Application of trichoderma hamatum T21 antibacterial peptide BII in prevention and treatment of Chinese cabbage soft rot

Abstract

The invention relates to the technical field of plant disease prevention and treatment, in particular to application of a trichoderma hamatum T21 antibacterial peptide BII in prevention and treatment of Chinese cabbage soft rot, experiments prove that the trichoderma hamatum T21 antibacterial peptide BII has a remarkable antibacterial effect on pectobacterium carotovorum, the minimum inhibitory concentration is 102.4 mu g / mL, and the antibacterial peptide BII has a remarkable antibacterial effect on pectobacterium carotovorum. The Chinese cabbage soft rot caused by pectobacterium carotovorum can be effectively prevented and treated.

Description

Technical Field

[0001] The invention relates to the technical field of plant disease prevention and control, and particularly to application of antimicrobial peptide BII of hooked Trichoderma T21 in preventing and controlling soft rot of Chinese cabbage. Background Art

[0002] Cabbage soft rot is a serious bacterial disease caused by infection with Pectobacterium carotovorum subsp. carotovorum. It mainly harms the roots, stems, and leaves of cabbage, invading from the wounds at the base of the stalks, forming yellowish water-soaked lesions, and then the affected areas become slimy, softened, and rotten. Severely affected plants will also develop from the base of the stalks to the roots, causing the pith tissue at the rhizomes to rot. Soft rot can occur during the growth period, transportation process, and storage period of Chinese cabbage, seriously affecting the yield and quality of Chinese cabbage. When the disease is severe, the yield can be reduced by more than 50%, posing a serious threat to the safe production of the cabbage industry.

[0003] At present, the main prevention and control measures for cabbage soft rot include agricultural prevention, physical prevention, chemical prevention and integrated prevention, but these measures have limitations. Among them, chemical agents such as copper succinate, thiophanate-copper, quinolinone, etc. will cause environmental pollution, and the long-term use of antibiotics such as agricultural streptomycin, sinomenine, and kasugamycin will lead to drug resistance in pathogens. Microbial-derived agents have become a research hotspot due to their safety, high efficiency, economy, and environmental protection. However, in current research, there are few agents that can effectively prevent and control cabbage soft rot. Antimicrobial peptides, as a microbial-derived agent, can effectively prevent and control plant diseases and insect pests, which is in line with the green concept of sustainable agricultural development at this stage. With the national policy of "reducing the amount of pesticides and increasing their efficiency" and people's increasing attention to food safety issues, the demand for the development of an antimicrobial peptide drug that can effectively prevent and control cabbage soft rot is increasing. Summary of the invention

[0004] In view of the above technical problems, the present invention provides the use of the antimicrobial peptide BII of Trichoderma hooked T21 in preventing and treating soft rot of cabbage.

[0005] The present invention adopts the following technical solution:

[0006] The invention provides application of the antimicrobial peptide BII of hooked Trichoderma T21 in preventing and treating soft rot of Chinese cabbage.

[0007] The present invention discloses for the first time the application of the antimicrobial peptide BII of Trichoderma hamata T21 in preventing and treating soft rot of cabbage. The antimicrobial peptide BII of Trichoderma hamata T21 can be directly used to prevent and treat soft rot of cabbage, and the minimum inhibitory concentration for carrot soft rot pectinobacterium is 102.4 μg / mL, with significant antibacterial activity.

[0008] Furthermore, the pathogenic bacteria of the cabbage soft rot include Pectobacterium carotovora.

[0009] The present invention also provides the use of the hooked Trichoderma T21 antimicrobial peptide BII in preparing a product for preventing and treating soft rot of cabbage, wherein the active ingredient of the product is the hooked Trichoderma T21 antimicrobial peptide BII.

[0010] Furthermore, the cabbage soft rot pathogenic bacteria include carrot soft rot pectinobacterium.

[0011] Furthermore, the product is a pesticide or a preservative.

[0012] Furthermore, the product is in solution dosage form.

[0013] Furthermore, the solvent of the product is water or glycerol.

[0014] Furthermore, the concentration of the antimicrobial peptide BII of Trichoderma hooked T21 in the product is not less than 102.4 μg / mL.

[0015] The present invention has the following beneficial effects:

[0016] Experiments have shown that the antimicrobial peptide BII of the hooked Trichoderma T21 in the present invention has a significant antibacterial effect on carrot soft rot pectinobacterium, with a minimum inhibitory concentration of 102.4 μg / mL, which can effectively inhibit the growth of carrot soft rot pectinobacterium, providing a new product for preventing and controlling cabbage soft rot. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The inhibitory effect of the tested drugs on carrot soft rot pectinobacterium. Note: a is the antimicrobial peptide BII of Trichoderma hamata T21; b is DMSO; c is H2O; d is kanamycin.

[0018] Figure 2 To determine the minimum inhibitory concentration of the antimicrobial peptide BII from hooked Trichoderma T21 against carrot soft rot pectinobacterium. DETAILED DESCRIPTION

[0019] The present invention is described in detail below in conjunction with the accompanying drawings and specific examples, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0020] The amino acid sequence and preparation method of the antimicrobial peptide BII of Trichoderma hooked T21 used in the following examples are recorded in Chinese Patent Publication No. CN117986326A.

[0021] The pathogenic bacteria Pectobacterium carotovora in the following examples are specifically Eack1, which was isolated from rotten leaves of cabbage and donated to the Disease Group of the Vegetable and Flower Research Institute of the Chinese Academy of Agricultural Sciences.

[0022] Implementation 1

[0023] 1. Determination of antibacterial activity

[0024] 1. Activation of pathogens. Take 10 μL from the frozen tube of pathogenic bacteria and drop it in the center of the LB culture dish. Then use a spreading stick to cover the entire culture medium and place it in a 28°C incubator for 24 hours.

[0025] 2. Prepare bacterial plates. Use a toothpick to pick a single colony and place it in a 50mL centrifuge tube filled with LB liquid culture medium. Place it in a shaker at 28°C and 220rpm for 12 hours. Use a spectrophotometer to measure the OD value of the bacterial culture solution, and dilute it with LB culture solution to adjust the OD value. 600 = 0.2. Pipette 1 mL of bacterial solution and add 49 mL of warm liquid LB solid culture medium until the final concentration of the bacterial solution is 2%, mix thoroughly, and pour the plate.

[0026] 3. Add the drug to be tested and the control. Draw a "cross" with the center of the culture dish as the intersection point, take two points on each straight line, and make the distance from the four points to the intersection point 2mm. Use the part of the disposable syringe without the needle to make holes at the four points, and then drop the sample into the hole. Add the antimicrobial peptide BII of Trichoderma hooked T21, negative control, blank control and positive control in a clockwise direction.

[0027] 4. Cultivation observation and record. Place in a 28℃ incubator and observe every 12 hours. Take photos and record when obvious inhibition zones appear.

[0028] 5. By observing the growth of bacteria, the experimental results are as follows Figure 1 As shown in the figure, the growth of pathogenic bacteria near the orifice where the antimicrobial peptide BII of hooked Trichoderma T21 was added was inhibited, and an obvious inhibition zone appeared. The results showed that the antimicrobial peptide BII of hooked Trichoderma T21 had a significant inhibitory effect on carrot soft rot pectinobacterium.

[0029] 2. Minimum inhibitory concentration determination

[0030] 1. Use a sterile pipette to add 180 μL of pathogenic bacteria solution to the sample wells in the first column, and then add 100 μL of bacteria solution to the sample wells in columns 2 to 11. Column 11 is the positive control group with bacteria solution, and column 12 is added with 100 μL of liquid LB medium as a blank control. Rows 1 to 3 are repeated three times, and row 4 is the positive drug control group.

[0031] 2. Prepare the stock solution of the drug to be tested and the positive control kanamycin at a concentration of 2.048 mg / mL. Pipette 20 μL and add it to the first column, then dilute it half-fold from the sample well of the first column to other columns: adjust the pipette to 100 μL, and after evenly aspirating the sample wells of the first column, take 100 μL of the mixed solution and move it to the next column, and then proceed to the 10th column. The 100 μL mixed solution drawn from the 10th column is discarded. The drug concentrations in columns 1 to 10 are 204.8 μg / mL, 102.4 μg / mL, 51.2 μg / mL, 25.6 μg / mL, 12.8 μg / mL, 6.4 μg / mL, 3.2 μg / mL, 1.6 μg / mL, 0.8 μg / mL, and 0.4 μg / mL, respectively.

[0032] 3. Observe and record. Place the 96-well plate with the sample in the incubator and observe every 6 hours. After 24 hours of incubation, add 10 μL of PrestoBlue resazurin dye to each well for dyeing. After dyeing for 30 minutes, find the well at the border between blue and red to determine the minimum inhibitory concentration. Take photos and record.

[0033] 4. After treatment with the antimicrobial peptide BII of Trichoderma hooked, add the cell activity colorimetric solution. Figure 2 As shown, the 1st to 2nd columns of the 1st to 3rd rows are blue, the 3rd column is purple, and the 4th to 10th columns are red. The 4th row is the positive control kanamycin, the 1st to 6th columns are blue, the 7th column is purple, and the 8th to 10th columns are red, indicating that there are surviving bacterial cells starting from the 3rd column. The minimum inhibitory concentration of the antimicrobial peptide BII of the hooked Trichoderma T21 against the soft rot of cabbage is the concentration in the 2nd column, that is, the minimum inhibitory concentration is 102.4μg / mL.

[0034] The hooked Trichoderma T21 antimicrobial peptide BII is separated from the hooked Trichoderma T21 with a preservation number of CGMCC NO.10923. It has been proved that it has antibacterial activity against wild campestris and can be used to prevent and treat cabbage black rot, but whether it has the function of preventing and treating cabbage soft rot is still uncertain. The present invention detects the antibacterial effect of the hooked Trichoderma T21 antimicrobial peptide BII on carrot soft rot pectinobacterium through an experiment to detect the antibacterial effect of the hooked Trichoderma T21 antimicrobial peptide BII on carrot soft rot pectinobacterium, and the minimum inhibitory concentration is 102.4 μg / mL, which can effectively inhibit the growth of carrot soft rot pectinobacterium, and provides a new product for preventing and treating cabbage soft rot.

[0035] It should be noted that when the claims of the present invention involve numerical ranges, it should be understood that the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. In order to avoid redundancy, the present invention describes a preferred embodiment.

[0036] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0037] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. Application of the antimicrobial peptide BII from Trichoderma hookedii T21 in the prevention and control of soft rot of cabbage.

2. The use of the antimicrobial peptide BII of hooked Trichoderma T21 in preventing and treating soft rot of cabbage according to claim 1, characterized in that: The pathogenic bacteria of the Chinese cabbage soft rot include Pectobacterium carotovora.

3. Application of the antimicrobial peptide BII of Trichoderma hooked T21 in the preparation of products for preventing and treating soft rot of Chinese cabbage, characterized in that: The active ingredient of the product is the antimicrobial peptide BII of Trichoderma hamata T21 as claimed in claim 1.

4. The use of the antimicrobial peptide BII of Trichoderma hooked T21 according to claim 3 in the preparation of a product for preventing and treating soft rot of Chinese cabbage, characterized in that: The cabbage soft rot pathogenic bacteria include carrot soft rot pectinobacterium.

5. The use of the antimicrobial peptide BII of Trichoderma hooked T21 according to claim 4 in the preparation of a product for preventing and treating soft rot of Chinese cabbage, characterized in that: The product described is a pesticide or a preservative.

6. The use of the antimicrobial peptide BII of Trichoderma hooked T21 according to claim 5 in the preparation of a product for preventing and treating soft rot of Chinese cabbage, characterized in that: The product is in solution dosage form.

7. The use of the antimicrobial peptide BII of Trichoderma hooked T21 according to claim 6 in the preparation of a product for preventing and treating soft rot of Chinese cabbage, characterized in that: The solvent of the product is water or glycerol.

8. The use of the antimicrobial peptide BII of Trichoderma hooked T21 according to claim 7 in the preparation of a product for preventing and treating soft rot of Chinese cabbage, characterized in that: The concentration of the antimicrobial peptide BII of hooked Trichoderma T21 in the product is not less than 102.4 μg / mL.

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