Application of kaempferol in preventing and treating p. insularis leaf spot disease

Abstract

This invention relates to the field of plant protection technology, specifically providing the application of kaempferol in the control of areca leaf spot disease. The pathogen causing areca leaf spot disease is *Pseudomonas stylosa*. Diaporthe pseudophoenicicola Experiments have shown that kaempferol can significantly inhibit the mycelial growth of this pathogen at low concentrations, and can significantly reduce its pathogenicity and lesion area by pretreating areca nut leaves. As a plant-derived active ingredient, kaempferol has the advantages of being environmentally friendly and not prone to developing resistance, providing a new technical solution and candidate agent for the green and sustainable control of areca nut leaf spot disease.

Description

Technical Field

[0001] This invention belongs to the field of plant protection technology and relates to the application of kaempferol in the prevention and control of areca leaf spot disease. Background Technology

[0002] In recent years, fungi of the genus *Pseudomonas* have been found to be... Diaporthe Leaf spot disease caused by fungi (spp.) has become one of the major diseases affecting the growth of areca nut. In the early stage of the disease, small brown spots appear on the leaves, which then rapidly expand and merge, leading to the death of large areas of leaves. In severe cases, the entire plant's leaves wither and fall off, weakening the tree and significantly affecting the yield and quality of areca nuts.

[0003] Currently, the prevention and control of this disease still mainly relies on chemical pesticides. However, long-term use can easily lead to problems such as pathogen resistance, pesticide residues, and environmental pollution. With the deepening of the concepts of green agriculture and sustainable development, the development of highly efficient, low-toxicity, and environmentally compatible plant-derived pesticides has become an important research direction.

[0004] Kaempferol is a flavonol compound widely found in plants, known to possess various biological activities such as antioxidant and anti-inflammatory effects. It has also been reported to have inhibitory effects on certain plant pathogens. However, to date, no published literature or patents have reported any effect of kaempferol on the specific pathogen affecting areca nut—*Pseudomonas stylosa*. Diaporthe pseudophoenicicola It has a preventive effect, but no relevant technical solutions for its application in the prevention and control of areca leaf spot disease have been found. Summary of the Invention

[0005] The present invention aims to solve the technical problem of the lack of efficient and green control methods for areca leaf spot disease in the existing technology.

[0006] Specifically, the first objective of this invention is to provide a novel use for the natural compound kaempferol, namely, its role in the prevention and control of diseases caused by a specific pathogen—*Pseudomonas stolonifera*. Diaporthe pseudophoenicicola Application in the treatment of areca leaf spot disease.

[0007] A second objective of this invention is to provide a medicament containing kaempferol as an active ingredient for preventing and treating the aforementioned areca leaf spot disease.

[0008] The third objective of this invention is to provide a method for preventing and controlling the aforementioned areca leaf spot disease based on kaempferol.

[0009] To achieve the above-mentioned objectives, the present invention adopts the following technical solution:

[0010] The first aspect of this invention provides the application of the chemical substance kaempferol, said application being any of the following:

[0011] A1) Application in the prevention and control of areca leaf spot disease, wherein the pathogen of areca leaf spot disease is Diaporthe pseudophoenicicola;

[0012] A2) Use in the preparation of products for the prevention and control of areca leaf spot disease caused by the fungus Diaporthe pseudophoenicicola.

[0013] Kaempferol can be used directly for disease control, or it can be used as a core active ingredient in the preparation of corresponding fungicides (such as pesticides and formulations). This application is based on the first discovery of kaempferol's effect on the pathogen of areca leaf spot disease. Diaporthe pseudophoenicicola Specific inhibitory effect.

[0014] Preferably, to facilitate the preparation and application of the aqueous system, kaempferol can be dissolved in sodium hydroxide solution before use to form its water-soluble salt form.

[0015] Experimental results showed that the effective concentration of kaempferol for inhibiting the growth of *Pseudomonas stylosa* mycelium was 5 µg / mL or higher. In detached leaf control experiments, the effective concentration of kaempferol for reducing the pathogenicity of *Pseudomonas stylosa* to areca nut was 2 µg / mL or higher.

[0016] A second aspect of this invention provides a medicament for preventing and controlling areca leaf spot disease, wherein the active ingredient of the medicament is kaempferol; and the pathogen of areca leaf spot disease is *Pseudomonas stolonifera*. Diaporthe pseudophoenicicola .

[0017] Preferably, the preparation method of the agent may be as follows: dissolving kaempferol powder in a sodium hydroxide solution of appropriate concentration and making up to a final volume to obtain a high-concentration kaempferol stock solution for easy storage and dilution. For example, 500 mg of kaempferol powder can be dissolved in 0.1 mol / L sodium hydroxide solution and made up to a final volume of 10 mL to obtain a 50 mg / mL kaempferol stock solution, which can be stored at low temperature (e.g., 4°C) for later use.

[0018] A third aspect of this invention provides a method for controlling areca leaf spot disease, comprising applying an agent containing kaempferol as an active ingredient (e.g., a diluted agent prepared by the aforementioned method) to areca plants, for example, by foliar spraying, to inhibit *Pseudomonas stearothermii*. Diaporthe pseudophoenicicola The purpose is to control mycelial growth and infection, and to prevent leaf spot disease.

[0019] The beneficial effects of this invention are:

[0020] (1) It provides a brand-new prevention and control strategy: For the first time, it has been revealed that the natural flavonoid compound kaempferol has a highly effective inhibitory effect on specific pathogens that harm areca nut, opening up a brand-new technical path based on plant-derived active ingredients for the prevention and control of areca leaf spot disease.

[0021] (2) Significant control effect: Kaempferol exhibits excellent biological activity even at low concentrations (µg / mL). In vitro experiments show that a concentration ≥5 µg / mL can significantly inhibit the mycelial growth of pathogens; in vivo (exposed leaf) experiments show that a concentration ≥2 µg / mL can significantly reduce the pathogenicity of pathogens and reduce the area of ​​lesions.

[0022] (3) Environmentally friendly and highly safe: Kaempferol is a natural component that is widely found in a variety of plants. It is easily biodegradable, has good environmental compatibility, is safe for non-target organisms, helps reduce chemical pesticide residues and environmental pollution, and meets the requirements of green agriculture and sustainable development.

[0023] (4) Clear application prospects: This invention not only clarifies the activity of kaempferol, but also provides specific examples of drug preparation and application methods, which have good operability and industrial application potential, laying a solid foundation for the development of new and efficient plant-derived fungicide products. Attached Figure Description

[0024] Figure 1 The effect of different concentrations of kaempferol on the growth rate of Pseudomonas aeruginosa.

[0025] Figure 2 Kaempferol reduces the pathogenicity of Pseudomonas stolonifera XJ5 to areca nut.

[0026] Figure 3 The image shows the control effect of kaempferol on leaf spot disease in areca nut plants. Detailed Implementation

[0027] The specific embodiments of the present invention are described below to enable those skilled in the art to understand the present invention. However, it should be understood that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, various changes are obvious as long as they are within the spirit and scope of the present invention as defined and determined by the appended claims. All inventions utilizing the concept of the present invention are protected.

[0028] The *Pseudomonas stolonifera* XJ1 involved in this invention is Diaporthe pseudophoenicicola It is stored in this laboratory. Kaempferol was purchased from Ron Reagent Company (catalog number R005378).

[0029] Example 1: Preparation of Kaempferol Mother Solution

[0030] Weigh 500 mg of kaempferol powder, dissolve it in 0.1 mol / L NaOH solution and bring the volume up to 10 mL to obtain a 50 mg / mL kaempferol stock solution. After dispensing, store the stock solution in a 4°C refrigerator. Dilute it with ddH2O to different concentrations for use in experiments.

[0031] Example 2: Effect of kaempferol on the growth rate of *Pseudomonas stolonifera*

[0032] PDA medium was heated and melted, and kaempferol stock solution was added to achieve final concentrations of 0, 0.2, 2, 5, 10, and 20 μg / mL. Five plates were prepared for each concentration of PDA. Activated *Pseudomonas aeruginosa* XJ1 was inoculated into 0.5 cm diameter mycelial discs, with the mycelial side down, onto the PDA plates of different concentrations. Three independent replicate experiments were performed. Colony diameter was measured and photographed after 4 days. Growth rate = (measured diameter - mycelial disc diameter) / number of days of culture. Results showed that kaempferol concentrations above 5 μg / mL significantly inhibited the growth rate of XJ1 (p < 0.0001). Figure 1 ).

[0033] Example 3: Kaempferol reduces the pathogenicity of *Stemona stolonifer*

[0034] For *Pseudomonas aeruginosa* strains, an in vitro leaf method was used. Wild-type *Pseudomonas aeruginosa* strain XJ5 was activated on PDA plates for 3-4 days. Several mycelial blocks were punched at the edge of the colony using a 5 mm diameter punch. Areca nut leaves of uniform size, consistent growth stage, and without surface damage or lesions were collected. The leaf surfaces were rinsed with ddH2O and dried. At the site of inoculation of the mycelial blocks, wounds of the same size were made with a needle. Moistened absorbent paper was placed at the bottom of a suitably sized transparent storage box, and areca nut leaves were placed on top. Different concentrations of kaempferol solution diluted with ddH2O were dripped into the puncture sites, with the ddH2O-added solution serving as a blank control. Subsequently, XJ5 mycelial cakes of the same size were inoculated onto the puncture sites. A layer of ddH2O was sprayed onto the leaf surface, taking care not to form large water droplets, just enough to keep the leaf surface moist. The storage box was covered and placed in a 28 ℃ incubator. The leaf surface was sprayed with ddH2O twice daily to prevent the mycelial cakes from drying out. After 14 days, photographs were taken, and the lesion area was calculated using ImageJ software. Ten leaves were inoculated each time, and the experiment was repeated three times. Statistical results showed that kaempferol at 2 μg / mL significantly reduced the pathogenicity of *Pseudomonas aeruginosa*, and the lesion area was significantly smaller than that of leaves without kaempferol (p<0.05). Figure 2 ).

[0035] Example 4: Live Plant Test

[0036] Select healthy areca palm plants that grow uniformly, and gently wipe the leaves with a sterile cotton cloth or gauze moistened with sterile water to remove surface dust.

[0037] Sterile inoculation needles were used to prick wounds of the same size. Kaempferol solution at a concentration of 20 μg / mL was sprayed onto each wound, while the control group was sprayed with ddH2O. From the edge of a cultured *Pseudomonas aeruginosa* plate, *Pseudomonas aeruginosa* cakes were punched using a sterile punch of the same diameter. Using sterile tweezers, the mycelial side of the *Pseudomonas aeruginosa* cakes were placed tightly against the pre-sprayed wounds, ensuring complete coverage. ddH2O was sprayed onto the inoculation site, covered with a layer of plastic wrap, and then sealed with a transparent plastic bag for moisture retention. Disease incidence was recorded by photographing 30 days after inoculation.

[0038] The results showed that the incidence of areca nut disease was significantly reduced after pre-spraying with kaempferol compared to the control group. Figure 3 This indicates that kaempferol has a preventive effect on areca leaf spot disease.

Claims

1. The application of kaempferol in the control of areca leaf spot disease, characterized in that, The pathogen causing the areca leaf spot disease is Diaporthe pseudophoenicicola .

2. Kaempferol in the preparation of drugs for the prevention and treatment of diseases caused by... Diaporthe pseudophoenicicola Application in products that cause areca leaf spot disease.

3. The application according to claim 1, characterized in that, Kaempferol is used after being dissolved in sodium hydroxide solution.

4. The application according to claim 1, characterized in that, Suppress the Diaporthe pseudophoenicicola The effective concentration of kaempferol for mycelial growth is above 5 µg / mL.

5. The application according to claim 1, characterized in that, Reduce the Diaporthe pseudophoenicicola The effective concentration of kaempferol for the pathogenicity of areca nut is above 2 µg / mL.

6. A method for preventing and controlling areca leaf spot disease, characterized in that, The agent was sprayed onto the leaves of the areca nut; the control of areca nut leaf spot disease was to inhibit... Diaporthe pseudophoenicicola The mycelial growth of the agent; the preparation method of the agent is as follows: 500 mg of kaempferol powder is dissolved in 0.1 mol / L sodium hydroxide solution and the volume is adjusted to 10 mL to obtain a 50 mg / mL mother liquor, which is then stored at low temperature in a refrigerator at 4℃.

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