A natural tobacco extract and its application in preventing and controlling plant viruses

Abstract

The present invention discloses a natural tobacco extract and its application in preventing and controlling plant viruses, belonging to the fields of agricultural drugs and biotechnology. The present invention obtains a natural extract from tobacco leaves slightly infected with viruses. Experiments have found that spraying the natural tobacco extract on virus-infected plant leaves can not only induce the immune defense ability of the plant, but also effectively inhibit the spread and replication of the virus, enhance the disease resistance of the plant, thereby achieving the purpose of improving crop yield and quality. The natural tobacco extract can be used as an effective ingredient of a plant antiviral agent, providing a new direction for the research and development of plant antiviral agents. The method of using an exogenous spray of the natural tobacco extract to prevent and control plant viruses has the advantages of being green, environmentally friendly, non-toxic and harmless, and is an ideal plant virus prevention and control technology. It is simple and efficient, and can maintain high stability and practicality in field applications, meeting the current requirements for ecological safety and sustainable development.

Description

Technical Field

[0001] The invention relates to the fields of agricultural chemicals and biotechnology, and in particular to a natural tobacco extract and application thereof in preventing and controlling plant viruses. Background Art

[0002] In agricultural production, plant viral diseases are an important factor leading to crop yield reduction, among which RNA viruses account for the main types of plant viruses, and their severity cannot be ignored. Since plant viruses can be transmitted through a variety of pathways such as vectors, soil, seeds, and pollen, once plant viral diseases occur, they are often difficult to completely eradicate. At present, there is a lack of highly effective broad-spectrum inhibitors for plant viruses on the market. Traditional prevention and control methods mainly rely on the selection and breeding of antiviral varieties, the use of chemical agents, and biological control strategies. However, these methods have certain limitations, such as the long cultivation cycle of resistant varieties, the easy mutation of viruses leading to loss of resistance, and the possible environmental pollution and toxicity risks caused by chemical agents.

[0003] As a natural antiviral resource, natural plant extracts are gaining more and more attention due to their high efficiency, safety and fewer side effects. The active ingredients in some plant extracts have significant antiviral effects. They can effectively prevent and control plant viral diseases by regulating the plant immune system, enhancing plant stress resistance, and inhibiting viral replication. These natural extracts can not only improve the disease resistance of crops, but also reduce the use of chemical pesticides and environmental pollution, and are in line with the current development direction of sustainable agriculture. Summary of the invention

[0004] The purpose of the present invention is to provide a natural tobacco extract and its application in preventing and controlling plant viruses to solve the problems existing in the above-mentioned prior art. The present invention extracts components with antiviral activity from natural plants and applies them to plant virus prevention and control, which can not only induce the immune defense ability of plants, but also effectively inhibit the spread and replication of viruses, enhance the disease resistance of plants, and thus achieve the purpose of improving crop yield and quality.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides a natural tobacco extract for plant antiviral agent, which is obtained by the following preparation method:

[0007] The tobacco leaves infected with Chinese wheat mosaic virus (CWMV) are dried and crushed, and then water is added for the first extraction at room temperature, and the first extract and precipitate are collected; water is added to the precipitate, and high-temperature concentration extraction is performed, and the second extract and the remaining precipitate are collected; methanol is added to the remaining precipitate for immersion extraction, and the third extract is collected; the three extracts are combined, and rotary evaporation concentration is performed to obtain the tobacco natural extract.

[0008] Optionally, during the first extraction, the mass volume ratio of the crushed tobacco leaves to water is 1 g:10 mL, and the extraction time is 2 h.

[0009] Optionally, during the high-temperature concentrated extraction, the mass ratio of the precipitate to water is 1:1.5-2.5, the extraction temperature is 90-100°C, and the extraction time is 2h.

[0010] Optionally, during the immersion extraction, the mass ratio of the remaining precipitate to methanol is 1:1, the extraction temperature is 0-4°C, and the extraction time is 12h. This low-temperature extraction helps to protect the active ingredients and improve the extraction effect. Gentle stirring should be maintained during the extraction process to ensure maximum release of the active ingredients.

[0011] Optionally, during the rotary evaporation concentration, the temperature is 40° C. and the volume is concentrated to 1 / 10 to 1 / 5 of the original volume.

[0012] Optionally, the active ingredients include spermidine, apigenin and astragaloside.

[0013] The present invention also provides the use of the natural tobacco extract in any one of the following A1)-A4):

[0014] A1) Preparation of plant antiviral agents;

[0015] A2) Inhibit virus infection of plants;

[0016] A3) Improve plant resistance to viruses;

[0017] A4) Prevent and control plant virus infections.

[0018] The natural tobacco extract can efficiently penetrate plant tissues and induce antiviral immune responses, thereby inhibiting the replication and amplification of multiple RNA viruses at the molecular level.

[0019] Optionally, the virus is an RNA virus.

[0020] Optionally, the RNA virus includes tobacco mosaic virus, tomato mosaic virus and turnip mosaic virus.

[0021] Optionally, the plant comprises tobacco.

[0022] The invention also provides a plant antiviral agent, comprising the natural tobacco extract.

[0023] The present invention also provides a method for preventing and controlling plant virus infection, comprising the step of spraying the natural tobacco extract or the plant antiviral agent on the plant.

[0024] The present invention discloses the following technical effects:

[0025] The present invention obtains a natural extract from tobacco leaves infected with the CWMV virus. Experiments have found that spraying the tobacco natural extract on virus-infected plant leaves can significantly improve the plant's ability to resist the virus. The natural extract can be used as an effective ingredient for plant antiviral agents, providing a new direction for the research and development of plant antiviral agents.

[0026] The present invention provides an antiviral method based on natural plant extracts. By extracting components with antiviral activity from natural plants and applying them to plant virus control, it is not only possible to induce the immune defense ability of plants, but also to effectively inhibit the spread and replication of viruses, enhance the disease resistance of plants, and thus achieve the purpose of improving crop yield and quality. The method has the advantages of being green, environmentally friendly, non-toxic and harmless, is an ideal plant virus control technology, is simple and efficient, and can maintain high stability and practicality in field applications, meeting the current requirements for ecological safety and sustainable development. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0028] Figure 1 This is the HPLC test chart of the active ingredient apigenin;

[0029] Figure 2 This is the HPLC test chart of the active ingredient spermidine;

[0030] Figure 3 This is the HPLC test chart of the active ingredient Astragalus glycoside;

[0031] Figure 4 The figure shows the virus infection in the control group (Mock) and crude extract treatment group observed under ultraviolet light;

[0032] Figure 5 Quantitative graphs of the fluorescent lesion areas on leaves infected by TuMV (A), ToMV (B), and TMV (C);

[0033] Figure 6 The accumulation of viral proteins in the control group (Mock) and crude extract treatment group detected by Western blotting. DETAILED DESCRIPTION

[0034] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0035] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0036] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0037] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0038] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0039] The present invention obtains an extract from tobacco leaves infected with Chinese wheat mosaic virus (CWMV). It is identified that the active ingredients in the extract include spermidine, apigenin and astragaloside. Experiments have confirmed that exogenous application of the extract can reduce the infection of various plant RNA viruses including TMV, ToMV and TuMV to plants, thereby improving the disease resistance of plants to RNA viruses.

[0040] The preparation method of the above-mentioned extract includes drying and crushing tobacco leaves infected with CWMV virus, adding water for extraction, and collecting the first extract; adding water to the remaining precipitate for concentration extraction at high temperature, and collecting the second extract; adding methanol solution to the remaining precipitate at a mass ratio of 1:1 for immersion extraction, and collecting the third extract; combining the three extracts, concentrating by rotary evaporation, and obtaining the above-mentioned extract.

[0041] Optionally, during the above-mentioned high-temperature concentrated extraction, the mass of the extraction solvent is 1.5-2.5 times the mass of the precipitate, the extraction temperature is 90-100° C., and the extraction time is 2 hours.

[0042] In a specific embodiment, the mass of the extraction solvent is twice the mass of the precipitate, and the extraction temperature is 100°C.

[0043] Optionally, during the above-mentioned immersion extraction, the volume fraction of the methanol solution is 85% to 100%.

[0044] In a specific embodiment, the volume fraction of the methanol solution is 100%.

[0045] In a specific embodiment, the mass of the extraction solvent is 1 times the mass of the precipitate, and the extraction temperature is 4°C.

[0046] Optionally, when the three extracts are combined and concentrated by rotary evaporation, the volume is concentrated to 1 / 5 to 1 / 10 of the original solution.

[0047] In a specific embodiment, the volume of the concentrate is 1 / 10 of the original solution, and the rotary evaporation temperature is 40°C.

[0048] In the embodiments of the present invention, the viruses used to infect tobacco include tobacco mosaic virus (TMV), tomato mosaic virus (ToMV) and turnip mosaic virus (TuMV), and the virus inoculation treatment is achieved by infecting tobacco with Agrobacterium carrying the infectious clones of the above viruses. The construction method of ToMV-GFP infectious clone refers to the non-patent literature "Liu Ni et al. Construction and expression of Agrobacterium infectious clone of ToMV tomato necrosis strain. Journal of Agricultural Biotechnology, 2014, 22(8): 1027-1034"; the construction method of TuMV-GFP infectious clone refers to the non-patent literature "Chen Shuxia et al. Construction of TuMv virus expression vector of green fluorescent protein gene. Northwest Botanical Journal, 2005, 8: 1395-1398"; the construction method of PVX-GFP infectious clone refers to the non-patent literature "Sean, et. Potato virus X as a vector for gene expression in plants. The plant journal. 1992"; the construction method of TMV-GFP infectious clone refers to the non-patent literature "An Mengnan et al. A TMV infectious cloning vector and its construction method. 2021".

[0049] Unless otherwise specified, the materials used in the embodiments of the present invention are all conventional materials in the art and can be obtained through conventional means. The related methods involved in the embodiments are all common methods in the art and can be implemented through conventional operations in the art.

[0050] Example 1 Preparation of extract

[0051] Raw material preparation: Select tobacco infected with CWMV virus symptoms and collect its leaves. Wash the leaves, remove impurities, dry them until the moisture content is less than 10%, and grind them in a mortar to obtain tobacco ultrafine powder.

[0052] Preparation of extracts:

[0053] (1) Add an appropriate amount of purified water to the tobacco ultrafine powder as the preliminary extraction solvent. The specific amount of addition is: add 10 mL of purified water per gram of tobacco ultrafine powder. This step is carried out at room temperature and the extraction time is 2 hours to ensure the maximum extraction of effective ingredients. The extract is centrifuged at a speed of 3000 rpm for 10 minutes. Collect the precipitate after centrifugation.

[0054] (2) Add an appropriate amount of purified water to the precipitate collected in step (1) for re-extraction. The specific amount added is: the mass of the extraction solvent is twice the mass of the precipitate. This step is performed at a high temperature, the temperature is 100°C, and the extraction time is 2 hours. The concentrated extract obtained after the concentrated extraction operation is cooled and centrifuged at a centrifugal temperature of 10°C, a centrifugal time of 1.5 hours, and a rotation speed of 3000 rpm, and the precipitate after centrifugation is collected.

[0055] (3) Add 100% methanol solution as the extraction solvent to the precipitate obtained in step (2) for immersion extraction. The mass of the extraction solvent is 1 times the mass of the precipitate, the extraction temperature is set to 4°C, and the extraction time is 12 hours. This low-temperature extraction helps to protect the active ingredients and improve the extraction effect. Maintain slight stirring during the extraction process to ensure maximum release of the active ingredients. After the immersion extraction is completed, centrifuge again, the centrifugal speed is maintained at 3000 rpm, and the centrifugation time is 10 minutes. Collect the clear supernatant and remove solid impurities and undissolved parts.

[0056] (4) The supernatants collected in steps (1), (2) and (3) are combined and concentrated by rotary evaporation to remove the methanol solvent, thereby obtaining a concentrated solution. The temperature of the rotary evaporation process is controlled at 40°C and the solution is concentrated to 1 / 10 of the volume of the original solution to ensure that the effective components of the extract are concentrated. A concentrated crude extract is obtained.

[0057] Example 2 Identification of extract components

[0058] In order to further confirm the main effective components of the crude extract, the present invention performs component analysis and identification on the crude extract.

[0059] High performance liquid chromatography (HPLC) was used to qualitatively and quantitatively analyze the crude extract to confirm the content of the main active ingredients. The contents of the main active ingredients in the crude tobacco extract were: spermidine 12.5 mg / g, apigenin 8.3 mg / g, and astragaloside 5.2 mg / g. These data show that the concentration of the extracted main active ingredients in the extract is high and meets the quality requirements of the standard. By comparing the chromatogram of the standard, it was determined that the main chemical components in the crude tobacco extract were apigenin, spermidine and astragaloside. The chromatogram identification diagram is shown in Figure 1-Figure 3 .

[0060] Example 3 Inhibitory effect of the extract on plant viruses

[0061] (1) The crude extract of tobacco leaves prepared in Example 1 was used as the active ingredient in the plant virus inhibitor. The crude extract was first dissolved and diluted with sterile water to a working concentration of 500 μM for later use.

[0062] (2) Inoculate Nicotiana benthamiana with viruses (including TMV, ToMV and TuMV) by Agrobacterium infection. The specific steps are as follows:

[0063] Agrobacterium carrying ToMV-GFP, TuMV-GFP and TMV-GFP infectious clones were inoculated into 2 mL YEP liquid medium, shaken and cultured at 28°C for 14 hours, and then centrifuged at 5000 rpm for 2 minutes to collect the bacteria. The bacteria were suspended in the infection solution (containing 10 mM MES, 10 mM MgCl2, 200 μM As) and the concentration of the bacterial solution was adjusted to OD 600 =0.0001.

[0064] (3) Use a sterile syringe to inject 0.2 mL of the Agrobacterium solution obtained in step (2) into the leaves (the injected leaves are the inoculated leaves). After the injection, the virus-injected leaves are divided into two groups, each group containing 3 biological replicates. One group is sprayed with sterile water as the control group, and the other group is sprayed with 500 μM of the crude extract solution diluted in step (1).

[0065] (4) After completing step (3), the leaves were placed in an artificial culture chamber at 22° C. for 3 days, and the virus accumulation in the inoculated leaves was observed under ultraviolet light on the 4th day to evaluate the inhibitory activity of the tobacco leaf extract against plant viruses;

[0066] Observation results are shown in Figure 4 ,The results showed that the amount of virus accumulation in the crude extract treatment group was significantly reduced compared with the control group (Mock).

[0067] (5) Use ImageJ software to quantify the GFP fluorescent spot area of ​​leaves with different treatments and calculate the ratio of leaf area infected by each virus. Figure 5 . Figure 5 A in the figure is a quantitative graph of the fluorescent lesion area on tobacco leaves infected with Agrobacterium carrying TuMV-GFP. Figure 5 B in the figure is a quantitative graph of the fluorescent lesion area on the leaf surface infected by Agrobacterium carrying ToMV-GFP. Figure 5 C in the figure is a quantitative graph of the fluorescent lesion area on the leaf surface infected by Agrobacterium carrying TMV-GFP. The results showed that compared with the control group (Mock), the GFP infection area ratios of TuMV, ToMV and TMV were significantly reduced after crude extract treatment. Statistical results showed that crude extract treatment effectively reduced the area of ​​virus infection.

[0068] (6) Take the systemic leaves of Nicotiana benthamiana (systemic leaves are the upper leaves of the inoculated leaves) of the control group and the crude extract treatment group, grind them into powder in liquid nitrogen; then take 0.2g of the powder in a 2mL sterile centrifuge tube, add 0.2mL of cell lysis buffer (buffer containing 200mM NaCl, 1.0mM EDTA, 1.0mM DTT, 20mM Tris-HCl, pH 7.4), vortex mix, and let stand on ice for 20min; then centrifuge at 4℃ and 14000g for 10min, collect the supernatant; mix 0.2mL of supernatant with 50μL of loading buffer (GenScript, Beijing) in a 1.5mL sterile centrifuge tube to obtain a mixture; boil the mixture in a boiling water bath for 5min, then let stand on ice for 5min, and centrifuge at 4℃ and 14000g for 10min to obtain the treated protein mixed sample. The accumulation level of viral protein in the sample was detected by western blot using GFP antibody. The test results are shown in Figure 6 The results showed that the accumulation of viral proteins in the crude extract treatment group was significantly reduced compared with the control group.

[0069] The above examples prove that the active ingredients extracted from tobacco leaves with mild virus infection symptoms can inhibit a variety of RNA viruses, and the above active ingredients can efficiently penetrate plant tissues and induce antiviral immune responses in plants, thereby inhibiting the replication and amplification of a variety of RNA viruses at the molecular level. Exogenous spraying of substances containing the above active ingredients can effectively reduce the infection and colonization of viruses in plants, thereby significantly improving the resistance of plants to viruses.

[0070] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. Application of natural tobacco extract in any of the following A1)-A4): A1) Preparation of plant antiviral agents; A2) Inhibit virus infection of plants; A3) Improve plant resistance to viruses; A4) Prevent and control plant virus infections; The viruses include tobacco mosaic virus, tomato mosaic virus and turnip mosaic virus; The natural tobacco extract is obtained by the following preparation method: The tobacco leaves infected with CWMV virus are dried and crushed, and then water is added for the first extraction at room temperature, and the first extract and precipitate are collected; the precipitate is added with water, and high-temperature concentrated extraction is performed, and the second extract and the remaining precipitate are collected; the remaining precipitate is added with methanol for immersion extraction, and the third extract is collected; the three extracts are combined, and rotary evaporation is performed to obtain the tobacco natural extract; The effective components of the natural tobacco extract include spermidine, apigenin and astragaloside.

2. A natural tobacco extract for preparing a plant antiviral agent, characterized in that: Obtained by the following preparation method: The tobacco leaves infected with CWMV virus are dried and crushed, and then water is added for the first extraction at room temperature, and the first extract and precipitate are collected; the precipitate is added with water, and high-temperature concentrated extraction is performed, and the second extract and the remaining precipitate are collected; the remaining precipitate is added with methanol for immersion extraction, and the third extract is collected; the three extracts are combined, and rotary evaporation is performed to obtain the tobacco natural extract; The effective components of the natural tobacco extract include spermidine, apigenin and astragaloside.

3. The natural tobacco extract according to claim 2, characterized in that During the first extraction, the mass volume ratio of the crushed tobacco leaves to water was 1 g:10 mL, and the extraction time was 2 h.

4. The natural tobacco extract according to claim 2, characterized in that During the high-temperature concentrated extraction, the mass ratio of the precipitate to water is 1:1.5-2.5, the extraction temperature is 90-100°C, and the extraction time is 2h.

5. The natural tobacco extract according to claim 2, characterized in that During the immersion extraction, the mass ratio of the remaining precipitate to methanol is 1:1, the extraction temperature is 0-4° C., and the extraction time is 12 h.

6. The natural tobacco extract according to claim 2, characterized in that During the rotary evaporation concentration, the temperature is 40° C. and the volume is concentrated to 1 / 10 to 1 / 5 of the original volume.

7. A plant antiviral agent, characterized in that: The method comprises the natural tobacco extract according to any one of claims 2 to 6.

8. A method for preventing and controlling plant virus infection, characterized in that: The method comprises the step of spraying the natural tobacco extract according to any one of claims 2 to 6 or the plant antiviral agent according to claim 7 onto the plant; The viruses include tobacco mosaic virus, tomato mosaic virus and turnip mosaic virus.

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