Composition for preventing and treating tobacco black shank and application thereof

Abstract

The invention discloses a composition for preventing and treating tobacco black shank and application thereof, and the composition comprises the following components in percentage by mass: 20-85% of a microbial diluent, 18-80% of an auxiliary agent and 0.05% of an active agent. According to the composition for preventing and treating the tobacco black shank, the microbial agent and the tetramycin are used for preventing and treating the tobacco black shank together, so that the composition has a very strong inhibition effect on pathogenic bacteria of the tobacco black shank and shows a very good disease prevention effect on the tobacco black shank; the composition also has the advantages of low possibility of generating drug resistance of germs, obvious synergistic effect, low cost, good effect and environmental friendliness; on the other hand, the composition controls the tobacco black shank through the microbial agent and the tetramycin together, so that the use of chemical pesticides can be reduced to a great extent, the environmental pollution is reduced, and the quality and safety of crops are improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of pesticides, in particular to a composition for preventing and treating tobacco black shank and application thereof. BACKGROUND

[0002] The comprehensive prevention and treatment of tobacco diseases has been an important link in tobacco production. For a long time, chemical prevention and treatment has played a very important role in the prevention and treatment strategy of tobacco diseases. The large and long-term continuous use of chemical pesticides in tobacco production and the unreasonable use of technology have caused the tobacco black shank fungus to develop drug resistance, aggravated the pollution of the environment by the use of pesticides, damaged the ecological system, and also directly affected the quality of tobacco leaves. In overcoming the drug resistance of plant diseases, reducing the pollution of the environment by chemical fungicides, damaging the ecology, and the residues of chemical pesticides in agricultural and sideline products, biocontrol microorganisms have unique advantages. The biopesticides prepared by using actinomycetes have the characteristics of no pollution, no residues, not easy to cause drug resistance of harmful organisms, high compatibility with the environment, safety to humans and animals, etc., and have become the main body of green pesticides without pollution and the development direction of future pesticides.

[0003] Therefore, vigorously developing the biological control research of biocontrol microorganisms is an important way for the sustainable development of agriculture, and has important practical significance in production. SUMMARY

[0004] The purpose of the present application is to provide a composition for preventing and treating tobacco black shank and application thereof, which solves the technical problem of how to reduce the amount of chemical pesticides in the prevention and treatment of tobacco black shank. In order to achieve the above-mentioned purpose, the present application provides the following technical solutions: In a first aspect, the present application provides a composition for preventing and treating tobacco black shank, which comprises 20-85% of microbial inoculant, 18-80% of adjuvant and 0.5‰ of active agent by mass percentage.

[0005] In a second aspect, the present application provides the application of a composition for preventing and treating tobacco black shank, which is sprayed on the front and back of tobacco leaves.

[0006] Beneficial technical effects: The composition for preventing and treating tobacco black shank of the present application has a strong inhibitory effect on the pathogenic fungus of tobacco black shank by using microbial inoculant and tetramycin to prevent and treat tobacco black shank, and shows a good disease prevention effect on tobacco black shank. Moreover, the composition has the advantages of not easy to cause drug resistance of pathogenic bacteria, obvious synergistic effect, low cost, good effect, and environmental friendliness. On the other hand, the composition can greatly reduce the use of chemical pesticides, reduce environmental pollution, and improve the quality and safety of crops by using microbial inoculant and tetramycin to prevent and treat tobacco black shank. DETAILED DESCRIPTION

[0007] In order to make the objectives, technical solutions and advantages of the present application clearer, the technical solutions of the present application will be described in detail below. Obviously, the described embodiments are only some of the embodiments of the present application, not all. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative labor fall within the scope of protection of the present application.

[0008] A composition for preventing and treating tobacco black foot disease, the composition comprising a microbial agent diluent, an auxiliary agent and an active agent, the ratio of the three being 20-85%:18-80%:0.5‰ in terms of mass percentage.

[0009] The microbial agent is a fermentation broth prepared by activating and fermenting Bacillus velezensis YWF-17-0005, and the effective bacterial viable count of the fermentation broth is not less than 5.0x10 9 cfu / mL.

[0010] The microbial agent diluent is obtained by diluting the microbial agent stock solution by 300-500 times.

[0011] The auxiliary agent is obtained by diluting the tetramycin stock solution by 800-1000 times.

[0012] The active agent is Tween 20.

[0013] The preservation accession number of Bacillus velezensis YWF-17-0005 is CCTCC No: M 2022753, the preservation date is May 30, 2022, the preservation unit is China Center for Type Culture Collection, the preservation address is Wuhan University, Wuhan, Hubei Province, China, and the state is alive.

[0014] The technical solutions of the present application will be further described below in combination with embodiments. Example 1

[0015] Preparation method of the composition for preventing and treating tobacco black foot disease: S1, diluting the Bacillus velezensis YWF-17-0005 fermentation broth with water by 300-500 times to obtain a microbial agent diluent, and reserving it; S2, diluting the tetramycin stock solution with water by 800-1000 times to obtain an auxiliary agent, and reserving it; S3, taking the "liquid medicine" prepared in step S1 in the ratio of 20-85%:18-50%:0.5‰ in terms of mass percentage, putting the active agent (Tween 20) and the auxiliary agent prepared in step S2 into a container, and stirring thoroughly to obtain the composition for preventing and treating tobacco black foot disease.

[0016] The preparation method of Bacillus belyssus YWF-17-0005 fermentation broth is carried out according to the following steps: (1) Activation of strain: The Bacillus berleis strain YWF-17-0005 stored at -80°C was activated on LB solid medium for 18~24h to obtain activated Bacillus berleis strain YWF-17-0005.

[0017] (2) Seed culture preparation: The Bacillus berleis strain YWF-17-0005 with better growth in step 1 was picked and inoculated into LB liquid medium using aseptic inoculation method. It was cultured for 17 h at a temperature of 35°C and a rotation speed of 180 rpm / min to obtain Bacillus berleis strain YWF-17-0005 seed culture.

[0018] (3) Preparation of fermentation broth: The Bacillus berleis YWF-17-0005 seed culture obtained in step 2 was inoculated into the fermentation medium in the workshop at a ratio of seed culture to culture medium of 1:10. The culture was carried out at 35±0.5°C and 180 rpm / min for 20-24 hours to obtain the Bacillus berleis YWF-17-0005 fermentation broth. This fermentation broth is the original microbial culture medium, and the effective viable count of the Bacillus berleis YWF-17-0005 fermentation broth is 5.0 × 10⁻⁶. 9 CFU / g or higher.

[0019] (4) The composition of LB liquid culture medium is: 10 g / L bacteriological peptone, 5 g / L yeast extract powder, 10 g / L sodium chloride, and 1 L water to obtain liquid culture medium; 20 g / L agar powder is added to the above liquid culture medium to obtain solid culture medium.

[0020] (5) By mass, the composition of the fermentation medium in the workshop is as follows: molasses solution 1.5~3%, glucose 0.3~0.5%, yeast powder 1.0~1.5%, soybean meal powder 1.8~2.5%, calcium carbonate 0.2~0.5%, potassium dihydrogen phosphate 0.08~0.12%, magnesium sulfate 0.03~0.06%, and the balance is water. The pH of the fermentation medium in the workshop is 7.2~7.5. Example 2

[0021] This embodiment measures the indoor toxicity of the composition for preventing tobacco black shank of the present invention.

[0022] I. Indoor toxicity determination of microbial stock solution and low-toxicity chemical pesticides against tobacco black shank.

[0023] (1) Measure 0.8 g of 0.15% tetracycline AS technical grade into a 10 mL volumetric flask, add 2 mL of sterile water to dissolve it completely, and then dilute to the mark with sterile pure water to prepare an 80 mg / mL AS stock solution for later use. Then dilute the 80 mg / mL stock solution to prepare sterile pure water solutions of 40.0 mg / mL, 13.3 mg / mL, and 8.0 mg / mL for later use.

[0024] Accurately pipette 1.0 g of the microbial stock solution into a 10 mL volumetric flask, add sterile pure water to the mark, and prepare a 100 mg / mL stock solution for later use.

[0025] (2) Take 3 portions of sterilized PDA culture medium (78 mL each), and when the temperature drops to about 50℃, add 2 mL of sterile pure water solution of 80% aluminum tris(ethyl phosphonate) WP containing 40.0 mg / mL, 13.3 mg / mL, and 8.0 mg / mL respectively. Stir and mix with a sterile glass rod to prepare drug-loaded plates containing AS 1000, 333, and 200 μg / mL.

[0026] Bacterial suspensions were added to PDA medium at concentrations of 1000.0 μg / mL, 10000 μg / mL, and 100000 μg / mL to prepare bacterial culture media.

[0027] (3) The virulence determination of tetracycline WP and microbial stock solution was based on the treatment of only inoculating black shank mycelial blocks as a control. The culture was carried out at 28°C. When the blank control colonies reached the edge of the plate, the colony diameter was measured using a vernier caliper using the cross-crossing method. The inhibition rate was calculated based on different concentration gradients. The inhibition rate was converted into a probability value as the dependent variable y, and the logarithm of the mass concentration was used as the independent variable x. A linear equation of "logarithm of mass concentration - probability value" was established, i.e., y = a + bx, which is the virulence regression equation. When y = 5, x was obtained, and the EC50 value was calculated. Inhibition rate (%) = [(control colony diameter - treated colony diameter) ÷ control colony diameter] × 100.

[0028] Table 1. Virulence determination of test agents against black shank pathogen.

[0029] II. Determination of the Combined Toxicity of Microbial Stock Solution and Tetramycin Aqueous Solution against Tobacco Black Shank Fever Based on the above experimental results, the EC50 value of the biocontrol agent alone was 529 μg / mL, and the EC50 value of the chemical agent tetramycin WP alone was 216 μg / mL. To determine the combined toxicity of the microbial stock solution and the chemical pesticide tetramycin aqueous solution, mixed stock solutions were prepared with volume ratios of microbial stock solution to chemical pesticide of 20%∶80%∶0.5‰, 50%∶50%∶0.5‰, and 80%∶18%∶0.5‰. Subsequent inoculation with pathogens and the calculation of relevant data were conducted using the same method as the single-agent toxicity determination method described above. The chemical pesticide tetracycline aqueous solution was selected as the standard pesticide, and the microbial stock solution and mixture were selected as the test "agents". The EC50 value was calculated based on the measured inhibition rate. The toxicity coefficient (TI) and co-toxicity coefficient (CTC) of the standard agent and the test agent were calculated based on the EC50. The effect of the compound preparation was determined based on the co-toxicity coefficient. CTC less than 80 indicates antagonism, CTC greater than 80 and less than 120 indicates addition, and CTC greater than 120 indicates synergistic effect. The formulas for calculating the toxicity index and co-toxicity coefficient are as follows: Toxicity Index (TI) = EC50 value of standard reagent / EC50 value of test reagent * 100; Measured Toxicity Index (ATI) of mixture = EC50 value of standard reagent / EC50 value of mixture * 100; Theoretical Toxicity Index (TTI) of mixture = TIA × PA + TIB × PB; where TIA is the toxicity index of reagent A; PA is the percentage content of reagent A in the mixture; TIB is the toxicity index of reagent B; and PB is the percentage content of reagent B in the mixture. Co-toxicity Coefficient (CTC) of mixture = Measured Toxicity Index of mixture / Theoretical Toxicity Index of mixture * 100.

[0030] Table 2 Record of Virulence Determination of Pathogens by Combination of Biocontrol Agent and Pesticide Tetracycline

[0031] Note: A is 20%∶80%∶0.5‰, B is 50%∶50%∶0.5‰, and C is 80%∶18%∶0.5‰.

[0032] The results showed that the inhibitory effects of the compound formulations differed from those of the single agents based on the co-toxicity coefficient, which ranged from 47 to 289. Specifically, when the volume ratio of microbial stock solution to chemical pesticide was 20%:80, the co-toxicity coefficient was <80, indicating an antagonistic effect. However, when the mixtures were formulated at ratios of 50%:50% and 80%:18%, the co-toxicity coefficients were both higher than 120, indicating a synergistic effect. The highest co-toxicity coefficient and the greatest synergistic effect were observed when the volume ratio of biocontrol agent to chemical pesticide was 50%:50%. Example 3

[0033] This embodiment measures the field effect of the composition for controlling tobacco black shank of the present invention. The field effect was measured by using microbial strain YWF-17-0005 in combination with the low-toxicity chemical pesticide tetramycin AS.

[0034] Test spraying treatment: Treatment was carried out by spraying. The following four treatments were set up for the experiment: a. Full dose of tetracycline AS (diluted 800 times); b. Mix tetracycline and microbial diluent at a volume ratio of 50%:50% (dilute 800 times); c. Microbial diluent YWF-17-0005 (diluted 500 times); d. Blank control. Each treatment was repeated 3 times. Control measures were initiated at the mid-stage of the plant growth, with 3 consecutive applications, each 5 days apart.

[0035] (2) Investigation Methods: Disease surveys were conducted before treatment and 5, 10, and 15 days after 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 leaves at each level × Representative value at each level) / (Total number of leaves surveyed × Highest representative value)] × 100 Control effect (%) = [(1 - (disease index before application in the blank control area × disease index after application in the treatment area) / (disease index after application in the blank control area × disease index before application in the treatment area)] × 100.

[0036] (3) Statistical results of tobacco black shank control in Ludian County, Zhaotong City, Yunnan Province. As can be seen from Table 3, spraying microbial inoculants and low-toxicity chemical pesticides alone both have certain effects on the control of tobacco black shank, with control effects of 27.11% and 25.73%, respectively. However, when microbial inoculants and low-toxicity chemical pesticides are mixed at a volume ratio of 50%:50% and sprayed, the control effect reaches 45.31%, indicating that the combination of the two has a synergistic effect and can improve the control effect on tobacco black shank.

[0037] Table 3. Statistical results of the control efficacy of biocontrol agents and low-toxicity chemical pesticides in combination for tobacco black shank disease.

[0038] (4) Statistical results of tobacco black shank control in Malong County, Qujing City, Yunnan Province. As can be seen from Table 4, spraying microbial inoculants and low-toxicity chemical pesticides alone both have certain effects on the control of tobacco black shank, with control effects of 33.67% and 28.87%, respectively. However, when microbial inoculants and low-toxicity chemical pesticides are mixed at a volume ratio of 50%:50% and sprayed, the control effect reaches 47.19%, indicating that the combination of the two has a synergistic effect and can improve the control effect on tobacco black shank.

[0039] Table 4. Statistical results of the control efficacy of different treatments against tobacco black shank.

[0040] Note: The treatments in Tables 3 and 4 are the average values ​​of three replicates, and " / " indicates no preventive effect.

[0041] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A composition for preventing and treating tobacco black shank disease, characterized in that: The composition comprises, by weight percentage, 20-85% microbial diluent, 18-80% adjuvants, and 0.5‰ activator.

2. The composition for preventing and treating tobacco black shank according to claim 1, characterized in that: The microbial agent is a fermentation broth obtained by activating and fermenting Bacillus belye YWF-17-0005, and the effective bacterial count of the fermentation broth is not less than 5.0 × 10⁻⁶. 9 The concentration of cfu / mL is specified, and the Bacillus velezensis YWF-17-0005 is registered under the CCTCC No: M 2022753.

3. The composition for preventing and treating tobacco black shank according to claim 1, characterized in that: The microbial diluent is obtained by diluting the original microbial solution by 300 to 500 times.

4. The composition for preventing and treating tobacco black shank according to claim 1, characterized in that: The adjuvant is obtained by diluting tetracycline 800-1000 times.

5. The composition for preventing and treating tobacco black shank according to claim 1, characterized in that: The active agent is Tween 20.

6. The application of the composition for preventing tobacco black shank according to any one of claims 1 to 5, wherein the composition is sprayed onto the front and back of tobacco leaves.

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