Application of Epigallocatechin Gallate (EGCG) in the Prevention and Treatment of Potato Late Blight

Abstract

The application belongs to the technical field of agricultural plant protection, and discloses application of epigallocatechin gallate in prevention and treatment of potato late blight. The application discloses application of epigallocatechin gallate in preparation of a product for preventing and treating potato late blight. By formulating the active ingredient into a product with an effective use concentration of 0.01-0.06 mg / mL, the application can significantly inhibit the mycelium growth and spore germination of the pathogenic Phytophthora. The application uses a natural extract as a core active ingredient, has the advantages of environmental friendliness, novel action mechanism, significant prevention and treatment effect, and plant stress resistance induction function, and provides a new technical means for green prevention and treatment of potato late blight.

Description

Technical Field

[0001] This invention belongs to the field of agricultural plant protection technology, specifically relating to the application of epigallocatechin gallate (EGCG) in the control of potato late blight. Background Technology

[0002] Potato late blight, caused by the oomycete *Phytophthora infestans*, seriously threatens the yield and quality of potatoes, the fourth largest staple food crop. This disease spreads rapidly and is highly destructive, making it one of the main factors restricting the development of the potato industry.

[0003] Currently, the control of this disease in agricultural production mainly relies on chemical fungicides. However, the long-term and excessive use of chemical pesticides has not only led to severe drug resistance and cross-resistance in pathogens, resulting in a year-on-year decline in control effectiveness, but also caused pesticide residues, environmental pollution, and threats to the safety of non-target organisms. Therefore, developing novel, highly efficient, low-toxicity, and environmentally friendly plant-derived fungicides to overcome the shortcomings of existing chemical pesticides has become a hot topic and urgent need in agricultural plant protection research.

[0004] Epigallocatechin gallate is the most abundant and biologically active core component of tea polyphenols. It has been proven to have broad-spectrum biological activity in the pharmaceutical field, but there are no publicly available technical solutions for its use in agricultural disease control, especially for the control of potato late blight. Summary of the Invention

[0005] Based on this, the purpose of this invention is to provide the application of epigallocatechin gallate in the control of potato late blight, that is, to use it to control potato late blight caused by pathogenic Phytophthora, so as to overcome the problems of existing chemical pesticides that are prone to resistance, environmental pollution and pesticide residues.

[0006] The present invention solves the above-mentioned technical problems through the following technical means:

[0007] In a first aspect, the present invention provides the application of epigallocatechin gallate in the preparation of products for the prevention and control of potato late blight.

[0008] Preferably, the potato late blight is caused by Phytophthora.

[0009] Preferably, the purity of the epigallocatechin gallate is not less than 90%.

[0010] Preferably, the product is used to inhibit the mycelial growth of Phytophthora infestans, inhibit the spore germination of Phytophthora infestans, or reduce the diameter of lesions on potato leaves infected with Phytophthora infestans.

[0011] Secondly, the present invention provides a plant-derived product for the prevention and control of potato late blight, wherein the active ingredient of the product is epigallocatechin gallate.

[0012] Preferably, the product further includes agriculturally acceptable adjuvants or carriers, wherein the adjuvants include one or more of dispersants, wetting agents, adhesives, or stabilizers.

[0013] Preferably, the dosage form of the product is an aqueous solution, an emulsifiable concentrate, a wettable powder, or a water-dispersible granule.

[0014] Preferably, the effective concentration of the epigallocatechin gallate after dilution of the product is 0.01-0.06 mg / mL.

[0015] More preferably, the effective concentration of the epigallocatechin gallate after dilution of the product is 0.02-0.05 mg / mL.

[0016] Thirdly, the present invention provides a method for preventing and controlling potato late blight, comprising spraying a product containing the active ingredient onto potato plants or leaves.

[0017] Preferably, the plant-derived product is also used to induce potato plants to enhance their disease resistance after spraying.

[0018] The beneficial effects of this invention are:

[0019] 1. The active ingredient epigallocatechin gallate used in this invention is derived from natural tea leaves and is a plant-based natural product. This ingredient is characterized by its easy degradation and low residue in the natural environment, exhibiting extremely high safety performance for humans, livestock, and non-target organisms in the ecosystem, fully meeting the development needs of modern green agriculture and environmentally friendly pesticides.

[0020] 2. Unlike traditional chemical fungicides with a single site of action, this invention utilizes the unique mechanism of action of epigallocatechin gallate ester, exerting its efficacy by disrupting the cell membrane permeability of pathogenic Phytophthora or interfering with its specific metabolic pathways. This novel mechanism of action makes it less likely for this component to develop cross-resistance with currently available conventional chemical pesticides, providing an effective technical means to solve the increasingly serious problem of drug resistance in pathogenic Phytophthora.

[0021] 3. This invention exhibits remarkable bioactivity in controlling potato late blight, effectively inhibiting the mycelial growth and spore germination of the pathogenic fungus *Phytophthora infestans*. Experimental data show that when the concentration of epigallocatechin gallate is 0.06 mg / mL, the inhibition rate on mycelial growth is as high as 85.94%, EC50. 50The concentration was 0.0182 mg / mL; EGCG treatment significantly delayed disease development and resulted in smaller lesions. The 10 mg / L treatment group achieved a control efficacy of over 68%. It significantly delayed disease development and substantially reduced lesion area, demonstrating significant efficacy and strong specificity.

[0022] 4. In addition to its direct bactericidal effect, epigallocatechin gallate, as a potent natural antioxidant, also provides multiple plant health benefits after spraying. It can induce a systemic stress response in potato plants, enhance their immunity, and provide some preservation, thus effectively maintaining the physiological activity of the crop and improving its growth quality while controlling diseases. Attached Figure Description

[0023] Figure 1 EGCG inhibits the growth of Late Pythium blight strains on MZ plates;

[0024] Figure 2 The effect of EGCG treatment on the activity of pathogenic Phytophthora hyphae and spores;

[0025] Figure 3 This involves nucleic acid leakage and changes in cell membrane potential in pathogenic Phytophthora after EGCG treatment;

[0026] Figure 4 The indoor control effect of EGCG on potato late blight;

[0027] Figure 5 It is the growth-promoting effect of EGCG on Arabidopsis thaliana. Detailed Implementation

[0028] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments.

[0029] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by those skilled in the art. The technical terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the scope of the invention; unless otherwise specified, all raw materials, reagents, instruments, and equipment used in this invention are commercially available or can be prepared by existing methods.

[0030] This invention addresses existing problems by providing the application of epigallocatechin gallate in the preparation of products for the prevention and control of potato late blight.

[0031] Example 1: Determination of the inhibitory effect of EGCG on the growth of pathogenic Phytophthora mycelia

[0032] Test reagent: EGCG standard (purity ≥98%), dissolved in a small amount of sterile water to prepare a stock solution for later use.

[0033] Experimental Method: The mycelial growth rate method was used. EGCG stock solution was added to melted rye medium at specific ratios to prepare plates with final concentrations of 0 (CK), 0.01, 0.02, 0.03, 0.04, 0.05, and 0.06 mg / mL. A 5 mm diameter *Phytophthora infestans* scab was inoculated in the center of each plate. After incubation at 20°C for 7-8 days, the colony diameter was measured using the cross-crossing method, and the inhibition rate was calculated.

[0034] The results show (see) Figure 1 As the concentration of EGCG increased, its inhibitory effect on mycelial growth gradually strengthened. At an EGCG concentration of 0.06 mg / mL, the inhibition rate reached 85.94%, EC... 50 The value was 0.0182 mg / mL.

[0035] Example 2: Effect of EGCG on the permeability of pathogenic Phytophthora cell membranes

[0036] (1) Trypan blue staining

[0037] Experimental method: Take 3 mL of the prepared trypan blue staining solution from a 14-day-old pathogenic *Phytophthora indicum* strain and spread it evenly on a petri dish. After standing for 10 minutes, gently pour off the staining solution, carefully add water, and let it stand for 10 minutes before removing the staining solution. Photograph the staining of the entire petri dish, and pick hyphae and sporangia for microscopic examination and photographing under an optical microscope.

[0038] Experimental Results: Trypan blue staining results showed that, compared with the water control, EGCG treatment significantly inhibited the activity of *Phytophthora infestans* hyphae and sporangia. Furthermore, the activity of *Phytophthora infestans* and sporangia decreased with increasing concentration (see [link to results]). Figure 2 ).

[0039] (2) Nucleic acid and protein leakage and changes in cell membrane potential

[0040] Experimental Methods: Pathogenic *Phytophthora* strains grown for 14 days were collected and placed in 10 mL centrifuge tubes. EGCG was prepared into working solutions with final concentrations of 0 (CK), 0.05, and 0.1 mg / mL. 5 mL of each solution was added to the 10 mL centrifuge tubes from the previous step. After mixing, the DNA, RNA, and protein content in the supernatant of the three treatments was measured at 0 h, 5 h, and 10 h. This experiment was repeated three times. Pathogenic *Phytophthora* strains grown for 14 days were collected and placed in 10 mL tubes, one plate per tube (three replicates). The samples were incubated at room temperature for 3 h. Standardization was performed using the standard solution. The A value of the first batch was measured, and the average value was calculated. The samples were boiled for 25 min, and timing was started after the water boiled. After the samples cooled to room temperature, the B value was measured, and the average value was calculated. Conductivity formula: A / B × 100%.

[0041] Experimental Results: By measuring the DNA and RNA content of *Phytophthora infestans* treated at different time points, the results showed that the leakage of DNA and RNA increased with increasing EGCG concentration, indicating that EGCG treatment increased the membrane permeability of *Phytophthora infestans*, leading to leakage of its contents. Regarding protein leakage, the leakage also increased with increasing EGCG concentration. Conductivity results showed that compared to water treatment, EGCG treatment caused more significant changes in the cell membrane potential of *Phytophthora infestans*, indicating more severe damage to the cell membrane (see...). Figure 3 ).

[0042] Example 3: Indoor control effect of EGCG on potato late blight

[0043] Experimental Method: Healthy, uniformly sized potato leaves were collected, retaining approximately 1 cm of petiole. A sterile, water-moistened cotton ball was wrapped around the petiole, and the underside of the leaf was placed on six layers of sterile, water-moistened crepe paper. Two concentrations of EGCG stock solution were prepared: 0.5 mg / mL and 1 mg / mL. These solutions were poured into small spray bottles and sprayed onto the underside of the leaves until fine water droplets formed that did not disperse. The control treatment was sprayed with sterile water. After placing the treated leaves in a humidified incubator for 12 hours, the water droplets on the underside of the leaves were wiped off with crepe paper, and 10 μL of a suspension of Phytophthora zoospores (1×10⁻⁶) was dropped into the center of the leaf. 5 (Number of lesions per mL). Incubate at 20°C in the dark for 24 hours. On the second day, transfer the incubator to 20°C under 16 hours of light / 8 hours of darkness for 3-5 days. Measure the diameter of the lesions afterward.

[0044] Results: Leaves in the control group developed the disease rapidly after inoculation, with lesions expanding quickly. Leaves treated with EGCG showed significantly delayed disease development and smaller lesions (see...). Figure 4 Among them, the 1 mg / mL treatment group achieved a control efficacy of over 68%.

[0045] Example 4: The promoting effect of EGCG on plant growth

[0046] Experimental Methods: To investigate the effects of EGCG on plant growth, Arabidopsis thaliana was cultured in 1 / 2 MS medium containing EGCG. The root growth of Arabidopsis seedlings was observed to assess the effect of EGCG on Arabidopsis growth. Wild-type seeds were first cultured vertically and aseptically in a medium without EGCG for approximately 5 days until most seeds developed roots approximately 5 mm in length. Seedlings with similar root length and growth status were selected and transferred to media containing different concentrations of EGCG (1, 10, 50, 100, and 1 μM). After approximately 7 days of vertical aseptic culture, photos were taken and root length and fresh weight were recorded.

[0047] Experimental results: The results showed that 1 μM EGCG treatment significantly promoted the growth of mustard roots and increased fresh weight.

[0048] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or essential characteristics. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, all variations falling within the meaning and scope of equivalents of the claims are intended to be included within the present invention. No reference numerals in the claims should be construed as limiting the scope of the claims.

Claims

1. Application of epigallocatechin gallate in the preparation of products for the prevention and control of potato late blight.

2. The application according to claim 1, characterized in that, The potato late blight is caused by the pathogenic fungus Phytophthora.

3. The application according to claim 1, characterized in that, The purity of the epigallocatechin gallate is not less than 90%.

4. The application according to claim 1, characterized in that, The product is used to inhibit the mycelial growth of Phytophthora infestans, inhibit the spore germination of Phytophthora infestans, or reduce the diameter of lesions on potato leaves infected with Phytophthora infestans.

5. A plant-derived product for controlling potato late blight, characterized in that, The active ingredient in the product is epigallocatechin gallate.

6. The plant-derived product according to claim 5, characterized in that, The product also includes agriculturally acceptable adjuvants or carriers, wherein the adjuvants include one or more of dispersants, wetting agents, adhesives, or stabilizers.

7. The plant-derived product according to claim 5 or 6, characterized in that, The product is available in the form of an aqueous solution, emulsifiable concentrate, wettable powder, or water-dispersible granules.

8. The plant-derived product according to claim 5, characterized in that, The effective concentration of the epigallocatechin gallate after dilution of the product is 0.01-0.06 mg / mL.

9. A method for controlling potato late blight, characterized in that, This includes spraying the product containing the active ingredient of claim 5 onto potato plants or leaves.

10. The method according to claim 9, characterized in that, The plant-derived product, after being sprayed, is also used to induce potato plants to enhance their disease resistance.

Images