.02-0.05 MPa, temperature 30-37℃, rotation speed 180-400 r / min, dissolved oxygen 30%, and culture time 15-36 h; (4) The liquid or solid inoculum of the highly effective antagonistic fungal agent against Verticillium wilt is further prepared according to the following method: Liquid bacterial agent: Bacillus subtilis and Bacillus belye bacterial solutions are mixed according to the ratio of effective viable bacteria; Solid bacterial agent: The bacterial solutions of the two strains obtained in step (3) are spray-dried separately with an outlet air temperature of 85°C and an inlet air temperature of 180°C. The carrier is Jia Yi powder, and the amount is 5-10%. The bacterial powder is prepared and then mixed according to the effective live bacteria ratio.

Images