Application of modified tobacco leaf polysaccharide in resisting tobacco mosaic virus

Abstract

The invention provides an application of modified tobacco leaf polysaccharide in resisting tobacco mosaic virus. According to the application of the modified tobacco polysaccharide in resisting the tobacco mosaic virus, the modified tobacco polysaccharide comprises one or more of acetylated tobacco polysaccharide, carboxymethylated tobacco polysaccharide and phosphorylated tobacco polysaccharide. The modified tobacco leaf polysaccharide is used for preventing and treating tobacco mosaic viruses by improving the protection effect on tobacco plants and the passivation effect on the tobacco mosaic viruses. Tobacco polysaccharide is used as a raw material, three chemical derivatization methods of acetylation, carboxymethylation and phosphorylation are adopted for modifying the tobacco polysaccharide, active functional groups are introduced, the solubility of the modified tobacco polysaccharide is remarkably improved, and the activity of the tobacco polysaccharide for resisting tobacco mosaic viruses is remarkably improved; in-vitro polymerization of the tobacco mosaic virus coat protein is inhibited, the structure of the tobacco mosaic virus is destroyed, and a foundation is laid for application of the tobacco mosaic virus in the field of botanical pesticides.

Description

Technical Field

[0001] The present application belongs to the field of tobacco technology, and specifically relates to the application of modified tobacco polysaccharides in resisting tobacco mosaic virus. Background Art

[0002] Plant viral diseases, often called "plant cancers," cause enormous losses to agricultural production. When tobacco plants are infected with tobacco mosaic virus (TMV), localized leaf tissue becomes mottled, leading to leaf deformities, stunted growth, and a significant decrease in tobacco yield and quality, severely impacting the development of the tobacco industry and the stability of the supply chain.

[0003] Currently, chemical pesticides remain the primary method for controlling tobacco mosaic virus (TMV). However, long-term use of chemical pesticides not only leads to resistance but also poses significant risks to plants, the environment, and humans. my country has made significant progress in the development and application of pesticides for controlling plant viral diseases, with the gradual discovery of several plant-derived active substances that are effective against TMV, including macromolecules like polysaccharides and proteins, as well as small molecules like alkaloids and sesquiterpenes.

[0004] Polysaccharides have demonstrated excellent resistance to tobacco mosaic virus (TMV). However, as large active molecules, most polysaccharides have poor water solubility, which limits their effectiveness. Numerous studies have confirmed that chemical modification of polysaccharides significantly enhances their biological activity. However, limited research has been conducted on the modification of tobacco polysaccharides, particularly regarding their application in the prevention and control of TMV. Summary of the Invention

[0005] In view of this, the present application provides the application of modified tobacco polysaccharides in resistance to tobacco mosaic virus, which significantly improves the solubility and anti-tobacco mosaic virus activity of tobacco polysaccharides and enhances the resource utilization level of tobacco polysaccharides.

[0006] In order to achieve the above objectives, this application provides the following technical solutions:

[0007] The invention relates to an application of modified tobacco polysaccharide in resisting tobacco mosaic virus. The modified tobacco polysaccharide comprises one or more of acetylated tobacco polysaccharide, carboxymethylated tobacco polysaccharide or phosphorylated tobacco polysaccharide.

[0008] In the present application, the modified tobacco polysaccharide prevents and controls tobacco mosaic virus by improving the protective effect on tobacco plants and the passivation effect on tobacco mosaic virus.

[0009] In the present application, the preparation method of the acetylated tobacco polysaccharide includes: dissolving the tobacco polysaccharide in deionized water, adjusting the pH to 9.0-10.0, slowly adding acetic anhydride thereto while stirring, and adjusting the pH of the entire reaction system to maintain at 8.0-10.0 during the addition process; after the addition is completed, placing the mixture in a water bath at 55-65°C for shaking, adjusting the pH to 7.0, dialyzing, and drying to obtain the acetylated tobacco polysaccharide.

[0010] Preferably, the volume ratio of the tobacco polysaccharide to the acetic anhydride is 1 g:(18-22) mL.

[0011] In the present application, the preparation method of the carboxymethylated tobacco polysaccharide includes: placing the tobacco polysaccharide in isopropanol, stirring and mixing at room temperature, slowly adding a 20% mass concentration NaOH solution, continuing to stir, slowly adding a 4 mol / L chloroacetic acid solution, stirring and reacting at 55-65°C for 4 hours, cooling to room temperature, adjusting the pH to 7.0, dialyzing, and drying to obtain carboxymethylated tobacco polysaccharide.

[0012] Preferably, the volume ratio of the mass of the tobacco polysaccharide to the NaOH solution is 1 g:(140-160) mL, and the volume ratio of the NaOH solution to the chloroacetic acid solution is (7-8):1.

[0013] In the present application, the preparation method of the phosphorylated tobacco polysaccharide includes: dissolving sodium tripolyphosphate and sodium trimetaphosphate in water to prepare a phosphorylation reagent; adding sodium sulfate and the tobacco polysaccharide to the phosphorylation reagent in sequence; adjusting the pH to 7.0 after mixing, and then stirring the reaction at 75-85°C, adding 95% ethanol solution to the reaction product, standing at 4°C, centrifuging, collecting and dissolving the precipitate, dialyzing, and drying to obtain the phosphorylated tobacco polysaccharide.

[0014] Preferably, the mass ratio of the sodium tripolyphosphate to the sodium trimetaphosphate is (5-7):1, the volume ratio of the mass of the tobacco polysaccharide to the phosphorylation reagent is 1g:(980-1200)mL, and the volume ratio of the mass of the sodium sulfate to the phosphorylation reagent is 1g:(150-250)mL.

[0015] A preparation for preventing and treating tobacco mosaic virus, comprising the modified tobacco polysaccharide in any of the above applications.

[0016] This application has the following advantages:

[0017] (1) The present application uses tobacco leaf polysaccharide as raw material and adopts three chemical derivatization methods, namely acetylation, carboxymethylation and phosphorylation, to modify the tobacco leaf polysaccharide, introduce active functional groups, significantly enhance the solubility of the modified tobacco leaf polysaccharide, significantly improve its anti-tobacco mosaic virus activity, inhibit the in vitro polymerization of tobacco mosaic virus coat protein, destroy the structure of tobacco mosaic virus, and lay the foundation for its application in the field of botanical pesticides.

[0018] (2) This application improves the comprehensive utilization of tobacco polysaccharides by studying the anti-tobacco mosaic virus activity of tobacco polysaccharide derivatives and their effects on the morphology of tobacco mosaic virus, provides new ideas and methods for the development and research of environmentally friendly pesticides, and explores the potential of acetylated, carboxymethylated and phosphorylated polysaccharides in antiviral drugs.

[0019] (3) The preparation of the present application can significantly enhance the ability of tobacco leaf polysaccharides to resist tobacco mosaic virus, play a certain protective role on tobacco plants, and destroy the morphology of tobacco mosaic virus, thereby preventing the spread of the virus. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the following briefly introduces the drawings required for use in the embodiments.

[0021] Figure 1 This is the infrared comparison of tobacco leaf polysaccharides before and after modification;

[0022] Figure 2 The scanning electron micrographs of tobacco leaf polysaccharides before and after modification are shown in Figure 2, including tobacco leaf polysaccharides: A: 300×, B: 500×; acetylated tobacco leaf polysaccharides: C: 300×, D: 500×; carboxymethylated tobacco leaf polysaccharides: E: 300×, F: 500×; phosphorylated tobacco leaf polysaccharides: G: 300×, H: 500×;

[0023] Figure 3 This is a comparison chart of the maximum solubility of tobacco polysaccharides before and after modification;

[0024] Figure 4 The protective effect of tobacco leaf polysaccharides on protoplasts in tobacco leaves before and after modification;

[0025] Figure 5 The figure shows the destructive effect of tobacco leaf polysaccharides on the morphology of tobacco mosaic virus before and after modification, where A: blank control; B: tobacco leaf polysaccharide; C: acetylated tobacco leaf polysaccharide; D: carboxymethylated tobacco leaf polysaccharide; E: phosphorylated tobacco leaf polysaccharide. DETAILED DESCRIPTION

[0026] To make the purpose, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below. The following embodiments are merely examples for clearly illustrating the technical solutions of the present application and are not intended to limit the present application.

[0027] Modified tobacco polysaccharides are used in tobacco mosaic virus (TMV) resistance. Modified tobacco polysaccharides include one or more of acetylated, carboxymethylated, or phosphorylated tobacco polysaccharides. Modified tobacco polysaccharides prevent and control TMV by enhancing its protective effect on tobacco plants and inactivating TMV.

[0028] Unless otherwise specified, the methods in this embodiment are operated according to conventional methods. Unless otherwise specified, the reagents used are conventional reagents or reagents prepared according to conventional methods. The tobacco leaves in the embodiment were collected from Chengjiang County, Yunnan Province in July 2022, and tobacco mosaic virus (TMV) and heartleaf tobacco seeds were provided by the Yuxi City Company of Yunnan Tobacco Company.

[0029] Example 1: Preparation of modified tobacco polysaccharides

[0030] 1. Preparation of tobacco polysaccharides

[0031] (1) The tobacco leaves were dried in an oven at 50°C, crushed with a traditional Chinese medicine grinder, and then passed through a 40-mesh sieve to obtain tobacco leaf powder. An 80% ethanol solution was added thereto, and the mixture was soaked and ultrasonicated for 15 minutes. The supernatant was discarded after centrifugation, and the tobacco leaf powder was placed in a ventilated place to evaporate and set aside;

[0032] (2) Tobacco leaf powder was mixed with ultrapure water at a ratio of 1 g:30 mL, extracted in a water bath at 80°C for 3 h, centrifuged at 4200 rpm for 20 min, and the solid was extracted twice. The combined extracts were collected and concentrated to 1 / 5 of the original volume using a rotary evaporator at 50°C.

[0033] (3) adding 95% ethanol to the concentrate three times to adjust the ethanol volume concentration in the concentrate to 40%, 60%, and 80% in sequence, and then performing alcohol precipitation. The concentrate was placed in a refrigerator at 4°C for 24 hours, and the supernatant was discarded by centrifugation. The precipitate was the crude tobacco leaf polysaccharide.

[0034] (4) The crude tobacco polysaccharide precipitate was dissolved again in deionized water until the precipitate was completely dissolved to obtain a crude tobacco polysaccharide solution. Sevag solution (chloroform: n-butanol = 4:1, v / v) was added to the obtained solution, stirred for 30 minutes using a magnetic stirrer, and then allowed to stand for 1 hour. After the aqueous phase and the organic phase were completely separated, the aqueous phase was collected using a separatory funnel, and the Sevag solution was repeatedly treated twice. The aqueous phases were combined, and the combined aqueous phases were centrifuged to obtain the supernatant, and freeze-dried to obtain the deproteinized crude tobacco polysaccharide.

[0035] (5) The deproteinized tobacco leaf crude polysaccharide was redissolved in water and passed through a 2.6 cm × 30 cm DEAE cellulose column. The column was eluted with deionized water, 0.1 M, 0.3 M, and 0.5 M NaCl solutions in sequence. The eluate from the 0.3 M NaCl solution was collected and freeze-dried to obtain tobacco leaf polysaccharide.

[0036] 2. Acetylation of tobacco polysaccharides

[0037] 100 mg of tobacco polysaccharide was dissolved in 5 mL of deionized water, the pH was adjusted to 9.0-10.0 with 0.5 mol / L NaOH, and 2 mL of acetic anhydride was slowly added dropwise while stirring. NaOH was also added dropwise during the addition process to maintain the pH of the entire reaction system at 8.0-10.0. Subsequently, the tobacco polysaccharide solution to which acetic anhydride was added was placed in a 60°C water bath for shaking reaction for 2 hours. After the reaction was completed, the pH was adjusted to 7.0 with 0.5 mol / L HCl, dialyzed for 2 days using a dialysis bag with a molecular weight cutoff of 500 Da, and freeze-dried to obtain acetylated tobacco polysaccharide.

[0038] 3. Carboxymethylation of tobacco polysaccharides

[0039] 100 mg of tobacco polysaccharide was placed in 40 mL of isopropanol and stirred at room temperature for 20 min. Then, 15 mL of 20% NaOH solution was slowly added thereto and stirring was continued for 1 h. 2 mL of 4 mol / L chloroacetic acid solution was slowly added to the solution and stirred at 60 ° C for 4 h. After the reaction was completed, the solution was cooled to room temperature and the pH was adjusted to 7.0 with HCl. The solution was dialyzed for 2 days using a dialysis bag with a molecular weight cutoff of 500 Da and freeze-dried to obtain carboxymethylated tobacco polysaccharide.

[0040] 4. Phosphorylation of tobacco polysaccharides

[0041] 6 g of sodium tripolyphosphate and 1 g of sodium trimetaphosphate were dissolved in 100 mL of distilled water as a phosphorylation reagent; subsequently, 500 mg of sodium sulfate was added to the 100 mL phosphorylation reagent solution, and then 100 mg of tobacco polysaccharide was added; the pH of the mixture was adjusted to 7.0 using 0.5 mol / L NaOH, and the mixture was stirred and reacted at 80° C. for 4 h. After that, 95% ethanol (4 times the volume of the reaction solution) was added thereto, and the mixture was allowed to stand at 4° C. for 12 h. The supernatant was discarded after centrifugation, the precipitate was re-dissolved with water, and dialyzed using a dialysis bag with a molecular weight cutoff of 500 Da for 2 days. The mixture was freeze-dried to obtain phosphorylated tobacco polysaccharide.

[0042] Weigh 5.0 mg each of tobacco polysaccharide, acetylated tobacco polysaccharide, carboxymethylated tobacco polysaccharide and phosphorylated tobacco polysaccharide prepared by the above method, add 500.0 mg KBr, mix and grind, and analyze the mixture at a wave number range of 4000-400 cm 1 Perform infrared scanning and draw infrared spectrum;

[0043] See the results Figure 1 As can be seen from the figure, the infrared spectra of the four polysaccharides have roughly the same shape, among which 3400cm -1 The strong absorption peak near 2920-3000cm -1 The weak absorption peaks between the two correspond to the asymmetric CH stretching vibration of sugars, which are characteristic absorption peaks of polysaccharides. -1 The OH absorption peak near the -1 A new peak appeared at 1257 cm -1 Enhanced CO stretching vibrations were observed near 3427 cm. These changes confirmed the successful introduction of acetyl groups. -1 The OH absorption peak at 1580 cm-1 becomes broad, indicating that the hydrogen bond is strengthened. -1 , 1422cm -1 New absorption bands were observed near 1295 cm-1, corresponding to the asymmetric stretching vibration of C=O and the stretching vibration of methyl groups. These peaks are characteristic of carboxymethyl groups, confirming the successful carboxymethylation of polysaccharides. -1 A new absorption peak appeared at , which was due to the P=O stretching vibration of the phosphate group, indicating that the phosphate group was successfully introduced into the polysaccharide structure.

[0044] The scanning electron micrograph of the above polysaccharide is shown in Figure 2 As can be seen from the figure, the surface structures of tobacco polysaccharides before and after modification are significantly different. Unmodified tobacco polysaccharides have a layered structure and a relatively smooth surface morphology. Acetylated polysaccharides, on the other hand, show a transition from fragmented particles to massive aggregates, exhibiting a certain degree of aggregation. Carboxymethylated polysaccharides have obvious fibers on their surface, showing a certain pore structure. Phosphorylated polysaccharides have a certain similarity in surface morphology to unmodified tobacco polysaccharides, but have more depressions and sharper edges. These changes in the surface morphology of tobacco polysaccharides may be due to chemical modification, which changes the internal structure of tobacco polysaccharides, thereby causing changes in surface morphology.

[0045] Example 2: Determination of the maximum solubility of modified tobacco polysaccharides

[0046] 100 mg of tobacco leaf polysaccharide, acetylated tobacco leaf polysaccharide, carboxymethylated tobacco leaf polysaccharide, and phosphorylated tobacco leaf polysaccharide were weighed and dissolved in 5 mL of deionized water. The mixture was stirred and dissolved at room temperature for 30 min. Subsequently, the mixture was centrifuged at 4500 r / min for 10 min. The resulting precipitate was placed in a 45°C oven until constant weight was reached. The mass of the precipitate was weighed to calculate the maximum solubility. The calculation formula is as follows:

[0047]

[0048] Wherein, A is the mass of the final precipitate.

[0049] See the results Figure 3 As can be seen from the figure, compared with tobacco polysaccharides, the solubility of acetylated tobacco polysaccharides, carboxymethylated tobacco polysaccharides and phosphorylated tobacco polysaccharides after modification is significantly improved. Therefore, it can be proved that chemical modification can significantly improve the solubility of polysaccharides, which is of great significance in the further development and application of tobacco polysaccharides.

[0050] Example 3: Experiment on the protective, therapeutic and inactivation effects of modified tobacco polysaccharides on tobacco mosaic virus

[0051] 1 mg of tobacco leaf polysaccharide, acetylated tobacco leaf polysaccharide, carboxymethylated tobacco leaf polysaccharide, and phosphorylated tobacco leaf polysaccharide powders were weighed and dissolved in 2 mL of deionized water to obtain 500 μg / mL polysaccharide sample solutions; 500 μg / mL Ningnanmycin solution was used as a positive control;

[0052] The half-leaf spot method was used, with healthy, uniformly growing heartleaf tobacco plants at the 5-6 leaf stage as the research subjects. Twelve heartleaf tobacco plants were selected and divided into three groups: protective, desensitizing, and curative. Each group contained four plants inoculated with four polysaccharide samples: tobacco leaf polysaccharide, acetylated tobacco leaf polysaccharide, carboxymethylated tobacco leaf polysaccharide, and phosphorylated tobacco leaf polysaccharide. TMV and sample solutions were applied using diamond abrasive friction inoculation. Three leaves of similar size were selected from each plant as biological replicates. The specific experimental steps are as follows:

[0053] (1) Protective effect: The left half of the leaf of Nicotiana tabacum was friction-inoculated with a polysaccharide sample solution (500 μg / mL, 100 μL / leaf) as the experimental group; the right half of the same leaf was friction-inoculated with deionized water as the blank control group; 24 h after inoculation with the sample and deionized water, TMV (32 μg / mL, 100 μL / leaf) was inoculated using the same friction inoculation method, and repeated three times.

[0054] (2) Therapeutic effect: The whole leaf of Nicotiana tabacum was inoculated with TMV (32 μg / mL, 100 μL / leaf) by friction. After 6 h of TMV inoculation, the left half of the leaf was inoculated with polysaccharide sample solution (500 μg / mL, 100 μL / leaf) by friction, which served as the experimental group. The right half of the same leaf was inoculated with deionized water by friction, which served as the blank control group. This was repeated three times.

[0055] (3) Passivation effect: TMV (32 μg / mL) was mixed with equal volumes of polysaccharide sample solution (500 μg / mL) and deionized water, respectively. After being placed at 25°C for 30 min, the left half of the leaf of Nicotiana chinensis was rub-inoculated with the mixed solution of polysaccharide sample and TMV (100 μL / leaf) as the experimental group; the right half of the same leaf was rub-inoculated with the mixed solution of deionized water and TMV (100 μL / leaf) as the blank control group. This was repeated three times.

[0056] The treated heartleaf tobacco leaves were placed in a greenhouse and the number of dead spots on the leaves was observed after 5-7 days. The protective effect, therapeutic effect, and passivation effect of the sample on TMV were calculated using the following formula:

[0057]

[0058] Wherein, C: the average number of necrosis spots in the blank control group; T: the average number of necrosis spots in the experimental group.

[0059] Table 1 Protective, therapeutic and passivating effects of tobacco polysaccharides before and after modification

[0060] Inhibition rate (%) Tobacco polysaccharides Acetylated tobacco polysaccharide Carboxymethylated tobacco polysaccharide Phosphorylated tobacco polysaccharide Protective effect <![CDATA[45.51±0.68 d ]]> <![CDATA[83.30±0.87 c ]]> <![CDATA[95.77±0.44 a ]]> <![CDATA[87.29±1.02 b ]]> therapeutic effects <![CDATA[42.53±0.64 e ]]> <![CDATA[68.39±0.72 c ]]> <![CDATA[86.70±1.25 a ]]> <![CDATA[65.20±0.91 d ]]> Passivation <![CDATA[36.53±1.14 c ]]> <![CDATA[75.92±1.23 b ]]> <![CDATA[87.76±0.39 a ]]> <![CDATA[76.54±1.16 b ]]>

[0061] From the results in Table 1, it can be seen that the protective effect, therapeutic effect and passivation effect of the three chemically modified tobacco polysaccharides are significantly higher than those of the tobacco polysaccharides before modification, indicating that the modified tobacco polysaccharides have better anti-TMV activity. It can be seen from the results that the inhibition rates of the protective effect and passivation effect are higher than those of the therapeutic effect, indicating that the modified polysaccharides control tobacco mosaic virus mainly through the protective effect on tobacco plants and the passivation effect on TMV.

[0062] Example 4: Protective Effect of Modified Tobacco Polysaccharides on Protoplasts in Tobacco Leaves

[0063] Randomly select healthy and uniformly growing K 326 Systemic tobacco was used as the research object, and three leaves of similar size were selected from each plant. 500 μg / mL tobacco leaf polysaccharide, acetylated tobacco leaf polysaccharide, carboxymethylated tobacco leaf polysaccharide, and phosphorylated tobacco leaf polysaccharide solutions were injected into the leaves using the infiltration method through a syringe with the needle removed. 200 μL of polysaccharide solution was injected into each leaf. After 24 hours of incubation, TMV (32 μg / mL) was inoculated using the same method. 24 hours after virus injection, the leaves were enzymatically hydrolyzed to obtain protoplasts, and the number and morphological changes of the protoplasts were observed under an inverted fluorescence microscope. Healthy tobacco leaves that were not inoculated with the virus served as blank controls, and virus-infected tobacco leaves served as positive controls.

[0064] The specific steps of protoplast enzymatic hydrolysis are as follows:

[0065] Place 1g of fresh tobacco leaves on a Petri dish and cut into 0.1-0.5mm strips. Incubate the leaves in an enzymatic hydrolysis solution at 25°C in the dark for 1-2 hours. Filter the hydrolysis solution through a cell strainer to remove any solids and impurities. Centrifuge the solution at 4000 rpm for 5 minutes and discard the supernatant. Add 5mL of 13% CPW wash solution to the precipitate and centrifuge at 4000 rpm for 5 minutes to collect the precipitate. Add 1mL of 13% CPW wash solution to the precipitate again to obtain a protoplast suspension.

[0066] 1L of 13% CPW wash solution is prepared as follows: 27.2mg potassium dihydrogen phosphate (KHPO4) + 101.0mg potassium nitrate (KNO3) + 1480mg calcium chloride (CaCl2) + 246mg magnesium sulfate (MgSO4) + 0.16mg potassium iodide (KI) + 0.025mg sulfuric acid (CuSO4) + 13% mannitol (w / v);

[0067] The results are as follows Figure 4 As shown, the protoplasts in healthy tobacco (blank) are oval or spherical, while the protoplasts in tobacco infected with TMV are significantly reduced in number compared to those in healthy tobacco, and many irregular cell fragments are generated due to the rupture of the protoplast membrane. After treatment with tobacco polysaccharide samples, there is a significant protective effect on the protoplasts in tobacco leaves, and the protective effect of the modified tobacco polysaccharide is significantly better than that of the tobacco polysaccharide before modification. The protective effect of tobacco polysaccharides and their modified tobacco polysaccharides on protoplasts corresponds to the protective effect shown in Example 1.

[0068] Example 5: Destructive effect of modified tobacco polysaccharides on tobacco mosaic virus morphology

[0069] 1 mg of tobacco leaf polysaccharide, acetylated tobacco leaf polysaccharide, carboxymethylated tobacco leaf polysaccharide, and phosphorylated tobacco leaf polysaccharide powder was weighed and dissolved in 2 mL of deionized water to prepare 500 μg / mL polysaccharide sample solutions. A 32 μg / mL TMV solution was mixed with equal volumes of the 500 μg / mL tobacco leaf polysaccharide, acetylated tobacco leaf polysaccharide, carboxymethylated tobacco leaf polysaccharide, and phosphorylated tobacco leaf polysaccharide solutions, respectively. The mixtures were allowed to stand at 25°C for 1 hour. A mixture of 32 μg / mL TMV and sterile distilled water was used as a blank control.

[0070] The specific steps are as follows: a copper mesh is clamped onto a silica gel staining plate, and the sample is then dropped onto the front of the mesh and dried at 24°C. Once the sample is dry, phosphotungstic acid is added for staining for 1-2 minutes. The sample is then gently rinsed three times in ddH2O and dried. The morphology of the TMV is then observed using a transmission electron microscope.

[0071] from Figure 5It can be seen that the intact TMV in the blank control presents a complete rod-shaped structure, while the TMV after mixing with the polysaccharide presents a broken and fractured rod-shaped structure, and the damage of the modified polysaccharide to TMV is more serious than that of the polysaccharide before modification, indicating that the modified tobacco polysaccharide can have a certain destructive effect on the morphology of TMV, confirming the inactivation effect of the modified tobacco polysaccharide on the virus in Example 3.

[0072] It should be pointed out that for ordinary technicians in this technical field, any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of this application should be included in the scope of protection of this application.

Claims

1. Application of modified tobacco polysaccharide in resisting tobacco mosaic virus, characterized in that: The modified tobacco leaf polysaccharide includes one or more of acetylated tobacco leaf polysaccharide, carboxymethylated tobacco leaf polysaccharide or phosphorylated tobacco leaf polysaccharide.

2. The use according to claim 1, characterized in that The modified tobacco leaf polysaccharide prevents and controls tobacco mosaic virus by improving the protective effect on tobacco plants and the passivation effect on tobacco mosaic virus.

3. The use according to claim 1, characterized in that The preparation method of the acetylated tobacco polysaccharide comprises the following steps: dissolving the tobacco polysaccharide in deionized water, adjusting the pH to 9.0-10.0, slowly adding acetic anhydride dropwise thereto while stirring, and simultaneously adjusting the pH of the entire reaction system to maintain at 8.0-10.0 during the addition process; after the addition is completed, placing the solution in a water bath at 55-65° C. under shaking conditions for reaction, adjusting the pH to 7.0, dialyzing, and drying to obtain the acetylated tobacco polysaccharide.

4. The use according to claim 3, characterized in that The volume ratio of the tobacco polysaccharide to the acetic anhydride is 1 g:(18-22) mL.

5. The use according to claim 1, characterized in that The preparation method of the carboxymethylated tobacco polysaccharide comprises: placing the tobacco polysaccharide in isopropyl alcohol, stirring and mixing at room temperature, slowly adding a 20% mass concentration NaOH solution, continuing stirring, slowly adding a 4 mol / L chloroacetic acid solution, stirring and reacting at 55-65° C. for 4 hours, cooling to room temperature, adjusting the pH to 7.0, dialyzing, and drying to obtain the carboxymethylated tobacco polysaccharide.

6. The use according to claim 5, characterized in that The volume ratio of the tobacco polysaccharide to the NaOH solution is 1 g:(140-160) mL, and the volume ratio of the NaOH solution to the chloroacetic acid solution is (7-8):

1.

7. The use according to claim 1, characterized in that The preparation method of the phosphorylated tobacco polysaccharide comprises: dissolving sodium tripolyphosphate and sodium trimetaphosphate in water to prepare a phosphorylation reagent; sequentially adding sodium sulfate and the tobacco polysaccharide to the phosphorylation reagent; adjusting the pH to 7.0 after mixing, then stirring and reacting at 75-85°C, adding 95% ethanol solution to the reaction product, standing at 4°C, centrifuging, collecting and dissolving the precipitate, dialyzing, and drying to obtain the phosphorylated tobacco polysaccharide.

8. The use according to claim 7, characterized in that The mass ratio of the sodium tripolyphosphate to the sodium trimetaphosphate is (5-7):1, the volume ratio of the tobacco polysaccharide to the phosphorylation reagent is 1g:(980-1200)mL, and the volume ratio of the sodium sulfate to the phosphorylation reagent is 1g:(150-250)mL.

9. A preparation for preventing and treating tobacco mosaic virus, characterized in that: The invention comprises the modified tobacco polysaccharide in the application according to any one of claims 1 to 8.

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