Preparation method and application of biocontrol bacterium inducer for preventing both tobacco vein necrosis and wildfire

Abstract

The invention discloses a preparation method and application of a biocontrol bacterium inducer for preventing both tobacco vein necrosis and wildfire. Belongs to the technical field of tobacco disease biological control. The biocontrol bacterium inducer disclosed by the invention is prepared by the following steps: mixing a bacillus subtilis bacterium solution, an enterobacter bacterium solution and a Pseudomonas aeruginosa bacterium solution, sequentially carrying out mitomycin C and ultraviolet induction, and carrying out membrane filtration. Aiming at the problems of pathogenic bacterium drug resistance, secondary pollution and the like caused by the existing chemical prevention and treatment method for the tobacco vein necrosis disease and the wildfire disease, the method disclosed by the invention is mainly used for preventing and treating the tobacco phyllospheric diseases by using a biocontrol bacterium inducer, and has the advantages that the morbidity of the tobacco vein necrosis disease and the wildfire disease is reduced; no pathogenic bacteria drug resistance is generated, and secondary pollution is not caused; the treatment capacity is large and the operation is simple.

Description

Technical Field

[0001] This invention relates to the field of biological control technology for tobacco diseases, and more specifically to a method for preparing and applying a biocontrol agent that can prevent both tobacco vein necrosis and wildfire disease. Background Technology

[0002] Tobacco is an important economic crop, and the tobacco industry is a vital pillar industry in my country. Potatoes are a widely cultivated and important food crop globally. Potatoes carry Potato Virus Y (PVY), which can be transmitted by aphids, causing tobacco to suffer from tobacco PVY disease, also known as vein necrosis. Tobacco wildfire disease is caused by Pseudomonas aeruginosa. In recent years, with the increasing area of ​​tobacco cultivation, the occurrence of tobacco diseases has also shown a trend of aggravation, with both the affected area and the severity of the disease increasing.

[0003] Tobacco vein necrosis and wildfire disease have high incidence rates and wide affected areas, severely impacting the yield and quality of flue-cured tobacco. Vein necrosis initially causes prominent veins on new leaves, later developing into systemic mosaic patterns, and in severe cases, it leads to stunted growth. Wildfire disease can break out in seedlings, mature plants, and at maturity; under high temperature and humidity, lesions rapidly merge, and bacterial ooze occurs. The occurrence of these diseases is closely related to varietal resistance, climatic conditions, fertilization levels, and cropping systems, making complete control difficult with a single treatment method.

[0004] Currently, the main method for controlling tobacco vein necrosis and wildfire disease is the application of chemical agents. Although chemical agents can suppress tobacco vein necrosis and wildfire disease in a short period of time, long-term use can lead to drug resistance and secondary pollution.

[0005] Biological control, with its advantages of being environmentally friendly, residue-free, having low resistance risk, and inducing plant immunity, is becoming a new direction for integrated tobacco disease management. This integrated and sustainable biological control strategy is precisely the key technological bottleneck to solving the problem of safe and efficient tobacco disease control. However, there are currently few reports on tobacco biological control technologies.

[0006] In summary, how to provide a biological control agent that can prevent both tobacco vein necrosis and wildfire disease is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0007] In view of this, the present invention provides a method for preparing a biocontrol agent that can prevent both tobacco vein necrosis and wildfire disease, and its application.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] A method for preparing a biocontrol bacterial inducer that can prevent both tobacco vein necrosis and wildfire disease involves mixing Bacillus subtilis bacterial suspension, Enterobacter bacterial suspension, and Pseudomonas geniculata bacterial suspension, and then sequentially passing the mixture through mitomycin C and ultraviolet induction followed by membrane filtration.

[0010] Further,

[0011] The Bacillus subtilis has the accession number CCTCC AB 130001;

[0012] The accession number of the Enterobacter is CCTCC AB 206648;

[0013] The accession number of the geniculate pseudomonocyte is CCTCC AB 205694.

[0014] Furthermore, the volume ratio of Bacillus subtilis culture, Enterobacter culture, and Pseudomonas geniculatus culture was (35-50):(18-25):(30-47).

[0015] Furthermore, the volume ratio of Bacillus subtilis culture, Enterobacter culture, and Pseudomonas geniculatus culture was 50:20:30.

[0016] Furthermore, the viable counts of Bacillus subtilis, Enterobacter, and Pseudomonas geniculatus bacterial suspensions were 10. 9 ~10 10 per ml.

[0017] Furthermore, the specific procedure for mitomycin C induction is as follows: add 0.5–2 μg / ml of mitomycin C to the bacterial culture, and then incubate in a shaker at 35–40°C for 4–6 h.

[0018] Furthermore, the specific operation of the ultraviolet induction is as follows: 1~3 mW / cm 2 Induce for 10–30 seconds, then incubate at 35–40°C in a shaker for 1 hour.

[0019] The biocontrol bacteria inducer prepared by the above preparation method.

[0020] The above-mentioned biocontrol bacteria inducers are used in the prevention and control of tobacco vein necrosis and wildfire disease.

[0021] Furthermore, the tobacco is Yunyan 87.

[0022] As can be seen from the above technical solution, compared with the prior art, the beneficial effects achieved by the present invention are as follows:

[0023] In view of the problems of pathogen resistance and secondary pollution caused by the current chemical control methods for tobacco vein necrosis and wildfire disease, the method of the present invention mainly uses biocontrol bacteria inducers to control tobacco leaf diseases. The advantages are as follows: (1) reducing the incidence of tobacco vein necrosis and wildfire disease; (2) no pathogen resistance is generated and no secondary pollution is caused; (3) large processing capacity and simple operation. Detailed Implementation

[0024] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0025] The reagents required for this invention are conventional experimental reagents, purchased from commercially available channels; the experimental methods not mentioned are conventional experimental methods, and will not be described in detail here.

[0026] The tobacco variety used in the following examples is Yunyan 87.

[0027] Example 1

[0028] (1) Select Bacillus subtilis (accession number: CCTCC AB 130001), Enterobacter p. (accession number: CCTCC AB 206648), and Pseudomonas geniculata (accession number: CCTCC AB 205694).

[0029] (2) Liquid culture medium components: including 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride and water; the pH value of the liquid culture medium is 7.0 to 7.3.

[0030] (3) Activation and expansion culture of biocontrol bacteria: After activating the bacterial powder in (1) into a primary strain through a solid slant, the activated strains were inoculated into liquid culture medium and cultured until the initial bacterial concentration was 1×10⁻⁶. 6 Seed culture was obtained by inoculating at a concentration of 15% (v / v) per mL, and then transferred to a new liquid culture medium for further cultivation. The culture was carried out at 35–40°C and 180–220 rpm until the microbial count reached 10⁻⁶. 9 ~10 10 The bacterial culture was collected at a rate of 100 cells / ml, and then the bacterial solution of each strain was collected in a storage tank and stored at 4°C.

[0031] Example 2

[0032] The preparation method of the biocontrol bacteria inducer is as follows:

[0033] The bacterial suspensions of the three strains prepared in Example 1 were selected and mixed at a volume ratio of 25% Enterobacter, 40% Bacillus subtilis, and 35% Pseudomonas geniculates. The mixture was incubated at 37°C and 200 rpm for 12 h on a shaker. After adding 0.8 μg / ml mitomycin C, the mixture was incubated at 37°C and 200 rpm for 5.5 h on a shaker. Subsequently, it was incubated under UV light at 2 mW / cm². 2 After induction for 30 seconds, the culture was continued at 37℃ and 200 rpm for 1 hour. The bacterial solution was then passed through a 0.22 μm filter membrane to obtain the biocontrol bacteria inducer.

[0034] 40% dimethoate diluted 2000 times and 0.6% (v / v) kasugamycin were selected as chemical control reagents for tobacco vein necrosis and wildfire disease.

[0035] Next, a potted plant experiment was conducted, with the following groupings:

[0036] ① Biological control group A for necrotic tuberculosis (tobacco plants + aphids carrying potato virus Y);

[0037] ② Chemical control group A for necrotic tuberculosis (tobacco plants + aphids carrying potato virus Y, with corresponding chemical reagents added after the disease occurs);

[0038] ③ Experimental group A of biocontrol bacteria inducer for necrotic tuberculosis (tobacco plant + aphids carrying potato virus Y, biocontrol bacteria inducer added after disease onset);

[0039] ④ Biological control group B for necrotic tuberculosis (tobacco plants + aphids carrying potato virus Y (time is the same as group B));

[0040] ⑤ Chemical control group B for necrotic tuberculosis (tobacco plants + corresponding chemical reagents, followed by the addition of aphids carrying potato virus Y);

[0041] ⑥ Experimental group B of biocontrol bacteria inducer for necrosis of the vein (tobacco plant + added biocontrol bacteria inducer, followed by the addition of aphids carrying potato virus Y);

[0042] ⑦ Wildfire disease biological control group A (tobacco plants + wildfire disease pathogen liquid);

[0043] ⑧ Chemical control group A for wildfire disease (tobacco plant + wildfire pathogen liquid, with corresponding chemical reagents added after the onset of the disease);

[0044] ⑨ Wildfire disease biocontrol inducer experimental group A (tobacco plant + wildfire disease pathogen liquid, biocontrol inducer added after disease onset);

[0045] ⑩ Wildfire disease biological control group B (tobacco plants + wildfire disease pathogen liquid (time is the same as group B));

[0046] Wildfire disease chemical control group B (tobacco plants + corresponding chemical reagents, followed by wildfire disease pathogen solution);

[0047] Wildfire disease biocontrol inducer experimental group B (tobacco plants + added biocontrol inducer, followed by wildfire disease pathogen liquid).

[0048] Each group has 15 parallels.

[0049] Select tobacco plants with similar growth conditions and transplant them into pots. After the growth conditions stabilize, groups ① to ③ are first treated with aphids carrying Potato Virus Y, 5 to 10 aphids per pot, maintaining a temperature of 20 to 25℃ and humidity of 90%. After the tobacco plants show obvious symptoms, they are inoculated with the corresponding reagent. Groups ④ to ⑥ are first inoculated with the corresponding reagent, and then treated with aphids carrying Potato Virus Y, 5 to 10 aphids per pot, maintaining a temperature of 20 to 25℃ and humidity of 90%. Groups ⑦ to ⑨ are first treated with a fungicide concentration of 1×10⁶. 7 Spray the wildfire pathogen solution evenly onto the upper and lower surfaces of tobacco leaves (spray until the leaves are wet). After 15 minutes of infection, rinse with clean water to wash away any remaining pathogens on the surface. Once obvious symptoms appear, add the corresponding reagent and maintain a temperature of 20-25℃ and a humidity of 90%. After adding the corresponding reagents, the group was then used with a bacterial count of 1×10⁻⁶. 7 Spray the wildfire pathogen solution evenly onto the upper and lower surfaces of the tobacco leaves (until the leaves are wet). After 15 minutes of infection, rinse with clean water to wash away any remaining pathogens on the surface. Maintain a temperature of 20-25℃ and a humidity of 90%.

[0050] The above groups were sprayed with the corresponding chemical reagents or biocontrol bacteria inducers (20ml per leaf, 100ml per plant). The incidence rate and the sprayed reagents were recorded every 7 days (i.e., the reagents were sprayed on day 0 and day 7 of the entire experiment).

[0051] Table 1. Incidence of Vein Necrosis in Tobacco Plants

[0052] Incidence rate on day 0 (%) 7-day incidence rate (%) 14-day incidence rate (%) Biological control group A 93％ 93％ 100％ Chemical control group A 93％ 67％ 60％ Biocontrol bacteria inducer experimental group A 93％ 60％ 60％ Biological control group B 93％ 100％ 100％ Chemical control group B 93％ 60％ 40％ Biocontrol bacteria inducer experimental group B 93％ 54％ 40％

[0053] Table 2. Incidence of wildfire disease in tobacco plants

[0054] Incidence rate on day 0 (%) 7-day incidence rate (%) 14-day incidence rate (%) Biological control group A 93％ 93％ 100％ Chemical control group A 93％ 67％ 60％ Biocontrol bacteria inducer experimental group A 93％ 67％ 67％ Biological control group B 93％ 100％ 100％ Chemical control group B 93％ 60％ 40％ Biocontrol bacteria inducer experimental group B 93％ 54％ 47％

[0055] Tables 1 and 2 show that the biocontrol agent prepared in Example 2 has a certain inhibitory effect on necrosis of blood vessels and wildfire disease, and the preventive effect is better when it is carried out before the onset of the disease. However, its overall inhibitory effect is not significantly better than that of chemical reagents.

[0056] Example 3

[0057] The preparation method of the biocontrol bacteria inducer is as follows:

[0058] The bacterial suspensions of the three strains prepared in Example 1 were selected and mixed at a volume ratio of 18% Enterobacter, 35% Bacillus subtilis, and 47% Pseudomonas geniculatus. The mixture was incubated at 37°C and 200 rpm for 12 h on a shaker. After adding 0.8 μg / ml mitomycin C, the mixture was incubated at 37°C and 200 rpm for 5.5 h on a shaker. Subsequently, it was subjected to UV 2 mW / cm² UV light. 2 After induction for 30 seconds, the culture was continued at 37℃ and 200 rpm for 1 hour. The bacterial solution was then passed through a 0.22 μm filter membrane to obtain the biocontrol bacteria inducer.

[0059] 40% dimethoate diluted 2000 times and 0.6% (v / v) kasugamycin were selected as chemical control reagents for tobacco vein necrosis and wildfire disease.

[0060] Next, a potted plant experiment was conducted, with the following groupings:

[0061] ① Biological control group for tuberculosis (tobacco plants + aphids carrying potato virus Y);

[0062] ② Chemical control group for necrosis of the vein (tobacco plants + corresponding chemical reagents, followed by the addition of aphids carrying potato virus Y);

[0063] ③ Experimental group of biocontrol bacteria inducer for necrotic tuberculosis (tobacco plants + added biocontrol bacteria inducer, followed by the addition of aphids carrying potato virus Y);

[0064] ④ Wildfire disease biological control group (tobacco plants + wildfire disease pathogen liquid);

[0065] ⑤ Wildfire disease chemical control group (tobacco plants + corresponding chemical reagents, followed by wildfire disease pathogen solution);

[0066] ⑥ Wildfire disease biocontrol agent inducer experimental group (tobacco plant + added biocontrol agent, followed by wildfire disease pathogen liquid).

[0067] Each group has 15 parallels.

[0068] Select tobacco plants with similar growth conditions and transplant them into pots. After their growth stabilizes, groups ① to ③ are first inoculated with the corresponding reagents, followed by the use of aphids carrying potato virus Y, 5 to 10 aphids per pot, maintaining a temperature of 20 to 25℃ and humidity of 90%; groups ④ to ⑥ are first inoculated with the corresponding reagents, followed by the use of aphids with a bacterial count of 1×10⁶. 7 Spray the wildfire pathogen solution evenly onto the upper and lower surfaces of the tobacco leaves (until the leaves are wet). After 15 minutes of infection, rinse with clean water to wash away any remaining pathogens on the surface. Maintain a temperature of 20-25℃ and a humidity of 90%.

[0069] The above groups were sprayed with the corresponding chemical reagents or biocontrol bacteria inducers (20ml per leaf, 100ml per plant). The incidence rate and the sprayed reagents were recorded every 7 days (i.e., the reagents were sprayed on day 0 and day 7 of the entire experiment).

[0070] Table 3. Incidence of Vein Necrosis in Tobacco Plants

[0071]

[0072]

[0073] Table 4. Incidence of wildfire disease in tobacco plants

[0074] Incidence rate on day 0 (%) 7-day incidence rate (%) 14-day incidence rate (%) biological control group 93％ 93％ 100％ Chemical control group 93％ 67％ 40％ Biocontrol bacteria inducer experimental group 93％ 47％ 40％

[0075] The data in Tables 3 and 4 show that the biocontrol agent prepared in Example 3 has a slightly better inhibitory effect than the chemical reagent.

[0076] Example 4

[0077] The preparation method of the biocontrol bacteria inducer is as follows:

[0078] The bacterial suspensions of the three strains prepared in Example 1 were selected and mixed at a volume ratio of 20% Enterobacter, 50% Bacillus subtilis, and 30% Pseudomonas geniculates. The mixture was incubated at 37°C and 200 rpm for 12 h on a shaker. After adding 0.8 μg / ml mitomycin C, the mixture was incubated at 37°C and 200 rpm for 5.5 h on a shaker. Subsequently, it was incubated under UV light at 2 mW / cm². 2 After induction for 30 seconds, the culture was continued at 37℃ and 200 rpm for 1 hour. The bacterial solution was then passed through a 0.22 μm filter membrane to obtain the biocontrol bacteria inducer.

[0079] 40% dimethoate diluted 2000 times and 0.6% (v / v) kasugamycin were selected as chemical control reagents for tobacco vein necrosis and wildfire disease.

[0080] Next, a potted plant experiment was conducted, with the following groupings:

[0081] ① Biological control group for tuberculosis (tobacco plants + aphids carrying potato virus Y);

[0082] ② Chemical control group for necrosis of the vein (tobacco plants + corresponding chemical reagents, followed by the addition of aphids carrying potato virus Y);

[0083] ③ Experimental group of biocontrol bacteria inducer for necrotic tuberculosis (tobacco plants + added biocontrol bacteria inducer, followed by the addition of aphids carrying potato virus Y);

[0084] ④ Wildfire disease biological control group (tobacco plants + wildfire disease pathogen liquid);

[0085] ⑤ Wildfire disease chemical control group (tobacco plants + corresponding chemical reagents, followed by wildfire disease pathogen solution);

[0086] ⑥ Wildfire disease biocontrol agent inducer experimental group (tobacco plant + added biocontrol agent, followed by wildfire disease pathogen liquid).

[0087] Each group has 15 parallels.

[0088] Select tobacco plants with similar growth conditions and transplant them into pots. After their growth stabilizes, groups ① to ③ are first inoculated with the corresponding reagents, followed by the use of aphids carrying Potato Virus Y, 5 to 10 aphids per pot, maintaining a temperature of 20 to 25°C and humidity of 90%; groups ④ to ⑥ are first inoculated with the corresponding reagents, followed by the use of aphids with a bacterial count of 1×10⁻⁶. 7 Spray the wildfire pathogen solution evenly onto the upper and lower surfaces of the tobacco leaves (until the leaves are wet). After 15 minutes of infection, rinse with clean water to wash away any remaining pathogens on the surface. Maintain a temperature of 20-25℃ and a humidity of 90%.

[0089] The above groups were sprayed with the corresponding chemical reagents or biocontrol bacteria inducers (20ml per leaf, 100ml per plant). The incidence rate and the sprayed reagents were recorded every 7 days (i.e., the reagents were sprayed on day 0 and day 7 of the entire experiment).

[0090] Table 5. Incidence of Vein Necrosis in Tobacco Plants

[0091] Incidence rate on day 0 (%) 7-day incidence rate (%) 14-day incidence rate (%) biological control group 93％ 93％ 100％ Chemical control group 93％ 67％ 40％ Biocontrol bacteria inducer experimental group 93％ 40％ 13％

[0092] Table 6. Incidence of wildfire disease in tobacco plants

[0093] Incidence rate on day 0 (%) 7-day incidence rate (%) 14-day incidence rate (%) biological control group 93％ 93％ 100％ Chemical control group 93％ 67％ 47％ Biocontrol bacteria inducer experimental group 93％ 33％ 13％

[0094] The data in Tables 5 and 6 show that the biocontrol agent prepared in Example 4 has a significantly better inhibitory effect than chemical reagents.

[0095] Example 5

[0096] Datian Application

[0097] Strain preparation: Prepare Bacillus subtilis (accession number: CCTCC AB130001), Enterobacter sp. (accession number: CCTCC AB 206648), and Pseudomonas geniculata (accession number: CCTCC AB 205694).

[0098] Strain activation: Enterobacteriaceae, Pseudomonas geniculatus, and Bacillus subtilis were activated into original strains by solid slant culture.

[0099] Expanded culture: The activated strains were inoculated into liquid culture medium and cultured until the initial bacterial concentration reached 1×10⁻⁶. 6 The inoculum was prepared at a concentration of 15% (v / v) and then transferred to a new liquid culture medium. The culture temperature was 37℃, 200 rpm, and the culture was expanded for 24 h. When the bacterial count of each strain reached 1×10⁻⁶, the culture was considered successful. 9 When the bacterial count is 1000 cells / ml, the bacterial solution of each strain is collected using a storage tank.

[0100] The liquid culture medium comprises 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, and water; the pH of the liquid culture medium is 7.0.

[0101] Preparation of biocontrol bacterial inducers: The collected bacterial suspensions were mixed at a volume ratio of 20% Enterobacter, 50% Bacillus subtilis, and 30% Pseudomonas geniculata. The mixture was incubated at 37°C and 200 rpm for 12 h on a shaker. Then, 0.8 μg / ml mitomycin C was added, and the mixture was incubated at 37°C and 200 rpm for 5.5 h on a shaker. Finally, the mixture was subjected to UV 2 mW / cm². 2 After induction for 30 seconds, the culture was continued at 37℃ and 200 rpm for 1 hour. The bacterial solution was then passed through a 0.22 μm filter membrane to obtain the biocontrol bacteria inducer.

[0102] 40% dimethoate diluted 2000 times and 0.6% (v / v) kasugamycin were selected as chemical control reagents for tobacco vein necrosis and wildfire disease.

[0103] Following this, a field experiment was conducted (at the tobacco field base in Shimen County, Changde City). Six fields were selected for the experiment, and the groups were as follows:

[0104] Field No. 1: Biological control group for tuberculosis (tobacco plants + aphids carrying potato virus Y);

[0105] Field No. 2: Chemical control group for vein necrosis (tobacco plants + corresponding chemical reagents, followed by the addition of aphids carrying potato virus Y);

[0106] Field No. 3: Experimental group of biocontrol bacteria inducer for necrotic tuberculosis (tobacco plants + added biocontrol bacteria inducer, followed by the addition of aphids carrying potato virus Y);

[0107] Field No. 4: Wildfire disease biological control group (tobacco plants + wildfire disease pathogen liquid);

[0108] Field No. 5: Wildfire disease chemical control group (tobacco plants + corresponding chemical reagents, followed by wildfire disease pathogen solution);

[0109] Field No. 6: Wildfire disease biocontrol inducer experimental group (tobacco plants + added biocontrol inducer, followed by wildfire disease pathogen liquid).

[0110] Fields 1-3 were first inoculated with the corresponding reagent, followed by the use of 5 aphids carrying potato virus Y per plant; fields 4-6 were first inoculated with the corresponding reagent, followed by the use of aphids with a bacterial count of 1×10⁶. 7 Spray the inoculum of wildfire pathogen evenly onto the upper and lower surfaces of tobacco leaves (until the leaves are moist). After 15 minutes of infection, rinse with clean water to wash away any remaining pathogens on the surface.

[0111] The above groups were sprayed with the corresponding chemical reagents or biocontrol bacteria inducers (20ml per leaf, 100ml per plant). The incidence rate and the sprayed reagents were recorded every 7 days (i.e., the reagents were sprayed on day 0 and day 7 of the entire experiment).

[0112] Table 7. Incidence of Vein Necrosis Disease in Tobacco Plants in the Field

[0113] Incidence rate on day 0 (%) 7-day incidence rate (%) 14-day incidence rate (%) biological control group 76.4％ 79.6％ 82.4％ Chemical control group 75.3％ 53.2％ 42.6％ Biocontrol bacteria inducer experimental group 76.8％ 46.9％ 27.9％

[0114] Table 8. Incidence of wildfire disease in tobacco plants in the field.

[0115] Incidence rate on day 0 (%) 7-day incidence rate (%) 14-day incidence rate (%) biological control group 74.1％ 77.8％ 85.4％ Chemical control group 75.1％ 56.4％ 43.6％ Biocontrol bacteria inducer experimental group 74.5％ 48.1％ 28.4％

[0116] The results in Tables 7 and 8 show that chemical reagents can inhibit tobacco vein necrosis and wildfire disease to a certain extent, but the effect is limited. Biocontrol agents can effectively control the spread of tobacco diseases in a short period of time and further reduce the incidence rate.

[0117] Comparative Example 1

[0118] The effect of a single biocontrol inducer on the control of tobacco vein necrosis and wildfire disease at different time points

[0119] The bacterial cultures of the three strains prepared in Example 1 were cultured at 37°C and 200 rpm for 12 h on a shaker. After adding 0.8 μg / ml mitomycin C, the cultures were cultured at 37°C and 200 rpm for 5.5 h on a shaker, followed by UV incubation at 2 mW / cm². 2 Induction was performed for 30 seconds, followed by incubation at 37℃ and 200 rpm for 1 hour. The bacterial culture was then passed through a 0.22 μm filter membrane to obtain Enterobacter inducers, Bacillus subtilis inducers, and Pseudomonas geniculatus inducers, respectively.

[0120] 40% dimethoate diluted 2000 times and 0.6% (v / v) kasugamycin were selected as chemical control reagents for tobacco vein necrosis and wildfire disease.

[0121] Next, a potted plant experiment was conducted, with the following groupings:

[0122] ① Biological control group A for necrotic tuberculosis (tobacco plants + aphids carrying potato virus Y);

[0123] ② Chemical control group A for necrotic tuberculosis (tobacco plants + aphids carrying potato virus Y, with corresponding chemical reagents added after the disease occurs);

[0124] ③ Experimental group A1 of the biocontrol agent for necrosis of the vein (tobacco plant + aphids carrying potato virus Y, with Enterobacter inducer added after the disease occurs);

[0125] ④ Experimental group A2 of biocontrol bacteria inducer for necrosis of the vein (tobacco plant + aphids carrying potato virus Y, with geniculate pseudomonocyte inducer added after disease onset);

[0126] ⑤ Experimental group A3 (tobacco plant + aphids carrying potato virus Y, Bacillus subtilis inducer added after disease onset);

[0127] ⑥ Biological control group B for necrotic tuberculosis (tobacco plants + aphids carrying potato virus Y (time same as control group B));

[0128] ⑦ Chemical control group B for necrotic tuberculosis (tobacco plants + corresponding chemical reagents, followed by the addition of aphids carrying potato virus Y);

[0129] ⑧ Experimental group B1 of biocontrol bacteria inducer for necrosis of the vein (tobacco plant + Enterobacter inducer, followed by aphids carrying potato virus Y);

[0130] ⑨ Experimental group B2 of biocontrol agent for necrosis of the vein (tobacco plant + added geniculate pseudomonocyte inducer, followed by the addition of aphids carrying potato virus Y);

[0131] ⑩ Experimental group B3 for the biocontrol agent inducer for necrosis of the vein (tobacco plant + Bacillus subtilis inducer, followed by the addition of aphids carrying potato virus Y);

[0132] Wildfire disease biological control group A (tobacco plants + wildfire disease pathogen liquid);

[0133] Wildfire disease chemical control group A (tobacco plants + wildfire disease pathogen liquid, with corresponding chemical reagents added after the onset of the disease);

[0134] Wildfire disease biocontrol agent inducer experimental group A1 (tobacco plant + wildfire disease pathogen liquid, Enterobacter inducer added after disease onset);

[0135] Wildfire disease biocontrol agent inducer experimental group A2 (tobacco plant + wildfire disease pathogen liquid, with geniculate pseudomonad inducer added after disease onset);

[0136] Wildfire disease biocontrol agent inducer experimental group A3 (tobacco plant + wildfire disease pathogen liquid, Bacillus subtilis inducer added after disease onset);

[0137] Wildfire disease biological control group B (tobacco plants + wildfire disease pathogen liquid (time same as control group B));

[0138] Wildfire disease chemical control group B (tobacco plants + corresponding chemical reagents, followed by wildfire disease pathogen solution);

[0139] Wildfire disease biocontrol agent inducer experimental group B1 (tobacco plant + Enterobacter inducer, followed by wildfire disease pathogen liquid);

[0140] Wildfire disease biocontrol agent inducer experimental group B2 (tobacco plant + added geniculate pseudomonad inducer, followed by wildfire disease pathogen liquid);

[0141] Wildfire disease biocontrol agent experimental group B3 (tobacco plant + Bacillus subtilis inducer, followed by wildfire disease pathogen liquid).

[0142] Each group has 15 parallels.

[0143] Select tobacco plants with similar growth conditions and transplant them into pots. After the growth conditions stabilize, groups ① to ⑤ first use aphids carrying potato virus Y, 5 to 10 aphids per pot, and maintain a temperature of 20 to 25°C and a humidity of 90%. After the tobacco plants show obvious symptoms, they are inoculated with the corresponding reagent. Groups ⑥ to ⑩ are first inoculated with the corresponding reagent, and then use aphids carrying potato virus Y, 5 to 10 aphids per pot, and maintain a temperature of 20 to 25°C and a humidity of 90%. The initial bacterial count used in the group was 1×10⁶. 7 Spray the wildfire pathogen solution evenly onto the upper and lower surfaces of tobacco leaves (until the leaves are wet). After 15 minutes of infection, rinse with clean water to wash away any remaining pathogens on the surface. Maintain a temperature of 20-25℃ and humidity of 90% until obvious symptoms appear. Once symptoms appear, add the corresponding reagent. First, add the corresponding reagents to the group, then use a bacterial count of 1×10⁻⁶. 7 Spray the wildfire pathogen solution evenly onto the upper and lower surfaces of the tobacco leaves (until the leaves are wet). After 15 minutes of infection, rinse with clean water to wash away any remaining pathogens on the surface. Maintain a temperature of 20-25℃ and a humidity of 90%.

[0144] The above groups were sprayed with the corresponding chemical reagents or biocontrol bacteria inducers (20ml per leaf, 100ml per plant). The incidence rate and the sprayed reagents were recorded every 7 days (i.e., the reagents were sprayed on day 0 and day 7 of the entire experiment).

[0145] Table 9. Incidence of Vein Necrosis in Tobacco Plants

[0146]

[0147]

[0148] Table 10 Incidence of wildfire disease in tobacco plants

[0149] Incidence rate on day 0 (%) 7-day incidence rate (%) 14-day incidence rate (%) Biological control group A 87％ 93％ 100％ Chemical control group A 87％ 67％ 60％ Biocontrol bacteria inducer experimental group A1 87％ 100％ 100％ Biocontrol bacteria inducer experimental group A2 87％ 100％ 100％ Biocontrol bacteria inducer experimental group A3 87％ 67％ 60％ Biological control group B 93％ 100％ 100％ Chemical control group B 93％ 67％ 53％ Biocontrol bacteria inducer experimental group B1 93％ 93％ 100％ Biocontrol bacteria inducer experimental group B2 93％ 93％ 100％ Biocontrol bacteria inducer experimental group B3 93％ 67％ 67％

[0150] As shown in Tables 9 and 10, both single-strain inducers and chemical reagents are more effective when used before the onset of disease. Chemical reagents are more effective than single-strain inducers, and only Bacillus subtilis inducers have a certain control effect.

[0151] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0152] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for preparing a biocontrol agent that simultaneously prevents tobacco vein necrosis and wildfire disease, characterized in that, The mixture of Bacillus subtilis culture, Enterobacter culture, and Pseudomonas geniculatus culture was then subjected to mitomycin C and UV induction followed by membrane filtration to obtain the final product.

2. The preparation method according to claim 1, characterized in that, The Bacillus subtilis has the accession number CCTCC AB 130001; The accession number of the Enterobacter is CCTCC AB 206648; The accession number of the geniculate pseudomonocyte is CCTCC AB 205694.

3. The preparation method according to claim 1, characterized in that, The volume ratio of Bacillus subtilis culture, Enterobacter culture, and Pseudomonas geniculatus culture was (35-50):(18-25):(30-47).

4. The preparation method according to claim 1, characterized in that, The volume ratio of Bacillus subtilis culture, Enterobacter culture, and Pseudomonas geniculatus culture was 50:20:

30.

5. The preparation method according to claim 1, characterized in that, The viable counts of Bacillus subtilis, Enterobacter, and Pseudomonas geniculatus bacterial suspensions were 10. 9 ~10 10 per ml.

6. The preparation method according to claim 1, characterized in that, The specific procedure for mitomycin C induction is as follows: add 0.5–2 μg / ml of mitomycin C to the bacterial culture, and then incubate in a shaker at 35–40°C for 4–6 hours.

7. The preparation method according to claim 1, characterized in that, The specific operation of the ultraviolet induction is as follows: 1~3mW / cm 2 Induce for 10–30 seconds, then incubate at 35–40°C in a shaker for 1 hour.

8. The biocontrol bacteria inducer prepared by any one of the preparation methods according to claims 1 to 7.

9. The application of the biocontrol agent according to claim 8 in the prevention and control of tobacco vein necrosis and wildfire disease.

10. The application as described in claim 9, characterized in that, The tobacco in question is Yunyan 87.

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