Bacillus subtilis for preventing and controlling tobacco anthrax and its application

Abstract

The present invention provides a strain of Bacillus subtilis for preventing and controlling tobacco anthrax and its application, which belongs to the technical field of microbial technology and tobacco disease control. The Bacillus subtilis is named K11, which has been deposited in the China Center for Type Culture Collection on March 26, 2021, with a deposit number of CCTCC NO: M 2021284. The bacterial agent prepared by the Bacillus subtilis K11 is used in the prevention and control technology of tobacco anthrax, which can reduce the amount of chemical pesticides by at least 52.8%; 75 days after tobacco transplanting, the prevention and control effect of the present invention on tobacco anthrax is at least 50.00% higher than that of conventional prevention and control measures; 100 days after tobacco transplanting, the prevention and control effect of the present invention on tobacco anthrax is at least 59.49% higher than that of conventional prevention and control measures.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial technology and tobacco disease prevention and control, and specifically relates to a Bacillus subtilis strain for preventing and controlling tobacco anthrax and application thereof. Background Art

[0002] Tobacco anthracnose is a fungal leaf disease caused by infection by Colletotrichum spp. The disease can occur throughout the tobacco seedling stage and field growth period. In recent years, the damage of tobacco anthracnose during the field growth period of tobacco leaves has gradually increased. In the early stage of the disease, small dark green, water-soaked spots will appear on tobacco leaves, which will gradually expand into round or oval lesions. When the weather is dry, the central part of the circular lesions is grayish white with yellow-brown or brown edges. In the later stage of the disease, the leaves at the lesions will be broken and even perforated; when the weather is humid, small black spots will appear on the lesions. Anthrax spreads quickly and reproduces at a high rate under suitable conditions. The foot leaves of tobacco plants generally get sick first, and then gradually spread to the upper leaves. When the disease is serious, the entire tobacco field will be destroyed. If the disease prevention and control work is not timely, it will also cause a pandemic of the disease, causing huge economic losses to tobacco production. Therefore, the prevention and control of tobacco anthracnose is imminent.

[0003] At present, the prevention and control methods of anthrax are mainly based on chemical pesticides. However, long-term use of chemical pesticides can easily make the pathogens resistant to drugs, which will cause more serious harm. At the same time, it will also cause problems such as increased pesticide residues in tobacco leaves and environmental pollution. Bacillus has the function of preventing diseases and promoting growth. It has strong adaptability and stability, and has an absolute advantage in occupying space and survival competition. It also has the advantages of low toxicity to humans and animals and is green and environmentally friendly. It has attracted the attention of microbial preparation researchers. However, there are very few Bacillus that have been developed for the prevention and control of tobacco anthrax, and the research technology is not mature yet, which makes the range of options for biological control of tobacco anthrax small and difficult to promote. Therefore, it is necessary to develop more biological control strains suitable for the prevention and control of tobacco anthrax. Summary of the invention

[0004] The first object of the present invention is to provide a strain of Bacillus subtilis that can be used to prevent and treat tobacco anthrax; the second object is to prepare the Bacillus subtilis into a bacterial agent for preventing and treating tobacco anthrax; and the third object is to provide a method for applying the bacterial agent to prevent and treat tobacco anthrax.

[0005] In order to achieve the first objective, the present invention provides a Bacillus subtilis strain for preventing and treating tobacco anthrax. The deposit number of the Bacillus subtilis strain is CCTCC NO: M 2021284.

[0006] The Bacillus subtilis (Bacillus sp.) of the present invention is named K11. The rhizosphere soil of healthy tobacco plants is taken from tobacco fields with tobacco anthrax (tobacco fields in Luoquanyan Village, Jiaoyuan Town, Xuanen County, Enshi Prefecture, Hubei Province). The soil microbial suspension is applied to NA culture medium by dilution and coating method to separate and purify bacteria. The pathogen of tobacco anthrax is used as the target, and the plate test is used for screening to obtain a strain with good prevention and control effect on tobacco anthrax. The strain has been deposited in the China Center for Type Culture Collection (Address: Wuhan University, Wuhan, China) on March 26, 2021, and the deposit number is: CCTCCNO: M 2021284.

[0007] In order to achieve the above second purpose, the present invention provides a bacterial agent for preventing and treating tobacco anthrax, wherein the bacterial agent is prepared from the above Bacillus subtilis K11 with the deposit number of CCTCC NO: M 2021284.

[0008] The preparation method of the bacterial agent comprises the following steps:

[0009] S1, activating the Bacillus subtilis for preventing and controlling tobacco anthrax on LB solid plate culture medium;

[0010] S2, picking a single bacterial colony from the LB solid plate medium in step S1 and inoculating it into LB liquid medium for cultivation to obtain a seed solution;

[0011] S3. Inoculate the seed liquid into the fermentation medium at an inoculation rate of 2 v / v% to 3 v / v% to obtain a fermentation liquid, and adjust the bacterial liquid concentration in the fermentation liquid to a desired range to obtain the bacterial agent for preventing and controlling tobacco anthrax.

[0012] Preferably, the ingredients of the LB solid plate culture medium include: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, 15 g / L agar powder, and a pH of 7.2.

[0013] Preferably, in step S2, the seed solution is obtained by shaking and culturing at 30±2° C. and 150 r / min for 20 hours.

[0014] Preferably, the ingredients of the LB liquid culture medium include: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, and a pH of 7.2.

[0015] Preferably, the fermentation medium comprises: 40.0 g / L corn starch, 50.0 g / L soybean meal, 2.00 g / L KH2PO4, 0.50 g / L MgSO4·7H2O, 0.50 g / L KCl, 0.10 g / L FeSO4·7H2O, and a pH of 6.8-7.0.

[0016] Preferably, in step S3, the fermentation liquid is obtained by shaking and culturing at 30±2° C. and 150 r / min for 30 to 36 hours.

[0017] Preferably, in step S3, the bacterial concentration in the fermentation broth is adjusted to 1×10 8 ~1×10 9 CFU / mL.

[0018] In order to achieve the third object, the method of using the above-mentioned bacterial agent to prevent and treat tobacco anthrax comprises the following steps:

[0019] P1. When transplanting tobacco seedlings, plant the seedlings of Dahuangjin at the edge of the tobacco field, and plant the seedlings of the main tobacco varieties in other areas of the tobacco field; the ratio of the number of Dahuangjin to the number of the main tobacco varieties is 1:(40-50);

[0020] P2. 30 days after transplanting tobacco seedlings, observe the incidence of anthracnose in the large golden tobacco plants every day; when anthracnose symptoms are found in the large golden tobacco plants, spray the leaves of the main tobacco varieties with a diluted solution of the fungicide at a dosage of 40-50 kg / mu; when anthracnose occurs in the main varieties, spray the leaves of the main tobacco varieties with a diluted solution of the compound fungicide twice, with each dosage of the compound fungicide being 100-120 mL / mu, and the interval between two sprayings being 10-15 days; the diluted solution of the fungicide is prepared with the fungicide, sodium lignin sulfonate, sodium dodecyl sulfate and water; the compound fungicide is prepared with the fungicide, triazole fungicide, sodium lignin sulfonate and sodium dodecyl sulfate.

[0021] Preferably, the preparation method of the bacterial agent dilution is as follows: sodium lignin sulfonate and sodium dodecyl sulfate are added to the bacterial agent to obtain the active ingredient of the bacterial agent, the active ingredient of the bacterial agent is mixed with water in a mass ratio of (102-152):(40000-50000), stirred evenly, and allowed to stand for 45-60 minutes to obtain the bacterial agent dilution; the mass ratio of the sodium lignin sulfonate, sodium dodecyl sulfate and the bacterial agent is 1:1:(100-150).

[0022] Preferably, the preparation method of the compound fungicide is as follows: the fungicide and the triazole fungicide are mixed in a mass ratio of (45-48):(2-5) to obtain active ingredient A; sodium lignin sulfonate and sodium dodecyl sulfate are mixed in a mass ratio of 1:1 to obtain adjuvant B; the adjuvant B and the active ingredient A are mixed in a mass ratio of 2:(100-120) to obtain the compound fungicide; when using the compound fungicide, the compound fungicide and water are mixed in a mass ratio of 1:(1500-2000) to obtain a diluted solution of the compound fungicide, and the diluted solution of the compound fungicide is sprayed on the tobacco leaf surface.

[0023] Preferably, the adjuvant B and the active ingredient A are mixed in a mass ratio of 2:120 to obtain the compound fungicide.

[0024] Preferably, the triazole fungicide is 25% propiconazole emulsifiable concentrate or 12.5% ​​diniconazole wettable powder.

[0025] The beneficial effects of the present invention are:

[0026] (1) The Bacillus subtilis K11 obtained by the present invention is applied to the prevention and control of anthrax of the main tobacco varieties, and can effectively eliminate most of the pathogen spores before germination; the Bacillus subtilis K11 is used in combination with a triazole fungicide to prevent and control anthrax of the main tobacco varieties. By combining the biocontrol agent with the chemical agent, the drug resistance problem caused by the single application of the biocontrol agent or the single application of the chemical agent can be overcome, the prevention and control effect can be improved, and the application amount of chemical pesticides can be reduced by at least 52.8%, thereby improving product safety and reducing environmental pollution; 75 days after tobacco transplanting, the prevention and control effect of the present invention on tobacco anthrax is improved by at least 50.00% compared with conventional prevention and control measures; 100 days after tobacco transplanting, the prevention and control effect of the present invention on tobacco anthrax is improved by at least 59.49% compared with conventional prevention and control measures.

[0027] (2) The present invention selects the anthrax-susceptible tobacco variety Dahuangjin as the indicator tobacco plant because, compared with the main tobacco varieties, the onset period of anthrax on Dahuangjin is usually 7 to 10 days earlier than that of the main tobacco varieties. When anthrax is observed on the indicator tobacco plant Dahuangjin, the conidia of the pathogen are spread by airflow or raindrop splashing, and anthrax will also occur on the main tobacco varieties 7 to 10 days later. Therefore, when transplanting tobacco, the tobacco seedlings of the anthrax-susceptible tobacco variety Dahuangjin are planted on the edge of the tobacco field. According to the occurrence of anthrax, the occurrence period of anthrax on the main tobacco varieties can be accurately predicted, and guidance can be provided for the accurate prevention and control of anthrax on the main tobacco varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is a schematic diagram of the inhibitory effect of K11 strain on tobacco anthrax;

[0029] Figure 2 This is the phylogenetic tree of K11 strain based on 16S rDNA gene sequence. DETAILED DESCRIPTION

[0030] The following is a clear and complete description of the technical solution of the present invention in conjunction with the embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work belong to the scope of protection of the present invention. Any equivalent transformation or substitution made by those skilled in the art based on the following implementation methods belongs to the protection scope of the present invention.

[0031] The screening and identification methods of Bacillus subtilis K11 used in the present invention are as follows:

[0032] (1) Screening of strains

[0033] In July 2019, 1 g of rhizosphere soil from healthy tobacco plants was collected from the tobacco fields in Luoquanyan Village, Jiaoyuan Town, Xuanen County, Enshi Prefecture, Hubei Province (tobacco anthrax-affected tobacco fields), added to 99 mL of sterile saline (NaCl concentration of 0.85 wt%) containing glass beads, and shaken at 150 r / min for 30 min. 1 mL of the obtained soil suspension was added to 999 mL of 0.85 wt% saline and mixed thoroughly to obtain 10 -5 Prepare 10 times soil sample dilution in the same way -6 , 10 -7 Three times soil sample dilution. Take out 100μL of each of the three concentration multiples of soil sample dilution and evenly spread them on NA plates. All NA plates were cultured in a 30℃ incubator for 48h, and single colonies were picked as biocontrol bacteria according to different colony morphological characteristics. The biocontrol bacteria was named K11. Among them, the culture medium components of NA plates include: peptone 10.0g / L, beef powder 3.0g / L, NaCl 5.0g / L, agar 15.0g / L, and pH value is 7.2.

[0034] (2) Determination of the antibacterial effect of biocontrol bacteria K11 on tobacco anthracnose

[0035] The plate confrontation method was used to determine the antibacterial effect of the above-mentioned biocontrol bacteria K11 on tobacco anthrax. 5 μL of the biocontrol bacteria K11 was inoculated on the central line of the PDA plate, and at the same time, the mycelium blocks of the test tobacco anthrax bacteria were inoculated in a cross pattern on both sides of the straight line (30 mm away from the straight line) as the experimental group, and the culture dish without the biocontrol bacteria but only inoculated with the mycelium blocks of the test tobacco anthrax bacteria was used as the control group. The size of the antibacterial zone was determined. The results are shown in the figure. Figure 1 shown. Figure 1 The left picture is the experimental group, and the right picture is the control group. Figure 1 It can be seen that the screened biocontrol bacteria K11 has a good inhibitory effect on tobacco anthrax, and the average inhibition zone is 19.33 mm.

[0036] (3) Identification of biocontrol bacteria K11

[0037] The genomic DNA of K11 strain was used as a template, and the universal primers 27F (5'-AGAGTTTGATCCTGGCT CAG-3') and 1492R (5'-TACGGTTACCTTGTTACGACTT-3') were used as primers for PCR amplification. The PCR amplification used a 50μL reaction system, and the reaction solution included 43μL Green Taq Mix, 25μmol / L primers 27F and 1492R, 2μL each, and 3μL K11 bacterial solution. The PCR reaction program was: 95℃ pre-denaturation for 15min; 95℃ denaturation for 30s, 50℃ annealing for 60s, 72℃ extension for 90s, 30 cycles; 72℃ extension for 10min. The obtained PCR amplification product was subjected to 2% agarose gel electrophoresis and then handed over to Beijing Aoke Biotechnology Co., Ltd. for sequencing, and the 16S rDNA sequence of K11 strain was obtained as shown in SEQ ID No.1.

[0038] The sequence of SEQ ID No.1 was compared with the NCBI database by Blast, and the strain was subjected to phylogenetic analysis using MEGA 7.0 software. A phylogenetic tree based on the 16S rDNA gene sequence was constructed using the neighbor-joining method. The results are shown in Figure 2 As shown, it was found that the biocontrol bacteria K11 was clustered with Bacillus subtilis, indicating that the biocontrol bacteria K11 was Bacillus subtilis.

[0039] (4) Deposit of Bacillus subtilis K11

[0040] The above-mentioned K11 strain identified as Bacillus subtilis was deposited in the China Center for Type Culture Collection (address: Wuhan University, Wuhan, China) on March 26, 2021, with the deposit number: CCTCC NO: M 2021284.

[0041] The tobacco seedlings of the tobacco anthrax susceptible variety Dahuangjin used in the following examples were cultivated by a known floating seedling method (see GB / T 25241.1-2010 "Technical Regulations for Intensive Tobacco Seedling Cultivation Part 1: Floating Seedling Cultivation"). The formula of the LB solid plate medium used was: 10 g tryptone, 5 g yeast extract, 10 g NaCl, 15 g agar powder, 1000 mL distilled water, pH 7.2. The formula of the LB liquid medium used was: 10 g tryptone, 5 g yeast extract, 10 g NaCl, 1000 mL distilled water, pH 7.2. The formula of the fermentation medium used is: corn starch 40.0 g / L, soybean meal 50.0 g / L, KH2PO4 2.00 g / L, MgSO4·7H2O 0.50 g / L, KCl 0.50 g / L, FeSO4·7H2O0.10 g / L, pH 6.8-7.0.

[0042] Example 1

[0043] In this embodiment, Bacillus subtilis K11 is prepared into a microbial agent, and the method for preventing and controlling tobacco anthrax comprises the following steps:

[0044] P1. When transplanting tobacco seedlings, plant the seedlings of Dahuangjin at the edge of the tobacco field, and plant the seedlings of the main varieties in other areas of the tobacco field; the ratio of Dahuangjin to the number of main varieties is 1:40; perform routine management of the tobacco field;

[0045] P2. 30 days after the tobacco seedlings were transplanted, the incidence of anthracnose in the Dahuangjin tobacco plants was observed every day; when the Dahuangjin showed anthracnose symptoms, the main tobacco varieties were sprayed with a diluted solution of the fungicide at a dosage of 40 kg / mu (including 100 g / mu of Bacillus subtilis K11 microbial agent, 1 g / mu of sodium lignin sulfonate, and 1 g / mu of sodium dodecyl sulfate); when anthracnose appeared in the main varieties, the main tobacco varieties were sprayed with a diluted solution of the compound fungicide at a dosage of 100 mL / mu, and after an interval of 10 days, the main tobacco varieties were sprayed with a diluted solution of the compound fungicide at a dosage of 100 mL / mu again;

[0046] The preparation method of the bacterial agent dilution comprises the following steps:

[0047] S1. Activate the strain of Bacillus subtilis K11 on LB solid plate medium;

[0048] S2, picking a single colony from the LB solid plate medium in step S1 and inoculating it into LB liquid medium, and shaking and culturing at 30±2°C and 150r / min for 20 hours to obtain seed solution;

[0049] S3, inoculate the seed liquid into the fermentation medium at an inoculation rate of 2v / v%, place it in a shaker at 30±2°C and a rotation speed of 150r / min for 35 hours to obtain a fermentation liquid; adjust the bacterial liquid concentration in the fermentation liquid to 1×10 8 ~1×10 9 CFU / mL, and obtained the Bacillus subtilis K11 microbial agent.

[0050] S4. Mix sodium lignin sulfonate, sodium dodecyl sulfate, and Bacillus subtilis K11 microbial agent in a mass ratio of 1:1:100 to obtain an agent active ingredient; mix the agent active ingredient with water in a mass ratio of 102:40000, stir evenly, and let stand for 45 minutes to obtain the agent dilution solution.

[0051] The preparation method of the compound fungicide comprises the following steps:

[0052] The Bacillus subtilis K11 microbial agent in the above step S3 and the triazole fungicide are evenly mixed in a mass ratio of 48:2 to obtain active ingredient A; sodium lignin sulfonate and sodium dodecyl sulfate are mixed in a mass ratio of 1:1 to obtain adjuvant B; the adjuvant B and the active ingredient A are mixed in a mass ratio of 2:100 to obtain the composite fungicide; the triazole fungicide is 25% benzopropiconazole emulsifiable concentrate, and the total amount of 25% benzopropiconazole emulsifiable concentrate in the composite fungicide applied twice in step P2 is 7.8 mL / mu.

[0053] When the compound fungicide is used, the compound fungicide is uniformly mixed with water at a mass ratio of 1:1500 to obtain a diluted solution of the compound fungicide, and the diluted solution of the compound fungicide is sprayed on the surface of tobacco leaves.

[0054] Example 2

[0055] In this embodiment, Bacillus subtilis K11 is prepared into a microbial agent, and the method for preventing and controlling tobacco anthrax comprises the following steps:

[0056] P1. When transplanting tobacco seedlings, plant the seedlings of Dahuangjin on the edge of the tobacco field, and plant the seedlings of the main varieties in other areas of the tobacco field; the ratio of Dahuangjin to the number of main varieties is 1:50; carry out routine management of the tobacco field;

[0057] P2. 30 days after the tobacco seedlings were transplanted, the incidence of anthrax in the Dahuangjin tobacco plants was observed every day; when the Dahuangjin showed anthrax symptoms, the main tobacco varieties were sprayed with a diluted solution of the fungicide at a dosage of 50 kg / mu (including 150 g / mu of Bacillus subtilis K11 microbial agent, 1 g / mu of sodium lignin sulfonate, and 1 g / mu of sodium dodecyl sulfate); when anthrax appeared in the main varieties, the main tobacco varieties were sprayed with a diluted solution of the compound fungicide at a dosage of 120 mL / mu, and after an interval of 15 days, the main tobacco varieties were sprayed with a diluted solution of the compound fungicide at a dosage of 120 mL / mu again;

[0058] The preparation method of the bacterial agent dilution comprises the following steps:

[0059] S1. Activate the strain of Bacillus subtilis K11 on LB solid plate medium;

[0060] S2, picking a single colony from the LB solid plate medium in step S1 and inoculating it into LB liquid medium, and culturing it at 30±2°C and 150r / min for 20 hours to obtain a seed solution;

[0061] S3, inoculate the seed liquid into the fermentation medium at an inoculation rate of 2v / v%, place it in a shaker at 30±2°C and a rotation speed of 150r / min for 35 hours to obtain a fermentation liquid; adjust the bacterial liquid concentration in the fermentation liquid to 1×10 8 ~1×10 9 CFU / mL, and obtained the Bacillus subtilis K11 microbial agent.

[0062] S4. Mix sodium lignin sulfonate, sodium dodecyl sulfate, and Bacillus subtilis K11 microbial agent in a mass ratio of 1:1:150 to obtain an agent active ingredient; mix the agent active ingredient with water in a mass ratio of 152:50000, stir evenly, and let stand for 60 minutes to obtain the agent dilution solution.

[0063] The preparation method of the compound fungicide comprises the following steps:

[0064] The Bacillus subtilis K11 microbial agent in the above step S3 and the triazole fungicide are evenly mixed in a mass ratio of 48:2 to obtain active ingredient A; sodium lignin sulfonate and sodium dodecyl sulfate are mixed in a mass ratio of 1:1 to obtain adjuvant B; the adjuvant B and the active ingredient A are mixed in a mass ratio of 2:120 to obtain the composite fungicide; the triazole fungicide is 25% benzopropiconazole emulsifiable concentrate, and the total amount of 25% benzopropiconazole emulsifiable concentrate in the composite fungicide applied twice in step P2 is 9.4 mL / mu.

[0065] When the compound fungicide is used, the compound fungicide is uniformly mixed with water at a mass ratio of 1:2000 to obtain a diluted solution of the compound fungicide, and the diluted solution of the compound fungicide is sprayed on the surface of tobacco leaves.

[0066] Example 3

[0067] In this embodiment, Bacillus subtilis K11 is prepared into a microbial agent, and the method for preventing and controlling tobacco anthrax comprises the following steps:

[0068] P1. When transplanting tobacco seedlings, plant the seedlings of Dahuangjin at the edge of the tobacco field, and plant seedlings of the main tobacco varieties in other areas of the tobacco field; the ratio of Dahuangjin to the main tobacco varieties is 1:45; perform routine management of the tobacco fields;

[0069] P2. 30 days after the tobacco seedlings were transplanted, the incidence of anthracnose in the Dahuangjin tobacco plants was observed every day; when the Dahuangjin showed anthracnose symptoms, the main tobacco varieties were sprayed with a diluted solution of the fungicide at a dosage of 45kg / mu (including 118g / mu of Bacillus subtilis K11 microbial agent, 1g / mu of sodium lignin sulfonate, and 1g / mu of sodium dodecyl sulfate); when anthracnose appeared in the main varieties, the main tobacco varieties were sprayed with a diluted solution of the compound fungicide at a dosage of 100mL / mu for the first time, and after an interval of 12 days, the main tobacco varieties were sprayed with a diluted solution of the compound fungicide at a dosage of 100mL / mu for the second time;

[0070] The preparation method of the bacterial agent dilution comprises the following steps:

[0071] S1. Activate the strain of Bacillus subtilis K11 on LB solid plate medium;

[0072] S2, picking a single colony from the LB solid plate medium in step S1 and inoculating it into LB liquid medium, and culturing it at 30±2°C and 150r / min for 20 hours to obtain a seed solution;

[0073] S3, inoculate the seed liquid into the fermentation medium at an inoculation rate of 3v / v%, place it in a shaker at 30±2°C and a rotation speed of 150r / min for 36 hours to obtain a fermentation liquid; adjust the bacterial liquid concentration in the fermentation liquid to 1×10 8 ~1×10 9 CFU / mL, and obtained the Bacillus subtilis K11 microbial agent.

[0074] S4. Mix sodium lignin sulfonate, sodium dodecyl sulfate, and Bacillus subtilis K11 microbial agent in a mass ratio of 1:1:120 to obtain an agent active ingredient; mix the agent active ingredient with water in a mass ratio of 120:45000, stir evenly, and let stand for 50 minutes to obtain the agent dilution solution.

[0075] The preparation method of the compound fungicide comprises the following steps:

[0076] The Bacillus subtilis K11 microbial agent and the triazole fungicide in the above step S3 are uniformly mixed in a mass ratio of 45:5 to obtain active ingredient A; sodium lignin sulfonate and sodium dodecyl sulfate are mixed in a mass ratio of 1:1 to obtain adjuvant B; the adjuvant B and the active ingredient A are mixed in a mass ratio of 2:100 to obtain the compound fungicide; the triazole fungicide is 12.5% ​​diniconazole wettable powder, and the total amount of 12.5% ​​diniconazole wettable powder in the compound fungicide applied twice in step P2 is 19.6 g / mu.

[0077] When the compound fungicide is used for the first time, the compound fungicide and water are mixed evenly at a mass ratio of 1:1500 to obtain a diluted solution of the compound fungicide; when the compound fungicide is used for the second time, the compound fungicide and water are mixed evenly at a mass ratio of 1:2000 to obtain a diluted solution of the compound fungicide.

[0078] Example 4

[0079] In this example, Bacillus subtilis K11 is prepared into a microbial agent, and the method for preventing and controlling tobacco anthrax is basically the same as that in Example 1, except that:

[0080] In step S3, the seed liquid is inoculated into the fermentation medium at an inoculation rate of 2.5 v / v%, and the medium is placed in a shaker at 30±2° C. and a rotation speed of 150 r / min for 30 hours to obtain a fermentation liquid;

[0081] In step P2, when anthrax occurs in the main cultivated variety, the diluted solution of the compound fungicide is sprayed on the tobacco leaf surface of the main cultivated variety at a dosage of 110 mL / mu of the compound fungicide for the first time, and after an interval of 13 days, the diluted solution of the compound fungicide is sprayed on the tobacco leaf surface of the main cultivated variety at a dosage of 110 mL / mu of the compound fungicide for the second time;

[0082] The preparation method of the compound fungicide comprises the following steps:

[0083] The Bacillus subtilis K11 microbial agent in the above step S3 and the triazole fungicide are evenly mixed in a mass ratio of 46:4 to obtain active ingredient A; sodium lignin sulfonate and sodium dodecyl sulfate are mixed in a mass ratio of 1:1 to obtain adjuvant B; the adjuvant B and the active ingredient A are mixed in a mass ratio of 2:110 to obtain the compound fungicide; the triazole fungicide is 12.5% ​​diniconazole wettable powder, and the total amount of 12.5% ​​diniconazole wettable powder in the compound fungicide sprayed twice in step P2 is 17.3 g / mu.

[0084] When the compound fungicide is used, the compound fungicide and water are uniformly mixed at a mass ratio of 1:1800 to obtain a diluted solution of the compound fungicide.

[0085] It should be noted that in step P2 of the above embodiments 1 to 4, the amount of the diluted compound fungicide applied twice may also be different; for example, the diluted compound fungicide is sprayed on the leaf surface of the main tobacco variety at a dosage of 100 mL / mu for the first time, and after an interval of 10 to 15 days, the diluted compound fungicide is sprayed on the leaf surface of the main tobacco variety at a dosage of 120 mL / mu for the second time.

[0086] Verification of the efficacy of Bacillus subtilis K11 in controlling anthrax

[0087] Blank control: No sowing or transplanting of indicator tobacco plants Dahuangjin was performed. When the diluted solution of Bacillus subtilis K11 inoculant and the diluted solution of the compound fungicide were sprayed in Example 1, the main cultivated varieties were sprayed with the same amount of clean water.

[0088] Positive control 1: Based on production experience, 45 days after the main tobacco varieties were transplanted, tobacco anthracnose prevention (100-150g / mu of Jinggangmycin, diluted with 40-50kg / mu of water) was carried out; when anthracnose appeared in the main tobacco varieties, fungicide (25% benzoproconazole emulsifiable concentrate, the dosage was 10mL / mu) was sprayed; after an interval of 10 days, fungicide (25% benzoproconazole emulsifiable concentrate, the dosage was 10mL / mu) was sprayed again; the total dosage of 25% benzoproconazole emulsifiable concentrate applied twice was 20mL / mu.

[0089] Positive control 2: Based on production experience, 45 days after the main tobacco varieties were transplanted, tobacco anthracnose prevention was carried out (100-150g / mu of Jinggangmycin, diluted with 40-50kg / mu of water); when anthracnose appeared in the main tobacco varieties, fungicide (12.5% ​​diniconazole wettable powder, the dosage was 25g / mu) was sprayed; after an interval of 10 days, fungicide (12.5% ​​diniconazole wettable powder, the dosage was 25g / mu) was sprayed again; the total dosage of 12.5% ​​diniconazole wettable powder applied twice was 50g / mu.

[0090] Tobacco anthracnose was controlled according to the schemes of blank control, positive control 1, positive control 2, and Examples 1 to 4. 100 tobacco plants were treated with each scheme, and each scheme was repeated 3 times. The occurrence of anthracnose in the main tobacco varieties was investigated 75 days and 100 days after tobacco transplanting, and the disease index and control effect were calculated. The specific test results are shown in Table 1 below.

[0091] Control effect (%) = (blank control disease index - Example / positive control disease index) ÷ blank control disease index × 100%

[0092] Table 1 Occurrence of tobacco anthrax

[0093]

[0094] In Table 1, a, b, and c represent multiple comparison significance (p<0.05) difference analysis markers. It can be seen from Table 1 that 75 days after tobacco transplanting, the tobacco anthracnose disease index in Examples 1 to 4 was 0.81 to 1.00, which was significantly lower than the positive control and blank control of the comparative example; relative to the blank control, the prevention efficacy of Examples 1 to 4 was 83.02 to 86.25%, which was significantly higher than the prevention efficacy of positive control 1 (66.04%) and positive control 2 (56.03%); the prevention efficacy of Examples 1 to 4 was 50.00 to 59.50% higher than that of positive control 1, and 61.39 to 68.73% higher than that of positive control 2. 100 days after tobacco transplanting, the disease index of tobacco anthracnose in Examples 1 to 4 was 1.37 to 2.07, which was significantly lower than that of the positive control and the blank control; relative to the blank control, the control efficacy of Examples 1 to 4 was 82.96 to 88.72%, which was significantly higher than that of the positive control 1 (57.94%) and the positive control 2 (57.04%); the control efficacy of Examples 1 to 4 was 59.49 to 73.19% higher than that of the positive control 1, and 60.34 to 73.75% higher than that of the positive control 2. Among Examples 1 to 4, Example 2 had the highest control efficacy against tobacco anthracnose.

[0095] The above description is only a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. For any person skilled in the art, the present invention may have various changes and modifications. Any simple equivalent changes and modifications made according to the scope of protection of the present invention and the contents of the specification should be included in the scope of protection of the present invention. Sequence Listing <110> Hubei Tobacco Science Research Institute <120> Bacillus subtilis for preventing and controlling tobacco anthrax and its application <160> 1 <170> SIPO Sequence Listing 1.0 <210> 1 <211> 1459 <212> DNA <213> Artificial Sequence <400> 1 ctcccgtgcc gcgtgcccga ttccttgcag ttcgagcgga cagatgggag cttgctccct 60 gatgttagcg gcggacgggt gagtaacacg tgggtaacct gcctgtaaga ctgggataac 120 tccgggaaac cggggctaat accggatggt tgtctgaacc gcatggttca gacataaaag 180 gtggcttcgg ctaccactta cagatggacc cgcggcgcat tagctagttg gtgaggtaac 240 ggctcaccaa ggcgacgatg cgtagccgac ctgagagggt gatcggccac actgggactg 300 agacacggcc cagactccta cgggaggcag cagtagggaa tcttccgcaa tggacgaaag 360 tctgacggag caacgccgcg tgagtgatga aggttttcgg atcgtaaagc tctgttgtta 420 gggaagaaca agtgccgttc aaatagggcg gcaccttgac ggtacctaac cagaaagcca 480 cggctaacta cgtgccagca gccgcggtaa tacgtaggtg gcaagcgttg tccggaatta 540 ttgggcgtaa agggctcgca ggcggtttct taagtctgat gtgaaagccc ccggctcaac 600 cggggagggt cattggaaac tgggaactt gagtgcagaa gaggagtg gattccacg 660 tgtagcggtg aaatgcgtag agatgtggag gaacaccagt ggcgaggcg actctctggt 720 ctgtaactga cgctgaggag cgaagcgtg gggagcgaac aggattagat accctggtag 780 tccacgccgt aaacgatgag tgctaagtgt taggggtt ccgcccctta gtgctgcagc 840 taacgcatta agcactccgc ctggggagta cggtcgcaag actgaactc aaggaattg 900 acggggggccc gcacaagcgg tggagcatgt ggttaatttc gaagcaacgc gagaacctt 960 accaggtctt gataccctct gataccta gagataggac gtccccttcg ggggcagagt 1020 gandaggtggt gcatggttgt cgtcagctcg tgtcgtgaga tgttggtt agtcccgcaa 1080 cgagcgcaac ccttgatctt agttgccacc attcagttgg gcactctaag gtgactgccg 1140 gtgacaaacc ggaggaggt ggggatgacg tcaatcatc atgcccctta tgacctgggc 1200 tacacacgtg ctacaatgga cagacaaag ggcagcgaaa ccgcgaggtt aagccaatcc 1260 cacaatctg ttctcagttc ggatcgcagt ctgcaactcg actgcgtgaa gctggaatcg 1320 ctagtaatcg cggatcagca tgccgcggtg aatacgttcc cgggccttgt acacaccgcc 1380 cgtcacacca cgagagtttg taacacccga agtcggtgag gtaaccttta tggagccagc 1440 cgccgaaggt gacagaggt 1459

Claims

1. A method for preventing and treating tobacco anthrax using a bacterial agent, characterized in that: The preparation method of the bacterial agent comprises the following steps: S1, Bacillus subtilis for preventing and controlling tobacco anthrax ( Bacillus sp. ) K11 was activated on LB solid plate medium; S2, picking a single colony from the LB solid plate medium in step S1 and inoculating it into LB liquid medium, and culturing at 30±2°C and 150r / min shaking for 20 hours to obtain seed solution; S3, inoculate the seed liquid into the fermentation medium at an inoculum rate of 2v / v%~3v / v%, and culture at 30±2℃ and 150r / min for 30~36 hours to obtain the fermentation liquid, and adjust the bacterial liquid concentration in the fermentation liquid to 1×10 8 ~1×10 9 CFU / mL, and the bacterial agent is obtained; The Bacillus subtilis ( Bacillus sp. )K11 was deposited in China Center for Type Culture Collection on March 26, 2021, with the deposit number CCTCC NO: M 2021284; The method for preventing and treating tobacco anthrax comprises the following steps: P1. When transplanting tobacco seedlings, plant the seedlings of Dahuangjin at the edge of the tobacco field, and plant seedlings of the main tobacco varieties in other areas of the tobacco field; the ratio of the number of Dahuangjin to the number of the main tobacco varieties is 1:(40-50); P2. 30 days after the tobacco seedlings were transplanted, the incidence of anthrax in the Dahuangjin tobacco plants was observed every day; when the Dahuangjin showed symptoms of anthrax, the main tobacco varieties were sprayed with a diluted solution of the fungicide at a dosage of 40-50 kg / mu; when anthrax appeared in the main varieties, the main tobacco varieties were sprayed with a diluted solution of the compound fungicide twice, with the dosage of the compound fungicide being 100-120 mL / mu each time, and the interval between the two sprayings being 10-15 days; the diluted solution of the fungicide was prepared with the fungicide, sodium lignin sulfonate, sodium dodecyl sulfate and water; the compound fungicide was prepared with the fungicide, triazole fungicide, sodium lignin sulfonate and sodium dodecyl sulfate; the triazole fungicide was 25% propiconazole emulsifiable concentrate or 12.5% ​​diniconazole wettable powder; The preparation method of the bacterial agent dilution is as follows: sodium lignin sulfonate and sodium dodecyl sulfate are added to the bacterial agent to obtain the active ingredient of the bacterial agent, the active ingredient of the bacterial agent is mixed with water in a mass ratio of (102-152): (40000-50000), stirred evenly, and allowed to stand for 45-60 minutes to obtain the bacterial agent dilution; the mass ratio of the sodium lignin sulfonate, sodium dodecyl sulfate and the bacterial agent is 1:1: (100-150); the preparation method of the compound fungicide is as follows : The fungicide and the triazole fungicide are mixed in a mass ratio of (45-48):(2-5) to obtain an active ingredient A; sodium lignin sulfonate and sodium dodecyl sulfate are mixed in a mass ratio of 1:1 to obtain an adjuvant B; the adjuvant B and the active ingredient A are mixed in a mass ratio of 2:(100-120) to obtain the composite fungicide; the dilution of the composite fungicide is obtained by uniformly mixing the composite fungicide and water in a mass ratio of 1:(1500-2000).

2. The method for preventing and treating tobacco anthrax according to claim 1, characterized in that: The auxiliary agent B and the active ingredient A are mixed in a mass ratio of 2:120 to obtain the compound fungicide.

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