Product for preventing and controlling rhizopus soft rot, application of product and fruit and vegetable fresh-keeping method

Abstract

The invention discloses a product for preventing and controlling rhizopus soft rot, application of the product and a fruit and vegetable fresh-keeping method, and belongs to the field of disease prevention and control. The product for preventing and controlling the rhizopus soft rot is prepared from any one of oregano essential oil, 2, 3-butanedione, nerol and clove essential oil and methyl salicylate. The rhizopus soft rot disease is caused by rhizopus stolonifer, and the rhizopus soft rot disease is caused by rhizopus stolonifer. Through compounding, the use amount of the essential oil can be reduced, the total antibacterial effect can be improved, the medication cost is reduced, the medication selectivity is improved, the operation is convenient, and the storability and the edible safety of picked fruits and vegetables are enhanced. And the methyl salicylate, the oregano essential oil, the 2, 3-butanedione, the nerol and the clove essential oil are all natural plant essential oil, can be applied to the food industry, has the advantages of low toxicity, high efficiency, low price, easily available raw materials and the like, and can ensure the safety of fruits and vegetables. The product disclosed by the invention is used for preventing and treating the rhizopus soft rot after the fruits are picked.

Description

Technical Field

[0001] The invention belongs to the field of disease prevention and control, and specifically relates to a product for preventing and controlling root mold soft rot, an application thereof, and a method for preserving fruits and vegetables. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Rhizopus stolonifer Rhizopus stolonifer ), belonging to the class Zygomycetes, the orders Mucorales, the family Mucoraceae, the genus Rhizopus, and the stolonifer, is the most common and fastest growing species of Zygomycetes. Stolonifer spores are widely present everywhere, can be spread by air currents, and grow rapidly in humid environments, causing soft rot in many fruits such as peaches, strawberries, pears, grapes, and tomatoes. When the fruit is infected, the infected area first becomes watery, and then becomes soft and rotten. Under appropriate temperature and humidity, the initial symptoms appear about 1 day after infection. Rapid growth and a large amount of mycelium are observed on the surface of the fruit, and the wound part is covered by the production of filamentous gray sporangia. The infected tissue eventually decomposes into watery rot, and its wound surface becomes very soft. After about 2-3 days, the infected fruit may release juice with a fermented or acidic smell. In addition, the infected fruit will also infect the surrounding healthy fruits, causing huge losses.

[0004] At present, the methods used to control postharvest soft rot of fruits mainly include physical control methods and chemical control methods. Physical control methods are usually combined with other antibacterial methods for fungal inhibition in food, and have the characteristics of low energy consumption, high efficiency and no secondary pollution. Physical control methods include low temperature storage, irradiation treatment, heat treatment, ozone treatment, ultrasonic treatment, microwave treatment, etc. Physical control methods are one-time sterilization, which has a strong effect on pathogens and will not produce drug resistance. No chemical substances are added during the treatment process, and there are no side effects on the environment. The sterilization conditions are easy to control, the operation is simple, and it is less affected by the external environment. However, the control intensity of physical control methods is limited, it is not universally applicable, and the effect is mostly unsatisfactory. It needs to be combined with other technologies to achieve a better prevention and control effect. Chemical control methods have a wide range of effects and obvious effects. They are commonly used in fruit postharvest management strategies. Chemical control methods include fungicides, calcium chloride, salicylic acid, peracetic acid, nanomaterials, etc. Chemical control methods are effective, low cost, and easy to implement, and are widely used in postharvest preservation of fruits and vegetables. However, the use of chemicals such as fungicides has always been controversial. Excessive use may lead to the residue of certain chemicals, posing a certain threat to human health and the environment. At the same time, excessive use of chemical fungicides may also cause pathogens to develop resistance, which is one of the reasons why consumers find it difficult to accept. Some chemical control methods require coating, and some agents will form a film on fruits and vegetables with rough surfaces, thereby accelerating the deterioration process. Therefore, it is necessary to find a safer and more effective alternative method to control the infection of Rhizopus stolonifer in post-harvest fruits. Summary of the invention

[0005] In order to solve the deficiencies of the prior art, the purpose of the present invention is to provide a product for preventing and controlling root mold soft rot and its application and a method for preserving fruits and vegetables. The product for preventing and controlling root mold soft rot provided by the present invention is effective against the pathogenic bacteria of fruit root mold soft rot. Rhizopus stolonifer It can control the disease and reduce the amount of antibacterial agents used. It has a synergistic effect on the prevention and treatment of fruit soft rot. When used in food, it can also ensure the safety of fruits and vegetables, and provide a new solution for the prevention and treatment of fruit root mold soft rot in the future.

[0006] In order to achieve the above object, the technical solution of the present invention is: The first aspect of the present invention provides a product for preventing and controlling root mold soft rot, the product comprising oregano essential oil, any one of 2,3-butanedione, nerol and clove essential oil and methyl salicylate (MeSA); The root mold soft rot is Rhizopus stolonifer ( Rhizopus stolonifer ) caused by.

[0007] In some embodiments of the present invention, the product consists of oregano essential oil and methyl salicylate, and the methyl salicylate and oregano essential oil are compounded at a ratio of 1 / 4-1:1 / 4-1 of their respective half-effective concentrations.

[0008] In some embodiments of the present invention, the product consists of 2,3-butanedione and methyl salicylate, and the methyl salicylate and 2,3-butanedione are compounded at a ratio of 1 / 4-1:1 / 4-1 of their respective half-effective concentrations.

[0009] In some embodiments of the present invention, the methyl salicylate and 2,3-butanedione are compounded at 1 / 4-1 / 2:1 / 4-1 / 2 of their respective half-effective concentrations.

[0010] In some embodiments of the present invention, the methyl salicylate and 2,3-butanedione are compounded at 1 / 2:1 / 4 of their respective half-maximal effective concentrations.

[0011] In some embodiments of the present invention, the product consists of clove essential oil and methyl salicylate, and the methyl salicylate and clove essential oil are compounded at a ratio of 1 / 4-1:1 / 4-1 of their respective half-effective concentrations.

[0012] In some embodiments of the present invention, the product consists of nerol and methyl salicylate, and the methyl salicylate and nerol are compounded at a half-effective concentration of 1 / 2:1 / 2-1 or 1 / 4:1 / 4-1.

[0013] In some embodiments of the present invention, the dosage form of the product includes any one of aqueous emulsion, emulsion, oil, suspension or solid preparation; The solid preparation includes any one of granules, powders, tablets, capsules and seed coatings.

[0014] The second aspect of the present invention provides an application of the above product in preventing and treating Rhizopus soft rot of fruits and vegetables; The fruits and vegetables include any one or more of apples, pears, strawberries, figs, grapes, tomatoes, peaches, green peppers, and sweet potatoes.

[0015] In some embodiments of the present invention, the root mold soft rot is Rhizopus stolonifer ( Rhizopus stolonifer ) caused by.

[0016] The third aspect of the present invention provides a method for preserving fruits and vegetables, comprising: fumigating the fruits and vegetables with the above-mentioned product; The fruits and vegetables include any one or more of apples, pears, strawberries, figs, grapes, tomatoes, peaches, green peppers, and sweet potatoes.

[0017] The beneficial effects of the present invention are: The present invention provides a product for preventing and controlling root mold soft rot, which is an antibacterial agent obtained by compounding methyl salicylate with oregano essential oil, 2,3-butanedione, nerol or clove essential oil, which can achieve a better prevention or treatment effect on root mold soft rot while reducing the usage of each part, and achieve a better effect when the usage is small. Methyl salicylate, oregano essential oil, 2,3-butanedione, nerol and clove essential oil are all natural plant essential oils, which can be applied to the food industry, have the advantages of low toxicity, high efficiency, low price and easy availability of raw materials, and can ensure the safety of fruits and vegetables. The product of the present invention is used to prevent and control root mold soft rot after fruit harvest, which can not only reduce the usage of essential oil, but also improve the overall antibacterial effect, reduce the cost of medication, increase the selectivity of medication, facilitate operation, and enhance the storage stability and edible safety of fruits and vegetables after harvest.

[0018] In particular, when methyl salicylate and 2,3-butanedione are combined, they have a synergistic effect in inhibiting the growth of Rhizopus soft rot in fruits. When the two are combined at their respective optimal concentrations, the incidence of Rhizopus soft rot is significantly reduced compared with the control group. On the basis of reducing the use of the two natural essential oils, the overall antibacterial effect and fruit quality are improved, the cost of medication is reduced, the selectivity of medication is increased, and the post-harvest storage stability and edible safety of fruits and vegetables are enhanced. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0020] Figure 1 The inhibitory effects of different preservatives on Rhizopus stolonifer in Example 1 of the present invention are shown in FIG. Figure 2 The inhibitory effects of different volatile compounds on Rhizopus stolonifer in Example 1 of the present invention; Figure 3 The in vivo antibacterial effect of different essential oil treatments on figs in Example 1 of the present invention; Figure 4 The diameter of the lesions and the decay rate of the figs treated with different concentrations of MeSA and oregano essential oil in Example 1 of the present invention; Figure 5 The diameter of the lesions and the decay rate of the figs treated with different concentrations of MeSA and clove essential oil in Example 1 of the present invention; Figure 6 The diameter of the lesions and the decay rate of the figs treated with different concentrations of MeSA and 2,3-butanedione in Example 1 of the present invention; Figure 7 The in vivo antibacterial effect of different essential oil treatments on peach in Example 1 of the present invention; Figure 8 The diameter of the lesions and the decay rate of peaches treated with different concentrations of MeSA and oregano essential oil in Example 1 of the present invention; Figure 9 The diameter of the lesions and the decay rate of peaches treated with different concentrations of MeSA and clove essential oil in Example 1 of the present invention; Figure 10 The diameter of the lesions and the decay rate of peaches treated with different concentrations of MeSA and 2,3-butanedione in Example 1 of the present invention; Figure 11 The in vivo antibacterial effect of different essential oil treatments on tomatoes in Example 1 of the present invention; Figure 12 The diameter of the lesions and the rot rate of tomatoes treated with different concentrations of MeSA and oregano essential oil in Example 1 of the present invention; Figure 13 The diameter of the lesions and the rot rate of tomatoes treated with different concentrations of MeSA and clove essential oil in Example 1 of the present invention; Figure 14 The diameter of the lesions and the rot rate of tomatoes treated with different concentrations of MeSA and 2,3-butanedione in Example 1 of the present invention; Figure 15 The in vivo antibacterial effect of different essential oil treatments on green peppers in Example 1 of the present invention; Figure 16 The diameter of the lesions and the decay rate of green peppers treated with different concentrations of MeSA and oregano essential oil in Example 1 of the present invention; Figure 17 The diameter of the lesions and the decay rate of green peppers treated with different concentrations of MeSA and clove essential oil in Example 1 of the present invention; Figure 18 The diameter of lesions and the decay rate of green peppers treated with different concentrations of MeSA and 2,3-butanedione in Example 1 of the present invention are shown.

[0021] Note: Figures 3 - 18In the table, 1+1 refers to the original concentration of MeSA (166.8 μL / L) mixed with oregano essential oil, clove essential oil, or 2,3-butanedione (155.2 μL / L, 148 μL / L, 299.2 μL / L, respectively); 1+1 / 2 refers to the original concentration of MeSA (166.8 μL / L) mixed with 1 / 2 of the original concentration of oregano essential oil, clove essential oil, or 2,3-butanedione (155.2 μL / L, 148 μL / L, 299.2 μL / L, respectively); 1+1 / 4 refers to the original concentration of MeSA (166.8 μL / L) mixed with oregano essential oil, clove essential oil, or 2,3-butanedione (155.2 μL / L, 148 μL / L, 299.2 μL / L, respectively). =1 / 4 of the original MeSA concentration (166.8 μL / L) was mixed with oregano essential oil, clove essential oil or 2,3-butanedione (155.2 μL / L, 148 μL / L, 299.2 μL / L, respectively); 1 / 2+1 / 2: refers to the mixing of 1 / 2 of the original MeSA concentration (166.8 μL / L) with oregano essential oil, clove essential oil or 2,3-butanedione (155.2 μL / L, 148 μL / L, 299.2 μL / L, respectively); 1 / 2+1 / 4: refers to the mixing of 1 / 2 of the original MeSA concentration (166.8 μL / L) with oregano essential oil, clove essential oil or 2,3-butanedione (155.2 μL / L, 148 μL / L, 299.2 μL / L, respectively). =1 / 4 of the original MeSA concentration (166.8 μL / L) was mixed with 1 / 4 of the original concentration of oregano essential oil, clove essential oil, or 2,3-butanedione (155.2 μL / L, 148 μL / L, 299.2 μL / L, respectively); 1 / 4+1 / 2: refers to the mixing of 1 / 4 of the original MeSA concentration (166.8 μL / L) with 1 / 2 of the original concentration of oregano essential oil, clove essential oil, or 2,3-butanedione (155.2 μL / L, 148 μL / L, 299.2 μL / L, respectively); 1 / 4+1 / 4: refers to the mixing of 1 / 4 of the original MeSA concentration (166.8 μL / L) with 1 / 4 of the original concentration of oregano essential oil, clove essential oil, or 2,3-butanedione (155.2 μL / L, 148 μL / L, 299.2 μL / L, respectively). Mix 1 / 4 of the 1 μL / L. DETAILED DESCRIPTION

[0022] The pathogen of root mold soft rot grows fast, and has fast penetration and colonization speed. At present, once the fruit is infected by the pathogen, it is basically impossible to be completely eliminated. Once the fruit is infected, it will spread rapidly to other fruits, resulting in significant losses. Some commonly used prevention and control methods are usually chemical methods, mainly agricultural prevention, pesticides and chemical fungicides, etc., but the use of chemical methods may lead to the residue of certain chemical substances, and excessive use of chemical fungicides may also cause pathogens to develop drug resistance, posing a certain threat to human health and the environment, which is difficult for consumers to accept. Therefore, it is necessary to find a safer and more effective alternative method to control the infection of creeping root mold in post-harvest fruits. Based on this, the present invention provides a product for preventing and controlling root mold soft rot and its application and a method for preserving fruits and vegetables.

[0023] A typical embodiment of the present invention provides a product for preventing and controlling root mold soft rot, the product comprising oregano essential oil, any one of 2,3-butanedione, nerol and clove essential oil, and methyl salicylate; The root mold soft rot is Rhizopus stolonifer ( Rhizopus stolonifer ) caused by.

[0024] Plant essential oils are natural volatile aromatic compounds and secondary metabolites obtained from different organs of plants. Due to their biological properties, especially antifungal properties, they have the potential to be used as a substitute for many traditional antifungal agents for preserving fruit and vegetable products. There are many studies on their application in fruit and vegetable preservation. In addition, fumigation as a way to use essential oils in fruit and vegetable preservation can achieve better antibacterial and preservation effects while keeping the fruit surface clean.

[0025] The present invention first determines the effects of different food preservatives and volatile compounds on Rhizopus stolonifer The mycelium inhibition rate was used to determine the antibacterial effect of each antibacterial agent. The maximum concentration of different food preservatives and volatile compounds that can be added to food was used for screening. Further, the maximum concentration of different food preservatives and volatile compounds used for Rhizopus stolonifer R was 1 / 2 of the maximum concentration. hizopus stolonifer To suppress.

[0026] It should be noted that the screening is a commonly used screening method in the art. Rhizopus stolonifer The culture medium is inoculated into potato dextrose agar (PDA) medium containing food preservatives and that without food preservatives for cultivation. After the control group is full, the colony diameter is measured by the cross method to compare the antibacterial effect. The screening method can also be: Rhizopus stolonifer Rhizopus stoloniferThe culture medium was inoculated into potato dextrose agar (PDA) and volatile organic compounds and sterile water were added by fumigation method. After the control group was full, the colony diameter was measured by cross method to compare the antibacterial effect.

[0027] In some examples of this embodiment, the screening comprises the following steps: S1: Activation of strains: Rhizopus stolonifer Rhizopus stolonifer The activation was carried out according to the ATCC guidelines. The isolated and purified strains were stored in a 4°C refrigerator for future use. Each time they were used, a single colony was picked out with an inoculation needle and stored for future use, and inoculated into the sterilized PDA medium by streaking, and then placed in a constant temperature incubator at 25°C for about 2-5 days for future use.

[0028] S2: Preparation of culture medium: Potato dextrose agar (PDA) culture medium: Weigh a certain mass of PDA culture medium powder according to the required volume, add a certain amount of deionized water, heat on an electric stove, stir while heating until boiling, cool, slowly dispense into the prepared conical bottles, seal with sealing film, sterilize in an autoclave at 121°C for 15 min, and set aside.

[0029] S3: Preparation of PDA medium containing food preservatives: After preparing the above PDA medium, weigh the corresponding concentrations of each food preservative, put it into a conical flask, and shake it evenly. Pay attention to the properties of the food preservatives during this process. For example, products that are easily decomposed by high temperatures must be sterilized and cooled before adding food preservatives. Acidic food preservatives require the pH of the culture medium to be adjusted to appropriately exert acidity. Ester food preservatives can be added with small molecule cosolvents such as dimethyl sulfoxide (DMSO). Each group of experiments should set up negative and positive controls as needed, with three parallels in each group.

[0030] S4: Preparation of plates containing volatile compounds: After the PDA medium is prepared, pour it into a disposable sterile culture dish, transfer the Rhizopus stolonifer cake to the solidified culture medium, place a sterile 6 mm diameter filter paper disc in the center of the dish cover, and drop 5 μL of volatile compounds on the filter paper disc. Set up negative and positive controls for each group of experiments as needed, with three parallels in each group.

[0031] S5: Determination of the antibacterial effect of different antibacterial agents: The cross-cross method was used to determine the antibacterial effect of different food preservatives and volatile compounds on Rhizopus stolonifer Rhizopus stolonifer Mycelial growth inhibition rate MGI.

[0032] A variety of food preservatives with antibacterial effects were screened (ethylenediaminetetraacetic acid EDTA, disodium ethylenediaminetetraacetic acid EDTA, β-naphthol, benzoic acid, natamycin, diphenyl ether, sodium sulfite, sodium nitrite, sodium acetate, ethoxyquin, ε-polylysine salt, sodium benzoate, nisin, sodium diacetate, sodium bisulfite, calcium propionate, potassium sorbate, lysozyme, ε-polylysine, 2,4-dichlorophenoxyacetic acid, pyroacetic acid The selected antibacterial agents are then screened in the fruit body to select the antibacterial agents with small amount and good effect. The fruits are fumigated with their respective antibacterial concentrations and compounded according to different volume ratios to accurately screen the antibacterial agents against Rhizopus soft rot. Rhizopus stolonifer The screening method is simple and accurate, and the screened antibacterial agent has high efficacy. The screened essential oils with inhibitory effects were compounded at different concentrations, which reduced the amount of antibacterial agent used and had an inhibitory effect on Rhizopus stolonifer. Rhizopus stolonifer The control played a synergistic role.

[0033] In some examples of this embodiment, the product consists of oregano essential oil and methyl salicylate, and the methyl salicylate and oregano essential oil are compounded at a ratio of 1 / 4-1:1 / 4-1 of their respective half-maximal effective concentrations.

[0034] It should be noted that the term median effective concentration (EC 50 ) refers to the drug concentration that can cause 50% of the test organisms to produce a certain effect in the toxicity test of exogenous substances. In quantitative reaction, it refers to the drug dose that can cause 50% of the maximum reaction intensity; in qualitative reaction, it refers to the drug dose that only causes 50% of the experimental animals to have a positive reaction. The larger the median lethal dose and the smaller the median effective dose, the higher the safety of the drug. After testing, the EC values ​​of methyl salicylate, oregano essential oil, 2,3-butanedione, clove essential oil and nerol were 50 They were 41.7 μL / L, 38.8 μL / L, 74.8 μL / L, 37.0 μL / L and 49.7 μL / L respectively.

[0035] In some examples of this implementation mode, the product consists of 2,3-butanedione and methyl salicylate, and the methyl salicylate and 2,3-butanedione are compounded at a ratio of 1 / 4-1:1 / 4-1 of their respective half-effective concentrations.

[0036] In some examples of this embodiment, the methyl salicylate and 2,3-butanedione are compounded at 1 / 4-1 / 2:1 / 4-1 / 2 of their respective half-maximal effective concentrations.

[0037] In some examples of this embodiment, the methyl salicylate and 2,3-butanedione are compounded at 1 / 2:1 / 4 of their respective half-maximal effective concentrations.

[0038] In some examples of this implementation manner, the product consists of clove essential oil and methyl salicylate, and the methyl salicylate and clove essential oil are compounded at a ratio of 1 / 4-1:1 / 4-1 of their respective half-effective concentrations.

[0039] In some examples of this implementation mode, the product consists of nerol and methyl salicylate, and the methyl salicylate and nerol are compounded at a half-effective concentration of 1 / 2:1 / 2-1 or 1 / 4:1 / 4-1.

[0040] In some examples of this embodiment, the dosage form of the product includes but is not limited to any one of aqueous emulsion, emulsion, oil, suspension or solid preparation; The solid preparation includes, but is not limited to, any one of granules, powders, tablets, capsules and seed coatings.

[0041] A second typical embodiment of the present invention provides a use of the above-mentioned product in preventing and treating root mold soft rot of fruits and vegetables; The fruits and vegetables include any one or more of apples, pears, strawberries, figs, grapes, tomatoes, peaches, green peppers, and sweet potatoes.

[0042] In some examples of this embodiment, the root mold soft rot is Rhizopus stolonifer ( Rhizopus stolonifer ) caused by.

[0043] A third typical embodiment of the present invention provides a method for preserving fruits and vegetables, comprising: fumigating the fruits and vegetables with the above-mentioned product; The fruits and vegetables include any one or more of apples, pears, strawberries, figs, grapes, tomatoes, peaches, green peppers, and sweet potatoes.

[0044] The product provided by the invention can be used in the field of fruit and vegetable disease prevention and control, and is a method for reducing the amount of use while controlling Rhizopus stolonifer. Rhizopus stoloniferAmong them, MeSA and 2,3-butanedione, when used alone, have a synergistic effect on Rhizopus stolonifer. Rhizopus stolonifer The growth of both strains was significantly inhibited, and the combination of the two had a synergistic effect on inhibiting the occurrence of bacterial soft rot. When MeSA and 2,3-butanedione were added according to their respective EC 50 When the ratio of MeSA to 2,3-butanedione is 1 / 2:1 / 4, the occurrence of root mold soft rot can be basically inhibited. In the present invention, MeSA and 2,3-butanedione with different sources and chemical structures are used in combination to inhibit the occurrence of root mold soft rot. It can reduce the usage of the two antibacterial agents, improve the overall antibacterial effect, and enhance the storage stability and edible safety of fruits and vegetables after harvest.

[0045] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0046] Example 1 1. Screening of binary antibacterial agent combinations with synergistic effects on inhibiting root mold soft rot 1. Soft rot pathogens Rhizopus stolonifer In vitro screening of antibacterial agents Pick a single colony of Rhizopus stolonifer on the plate and inoculate it into the sterilized PDA medium by streaking, then place it in a constant temperature incubator and culture it at 25°C for about 2-5 days for use. According to the maximum concentration used in food obtained from the literature (Table 1), the maximum concentration is Max, and Max is measured. Rhizopus stolonifer is treated at 1 / 2Max concentration. Rhizopus stolonifer The inhibitory effect was measured by the cross method, that is, the colony diameter was measured and the mycelium growth inhibition rate (MGI) was calculated. For the concentration of natural essential oils not specified in the national standard, combined with the reference, a preliminary method was selected to add 5 μL of plant essential oil to the filter paper disc to treat the determination of Rhizopus stolonifer. Rhizopus stolonifer The calculation formula of MGI is as follows:

[0047] Table 1 Maximum concentration of each food preservative (Max)

[0048] 2. Determination of mycelial growth inhibition rate Under sterile conditions, pour the melted culture medium onto the plate. After transferring the Rhizopus stolonifer cake to the solidified culture medium, place a sterile 6 mm diameter filter paper disc in the center of the dish lid, and drip volatile compounds onto the filter paper disc to make the concentrations 25, 50, 75, 100, 150, and 200 μL / L, respectively. The plate without the drug dripped onto the filter paper disc was used as the control. Seal the plate and culture it at 25 ℃ for 2-5 days before observing the inhibition results.

[0049] 3. EC 50 Calculation The concentration of the antibacterial agent in the PDA medium was converted into logarithm, and the corresponding probability value was obtained by looking up the biostatistical probability value conversion table according to the mycelium growth inhibition rate. The logarithm of the concentration was used as the abscissa, and the probability value obtained from the table was used as the ordinate. Linear regression was performed to obtain the effects of MeSA, oregano essential oil, 2,3-butanedione, clove essential oil, and nerol on Rhizopus stolonifer. Rhizopus stolonifer The antibacterial toxicity equation was used to calculate the half effective concentration (EC) of each agent on Rhizopus stolonifer. 50 ).

[0050] 4. Evaluation of synergistic effect 4.1 Compounding scheme design MeSA was mixed with oregano essential oil, 2,3-butanedione, clove essential oil and nerol to find out the combination with MeSA that has a synergistic effect on the growth of Rhizopus stolonifer. 50 Set as a, the other four antibacterial agents EC 50 Set it as b, and set the compounding ratio to a+b, a+b / 2, a+b / 4, a / 2+b, a / 2+b / 2, a / 2+b / 4, a / 4+b, a / 4+b / 2, and a / 4+b / 4. Treat them according to the compounding ratios, with 3 parallels for each treatment. After 2-5 days, the colony diameter was measured by the cross method.

[0051] 4.2 Evaluation of synergistic effect According to "1. Soft rot pathogens Rhizopus stolonifer The actual mycelial growth inhibition rate of the compound combination was determined by the method described in "In vitro screening of antibacterial agents", and the co-toxicity factor method was used to evaluate whether the compound combination had a synergistic effect.

[0052]

[0053] In the above formula: F: Co-toxic factor M: Actual inhibition rate of mixed antibacterial agent (%) m: Theoretical inhibition rate of mixed antibacterial agent (%) This method compares the actual inhibition rate obtained from the compound combination experiment with the calculated theoretical inhibition rate, and uses the co-toxicity factor F value to evaluate whether the binary antibacterial agent combination has a synergistic effect on inhibiting the growth of Rhizopus stolonifer. Its scientificity has been proven many times. When F>20, it indicates that the two mixed antibacterial agents have a synergistic effect; F≤-20 indicates that the combination of the two has an antagonistic effect; F is between -20 and 20, indicating that the two mixed antibacterial agents have an additive effect.

[0054] 5. In vivo antibacterial effect of antibacterial agents 5.1 Fruit selection and cleaning Select figs, peaches, tomatoes, and green peppers of uniform size, uniform maturity, no mechanical damage, and no diseases or insect pests. Soak them in 0.5% NaClO solution for 30 s, then rinse them with clean water, dry them naturally in the shade, and set aside.

[0055] 5.2 Determination of antibacterial effect Use sterile steel nails to punch holes in the skin of figs, peaches, and tomatoes. For green peppers, use a sterile puncher with a diameter of 1 cm to gently press the center of the surface to leave a circular mark. Then use a sterile blade to scrape off a thin layer of the green pepper skin along the circular mark to form a wound, but do not pierce the skin to prevent the solution from flowing into the green pepper cavity. Take 10 μL Rhizopus stolonifer Use a pipette to punch into the hole or wound and air dry at room temperature. Put the fruit into a plastic basket (340 mm×240 mm×100 mm) and cover it with a polyethylene bag. Drop essential oils of different concentrations (four times the compound concentration in 4.1) on sterile filter paper. The sterile filter paper is fixed on the bag. Use wet gauze to keep the humidity in the frame at 90-95%, and then seal it. Then place the fruit in a 25℃ observation room to observe the disease. After 4 days, the cross method is used to measure the diameter of the lesions and the incidence rate is calculated. The experiment is repeated three times.

[0056]

[0057] 6. Data processing SPSS 25.0 was used to analyze the data, and Duncan's multiple comparison was used to analyze the significant differences between the values ​​(P<0.05). All charts were drawn using origin 2018 software.

[0058] 7. Experimental Results 7.1 Pathogens of Rhizopus soft rot Rhizopus stolonifer In vitro screening of antibacterial agents By consulting the literature and conducting preliminary experiments, the concentrations of each food preservative were selected in Table 1 for antibacterial experiments. Figure 1 ) It can be seen that benzoic acid, sodium sulfite, sodium nitrite, nisin, sodium bisulfite, lysozyme Rhizopus stolonifer No inhibitory effect, EDTA, disodium EDTA, natamycin, diphenyl ether, ethoxyquin, sodium diacetate, potassium sorbate, ε-polylysine Rhizopus stolonifer had a significant inhibitory effect, while β-naphthol and ε-polylysine could completely inhibit Rhizopus stolonifer growth.

[0059] For volatile compounds, 5 μL was used for fumigation treatment. Figure 2 It can be seen that ethanol, 2-phenylethanol, rosemary essential oil, and perilla leaf essential oil have Rhizopus stolonifer There was no inhibitory effect on peppermint essential oil, citronella essential oil, nerol, L-carvone, tea tree essential oil, and linalool. Rhizopus stolonifer 2,3-Butanedione, 4-propylphenol, clove essential oil, eugenol, thyme essential oil, oregano essential oil, wintergreen essential oil, and methyl salicylate can completely inhibit Rhizopus stolonifer growth.

[0060] Taking into account the safety and actual price factors of food preservatives and the flavor effects of volatile compounds, MeSA, 2,3-butanedione, oregano essential oil, clove essential oil and nerol fumigation treatments were selected for subsequent experiments.

[0061] 7.2 Plant essential oils Rhizopus stolonifer Inhibition of mycelial growth and EC 50 Plant essential oil Rhizopus stolonifer The inhibitory effects of MeSA, oregano essential oil, clove essential oil, 2,3-butanedione and nerol on the growth of mycelium are shown in Table 2. Rhizopus stolonifer Mycelial growth showed different degrees of inhibition, and the mycelial inhibition rate could reach more than 90% when the concentrations of MeSA, oregano essential oil, clove essential oil and nerol were 75, 100, 75 and 100 μL / L, respectively. From the overall trend, the antibacterial effect of these five plant essential oils was closely related to the concentration and increased with the increase of concentration.

[0062] The concentrations of each plant essential oil in Table 2 were converted into logarithms, and the mycelium growth inhibition rate was converted into probability values ​​for linear regression, and the results in Table 3 were obtained. It can be seen from Table 3 that there is a close correlation between the concentration of each plant essential oil and the antibacterial effect. As the concentration increases, the inhibition rate increases significantly. The correlation between the concentration of five plant essential oils, MeSA, oregano essential oil, clove essential oil, 2,3-butanedione and nerol, and the antibacterial effect is high, with correlation coefficients of 0.9934, 0.9981, 0.9957, 0.9988, and 0.9975, respectively. The EC values ​​of MeSA, oregano essential oil and clove essential oil are 0.994, 0.998, 0.998, and 0.998, respectively. 50 are relatively close, which are 41.7 μL / L, 38.8 μL / L, and 37.0 μL / L, respectively, while the EC values ​​of 2,3-butanedione and nerol are 50 Higher, 74.8 μL / L and 49.7 μL / L respectively. 50 , each plant essential oil Rhizopus stolonifer The antibacterial strength is clove essential oil>oregano essential oil>MeSA>nerol>2,3-butanedione.

[0063] Table 2 Inhibition rate and EC of plant essential oils on mycelium of Rhizopus stolonifer 50

[0064] Table 3 The inhibitory toxicity equations and EC of different plant essential oils against Rhizopus stolonifer 50

[0065] 7.3. Essential oil combination Rhizopus stolonifer Synergistic effect of mycelial growth On the basis of determining the inhibitory effect of each plant essential oil on Rhizopus stolonifer, the co-toxicity factor method was used to determine the synergistic effect of each essential oil combination. As shown in Table 4, when the concentration combination of MeSA and nerol was 41.7+12.425 μL / L, the actual inhibition rate of both on the growth of Rhizopus stolonifer was significantly lower than the theoretical inhibition rate, showing an antagonistic effect. However, the actual inhibition rate of the combination of MeSA with oregano essential oil, 2,3-butanedione, and clove essential oil was significantly higher than the theoretical inhibition rate.

[0066] When the concentration combination of MeSA and 2,3-butanedione was 10.425+74.8 μL / L, the co-toxicity factor was between -20 and 20, indicating that the two mixed antibacterial agents had an additive effect, while the co-toxicity factors of other concentration combinations were all greater than 20, indicating that the two mixed antibacterial agents had a synergistic effect. When the concentration combination of MeSA and 2,3-butanedione was 20.85+18.7 μL / L, the co-toxicity factor was 165.15, which was significantly higher than that of other treatment groups. When the concentration combination of MeSA and 2,3-butanedione was 41.7+37.4 μL / L, the actual inhibition rate reached 100%, and it played a role of reducing the amount and increasing the effect.

[0067] Table 4 Co-toxic factors of essential oil combinations against Rhizopus stolonifer

[0068] 7.4. Control effect of plant essential oil complex on Rhizopus soft rot in fruits Combined with the synergistic effect evaluation results, the combination of MeSA with oregano essential oil, 2,3-butanedione, and clove essential oil was selected to determine the fruit rot rate and lesion diameter. Figures 3 - 18 It can be seen that compared with the control group, the combination of MeSA with oregano essential oil, 2,3-butanedione, and clove essential oil can effectively reduce the incidence of fig, peach, tomato, and green pepper fruits, and reduce the diameter of lesions on damaged inoculated fruits.

[0069] Overall, the effects of MeSA combined with oregano essential oil and clove essential oil were similar, and the higher the concentration, the lower the incidence rate and the smaller the lesion diameter. In contrast, the combination of MeSA and 2,3-butanedione had the best effect. Figures 3 - 6 It can be seen that for fig fruits, after injury inoculation, the morbidity of the control group reached 100%, while when the combination of MeSA and 2,3-butanedione was 1 / 2+1 / 4, the morbidity dropped to 11.11%. Similarly, for tomato fruits, the morbidity of the control group reached 100%, and when the combination of MeSA and 2,3-butanedione was 1 / 2+1 / 4, the morbidity dropped to 8.33%. Therefore, the combination of MeSA and 2,3-butanedione can significantly reduce the morbidity and lesion diameter of fruit root mold soft rot, and when the combination is 1 / 2+1 / 4, the morbidity can be reduced by more than 80%, achieving the effect of reducing the amount and increasing the efficiency. The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A product for preventing and controlling root mold soft rot, characterized in that, The product is composed of oregano essential oil, any one of 2,3-butanedione, nerol and clove essential oil and methyl salicylate; The root mold soft rot is Rhizopus stolonifer ( Rhizopus stolonifer ) caused by; When the product is composed of oregano essential oil, any one of 2,3-butanedione and clove essential oil and methyl salicylate, the oregano essential oil, any one of 2,3-butanedione and clove essential oil and methyl salicylate are compounded at 1 / 4-1:1 / 4-1 of their respective half-effective concentrations; When the product is composed of nerol and methyl salicylate, the methyl salicylate and nerol are compounded at a half-effective concentration of 1 / 2:1 / 2-1 or 1 / 4:1 / 4-1.

2. The product according to claim 1, characterized in that The methyl salicylate and 2,3-butanedione are compounded at 1 / 4-1 / 2:1 / 4-1 / 2 of their respective half-effective concentrations.

3. The product according to claim 1, characterized in that The methyl salicylate and 2,3-butanedione are compounded at 1 / 2:1 / 4 of their respective half-effective concentrations.

4. The product according to claim 1, characterized in that The dosage form of the product includes any one of aqueous emulsion, emulsion, oil, suspension or solid preparation.

5. The product according to claim 4, characterized in that The solid preparation includes any one of granules, powders, tablets, capsules and seed coatings.

6. Use of the product according to any one of claims 1 to 5 in preventing and treating Rhizopus soft rot of fruits and vegetables.

7. The use according to claim 6, characterized in that The fruits and vegetables include any one or more of apples, pears, strawberries, figs, grapes, tomatoes, peaches, green peppers, and sweet potatoes.

8. The use according to claim 6, characterized in that The root mold soft rot is Rhizopus stolonifer ( Rhizopus stolonifer ) caused by.

9. A method for preserving fruits and vegetables, characterized in that: include: The product according to any one of claims 1 to 5 is used to fumigate fruits and vegetables.

10. The method for preserving fruits and vegetables as claimed in claim 9, characterized in that: The fruits and vegetables include any one or more of apples, pears, strawberries, figs, grapes, tomatoes, peaches, green peppers, and sweet potatoes.

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