Application of natural compound in prevention and treatment of gray mold of fruits and vegetables

Abstract

The invention provides application of a natural compound in prevention and treatment of gray mold of fruits and vegetables. In the invention, botrytis cinerea can be inhibited by using the natural compound kaempferol. Experimental results show that the inhibitor has an inhibition effect on botrytis cinerea, has an IC50 value of 64 [mu] g / mL, has a good prevention and treatment effect on gray mold of cherry tomatoes, delays rotting and deterioration of the cherry tomatoes and prolongs the shelf life. The effect of the natural compound kaempferol in inhibiting the toxicity of botrytis cinerea is expected to become a novel prevention and treatment means for gray mold of fruits and vegetables.

Description

Technical Field

[0001] The invention relates to application of a natural compound, kaempferol, in preventing and controlling gray mold of fruits and vegetables. Background Art

[0002] Botrytis cinerea belongs to the genus Botrytis, a typical filamentous plant pathogenic fungus that can cause gray mold disease in plants. It has a wide variety of hosts, mainly economic crops such as tomatoes, pears, apples, rubber trees, pecans, peppers, peaches, avocados, strawberries, and citrus. It causes serious gray mold disease and is widely distributed throughout the world, causing serious economic losses to agricultural production.

[0003] Botrytis cinerea infection is mainly caused by 50-75μm hyphae germinating into oval conidia, which attach to the plant surface and germinate. The germ tube or more slender hyphae distinguish simple compression and infection pads. Both have special structures that facilitate host penetration. Botrytis cinerea has been shown to enter the host through stomata or directly penetrate the cuticle through short conidial germ tubes. After the initial contact with the host, there are three different infection stages: early, middle and late.

[0004] In current production practice, we still mainly rely on chemical fungicides to prevent and control the occurrence of diseases. Common fungicides for the prevention and control of gray mold include carbendazim, prochloraz, and difenoconazole. Chemical control has the advantages of convenient operation, fast and efficient, and high economic benefits, but the abuse and misuse of chemical pesticides often cause environmental pollution, poisoning of humans and animals, killing beneficial microorganisms, and causing pathogens to develop drug resistance. Therefore, it is imperative to use pesticides scientifically and rationally, reduce pesticide residues, reduce environmental pollution, and delay pathogen resistance. With the rapid development of agricultural technology, pesticide reduction, pest control and efficiency enhancement technology has begun to be widely used, and new high-efficiency, low-toxic and environmentally friendly pesticides have begun to be developed, which is of great significance for the development of agricultural ecological systems, the improvement of the ecological environment and the increase of crop yields.

[0005] Kaempferol is widely distributed in many fruits and vegetables such as tomatoes, strawberries, leeks, onions, and traditional Chinese herbal medicines such as forsythia, rosemary, and acacia. Kaempferol has anti-inflammatory and antioxidant properties. The anti-inflammatory mechanism of kaempferol mainly includes regulating the activity of pro-inflammatory enzymes and the expression of inflammation-related genes, inhibiting transcription factors, adhesion molecules, and matrix metalloproteinases. Its antioxidant mechanism is manifested in significantly inhibiting the expression of antioxidant enzymes such as reduced coenzyme II, catalase, glutathione peroxidase, and glutathione-S transferase. In addition, kaempferol has a very wide range of biological activities, but its application in inhibiting gray mold of fruits and vegetables has not been reported.

[0006] The present invention uses the natural compound kaempferol to inhibit the mycelial growth and spore germination of Botrytis cinerea, thereby preventing and controlling Botrytis cinerea. The inhibitory effect of kaempferol on the toxicity of Botrytis cinerea is determined by experiments of inhibiting mycelial growth and spore germination of Botrytis cinerea with different concentrations, and then the preservation effect of kaempferol on fruits and vegetables is verified by cherry tomato preservation experiments. This inhibitor is safe and non-toxic and is not easy to induce drug resistance in strains. It can be promoted and used as a new toxicity inhibitor, providing a new idea for preventing and controlling gray mold of fruits and vegetables. Summary of the invention

[0007] The purpose of the present invention is to provide a new method for inhibiting the growth of Botrytis cinerea and preventing and controlling gray mold of fruits and vegetables, so as to solve the problem of fruit and vegetable rot and deterioration caused by gray mold of fruits and vegetables and extend the shelf life of fruits and vegetables. In order to achieve the above purpose, the technical solution adopted by the present invention is to use a natural compound as a toxicity inhibitor of Botrytis cinerea and prepare it into an antibacterial coating liquid to treat gray mold of cherry tomatoes. The present invention is achieved by the following technical solutions:

[0008] The present invention provides a natural compound as a toxicity inhibitor of Botrytis cinerea and a method for preventing and controlling gray mold of fruits and vegetables, wherein the natural compound is kaempferol; the method for preventing and controlling gray mold of fruits and vegetables is to prepare an antibacterial coating liquid to preserve cherry tomatoes. Accordingly, a natural compound as a toxicity inhibitor of Botrytis cinerea and a method for preventing and controlling gray mold of fruits and vegetables comprises the following steps:

[0009] (1) Natural compounds as toxicity inhibitors of Botrytis cinerea: Kaempferol was dissolved in water to prepare a solution with a concentration of 96 μg / mL.

[0010] (2) Preparation of antibacterial coating solution: Kaempferol was dissolved in water to prepare a gradient concentration solution, and a certain proportion of glycerol was added to increase the viscosity of the coating solution, and citric acid was added to make its pH closer to the internal environment of cherry tomatoes.

[0011] Beneficial effects: The present invention adopts a convenient and simple preparation method, and prepares a natural compound kaempferol anti-Botrytis cinerea toxicity inhibitor to address the problem that traditional fungicides are prone to residue and drug resistance induction, which has a significant inhibitory effect on inhibiting the growth of Botrytis cinerea (IC50 value is 64μg / mL). In addition, by constructing a model of Botrytis cinerea infecting cherry tomatoes and making kaempferol into an antibacterial coating liquid, it is possible to prevent and treat gray mold on cherry tomatoes, delay the decay and deterioration of cherry tomatoes, and achieve the purpose of protecting quality and yield. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 Effects of kaempferol treatment on the mycelial growth of Botrytis cinerea

[0013] Figure 2Effect of kaempferol on the inhibition rate of Botrytis cinerea hyphae after treatment

[0014] Figure 3 Effects of kaempferol on the mycelial morphology of Botrytis cinerea after treatment

[0015] Figure 4 .Inhibitory kinetics of kaempferol on hyphae growth of Botrytis cinerea after treatment

[0016] Figure 5 Effects of kaempferol on the microscopic morphology of Botrytis cinerea after treatment

[0017] Figure 6 Effects of kaempferol on cell membrane permeability of Botrytis cinerea after treatment

[0018] Figure 7 .Comparison chart of strawberry anthracnose control

[0019] Figure 8 Sensory evaluation of cherry tomatoes treated with kaempferol during storage

[0020] Fig. 9 Effects of kaempferol treatment on weight loss rate of cherry tomatoes during storage

[0021] Fig.10 Effects of kaempferol treatment on the firmness of cherry tomatoes during storage

[0022] Fig.11 . Effects of kaempferol treatment on L* of cherry tomatoes during storage

[0023] Fig.12 Effects of kaempferol treatment on a* of cherry tomatoes during storage DETAILED DESCRIPTION

[0024] In order to further explain the technical means and effects adopted by the present invention to achieve the predetermined purpose, the specific operation mode and effect results of the present invention are described in detail below in combination with the accompanying drawings and embodiments.

[0025] Embodiment 1:

[0026] Step 1: Preparation of anti-Botrytis cinerea toxicity inhibitors

[0027] The natural compound kaempferol is dissolved in dimethyl sulfoxide, stored in a refrigerator away from light, and used for standby. When used, the PBS solution is used to prepare the required concentration gradient by multiple dilution method. Among them, the final concentration of the test solution after being dissolved in dimethyl sulfoxide is 20 mg / mL.

[0028] Step 2: Calculate the half-maximal effect concentration (EC50)

[0029] In a sterile clean bench, add the candidate drug to 3mL PDA and mix well to a drug concentration of 16, 32, 64, 128, and 256 μg / mL. Use an equal amount of sterile PBS buffer as a negative control. After the PDA cools and solidifies, inoculate a 6-mm diameter Botrytis cinerea mycelium disk into the center of the culture dish, cover the culture dish lid, seal it with a sealing film, and culture it at 28°C until the mycelium of the negative control group covers the entire culture dish. Use SPSS Statistics 21.0's Probit analysis to calculate the EC50 value. All experiments were performed in triplicate.

[0030] Step 3: Determine the growth of gray mold hyphae

[0031] The cultivation and treatment of mycelium were the same as step 2. The inoculated mycelium of Botrytis cinerea was cultured at 28°C until the mycelium of the negative control group covered the entire culture dish. The mycelium growth diameter under different kaempferol concentrations was determined by the cross method, and the EC50 value of inhibiting mycelium growth was calculated by Probit analysis of SPSS21.

[0032] Step 4: Observation of the spore morphology of Botrytis cinerea after kaempferol treatment

[0033] In a sterile clean bench, gray mold was inoculated into PDB medium and cultured at 28°C. After it became turbid, it was filtered through four layers of sterile gauze and the supernatant was discarded. The remaining liquid was diluted with sterile water to 106 spores / mL. Kaempferol was diluted to 16, 32, 64 and 128 μg / mL with PDB medium. Different concentrations of drugs were mixed with the diluted spore solution at a volume ratio of 1:1, and 20-30 μL was drawn and dropped onto the center of the slide. After the coverslip was covered, it was placed in a culture dish and cultured at 28°C for 8 hours. In order to prevent the bacterial solution from drying out during the culture process, sterile wet filter paper was placed in the culture dish. An equal amount of PBS buffer was used as a negative control, and an equal amount of 128 μg / mL pyrimethanil was used as a positive control. Each group was repeated three times. Under an inverted microscope with a 20× objective lens, at least 100 spores were observed in each group and the number of spore germination was recorded. The EC50 value of inhibiting spore germination was calculated using Probit analysis of SPSS21.

[0034] Embodiment 2:

[0035] Step 1: TEM observation of the changes in the internal structure of Botrytis cinerea after kaempferol treatment

[0036] In a sterile clean bench, kaempferol was diluted to 32 μg / mL with 3 mL PDA medium. After the PDA medium solidified, a 6 mm diameter gray mold cake was inoculated into the center of the culture dish and cultured at 28 ° C for 3 days. The cake was cut off and an equal amount of PBS buffer was used as a blank control. The cut cake was washed repeatedly with PBS buffer 2-3 times until it was clean. After centrifugation at 3000 r / min for 20 minutes in a centrifuge, the sample was placed in a fixative (3% glutaraldehyde, 1% osmium tetroxide) and fixed. Dehydrated with ethanol solutions of different concentration gradients, then embedded with resin and placed at 37 ° C, 45 ° C and 60 ° C respectively, and the embedded head was trimmed after polymerization for 48 hours. Finally, the sample was cut into thin slices with a thickness of 70 nm, and double-stained with uranyl acetate and lead citrate, and the ultrastructural changes on the surface of gray mold hyphae were observed with TEM.

[0037] Step 2: Propidium iodide staining to observe the effect of kaempferol on the cell membrane permeability of Botrytis cinerea

[0038] In a sterile clean bench, kaempferol was diluted to 16, 32, 64 and 128 μg / mL with 1 mL PDB medium, and 0.4 g of mycelium was added to each group. An equal amount of PBS buffer was used as a negative control, and an equal amount of 128 μg / mL ACZ was used as a positive control. The mycelial suspension was cultured at 28°C for 8 hours and then washed twice with PBS buffer by centrifugation. 200 μL of propidium iodide (PI, 100 μg / mL) was added to the mycelial precipitate and stained in the dark for 15 minutes at room temperature, and then washed twice with PBS buffer by centrifugation to remove the fluorescent dye. The sample was placed under a 40× objective lens of a fluorescence microscope to observe and photograph the fluorescent dye of each group of mycelium. The sample was placed under a 40× objective lens of a fluorescence microscope to observe and photograph the fluorescence intensity of each group of mycelium.

[0039] Embodiment 3:

[0040] Step 1: Treat cherry tomatoes with antibacterial coating liquid

[0041] After inoculation of spores, the cherry tomatoes were immersed in the prepared 64, 96, and 128 μg / mL kaempferol antibacterial agent. After 1 minute, the surface moisture was gently absorbed with clean filter paper to keep it dry. All cherry tomatoes were grouped and stored in plastic trays at room temperature. Afterwards, the quality changes of cherry tomatoes were observed, photographed, and recorded on days 0, 4, 8, 12, and 16.

[0042] Step 2: Preparation of Botrytis cinerea spore suspension

[0043] The strain of Botrytis cinerea was inoculated on potato dextrose agar by the partition and streak method and cultured in a constant temperature incubator for later use. Sterile distilled water was added to the cultured bacterial plate, hyphae were scraped by streak, and the original spore suspension was obtained by filtering through four layers of gauze, and the concentration of the spore suspension was 1×106 / mL.

[0044] Step 3: Determination of weight loss rate of cherry tomatoes

[0045] The weight of each group of cherry tomatoes was weighed on the 0th, 4th, 8th, 12th and 16th days respectively, and the weight loss rate was calculated according to the following formula, which was used as the basis for judging the preservation effect of cherry tomatoes.

[0046] Step 4: Determination of Chromatic Value of Cherry Tomato

[0047] The chromaticity of cherry tomatoes was measured with the help of a CR-400 colorimeter, and the indicators were L* and a*. Among them, the L* value was used to indicate the lightness and darkness of cherry tomatoes. When L*=0, the darkest lightness is black; when L*=100, the lightest lightness is white; a* was used to indicate the degree of red and green of cherry tomatoes, +a* represents reddish, and -a* represents greenish. On the 0th, 4th, 8th, 12th, and 16th days, after calibrating the L* as close to 100 as possible using a white calibration plate, three parts at the equator of the cherry tomatoes were randomly selected to measure their L* and a* values, and their average values ​​were calculated.

[0048] Step 5: Determination of cherry tomato hardness

[0049] Three parts at the equator of cherry tomatoes were randomly selected on days 0, 4, 8, 12, and 16, and the hardness of the cherry tomatoes was measured using the TPA texture analysis of the texture analyzer. The probe was TA43, the trigger point load was 5g, the speed was 1mm / s, and a total of 2 cycles were performed to calculate the average value.

[0050] Step 6: Sensory evaluation of cherry tomatoes

[0051] Photographs were taken on days 0, 2, 4, 6, and 8 of storage of cherry tomatoes, and their color, taste, texture, and appearance were observed and sensory scores were performed.

[0052] The results of mycelium inhibition experiment are as follows Figure 1 and Figure 2 As shown in the figure, with the increase of drug concentration, the inhibitory effect of kaempferol also increased, showing obvious concentration dependence, and the mycelial growth inhibition rate at different concentrations was significant. The mycelial growth inhibition rate at 128μg / mL can reach 71.86%. By calculation, the EC50 value of kaempferol on mycelial growth inhibition was 64μg / mL. The outer edge morphology changes during the growth of gray mold mycelium are shown in the figure. Figure 3As shown in the figure. In the negative control group, the hyphae of Botrytis cinerea were longer and had a complete structure. There was no diaphragm inside the hyphae, and the hyphae spread and grew in all directions. When the concentration was > 64 μg / mL, the hyphae would aggregate and shrink severely, and would no longer spread and grow in all directions. An obvious diaphragm appeared inside the hyphae. Figure 4 The higher the concentration of kaempferol, the higher the inhibition rate on the mycelial growth of Botrytis cinerea. Figure 5 It can be seen that the internal morphology of the mycelium in the blank group was normal and the cell structure was intact. However, after the mycelium was treated with 96 μg / mL kaempferol, it was clearly seen that cytoplasm leakage and cavitation occurred inside the mycelium. Figure 6 After staining with PI, as the concentration of kaempferol increased, the number of red fluorescent hyphae gradually increased. No hyphae in the blank group produced fluorescence, indicating that the cell membrane was not damaged and was intact. When the concentration of kaempferol was 64μg / mL, more hyphae emitted red fluorescence, and the fluorescence intensity was also enhanced; when the concentration of kaempferol was 128μg / mL, almost all hyphae showed strong red fluorescence, and the intensity and quantity exceeded the positive control group. Figure 7 As can be seen in the figure, the cherry tomatoes inoculated with gray mold spores will develop black spots, internal rot and softening, and juice leakage as the spores spread. There was no growth and spread of gray mold hyphae within 16 days. As can be seen in Figure 9, the weight loss rate of all groups increased, and there was a significant difference in the weight loss rate of each group on the last day of storage. Fig.10 The results show the changes in the fruit hardness of cherry tomatoes after kaempferol treatment, and the hardness reduction rate on the last day of storage has significant differences. Fig.11 It can be seen that the L* values ​​of cherry tomatoes decreased during storage. Fig.12 It can be seen that the a* value shows a trend of increasing first and then decreasing. The ripening process of the blank group and the 1% citric acid group was normal, and kaempferol inhibited the ripening of cherry tomatoes, which was beneficial to the preservation of cherry tomatoes.

Claims

1. An application of a natural compound in the prevention and treatment of gray mold in fruits and vegetables, characterized in that: Application of a natural compound, kaempferol, in the prevention and treatment of gray mold in fruits and vegetables.

2. The use of a natural compound according to claim 1 in the prevention and treatment of gray mold in fruits and vegetables, characterized in that: The natural compound kaempferol is used in inhibiting the toxicity of Botrytis cinerea.

3. The use of a natural compound according to claim 2 in the prevention and treatment of gray mold in fruits and vegetables, characterized in that: The toxicity inhibition concentrations of the natural compound kaempferol were 16, 32, 64, 128, and 256 μg / mL, respectively.

4. A method for preventing and controlling gray mold of fruits and vegetables, characterized in that: The method for preventing and controlling gray mold of fruits and vegetables is to prepare an antibacterial coating liquid to inhibit bacteria and preserve cherry tomatoes; the antibacterial coating liquid is prepared by using the natural compound kaempferol described in claim 2.

5. A method for preventing and controlling gray mold of fruits and vegetables according to claim 4, characterized in that: The concentrations of the natural compound kaempferol in the coating solution are 32, 64, 96 and 128 μg / mL respectively, and the coating solution contains 1% of glycerol and citric acid.

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