Preparation method of plant disease-resistant agent based on grape leaf extract and application of plant disease-resistant agent to prevention and treatment of grape gray mold

Abstract

The invention provides a preparation method of a plant disease-resistant agent based on a grape leaf extract and application of the plant disease-resistant agent to prevention and treatment of grape gray mold, and particularly belongs to the technical field of botanical pesticides. The invention provides an application of a grape leaf extract in preparation of a bacteriostatic agent. Grape leaves comprise leaves of Beibinghong grapes. The effective components (disease-resistant flavonoid substances) of the extract contain catechin, isoquercitrin and phloretin, and botrytis cinerea, specifically botrytis cinerea, can be efficiently inhibited.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant-derived pesticides, and particularly relates to a preparation method of a plant disease-resistant agent based on grape leaf extract and its application in controlling grape gray mold disease. Background Art

[0002] Grape (Vitis vinifera L.) is a perennial woody vine fruit tree widely cultivated globally. However, the healthy and sustainable development of the grape industry faces many challenges, among which the threat of diseases is particularly prominent. Currently, there are more than forty known diseases on grapes, mainly including gray mold disease, downy mildew disease, etc. Grape gray mold caused by Botrytis cinerea is one of the main diseases in grape production. It has strong infectivity and a wide host range, posing a serious threat to the yield and quality of grapes. Gray mold not only affects the growth and development of grapes but may also be transmitted among different hosts through spores, resulting in serious losses to grapes.

[0003] In the face of these challenges, scientific researchers and agricultural technicians have taken various measures for prevention and control. These include selecting disease-resistant varieties, improving cultivation management measures, rational fertilization and irrigation, and timely application of chemical and biological pesticides. In traditional grape disease management, chemical pesticide spraying is the main prevention and control method. Since the late 1960s, benzimidazole fungicides (such as carbendazim) have been widely used due to their high efficiency and low toxicity, effectively controlling diseases such as gray mold. However, long-term and single use has led to obvious drug resistance in gray mold (Botrytis cinerea), greatly reducing the control effect of these agents. The dicarboximide fungicides introduced in the 1970s, such as procymidone, had significant initial control effects but also could not escape the problem of resistance. Over time, new fungicides such as phenylpyrroles, nicotinamides, pyrroles, and imidazoles have been successively developed and put into use, bringing new options for grape disease control. However, with the widespread and repeated use of fungicides, the problem of drug resistance has become a key factor restricting the healthy development of the grape industry.

[0004] Biological control technology has been increasingly emphasized due to its environmental friendliness, especially in the context of the increasingly prominent problems of pesticide residues and pest drug resistance. Grapes are important economic crops, and disease prevention and control in their production is a key link. Although traditional chemical control methods have direct effects, long-term and large-scale use of chemical pesticides will cause environmental pollution and damage to the ecological balance, and at the same time increase production costs. Therefore, exploring safe and environmentally friendly biological control methods is of great significance for the sustainable development of the grape industry. Summary of the Invention

[0005] The object of the present invention is to provide a preparation method of a plant disease-resistant medicament based on grape leaf extract and its application in preventing and controlling grape gray mold. The disease-resistant natural active product extracted from grape leaves in the present invention has an antibacterial and disease-resistant effect. Specifically, when sprayed on grape leaves, it can assist grapes in resisting Botrytis cinerea.

[0006] The present invention provides the application of grape leaf extract in the preparation of antibacterial medicaments, and the grape leaves include the leaves of Beibinghong grapes.

[0007] Preferably, the bacterium includes Botrytis cinerea.

[0008] The present invention also provides the application of grape leaf extract in the preparation of medicaments for improving plant disease resistance, and the grape leaves include the leaves of Beibinghong grapes.

[0009] Preferably, the plant includes grapes, strawberries or raspberries.

[0010] Preferably, the disease includes gray mold.

[0011] Preferably, the grape leaf extract includes the ethanol extract of grape leaves.

[0012] The present invention also provides a preparation method of an extract of Beibinghong grape leaves, including the following steps:

[0013] Use an ethanol aqueous solution to extract Beibinghong grape leaves, perform ultrasonic treatment, take the supernatant, and centrifuge to take the supernatant to obtain the extract of Beibinghong grape leaves.

[0014] Preferably, the volume percentage content of ethanol in the ethanol aqueous solution is 70-90%; the mass ratio of grape leaves to the volume of the ethanol aqueous solution is 1 g:(3-5) mL; the ultrasonic treatment time is 60-90 min.

[0015] The present invention also provides the extract of Beibinghong grape leaves prepared by the preparation method described in the above technical solution.

[0016] The present invention also provides the use method of the extract of Beibinghong grape leaves prepared by the preparation method described in the above technical solution or the extract of Beibinghong grape leaves described in the above technical solution, including the following steps:

[0017] Dilute the extract of Beibinghong grape leaves and spray it on plants.

[0018] The present invention provides the application of grape leaf extract in the preparation of antibacterial agents. As a plant-derived agent, the grape leaf extract has a low risk of drug resistance, is environmentally friendly, has a low residue risk, and can achieve the efficient utilization of active substances in grape leaves and green prevention and control. The active ingredients (disease-resistant flavonoid substances) of the extract of the present invention include catechin, isoquercitrin, and phloretin, and can effectively inhibit Botrytis cinerea, specifically including Botrytis cinerea on grapes. The test results show that treating diseased grape leaves with the grape leaf extract (grape disease-resistant natural active product) of the present invention can effectively inhibit the spread of Botrytis cinerea on grape leaves, reduce the use of disease-resistant and bactericidal drugs after use, effectively avoid the development of drug resistance and pesticide residue problems, and conform to the environmental protection concept of modern green agriculture.

[0019] In addition, the preparation method and process of the present invention are simple, with a high extraction rate, wide and easily available raw material sources, low cost, and are suitable for popularization and use. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0021] Figure 1 It is a schematic diagram of the efficient extraction method of the grape disease-resistant natural active product provided by the present invention;

[0022] Figure 2 It is an effect diagram of the grape disease-resistant natural active product provided by the present invention assisting different grape varieties of leaves to resist Botrytis cinerea;

[0023] Figure 3 It is a result diagram of the effects of spraying the grape disease-resistant natural active product or inoculating Botrytis cinerea on the physiological indexes and the expression of genes related to the flavonoid synthesis pathway of different grape varieties; among them, A: a result diagram of the change in the diseased area of different grape varieties after spraying the grape disease-resistant natural active product; B: a result diagram of the change in the electrolyte leakage rate of different grape varieties before and after spraying the grape disease-resistant natural active product or inoculating Botrytis cinerea; C: a result diagram of the change in the chlorophyll content of different grape varieties before and after spraying the grape disease-resistant natural active product or inoculating Botrytis cinerea; D: a result diagram of the change in the expression of genes related to the flavonoid synthesis pathway of different grape varieties after spraying the grape disease-resistant natural active product. DETAILED DESCRIPTION OF THE INVENTION

[0024] The present invention provides the application of grape leaf extract in the preparation of antibacterial agents, and the grape leaves include the leaves of Beibinghong grapes. In the present invention, the bacteria include Botrytis cinerea. In the present invention, the grape leaf extract includes the ethanol extract of grape leaves. The present invention uses the leaves of Beibinghong grapes as raw materials and can obtain antibacterial green agents through ethanol extraction. The active ingredients (disease-resistant flavonoid substances) of the extract of the present invention include catechin, isoquercitrin and phloretin, which can effectively inhibit Botrytis cinerea, specifically including Botrytis cinerea of grapes.

[0025] The present invention also provides the application of grape leaf extract in the preparation of agents for improving plant disease resistance, and the grape leaves include the leaves of Beibinghong grapes. In the present invention, the plants include grapes, strawberries or raspberries. In the present invention, the grape varieties include Beibinghong, Xin Yu, Thompson Seedless, Kyoho, that is, the grape leaf extract can enhance the disease resistance of Beibinghong, Xin Yu, Thompson Seedless and Kyoho. In the present invention, the diseases include gray mold. In the present invention, the grape leaf extract includes the ethanol extract of grape leaves. The test results show that treating the diseased grape leaves with the grape leaf extract of the present invention can effectively inhibit the spread of Botrytis cinerea on grape leaves. After use, it can reduce the use of disease-resistant and bactericidal drugs, effectively avoid the development of drug resistance and the problem of pesticide residues, and conform to the environmental protection concept of modern green agriculture. The raw materials for preparing the agent of the present invention are widely available, low in cost and convenient for popularization and application.

[0026] The present invention also provides a preparation method of an extract of Beibinghong grape leaves, comprising the following steps:

[0027] Use an ethanol aqueous solution to extract the Beibinghong grape leaves, perform ultrasonic treatment, take the supernatant, and centrifuge to take the supernatant to obtain the extract of Beibinghong grape leaves.

[0028] In the present invention, the leaves of Beibinghong grapes include the leaves from the fruit-drop stage to the mature stage of Beibinghong grapes, or the redundant grape leaves after harvesting the grape fruits. Before extraction, the leaves of Beibinghong grapes in the present invention also include a pretreatment, and the pretreatment includes grinding into powder. In the present invention, the powder is ground under the protection of liquid nitrogen. In the present invention, the volume percentage content of ethanol in the ethanol aqueous solution is 70-90%. In a specific embodiment, the volume percentage content of ethanol can be 70%, 75%, 80%, 85% or 90%. In the present invention, the mass ratio of the grape leaves to the volume of the ethanol aqueous solution is 1 g:(3-5) mL. In a specific embodiment, the mass ratio of the grape leaves to the volume of the ethanol aqueous solution can be 1 g:3 mL, 1 g:4 mL or 1 g:5 mL. In the present invention, the time of ultrasonic treatment is 60-90 min. In a specific embodiment, the time of ultrasonic treatment can be 60 min, 70 min, 80 min or 90 min. In a specific embodiment, the conditions for centrifugation are 10000-12000 rpm and 10 min.

[0029] The preparation method of the present invention can extract the natural disease-resistant active ingredients from the leaves of Beibinghong grapes and can be applied to the biological control reagents for plant disease resistance.

[0030] The present invention also provides an extract of Beibinghong grape leaves prepared by the preparation method described in the above technical solution. The extract of Beibinghong grape leaves in the present invention contains natural active products, and the natural active products contain catechin, isoquercitrin and phloretin. The present invention provides a biological control reagent for plant disease resistance (including grape resistance to Botrytis cinerea). The extract of Beibinghong grape leaves in the present invention has significant anti-Botrytis cinerea activity and can be used to control Botrytis cinerea on species such as strawberries and raspberries. As a plant-derived medicament, the extract of Beibinghong grape leaves in the present invention has a low risk of drug resistance, is environmentally friendly, and has a low residue risk, realizing the efficient utilization and green prevention and control of the active substances in grape leaves. In the present invention, the extract of Beibinghong grape leaves is stored for standby at -20°C for 6 months or at -80°C for more than 6 months, and the substance structure is stable and not easily inactivated.

[0031] The present invention also provides the extract of Vitis amurensis Rupr. cv. Beibinghong grape leaves prepared by the preparation method described in the above technical solution, or the use method of the extract of Vitis amurensis Rupr. cv. Beibinghong grape leaves described in the above technical solution, including the following steps: diluting the extract of Vitis amurensis Rupr. cv. Beibinghong grape leaves and spraying it onto plants. In the present invention, water is used for dilution. In a specific embodiment, the water includes distilled water. In a specific embodiment, the dilution multiple is 5 to 10 times, specifically 10 times. The diluted solution obtained after dilution can be stored refrigerated or frozen. In a specific embodiment, the extract of Vitis amurensis Rupr. cv. Beibinghong grape leaves of the present invention is stored at 4°C after dilution, and the diluted solution is used within 2 days. In a specific embodiment, the spraying includes spraying onto the front and back sides of the expanded leaves and young buds of the plants, so that the solution reaches the state of being about to drip but not dripping on the leaves. In a specific embodiment, the spraying frequency can be 2 to 3 times a week. The method of the present invention can not only prevent and control Botrytis cinerea, but also improve the disease resistance of plants.

[0032] In order to further illustrate the present invention, the preparation method of a plant disease-resistant medicament based on grape leaf extract provided by the present invention and its application in the prevention and control of grape Botrytis cinerea will be described in detail below with reference to embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0033] Example 1

[0034] The schematic diagram of the efficient extraction method of grape disease-resistant natural active products is as Figure 1 shown. Using the mycelial disc inoculation method, select well-grown and consistent "Beibinghong" soil-cultivated seedlings as the test hosts for the experiment. When inoculating the treatment group, after culturing the bacteria on the PDA medium at 28°C for 7 days, use a punch to punch out mycelial discs with a diameter of 5 mm from the edge of the colony. The control group is inoculated with a PDA medium disc with a diameter of 5 mm. Each treatment includes 3 plants, each plant is inoculated with 8 mycelial discs on 8 leaves, and each leaf is inoculated with 3 mycelial discs. After 72 hours of inoculation, take the leaves of the treatment group and the control group respectively, and use the following method to extract and screen their active substances: accurately weigh 1 g of grape leaves, grind them into powder under liquid nitrogen protection, use 70% ethanol, with a material-liquid ratio of 1 g:5 ml, extract for 24 hours, ultrasonicate for 60 minutes, take the supernatant, centrifuge at 12000 rpm·min -1 for 10 minutes, take the supernatant, filter it through a 0.45 μm organic microporous filter membrane, and then determine it using a high performance liquid chromatograph.

[0035] Example 2

[0036] The difference from Example 1 is that the grape variety is "Xinyu", and the rest are the same as in Example 1.

[0037] Example 3

[0038] The difference from Example 1 is that the grape variety is "Thompson Seedless", and the rest are the same as in Example 1.

[0039] Example 4

[0040] It is different from Example 1 in that the grape variety is "Kyoho", and the rest are the same as in Example 1.

[0041] The results of the active substances in the crude extracts obtained in Examples 1 to 4 are shown in Table 1.

[0042] The disease incidence and the changes in active substances before and after inoculation of four grape varieties were statistically analyzed, and the active substances with significant changes were screened out for disease-resistant varieties, namely isoquercitrin, phloretin, and catechin, with the unit of mg·g -1 , disease incidence = diseased leaf area / total leaf area × 100%, change rate = (active substance content after inoculation - active substance content before inoculation) / active substance content before inoculation × 100%.

[0043] Table 1 Changes in disease incidence and active substances of four grape varieties before and after inoculation

[0044]

[0045] As can be seen from Table 1, the change rates of isoquercitrin, phloretin, and catechin in the diseased varieties "Xinyu", "Thompson Seedless", and "Kyoho" and the disease-resistant variety "Beibinghong" were all relatively high after inoculation, and among them, the changes in the three substances in the leaves of "Beibinghong" were the most significant.

[0046] Example 5

[0047] It is different from Example 1 in that methanol is used, and the rest are the same as in Example 1.

[0048] Example 6

[0049] It is different from Example 1 in that 50% ethanol is used, and the rest are the same as in Example 1.

[0050] Example 7

[0051] It is different from Example 1 in that 90% ethanol is used, and the rest are the same as in Example 1.

[0052] The extraction results of the active substances obtained in Examples 1, 5, 6, and 7 are shown in Table 2.

[0053] Table 2 Extraction amounts of active substances extracted with methanol and ethanol

[0054] Solvent type Catechin extraction amount (mg·g-1) Isoquercitrin content (mg·g-1) Phloretin content (mg·g-1) Example 1 70% ethanol 1.60±0.19a 0.88±0.06a 0.68±0.07a Example 5 70% methanol 0.83±0.05c 0.31±0.06c 0.28±0.03b Example 6 50% ethanol 1.14±0.04b 0.67±0.03b 0.56±0.03a Example 7 90% ethanol 1.69±0.13a 0.91±0.17a 0.71±0.10a

[0055] Note: The data are mean ± standard error, and different letters indicate significant differences at the 0.05 level by Duncan's multiple range test using SPSS software.

[0056] As can be seen from Table 2, the effect of extracting active substances with 70% ethanol is the best. The extraction amount with 90% ethanol is the highest, but there is no significant difference from that with 70% ethanol, and it consumes more reagents.

[0057] Example 8

[0058] The difference from Example 1 is that the solid-liquid ratio is 1 g:10 ml, and the rest is the same as in Example 1.

[0059] Example 9

[0060] The difference from Example 5 is that the solid-liquid ratio is 1 g:15 ml, and the rest is the same as in Example 1.

[0061] The extraction amounts of the active substances obtained in Examples 1, 8, and 9 are shown in Table 3.

[0062] Table 3 Extraction amounts of active substances with three different solid-liquid ratios

[0063] Solid-liquid ratio Catechin extraction amount (mg·g-1) Isoquercitrin content (mg·g-1) Phloretin content (mg·g-1) Example 1 1g:5ml 1.63±0.10a 0.87±0.03a 0.69±0.06a Example 8 1g:10ml 0.54±0.05b 0.25±0.03b 0.18±0.04b Example 9 1g:15ml 0.21±0.09c 0.15±0.03c 0.11±0.01c

[0064] Note: The data are mean ± standard error. Different letters indicate significant differences at the 0.05 level by Duncan's multiple range test using SPSS software for the control effect.

[0065] As can be seen from Table 3, the effect of extracting active substances with a solid-liquid ratio of 1 g:5 ml is the best, which is significantly higher than those with solid-liquid ratios of 1 g:10 ml and 1 g:15 ml.

[0066] Example 10

[0067] The difference from Example 1 is that the extraction time is 12 h, and the rest is the same as in Example 1.

[0068] Example 11

[0069] The difference from Example 1 is that the extraction time is 36 h, and the rest is the same as in Example 1.

[0070] The extraction results of the active substances obtained in Examples 1, 10, and 11 are shown in Table 4.

[0071] Table 4 Extraction amounts of active substances with three different extraction times

[0072] Extraction time Catechin extraction amount (mg·g-1) Isoquercitrin content (mg·g-1) Phloretin content (mg·g-1) Example 1 24h 1.67±0.08a 0.84±0.06a 0.70±0.06a Example 10 12h 0.93±0.03b 0.41±0.05b 0.38±0.03b Example 11 36h 1.75±0.15a 0.91±0.06a 0.78±0.06a

[0073] Note: The data are mean ± standard error. Different letters indicate significant differences at the 0.05 level by Duncan's multiple range test using SPSS software for the control effect.

[0074] As can be seen from Table 4, the effect of extracting active substances is the best at an extraction time of 24 h. The extraction yield of active substances is the highest at an extraction time of 36 h, but it is not significantly higher than that at 24 h and is more time-consuming.

[0075] Example 12

[0076] It is different from Example 1 in that the ultrasonic time is 30 min, and the rest are the same as in Example 1.

[0077] Example 13

[0078] It is different from Example 1 in that the ultrasonic time is 90 min, and the rest are the same as in Example 1.

[0079] The extraction results of the active substances obtained in Examples 1, 12, and 13 are shown in Table 5.

[0080] Table 5 Extraction yields of active substances extracted at three different ultrasonic times

[0081] Ultrasonic time Catechin extraction amount (mg·g-1) Isoquercitrin content (mg·g-1) Phloretin content (mg·g-1) Example 1 60min 1.63±0.13a 0.81±0.11a 0.66±0.07a Example 12 30min 0.66±0.04b 0.51±0.06a 0.40±0.02b Example 13 90min 1.69±0.12a 0.87±0.13a 0.72±0.01a

[0082] Note: The data are mean ± standard error. Different letters indicate significant differences at the 0.05 level by Duncan's multiple range test using SPSS software for the control effect.

[0083] In summary, as can be seen from Tables 1 to 5, the crude extract obtained in Example 1 has a relatively high flavonoid extraction yield, saves reagents, and takes less time, and its effect is better than that of other examples. Finally, it is determined that the mass ratio of grape leaves to the volume of the ethanol aqueous solution is 1 g:(3 - 5) mL; the ultrasonic time is 60 - 90 min.

[0084] Example 14

[0085] Using the mycelial disc inoculation method, well - growing and consistent "Beibinghong" soil - cultivated seedlings were selected as the test hosts for the experiment. In the treatment group, the crude extract of "Beibinghong" leaves prepared in Example 1 was sprayed on the front and back of the expanded leaves and young buds of the grapevines for each soil - cultivated seedling. In the control group, an equal amount of solvent solution was sprayed to make the solution reach the state of being about to drip but not dripping on the leaves, twice a week. After 24 h, Botrytis cinerea was inoculated. After culturing the fungus on PDA medium at 28 °C for 7 d, a mycelial disc with a diameter of 5 mm was punched from the edge of the colony. Each treatment included 3 plants, each plant was inoculated with 8 mycelial discs on the leaves, and each leaf was inoculated with 3 mycelial discs. After 3 days, the number of disease spots was counted, and the inhibition rate was calculated according to the following formula. The whole experiment was repeated 3 times, and the measurement results are shown in Table 6, and the control effect is as Figure 2 shown.

[0086] Inhibition rate (%) = (control diseased area - treated diseased area) / control diseased area × 100%.

[0087] Example 15

[0088] It is different from Example 14 in that the crude extract is sprayed on the leaves of Xin Yu grapes, and the rest are the same as in Example 14.

[0089] Example 16

[0090] It is different from Example 14 in that the crude extract is sprayed on the leaves of Thompson Seedless grapes, and the rest are the same as in Example 14.

[0091] Example 17

[0092] It is different from Example 14 in that the crude extract is sprayed on the leaves of Kyoho grapes, and the rest are the same as in Example 14.

[0093] Comparative Example 1

[0094] It is different from Example 14 in that the crude extract of "Xin Yu" leaves prepared by the same method is used, and the rest are the same as in Example 14.

[0095] Comparative Example 2

[0096] It is different from Example 14 in that the crude extract of "Thompson Seedless" leaves prepared by the same method is used, and the rest are the same as in Example 14.

[0097] Comparative Example 3

[0098] It is different from Example 14 in that the crude extract of "Kyoho" leaves prepared by the same method is used, and the rest are the same as in Example 14.

[0099] Comparative Example 4

[0100] It is different from Example 15 in that the crude extract of "Xin Yu" leaves prepared by the same method is used, and the rest are the same as in Example 15.

[0101] Comparative Example 5

[0102] It is different from Example 15 in that the crude extract of "Thompson Seedless" leaves prepared by the same method is used, and the rest are the same as in Example 15.

[0103] Comparative Example 6

[0104] It is different from Example 15 in that the crude extract of "Kyoho" leaves prepared by the same method is used, and the rest are the same as in Example 15.

[0105] Comparative Example 7

[0106] It is different from Example 16 in that the crude extract of "Xin Yu" leaves prepared by the same method is used, and the rest are the same as in Example 16.

[0107] Comparative Example 8

[0108] It is different from Example 16 in that the crude extract of "Thompson Seedless" leaves prepared by the same method is used, and the rest are the same as in Example 16.

[0109] Comparative Example 9

[0110] It is different from Example 16 in that the crude extract of "Kyoho" leaves prepared by the same method is used, and the rest are the same as in Example 16.

[0111] Comparative Example 10

[0112] It is different from Example 17 in that the crude extract of "Xinyu" leaves prepared by the same method is used, and the rest are the same as in Example 17.

[0113] Comparative Example 11

[0114] It is different from Example 17 in that the crude extract of "Thompson Seedless" leaves prepared by the same method is used, and the rest are the same as in Example 17.

[0115] Comparative Example 12

[0116] It is different from Example 17 in that the crude extract of "Kyoho" leaves prepared by the same method is used, and the rest are the same as in Example 17.

[0117] The inhibition rates obtained in Examples 14 to 17 and Comparative Examples 1 to 12 are shown in Table 6.

[0118] Table 6 Effects of different test agent source varieties on different grape leaves

[0119]

[0120] Note: The data are mean ± standard error, and different letters indicate significant differences in control effects at the 0.05 level by Duncan's multiple range test using SPSS software.

[0121] In summary, from Figure 2 , Table 6 shows that the crude extract of "Beibinghong" leaves sprayed in Examples 14 to 17 has good inhibitory activity against Botrytis cinerea on different grape varieties, and its effect is better than that of the crude extracts of "Xinyu", "Thompson Seedless", and "Kyoho" prepared by the same method.

[0122] Comparative Example 13

[0123] It is different from Example 14 in that a commercially available catechin solution of the same concentration is used, and the rest are the same as in Example 14.

[0124] Comparative Example 14

[0125] It is different from Example 14 in that a commercially available isoquercitrin solution of the same concentration is used, and the rest are the same as in Example 14.

[0126] Comparative Example 15

[0127] It is different from Example 14 in that a commercially available phloretin solution with the same concentration is used, and the rest are the same as in Example 14.

[0128] Comparative Example 16

[0129] It is different from Example 15 in that a commercially available catechin solution with the same concentration is used, and the rest are the same as in Example 15.

[0130] Comparative Example 17

[0131] It is different from Example 15 in that a commercially available isoquercitrin solution with the same concentration is used, and the rest are the same as in Example 15.

[0132] Comparative Example 18

[0133] It is different from Example 15 in that a commercially available phloretin solution with the same concentration is used, and the rest are the same as in Example 15.

[0134] Comparative Example 19

[0135] It is different from Example 16 in that a commercially available catechin solution with the same concentration is used, and the rest are the same as in Example 16.

[0136] Comparative Example 20

[0137] It is different from Example 16 in that a commercially available isoquercitrin solution with the same concentration is used, and the rest are the same as in Example 16.

[0138] Comparative Example 21

[0139] It is different from Example 16 in that a commercially available isoquercitrin solution with the same concentration is used, and the rest are the same as in Example 16.

[0140] Comparative Example 22

[0141] It is different from Example 17 in that a commercially available catechin solution with the same concentration is used, and the rest are the same as in Example 17.

[0142] Comparative Example 23

[0143] It is different from Example 17 in that a commercially available isoquercitrin solution with the same concentration is used, and the rest are the same as in Example 17.

[0144] Comparative Example 24

[0145] It is different from Example 17 in that a commercially available isoquercitrin solution with the same concentration is used, and the rest are the same as in Example 17.

[0146] The inhibition rates obtained in Examples 14 to 17 and Comparative Examples 13 to 24 are shown in Table 7.

[0147] Table 7 Effects of Different Test Agents on Different Grape Leaves

[0148]

[0149] Note: The data are mean ± standard error. Different letters indicate significant differences at the 0.05 level by Duncan's multiple range test using SPSS software for the control effect.

[0150] In summary, from Figure 2 , Table 7 shows that the crude extracts of "Beibinghong" leaves sprayed in Examples 14 to 17 have good inhibitory activity against Botrytis cinerea, and the antibacterial effects on different susceptible grapes are better than those of commercially available catechin, isoquercetin or phloretin solutions at the same concentration.

[0151] Example 18

[0152] Using the mycelial disc inoculation method, select well-grown and consistent "Beibinghong" soil-cultivated seedlings as the test hosts for the experiment. In the treatment group, each soil-cultivated seedling was sprayed with the crude extract of Beibinghong diluted 10 times prepared in Example 1 on the front and back of the expanded leaves and young buds of the grapes. In the control group, an equal amount of solvent solution was sprayed to make the solution reach the state of being about to drip but not drip on the leaves, twice a week. After 24 hours, Botrytis cinerea was inoculated. After the strain was cultured on PDA medium at 28°C for 7 days, a mycelial disc with a diameter of 5 mm was punched from the edge of the colony. Each treatment included 3 plants, 8 leaves were inoculated with mycelial discs per plant, and 3 mycelial discs were inoculated per leaf. After 3 days, the number of disease spots was counted, and the inhibition rate was calculated according to the following formula. The whole experiment was repeated 3 times. After treatment, the electrolyte leakage rate, chlorophyll concentration, and changes in the expression level of flavonoid-related genes were measured and compared. The measurement results are shown in Table 8 Figure 3 as shown

[0153] Inhibition rate (%) = (control diseased area - treated diseased area) / control diseased area × 100%.

[0154] Determination of electrolyte leakage

[0155] The electrolyte leakage rate was measured using a conductivity meter to measure the blank conductivity C0 of double-distilled water; 5 mL of distilled water was added to a test tube, and then 10 leaf discs were punched and placed in the test tube and soaked at room temperature for 10 h, and mixed several times during the process, and the conductivity C1 was measured; then the test tube was placed in a water bath and boiled for 20 min, and after the temperature dropped to room temperature, it was mixed evenly, and the conductivity reading C2 was recorded.

[0156] Relative electrolyte leakage rate REL (%) of leaves = (C1 - C0) / (C2 - C0) × 100%.

[0157] Determination of chlorophyll content

[0158] Use a hole punch to cut the leaves into leaf discs. Weigh 0.1 g of leaf discs, add 10 mL of 80% acetone, and extract for 24 h at room temperature in the dark, shaking several times during this period. Measure the absorbance values at 663 nm and 646 nm. Chlorophyll content = 17.32A646 + 7.18A663.

[0159] Example 19

[0160] The difference from Example 18 is that the crude extract diluted 10 times was sprayed on the leaves of Xin Yu grapes, and the rest was the same as in Example 18.

[0161] Example 20

[0162] The difference from Example 18 is that the crude extract diluted 10 times was sprayed on the leaves of Thompson Seedless grapes, and the rest was the same as in Example 18.

[0163] Example 21

[0164] The difference from Example 18 is that the crude extract diluted 10 times was sprayed on the leaves of Kyoho grapes, and the rest was the same as in Example 18.

[0165] Comparative Example 25

[0166] The difference from Example 18 is that the mother liquor was diluted 5 times, and the rest was the same as in Example 18.

[0167] Comparative Example 26

[0168] The difference from Example 18 is that the mother liquor was diluted 15 times, and the rest was the same as in Example 18.

[0169] Comparative Example 27

[0170] The difference from Example 19 is that the mother liquor was diluted 5 times, and the rest was the same as in Example 19.

[0171] Comparative Example 28

[0172] The difference from Example 19 is that the mother liquor was diluted 15 times, and the rest was the same as in Example 19.

[0173] Comparative Example 29

[0174] The difference from Example 20 is that the mother liquor was diluted 5 times, and the rest was the same as in Example 20.

[0175] Comparative Example 30

[0176] The difference from Example 20 is that the mother liquor was diluted 15 times, and the rest was the same as in Example 20.

[0177] Comparative Example 31

[0178] It is different from Example 21 in that the mother liquor is diluted 5 times, and the rest are the same as in Example 21.

[0179] Comparative Example 32

[0180] It is different from Example 21 in that the mother liquor is diluted 15 times, and the rest are the same as in Example 21.

[0181] The inhibition rates and physiological data obtained in Examples 18 to 21 and Comparative Examples 25 to 32 are shown in Table 8.

[0182] Table 8 Effects of Different Grape Varieties and Dilution Ratios of Mother Liquor on Grape Leaves

[0183]

[0184] Note: The data are mean ± standard error. Different letters indicate significant differences in the control effect at the 0.05 level by Duncan's multiple range test using SPSS software.

[0185] From Figure 3 A in Figure 3 and D in Figure 3 it can be seen that after spraying the crude extract of "Beibinghong" leaves, the diseased areas of different grape varieties were significantly reduced, and the expression of genes related to the flavonoid synthesis pathway was significantly up-regulated. As shown in Table 8, after inoculation with Botrytis cinerea, the chlorophyll content of the leaves of the plants treated with the crude extract solution of "Beibinghong" leaves was higher, and the electrolyte leakage rate was lower ( Figure 3 B in

[0186] Example 22

[0187] Using the mycelial disc inoculation method, select well-grown and consistent "Beibinghong" soil-cultivated seedlings as the test hosts for the experiment. In the treatment group, each soil-cultivated seedling was sprayed with the crude extract of Beibinghong diluted 10 times prepared in Example 1 on the front and back of the expanded leaves and young buds of the grapes. In the control group, an equal amount of solvent solution was sprayed to make the solution reach the state of being about to drip but not drip on the leaves, twice a week. After 24 h, inoculate with Botrytis cinerea. After culturing the bacteria on PDA medium at 28 °C for 7 d, use a puncher to punch mycelial discs with a diameter of 5 mm from the edge of the colony. Each treatment included 3 plants, 8 leaves were inoculated with mycelial discs per plant, and 3 mycelial discs were inoculated per leaf. After 3 days, count the number of disease spots and calculate the inhibition rate according to the following formula. The whole experiment was repeated 3 times. After treatment, measure and compare the leaf status, and the measurement results are shown in Table 9.

[0188] Inhibition rate (%) = (control diseased area - treated diseased area) / control diseased area × 100%.

[0189] Example 23

[0190] It is different from Example 22 in that the diluted mother liquor is sprayed on the leaves of Xin Yu grapes, and the rest are the same as in Example 22.

[0191] Example 24

[0192] It is different from Example 22 in that the diluted mother liquor is sprayed on the leaves of Thompson Seedless grapes, and the rest are the same as in Example 22.

[0193] Example 25

[0194] It is different from Example 22 in that the diluted mother liquor is sprayed on the leaves of Kyoho grapes, and the rest are the same as in Example 22.

[0195] Comparative Example 33

[0196] It is different from Example 22 in that the crude extract is sprayed once a week, and the rest are the same as in Example 22.

[0197] Comparative Example 34

[0198] It is different from Example 22 in that the crude extract is sprayed three times a week, and the rest are the same as in Example 22.

[0199] Comparative Example 35

[0200] It is different from Example 22 in that the crude extract is sprayed four times a week, and the rest are the same as in Example 22.

[0201] Comparative Example 36

[0202] It is different from Example 23 in that the crude extract is sprayed once a week, and the rest are the same as in Example 23.

[0203] Comparative Example 37

[0204] It is different from Example 23 in that the crude extract is sprayed three times a week, and the rest are the same as in Example 23.

[0205] Comparative Example 38

[0206] It is different from Example 23 in that the crude extract is sprayed four times a week, and the rest are the same as in Example 23.

[0207] Comparative Example 39

[0208] It is different from Example 24 in that the crude extract is sprayed once a week, and the rest are the same as in Example 24.

[0209] Comparative Example 40

[0210] It is different from Example 24 in that the crude extract is sprayed 3 times a week, and the rest are the same as in Example 24.

[0211] Comparative Example 41

[0212] It is different from Example 24 in that the crude extract is sprayed 4 times a week, and the rest are the same as in Example 24.

[0213] Comparative Example 42

[0214] It is different from Example 25 in that the crude extract is sprayed 1 time a week, and the rest are the same as in Example 25.

[0215] Comparative Example 43

[0216] It is different from Example 25 in that the crude extract is sprayed 3 times a week, and the rest are the same as in Example 25.

[0217] Comparative Example 44

[0218] It is different from Example 25 in that the crude extract is sprayed 4 times a week, and the rest are the same as in Example 25.

[0219] The inhibition rates and physiological data obtained in Examples 22 - 25 and Comparative Examples 33 - 44 are shown in Table 9.

[0220] Table 9 Effects of different spraying frequencies on the state of grape leaves

[0221] Grape variety Spraying frequency Inhibition rate (%) Leaf condition Example 22 Beibinghong Twice a week 59.39±1.45a Leaves are flat, with a low degree of infection Control 33 Beibinghong Once a week 37.41±2.58b Leaves are flat, with a high degree of infection Control 34 Beibinghong Three times a week 58.41±1.46a Leaves are flat, with a low degree of infection Control 35 Beibinghong Four times a week 62.54±2.16a Leaves are slightly curled at the edges, with a low degree of infection Example 23 Xinyu Twice a week 72.19±1.79a Leaves are flat, with a low degree of infection Control 36 Xinyu Once a week 49.86±1.14b Leaves are flat, with a high degree of infection Control 37 Xinyu Three times a week 79.79±1.17a Leaves are flat, with a low degree of infection Control 38 Xinyu Four times a week 78.78±1.15a Leaves are slightly curled at the edges, with a low degree of infection Example 24 Thompson Seedless Twice a week 69.74±0.73a Leaves are flat, with a low degree of infection Control 39 Thompson Seedless Once a week 48.32±2.29b Leaves are flat, with a high degree of infection Control 40 Thompson Seedless Three times a week 74.78±1.84a Leaves are flat, with a low degree of infection Control 41 Thompson Seedless Four times a week 75.97±1.94a Leaves are slightly curled at the edges, with a low degree of infection Example 25 Kyoho Twice a week 62.88±2.47a Leaves are flat, with a low degree of infection Control 42 Kyoho Once a week 40.74±0.58b Leaves are flat, with a high degree of infection Control 43 Kyoho Three times a week 64.79±1.51a Leaves are flat, with a low degree of infection Control 44 Kyoho Four times a week 66.01±1.79a Leaves are slightly curled at the edges, with a low degree of infection

[0222] Note: The data are mean ± standard error. Different letters indicate significant differences in the control effect at the 0.05 level by Duncan's multiple range test using SPSS software.

[0223] In summary, as can be seen from Table 9, for different grape varieties, spraying 2 - 3 times a week can better inhibit Botrytis cinerea. Although spraying 4 times a week has a better antibacterial effect, it will cause leaf curling and has no obvious advantage. Therefore, spraying 2 - 3 times a week is the best spraying frequency.

[0224] Example 26

[0225] Using the mycelial cake inoculation method, raspberry soil-cultivated seedlings with good and consistent growth were selected as the host to be tested for the experiment. In the treatment group, the diluted 10-fold Beibinghong crude extract prepared in Example 1 was sprayed on the front and back of the expanded leaves and young buds of the grapes for each soil-cultivated seedling. In the control group, an equal amount of solvent solution was sprayed to make the solution reach the state of being about to drip but not dripping on the leaves, twice a week. After 24 hours, Botrytis cinerea was inoculated. After the bacterial strain was cultured on the PDA medium at 28°C for 7 days, a mycelial cake with a diameter of 5 mm was punched from the edge of the colony. Each treatment included 3 plants, each plant was inoculated with 8 mycelial cakes per leaf, and 3 mycelial cakes were inoculated per leaf. After 3 days, the number of disease spots was counted, and the inhibition rate was calculated according to the following formula. The whole experiment was repeated 3 times, and the measurement results are shown in Table 10.

[0226] Inhibition rate (%) = (control diseased area - treated diseased area) / control diseased area × 100%.

[0227] Comparative Example 45

[0228] The difference from Example 26 is that the crude extract of "Xinyu" leaves was used, and the rest was the same as in Example 26.

[0229] Comparative Example 46

[0230] The difference from Example 26 is that the crude extract of "Thompson Seedless" leaves was used, and the rest was the same as in Example 26.

[0231] Comparative Example 47

[0232] The difference from Example 26 is that the crude extract of "Kyoho" leaves was used, and the rest was the same as in Example 26.

[0233] Example 27

[0234] Using the mycelial cake inoculation method, strawberry soil-cultivated seedlings with good and consistent growth were selected as the host to be tested for the experiment. In the treatment group, the diluted 10-fold Beibinghong crude extract prepared in Example 1 was sprayed on the front and back of the expanded leaves and young buds of the grapes for each soil-cultivated seedling. In the control group, an equal amount of solvent solution was sprayed to make the solution reach the state of being about to drip but not dripping on the leaves, twice a week. After 24 hours, Botrytis cinerea was inoculated. After the bacterial strain was cultured on the PDA medium at 28°C for 7 days, a mycelial cake with a diameter of 5 mm was punched from the edge of the colony. Each treatment included 3 plants, each plant was inoculated with 8 mycelial cakes per leaf, and 3 mycelial cakes were inoculated per leaf. After 3 days, the number of disease spots was counted, and the inhibition rate was calculated according to the following formula. The whole experiment was repeated 3 times, and the measurement results are shown in Table 10.

[0235] Inhibition rate (%) = (control diseased area - treated diseased area) / control diseased area × 100%.

[0236] Comparative Example 48

[0237] It is different from Example 27 in that the crude extract of "Xinyu" leaves is used, and the rest are the same as in Example 27.

[0238] Comparative Example 49

[0239] It is different from Example 27 in that the crude extract of "Thompson Seedless" leaves is used, and the rest are the same as in Example 27.

[0240] Comparative Example 50

[0241] It is different from Example 27 in that the crude extract of "Kyoho" leaves is used, and the rest are the same as in Example 27.

[0242] The inhibition rates obtained in Examples 26 - 27 and Comparative Examples 45 - 50 are shown in Table 10.

[0243] Table 10 Effects of crude extracts of different grape leaves on different species

[0244] Test agents Diseased species Inhibition rate (%) Example 26 Beibinghong Raspberry 69.59±1.75a Control 45 Xinyu Raspberry 41.32±1.14b Control 46 Thompson Seedless Raspberry 31.32±2.41b Control 47 Kyoho Raspberry 30.01±1.02b Example 27 Beibinghong Strawberry 73.45±1.11 Control 48 Xinyu Strawberry 45.80±1.14b Control 49 Thompson Seedless Strawberry 31.51±1.33b Control 50 Kyoho Strawberry 34.67±2.80b

[0245] As can be seen from Table 10, the crude extract of "Beibinghong" leaves also has strong inhibitory ability against raspberry and strawberry leaves inoculated with Botrytis cinerea.

[0246] To sum up: The design of the present invention is reasonable, and the preparation method is simple, having the following advantages:

[0247] (1) The crude extract of "Beibinghong" leaves of the present invention has significant anti - Botrytis cinerea activity and can be used to control Botrytis cinerea on species such as strawberries and raspberries;

[0248] (2) As a plant - derived medicament, the crude extract of "Beibinghong" leaves of the present invention has a low risk of drug resistance, is environmentally friendly, has a low residue risk, and realizes the efficient utilization and green prevention and control of active substances in grape leaves;

[0249] (3) The preparation method and process of the present invention are simple, with a high extraction rate, wide and easily available raw material sources, low cost, and are suitable for popularization and use.

[0250] Although the above - mentioned embodiments have made a detailed description of the present invention, they are only a part of the embodiments of the present invention, not all of them. People can also obtain other embodiments without creative efforts based on these embodiments, and these embodiments all belong to the protection scope of the present invention.

Claims

1. Application of grape leaf extract in the preparation of antibacterial agents, wherein the grape leaves include leaves of Arctic Red Grape.

2. The use according to claim 1, characterized in that: The fungi include Botrytis cinerea.

3. Application of grape leaf extract in the preparation of a medicament for improving plant disease resistance, wherein the grape leaves include leaves of Arctic Red Grape.

4. The use according to claim 3, characterized in that: The plants include grapes, strawberries or raspberries.

5. The use according to claim 3, characterized in that: Such diseases include gray mold.

6. The use according to claim 1 or 3, characterized in that: The grape leaf extract comprises grape leaf ethanol extract.

7. A method for preparing an extract of red grape leaves, characterized in that: The following steps are involved: The leaves of Beibinghong grape were extracted with ethanol aqueous solution, ultrasonicated, and the supernatant was taken. The supernatant was taken by centrifugation to obtain the Beibinghong grape leaf extract.

8. The preparation method according to claim 7, characterized in that: The volume percentage of ethanol in the ethanol aqueous solution is 70-90%; the volume ratio of the mass of the grape leaves to the ethanol aqueous solution is 1g:(3-5)mL; and the ultrasonic time is 60-90min.

9. The Beibinghong grape leaf extract prepared by the preparation method according to claim 7 or 8.

10. A method for using the Beibinghong Grape leaf extract prepared by the preparation method of claim 7 or 8 or the Beibinghong Grape leaf extract of claim 9, characterized in that: The following steps are involved: Dilute the Arctic Red Grape leaf extract and spray it on the plants.

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