Wound sealant for preventing and treating kiwifruit tree canker as well as preparation method and application of wound sealant

Abstract

The invention relates to the field of biopesticides, and discloses a wound sealant for preventing and treating kiwifruit tree canker as well as a preparation method and application of the wound sealant. The wound sealant provided by the invention is prepared from polylysine, egg white lysozyme, activated carbon, white vaseline, glycerin monostearate, peregal, octadecanol, glycerol and water. The invention also provides a preparation method of the wound sealant. The wound sealing agent has the effects of sealing trimmed wounds and frostbite wounds, can promote wound healing, enrich pathogenic bacteria and kill the pathogenic bacteria, does not cause generation of drug-resistant bacteria, is non-toxic and harmless to the environment, is degradable, is an environment-friendly, green and sustainable development biological agent, can achieve the sealing and sterilizing effects with the optimal effect, and has a wide application prospect. The effective prevention and treatment of the kiwifruit canker are realized.

Description

Technical Field

[0001] The present disclosure relates to the field of biological pesticides, and in particular to a wound sealant for preventing and treating kiwifruit tree canker, and a preparation method and application thereof. Background Art

[0002] Kiwifruit is a crop of the genus Actinidia in the family Actinidia. The fruit is rich in nutrition, has a rich and sweet taste, is rich in vitamin C, and has certain antioxidant effects. Kiwifruit can also be processed into kiwifruit juice, preserved fruit, canned fruit and other products, which have good edible and economic value. In addition, kiwifruit also has certain medicinal value. The fruit, leaves, stems, etc. of kiwifruit can be used as Chinese herbal medicine. With the development of science and technology and economy, the demand for kiwifruit is increasing day by day.

[0003] However, kiwifruit canker has become a major problem facing kiwifruit cultivation. Kiwifruit canker spreads quickly and is highly secretive. Once an orchard breaks out, it will cause huge losses. Kiwifruit canker is caused by Pseudomonas syringae pv. Actinidiae (PSA) infecting wounds on the trunk and main vine of kiwifruit trees. The bacteria multiply and exude white secretions at the lesion site, and the infected site turns into a rust-red lesion in the later stage. The presence of the lesion will cause the nutrient transport of kiwifruit seedlings to be blocked, affecting the quality of the fruit.

[0004] As for the existing kiwifruit canker control technology, chemical agents are mainly inorganic copper preparations and organic ketone preparations. Frequent and excessive use of chemical agents will lead to serious heavy metal pollution of soil and water and even the ecological environment; the abuse of antibiotics will lead to the emergence of drug resistance; and the high cost of biological control technology is not suitable for large-scale use. Therefore, an environmentally friendly, green and sustainable kiwifruit canker control agent is urgently needed.

[0005] In addition, kiwifruit canker is mainly prevented by pre-dose medication before the outbreak and medication after the outbreak, usually by spraying and drenching, which requires a large amount of pesticides. Since kiwifruit canker is mainly caused by pathogenic bacteria invading pruning wounds and frostbite wounds of fruit trees, avoiding pathogenic bacteria infestation of pruning wounds and frostbite wounds of fruit trees is a potential method for preventing and controlling kiwifruit canker from the first step of PSA invading kiwifruit seedlings. Summary of the invention

[0006] In view of this, the present invention aims to provide a wound sealant for preventing and treating kiwifruit tree canker disease, which has the function of sealing pruning wounds and frostbite wounds, promoting wound healing, and enriching and killing pathogenic bacteria. At the same time, it does not induce the production of drug-resistant bacteria, does not require large-scale application of drugs, is environmentally friendly, and has huge application potential and economic value.

[0007] Another object of the present invention is to provide a preparation method of the wound sealant and its application in preventing and treating kiwifruit tree canker.

[0008] In order to achieve the above object, the present invention provides the following technical solutions:

[0009] A wound sealant for preventing and treating kiwifruit tree canker disease comprises polylysine, egg white lysozyme, activated carbon, white vaseline, glyceryl monostearate, peregal, octadecyl alcohol and glycerol.

[0010] Preferably, the weight ratio of polylysine, egg white lysozyme, activated carbon, white vaseline, glyceryl monostearate, peregal, stearyl alcohol and glycerol is 1:0.25:0.25:1.25:0.24:0.24:0.54:2.02.

[0011] More preferably, the wound sealant is a cream or an ointment.

[0012] The present invention also provides a method for preparing the wound sealant, the method comprising the following steps:

[0013] (1) placing activated carbon, white vaseline, glyceryl monostearate, peregal, and octadecyl alcohol in a centrifuge tube and heating in a water bath until completely dissolved to obtain a blend;

[0014] (2) adding glycerol to the blend;

[0015] (3) Dissolve poly-lysine in ultrapure water and preheat in a water bath;

[0016] (4) adding the blend into a preheated poly-lysine aqueous solution and heating while stirring until completely emulsified;

[0017] (5) Cooling the reactants, adding egg white lysozyme and continuing stirring to obtain the wound sealing agent.

[0018] Preferably, in the preparation method, the weight ratio of polylysine, egg white lysozyme, activated carbon, white vaseline, glyceryl monostearate, peregal, stearyl alcohol and glycerol is 1:0.25:0.25:1.25:0.24:0.24:0.54:2.02.

[0019] Meanwhile, the invention also provides application of the wound sealant in preventing and treating kiwifruit tree canker.

[0020] Preferably, the pathogenic bacteria of kiwifruit canker disease is Pseudomonas syringaepv. Actinidiae.

[0021] Preferably, the wound sealant is applied to a wound of a kiwi fruit tree.

[0022] More preferably, the kiwifruit tree wound is a pruning wound or a frostbite wound of the kiwifruit tree.

[0023] Compared with the prior art, the present invention has the following advantages:

[0024] (1) The present inventors have discovered that poly-lysine exhibits excellent inhibitory effect on the growth of PSA bacteria in vitro.

[0025] (2) The wound sealant containing polylysine provided by the present invention has the function of sealing pruning wounds and frostbite wounds, promoting wound healing, and enriching and killing pathogenic bacteria. At the same time, it does not induce the production of drug-resistant bacteria, and can achieve the best sealing and bactericidal effects, thereby achieving effective prevention and treatment of kiwifruit canker.

[0026] (3) The wound sealant containing polylysine provided by the present invention is non-toxic and harmless to the environment, is degradable, and will not cause the problem of pesticide residues polluting farmland, which is in line with my country's green environmental protection and sustainable development strategy.

[0027] (4) The present invention deeply combines the field demand and the existing problems in production practice research, has certain practicality and promotion potential, can overcome the shortcomings of the existing technology, and provide a scientific basis for the prevention and control of ulcer disease in kiwifruit production. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The graph shows the results of the assay of the inhibitory effect of poly-lysine on Pseudomonas syringae in the present invention.

[0029] Figure 2 The figure shows the results of the test on the inhibition of Pseudomonas syringae by the wound sealant of the present invention.

[0030] Figure 3 A schematic diagram showing the wound sealant of the present invention for preventing and controlling kiwifruit canker in the field. DETAILED DESCRIPTION

[0031] In order to make those skilled in the art better understand the technical solution of the present invention, the present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments. The following embodiments are only used to more clearly illustrate the technical solution of the present invention, rather than to limit the protection scope of the present invention.

[0032] Example 1: Determination of the performance of poly-lysine in inhibiting Pseudomonas syringae

[0033] 1. Experimental strains: Pseudomonas syringae pv. kiwifruit PSA was purchased from China Center for Type Culture Collection (No. CCTCC AB 2018304). The most suitable culture temperature in the laboratory is 28°C.

[0034] 2. Activation of strains: Activate the PSA glycerol bacteria stored in the ultra-low temperature refrigerator, and use King's B agar medium as the activation medium. Perform preliminary activation of the bacteria taken out of the ultra-low temperature refrigerator by streaking, and culture them in a bacterial biochemical incubator at 28°C for 16 hours. Pick a single colony, inoculate it into King's B liquid culture medium, and culture it in a shaking incubator at 28°C for 16 hours.

[0035] 3. Dilution to obtain the number of colonies at 0.5 McFarland turbidity: Culture King's B liquid medium for 16 hours to obtain a certain amount of bacteria, centrifuge at 8000rpm for 15min to collect the bacterial precipitate, resuspend and wash twice with PBS. Dilute the resuspended bacteria appropriately, and when the OD625 is between 0.08-0.11, it is 0.5 McFarland turbidity, and the bacterial concentration is 1.5x10 8 CFU / mL. The above bacterial concentration was diluted 100 times for subsequent experiments.

[0036] 4. Inhibition zone determination:

[0037] 1). Pipette 20 μL PSA bacterial solution and inoculate it into King's B solid medium, and spread it evenly using a spreading rod.

[0038] 2). Stick a 6mm filter paper for drug sensitivity test that has been pre-soaked in sufficient amount of poly-lysine solution to the designated location.

[0039] 3). Place in a biochemical incubator at 28°C for 18 hours, then observe the inhibition zone.

[0040] 4). The antibacterial effect of poly-lysine on Pseudomonas syringae pv. Actinidiae (PSA) in vitro was observed by experiment. The experimental results are shown in Figure 1 In. Figure 1 As shown in (a), the inhibitory effect of poly-lysine on PSA was determined by the punching method. The sample in the lower right hole is poly-lysine, and the upper left hole, lower left hole, and upper right hole are different control groups, which are lysozyme-nanosilver complex, sterile water, and lysozyme, respectively. The experimental results show that lysozyme and sterile water have no inhibitory effect on PSA, lysozyme-nanosilver complex has a weak antibacterial effect on PSA, and poly-lysine has a larger inhibition zone on PSA. Figure 1As shown in (b), the inhibitory effect of polylysine on PSA was determined by the filter paper method. The upper left well was the blank control sterile water, and the samples in the lower left well, upper right well, and lower right well were all polylysine. The experimental results showed that polylysine had a large inhibition zone on PSA. Both inhibition zone methods proved that polylysine had a strong antibacterial effect on PSA. Therefore, in the future, polylysine will be used as the main component to inhibit PSA for wound sealing agent preparation.

[0041] 5. Minimum inhibitory concentration MIC determination:

[0042] 1) Different concentrations of poly-lysine were obtained by gradient dilution method, with the specific final concentrations being 2.5 mg / mL, 1.25 mg / mL, 0.625 mg / mL, 0.313 mg / mL, 0.156 mg / mL, 0.078 mg / mL, and 0 mg / mL.

[0043] 2) Take 100 μL of each of the above poly-lysine of different concentrations and add them into a 96-well plate.

[0044] 3) Add 100 μL of King's B liquid culture medium to each well where the drug is added.

[0045] 4) Add 20 μL of the drug and culture medium to each well to dilute to 1.5x10 6 PSA bacterial solution, set up a blank control group.

[0046] 5) Place the 96-well plate with the sample loaded in a biochemical incubator at 22° C. for 18 hours.

[0047] 6) After the culture is completed, measure OD600 nm The minimum inhibitory concentration was determined to be 0.078 mg / mL.

[0048] Example 2: Preparation of the wound sealant containing polylysine of the present invention

[0049] In order to make the wound sealant of the present invention better promote wound healing and have the functions of moisturizing and antifreezing, the inventors have added the following functional ingredients to the wound sealant: egg white lysozyme, which is a natural protein derived from egg white, can inhibit Gram-positive bacteria infection, anti-oxidation and promote wound healing; activated carbon, which is a common chemical adsorbent, can adsorb and enrich bacteria and other microorganisms; white vaseline, which is a commonly used chemical cream, can play a certain waterproof role; glycerol, which is a commonly used chemical moisturizer and antifreeze agent, can play a certain moisturizing and antifreezing role; glyceryl monostearate, which is a commonly used oil-in-water emulsifier, can promote the preparation of the cream of the present invention; peregrin, which is a non-ionic surfactant, can activate various components in the cream; octadecyl alcohol, which is an emulsion stabilizer, can promote the stable formation of the cream.

[0050] The preparation process of the wound sealant of the present invention is as follows:

[0051] 1) Weigh 0.5 g of activated carbon, 2.5 g of white vaseline, 0.48 g of glyceryl monostearate, 0.48 g of peregrin, and 1.08 g of octadecyl alcohol into a 50 mL centrifuge tube and heat the tube in a 88°C water bath while stirring until completely dissolved.

[0052] 2) Then, 3.2 mL of glycerol was added into the above reaction system.

[0053] 3) Weigh 2 g of poly-lysine and dissolve it in 6 ml of ultrapure water, and preheat it in a water bath at 88°C.

[0054] 4) Add the reactants prepared in steps 1, 2, and 3 to the preheated poly-lysine aqueous solution, and heat while stirring until completely emulsified.

[0055] 5) When the temperature of the reactants drops to 55° C., 0.5 g of egg white lysozyme is added, and stirring is continued during the process to obtain the wound sealing agent of the present invention.

[0056] Example 3: Determination of the performance of the wound sealant containing polylysine of the present invention in inhibiting Pseudomonas syringae

[0057] The antibacterial performance of the wound sealant provided by the present invention in inhibiting PSA is characterized in the form of an inhibition zone. The activation and dilution of PSA strains and the preparation steps of the wound sealant are the same as those in the above embodiment, and the obtained wound sealant is injected into a sterile syringe for convenient extrusion and use in subsequent experiments.

[0058] Determination process of inhibition zone:

[0059] 1) Inoculate 20 μL of PSA bacterial solution into King's B solid medium.

[0060] 2) After the PSA bacteria coating is completed, 200 μL of the wound sealant of the present invention is squeezed out with a syringe and placed in the test area.

[0061] 3) Place the culture dish in a 28°C biochemical incubator and culture for 18 hours.

[0062] 4) After the culture is completed, observe the inhibition zone.

[0063] After experimental verification, the experimental results are as follows Figure 2 As shown, the upper left sample is a control cream sample without adding polylysine, and the lower left, upper right, and lower right samples are all polylysine-containing wound sealants of the present invention. According to the results of the inhibition zone experiment, the wound sealant containing polylysine of the present invention has a large and obvious inhibition zone, so the wound sealant of the present invention has a strong antibacterial effect on PSA. This experimental result provides a strong theoretical basis for the subsequent application of the wound sealant of the present invention to actual production.

[0064] Example 4: Control of kiwifruit ulcers in kiwifruit fields

[0065] The specific use steps of the wound sealant for preventing and treating kiwifruit ulcers of the present invention are relatively simple. Figure 3 The specific scenario usage is as follows:

[0066] 1. Prepare the wound sealant according to the method in Example 2.

[0067] 2. After pruning fruit trees in the field, apply the wound sealant to the pruning wound in an amount that can cover the wound.

[0068] 3. If the seedlings are found to be slightly infected, you can consider applying the wound sealant.

[0069] 4. If you find that the seedlings have frozen cracks, you can apply an appropriate amount of wound sealant.

[0070] The above are only preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be regarded as limiting the present invention, and the protection scope of the present invention should be based on the scope defined by the claims. For ordinary technicians in this technical field, several improvements and modifications can be made without departing from the spirit and scope of the present invention, which should also be regarded as falling within the protection scope of the present invention.

Claims

1. A wound sealant for preventing and treating kiwifruit canker, It is characterized in that The wound sealant comprises poly-lysine, egg white lysozyme, activated carbon, white petrolatum, glyceryl monostearate, peregal, stearyl alcohol and glycerin.

2. The wound sealant according to claim 1, It is characterized in that The weight ratio of the polylysine, egg white lysozyme, activated carbon, white vaseline, glyceryl monostearate, peregal, octadecyl alcohol and glycerol is 1:0.25:0.25:1.25:0.24:0.24:0.54:2.

02. The wound sealant according to claim 1 , which is a cream or a paste.

4. A method for preparing the wound sealant according to claim 1, comprising the following steps: (1) placing activated carbon, white vaseline, glyceryl monostearate, peregal, and octadecyl alcohol in a centrifuge tube and heating in a water bath until completely dissolved to obtain a blend; (2) adding glycerol to the blend; (3) Dissolve poly-lysine in ultrapure water and preheat in a water bath; (4) adding the blend into a preheated poly-lysine aqueous solution and heating while stirring until completely emulsified; (5) Cooling the reactants, adding egg white lysozyme and continuing stirring to obtain the wound sealing agent.

5. The method according to claim 4, It is characterized in that The weight ratio of the polylysine, egg white lysozyme, activated carbon, white vaseline, glyceryl monostearate, peregal, octadecyl alcohol and glycerol is 1:0.25:0.25:1.25:0.24:0.24:0.54:2.

02.

6. Use of the wound sealant according to claim 1 in preventing and treating kiwifruit canker.

7. The use according to claim 6, It is characterized in that The pathogenic bacteria of the kiwifruit canker disease is Pseudomonas syringaepv. Actinidiae.

8. The use according to claim 7, It is characterized in that The wound sealant is applied to wounds of kiwi fruit trees.

9. The use according to claim 8, It is characterized in that The kiwi fruit tree wound is a pruning wound or a frostbite wound of the kiwi fruit tree.

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