Application of luteolin in prevention and treatment of areca leaf spot disease

Abstract

The invention discloses application of luteolin to prevention and treatment of areca leaf spot disease, and belongs to the technical field of biological prevention and treatment. By adding luteolin into the culture medium, the growth rate of phomopsis is remarkably reduced. The areca seeds are pretreated by luteolin solution and then inoculated with pathogenic fungi, compared with unpretreated plants, germs show lower pathogenicity, effective prevention and treatment of plant pathogenic fungal diseases are achieved, meanwhile, the effects of environmental pollution and harm to human bodies are reduced, and compared with traditional chemical pesticides, the areca seeds have the advantages that the cost is low, and the effect is obvious. And the toxicity to a human body and the environment is lower. Experiments show that when the final concentration of luteolin reaches 50 [mu] g / mL, the growth rate of phomopsis is significantly reduced; and when the concentration of the luteolin reaches 0.2 mu g / mL, the pathogenicity of phomopsis can be obviously reduced. The invention provides a novel biological prevention and control method for preventing and controlling the leaf spot disease of the areca seeds.

Description

Technical Field

[0001] This invention belongs to the field of biological control technology, specifically, it relates to the application of luteolin in the control of areca leaf spot disease. Background Technology

[0002] By *Pseudomonas stolonifera* ( Diaporthe pseudophoenicicola Leaf spot disease caused by *Pseudomonas stolonifera* is a major disease affecting areca palms. Severely infected leaves wither and droop over large areas, eventually leading to the death of the areca palm leaves. This disease is more prevalent in areca palm orchards with high humidity, and is also more severe in young orchards. It seriously impacts the sustainable development of the areca palm industry. Therefore, in-depth research into biological control methods for *Pseudomonas stolonifera* leaf spot is of great significance for disease control.

[0003] For a long time, chemical agents (such as benzimidazole) have played an important role in controlling plant fungal diseases and reducing agricultural production losses, and have been widely accepted for a considerable period. However, research has found that long-term and frequent use can easily lead to drug resistance in pathogens, and their residues on plants can pose a health hazard to humans and animals. Therefore, it is urgent to find novel compounds that are highly effective, low in toxicity, environmentally friendly, and suitable for sustainable development in controlling plant fungal diseases. In recent years, the use of natural products to control plant fungal diseases has gradually become a research hotspot. Plant extracts contain a variety of natural products, possess diverse chemical structures, unique biological activity mechanisms, and the ability to enhance crop resistance. They also have good environmental compatibility, delay the development of drug resistance, and promote healthy crop growth, making them an important resource for pharmaceutical and pesticide development. Developing plant-derived pesticides based on plant extracts for the prevention and treatment of plant fungal diseases is of paramount importance. Summary of the Invention

[0004] The purpose of this invention is to provide an application of luteolin for the control of areca leaf spot disease, which solves two key problems existing in the control of areca leaf spot disease by traditional chemical pesticides: first, the pathogen is prone to developing resistance; second, chemical pesticide residues cause harm to the environment and human health. It provides a new way to control plant diseases with high efficiency, low toxicity and environmental friendliness, and lays the foundation for the development of natural and environmentally friendly plant-derived pesticides.

[0005] To achieve the above-mentioned objectives, the present invention adopts the following technical solution: This invention provides the application of luteolin in the prevention and control of areca leaf spot disease caused by *Pseudomonas stolonifera*.

[0006] Furthermore, the concentration of luteolin used is 0.2-50 μg / mL.

[0007] Furthermore, the application includes pretreatment of areca leaves with luteolin before inoculation with *Pseudomonas stolonifera*.

[0008] Furthermore, the pretreatment includes dripping or spraying a luteolin solution onto the wounds on the areca leaves.

[0009] Furthermore, the luteolin controls areca leaf spot disease by inhibiting the growth rate of *Pseudomonas stolonifera*.

[0010] Furthermore, the luteolin controls areca leaf spot disease by reducing the pathogenicity of Pseudomonas stolonifera to areca.

[0011] Furthermore, the *Pseudomonas stolonifera* is... Diaporthe pseudophoenicicola.

[0012] Furthermore, the luteolin is prepared by dissolving luteolin powder in 0.1 mol / L NaOH solution to form a mother liquor, which is then diluted with ddH2O to the required concentration before use.

[0013] Furthermore, the concentration of the luteolin stock solution is 50 mg / mL, and it is aliquoted and stored in a refrigerator at 4°C.

[0014] Beneficial Effects: This invention utilizes luteolin, a natural flavonoid compound. Adding luteolin at a concentration of 50 μg / mL to the culture medium significantly inhibits the growth rate of *Pseudomonas aeruginosa*. Pretreatment of areca nut leaves with luteolin at a concentration as low as 0.2 μg / mL significantly reduces the pathogenicity of *Pseudomonas aeruginosa* and noticeably reduces the area of ​​lesions. This biological control method not only effectively controls areca nut leaf spot disease but also avoids the negative impacts of chemical pesticides, providing a new, highly efficient, low-toxicity, and environmentally friendly approach to plant disease control, laying the foundation for the development of natural and environmentally friendly plant-derived pesticides. Attached Figure Description

[0015] Figure 1 Figure 1 shows the effect of different concentrations of luteolin on the growth rate of Pseudomonas aeruginosa XJ1.

[0016] Figure 2 The effect of different concentrations of luteolin on the pathogenicity of Areca catechu was shown in the figure.

[0017] Figure 3 This image shows the control effect of luteolin on leaf spot disease in areca nut plants. Detailed Implementation

[0018] To enable those skilled in the art to better understand the technical solutions of this invention, the present application will be further described in detail below with reference to embodiments.

[0019] The *Pseudomonas stolonifer* XJ1 involved in this invention is a fungus containing *Pseudomonas stolonifer* (… Phomopsis asparagi The strain obtained after detoxification treatment of XJ5 (using ribavirin to detoxify the hyphal tips of XJ5) was preserved in our laboratory. Luteolin was purchased from Ron Reagent Company (catalog number R005493).

[0020] Example 1: Preparation of luteolin stock solution Weigh 500 mg of luteolin powder, dissolve it in 0.1 mol / L NaOH solution, and bring the volume to 10 mL to obtain a 50 mg / mL luteolin stock solution. Aliquot the stock solution and store it at 4°C. Dilute with ddH₂O to the required concentration before use in experiments.

[0021] Example 2: Effect of luteolin on the growth rate of Pseudomonas stearans PDA medium was heated and melted, and luteolin stock solution was added to achieve final concentrations of 0, 0.2, 2, and 50 μg / mL. Five plates were prepared for each concentration of PDA containing luteolin. Activated strain *Pseudomonas aeruginosa* XJ1 was used to create 0.5 cm diameter mycelial discs, which were then inoculated onto the different concentrations of PDA plates with the mycelial side down. Three independent replicate experiments were performed. Colony diameter was measured and photographed after 4 days. Growth rate = (measured diameter - mycelial disc diameter) / number of incubation days.

[0022] The results showed that when the concentration of luteolin reached 50 μg / mL, it significantly inhibited the growth rate of XJ1 (p<0.05). Figure 1 ).

[0023] Example 3: Luteolin reduces the pathogenicity of *Pseudomonas stylosa*. For the *Pseudomonas stolonifer* strain, the in vitro leaf method was used. The wild-type *Pseudomonas stolonifer* strain XJ1 was activated on a PDA plate for 3-4 days. Several mycelial blocks were punched at the edge of the colony using a puncher (5 mm in diameter). Areca leaves of uniform size, consistent growth period, and no surface damage or lesions were collected. The leaf surface was rinsed with ddH2O and dried. At the site of inoculation of mycelial blocks, wounds of the same size were punctured with a needle.

[0024] Place a damp absorbent paper at the bottom of a suitably sized transparent storage box, then place areca leaves inside. Drip different concentrations of luteolin solution diluted with ddH2O onto the puncture site, with the ddH2O-added solution serving as a blank control.

[0025] Then, inoculate the punctured areas with XJ1 mycelial cakes of the same size. Spray a layer of ddH2O on the leaf surface, being careful not to form large water droplets; just keep the leaf surface moist. Cover the storage box and place it in a 28 ℃ incubator, spraying the leaf surface with ddH2O twice a day to prevent the mycelial cake from drying out. After 14 days, take photos and calculate the lesion area using ImageJ software. Inoculate 10 leaves each time, and repeat the experiment 3 times.

[0026] Statistical results showed that luteolin at a concentration of 0.2 μg / mL significantly reduced the pathogenicity of *Pseudomonas aeruginosa*, and the lesion area was significantly smaller than that without luteolin (p<0.05). Figure 2 ).

[0027] Example 4: Live Plant Test 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.

[0028] Sterilized inoculation needles were used to prick wounds of the same size. A 50 μg / mL luteolin solution was sprayed onto the wounds, while the control group was sprayed with ddH2O. From the edge of the cultured *Pseudomonas aeruginosa* agar plates, *Pseudomonas aeruginosa* cakes were punched with a sterilized punch of the same diameter. Using sterilized tweezers, the mycelial side of the *Pseudomonas aeruginosa* cakes were placed tightly against the pre-sprayed wounds, ensuring that the *Pseudomonas aeruginosa* cakes completely covered the wounds. ddH2O was sprayed onto the inoculation site, a layer of plastic wrap was applied, and then a transparent plastic bag was placed over it to maintain moisture. The disease incidence was recorded by taking photos 30 days after inoculation.

[0029] The results showed that the incidence of areca nut disease was significantly reduced after pre-spraying with luteolin compared to the control group. Figure 3 This indicates that luteolin has a preventive effect on areca leaf spot disease.

Claims

1. The application of luteolin in the control of areca leaf spot disease, characterized in that, The areca leaf spot disease is caused by Pseudomonas stolonifera, and the concentration of luteolin used is 0.2-50 μg / mL.

2. The application according to claim 1, characterized in that, The application involves pretreating areca leaves with luteolin before inoculating them with Pseudomonas stolonifera.

3. The application according to claim 2, characterized in that, The pretreatment includes dripping or spraying a luteolin solution onto the wounds on the areca leaves.

4. The application according to any one of claims 1-3, characterized in that, The luteolin controls areca leaf spot disease by inhibiting the growth rate of Pseudomonas stolonifera.

5. The application according to any one of claims 1-3, characterized in that, The luteolin mentioned above controls areca leaf spot disease by reducing the pathogenicity of Pseudomonas stolonifera to areca.

6. The application according to claim 1, characterized in that, The *Pseudomonas* species is Diaporthe pseudophoenicicola.

7. The application according to claim 1, characterized in that, The luteolin is prepared by dissolving luteolin powder in 0.1 mol / L NaOH solution to form a mother liquor, which is then diluted with ddH2O to the required concentration before use.

8. The application according to claim 7, characterized in that, The concentration of the luteolin stock solution was 50 mg / mL, and it was aliquoted and stored in a refrigerator at 4°C.

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