A preparation method and application of a nanogel preparation for preventing and treating tobacco diseases

Abstract

The application discloses a preparation method of a nanogel preparation for preventing and treating tobacco diseases, and specifically comprises the following steps: (1) preparing bacillus licheniformis bacterial liquid; (2) preparing nanometer selenium precipitate; (3) preparing purified nanometer selenium particles; (4) preparing streptomycin-nanometer selenium solid; (5) preparing nanometer titanium dioxide photo-semiconductor sol; and (6) mixing low-melting agarose powder, the streptomycin-nanometer selenium solid and the nanometer titanium dioxide photo-semiconductor sol, centrifuging, washing, and drying to obtain the nanogel preparation. The nanogel preparation has significant bacteriostatic efficacy, can effectively prevent and treat tobacco diseases (especially tobacco bacterial wilt and tobacco wild fire disease), has a promoting effect on tobacco growth, is simple to make, has strong adhesion, and does not pollute the soil environment.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of tobacco disease prevention and control, and more particularly to a preparation method and application of a nanogel preparation for preventing and controlling tobacco diseases. BACKGROUND

[0002] Tobacco is an important economic crop in China, but in recent years, tobacco diseases have occurred frequently, are prone to occur under high temperature and high humidity conditions, spread very quickly, and often cause symptoms such as tobacco rot, wilting, and spots, resulting in a decrease in tobacco yield and quality and causing serious economic losses. Therefore, exploring a simple, safe, pollution-free, and efficient method for preventing and controlling tobacco diseases has become a major research focus.

[0003] In Chinese patent CN 113951279 A, a bacteriostatic combination liquid for preventing and controlling tobacco bacterial wilt and a preparation method are disclosed. The bacteriostatic combination liquid is prepared by mixing Lactobacillus bulgaricus lysate and Bacillus pumilus fermentation broth in a certain proportion to form a microbial liquid, and then mixing the microbial liquid with fulvic acid, mustard alkaloid, boron plus selenium, puerarin, ellagic acid, and chitosan oligosaccharide. The obtained combination liquid is diluted and sprayed on the whole plant. However, the method uses too many chemical compositions, which can easily pollute the environment, reduce the density of beneficial plant growth bacteria in the soil, and make the plant more resistant, which is not conducive to long-term use. The sprayed solution is easily washed away by rainwater, and the prevention and control effect is reduced.

[0004] In Chinese patent CN 115812491 A, a biological control method for preventing and controlling tobacco angular leaf spot and tobacco wildfire is disclosed. The method includes spraying a biocontrol agent once during the tobacco seedling stage and spraying the biocontrol agent again 15-20 days after transplanting in the field. The biocontrol agent includes diatomite, chitosan, sucrose, and a biocontrol bacteria culture solution. The biocontrol bacteria culture solution is obtained by mixing Bacillus polymyxa fermentation broth, Bacillus pumilus fermentation broth, Brevibacillus laterosporus fermentation broth, and Pseudomonas fluorescens fermentation broth. However, the bacterial agent has poor adhesion, and the use of too many bacterial species can easily cause contamination and death during the purification and culture process, which is not conducive to industrial production.

[0005] In Chinese patent CN 108293388 A, a method for preventing and controlling tobacco soil-borne diseases and improving tobacco yield and quality is disclosed. The method uses a combination of pesticides and fertilizers, and mixes and uses manganese-zinc, zhongshengmycin, and tobacco special fertilizer in a certain proportion, and then sprays and applies the fertilizer. However, the fertilizer is easily washed away by rainwater during the application stage, which greatly reduces the effect of topdressing and easily causes environmental pollution and waste.

[0006] Therefore, how to develop a tobacco disease prevention and control preparation with strong adhesion and no pollution to the soil environment is a problem that needs to be solved by those skilled in the art. SUMMARY

[0007] Therefore, the present application aims to provide a preparation method and application of a nanogel preparation for preventing and treating tobacco diseases to solve the problems in the prior art.

[0008] To achieve the above-mentioned purpose, the present application adopts the following technical solutions:

[0009] A preparation method of a nanogel preparation for preventing and treating tobacco diseases, specifically comprising the following steps:

[0010] (1) Bacillus licheniformis (Bl-001) is inoculated into modified LB solid medium for activation, and then inoculated into modified LB liquid medium for shock culture to obtain a Bacillus licheniformis bacterial liquid;

[0011] (2) A sodium selenite (Na2SeO3) solution is prepared, filtered, sterilized, added into the modified LB liquid medium, and then inoculated with the Bacillus licheniformis bacterial liquid for culture; after centrifugation of the bacterial liquid, the supernatant is discarded, and the precipitate is sequentially washed with sterile water, a NaCl solution, and a Tris-HCl buffer solution; sterile water is added to the washed precipitate, and ice bath ultrasonic crushing is performed, and after centrifugation, the precipitate is discarded, and the supernatant is centrifuged and the supernatant is discarded, and the precipitate is washed with a SDS Tris / HCl buffer solution, centrifuged, to obtain a nano selenium precipitate;

[0012] (3) The nano selenium precipitate is first resuspended in the first portion of ultrapure water, and n-octanol is added, shaken, centrifuged, and allowed to stand, and after layering, the upper layer solution is discarded, and the precipitate is sequentially washed with chloroform, ethanol, and sterile water to obtain nano selenium particles; finally, the nano selenium particles are resuspended in the second portion of ultrapure water, ultra-low temperature frozen, vacuum freeze-dried, and purified nano selenium particles (MSe NPs) are obtained;

[0013] (4) Streptomycin (SM) is added to anhydrous ethanol, ultrasonically treated, and then purified nano selenium particles are added, stirred, and after centrifugation, the supernatant is discarded, and the precipitate is washed with distilled water, dried, to obtain streptomycin-nano selenium solids (SM@MSe NPs) for standby use;

[0014] (5) A titanium sulfate (Ti(S04)2) solution is prepared, and while stirring, ammonia water is added dropwise, a white precipitate is generated, the pH value of the reaction system is adjusted to 7.0, the white precipitate is washed and suction-filtered with deionized water, diluted with pure water, and a H2O2 solution is added dropwise, and a nano titanium dioxide photo-semiconductor sol is obtained by hydrothermal reaction for standby use;

[0015] (6) The low melting point agarose LA powder is added into distilled water, and the streptomycin-nano selenium solid and nano titanium dioxide photo semiconductor sol are added while stirring, mixed, centrifuged, and the supernatant is discarded, the precipitate is washed with PBS buffer, dried, and the nano gel preparation (TiO2@SM@MSe@LANPs) for preventing and treating tobacco diseases is obtained.

[0016] Further, in the step (1), the formula of the modified LB solid culture medium is 1% peptone, 0.5% yeast powder, 1% NaCl, 0.5% soluble starch, 2% glucose, 0.5% beef extract and 2% agar powder, and the rest is water; the formula of the modified LB liquid culture medium is 1% peptone, 0.5% yeast powder, 1% NaCl, 0.5% soluble starch, 2% glucose and 0.5% beef extract, and the rest is water; the temperature of the shock culture is 30°C, the rotation speed is 150 r / min, and the time is 36 h.

[0017] Further, in the step (2), the concentration of the sodium selenite solution is 10 g / L; the pore size of the filter membrane for filtration is 0.22 μm; the concentration of sodium selenite in the modified LB liquid culture medium is 120 μg / mL; the inoculation amount of the bacillus licheniformis bacterial solution is 2% by volume fraction; the culture temperature is 30°C, the rotation speed is 150 r / min, and the time is 60 h; the centrifugation speed of the bacterial solution is 12000 r / min, and the time is 10 min; the mass concentration of the NaCl solution is 0.9%; the concentration of the Tris-HCl buffer solution is 0.1 mol / L; the washing times are 3 times; the ice bath ultrasonic crushing time is 30 min; the centrifugation speed is 3000 r / min, and the time is 10 min; the centrifugation speed of the supernatant is 12000 r / min, and the time is 10 min; in the SDS Tris / HCl buffer solution, the mass concentration of SDS is 1%, the concentration of the Tris / HCl buffer solution is 1.5 mol / L, and the pH value is 8.3; the washing times are 3 times.

[0018] Further, in the step (3), the mass / volume ratio of the nano selenium precipitate, the first ultrapure water, n-octanol, chloroform, ethanol, sterile water and the second ultrapure water is 2 g:10 mL:5 mL:10 mL:10 mL:10 mL:15 mL; the shock time is 5 min; the centrifugation speed is 3000 r / min, and the time is 5 min; the standing temperature is 4°C, and the time is 24 h; the ultralow temperature freezing temperature is -80°C, and the time is 12 h; the vacuum freeze-drying time is 36 h.

[0019] Further, in the step (4), the mass-volume ratio of streptomycin sulfate, anhydrous ethanol and purified nano-selenium particles is 20 mg:10 mL:10 mg; the ultrasonic treatment time is 10 min; the stirring temperature is room temperature, the stirring speed is 800 rpm, and the stirring time is 24 h; and the washing times are 3 times.

[0020] Further, in the step (5), the concentration of the titanium sulfate solution is 1.5 mol / L; the concentration of the ammonia water is 3 mol / L; the molar ratio of titanium sulfate to H2O2 is 1:4; and the mass concentration of the H2O2 solution is 30%.

[0021] Further, in the step (6), the mass-volume ratio of the low-melting-point agarose powder, distilled water, streptomycin-nano-selenium solid and nano-titanium dioxide photo-semiconductor sol is 20 mg:10 mL:30 mg:10 mg; the heating and stirring temperature is 35℃, and the heating and stirring time is 2 h; the mixing temperature is 35℃, and the mixing time is 6 h; the centrifugal speed is 8000 rpm, and the centrifugal time is 10 min; the concentration of the PBS buffer is 0.01 mol / L, and the pH is 7.4; the washing times are 3 times; and the drying temperature is room temperature.

[0022] The application also claims a nano-gel preparation prepared by the above preparation method and an application of the nano-gel preparation in preventing and treating tobacco diseases.

[0023] A use method of the nano-gel preparation prepared by the above preparation method, specifically including the following steps:

[0024] (1) dissolving the nano-gel preparation in water to obtain a nano-gel preparation solution;

[0025] (2) spraying the nano-gel preparation solution on the stems and leaves of the tobacco at the seedling stage to the rosette stage of the tobacco.

[0026] Further, in the step (1), the concentration of the nano-gel preparation solution is 2-6 mg / mL.

[0027] Further, in the step (2), the spraying times are 2-3 times, the interval between each spraying is 15 days, and the nano-gel preparation solution sprayed each time is 60-180 mL.

[0028] According to the above technical solution, compared with the prior art, the application has the following beneficial effects:

[0029] 1. The application uses bacillus licheniformis to prepare nano-selenium, the conversion rate of sodium selenite is high (91.52%), the yield of nano-selenium is high (74.83%), the obtained nano-selenium has small particle size (90-200 nm), high biological activity and small cytotoxicity.

[0030] 2. The low-melting-point agarose selected in this invention is a safe, biodegradable, temperature-responsive material with a large surface area. It can adhere to fungi to capture them and can also rapidly soften and release streptomycin-nano selenium and titanium dioxide when the temperature rises, enabling controlled-release drug delivery.

[0031] 3. The advantages of nano-titanium dioxide photocatalytic sol lie in its photocatalytic performance. It can not only directly kill bacteria, viruses, and other microorganisms in the environment by decomposing and destroying the structural substances of microbial cells, or weaken their vitality, but also degrade the toxins they release, resulting in a long-lasting effect. The nano-titanium dioxide photocatalytic sol prepared by this invention has good adhesion and film-forming properties, and can form a continuous and stable antibacterial film under natural drying conditions, thereby significantly reducing bacterial keratosis.

[0032] 4. The nanogel formulation of this invention has significant antibacterial effects, can effectively prevent and control tobacco diseases (especially tobacco bacterial wilt and tobacco wildfire), promotes tobacco growth, and is simple to make, has strong adhesion, and does not pollute the soil environment. Detailed Implementation

[0033] 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.

[0034] Example 1

[0035] The preparation method of the nanogel formulation for the prevention and control of tobacco diseases specifically includes the following steps:

[0036] (1) First, Bacillus licheniformis was inoculated into modified LB solid medium for activation, and then inoculated into modified LB liquid medium. The mixture was shaken and cultured for 36 hours at a temperature of 30℃ and a rotation speed of 150r / min to obtain Bacillus licheniformis bacterial solution.

[0037] The modified LB solid medium formula, by mass percentage, is: 1% peptone, 0.5% yeast extract, 1% NaCl, 0.5% soluble starch, 2% glucose, 0.5% beef extract and 2% agar powder, with the remainder being water;

[0038] The modified LB liquid medium formula, by weight percentage, is: 1% peptone, 0.5% yeast extract, 1% NaCl, 0.5% soluble starch, 2% glucose and 0.5% beef extract, with the remainder being water;

[0039] (2) Prepare a sodium selenite solution with a concentration of 10 g / L, filter it with a filter membrane with a pore size of 0.22 μm, sterilize it, add it to the modified LB liquid medium to a concentration of 120 μg / mL of sodium selenite, and then inoculate it with a Bacillus licheniformis bacterial solution at a volume fraction of 2%, and cultivate it at a temperature of 30°C and a rotation speed of 150 r / min for 60 h; centrifuge the bacterial solution at a rotation speed of 12000 r / min for 10 min, discard the supernatant, and sequentially wash the precipitate with sterile water, a 0.9% NaCl solution, and a 0.1 mol / L Tris-HCl buffer solution for 3 times respectively; add sterile water to the washed precipitate to adjust the volume of the precipitate to not less than 1 / 2 of the original sample volume, and perform ice bath ultrasonic crushing for 30 min; centrifuge at a rotation speed of 3000 r / min for 10 min, discard the precipitate, centrifuge the supernatant at a rotation speed of 12000 r / min for 10 min, discard the supernatant, wash the precipitate with a 1% SDS, 1.5 mol / L, pH 8.3 Tris / HCl buffer solution for 3 times, and centrifuge to obtain a nanometer selenium precipitate;

[0040] (3) First, resuspend 2 g of the nanometer selenium precipitate in 10 mL of ultrapure water, add 5 mL of n-octanol, shake for 5 min, centrifuge at a rotation speed of 3000 r / min for 5 min, place it in a 4°C refrigerator for 24 h, discard the upper layer after layering, and sequentially wash the precipitate with 10 mL of chloroform, 10 mL of ethanol, and 10 mL of sterile water to obtain nanometer selenium particles; finally, resuspend the nanometer selenium particles in 15 mL of ultrapure water, place it in a -80°C ultra-low temperature refrigerator for 12 h, vacuum freeze dry for 36 h, and obtain purified nanometer selenium particles;

[0041] (4) First, add 20 mg of streptomycin to 10 mL of anhydrous ethanol, ultrasonically treat for 10 min, then add 10 mg of purified nanometer selenium particles, stir at a rotation speed of 800 rpm at room temperature for 24 h, centrifuge to discard the supernatant, wash the precipitate with distilled water for 3 times, dry, and obtain streptomycin-nanometer selenium solids, which are resuspended in 3 mL of distilled water for standby use;

[0042] (5) Prepare a titanium sulfate solution with a concentration of 1.5 mol / L, while stirring, add concentrated ammonia water with a concentration of 3 mol / L drop by drop with a dropper, generate a white precipitate, adjust the pH value of the reaction system to 7.0, wash and suction filter with deionized water to remove NH4 + and SO4 2- , dilute with pure water, add a 30% H2O2 solution drop by drop with a dropper to gelatinize the precipitate, the molar ratio of titanium sulfate to H2O2 is 1:4, obtain a yellow transparent PTA sol after a certain period of stabilization, hydrothermally react to obtain a nanometer titanium dioxide photo-semiconductor sol, and standby use;

[0043] (6) 20mg low-melting agarose powder was added to 10ml distilled water, heated to 35°C and stirred for 2h to obtain a uniform gel solution; then 30mg streptomycin-nano selenium solid and 10mg nano titanium dioxide photo-semiconductor sol were added while stirring, mixed at 35°C for 6h, centrifuged at 8000rpm for 10min, the supernatant was discarded, the precipitate was washed with PBS buffer with a concentration of 0.01mol / L and pH of 7.4 for 3 times, and dried at room temperature to obtain a nano gel preparation for preventing and treating tobacco diseases.

[0044] Example 2

[0045] The method for using the nano gel preparation for preventing and treating tobacco diseases specifically includes the following steps:

[0046] (1) The nano gel preparation prepared in Example 1 was dissolved in water to obtain a nano gel preparation solution with a concentration of 2mg / mL;

[0047] (2) The nano gel preparation solution was sprayed on the stems and leaves of tobacco 3 times at an interval of 15 days from the seedling stage to the rosette stage of tobacco, and 180ml of the nano gel preparation solution was sprayed each time.

[0048] Example 3

[0049] The method for using the nano gel preparation for preventing and treating tobacco diseases specifically includes the following steps:

[0050] (1) The nano gel preparation prepared in Example 1 was dissolved in water to obtain a nano gel preparation solution with a concentration of 4mg / mL;

[0051] (2) The nano gel preparation solution was sprayed on the stems and leaves of tobacco 3 times at an interval of 15 days from the seedling stage to the rosette stage of tobacco, and 180ml of the nano gel preparation solution was sprayed each time.

[0052] Example 4

[0053] The method for using the nano gel preparation for preventing and treating tobacco diseases specifically includes the following steps:

[0054] (1) The nano gel preparation prepared in Example 1 was dissolved in water to obtain a nano gel preparation solution with a concentration of 6mg / mL;

[0055] (2) The nano gel preparation solution was sprayed on the stems and leaves of tobacco 3 times at an interval of 15 days from the seedling stage to the rosette stage of tobacco, and 180ml of the nano gel preparation solution was sprayed each time.

[0056] Performance test

[0057] 1. Bacteriostatic test

[0058] The nanogel preparation solution prepared in step (1) of each of Examples 2-4 was taken, and water was taken as a control group (CK), and the colony diameters (average value ± standard deviation) and growth inhibition rates of Pseudomonas syringae of the nanogel preparation solutions were tested.

[0059] The growth inhibition rate = (colony diameter of the control group - colony diameter of the treatment group) / colony diameter of the control group x 100%.

[0060] The results are shown in Table 1.

[0061] Table 1 Inhibition effect of the nanogel preparation on Pseudomonas syringae

[0062] Solution concentration (mg / mL) Colony diameter (cm) Inhibition rate (%) 2 5.1±0.2 28.2 4 2.6±0.1 63.4 6 1.1±0.0 84.5 CK 7.1±0.1 —

[0063] As can be seen from Table 1, as the concentration of the nanogel preparation solution increases, the colony diameter of Pseudomonas syringae decreases, and the growth inhibition rate increases.

[0064] The above tests show that the disease control effect of the nanogel preparation is preliminarily effective.

[0065] 2. Tobacco bacterial wilt control test

[0066] The nanogel preparation solution was sprayed on tobacco plants suffering from bacterial wilt using the methods of Examples 2-4, and water was taken as a control group (CK), and the bacterial wilt leaf rates (average value ± standard deviation) on the day of the third application, 10 days after the third application, and 15 days after the third application were tested.

[0067] The bacterial wilt leaf rate = number of tobacco plant leaves suffering from bacterial wilt / total number of tobacco plant leaves x 100%.

[0068] The results are shown in Table 2.

[0069] Table 2 Control effect of the nanogel preparation on tobacco bacterial wilt

[0070]

[0071]

[0072] As can be seen from Table 2, in the three tests, the bacterial wilt leaf rates of the three application groups were lower than that of the CK group, and decreased with increasing concentration. In the third test, the bacterial wilt leaf rate of the tobacco in the CK group was as high as 46.2%, while the bacterial wilt leaf rates of the three application groups were effectively controlled, and when the concentration of the nanogel preparation solution was 6 mg / mL, the bacterial wilt leaf rate was only 23.26%.

[0073] 3. Tobacco wild fire disease control test

[0074] The nanogel preparation solution was sprayed on the tobacco plants with wildfire disease using the use method of Examples 2-4, and water was used as a control group (CK), and the bacterial leaf blight rate (average ± standard deviation) was tested on the day of the third application, 10 days after the third application, and 15 days after the third application, respectively.

[0075] The wildfire disease leaf rate = the number of tobacco plant leaves with wildfire disease / the total number of tobacco plant leaves x 100%.

[0076] The results are shown in Table 3.

[0077] Table 3 Prevention and treatment effect of nanogel preparation on tobacco wildfire disease

[0078]

[0079] As shown in Table 3, in the three tests, the bacterial leaf blight rate of the three application groups was lower than that of the CK group, and decreased with the increase of the concentration. In the third test, the wildfire disease leaf rate of the CK group of tobacco was as high as 40.2%, while the bacterial leaf blight rate of the three application groups was effectively controlled, and when the concentration of the nanogel preparation solution was 6 mg / mL, the wildfire disease leaf rate was only 31.26%.

[0080] 4. Tobacco agronomic trait investigation

[0081] The nanogel preparation solution was sprayed on the tobacco plants with wildfire disease using the use method of Examples 2-4, and water was used as a control group (CK), and the bacterial leaf blight rate (average ± standard deviation) was tested on the day of the third application, 10 days after the third application, and 15 days after the third application, respectively.

[0082] The results are shown in Tables 4-6.

[0083] Table 4 Agronomic traits of tobacco at the rosette stage

[0084]

[0085] Table 5 Agronomic traits of tobacco at the vigorous growth stage

[0086]

[0087] Table 6 Agronomic traits of tobacco at the topping stage

[0088]

[0089] As shown in Tables 4-6, from the results of the investigation of the agronomic traits, the plant height, stem circumference, leaf number, leaf length, and leaf width of the application groups were significantly increased compared with the control group.

[0090] The above tests show that the application of the nanogel preparation has a promoting effect on the growth of tobacco.

[0091] The foregoing description of the disclosed embodiments enables a person skilled in the art to make or use the application. Numerous modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein can be applied to other embodiments without the use of the inventive faculty. Therefore, the present application is not intended 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 nanogel formulation for controlling tobacco diseases, characterized by, Specifically comprising the following steps: (1) First, Bacillus licheniformis is inoculated into modified LB solid medium for activation, and then inoculated into modified LB liquid medium for shaking culture to obtain Bacillus licheniformis liquid; (2) Prepare a sodium selenite solution, filter, sterilize, add to the modified LB liquid medium, then inoculate the Bacillus licheniformis liquid for culture; centrifuge the liquid, discard the supernatant, and sequentially wash the precipitate with sterile water, NaCl solution and Tris-HCl buffer; Add sterile water to the washed precipitate, ice-bath ultrasonic crushing, centrifuge and discard the precipitate, centrifuge the supernatant and discard the supernatant, and wash the precipitate with SDS Tris / HCl buffer, centrifuge to obtain nano-selenium precipitate; (3) First, resuspend the nano-selenium precipitate in the first ultrapure water, add n-octanol, shake, centrifuge, stand, discard the upper solution after layering, and sequentially wash the precipitate with chloroform, ethanol and sterile water to obtain nano-selenium particles; finally, resuspend the nano-selenium particles in the second ultrapure water, freeze at ultra-low temperature, vacuum freeze-dry to obtain purified nano-selenium particles; (4) First, add streptomycin to anhydrous ethanol, ultrasonic treatment, then add purified nano-selenium particles, stir, centrifuge and discard the supernatant, wash the precipitate with distilled water, dry to obtain streptomycin-nano-selenium solid, ready for use; (5) Prepare a titanium sulfate solution, while stirring, add ammonia water drop by drop, generate white precipitate, adjust the pH value of the reaction system to 7.0, wash with deionized water, dilute with pure water, add H2O2 solution drop by drop, hydrothermal reaction to obtain nano-titanium dioxide photo-semiconductor sol, ready for use; (6) Add low-melting agarose powder to distilled water, heat and stir while adding streptomycin-nano-selenium solid and nano-titanium dioxide photo-semiconductor sol for mixing, centrifuge and discard the supernatant, wash the precipitate with PBS buffer, dry to obtain the nano-gel preparation for preventing and treating tobacco diseases.

2. The method of claim 1, wherein the nanogel formulation for controlling tobacco diseases is prepared by the steps of: In step (1), the formula of the modified LB solid medium is 1% peptone, 0.5% yeast powder, 1% NaCl, 0.5% soluble starch, 2% glucose, 0.5% beef extract and 2% agar powder, with the balance being water; the formula of the modified LB liquid medium is 1% peptone, 0.5% yeast powder, 1% NaCl, 0.5% soluble starch, 2% glucose and 0.5% beef extract, with the balance being water; the shaking culture temperature is 30°C, the rotation speed is 150 r / min, and the time is 36 h.

3. The method of claim 1, wherein the nanogel formulation for controlling tobacco diseases is prepared by the steps of: In step (2), the concentration of the sodium selenite solution is 10 g / L; the pore size of the filter membrane is 0.22 μm; the concentration of sodium selenite in the improved LB liquid medium is 120 μg / mL; the inoculation amount of the bacillus licheniformis liquid is 2% by volume; the culture temperature is 30°C, the rotation speed is 150 r / min, and the culture time is 60 h; the centrifugation speed of the bacterial liquid is 12 000 r / min, and the centrifugation time is 10 min; the mass concentration of the NaCl solution is 0.9%; the concentration of the Tris-HCl buffer solution is 0.1 mol / L; the washing times are 3; the ice-bath ultrasonic crushing time is 30 min; the centrifugation speed is 3 000 r / min, and the centrifugation time is 10 min; the centrifugation speed of the supernatant is 12 000 r / min, and the centrifugation time is 10 min; in the SDS Tris / HCl buffer solution, the mass concentration of SDS is 1%, the concentration of the Tris / HCl buffer solution is 1.5 mol / L, and the pH value is 8.3; the washing times are 3.

4. The method of claim 1, wherein the nanogel formulation for controlling tobacco diseases is prepared by the steps of: In step (3), the mass / volume ratio of the nano-selenium precipitate, the first portion of ultrapure water, n-octanol, chloroform, ethanol, sterile water, and the second portion of ultrapure water is 2 g:10 mL:5 mL:10 mL:10 mL:10 mL:15 mL; the oscillation time is 5 min; the centrifugation speed is 3 000 r / min, and the centrifugation time is 5 min; the standing temperature is 4°C, and the standing time is 24 h; the ultra-low temperature freezing temperature is -80°C, and the ultra-low temperature freezing time is 12 h; the vacuum freeze-drying time is 36 h.

5. The method of claim 1, wherein the nanogel formulation for controlling tobacco diseases is prepared by the steps of: In step (4), the mass / volume ratio of the streptomycin, anhydrous ethanol, and the purified nano-selenium particles is 20 mg:10 mL:10 mg; the ultrasonic treatment time is 10 min; the stirring temperature is room temperature, the stirring speed is 800 rpm, and the stirring time is 24 h; the washing times are 3.

6. The method of claim 1, wherein the nanogel formulation for controlling tobacco diseases is prepared by the steps of: In step (5), the concentration of the titanium sulfate solution is 1.5 mol / L; the concentration of the ammonia water is 3 mol / L; the molar ratio of the titanium sulfate to H2O2 is 1:4; the mass concentration of the H2O2 solution is 30%.

7. The method for preparing a nanogel formulation for preventing and controlling tobacco diseases according to claim 1, characterized in that, In step (6), the mass / volume ratio of the low-melting-point agarose powder, distilled water, streptomycin-nano-selenium solid, and nano-titanium dioxide photo-semiconductor sol is 20 mg:10 mL:30 mg:10 mg; the heating and stirring temperature is 35°C, and the heating and stirring time is 2 h; the mixing temperature is 35°C, and the mixing time is 6 h; the centrifugation speed is 8 000 rpm, and the centrifugation time is 10 min; the concentration of the PBS buffer solution is 0.01 mol / L, and the pH value is 7.4; the washing times are 3; and the drying temperature is room temperature.

8. The application of the nanogel preparation prepared by the preparation method of claim 1 in the prevention and treatment of tobacco diseases.

9. A method of using a nanogel formulation prepared according to the method of claim 1, wherein, Specifically comprising the following steps: (1) dissolving the nanogel preparation in water to obtain a nanogel preparation solution; (2) spraying the nanogel preparation solution on the stems and leaves of the tobacco at the seedling stage to the rosette stage.

10. A method of using a nanogel formulation according to claim 9, wherein, In step (1), the concentration of the nanogel preparation solution is 2-6 mg / mL; in step (2), the spraying is performed 2-3 times with an interval of 15 days, and the mass of the nanogel preparation solution sprayed each time is 60-180 mL.

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