Botanical pesticide for preventing and treating tobacco pepper vein mottle virus, application of botanical pesticide and prevention and treatment method

Abstract

The invention discloses a botanical pesticide for preventing and treating tobacco pepper vein mottle virus, application of the botanical pesticide and a prevention and treatment method, and belongs to the technical field of pesticides. The botanical pesticide disclosed by the invention is a melaleuca cordifolia extract. The extract has remarkable inhibitory activity on the tobacco pepper vein mottle virus, the effect of the extract is superior to that of Eight Bao, Sedum aizoon, Artemisia argyi and other extracts, and the effect of the extract is equivalent to that of current chemical pesticides such as Xinjunan acetate, so that the extract not only shows important application prospects and values in the aspect of preventing and treating the tobacco pepper vein mottle virus, but also has a good application prospect in the aspect of preventing and treating the tobacco pepper vein mottle virus. In addition, the technical field of pesticides for preventing and treating the tobacco pepper vein mottle virus is expanded, and a new direction is provided for preventing and treating the virus.

Description

Technical Field

[0001] The invention belongs to the technical field of pesticides, and particularly relates to a botanical pesticide for preventing and treating tobacco and pepper vein mottle virus, and an application and prevention method thereof. Background Art

[0002] Tobacco pepper vein mottle virus (ChiVMV) is a species of the genus Potyvirus. Its natural hosts include peppers, tomatoes, tobacco, okra, nightshade, datura, buddleja, and Amaranthus caudatus. It is transmitted by aphids feeding and sap friction, causing viral diseases in peppers, tobacco, and other Solanaceae crops. When infected with ChiVMV, circular, bright spots appear on tobacco leaves that turn chlorotic and yellow. Subsequently, the entire leaf develops a chlorotic, yellowing, mottled pattern, blisters, and wrinkling. In severe cases, the circular bright spots become continuous, turning brown and dying, and the veins turn brown and necrotic, leading to a decline in tobacco yield and quality, or even crop failure. ChiVMV not only spreads between peppers and tobacco, but also has a large and widespread population of intermediate hosts and aphids that transmit the virus, making it a rapid disease and difficult to control. Currently, there are no antiviral agents specifically targeting ChiVMV. Therefore, developing a ChiVMV-specific agent is crucial for agricultural disease control and crop yield improvement. Summary of the Invention

[0003] In response to the problems of rapid spread, severe damage and difficulty in prevention and control of tobacco and pepper vein mottle virus (ChiVMV) in crop production, the present invention aims to provide a botanical pesticide for the prevention and control of tobacco and pepper vein mottle virus, expand ChiVMV pesticide prevention technology, and promote crop growth and production.

[0004] The technical solutions of the present invention are as follows: The invention provides a botanical pesticide for preventing and treating tobacco and pepper vein mottle virus. The active ingredient of the botanical pesticide is an extract of Helianthus annuus.

[0005] The above-mentioned Chinese gentiana flower extract is prepared by the following method: The branches and leaves of the heartleaf heliotrope are taken, crushed, and an organic solvent is added, firstly homogenized for extraction, and then ultrasonically extracted; the extract is filtered, the solvent is removed, and the obtained extract is the heartleaf heliotrope extract.

[0006] In the above preparation method, the organic solvent is ethanol.

[0007] In the above preparation method, the mass volume ratio of the heart-leaf sunflower branches and leaves to the organic solvent is selected from (1~3):(1~3), g:mL; preferably 1:1.

[0008] In the above preparation method, the time for the homogenate extraction is selected from 10 to 60 s, preferably 30 s.

[0009] In the above preparation method, the ultrasonic extraction conditions are selected from: ultrasonic extraction at 40-70 kHz for 10-60 min; preferably: extraction at 50 kHz for 30 min.

[0010] The dosage form of the botanical pesticide is a liquid preparation, which is prepared by dissolving the above-mentioned Zinnia cordifolia extract in a phosphate buffer solution.

[0011] In the above-mentioned botanical pesticide, the concentration of the liquid preparation is selected from 0.5 to 10 mg / mL, preferably 1 mg / mL.

[0012] The present invention provides application of the botanical pesticide in preventing and controlling tobacco and pepper vein mottle virus.

[0013] The invention provides a method for preventing and controlling tobacco and pepper vein mottle virus disease. The method comprises spraying the above-mentioned botanical pesticide liquid preparation on crop leaves to achieve the purpose of preventing and controlling tobacco and pepper vein mottle virus disease.

[0014] The beneficial effects of the present invention are: The present invention has developed a botanical pesticide, an extract of the Chinese hibiscus sabdariffa (Schisandra chinensis) plant. This extract exhibits significant inhibitory activity against tobacco and pepper vein mottle virus (TPV), surpassing extracts of babao, feicai, and mugwort, and comparable to current chemical pesticides such as cintamidine acetate. Consequently, the present invention not only demonstrates significant application prospects and value in preventing and treating TPV, but also expands the field of pesticide technology for the prevention and treatment of TPV, providing a new direction for the prevention and treatment of this type of virus. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 These are actual pictures of the heart-leaved helianthus; among them, A is an indoor plant of the heart-leaved helianthus, and B is a field plant of the heart-leaved helianthus.

[0016] Figure 2 The results are for the inactivation test of tobacco mosaic virus (TMV).

[0017] Figure 3 These are the results of the inactivation test on tobacco pepper vein mottle virus (ChiVMV); the upper row shows symptoms, the lower row shows the virus content in the test strips for rapid detection, A is the treatment solution inoculated with tobacco No. 208 (a mixture of crude extract of the heartleaf jasmine flower and an equal volume of ChiVMV-infected sap), and B is the control solution inoculated with tobacco No. 208 (a mixture of clean water and an equal volume of ChiVMV-infected sap).

[0018] Figure 4These are the results of the activity test against tobacco pepper vein mottle virus; A is the symptom picture of Nicotiana benthamiana sprayed with crude extract of heartleaf jasmine, B is the symptom picture of Nicotiana benthamiana sprayed with water, and C is the relative expression level of the virus detected by real-time fluorescence quantitative PCR.

[0019] Figure 5 The results are as follows: The results are as follows: The results are as follows: The results are as follows: The results are as follows: A is the crude extract of Heliconia odorata sprayed on Zhongchuan 208 tobacco, B is the clean water sprayed on Zhongchuan 208 tobacco, and C is the 500-fold diluted 20% cefotaxime acetate aqueous solution sprayed on Zhongchuan 208 tobacco. DETAILED DESCRIPTION

[0020] In the present invention, the heart-leaved sunflower used is a plant such as Figure 1 As shown. Heartleaf Sunflower is a perennial evergreen herb. Its stems are spreading, branched, and slightly fleshy. Its leaves are opposite, flat, and heart-ovate. Its flowers are reddish-purple and spoon-shaped. It is easily propagated by cuttings in greenhouses and is drought- and heat-resistant in the field. Both greenhouse and field cultivation provide easy access to large quantities of raw material.

[0021] The tobacco varieties and the raw materials of the following embodiments of the present invention are sourced from the Tobacco Research Institute of the Chinese Academy of Agricultural Sciences.

[0022] In the present invention, TMV diseased sap is obtained by grinding 1 g of diseased leaves, adding 60 mL of phosphate buffer to homogenize, and filtering through lens paper. ChiVMV diseased sap is obtained by grinding 1 g of diseased leaves, adding 20 mL of phosphate buffer to homogenize, and filtering through lens paper.

[0023] In the present invention, the preparation methods of the crude extract of Babao, the crude extract of Feicai and the crude extract of Artemisia argyi are the same as the preparation method of the crude extract of Zinnia cordifolia described below.

[0024] The other materials used in the present invention, unless otherwise stated, can be obtained through commercial channels. Unless otherwise specified, other terms used in the present invention generally have the meanings commonly understood by those of ordinary skill in the art. The present invention will be further described in detail below with reference to specific examples and data. The following examples are merely for illustration of the present invention and are not intended to limit the scope of the present invention in any way.

[0025] Example 1 The steps for preparing the extract of the Chinese hibiscus flower are as follows: 100 g of young branches and leaves of P. cordifolia were collected and placed in a juicer. 100 mL of anhydrous ethanol was added and homogenized for 30 s, followed by ultrasonic extraction at 50 kHz for 30 min. The extract was first filtered through lens paper to remove residual tissue. The filtrate was collected in a conical flask and then filtered through a 0.45 μm inorganic microporous membrane to remove particulate matter. The collected filtrate was transferred to a glass bottle and purged with nitrogen to remove ethanol and water. The extract at the bottom of the bottle was the P. cordifolia extract.

[0026] Dissolve the extract in phosphate buffer to obtain a crude extract of Z. cordifolia (1 mg / mL). Store at 4°C until use.

[0027] 1. Determination of the inactivation effect on tobacco mosaic virus (TMV) The half-leaf method was used to detect the inactivation effect of the crude extract of the heartleaf jasmine on tobacco mosaic virus.

[0028] The crude extract of Heliconia odorata (1 mg / mL) was mixed with an equal volume of TMV-infected sap to form the treatment solution, and the control solution was mixed with an equal volume of TMV-infected sap with water. After 15 minutes, the treatment solution was rubbed onto one side of leaves of 6-7-leaf-stage N genotype Nicotiana tabacum (TMV necrotic host) and the control solution was inoculated onto the other side. Each treatment involved 15 leaves, with three replicates.

[0029] At the same time, the following treatment groups were also set up: crude extract of Babao, crude extract of Feicai, crude extract of Artemisia argyi, 500 times diluted 5% amino oligosaccharide aqueous solution, 150 times diluted 5% carvacrol soluble solution, 150 times diluted 20% guanidine hydrochloride wettable powder, and 500 times diluted 20% cypermethrin acetate aqueous solution.

[0030] The test results are as follows Figure 2 As shown: Figure 2 The results of the inactivation test on tobacco mosaic virus (TMV) are shown. The upper row of leaves from left to right are crude extracts of heartleaf sunflower, eight treasures, feicai, and artemisia crude extracts, and the lower row of leaves from left to right are 500 times diluted 5% amino oligosaccharide aqueous solution, 150 times diluted 5% carvacrol soluble solution, 150 times diluted 20% morpholino hydrochloride wettable powder, and 500 times diluted 20% cintamidine acetate aqueous solution; the left side is rubbed with the treatment solution (a mixture of crude extract or drug solution and an equal volume of TMV disease sap), and the right side is rubbed with the control solution (a mixture of clean water and an equal volume of TMV disease sap). Symptoms were observed 2 days after inoculation.

[0031] like Figure 2As shown in the results, 2 days after inoculation, the TMV necrotic spot density on the side of the japanese schizonepeta treated with the solution was significantly lower than that on the side of the control solution. Furthermore, the passivation effect of the japanese schizonepeta crude extract on TMV was significantly greater than that of the crude extracts of Babao, Feicai, and Artemisia argyi. There was no significant difference in the passivation effect of the crude extracts of 5% amino oligosaccharides (500-fold solution), 5% carvacrol (150-fold solution), 20% morphoguanidine hydrochloride wettable powder (150-fold solution), and 20% cintamidine acetate (500-fold solution).

[0032] Therefore, the crude fluid of the heartleaf jasmine was selected for the next step of tobacco pepper vein mottle virus (ChiVMV) inactivation test.

[0033] 2. Determination of the inactivation effect on tobacco pepper vein mottle virus (ChiVMV) A crude extract of the heartleaf jasmine flower (1 mg / mL) was mixed with an equal volume of ChiVMV-infected sap as the treatment solution, while a control solution was mixed with an equal volume of ChiVMV-infected sap and water. After 15 minutes, the treatment and control solutions were rubbed onto 6- to 7-leaf-stage N genotype tobacco plants (Zhongchuan 208) in the treatment and control groups, respectively. Each treatment consisted of 15 plants, with the largest expanded leaf in the middle of each plant inoculated three times. Before inoculation, a small amount of quartz sand was evenly sprinkled on the leaf surface. During inoculation, a cotton swab dipped in the diseased sap was gently rubbed against the leaf surface to create a microwound. 15 minutes after inoculation, the leaves were sprayed with clean water to rinse away excess quartz sand. Fourteen days after inoculation, each plant was observed for disease symptoms. Three randomly selected plants per treatment had their inoculated leaves ground into sap, and the ChiVMV virus content was measured using a rapid ChiVMV test strip.

[0034] The test results are as follows Figure 3 As shown: Figure 3 The results of the inactivation test on tobacco pepper vein mottle virus (ChiVMV) are shown; the upper row shows symptoms, the lower row shows the virus content in the test strips for rapid detection, A is the treatment solution inoculated with Zhongchuan 208 (a mixture of the crude extract of the heartleaf sunflower and an equal volume of ChiVMV diseased sap), and B is the control solution inoculated with Zhongchuan 208 (a mixture of clean water and an equal volume of ChiVMV diseased sap).

[0035] like Figure 3 As shown in the figure, 14 days after inoculation, the symptoms of ChiVMV in the group treated with R. cinnamomea were significantly milder, and the number of yellow spots was significantly lower than that in the control group. Moreover, the rapid test strips for detecting the virus content also showed that the detection line of the treatment group was weakly positive, and the brightness of the strip was significantly weaker than that of the control group, indicating that the crude extract of R. cinnamomea had a significant inactivating effect on tobacco and pepper vein mottle virus (ChiVMV).

[0036] 3. Determination of activity against tobacco pepper vein mottle virus Wild tobacco plants, Nicotiana benthamiana, at the 6- to 7-leaf stage were inoculated with ChiVMV diseased sap by friction. Before inoculation, a small amount of quartz sand was evenly sprinkled on the leaf surface. During inoculation, a cotton swab dipped in the diseased sap was gently rubbed against the leaf surface to create microwounds. Within 5-10 minutes after inoculation, the leaves were immediately sprayed with a crude extract of Heliconia citrifolia and water. The treatment group was sprayed with the crude extract of Heliconia citrifolia (1 mg / mL), while the control group was sprayed with water, 15 mL per plant. Each treatment consisted of 15 plants, with three replicates. Disease symptoms were observed for each plant 5-7 days after inoculation. Three randomly selected plants per treatment had their inoculated leaves taken from each plant for total RNA extraction, reverse transcription, and quantitative real-time PCR (qRT-PCR) analysis to determine relative viral expression.

[0037] The test results are as follows Figure 4 As shown: Figure 4 The results of the activity test against tobacco pepper vein mottle virus are shown; the upper row shows symptoms, and the lower row shows the relative expression of the virus detected by real-time fluorescence quantitative PCR. A is Nicotiana benthamiana sprayed with crude extract of Heliconia sinensis; B is Nicotiana benthamiana sprayed with water.

[0038] like Figure 4 As shown in the figure, the symptoms of Nicotiana benthamiana 5 days after inoculation showed that the curling and wrinkling symptoms on the top of the plants treated with the crude extract of N. benthamiana were significantly lower than those of the control plants sprayed with water; and the relative expression of viruses detected by qRT-PCR also showed that the virus content in the treated plants was significantly lower than that in the control plants, indicating that the crude extract of N. benthamiana has anti-tobacco pepper vein mottle virus (ChiVMV) activity.

[0039] Application Examples N genotype tobacco Zhongchuan 208 was raised and planted in greenhouse nursery beds according to conventional methods, with 20 plants per bed. A crude extract of 1 mg / mL of fennel seedling was sprayed on the leaves at the 6- to 7-leaf stage. Three sprays were applied 5 days apart. Water was sprayed as a blank control, while a 500-fold dilution of 20% fenpyroxan acetate solution was sprayed as a control. Four replicate beds were randomly arranged for each treatment. Fifteen to 30 minutes after the last spray, ChiVMV sap was inoculated by friction. Before inoculation, a small amount of quartz sand was evenly sprinkled on the leaf surfaces. During inoculation, a cotton swab dipped in the sap was gently rubbed against the leaf surface to create microwounds. Fifteen minutes after inoculation, the crude extract, water, and fenpyroxan acetate solution were sprayed on the leaves, and excess quartz sand was rinsed off. Watering was then continued as normal. Fourteen days after inoculation, the tobacco plants were observed for disease progression.

[0040] The ChiVMV disease severity level was recorded per plant, with level 0 indicating that the entire plant was disease-free; level 1 indicated that the heart leaves showed clear chlorosis or a slight yellow circular spot appeared on a leaf in the middle and lower parts, with no obvious dwarfing of the diseased plant; level 3 indicated that 1 / 3 of the leaves showed chlorotic mosaic or yellow circular spots, but did not curl or shrink, and the diseased plant was dwarfed to more than 3 / 4 of the normal plant height; level 5 indicated that 1 / 3 to 1 / 2 of the leaves showed chlorotic mosaic or yellow circular spots, with slight curling or shrinkage, and the diseased plant was dwarfed to 2 / 3 to 3 / 4 of the normal plant height; level 7 indicated that 1 / 2 to 2 / 3 of the leaves showed chlorotic mosaic or yellow circular spots, with obvious curling or blistering, and the diseased plant was dwarfed to 1 / 2 to 2 / 3 of the normal plant height; level 9 indicated that all leaves showed chlorotic mosaic or continuous yellow circular spots, with severe deformation or browning and necrosis, and the diseased plant was dwarfed to less than 1 / 2 of the normal plant height.

[0041] The incidence rate, disease index, and preventive effect were calculated according to the following formula: ; ; .

[0042] The test results are as follows Figure 5 As shown in Table 1: Table 1 Preventive effect of extracts from the Chinese hibiscus flower on tobacco and pepper vein mottle virus (ChiVMV) Fourteen days after inoculation, tobacco plants sprayed with the crude extract of Zhongchuan 208 showed significantly milder disease symptoms than those treated with water. Table 1 shows that the disease index of plants treated with the crude extract of 1 mg / mL (Centrifugal umbelliferae) was 34.31, which was not significantly different from the disease index of 34.86 for the control agent, 20% cintamidine acetate 500-fold aqueous solution. Both treatments were significantly lower than the disease index of 70.28 for the water control. The Centrifugal umbelliferae extract achieved a 51.18% preventive effect against ChiVMV, suggesting its potential for development as a botanical antiviral pesticide.

[0043] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any other manner. Any person skilled in the art may utilize the above-disclosed technical content to modify or modify the present invention into equivalent embodiments. However, any simple modifications, equivalent variations, and modifications to the above embodiments that do not depart from the technical content of the present invention and are based on the technical essence of the present invention remain within the scope of protection of the present invention.

Claims

1. A botanical pesticide for preventing and treating tobacco and pepper vein mottle virus, characterized in that: The active ingredient of the botanical pesticide is an extract of Helianthus annuus.

2. The botanical pesticide according to claim 1, characterized in that The extract of the Chinese gentiana odorata is prepared by the following method: The branches and leaves of the heartleaf heliotrope are taken, crushed, and an organic solvent is added, firstly homogenized for extraction, and then ultrasonically extracted; the extract is filtered, the solvent is removed, and the obtained extract is the heartleaf heliotrope extract.

3. The botanical pesticide according to claim 2, characterized in that The organic solvent is ethanol.

4. The botanical pesticide according to claim 2, characterized in that The mass volume ratio of the heartleaf sunflower branches and leaves to the organic solvent is selected from (1-3): (1-3), g:mL.

5. The botanical pesticide according to claim 2, characterized in that The time for the homogenate extraction is selected from 10 to 60 s.

6. The botanical pesticide according to claim 2, characterized in that The ultrasonic extraction conditions are selected from: ultrasonic extraction at 40-70 kHz for 10-60 min.

7. The botanical pesticide according to claim 1, characterized in that The formulation of the botanical pesticide is a liquid preparation, which is prepared by dissolving the extract of the herb Zinnia cordifolia in a phosphate buffer solution.

8. The botanical pesticide according to claim 7, characterized in that The concentration of the liquid preparation is selected from 0.5 to 10 mg / mL.

9. Use of the botanical pesticide according to claim 1 in preventing and controlling tobacco and pepper vein mottle virus.

10. A method for preventing and treating tobacco and pepper vein mottle virus disease, characterized in that: The method comprises spraying the botanical pesticide according to claim 8 on the leaves of crops to achieve the purpose of preventing and controlling tobacco and pepper vein mottle virus disease.

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