Bacillus velezensis YWF-25-15 and composition and application thereof
By combining Bacillus vesiculosus YWF-25-15 with aluminum tris(ethyl phosphonate) wettable powder, the problems of environmental dependence of biological agents and pollution from chemical pesticides in tobacco disease control have been solved, achieving efficient and environmentally friendly disease control.
Patent Information
- Application Number
- CN202511961679.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-24
- Publication Date
- 2026-04-24
AI Technical Summary
Existing biological agents are greatly affected by the environment and have unstable biological activity when controlling tobacco black shank and Fusarium root rot, resulting in limited field application. Furthermore, the use of chemical fungicides causes environmental pollution problems.
A combination of Bacillus vesiculosus YWF-25-15 and 80% aluminum tris(ethyl phosphonate) wettable powder was used to control tobacco diseases by mixing the diluted liquid at a volume ratio of 1-5:1-5, combining the synergistic application of biocontrol agents and fungicides.
It significantly improved the control of tobacco black shank and Fusarium root rot, reduced the use of chemical pesticides, reduced environmental pollution, enhanced disease prevention effects, and was less likely to induce drug resistance.
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Abstract
Description
Technical Field
[0001] This invention relates to the field of microbial pesticide technology, and in particular to Bacillus belye YWF-25-15, its compositions, and applications. Background Technology
[0002] In tobacco cultivation, black shank and Fusarium root rot are serious diseases that affect the entire growth cycle of tobacco, causing significant economic losses to the tobacco industry. Currently, there are increasingly more biological agents for controlling tobacco diseases, but due to their inherent limitations, such as severe susceptibility to environmental influences and unstable biological activity, their practical application in the field remains limited. The synergistic application of antagonistic microorganisms and chemical fungicides can overcome the shortcomings of both, representing an environmentally friendly approach to biological control. Summary of the Invention
[0003] Objective: To overcome the shortcomings of the existing technology, the present invention provides Bacillus belye YWF-25-15 and its composition and application for the prevention and control of tobacco black shank disease and / or Fusarium root rot, with good control effect and environmental friendliness.
[0004] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:
[0005] In a first aspect, the present invention provides a strain of Bacillus belyceae YWF-25-15, wherein the taxonomic name of Bacillus belyceae YWF-25-15 is... Bacillus velezensis It is deposited at the China Center for Type Culture Collection, with accession number CCTCC NO: M 20252734.
[0006] In a second aspect, the present invention provides a Bacillus vesiculus YWF-25-15 composition, comprising a Bacillus vesiculus YWF-25-15 diluent and a bactericide diluent; wherein the volume ratio of the Bacillus vesiculus YWF-25-15 diluent to the bactericide diluent is 1-5:1-5.
[0007] In some embodiments, the Bacillus vesiculus YWF-25-15 dilution is obtained by diluting the Bacillus vesiculus YWF-25-15 fermentation broth by 200-500 times.
[0008] In some embodiments, the *Bacillus vesiculus* YWF-25-15 fermentation broth is a fermentation broth obtained by activating and fermenting *Bacillus vesiculus* YWF-25-15; the effective viable count of the *Bacillus vesiculus* YWF-25-15 fermentation broth is ≥5.0 × 10⁻⁶. 9 cfu / mL.
[0009] In some embodiments, the bactericide dilution is obtained by diluting 80% aluminum tris(ethyl phosphonate) wettable powder by 800 to 1200 times.
[0010] Thirdly, the present invention provides a method for preparing a Bacillus belyssus YWF-25-15 composition, comprising: The Bacillus belye YWF-25-15 strain was activated on a solid culture medium, and the strain was picked and inoculated into a liquid culture medium to obtain a seed culture. The seed culture was inoculated into the fermentation medium in the workshop at a volume ratio of 1:40~100 to obtain Bacillus berberis YWF-25-15 fermentation broth. Dilute the fermentation broth of Bacillus vesiculus YWF-25-15 by 200 to 500 times to obtain the diluted fermentation broth of Bacillus vesiculus YWF-25-15. Dilute 80% aluminum tris(ethyl phosphonate) wettable powder 800-1200 times to obtain a bactericide dilution; The solution of Bacillus baileyi YWF-25-15 and the solution of bactericide are mixed evenly at a volume ratio of 1-5:1-5 to obtain the final product.
[0011] In some embodiments, the culture conditions for preparing the seed solution are culture at 35-37°C for 14-20 hours.
[0012] In some embodiments, the culture conditions for preparing the Bacillus belyss YWF-25-15 fermentation broth are as follows: culture at 35-37°C for 24-36 h.
[0013] In some embodiments, each liter of the solid culture medium comprises: 10 g of bacteriological peptone, 5 g of yeast extract, 5 g of sodium chloride, and 20 g of agar powder.
[0014] In some embodiments, each liter of the liquid culture medium comprises: 10 g of bacteriological peptone, 5 g of yeast extract powder, and 5 g of sodium chloride.
[0015] In some embodiments, the workshop fermentation medium comprises: 1.5-2.0 wt% glucose, 1.5-2.0 wt% soybean meal, 0.4-0.8 wt% yeast extract, 0.1-0.2 wt% dipotassium hydrogen phosphate, 0.5-0.8 wt% mannitol, 0.05-0.1 wt% magnesium sulfate, with the remainder being water; the pH of the workshop fermentation medium is 7.0-7.5.
[0016] Fourthly, the present invention provides the use of Bacillus belye YWF-25-15 composition in the prevention and control of tobacco black shank disease and / or Fusarium root rot.
[0017] In some embodiments, the Bacillus berberis YWF-25-15 composition is applied to the roots of tobacco plants via root irrigation.
[0018] Beneficial effects: The Bacillus vesiculus YWF-25-15 and its composition provided by this invention have a strong inhibitory effect on tobacco black shank and / or Fusarium root rot pathogens, showing good disease prevention effect. Moreover, the pathogens are not prone to developing drug resistance, and it has the advantages of significant synergistic effect, low cost, good effect and environmental friendliness. On the other hand, the Bacillus vesiculus YWF-25-15 composition controls tobacco black shank and Fusarium root rot through the joint control of biocontrol agents and fungicides. The fungicide is low in toxicity, which can greatly reduce the use of chemical pesticides, reduce environmental pollution and improve crop quality and safety. Detailed Implementation
[0019] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. The following description of at least one exemplary embodiment is merely illustrative and is in no way intended to limit the present invention or its application or use.
[0020] Unless otherwise specifically stated, the relative arrangement, numerical expressions, and values of the components and steps set forth in these embodiments do not limit the scope of the invention. It should also be understood that techniques, methods, and devices known to those skilled in the art may not be discussed in detail, but where appropriate, such techniques, methods, and devices should be considered part of the specification. In all examples shown and discussed herein, any specific values should be interpreted as merely exemplary and not as limitations. Therefore, other examples of exemplary embodiments may include different values.
[0021] The present invention will be further described below with reference to the embodiments.
[0022] In the following examples, the 80% aluminum tris(ethyl phosphonate) wettable powder used was purchased from Limin Chemical Co., Ltd.
[0023] Example 1: Preparation of Bacillus belyssus YWF-25-15 composition
[0024] This embodiment provides a method for preparing a Bacillus belyssus YWF-25-15 composition, including: Step 1: Activate the *Bacillus belye* YWF-25-15 strain on LB solid medium. Use a sterile inoculation loop to pick out the best-growing strains and inoculate them into LB liquid medium. Incubate at 35-37℃ for 14-20 h to obtain the seed culture. The LB solid medium consists of: 10 g bacteriological peptone, 5 g yeast extract, 5 g sodium chloride, 1 L water, and 20 g agar powder. The LB liquid medium consists of: 10 g bacteriological peptone, 5 g yeast extract, 5 g sodium chloride, and 1 L water. Step 2: Inoculate the seed culture into the fermentation medium in the workshop at a volume ratio of 1:40, and incubate at 35~37℃ for 24~36 hours to obtain the fermentation broth; this fermentation broth is the Bacillus belyssus YWF-25-15 fermentation broth, and the effective viable count in the fermentation broth is ≥5.0×10⁻⁶. 9 cfu / mL; wherein, the fermentation medium in the workshop includes: glucose 1.6wt%, soybean meal powder 1.5wt%, yeast extract 0.6wt%, dipotassium hydrogen phosphate 0.15wt%, mannitol 0.7wt%, magnesium sulfate 0.08wt%, and the remainder is water, and the pH of the fermentation medium in the workshop is 7.2; Step 3: Dilute the Bacillus vesiculus YWF-25-15 fermentation broth 200 times to obtain the diluted Bacillus vesiculus YWF-25-15 solution; Step 4: Dilute 80% aluminum tris(ethyl phosphonate) wettable powder 1000 times to obtain the bactericide diluted solution; Step 5: Mix the diluted solution of Bacillus baileyi YWF-25-15 with the diluted solution of the bactericide at a volume ratio of 4:1 until homogeneous.
[0025] Example 2: Combined virulence determination of Bacillus belyssus YWF-25-15 dilution and bactericide dilution
[0026] Dilute the fermentation broth of Bacillus vesiculus YWF-25-15 by 200 times to obtain a diluted solution of Bacillus vesiculus YWF-25-15; dilute 80% aluminum tris(ethyl phosphonate) wettable powder by 1000 times to obtain a diluted solution of bactericide.
[0027] Compositions A, B, and C were prepared by mixing Bacillus belye YWF-25-15 dilution with a fungicide dilution at volume ratios of 1:4, 5:5, and 4:1, respectively. Compositions A, B, and C were added to plates inoculated with mycelial blocks in the experimental groups, while the control group consisted of PDA plates inoculated only with mycelial blocks. The pathogens were *Blacklegs* and *Fusarium* root rot pathogens.
[0028] Incubate at 28°C. When the control group colonies reach the edge of the plate, measure the colony diameter using calipers with a cross-hatching method and calculate the inhibition rate. Inhibition rate (%) = [(control group colony diameter - experimental group colony diameter) ÷ control group colony diameter] × 100%.
[0029] The inhibition rate was converted into a probability value and used as the dependent variable y. The logarithm of the mass concentration of the compound YWF-25-15 fermentation broth and 80% aluminum tris(ethyl phosphonate) wettable powder was used as the independent variable x. A linear equation of "logarithm of mass concentration - probability value" was established, namely y = a + bx, which is a virulence regression equation. When y = 5, x was obtained, and then EC was calculated. 50 value.
[0030] A 1000-fold dilution of 80% aluminum tris(ethyl phosphonate) wettable powder was used as the standard reagent. A 200-fold dilution of Bacillus vesiculosus YWF-25-15 fermentation broth, Composition A, Composition B, and Composition C were used as test reagents. The EC values of the standard reagent and the test reagents were determined according to... 50 Calculate the toxicity index (TI) and co-toxicity coefficient (CTC), and determine the effects of compositions A, B, and C based on the CTC.
[0031] Toxicity Index (TI) = EC of the standard agent 50 EC value of the test reagent 50 Value * 100.
[0032] Actual toxicity index (ATI) = EC of the standard agent 50 EC value of the composition 50 Value * 100.
[0033] Theoretical toxicity index (TTI) = TI A ×P A +TI B ×P B In the formula, TI A The virulence index of Bacillus belyssus YWF-25-15 dilution; PA is the percentage content of Bacillus belyssus YWF-25-15 dilution in the composition; TI B The toxicity index of the diluted bactericide solution; P B This refers to the percentage content of the bactericide dilution in the mixture.
[0034] Cotoxicity coefficient (CTC) = Measured toxicity index / Theoretical toxicity index * 100.
[0035] CTC < 80 indicates an antagonistic effect, 80 ≤ CTC < 120 indicates an additive effect, and CTC ≥ 120 indicates a synergistic effect.
[0036] Table 1. Results of virulence assays of compositions A, B, and C against the black shank pathogen. Composition Regression equation (y=) <![CDATA[EC 50 Value (ug / mL)]]> Actual toxicity index Theoretical toxicity index Cotoxicity coefficient A 3.5594+0.4038X 3695 0.3031 0.9833 31 B 3.5265+0.6412X 199 5.6281 0.9583 587 C 4.4527+0.3694X 30 37.3333 0.9332 4000
[0037] Table 1 shows that the antibacterial effect of the combined solution of Bacillus vesiculosus YWF-25-15 and the bactericide dilution is different from that of the single agent, with a co-toxicity coefficient of 31-4000. When the volume ratio of Bacillus vesiculosus YWF-25-15 and the bactericide dilution is 1:4, the co-toxicity coefficient is <80, indicating an antagonistic effect. When the volume ratios of Bacillus vesiculosus YWF-25-15 and the bactericide dilution are 5:5 and 4:1, the co-toxicity coefficient is >120, indicating a synergistic effect. Among them, the 4:1 mixture has the highest co-toxicity coefficient and the greatest synergistic effect.
[0038] Table 2. Results of virulence assays of compositions A, B, and C against Fusarium root rot pathogen. Composition Regression equation (y=) <![CDATA[EC 50 Value (ug / mL)]]> Actual toxicity index Theoretical toxicity index Cotoxicity coefficient A -13.7309+6.3280X 912 1.3706 1.2508 110 B -0.6893+2.3133X 288 4.3403 1.6269 267 C 4.1762+0.4610X 61 20.4918 2.0031 1023
[0039] Table 2 shows that the antibacterial effect of the combined solution of Bacillus vesiculosus YWF-25-15 and the bactericide dilution is different from that of the single agent, with a co-toxicity coefficient of 110-1023. When the volume ratio of Bacillus vesiculosus YWF-25-15 and the bactericide dilution is 1:4, the co-toxicity coefficient is between 80 and 120, indicating an additive effect. When the volume ratios are 5:5 and 4:1, the co-toxicity coefficient is >120, indicating a synergistic effect.
[0040] Example 3
[0041] This embodiment measures the field effect of composition C in Example 2 on the control of tobacco black shank disease.
[0042] (1) Root irrigation treatment: Apply the medicine twice consecutively, with an interval of 7 days between each application; repeat 3 times.
[0043] The experiment was divided into the following 4 groups, with the following treatments: a. Composition C: Prepared by mixing Bacillus belye YWF-25-15 dilution and bactericide dilution at a volume ratio of 4:1; b. Diluent for fungicide: 1000 times dilution of 80% aluminum tris(ethyl phosphonate) wettable powder; c. Bacillus vesiculus YWF-25-15 dilution: A 200-fold dilution of Bacillus vesiculus YWF-25-15 fermentation broth; d. Blank control: water.
[0044] (2) Disease surveys were conducted on days 7 and 15 after the second treatment. The incidence of black shank was investigated and recorded according to the national standard GB / T23222-2008. The calculation formula is as follows: Disease index = ∑[(number of diseased plants at each level × representative value at each level) / (total number of plants surveyed × highest representative value)] × 100; Prevention and control effect (%) = [(control disease index - treatment disease index) / control disease index] × 100.
[0045] (3) The efficacy test for tobacco black shank was conducted in Nazhang Town, Malong District, Qujing City, Yunnan Province. As shown in Table 3, both the dilution of Bacillus vesicularis YWF-25-15 and the dilution of low-toxicity chemical pesticide fungicide had certain effects on the control of tobacco black shank, with control effects of 75.00% and 78.55%, respectively. After drenching the roots with composition C, the control effect reached 80.09%, indicating that the combination of Bacillus vesicularis YWF-25-15 dilution and fungicide dilution can not only greatly reduce the amount of chemical pesticides used, but also improve the control effect on tobacco black shank.
[0046] Table 3. Statistical results of the prevention and control efficacy of different treatments against black shank in Nazhang Town.
[0047] Note: The values in the table are the average of three repetitions, and " / " indicates no protective effect.
[0048] (4) The efficacy test for tobacco black shank disease was conducted in Dazhuang Township, Malong County, Qujing City, Yunnan Province. As shown in Table 4, both the dilution of Bacillus vesiculosus YWF-25-15 and the dilution of low-toxicity chemical pesticide fungicide had certain effects on the control of tobacco black shank disease, with control effects of 70.03% and 83.5%, respectively. After drenching the roots with composition C, the control effect reached 85.50%. The combination of Bacillus vesiculosus YWF-25-15 and the fungicide dilution can not only greatly reduce the amount of chemical pesticides used, but also improve the control effect on tobacco black shank disease.
[0049] Table 4. Statistical results of the prevention and control efficacy of different treatments against black shank disease in Dazhuang Township.
[0050] Note: The values in the table are the average of three repetitions, and " / " indicates no protective effect.
[0051] Example 4
[0052] This embodiment measures the field efficacy of composition C in Example 2 in controlling tobacco Fusarium root rot.
[0053] (1) Root irrigation treatment: Apply the medicine twice consecutively, with an interval of 7 days between each application; repeat 3 times.
[0054] The experiment was divided into the following 4 groups, with the following treatments: a. Composition C: Prepared by mixing Bacillus belye YWF-25-15 dilution and bactericide dilution at a volume ratio of 4:1; b. Diluent for fungicide: 1000 times dilution of 80% aluminum tris(ethyl phosphonate) wettable powder; c. Bacillus vesiculus YWF-25-15 dilution: A 200-fold dilution of Bacillus vesiculus YWF-25-15 fermentation broth; d. Blank control: water.
[0055] (2) On days 7 and 15 after the second treatment, a disease survey was conducted. The incidence of Fusarium root rot was investigated and recorded according to the national standard GB / T23222-2008. The calculation formula is as follows: Disease index = ∑[(number of diseased plants at each level × representative value at each level) / (total number of plants surveyed × highest representative value)] × 100; Prevention and control effect (%) = [(control disease index - treatment disease index) / control disease index] × 100.
[0056] (3) The efficacy test of Fusarium root rot of tobacco in Nazhang Town, Malong District, Qujing City, Yunnan Province, shows in Table 5 that the application of Bacillus vesiculosus YWF-25-15 dilution alone and the dilution of low-toxicity chemical pesticide fungicide both have certain effects on the control of Fusarium root rot of tobacco, with control effects of 65.93% and 76.92%, respectively. After drenching the roots with composition C, the control effect reached 78.02%, indicating that the combination of Bacillus vesiculosus YWF-25-15 dilution and fungicide dilution has a synergistic effect and can improve the control effect on Fusarium root rot of tobacco.
[0057] Table 5. Statistical results of the control efficacy of different treatments against tobacco Fusarium root rot in Nazhang Town.
[0058] Note: The values in the table are the average of three repetitions, and " / " indicates no protective effect.
[0059] (4) The efficacy test of Fusarium root rot of tobacco in Dazhuang Township, Malong District, Qujing City, Yunnan Province, can be seen from Table 6: the application of Bacillus vesiculosus YWF-25-15 dilution alone and the dilution of low-toxicity chemical pesticide fungicide both have certain effects on the control of Fusarium root rot of tobacco, with control effects of 68.10% and 79.50%, respectively. After drenching the roots with composition C, the control effect reached 80.65%, indicating that the use of Bacillus vesiculosus YWF-25-15 dilution and fungicide dilution together has a synergistic effect and can significantly improve the control effect on Fusarium root rot of tobacco.
[0060] Table 6. Statistical results of the control efficacy of different treatments against tobacco Fusarium root rot in Dazhuang Township.
[0061] Note: The values in the table are the average of three repetitions, and " / " indicates no protective effect.
[0062] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the technical principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.
Claims
1. A strain of Bacillus belye YWF-25-15, characterized in that, The classification name of the Bacillus belyssus YWF-25-15 is... Bacillus velezensis It is deposited at the China Center for Type Culture Collection, with accession number CCTCC NO: M20252734.
2. A Bacillus belyssus YWF-25-15 composition, characterized in that, It includes a diluted solution of Bacillus vesiculus YWF-25-15 and a diluted solution of a bactericide; the volume ratio of the diluted solution of Bacillus vesiculus YWF-25-15 to the diluted solution of the bactericide is 1-5:1-5.
3. The Bacillus belyssus YWF-25-15 composition according to claim 2, characterized in that, The diluted solution of Bacillus vesiculus YWF-25-15 was obtained by diluting the fermentation broth of Bacillus vesiculus YWF-25-15 by 200-500 times.
4. The Bacillus belyssus YWF-25-15 composition according to claim 3, characterized in that, The *Bacillus berberis* YWF-25-15 fermentation broth is a fermentation broth obtained by activating and fermenting *Bacillus berberis* YWF-25-15; the effective viable count of the *Bacillus berberis* YWF-25-15 fermentation broth is ≥5.0 × 10⁻⁶. 9 cfu / mL.
5. The Bacillus belyssus YWF-25-15 composition according to claim 2, characterized in that, The bactericide dilution is obtained by diluting 80% aluminum tris(ethyl phosphonate) wettable powder by 800-1200 times.
6. A method for preparing a Bacillus belye YWF-25-15 composition, characterized in that, include: The Bacillus belye YWF-25-15 strain was activated on a solid culture medium, and the strain was picked and inoculated into a liquid culture medium to obtain a seed culture. The seed culture was inoculated into the fermentation medium in the workshop at a volume ratio of 1:40~100 to obtain Bacillus berberis YWF-25-15 fermentation broth. Dilute the fermentation broth of Bacillus vesiculus YWF-25-15 by 200 to 500 times to obtain the diluted fermentation broth of Bacillus vesiculus YWF-25-15. Dilute 80% aluminum tris(ethyl phosphonate) wettable powder 800-1200 times to obtain a bactericide dilution; The solution of Bacillus baileyi YWF-25-15 and the solution of bactericide are mixed evenly at a volume ratio of 1-5:1-5 to obtain the final product.
7. The method for preparing the Bacillus belyssus YWF-25-15 composition according to claim 6, characterized in that, The culture conditions for the prepared seed solution are: culture at 35-37℃ for 14-20 h; The culture conditions for preparing the Bacillus berberis YWF-25-15 fermentation broth were 35-37℃ for 24-36 h.
8. The method for preparing the Bacillus belyssus YWF-25-15 composition according to claim 6, characterized in that, Each liter of the solid culture medium comprises: 10 g of bacteriological peptone, 5 g of yeast extract, 5 g of sodium chloride, and 20 g of agar powder; Each liter of the liquid culture medium comprises: 10 g of bacteriological peptone, 5 g of yeast extract, and 5 g of sodium chloride; The fermentation medium in the workshop comprises: 1.5-2.0 wt% glucose, 1.5-2.0 wt% soybean meal, 0.4-0.8 wt% yeast extract, 0.1-0.2 wt% dipotassium hydrogen phosphate, 0.5-0.8 wt% mannitol, 0.05-0.1 wt% magnesium sulfate, with the remainder being water; the pH of the fermentation medium in the workshop is 7.0-7.
5.
9. Application of Bacillus belyssus YWF-25-15 composition in the control of tobacco black shank and / or Fusarium root rot.
10. The application according to claim 8, characterized in that, The Bacillus berberis YWF-25-15 composition was applied to the roots of tobacco plants via root irrigation.