KR20070055195A - Antiviral material for plant virus containing the extract from the gall of rhus javanica and method thereof - Google Patents

Abstract

The present invention relates to a composition comprising a gall bladder extract showing a number of plant virus infection inhibitory activity, tomato mosaic virus (ToMV), cucumber mosaic virus (CMV), pepper mild mottle virus (PMMoV), zucchini macular mosaic virus (ZYMV) ), Watermelon mosaic virus (WMV), pumpkin mosaic virus (WMV2), potato virus Y (PVY), turnip mosaic virus (TuMV), cucumber nocturnal mosaic virus (CGMMV), melon necrotic virus (MNSV), potato leaf virus (MNSV) PLRV), narimosavirus virus (LMoV), narimu disease virus (LSV), ordontogrosoming spot virus (ORSV), symbiotic mosaic virus (CyMV), etc. It relates to a composition containing the gall bladder extract as an active ingredient.

The composition according to the present invention is non-toxic as a substance derived from natural products, and has an excellent effect on various plant viruses, and thus may be usefully used as a plant virus infection control agent.

Description

Antiviral material for plant virus containing the extract from the gall of Rhus javanica and method

1 is a schematic diagram illustrating a method for preparing hexane, ethyl acetate, butanol extract from methanol extract of dried gallnut.

Figure 2 (a) is a photograph showing the pepper Mild Mottle virus (PMMoV) particles before treatment of the gall bladder extract diluent,

Figure 2 (b) is a photograph showing the change of virus particles immediately after treatment of the gall bladder extract dilution to the virus,

Figure 2 (c) is a photograph showing the change in morphology of virus particles 10 minutes after the treatment as described above,

Figure 2 (d) is a photograph showing the change in the shape of the virus particle 20 minutes after the treatment as described above.

FIG. 3 is treated with half-leaf of tobacco N. glutinosa as a distilled extract diluent and control, respectively, and extracted and infected with only RNA of pepper mild virus (PMMoV) as an inoculum after 3 days of tobacco N. glutinosa. It is a picture of the symptoms expressed on the leaves.

The present invention relates to a plant virus control agent comprising a gall bladder extract exhibiting a control activity against plant viruses, and more particularly, contains a gall bladder extract exhibiting excellent antiviral activity against a plant virus having RNA as a gene. It relates to a plant virus control agent.

 Viruses are living organisms that infect and multiply host cells, and cannot exist in isolation from the host. There are various kinds of viruses, such as animal viruses, plant viruses, bacteriophages, and fungal viruses, depending on the host. Among them, plant viruses are generic to viruses that infect plant cells. About 1,000 plant viruses have been reported worldwide, and about 120 plant viruses have been reported in Korea. In Korea, since the late 80s, the importance of genetic resources and economic aspects of viruses has been reassessed, and various research teams are now actively researching plant viruses. Plant viruses cause disease in many plants and cause severe economic damages in agricultural production, including production and quality degradation of various crops and flowers and degradation of varieties. In particular, as the international trade volume of food and various plants has increased recently, foreign pathogenic plant viruses, which did not exist in Korea, have also been introduced, and the damage caused by them is gradually increasing. For this reason, the prevention, treatment and quarantine of viruses against cultivated plants is an important task in agriculture.

Plant virus is a pathogen that causes great damage to various economic crops. In Korea, tomato mosaic virus (ToMV), red pepper mittle virus (PMMoV), cucumber mosaic virus (CMV), potato Virus Y (PVY) and potato leaf virus (PLRV) are responsible for the largest decrease in yield, and Zucchini macular mosaic virus virus (ZYMV) and cucumber nocturnal mosaic virus (CGMMV) have been used for watermelons and crops such as cucumbers, pumpkins, watermelons and melons. ), Watermelon mosaic virus (WMV), pumpkin mosaic virus (WMV2), cucumber mosaic virus (CMV), melon gangrene spot virus (MNSV), and nari mosaic virus (LMoV), lily-free disease virus (LSV), Cucumber mosaic virus (CMV), odontogrosoming spot virus (ORSV), symbiosis mosaic virus (CyMV) cause significant damage in eggs.

Until now, the control of plant virus disease mainly used the seedling methods such as the control of plant cultivation time, disease-free plant cultivation, and the method of controlling insects, which are virus carriers, but these have limitations as an indirect control means. Meanwhile, sodium alginic acid, shiitake mushroom (Lentinus edodes) mycelium extract, and milk have been developed as a direct control against plant virus diseases, but the effectiveness of the control has not been significant. Also, Mirabilis jalapa , Kubo et al., 1990), proteins produced by Pytoraka americana, Boeravia diffus, and Pomerz formmentarius, and sugar protein components have been reported as antiviral agents with systemic effect, but have problems with productivity and toxicity. It was not commercialized as.

The gall bladder is a bug house made by the gall bladder parasitic on the leaves of Rhus javanica L., also known as anastomosis and baekchungchang. It is used to dry insect worms on the leaves of Rhus, a deciduous arborescent tree belonging to the family Sumac. Its main ingredients include large amounts of gallotannin, resins, fats, and starches. In pharmacological action, the 'gallotannin' component of the gall bladder is converging. In addition, there is an antimicrobial action, such as dysentery, Pseudomonas aeruginosa, etc., and shows a liver function protective action and antioxidant action. In clinical reports, a dose of 15 grams of this drug for gastrointestinal bleeding was shown to be effective in combination with Gaza, resulting in ulcerative colitis, radioactive proctitis, oil wells, and pulmonary tuberculosis, bleeding, dysentery, autosomnia, diabetes, and esophageal cancer. Therapeutic effects have been reported for hemorrhoids, burns, premature ejaculation, fertility drugs, pediatric diarrhea, and enteritis that are prevalent in the fall season (primary Korean herb book, Andeok-Gyunje).

Currently, the patent for the gall in Korea is powdery mildew control composition (registered patent 10-0480488-0000), powdery mildew control agent (registered patent 10-0505819-0000) of plants, nematicidal composition using herbal medicine (registered patent 10-0493364) -0000), natural dyeing method of the fiber (Patent Publication 10-2003-0078283) and the like have been reported. However, there is no report that the gall bladder extract exhibits an inhibitory effect on the plant virus.

The present invention has been proposed to solve the problems of the prior art as described above, an object of the present invention is to provide a plant virus control agent containing the gall bladder extract as an active ingredient.

Another object of the present invention is to provide a method for producing a practical plant virus infection control agent, which is nontoxic as a substance derived from a natural product containing a gall extract as an active ingredient and exhibits excellent effects against various plant viruses.

In order to achieve the above object, the present invention includes a plant virus control agent containing the gall bladder extract as an active ingredient.

In the present invention, the plant virus is an RNA virus, for example, tomato mosaic virus (ToMV), cucumber mosaic virus (CMV), pepper mild mottle virus (PMMoV), zucchini macular mosaic virus (ZYMV), watermelon mosaic virus (WMV) , Pumpkin mosaic virus (WMV2), potato virus Y (PVY), turnip mosaic virus (TuMV), melon blotch virus (MNSV), cucumber nocturnal mosaic virus (CGMMV), potato leaf virus (PLRV), narimosa virus (LMoV) ), Nali-free disease virus (LSV), ordontogrosoming spot virus (ORSV), symbiotic mosaic virus (CyMV), and the like.

The present invention provides a method for producing a plant virus control agent, characterized in that the gall extract is any one or more extracts selected from water extract, methanol, ethanol, ethyl acetate, butanol.

The present invention provides a method for producing a plant virus control agent, characterized in that the gall extract is contained in 10 to 90% by weight relative to the total weight of the composition.

In the present invention, while studying the antiviral activity against various plant viruses in the herbal medicines derived from natural products with little toxicity and side effects, the gall extract of the present invention confirmed the excellent antiviral effect against various plant viruses, the present invention Was completed.

Hereinafter, the content of the present invention in more detail as follows.

The present invention includes a plant virus control agent containing the gall bladder extract as an active ingredient.

The present invention provides a plant virus infection inhibitor containing a gall bladder extract having a wide range of antiviral activity against various plant viruses.

The gall bladder extract for the inhibition and treatment of plant viruses in the present invention can be obtained as follows.

The gallant of the present invention purchases the guaranteed one as a herbal medicine and grinds it to 30-50 mesh, and then the volume of water and carbon number such as methanol, ethanol butanol, etc. Is a polar solvent of 1-4 lower alcohols or a mixed solvent having a mixing ratio of about 1: 0.1-1: 10 for hot water extraction, cold extraction, reflux cooling extraction, ultrasonic extraction or ultra-sonic extraction for 3 hours to 3 days at room temperature. Extract can be obtained from the gall bladder by any one method selected from the critical fluid extraction methods. After extracting using the above extraction method, filtered with a filter of 0.1 ~ 10㎛, the filtrate can be reduced pressure, concentrated to obtain an extract. In addition, the non-polar solvent soluble extract of the present invention, after suspending the extract in distilled water, it is added to a non-polar solvent such as hexane, ethyl acetate, chloroform of about 1 to 5 times the volume of the non-polar solvent soluble layer is extracted 2 to 5 times, Can be obtained separately. In addition, a conventional fractionation process may be further performed.

The composition comprising the extract of the present invention may typically contain 10 to 90% by weight of any one or more selected from a suitable carrier, excipient, and diluent that can be used as a subsidiary material when preparing pesticides.

According to a preferred embodiment of the present invention, Nicotina glutinsa , which is a tobacco that forms a local lesion upon infection, is used to investigate the inhibitory activity of pepper mildworm virus infection of the extract. Chili MildMottle virus inoculum was dried with leaves of N. tabacum cv samsun infected with Chili MildMottle virus, added 200M weight 0.01M phosphate buffer, and then filtered through a sterilized inoculation and filtered to a final scale of 2,000 times. It is good to use after. Inhibitory activity of the Methanol extract of Mt. Glja was treated after mixing 1: 1 mixture of red pepper milt virus inoculum and dilution of extract, or after 24 hours of rubbing the dilution of the extract with a leaf of tobacco leaf ( N. glutinosa ). A pretreatment method for inoculating Pepper Mildttle Virus can be used. Sterilized distilled water was used as a control, and the anti-infection effect was calculated by comparing the number of local lesions between the extract treatment and the distilled water treatment two to four days after the inoculation.

In addition, we investigated the inhibitory effect of the gall bladder extract on Samsun tobacco seedlings, which are mosaic varieties that show mosaic symptom in the whole body against red pepper mitovirus. As a positive control, 10% and 1% aqueous solutions of skim milk and sodium alginate, which are currently used as antiviral agents in farms, were treated with 10% and 1% aqueous solutions, respectively, and 10mM phosphate buffer (pH 7.2) was used as a negative control. do. Three weeks after the virus inoculation, individuals with mosaic symptoms were examined and compared with the negative control to calculate the control effect.

The inhibitory activity of cucumber mosaic virus infection of five gall extracts was investigated in zucchini (breed zucchini) and zucchini (breed Seoul zucchini). In addition, the sustained effect of antiviral activity of Methanol methanol extract on zucchini (breed zucchini) and zucchini zucchini (breed Namgang zucchini) was investigated. The inhibitory activity of Zucchini macular mosaic virus infection of Methanol methanol extract was performed by the juice solution inoculation method. The virus inoculum uses two extracts of Cucumber Cucumber Virus Tobacco Leaf and Zucchini Squash Extract from Zucchini Macular Mosaic Virus.

In order to investigate the effect of the gall bladder extract on the morphological change and the RNA infectivity of the pepper mildworm virus (PMMoV) particles, the effect of gall bladder extract directly on viral RNA was investigated.

According to the results of the present invention, the gall bladder extract exhibits a high control effect of 90-100% against pepper mild mottle virus (PMMoV), cucumber mosaic virus, and zucchini macula mosaic virus, and the antiviral active ingredient of the extract is 8 Effective until 10 days, the control effect is maintained until about 4 weeks after the virus inoculation.

Therefore, according to the present invention it can be confirmed that the gall bladder extract exhibits an excellent antiviral effect against various plant viruses. In addition, the gall bladder extract acts directly on the red pepper milt virus virus particles to destroy the virus particles, it can be confirmed that the gall bladder extract directly affects the viral RNA to lose infectivity.

Hereinafter, the content of the present invention will be described in detail through examples and test examples. However, these are intended to explain the present invention in more detail, and the scope of the present invention is not limited to these examples.

Example 1 Preparation of Methanol Extract and Inhibitory Activity of Pepper Mild Mole Virus (PMMoV)

The extraction method was to pulverize the dried gallula into 40 mesh, 6 liters of methanol was added to 1 kg of powder, and then ultrasonically extracted at 40 ° C. for 3 hours to obtain a gallant extract which completely evaporated the liquid components.

Inhibitory activity of the extract was investigated using Nicotina glutinsa , a tobacco that forms local lesions upon infection. Chili MildMottle virus inoculum was dried with leaves of N. tabacum cv samsun infected with Chili MildMottle virus, added 200M weight 0.01M phosphate buffer, and then filtered through a sterilized inoculation and filtered to a final 2,000 times. It was used after. In order to induce sufficient infection prior to virus inoculation, carborundum was sprayed and the virus inoculation was smeared on cotton swabs and rubbed on tobacco leaves. Two to four days after inoculation, 200 to 300 local lesions were formed by the growth of pepper mild mottle virus.

Inhibitory activity of the Methanol extract of Mt. Glja was treated after mixing 1: 1 mixture of red pepper milt virus inoculum and dilution of extract, or after 24 hours of rubbing the dilution of the extract with a leaf of tobacco leaf ( N. glutinosa ). A pretreatment method of inoculating PMMoV was used. As a control, sterile distilled water was used, and after 2 to 4 days of inoculation, the effect of inhibition of infection was calculated by comparing the local lesions of the extract treatment and the distilled water treatment, and the results are shown in Table 1 below. As shown in Table 1, more than 97.9% of pepper mild mottle virus infection was inhibited when mixed and pretreated.

<Table 1> Inhibitory Activity of Pepper Mild Mole Virus Infection of Methanol Extract

Extract Treatment Method Extract Dilution Concentration (%) Inhibitory Activity (%) Mixing treatment One 99.4 0.5 98.8 Pretreatment One 98.4 0.5 97.9

Example 2 Inhibitory Effect of Methanol Extract of Mellitus Mica Extract on Systemic Infection

Inhibitory effects of the gall bladder extract on the pepper seedlings of samsun tobacco, the tobacco varieties with mosaic symptom on the whole, were investigated. In other words, 10 ml (20 seedlings per test zone) culture filtrate per week of 4-5 foliar seedlings were evenly spread over the leaves and left for 24 hours in a greenhouse condition. The leaf surface was infected by infecting the back surface or by absorbing the virus fluid with a cotton swab. As a positive control, skim milk and sodium alginate, which are currently used as antiviral agents in farms, were treated with 10% and 1% aqueous solutions, respectively. As a negative control, 10 mM phosphate buffer (pH 7.2) was used. Used. Three weeks after virus inoculation, the subjects with mosaic symptoms were examined and the control effect was calculated by comparing with the negative control. As a result, as shown in Table 2, it showed 100% infection prevention effect after 3 weeks of surface inoculation, showing superior control effect than skimmed milk powder and sodium alginate in the control.

Table 2. Inhibitory Effects of Methanol Extracts of Gallia japonica on Samsun Tobacco

Treatment solution Inhibition effect of onset after 3 weeks of inoculation (%) Ginseng Extract (1% aqueous solution) 100 Skim milk powder (10% aqueous solution) 35.0 Sodium alginate (1% aqueous solution) 30.0 Control 0

Example 3 Inhibitory Activity of Cucumber Methanol Extract on Cucumber Mosaic Virus (CMV) Infection

The inhibitory activity of cucumber mosaic virus infection of Methanol methanol extract was investigated in zucchini (variety zucchini) and zucchini zucchini (breed Seoul Jukini) grown with 6-7 sheets of main leaves. Inhibitory activity of gall methanol extract was treated by mixing 1: 1 mixture of cucumber mosaic virus inoculum and extract diluent, or spraying the diluted extract evenly on the surface of plant leaves and inoculating cucumber mosaic virus on leaf surface after 24 hours. A pretreatment method was used. On the other hand, in order to confirm the inhibitory effect of cucumber mosaic virus infection due to the penetration performance of the extract, the extract was sprayed on the surface of the leaf 40 ml per week, and after 24 hours in greenhouse conditions, the virus solution added with carborundum was swabed with a cotton swab. Injury was induced on the back of the wound. Three weeks after the inoculation, the number of seedlings with cucumber symptomatic virus symptoms was compared with the control group. Virus inoculum was used to extract the cucumber mosaic virus disease tobacco leaf, and extracted with buffer 5 times the weight.

Inhibition activity of cucumber mosaic virus against cucumber zucchini of Cucumber Methanol extract was superior to 85-100% infection prevention effect during mixing and pretreatment with Methanol methanol extract and virus solution. Although low at 45-55%, a slight penetrating effect of the extract was recognized in the zucchini leaves. In addition, as shown in Table 4, when zucchini squash was mixed and pretreated with Methanol methanol extract and virus solution, the infection prevention effect was excellent at 90 to 100%, and the backside treatment showed 45% to 60% infection inhibition rate. .

<Table 3> Inhibitory Effects of Methanol Extracts from Cucumbers on Cucumber Cucumber Virus (CMV) Infection

process Treatment concentration (%) Disease Survey  Infection Control Rate (%) after 2 weeks 3 weeks later after 2 weeks 3 weeks later Extract blend treatment One 0 0 20 100 100 0.5 3 3 20 85 85 Extract pretreatment One 0 0 20 100 100 0.5 3 3 20 85 85 Extract Back Treatment One 8 9 20 60 55 0.5 11 11 20 45 45 Control 20 20 20 0 0

Table 4. Effect of Cucumber Methanol Extracts on Cucumber Zucchini Against Prevention of Cucumber Mosaic Virus (CMV)

process Treatment concentration (%) Disease Survey  Infection inhibition rate (%) after 2 weeks 3 weeks later after 2 weeks 3 weeks later Extract blend treatment One 0 0 20 100 100 0.5 2 2 20 90 90 Extract pretreatment One 0 0 20 100 100 0.5 2 2 20 90 90 Extract Back Treatment One 7 8 20 65 60 0.5 11 11 20 45 45 Control 20 20 20 0 0

Example 4 Inhibitory Activity of Zucchini Macular Mosaic Virus (ZYMV) Infection of Methanol Extract

The inhibitory activity of Zucchini macular mosaic virus infection of Methanol methanol extract was performed by the juice solution inoculation method. First, the gall bladder extract was sprayed on the surface of Zucchini pumpkin (breed Seoul Jukini) and after 24 hours, the virus was inoculated on the leaf surface. The inoculum was used to extract dried zucchini zucchini leaves infected with Zucchini macular mosaic virus and extracted with 5 times buffer of dried zucchini. As a result, as shown in Table 5, even after 3 weeks of inoculation when treated with the culture filtrate, 100% infection prevention effect was shown.

<Table 5> Prevention of Zucchini Macular Mosaic Virus (ZYMV) Infection by Methanol Extract of Mellitus Extract in Zucchini Pumpkin

process Treatment concentration (%) Disease Survey Infection inhibition rate (%) after 3 weeks after 2 weeks 3 weeks later extract 0.7 0 0 20 100 Control 20 20 20 0

<Example 5> Sustained effect test of antiviral active ingredient of Methanol extract

Zucchini zucchini (Zucchini zucchini) and Zucchini zucchini (Zucchini Namgang zucchini) are packaged in a plastic house package. After diluting the gallant methanol extract with water, the solution is diluted with water to 0.7%. It was. In addition, virus (ZYMV) inoculation was carried out separately after spraying the drug, 2 days, 3 days, 5 days, 8 days later. Each test zone was carried out in three repetitions of 10 weeks. The effect test was determined by virus symptoms on leaves at 2, 3 and 4 weeks of inoculation for each treatment. As a result, as shown in Tables 6 and 7, all treatments of zucchini and zucchini were found to have high virus infection inhibitory effects. It was confirmed that the antiviral active ingredient of the extract lasted effectively for 8 days after side spraying.

<Table 6> Antiviral Activity Sustained Effect of Methanol Extract of Mellitus Squash on Zucchini

Number of days between treatment and inoculation Control effect (%) 2 weeks after inoculation  3 weeks later  4 weeks later Immediately 100 100 100 2 100 100 100 3 100 100 100 6 100 96.7 96.7 8 96.7 93.3 93.3 Control 0 0 0

<Table 7> Antiviral Activity Sustained Effect of Methanol Extract of Mellitus Rhizome on Zucchini Squash

Number of days between treatment and inoculation Control effect (%) 2 weeks after inoculation  3 weeks later  4 weeks later Immediately 100 100 100 2 100 100 100 3 100 100 100 6 100 96.7 96.7 8 0 96.7 93.3 Control 0 0 0

Example 6 Preparation of Solvent Extracts, such as Ethyl Acetate and Inhibitory Activity Against Pepper Mild Mottle Virus (PMMoV) Infection

200 g of the dry sample was ground and the extract obtained by extraction with methanol was concentrated under reduced pressure. The obtained concentrated solution was added to 800 ml of 80% methanol, and dissolved sufficiently. Then, 800 ml of hexane was added for extraction, followed by concentration under reduced pressure. (Sample 1: 5.1 g). The remaining 80% methanol layer was extracted with 800 ml of ethyl acetate, and the extract was concentrated under reduced pressure to use a sample (sample 2: 82.3 g). After extraction with ethyl acetate, the remaining layer was extracted with 800 ml of butanol, and the extract was concentrated under reduced pressure to obtain 4.8 g (sample 3). The remaining water layer was concentrated under reduced pressure to obtain 3.8 g (Sample 4) (Fig. 1, Table 8).

Four samples extracted according to the above method were each diluted 0.5% with sterile distilled water to prepare a sample. Inhibitory activity of Chilli Mild Mottle virus of each extract sample was investigated using Nicotina glutinsa , a tobacco that forms local lesions upon infection. Chili MildMottle virus inoculum was dried with leaves of N. tabacum cv samsun infected with Chili MildMottle virus, added 200M weight 0.01M phosphate buffer, and then filtered through a sterilized inoculation and filtered to a final scale of 2,000 times. It was used after. In order to induce sufficient infection prior to virus inoculation, carborundum was sprayed and the virus inoculation was smeared on cotton swabs and rubbed on tobacco leaves. Three days after the inoculation, 100 to 200 local lesions were formed by the growth of pepper mild mottle virus.

Inhibition activity assay was performed by pre-treatment of each dilution of the previously prepared extract by rubbing with a cotton swab on the leaves of tobacco leaves ( N. glutinosa ) first and inoculating pepper mild mottle virus after 24 hours. Sterilized distilled water was used as a control and the inhibitory effect was calculated by comparing the number of local lesions of the extract treatment and distilled water treatment two to four days after inoculation. As shown in Table 8, the ethyl acetate fraction fraction showed the highest inhibitory effect of pepper mildworm virus (98.6%).

<Table 8> Inhibitory Effects of Organic Solvent Fractions from Methanol Extracts of Pepper Mice on Pepper Mild Virus (PMMoV) Infection

Solvent extract Multiples (dog) Infection inhibition rate (%) No treatment process Hexane (Sample 1) 140 54 61.4 Ethyl Acetate (Sample 2) 147 2 98.6 Butanol (Sample 3) 119 25 79.0 Water layer (sample 4) 157 182 -15.9

<Example 7> Effects of the gall bladder extract on the morphology change and RNA infectivity of red pepper Mild Mottle virus (PMMoV) particles

Investigation of the effect of the gall bladder extract on the morphology of red pepper milt virus particles and the results are shown in FIG. 2, the effect of the gall bladder extract directly on the viral RNA was investigated and the results are shown in FIG. 3. .

As shown in FIG. 2, it was found that the Methanol methanol extract finely segmented pepper mildworm virus particles. Figure 2 (a) is a photograph showing the pepper mildworm virus particles before the treatment of the gall bladder methanol extract, Figure 2 (b) is a picture showing the particle change immediately after treatment with the gall bladder extract to the virus shows that the crack symptoms of the particles Could know. As shown in FIG. 2 (c), the particles were shredded by a photograph showing the particle change after 10 minutes after the treatment as described above. Figure 2 (d) is a photograph showing the particle change after 20 minutes after the treatment as described above, almost all of the virus particles were found to be finely broken and destroyed.

The destruction of PMMoV particles by the gall bladder extract was investigated through the following experiment. First, in order to obtain the purified red pepper mittle virus, 100 g of red pepper milt virus grown on tobacco (variety samsun) was repeatedly subjected to high-speed centrifugation (10,000 × g) and ultra-fast centrifugation (32,000 × g). The virus was purified and diluted in 0.1 mM pH phosphate buffer solution to a concentration of 10 mg / ml. Then, the Methanol methanol extract was diluted in distilled water (pH 7.2) to a concentration of 8,000 ppm, mixed 1: 1 with purified Mild Myrtle Virus purified solution (10 mg / ml), and 2% phosphotungstic acid. Reverse staining with sodium phosphotungstic acid (pH 6.8), virus particles were observed under a transmission electron microscope.

On the other hand, the loss of infectivity of the pepper mild mottle virus RNA by the gall bladder extract was investigated through the following experiment. First, the extract was rubbed with a cotton swab on the half leaf of tobacco leaf ( N. glutinosa ) and inoculated with pepper mild virus virus after 1 hour. Sterilized distilled water was used as a control, and after comparing the number of local lesions of the extract treatment and the distilled water treatment three days after inoculation, it was found that the infectivity of the pepper mild mottle virus RNA was completely lost due to the extract (Table 9, FIG. 9). 3).

The RNA purification method and the inoculation method of the pepper milde virus are as follows. Extract buffer (100mM Tris, pH7.4, 20mM EDTA, 100mM NaCl, 2mM DTT, 1% SDS) and proteinase K (100µg / ml) were added to the purified red pepper mildworm virus (1mg / ml). After reacting for 1 hour in the reaction tank, only red pepper mild virus RNA was recovered by two organic solvent treatments (phenol + chloroform + isoamyl alcohol) and ethanol precipitation. After diluting the purified RNA with diethyl pyrocarbamate (DEPC) -treated distilled water at a concentration of 100 ㎍ / ml, tobacco ( N. glutinosa ) Used as an inoculum of leaves. Inoculation method is sprayed with 200 mesh carborundum to make a weak wound on the leaves, and then 200 μl per half leaf of the developed leaf of tobacco ( N. glutinosa ) Capsicum mildevirus RNA (100 μg / ml) was plated with a sterile swab. As shown in Table 9, the gall bladder extract showed 99.1% infection inhibition in tobacco ( N. glutinosa ) infected with Pepper Milt Molecular Virus RNA.

<Table 9> Inhibitory Effects of Methanol Extracts of Mellitus Mellitus Extract on Pepper Mild Molecular Virus (PMMoV) RNA

Extract concentration (%) Local lesions forming (dogs) Infection Control Rate (%) Extract Treatment Half Leaf Distilled water One 0 327 100 0.5 3 326 99.1

From the above results, it was confirmed that the gall bladder extract acted directly on the red pepper milt virus virus particles to destroy the virus particles, and the gall bladder extract directly affected the viral RNA and thus lost infectivity.

As described above, the plant virus control agent, which is contained as an active ingredient of the gall bladder extract of the present invention, has an excellent antiviral effect against various plant viruses. This effect will be a useful material for industrializing the above-mentioned control agent is prepared as a foliar spray formulation of the crop, when used, it is excellent in the inhibitory effect on various plant viruses and can prevent plant viruses.

Claims (5)

Plant virus control agent containing the gall bladder extract as an active ingredient. The plant virus control agent according to claim 1, wherein the plant virus is an RNA virus. The method of claim 2, Tomato Mosaic Virus (ToMV), Cucumber Mosaic Virus (CMV), Chilli Mild Mottle Virus (PMMoV), Zucchini Macular Mosaic Virus (ZYMV), Watermelon Mosaic Virus (WMV), Pumpkin Mosaic Virus (WMV2), Potato Virus Y (PVY) , Turnip mosaic virus (TuMV), cucumber nocturnal mosaic virus (CGMMV), melon stromal spot virus (MNSV), potato leaf virus (PLRV), narimosa virus (LMoV), nari disease disease virus (LSV), ordontogrosome Ring spot virus (ORSV), Symbiotic mosaic virus (CyMV) RNA for a plant virus composition comprising the gene as a gene. The plant virus control agent according to claim 1, wherein the five gall extract is at least one selected from water extract, methanol, ethanol, ethyl acetate and butanol. The plant virus control agent according to claim 1, wherein the 5 g extract contains 10 to 90% by weight.

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