Plant protection film agent taking kaempferol as active ingredient as well as preparation method and application of plant protection film agent

Abstract

The invention relates to the technical field of plant protection, and particularly discloses a plant protection film agent with kaempferol as an active ingredient and a preparation method and application thereof, and the plant protection film agent is prepared from the following components: a kaempferol-hydroxypropyl-beta-cyclodextrin inclusion compound, a film forming substance, an emulsifier, an antifreezing agent, a preservative, a surfactant and purified water. Based on a plant protection film principle, the plant protection film agent taking kaempferol as an active component is successfully prepared. The film agent has multiple advantages that firstly, the inhibition effect of kaempferol on growth of rhizoctonia solani hyphae is remarkably enhanced, the activity of cellulase and pectinase released when rhizoctonia solani infects rice leaf sheaths can be more efficiently inhibited, and infection of rhizoctonia solani on rice is effectively prevented; 2, the film agent is stable in structure, good in uniformity and relatively strong in wettability, and the applicability of the film agent in actual agricultural production is remarkably enhanced; and 3, except for rhizoctonia solani, the compound can also be used for preventing and treating other pathogenic bacteria for releasing cell wall degrading enzyme to finish infection.

Description

Technical Field

[0001] The present invention relates to a plant protective film agent, and more specifically, to a plant protective film agent with kaempferol as an active ingredient, a preparation method thereof, and an application thereof. Background Art

[0002] Plant protection is crucial in agricultural production and is related to the yield and quality of crops. With the increasing attention of people to food safety and environmental protection, new green prevention and control technologies have become a research hotspot, and plant protective film agents have emerged as the times require. As a highly potential pesticide formulation, they are gradually changing the traditional pest and disease control mode.

[0003] The prevention and control principle of plant protective film agents is unique and environmentally friendly. On the one hand, it can form a barrier layer on the surface of the plant, interfering with the recognition of host plants by harmful organisms. After the harmful organisms come into contact with the film layer, they cannot accurately identify the host, thus blocking the infection process, but not directly causing harm to the harmful organisms. This prevention and control method based on behavioral biology greatly reduces the damage to the ecosystem. On the other hand, the film agent containing antibacterial substances forms a protective layer on the surface of the plant. When the pathogenic bacteria come into contact with the film layer, the active antibacterial substances play a role in inhibiting the growth and spread of the pathogenic bacteria, effectively blocking the spread of diseases and escorting the healthy growth of plants.

[0004] At present, plant protective film agents have been widely used in the fields of preventing and controlling storage diseases of fruits and vegetables and field plant diseases, especially with remarkable results in the storage of fruits and vegetables. However, when applied in the field, there are some problems to be solved urgently for traditional formulations of plant protective film agents. The active substances of traditional formulations are difficult to accurately control the release rate, resulting in a short effective period. For those diseases with a long occurrence cycle, in order to achieve an ideal prevention and control effect, it is often necessary to apply pesticides multiple times. Frequent pesticide application not only increases the production cost, but also significantly increases the risk of pesticide residues in agricultural products, causing great pressure on the ecological environment and threatening the balance of biodiversity and soil microbial communities.

[0005] Taking rice sheath blight as an example, this is a disease that seriously threatens rice production. The cellulase and pectinase produced by the sheath blight pathogen play a key role in degrading the rice cell wall and promoting the infection process of the pathogen. In the biotrophic stage of the sheath blight pathogen, these enzymes assist the pathogen in breaking through the defense barrier of rice and invading the host plant, thereby triggering a series of disease symptoms, resulting in reduced rice yield or even crop failure.

[0006] Kaempferol, a natural compound, has been proven to have an inhibitory effect on the infection of rice sheath blight pathogen on rice. It can effectively reduce the activities of cellulase and pectinase, block the damage of the pathogen to the rice sheath tissue, and provide a certain degree of protection for rice plants. However, the physicochemical properties of kaempferol itself limit its large-scale application in agricultural production. Kaempferol is insoluble in water, which poses a great challenge in formulating it into a pesticide formulation and makes it difficult to disperse evenly in the field. Moreover, its phenolic hydroxyl group has high activity and is extremely easy to be oxidized, resulting in poor stability during storage and use, seriously affecting its actual application effect. Summary of the Invention

[0007] The object of the present invention is to provide a plant protective film agent with kaempferol as the active ingredient, its preparation method and application, so as to overcome the deficiencies of the prior art.

[0008] Based on the first main aspect of the present invention, there is provided a plant protective film agent with kaempferol as the active ingredient, and the plant protective film agent is made of the following components: kaempferol-hydroxypropyl-β-cyclodextrin inclusion compound, film-forming substance, emulsifier, antifreeze, preservative, surfactant, pure water. In the above scheme, the kaempferol-hydroxypropyl-β-cyclodextrin inclusion compound is used to solve the problems of poor water solubility and easy oxidation of kaempferol, so that it can stably exist in the protective film agent. The film-forming substance forms a continuous protective film on the plant surface to block harmful organisms; the emulsifier helps the components to be evenly mixed; the antifreeze reduces the freezing point of the solution to prevent the film agent from being damaged by freezing in a low-temperature environment; the preservative inhibits the growth of microorganisms and extends the shelf life of the film agent; the surfactant enhances the wetting and spreading ability of the film agent on the plant surface; pure water is used as a solvent to dissolve and disperse the components. The components work together synergistically to form a plant protective film agent with good performance, laying a foundation for subsequent applications and providing a formulation idea for a stable and effective preparation for controlling rice sheath blight.

[0009] Furthermore, the plant protective film agent is made of the following components: 20 parts of kaempferol-hydroxypropyl-β-cyclodextrin inclusion compound, 40 parts of film-forming substance, 7 parts of emulsifier, 3 parts of antifreeze, 2 parts of preservative, 0.1 part of surfactant, 47.9 parts of pure water. The above scheme clarifies the specific weight parts of each component, which is a further quantification of the formulation. Through precise proportioning, it is ensured that each component plays the best synergistic role in the protective film agent. For example, determining 20 parts of kaempferol-hydroxypropyl-β-cyclodextrin inclusion compound can not only ensure the effective content of kaempferol but also make it compatible well with other components; 40 parts of the film-forming substance can form a stable film structure.

[0010] Among them, in the prepared plant protective film agent, the content of kaempferol is 3.7% by weight percentage. The above limitation further clarifies the content standard of the key active ingredient kaempferol. This content is verified through experiments to ensure the control effect while ensuring the overall stability of the film agent. The above limitation provides a clear quantitative index for product quality control, helps to ensure the consistency and effectiveness of different batches of products, facilitates users to accurately understand and use the products, and ensures the expected effect in the control of rice sheath blight.

[0011] In some embodiments, as a further preferred scheme, the kaempferol-hydroxypropyl-β-cyclodextrin inclusion complex is prepared by mixing kaempferol and hydroxypropyl-β-cyclodextrin. The kaempferol-hydroxypropyl-β-cyclodextrin inclusion complex is formed by mixing kaempferol and hydroxypropyl-β-cyclodextrin. Utilizing the special structure of hydroxypropyl-β-cyclodextrin, kaempferol is included therein. This inclusion effect can improve the solubility and stability of kaempferol, reduce the possibility of phenolic hydroxyl groups being oxidized, and improve the effective utilization rate of kaempferol in the protective film agent. It can improve the applicability of kaempferol in agricultural production, enable it to better play the role of inhibiting rice sheath blight pathogens, and enhance the control effect of the protective film agent on rice sheath blight.

[0012] In some embodiments, as a further preferred scheme, the film-forming substance is one or a combination of hydroxypropyl methylcellulose and sodium alginate; preferably, the film-forming substance is a combination of 2% hydroxypropyl methylcellulose and 2% sodium alginate in a weight ratio of 3:1. Hydroxypropyl methylcellulose and sodium alginate as film-forming substances have good film-forming properties. When used alone, they can each form a film with certain properties. When used in combination, the characteristics of the two are complementary. Formulated in a weight ratio of 3:1 can optimize the structure and properties of the film, such as improving the strength, flexibility and air permeability of the film, and better forming a protective barrier on the surface of rice.

[0013] In some embodiments, as a further preferred scheme, the emulsifier is OP-15. As an emulsifier, OP-15 has good emulsifying properties. It can reduce the surface tension at the oil-water interface, make components such as kaempferol-hydroxypropyl-β-cyclodextrin inclusion complex evenly dispersed in the aqueous phase, form a stable emulsion system, and ensure that the components of the protective film agent do not stratify or precipitate during storage and use.

[0014] In some embodiments, as a further preferred scheme, the antifreeze is glycerol. As an antifreeze, the aqueous solution of glycerol has a relatively low freezing point. In a low-temperature environment, glycerol can reduce the freezing point of the protective film agent, prevent the water in the film agent from freezing and expanding, avoid damaging the structure and properties of the film agent, and ensure that the film agent can still be used normally under cold conditions.

[0015] In some embodiments, as a further preferred solution, the preservative is sodium benzoate; more preferably, the surfactant is Silwet-L77. Sodium benzoate, as a preservative, can inhibit the growth and reproduction of microorganisms. It prevents the protective film agent from deteriorating due to microbial contamination during storage by interfering with the metabolic processes of microbial cells and damaging the cell membranes and enzyme systems of microorganisms. Silwet-L77, as a surfactant, has excellent wettability and spreading properties, can reduce the contact angle between the film agent and the rice surface, make the film agent more easily spread on the rice surface, and increase the coverage area. The above solutions extend the shelf life of the protective film agent, improve the adhesion and spreading effects of the film agent on the rice surface at the same time, make the protective film cover the rice more evenly, and enhance the control effect on sheath blight of rice.

[0016] Based on the second main aspect of the present invention, there is provided a preparation method of the aforementioned plant protective film agent with kaempferol as the active ingredient, comprising the following steps:

[0017] S1: By weight, weigh 100 parts of kaempferol, dissolve it in an appropriate amount of absolute ethanol, weigh 500 parts of hydroxypropyl-β-cyclodextrin in a round-bottom flask, dissolve it in an appropriate amount of distilled water, stir magnetically at a constant temperature of 60 °C, gradually add the kaempferol ethanol solution dropwise to the hydroxypropyl-β-cyclodextrin, stir at a constant temperature for 7 hours, let it cool to room temperature, centrifuge, refrigerate at -80 °C for 12 hours, freeze-dry for 24 hours, and grind with a mortar to obtain kaempferol-hydroxypropyl-β-cyclodextrin inclusion complex powder for later use;

[0018] S2: By weight, weigh 2 parts of hydroxypropyl methylcellulose and 98 parts of water and mix them to prepare 2% hydroxypropyl methylcellulose; weigh 2 parts of sodium alginate and 98 parts of water and mix them to prepare 2% sodium alginate.

[0019] S3: By weight, add 30 parts of 2% hydroxypropyl methylcellulose solution, 10 parts of 2% sodium alginate solution, 20 parts of kaempferol-hydroxypropyl-β-cyclodextrin inclusion complex, 7 parts of OP-15, 3 parts of glycerol, 2 parts of sodium benzoate, 0.1 part of silwet L77 to a beaker in sequence, and add 47.9 parts of pure water, stir evenly with magnetic force at room temperature to obtain the product.

[0020] Among them, in step S1, the kaempferol-hydroxypropyl-β-cyclodextrin inclusion complex is prepared by specific temperature, time and operation methods, and the inclusion reaction is promoted by stirring at a constant temperature. Centrifugation, refrigeration and freeze-drying are used to remove impurities and obtain a stable inclusion complex powder. In step S2, each component is mixed and stirred in proportion to make them fully and evenly dispersed, forming a protective film agent with stable performance. This preparation method has a reasonable process and feasible operation, can ensure the stable quality of the prepared protective film agent, and the components are evenly mixed, thus ensuring the consistency and effectiveness of the product, which is conducive to large-scale industrial production.

[0021] Based on the third main aspect of the present invention, there is provided the use of a plant protective film agent with kaempferol as the active ingredient, including the application of the plant protective film agent in controlling rice sheath blight; and including diluting the plant protective film agent 500 times with water and directly spraying it evenly on the surface of rice, including spraying on the leaf sheath part. Spraying the plant protective film agent diluted 500 times with water on the surface of rice, especially on the leaf sheath part, is based on the characteristic that the rice sheath blight pathogen mainly infects the leaf sheath part. The diluted film agent forms a protective film on the surface of rice. Kaempferol inhibits the activities of cellulase and pectinase of the sheath blight pathogen, preventing the pathogen from infecting. The film-forming substance blocks the pathogen, and other components act synergistically. Therefore, a simple and effective application method for controlling rice sheath blight is provided, which can effectively reduce the occurrence of rice sheath blight, improve the yield and quality of rice, and has good application value.

[0022] Compared with the prior art, by using the plant protective film agent with kaempferol as the active ingredient and the preparation method provided by the present invention, the following technical effects can be achieved: stronger wettability, better uniformity and stability, can efficiently inhibit the mycelial growth of the sheath blight pathogen and the activities of cellulase and pectinase secreted by the sheath blight pathogen, can effectively prevent the sheath blight pathogen from infecting rice and has a longer effective period. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Shows the wetting properties of the film agent at different dilution multiples in an embodiment of the present invention.

[0024] Figure 2 Shows the absorbance of the upper, middle and lower parts of the film agent at different dilution multiples in an embodiment of the present invention.

[0025] Figure 3 Shows the inhibitory effect of the plant protective film agent diluted at different dilution multiples on the mycelial growth of the sheath blight pathogen in an embodiment of the present invention.

[0026] Figure 4 Shows the inhibitory effect of the film agent diluted at different multiples on cellulase solution and pectinase in an embodiment of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0027] The following will elaborate on the preferred embodiments of the present invention in detail to more clearly understand the purpose, characteristics and advantages of the present invention. It should be understood that the following embodiments are not limitations on the scope of the present invention, but only to illustrate the essential spirit of the technical solution of the present invention.

[0028] In the following description, certain specific details are set forth in order to provide a thorough understanding of various disclosed embodiments. However, those skilled in the relevant art will recognize that the embodiments may be practiced without one or more of these specific details. In other instances, well-known technologies associated with the present application may not be shown or described in detail so as to avoid unnecessarily obscuring the description of the embodiments.

[0029] References to "one embodiment" or "an embodiment" in the course of the specification mean that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of "in one embodiment" or "in an embodiment" in various places throughout the specification are not necessarily all referring to the same embodiment. Additionally, the particular features, structures, or characteristics may be combined in any manner in one or more embodiments.

[0030] Embodiment 1:

[0031] The raw materials used in the embodiments of the present invention are all commercially available.

[0032] The parts in the embodiments of the present invention are in grams.

[0033] Plant protective film components:

[0034] 20 parts of kaempferol - hydroxypropyl - β - cyclodextrin inclusion compound, 40 parts of film - forming substance, 7 parts of emulsifier, 3 parts of antifreeze, 2 parts of preservative, 0.1 part of surfactant, 47.9 parts of pure water.

[0035] Preparation steps:

[0036] (1) Weigh 100 parts of kaempferol and dissolve it in an appropriate amount of absolute ethanol.

[0037] (2) Weigh 500 parts of hydroxypropyl - β - cyclodextrin into a round - bottom flask, dissolve it in an appropriate amount of distilled water, and stir magnetically at a constant temperature of 60°C.

[0038] (3) Gradually add the kaempferol ethanol solution drop - by - drop to the hydroxypropyl - β - cyclodextrin, stir at a constant temperature for 7 hours, let it cool to room temperature, centrifuge, refrigerate at - 80°C for 12 hours, freeze - dry for 24 hours, and grind with a mortar to obtain the kaempferol - hydroxypropyl - β - cyclodextrin inclusion compound powder for use.

[0039] (4) Weigh 2 parts of hydroxypropyl methylcellulose and add it to 98 parts of water to obtain a 2% hydroxypropyl methylcellulose solution, and weigh 2 parts of sodium alginate and add it to 98 parts of water to obtain a 2% sodium alginate solution for use.

[0040] (5) 30 parts of 2% hydroxypropyl methylcellulose solution, 10 parts of 2% sodium alginate solution, 20 parts of kaempferol-hydroxypropyl-β-cyclodextrin inclusion complex, 7 parts of OP-15, 3 parts of glycerol, 2 parts of sodium benzoate, 0.1 part of silwet L77, and 47.9 parts of purified water. Stir evenly with magnetic force at room temperature to obtain.

[0041] Determination of wetting performance in the example:

[0042] Test samples: Example samples, rice leaves, variety, Dali fragrant.

[0043] Test method:

[0044] (1) Under indoor normal temperature conditions, cut fresh and clean rice leaves (2×2 cm), without damaging the leaf surface structure and keeping the leaf surface in a natural state, and fix them flat on the stage of the contact angle measuring instrument.

[0045] (2) Preparation of plant protective film agent dilution: Dilute the film agent with sterile water to 500-fold, 800-fold, and 1000-fold solutions respectively for use.

[0046] (3) Use a micro syringe to inject 3 μL droplets onto the rice leaves respectively.

[0047] (4) Use the camera on the contact angle measuring instrument to take pictures of the droplets on the leaf surface, and use the fitting analysis method to calculate the contact angles of the droplets on the rice leaf surface at 1 s, 5 s, and 10 s.

[0048] Contact angle: The smaller the contact angle, the better the wetting performance of the droplet, and vice versa. The better the wetting performance, it indicates that the droplet loss is less after spraying in the field and the risk of off-target of the liquid medicine is low.

[0049] The results are shown in Figure 1 , which shows the wetting performance of the film agent. As Figure 1 shown, after the film agent drops on the surface of the rice leaf, with the increase of the dilution ratio (500-fold, 800-fold, 1000-fold), the contact angles gradually become larger, which are 21.52°, 22.71°, and 24.45° respectively (less than 30°), and can be completely spread on the surface of the rice leaf within 10 s, indicating that the film agent has good wetting and spreading properties.

[0050] Determination of dispersion performance in the example:

[0051] Test samples: Example samples, rice leaves, variety, Dali fragrant.

[0052] Test method:

[0053] (1) Dilute the film agent 200-fold, 400-fold, 600-fold, 800-fold, and 1000-fold with sterile water, stir and mix evenly, and let it stand at room temperature for 2 h.

[0054] (2) Use a 5 mL syringe to aspirate 1 mL of the membrane solution from the upper, middle, and lower parts of the membrane solution and the film-forming substance respectively.

[0055] (3) Use an ultraviolet spectrophotometer to detect the absorbance values of the membrane solution and the upper, middle, and lower parts of the film-forming substance at 367 nm respectively, and repeat 3 times.

[0056] Data investigation:

[0057] Statistically calculate the average value of the 3 repetitions, and compare the differences in absorbance values of the upper, middle, and lower parts of the membrane solution at the same dilution factor.

[0058] Absorbance value: The smaller the difference between the absorbance data, the more uniform the dilution of the membrane agent particles, and the better the dispersibility and stability. Good dispersion and stability indicate that the selection of membrane agent raw materials, the optimization of the formula, and the processing technology are appropriate, and the membrane agent has stronger applicability in the field.

[0059] The results are shown in Figure 2 , which shows the absorbance of the membrane agent. As can be seen from Figure 2 , when the membrane agent is diluted 200 - 1000 times, there is no significant difference in the absorbance at 367 nm of the upper, middle, and lower parts of the solutions at each dilution factor, proving that the membrane agent can be quickly and uniformly dispersed in the membrane solution after being diluted by different multiples, without aggregation or precipitation, and has good stability.

[0060] Test example 1: Antibacterial effect of the culture medium

[0061] Test samples: Samples of the examples, Strain: Rhizoctonia solani (preserved by the Institute of Crop Protection, Guizhou University).

[0062] Test method:

[0063] Activation and cultivation of the strain: Inoculate the preserved Rhizoctonia solani on a PDA medium (the composition is 200 g of potato, 20 g of glucose, 20 g of agar, and 1000 mL of distilled water, sterilized at high temperature and high pressure at 121 °C) for cultivation.

[0064] Preparation of the plant protection film agent-containing plate: Use sterile water to mix the membrane agent with the sterilized culture medium evenly, and dilute it into 100, 500, 800, 1000, and 2000-fold solutions. Use the culture medium without the plant protection film agent as a control, and let it stand at room temperature until the culture medium solidifies.

[0065] Take 5 mm fresh Rhizoctonia solani fungal cakes and inoculate them in the center of the plant protection film agent-containing plates and the control plates. Repeat each treatment 3 times, and place them in an artificial climate incubator for cultivation (cultivate under the condition of alternating light and darkness at 28 °C).

[0066] Data investigation:

[0067] (1) Colony diameter: Cultivate in an artificial climate chamber (under the condition of 25°C with alternating light and darkness) for 48 hours, and measure the net growth diameter of the Rhizoctonia solani colony on rice with a ruler (using the cross method).

[0068] (2) Inhibition rate (%): Reflects the inhibitory effect of the plant protective film on the colony growth. The higher the inhibition rate, the better the inhibitory effect.

[0069] Inhibition rate (%) = 100 * (net growth diameter of the control group - net growth diameter of the example group) / net growth diameter of the control group.

[0070] Data statistical analysis is shown in Table 1, and the results are shown in Figure 3 , which shows the inhibitory effect of the plant protective film agent diluted at different dilution multiples on the mycelial growth of Rhizoctonia solani.

[0071] Among them, Table 1 shows the inhibitory effect of the film agent at different dilution multiples on the mycelial growth of Rhizoctonia solani, Figure 3 the inhibitory effect of the film agent diluted at different dilution multiples on the mycelial growth of Rhizoctonia solani.

[0072] Table 1 Inhibitory effect of the film agent at different dilution multiples on the mycelial growth of Rhizoctonia solani

[0073]

[0074] From Figure 3 It can be seen that the plant protective film agent with kaempferol as the active ingredient has an obvious inhibitory effect on the mycelia of Rhizoctonia solani on rice. As can be seen from Table 1, when the plant protective film agent is diluted 100, 500, 800, 1000, and 2000 times respectively, the inhibition rates on mycelial growth are 88.31%, 80.67%, 56.32%, 51.07%, and 38.19% respectively. The results show that the plant protective film agent with kaempferol as the active ingredient can effectively inhibit the growth of Rhizoctonia solani mycelia.

[0075] Test Example 2: Inhibitory effect of the film agent on the activities of cellulase solution and pectinase

[0076] Test samples: Samples of the example, rice variety, Dali fragrant.

[0077] Test method:

[0078] (1) Preparation of the diluted solution of the plant protective film agent: Dilute the film agent with sterile water into 500, 800, and 1000-fold solutions respectively for use.

[0079] (2) Induction of cellulase solution and pectinase: In the modified Marcus culture solution (KNO 3 2 g, KCl 0.5 g, FeSO 4 0.01 g, K 2 HPO4 1 g of MgSO 4 ·7H 2 O, 0.5 g of L-asparagine, 0.2 mg of VB 1 (In 1000 mL of deionized water), 10 g of sodium carboxymethyl cellulose (to induce the production of cellulase by Rhizoctonia solani) or 10 g of pectin (to induce the production of pectinase by Rhizoctonia solani) was added respectively for the induction culture of Rhizoctonia solani.

[0080] (3) Preparation of purified cellulase solution and pectinase solution: 144 hours after the induction culture, the mycelium was filtered and centrifuged at 4°C for 30 min (10000 rpm) to obtain the supernatant. 60% saturated (NH 4 ) 2 SO 4 solution was added to the supernatant, refrigerated and allowed to stand, then centrifuged for 20 min (4°C, 15000 rpm). The precipitate was dissolved with acetic acid-sodium acetate buffer solution and dialyzed for 24 hours (changing the buffer solution twice) to obtain the purified enzyme solution.

[0081] (4) The diluted plant protective film agent was evenly sprayed on healthy rice leaf sheaths (sterilized with 75% ethanol, rinsed twice with sterile water, and micro-wounds were made by pricking with a No. 5 insect needle). The control group was sprayed with sterile water (without spraying the film agent), and the blank control group was not treated. It was left to stand at room temperature for 12 h.

[0082] (5) After the film agent was air-dried, 500 μL of the purified cellulase solution and pectinase solution prepared in method (3) were inoculated into the wound of the rice leaf sheath respectively. The control group was also inoculated with the enzyme solution, and the blank control group was not treated. It was cultured under moisturizing conditions at 28°C and repeated 3 times.

[0083] 5 days after inoculation, the changes in the inoculation site of the rice leaf sheath tissue were observed and photographed.

[0084] The results are shown in Figure 4 . Figure 4 shows the inhibitory effects of different dilution multiples of the film agent on cellulase solution and pectinase. As Figure 4 can be seen, 5 days after inoculation with cellulase solution and pectinase, obvious discolored lesions or brown cloud-like lesions had appeared on the surface of the rice leaf sheath in the sterile water control group, indicating that the cellulase solution and pectinase caused damage to the rice leaf sheath. There was no damage on the surface of the leaf sheath in the blank control group, excluding interference from other factors for the damage of the rice leaf sheath; under the treatment of 1000, 800, and 500-fold dilutions of the film agent, the damage on the surface of the rice leaf sheath gradually decreased, indicating that the plant protective film agent with kaempferol as the active ingredient can inhibit the activities of cellulase and pectinase and reduce their damage to the rice leaf sheath, thus protecting the rice.

[0085] Experimental Example 3: Antibacterial effect in potted plants

[0086] Test samples: Samples of examples, strains: Rhizoctonia solani (preserved by the Institute of Crop Protection, Guizhou University), rice variety: Dali xiang.

[0087] Test methods:

[0088] (1) Preparation of plant protective film agent dilutions: Dilute the film agent into 500-fold, 800-fold, and 1000-fold dilutions with sterile water respectively. Dilute the film-forming substances (hydroxypropyl methylcellulose solution, sodium alginate solution) into 800-fold dilution with sterile water, and dilute the kaempferol aqueous emulsion into 800-fold dilution with sterile water.

[0089] (2) Preparation of bacterial suspension: Take a 5-mm diameter Rhizoctonia solani agar disc and place it in 100 mL of PDB nutrient solution (the composition is 200 g of potato, 20 g of glucose, and 1000 mL of distilled water, sterilized at 121 °C under high temperature and high pressure), and place it in a constant temperature shaker at 28 °C with alternating light and darkness for 72 hours. Filter out the mycelium, break the mycelium with a tissue grinder (length ≤ 2 mm), and add 1000 mL of 10% glucose solution to prepare a mycelium suspension.

[0090] (3) Treatment of rice plants: Sow rice in trays with a length of 40 cm and a width of 25 cm, and then transplant them into pots. Wait until the seedlings grow to a height of 25 - 30 cm.

[0091] (4) Spray the plant protective film agent dilutions, film-forming substance dilutions, and kaempferol aqueous emulsion dilutions evenly on the rice plants respectively. Spray clear water on the control group, and let it stand at room temperature for 12 h until the film agent dries.

[0092] (5) Spray the mycelium suspension evenly on the surface of the rice leaf sheaths. There are 6 pots of rice for each treatment, and repeat three times. Place the inoculated rice under suitable conditions (temperature 28 °C, relative humidity 90%, alternating light and darkness for 12 h) for cultivation.

[0093] Data investigation:

[0094] Count the disease grades and the number of plants in each disease grade 3 days, 5 days, 7 days, and 14 days after inoculation.

[0095] Disease grades: Different grades divided according to the degree of disease manifestation on the plants (such as lesion area, number of diseased parts, etc.).

[0096] Disease grade classification standard: Grade 0: The whole plant is disease-free; Grade 1: The fourth leaf and the leaf sheaths and leaves below it are diseased (taking the flag leaf as the first leaf); Grade 3: The third leaf and the leaf sheaths and leaves below it are diseased; Grade 5: The second leaf and the leaf sheaths and leaves below it are diseased; Grade 7: The flag leaf and the leaf sheaths and leaves below it are diseased; Grade 9: The whole plant is diseased and withers prematurely.

[0097] Calculate the disease index.

[0098] Disease index: comprehensively reflects the degree of rice sheath blight.

[0099] Disease index = 100 × (Σ(number of diseased plants at each level × relative level value) / total number of investigations × 9)

[0100] Calculate the control effect.

[0101] Control effect (%): reflects the control effect of the plant protective film on rice sheath blight.

[0102] Control effect (%) = 100 × ((disease index after spraying in the control group - disease index after spraying in the film agent treatment group) / disease index after spraying in the control group)

[0103] Data statistical analysis is shown in Table 2. The data was subjected to normality test and homogeneity of variance test using IBM SPSS Stastistic 23 (SPSS Inc., Chicago, Ill., USA). The results are expressed as mean ± SE (standard error), and one-way analysis of variance and Duncan's new multiple range method were used to analyze the data significance (p < 0.05).

[0104] Table 2 Pot control effects of different dilution multiples of the film agent on rice sheath blight

[0105]

[0106] Note: The values in the table are mean ± SE. Different lowercase letters in the same column indicate significant differences (p < 0.05).

[0107] It can be seen from Table 2 that at 3d, 5d, 7d, and 14d after inoculation, the control effects after spraying the 500-fold solution of the film agent were 87.44%, 82.18%, 73.17%, and 67.25% respectively, which were significantly better than those after spraying the 800-fold solution of the film-forming substance and the 800-fold solution of kaempferol aqueous emulsion, indicating that the plant protective film agent with kaempferol as the active ingredient has a good control effect on rice sheath blight, and the control effect is the best when diluted 500 times.

[0108] Test Example 4: Field efficacy test

[0109] Test samples: Samples of the examples, rice variety, Dali fragrant.

[0110] Test site: Liupo Group, Huimin Village, Huishui County, Qiannan Prefecture, Guizhou Province (longitude 106.64°, latitude 26.11°). The test field has been planted with rice all year round, with medium soil fertility, good irrigation, and no other fungicides used during the entire growth period. Rice sheath blight has occurred for 3 consecutive years.

[0111] Test method:

[0112] (1) Preparation of the plant protective film agent diluent: Dilute the film agent with sterile water to 500-fold, 800-fold, and 1000-fold solutions, dilute the film-forming substances (hydroxypropyl methylcellulose solution, sodium alginate solution) with sterile water to 800-fold solution, dilute the kaempferol aqueous emulsion with sterile water to 800-fold solution, and dilute the 15% jinggangmycin wettable powder with sterile water to 1200-fold solution, with clear water as the control.

[0113] There were 28 plots in total for the experiment (7 treatments, 4 replicates), with each plot having an area of 20 m2, arranged randomly. A 1-m buffer zone was set up in the experimental field, and the pesticide application plan for the field experiment is shown in Table 3.

[0114] Table 3 Pesticide application plan for the field experiment

[0115]

[0116] The arrangement of the experimental plots is shown in Table 4.

[0117] Table 4 Arrangement of the experimental plots for the field experiment

[0118]

[0119] (2) Conduct field spraying using a sprayer, with key spraying on the leaf sheaths of rice.

[0120] (3) Investigate the disease incidence base before pesticide application, and conduct investigations and statistics 7 days and 14 days after pesticide application respectively. For each plot, take 5 samples at 5 points along the diagonal, investigate 5 rice plants at each point, and count the disease grade numbers and the number of plants at each disease grade.

[0121] Disease grade number: Different grades divided according to the degree of disease manifestation on the plant (such as lesion area, number of diseased parts, etc.).

[0122] Disease grade number classification standard: Grade 0: The whole plant is disease-free; Grade 1: The fourth leaf and the leaf sheaths and leaves below it are diseased (taking the flag leaf as the first leaf); Grade 3: The third leaf and the leaf sheaths and leaves below it are diseased; Grade 5: The second leaf and the leaf sheaths and leaves below it are diseased; Grade 7: The flag leaf and the leaf sheaths and leaves below it are diseased; Grade 9: The whole plant is diseased and withers prematurely.

[0123] Calculate the disease index.

[0124] Disease index: Comprehensively reflects the degree of rice sheath blight.

[0125] Disease index = 100×(Σ(number of diseased plants at each grade × relative grade value) / total number of investigated plants × 9)

[0126] Calculate the control effect.

[0127] Control effect (%): Reflects the control effect of the plant protective film on rice sheath blight.

[0128] Control effect (%) = 100 × ((disease index after spraying in the control group - disease index after spraying in the film agent treatment group) / disease index after spraying in the control group)

[0129] Data statistical analysis is shown in Table 5. The data were subjected to normality test and homogeneity of variance test using IBM SPSS Stastistic 23 (SPSS Inc., Chicago, Ill., USA). The results are expressed as mean ± SE (standard error), and one-way analysis of variance and Duncan's new multiple range method were used to analyze the data significance (p < 0.05).

[0130] Table 5 Field control effect of film agent on sheath blight of rice

[0131]

[0132] Note: The values in the table are mean ± SE. Different lowercase letters in the same column indicate significant differences (p < 0.05).

[0133] As can be seen from Table 5, the disease indices of field rice after spraying the film agent at 500 - fold, 800 - fold, and 1000 - fold dilutions were 8.19, 9.83, and 13.92 respectively, showing no significant difference from the 9.02 of the commercial preparation Jinggangmycin SP at 1200 - fold dilution. At this time, the control effects were 74.41%, 69.29%, and 56.54% respectively. Among them, the control effects of the film agent at 500 - fold and 800 - fold dilutions had no significant difference from the control effect of 71.82% of Jinggangmycin treatment, but were significantly higher than the control effects of 46.46% and 32.47% of kaempferol aqueous emulsion at 800 - fold dilution and film - forming substance treatment. The control effects of the film agent at 500 - fold dilution and Jinggangmycin SP at 1200 - fold dilution were 58.96% and 60.49% respectively, which were significantly higher than other treatments 14 days after spraying. The test results show that applying the plant protection film agent with kaempferol as the active ingredient in the early stage of sheath blight occurrence of rice has a good control effect on sheath blight of rice and a good persistent effect period.

[0134] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above - mentioned embodiments. What is described in the above - mentioned embodiments and the specification only illustrates the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention will have various changes and improvements, and these changes and improvements all fall within the scope of the present invention claimed. The scope of the present invention claimed is defined by the appended claims and their equivalents.

Claims

1. A plant protective film agent with kaempferol as an active ingredient, characterized in that: The plant protection film agent is prepared from the following components: kaempferol-hydroxypropyl-β-cyclodextrin inclusion compound, film-forming substance, emulsifier, antifreeze agent, preservative, surfactant and purified water.

2. The plant protective film agent with kaempferol as an active ingredient according to claim 1, characterized in that: The plant protection film agent is made of the following components based on 100 parts by total weight: 20 parts of kaempferol-hydroxypropyl-β-cyclodextrin inclusion compound, 40 parts of film-forming substance, 7 parts of emulsifier, 3 parts of antifreeze agent, 2 parts of preservative, 0.1 parts of surfactant and 47.9 parts of purified water.

3. The plant protective film agent with kaempferol as an active ingredient according to claim 1 or 2, characterized in that: In the prepared plant protection film agent, the content of kaempferol is 3.7% by weight.

4. The plant protective film agent with kaempferol as an active ingredient according to claim 1 or 2, characterized in that: The kaempferol-hydroxypropyl-β-cyclodextrin inclusion compound is prepared by mixing kaempferol and hydroxypropyl-β-cyclodextrin; Preferably, the kaempferol-hydroxypropyl-β-cyclodextrin inclusion complex is composed of kaempferol and hydroxypropyl-β-cyclodextrin in a weight ratio of 1:

5.

5. The plant protective film agent with kaempferol as an active ingredient according to claim 1 or 2, characterized in that: The film-forming substance is one of hydroxypropyl methylcellulose and sodium alginate or a combination thereof; Preferably, the film-forming substance is 2% hydroxypropyl methylcellulose and 2% sodium alginate in a weight ratio of 3:1; Preferably, in parts by weight, the 2% hydroxypropyl methylcellulose is prepared by mixing 2 parts of hydroxypropyl methylcellulose and 98 parts of water; the 2% sodium alginate is prepared by mixing 2 parts of sodium alginate and 98 parts of water.

6. The plant protective film agent with kaempferol as an active ingredient according to claim 1 or 2, characterized in that: The emulsifier was OP-15.

7. The plant protective film agent with kaempferol as an active ingredient according to claim 1 or 2, characterized in that: The antifreeze agent is glycerol.

8. The plant protective film agent with kaempferol as an active ingredient according to claim 1 or 2, characterized in that: The preservative is sodium benzoate; More preferably, the surfactant is Silwet-L77.

9. A method for preparing a plant protective film agent with kaempferol as an active ingredient as claimed in any one of claims 1 to 8, characterized in that: The steps include: S1: According to weight, weigh 100 parts of kaempferol, dissolve it in an appropriate amount of anhydrous ethanol, weigh 500 parts of hydroxypropyl-β-cyclodextrin in a round-bottom flask, dissolve it in an appropriate amount of distilled water, keep it at 60℃ with magnetic stirring, add the kaempferol ethanol solution dropwise to the hydroxypropyl-β-cyclodextrin, keep it at constant temperature and stir for 7 hours, let it cool to room temperature, centrifuge, refrigerate at -80℃ for 12 hours, freeze-dry for 24 hours, grind it in a mortar, and obtain kaempferol-hydroxypropyl-β-cyclodextrin inclusion compound powder for standby use; S2: In parts by weight, 2 parts of hydroxypropyl methylcellulose and 98 parts of water are mixed to prepare 2% hydroxypropyl methylcellulose; 2 parts of sodium alginate and 98 parts of water are mixed to prepare 2% sodium alginate; S3: Based on a total weight of 100 parts, add 30 parts of 2% hydroxypropyl methylcellulose solution, 10 parts of 2% sodium alginate solution, 20 parts of kaempferol-hydroxypropyl-β-cyclodextrin inclusion compound, 7 parts of OP-15, 3 parts of glycerol, 2 parts of sodium benzoate, 0.1 parts of Silwet L77, and 47.9 parts of purified water into a beaker in sequence, and stir evenly with a magnetic stirrer at room temperature to obtain the mixture.

10. The use of the plant protective film agent with kaempferol as an active ingredient as claimed in claim 1 or 2, characterized in that: Including the use of the plant protection film agent in preventing and controlling rice sheath blight; and The method comprises diluting the plant protection film agent with water by 500 times and then directly and evenly spraying the plant protection film agent on the surface of rice, including spraying the leaf sheath.

Images