Preparation method and use of nano-antibiotic for preventing and treating tobacco bacterial wilt

Abstract

The application discloses a preparation method and application of a novel nano-antibiotic agent for preventing and treating tobacco bacterial wilt, and belongs to the technical field of tobacco bacterial wilt prevention and treatment. Under the catalysis of N, N'-dicyclohexyl carbodiimide and 4-dimethylaminopyridine, daphnetin is condensed with carboxymethyl chitosan to form nanoparticles, and the prepared novel nanoparticles have high water solubility and good prevention and treatment effect.

Description

Technical Field

[0001] This invention relates to the field of tobacco bacterial wilt control technology, and in particular to a method for preparing and using a novel nano-inducer for controlling tobacco bacterial wilt. Background Technology

[0002] Since its first report in my country, tobacco bacterial wilt has been spreading and worsening, causing significant economic losses to the tobacco industry annually. Once infected, tobacco plants in the field quickly wilt and die under favorable conditions of high temperature and humidity. Currently, control of tobacco bacterial wilt primarily relies on chemical control, supplemented by other methods, including the selection of resistant varieties and agronomic management practices. While chemical control is effective against tobacco bacterial wilt, it can increase the pathogen's resistance to pesticides, reduce soil biodiversity in tobacco fields, and negatively impact soil environmental quality.

[0003] In recent years, induced resistance has become an effective plant disease resistance strategy. Enhancing plant stress resistance and disease resistance potential to produce sustained and broad-spectrum resistance to pathogens has gained increasing attention. Plant immune inducers are green pesticides that activate the plant immune system and improve plant disease resistance. Unlike traditional fungicides, plant immune inducers do not directly inhibit bacteria but induce plant defense mechanisms, endowing plants with the ability to resist infection by external pathogens and repair disease damage, thereby achieving the purpose of disease prevention. Daphne is a common secondary metabolite in plants, bacteria, and fungi, belonging to the hydroxycoumarin family. Previous studies have shown that daphne can be used as a plant resistance inducer to improve tobacco's resistance to bacterial wilt, making it a potential plant resistance inducer. However, its difficult extraction, poor water solubility, and easy decomposition in light have restricted its development and utilization. Therefore, improving the water solubility of daphne while maintaining its original disease resistance induction ability is an important issue in the development and utilization of coumarin compounds. Summary of the Invention

[0004] Therefore, the purpose of this invention is to provide a novel nano-inducer for controlling tobacco bacterial wilt by inducing resistance, while solving the problems of poor water solubility and low coupling rate of current inducers.

[0005] The present invention solves the above-mentioned technical problems through the following technical means:

[0006] A method for preparing a novel nano-inducer for controlling tobacco bacterial wilt involves the condensation reaction of daphne with carboxymethyl chitosan to form nanoparticles under the catalysis of N,N′-dicyclohexylcarbodiimide and 4-dimethylaminopyridine.

[0007] Compounds resulting from the coupling of carboxymethyl chitosan and daphne can enhance plant disease resistance within a specific concentration range and synergistically control bacterial wilt.

[0008] Furthermore, the mass ratio of daphne to carboxymethyl chitosan is (0.01-1):1.

[0009] The preferred ratio is (0.05-7):1, and the more preferred ratio is (0.1-7):1.

[0010] Furthermore, the mass ratio of N,N′-dicyclohexylcarbodiimide to 4-dimethylaminopyridine is 2:1.

[0011] Furthermore, the present invention also adds methyl formate and glyoxylic acid during the condensation process, wherein the mass ratio of methyl formate, glyoxylic acid and daphne is (1-2):(2-3):1.

[0012] Although nanoparticles are formed by the condensation reaction of daphne and carboxymethyl chitosan under the catalysis of N,N′-dicyclohexylcarbodiimide and 4-dimethylaminopyridine, the coupling rate between daphne and carboxymethyl chitosan is low, around 10%, and the control efficiency is about 70%. Therefore, this invention introduces methyl formate and glyoxylic acid under the catalysis of N,N′-dicyclohexylcarbodiimide and 4-dimethylaminopyridine to improve the coupling effect.

[0013] Furthermore, the specific steps are as follows:

[0014] (1) Dissolve carboxymethyl chitosan in water to obtain a carboxymethyl chitosan solution;

[0015] (2) Dissolve daphne in DMSO to obtain a daphne solution;

[0016] (3) Dissolve N,N′-dicyclohexylcarbodiimide and 4-dimethylaminopyridine in DMSO to obtain a mixed solution. Then add the mixed solution to a carboxymethyl chitosan solution and heat at 60°C for 1 h. Add daphne solution and stir at 60°C for 4 h. Add 3 times the volume of acetone solution and let stand for 6 h. Take out the brown precipitate, wash it with pure acetone, and freeze-dry for 24 h to obtain a novel nano-inducer.

[0017] The present invention also provides a preparation method, the specific steps of which are as follows:

[0018] (1) Dissolve carboxymethyl chitosan in water to obtain a carboxymethyl chitosan solution;

[0019] (2) Dissolve daphne in DMSO to obtain a daphne solution;

[0020] (3) Dissolve N,N′-dicyclohexylcarbodiimide and 4-dimethylaminopyridine in DMSO to obtain a mixed solution. Then add the mixed solution and glyoxylic acid to the carboxymethyl chitosan solution and heat at 60°C for 1 h. Then add daphne solution and methyl formate and stir at 60°C for 4 h. Add 3 times the volume of acetone solution and let stand for 6 h. Take out the brown precipitate and wash it with pure acetone. After freeze-drying for 24 h, a new nano-inducer is obtained.

[0021] Furthermore, the reaction process in step (3) is carried out under dark conditions.

[0022] The novel nano-inducer prepared in this invention has applications in improving plant disease resistance or agronomic traits.

[0023] The term "enhanced plant disease resistance" in this invention refers to enhanced plant disease resistance, including: delaying disease occurrence, resisting the invasion of Ralstonia solanacearum, increasing the activity of defense enzymes and the expression of resistance genes, and stimulating the tobacco plant's own disease resistance.

[0024] Furthermore, the novel nano-inducer is used for plants during the transplanting and / or rosette stages.

[0025] Of course, the novel nano-inducer prepared by this invention can be applied to crops and / or crop seeds including but not limited to: tobacco, eggplant, tomato, and potato; preferably tobacco, eggplant, and tomato; more preferably tobacco.

[0026] The conventional method of using the novel nano-inducer for tobacco bacterial wilt prepared in this invention is foliar spraying.

[0027] Beneficial effects:

[0028] A novel nano-inducer was prepared by coupling carboxymethyl chitosan with daphne. Compared with individual agents, the novel nano-inducer improved the water solubility of daphne and made it easier to use. At the same time, it also has the effect of delaying the occurrence of diseases, resisting the invasion of Ralstonia solanacearum, and improving the disease resistance of tobacco plants. In addition, it also increased the coupling rate of carboxymethyl chitosan and daphne, thus improving the control effect. Attached Figure Description

[0029] Figure 1 : A schematic diagram of the preparation of novel nano-inducers;

[0030] Figure 2 Characterization diagram of the novel nano-inducer;

[0031] Figure 3 The effects of novel nano-inducers on the activities of CAT, PPO and SOD. Detailed Implementation

[0032] The present invention will now be described in detail with reference to specific embodiments and accompanying drawings:

[0033] Example 1: Preparation of a novel nano-inducer

[0034] Weigh out 30 mg of daphne, 100 mg of carboxymethyl chitosan, 40 mg of N,N′-dicyclohexylcarbodiimide, and 20 mg of 4-dimethylaminopyridine.

[0035] Dissolve carboxymethyl chitosan in 10 mL of distilled water to obtain a carboxymethyl chitosan solution;

[0036] Daphne was dissolved in 20 mL of DMSO to obtain a daphne solution;

[0037] N,N′-dicyclohexylcarbodiimide and 4-dimethylaminopyridine were dissolved in 10 mL of DMSO to obtain a mixed solution. The mixed solution was then added to a carboxymethyl chitosan solution and heated at 60 °C under N2 protection for 1 h. Then, a daphne solution was added and stirred in the dark at 60 °C for 4 h. Three times the volume of the above solution in acetone solution was added and allowed to stand in the dark for 6 h. The brown precipitate was removed, washed with pure acetone, and freeze-dried at -80 °C for 24 h to obtain a novel nano-inducer.

[0038] Example 2: Preparation of a novel nano-inducer

[0039] Weigh out 50 mg of daphne, 100 mg of carboxymethyl chitosan, 40 mg of N,N′-dicyclohexylcarbodiimide, and 20 mg of 4-dimethylaminopyridine.

[0040] Dissolve carboxymethyl chitosan in 10 mL of distilled water to obtain a carboxymethyl chitosan solution;

[0041] Daphne was dissolved in 15 mL of DMSO to obtain a daphne solution;

[0042] N,N′-dicyclohexylcarbodiimide and 4-dimethylaminopyridine were dissolved in 10 mL of DMSO to obtain a mixed solution. The mixed solution was then added to a carboxymethyl chitosan solution and heated at 60 °C under N2 for 1 h. Then, a daphne solution was added and stirred in the dark at 60 °C for 4 h. Three times the volume of the above solution in acetone solution was added and allowed to stand in the dark for 6 h. The brown precipitate was removed, washed with pure acetone, and freeze-dried at -80 °C for 24 h to obtain a novel nano-inducer.

[0043] Example 3: Preparation of Novel Nano-Inducing Agents

[0044] Weigh out 0.1 mg of daphne, 10 mg of carboxymethyl chitosan, 0.4 mg of N,N′-dicyclohexylcarbodiimide, and 0.2 mg of 4-dimethylaminopyridine.

[0045] Dissolve carboxymethyl chitosan in 5 mL of distilled water to obtain a carboxymethyl chitosan solution;

[0046] Daphne was dissolved in 5 mL of DMSO to obtain a daphne solution;

[0047] N,N′-dicyclohexylcarbodiimide and 4-dimethylaminopyridine were dissolved in 5 mL of DMSO to obtain a mixed solution. The mixed solution was then added to a carboxymethyl chitosan solution and heated at 60 °C under N2 for 1 h. Then, a daphne solution was added and stirred in the dark at 60 °C for 4 h. Three times the volume of the above solution in acetone solution was added and allowed to stand in the dark for 6 h. The brown precipitate was removed, washed with pure acetone, and freeze-dried at -80 °C for 24 h to obtain a novel nano-inducer.

[0048] Preparation process as follows Figure 1 As shown.

[0049] The following tests were conducted on the novel nano-inducer prepared in Example 1:

[0050] I. Characterization Experiment

[0051] Solubility experiments were conducted on daphne and a novel nano-inducer. Both the novel nano-inducer and daphne were dissolved in water at a concentration of 10 mg / L, and the solubility was observed. The results are as follows: Figure 2 As shown in a.

[0052] The absorbance of daphne in the sample was measured using a spectrophotometer at a wavelength of 318 nm. The results are as follows: Figure 2 As shown in b.

[0053] The morphology and structure of the prepared novel nano-inducer were observed using a Tecnai 20 transmission electron microscope (TEM) and a JSM-6510LV tungsten filament scanning electron microscope (SEM). The results are as follows: Figure 2 As shown in c.

[0054] The Nano ZS90 particle size analyzer measured the average size and particle distribution of novel nano-inducer samples, and the results are as follows: Figure 2 As shown in d.

[0055] II. Enzyme Activity Assay

[0056] Tested variety: Tobacco (Tobacco 87 seedlings)

[0057] Experimental Methods: After spraying tobacco leaves with a novel nano-inducer, daphne, and carboxymethyl chitosan solution, 2 g of leaves from three tobacco plants were collected at 10 min, 6 h, 12 h, and 24 h, respectively. The samples were immediately placed at -80℃ for later use. Liquid nitrogen was added to the mortar containing the leaf samples, and the mixture was ground into powder. These samples were then aliquoted into 10 mL enzyme-free tubes. The samples were divided into three tubes, corresponding to PPO, CAT, and SOD activities, respectively. 1 mL of the corresponding enzyme activity extraction solution was added to each tube, mixed thoroughly, and centrifuged at 800×g for 10 min at 4℃ to obtain the enzyme solution. The optimal enzyme activity was then determined using PPO, SOD, and SOD enzyme activity reagents. The results are shown below. Figure 3 As shown.

[0058] III. Expression of Tobacco Defense-Related Genes

[0059] Tested variety: Tobacco (Tobacco 87 seedlings)

[0060] Test strains: Ralstonia solanacearum (CQPS-1), all provided by the Natural Products Research Laboratory of Southwest University.

[0061] Experimental Methods: After spraying tobacco leaves with a novel nano-inducer, daphne, and carboxymethyl chitosan solution, 2 g of leaves from three tobacco plants were collected 12 h later. The samples were immediately placed at -80℃ for later use. Then, qRT-PCR analysis was performed on 96-well plates with a reaction volume of 20 μL using a thermal cycler (Bio-Rad Corporation, Hercules, CA, USA). Normalized gene expression levels were calculated using Bio-Rad CFX, with EF-1 as the reference gene. The qRT-PCR procedure included an initial denaturation temperature of 95℃ for 3 min, followed by 40 cycles of 95℃ for 10 s, 55℃ for 20 s, and an extension temperature of 56℃ for 30 s. Using 2... -ΔΔCT The method normalizes the relative mRNA level of the target gene to its abundance, and the results are shown in Table 1.

[0062] Table 1. Effects of different treatments on the expression of tobacco resistance genes.

[0063]

[0064] IV. Induction of Disease Resistance Test

[0065] Experimental Methods: Tobacco seeds were cultivated in a greenhouse environment. After the seedlings reached the "three-leaf and one-heart" stage, foliar spraying was performed according to the recommended dosage of each pesticide (see Table 2), for a total of two applications. Two days after pesticide treatment, the roots were drenched with 10 mL / plant of *Ralstonia solanacearum* (OD200). 600=0.1), and cultured in a constant temperature incubator at 28℃, 75% humidity, and a light cycle of 14 h / 10 h. The disease incidence was recorded daily after inoculation.

[0066] Table 2 Concentrations of different pesticides for foliar spraying

[0067]

[0068] Tobacco bacterial wilt disease survey: The disease severity level of tobacco bacterial wilt was graded from 0 to 4 (where grade 0 is no symptoms; grade 1 is 1-25% leaf wilting; grade 2 is 26-50% leaf wilting; grade 3 is 51-75% leaf wilting; and grade 4 is 76-100% leaf wilting). Each treatment included at least 10 tobacco seedlings, and was repeated 3 times. The average disease index was calculated, and the disease index and relative control effect were calculated using formulas (1) and (2). The results are shown in Table 2.

[0069]

[0070] Table 3. Relative control efficacy of different treatments against tobacco bacterial wilt

[0071]

[0072] Analysis of the results of Experiments 1 through 4:

[0073] 1. By Figure 2 The results show that the prepared novel nano-inducer has good water solubility. TEM observation shows that the novel nano-inducer is in the form of spherical particles with uniform distribution and an average particle size of 349 nm.

[0074] 2. By Figure 3 The results showed that the novel nano-inducer significantly improved the activities of CAT, PPO and SOD defense enzymes, which were 386.37, 203.33 and 192.89 U / g, respectively, and were higher than those of the other treatments.

[0075] 4. As shown in Table 1, treatments with the novel nano-inducer, DA, and CMCS increased the expression of the PR family gene NtPR1 by 25.4, 12.9, and 10.3 times, respectively. The other three treatments significantly increased the transcriptional expression of NtNPR1, reaching 36.74, 23.68, and 11.23, respectively. This indicates that the novel nano-inducer enhances the expression of genes encoding proteins related to plant disease progression, thereby activating the plant's systemic immunity.

[0076] 5. As shown in Table 3, the novel nano-inducer achieved a synergistic effect in controlling tobacco bacterial wilt compared to single-agent treatment within the effective concentration range of 1.43-5.7.

[0077] Example 4: Preparation of Novel Nano-Inducing Agents

[0078] Weigh out 30 mg of daphne, 100 mg of carboxymethyl chitosan, 40 mg of N,N′-dicyclohexylcarbodiimide, 20 mg of 4-dimethylaminopyridine, 30 mg of methyl formate, and 60 mg of glyoxylic acid.

[0079] (1) Dissolve carboxymethyl chitosan in 10 mL of distilled water to obtain a carboxymethyl chitosan solution;

[0080] (2) Dissolve daphne in 20 mL of DMSO to obtain a daphne solution;

[0081] (3) Dissolve N,N′-dicyclohexylcarbodiimide and 4-dimethylaminopyridine in 10 mL of DMSO to obtain a mixed solution. Then add the mixed solution and glyoxylic acid to the carboxymethyl chitosan solution and heat for 1 h under the protection of N2 at 60 °C. Then add daphne solution and methyl formate and stir in the dark at 60 °C for 4 h. Add acetone solution with a volume of 3 times that of the above solution and let stand in the dark for 6 h. Take out the brown precipitate, wash it with pure acetone, and freeze dry for 24 h to obtain a novel nano-inducer.

[0082] Example 5: Preparation of Novel Nano-Inducing Agents

[0083] Weigh out 30 mg of daphne, 100 mg of carboxymethyl chitosan, 40 mg of N,N′-dicyclohexylcarbodiimide, 20 mg of 4-dimethylaminopyridine, 60 mg of methyl formate, and 60 mg of glyoxylic acid.

[0084] (1) Dissolve carboxymethyl chitosan in 10 mL of distilled water to obtain a carboxymethyl chitosan solution;

[0085] (2) Dissolve daphne in 20 mL of DMSO to obtain a daphne solution;

[0086] (3) Dissolve N,N′-dicyclohexylcarbodiimide and 4-dimethylaminopyridine in 10 mL of DMSO to obtain a mixed solution. Then add the mixed solution and glyoxylic acid to the carboxymethyl chitosan solution and heat at 60 °C under N2 protection for 1 h. Then add daphne solution and methyl formate and stir in the dark at 60 °C for 4 h. Add acetone solution with a volume of 3 times that of the above solution and let stand in the dark for 6 h. Take out the brown precipitate, wash it with pure acetone, and freeze-dry for 24 h to obtain a novel nano-inducer.

[0087] Example 6: Preparation of Novel Nano-Inducing Agents

[0088] Weigh out 30 mg of daphne, 100 mg of carboxymethyl chitosan, 40 mg of N,N′-dicyclohexylcarbodiimide, 20 mg of 4-dimethylaminopyridine, 60 mg of methyl formate, and 90 mg of glyoxylic acid.

[0089] (1) Dissolve carboxymethyl chitosan in 10 mL of distilled water to obtain a carboxymethyl chitosan solution;

[0090] (2) Dissolve daphne in 20 mL of DMSO to obtain a daphne solution;

[0091] (3) Dissolve N,N′-dicyclohexylcarbodiimide and 4-dimethylaminopyridine in 10 mL of DMSO to obtain a mixed solution. Then add the mixed solution and glyoxylic acid to the carboxymethyl chitosan solution and heat for 1 h under the protection of N2 at 60 °C. Then add daphne solution and methyl formate and stir in the dark at 60 °C for 4 h. Add acetone solution with a volume of 3 times that of the above solution and let stand in the dark for 6 h. Take out the brown precipitate, wash it with pure acetone, and freeze dry for 24 h to obtain a novel nano-inducer.

[0092] V. Determination of the coupling rate between carboxymethyl chitosan and daphne

[0093] Experimental Group 1: A novel nano-inducer was prepared according to the method in Example 4;

[0094] Experimental Group 2: A novel nano-inducer was prepared according to the method in Example 5;

[0095] Experimental Group 3: A novel nano-inducer was prepared according to the method in Example 6;

[0096] Control group 1: The difference from Example 4 is that methyl formate and glyoxylic acid are not added, and the preparation method is the same as that of Example 1;

[0097] Control group 2: The difference from Example 4 is that methyl formate is not added, and the preparation method is the same as in Example 4;

[0098] Control group 3: The difference from Example 4 is that glyoxylic acid is not added; the preparation method is the same as in Example 4.

[0099] Control group 4: The difference from Example 4 is that only potassium persulfate was added, with an addition amount of 1 mg. The specific preparation method is as follows:

[0100] N,N′-dicyclohexylcarbodiimide and 4-dimethylaminopyridine were dissolved in 10 mL of DMSO to obtain a mixed solution. The mixed solution was then added to a carboxymethyl chitosan solution and heated at 60 °C under N2 protection for 1 h. Then, daphne solution and potassium persulfate were added, and the mixture was stirred in the dark at 60 °C for 4 h. Three times the volume of the above solution was added to acetone solution, and the mixture was allowed to stand in the dark for 6 h. The brown precipitate was removed, washed with pure acetone, and freeze-dried at -80 °C for 24 h to obtain a novel nano-inducer.

[0101] The coupling rate of carboxymethyl chitosan and daphne in the novel nano-inducers obtained in Examples 4 (Experimental Group 1), 5 (Experimental Group 2), 6 (Experimental Group 3), and Control Groups 1-4 was determined. The specific measurement method is as follows:

[0102] Grafting rates were determined using an Agilent 1260 high-performance liquid chromatography (HPLC) system (Agilent Technologies, USA). First, standard solutions of daphne were prepared at concentrations of 10, 20, 40, 80, and 160 mg / L. Second, 0.1 g of the product was added to a methanol solution (100 ml, pH=4.0) and hydrolyzed for 2 h to extract all daphne from the conjugate. The solution was then diluted 10-fold for detection. Chromatography was performed at C1. 18 The chromatography was performed on a reversed-phase column with a mobile phase of methanol / 0.5% phosphoric acid (30:70, v / v), a flow rate of 0.8 mL / min, a column temperature of 25℃, and a test wavelength of 325 nm. The results are shown in Table 4.

[0103] Table 4

[0104]

[0105] VI. Induction of Disease Resistance Test (II)

[0106] The relative efficacy experiment was conducted using the same method as in Experiment 4, and the data obtained are shown in Table 5:

[0107] Table 5

[0108]

[0109] Analysis of the results of Experiments 5 and 6 shows that:

[0110] 1. The novel nano-inducer prepared in control group 1 achieved a coupling rate of 11.05%, and the highest control effect reached 93.41% at a relatively low concentration of 1.43 mg / L.

[0111] 2. Compared with the experimental group, the experimental group introduced methyl formate and glyoxylic acid, which significantly increased the coupling rate to 20.19-21.75%. At the same effective concentration, the relative efficacy was significantly increased, reaching a maximum of 96.74%-98.87%, and the relative control effect reached 78.56%-80.62% after 11 days.

[0112] 3. Compared with the experimental group, the control groups 2 and 3 did not add methyl formate or glyoxylic acid, and their coupling rate was still low at about 11%. Compared with control group 1, control group 4, which added potassium persulfate, had a slightly higher coupling rate due to the potassium persulfate initiator, but it was still lower than that of the experimental group. This may be because the addition of methyl formate and glyoxylic acid in the experimental group synergistically protected the active groups of daphne and adjusted the structure of the substrate, increasing its reactivity and making it more conducive to the condensation reaction between daphne and carboxymethyl chitosan, thereby increasing the coupling rate. The increase in coupling rate can significantly increase the control effect.

[0113] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention, and all such modifications and substitutions should be covered within the scope of the claims of the present invention. Technical aspects, shapes, and structures not described in detail in this invention are all well-known technologies.

Claims

1. A method for preparing a nano-inducer for controlling tobacco bacterial wilt, characterized in that, Nanoparticles are formed by the condensation reaction of daphne and carboxymethyl chitosan under the catalysis of N,N′-dicyclohexylcarbodiimide and 4-dimethylaminopyridine. Methyl formate and glyoxylic acid are also added during the condensation process. The mass ratio of methyl formate, glyoxylic acid and daphne is (1-2):(2-3):

1.

2. The method for preparing a nano-inducer for controlling tobacco bacterial wilt according to claim 1, characterized in that, The mass ratio of daphne to carboxymethyl chitosan is (0.01-1):

1.

3. The method for preparing a nano-inducer for controlling tobacco bacterial wilt according to claim 1, characterized in that, The mass ratio of N,N′-dicyclohexylcarbodiimide to 4-dimethylaminopyridine is 2:

1.

4. The method for preparing a nano-inducer for controlling tobacco bacterial wilt according to claim 3, characterized in that, The specific steps are as follows: (1) Dissolve carboxymethyl chitosan in water to obtain a carboxymethyl chitosan solution; (2) Dissolve daphne in DMSO to obtain a daphne solution; (3) Dissolve N,N′-dicyclohexylcarbodiimide and 4-dimethylaminopyridine in DMSO to obtain a mixed solution. Then add the mixed solution and glyoxylic acid to the carboxymethyl chitosan solution and heat at 60°C for 1 h. Then add daphne solution and methyl formate and stir at 60°C under N2 for 4 h. Add acetone solution and let stand for 6 h. Take out the brown precipitate and wash it with pure acetone. Freeze-dry for 24 h to obtain the nano-inducer.

5. The method for preparing a nano-inducer for controlling tobacco bacterial wilt according to claim 4, characterized in that, The reaction process in step (3) is carried out under dark conditions.

6. The use of a nano-inducer for preventing and controlling tobacco bacterial wilt prepared according to the preparation method described in claim 5, characterized in that, The nano-inducer is used to improve plant disease resistance or agronomic traits.

7. The use according to claim 6, characterized in that, The nano-inducer is used on plants during the transplanting and / or rosette stages.

8. The use according to claim 7, characterized in that, The nano-inducer is applied by foliar spraying.

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