A product for preventing and controlling Rhizopus soft rot, its application and a method for fresh-keeping fruits and vegetables

Abstract

The present invention discloses a product for preventing and controlling Rhizopus soft rot, its application and a method for preserving fruits and vegetables, belonging to the field of disease control. The product for preventing and controlling Rhizopus soft rot is composed of any one of oregano essential oil, 2,3-butanedione, nerol and clove essential oil and methyl salicylate; the Rhizopus soft rot is caused by Rhizopus stolonifer ( Rhizopus stolonifer ). Through compounding, the usage amount of essential oil can be reduced, the overall antibacterial effect can be improved, the drug cost can be reduced, the drug selection can be increased, the operation is convenient, and the postharvest storage tolerance and food safety of fruits and vegetables are enhanced. Moreover, 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, and have the advantages of low toxicity and 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 for preventing and controlling postharvest Rhizopus soft rot of fruits.

Description

Technical Field

[0001] The present invention belongs to the field of disease control, and particularly relates to a product for preventing and controlling Rhizopus soft rot, its application and a method for preserving fruits and vegetables fresh. Background Art

[0002] Disclosing the information of this background art section is only intended to enhance the overall understanding of the present invention, and it is not necessarily regarded as an admission or an implication in any form that this information constitutes the prior art already known to those of ordinary skill in the art.

[0003] Rhizopus stolonifer ( Rhizopus stolonifer ) belongs to the class Zygomycetes, order Mucorales, family Mucoraceae, genus Rhizopus, and is the most common and fastest-growing one among Zygomycetes. The spores of Rhizopus stolonifer exist widely everywhere, can be spread by air currents, and grow rapidly in a humid environment, resulting in soft rot of various fruits such as peaches, strawberries, pears, grapes, tomatoes, etc. When the fruit is infected, the initially infected area is watery and then becomes soft and rotten. At appropriate temperature and humidity, the initial symptoms appear about 1 day after infection, rapid growth and a large amount of mycelia are observed on the fruit surface, and the wound part is covered by producing filamentous gray sporangia. The infected tissue finally decomposes into a watery rot, and its wound surface becomes very soft. About 2 - 3 days later, 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 for controlling 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 foods, 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, have a strong effect on pathogenic bacteria, do not produce drug resistance, do not add chemical substances during the treatment process, have no side effects on the environment, the sterilization conditions are easy to control, the operation is simple, and are less affected by the external environment. However, the control intensity of physical control methods is limited, they do not have universal applicability, and most of the effects are not ideal. They need to be combined with other technologies to achieve better prevention and control effects. Chemical control methods have a wide range of effects and obvious effects, and are commonly used postharvest management strategies for fruits. Chemical control methods include fungicides, calcium chloride, salicylic acid, peracetic acid, nanomaterials, etc. Chemical control methods have good effects, low costs, and are easy to implement, and are widely used in the postharvest preservation of fruits and vegetables. However, the use of chemical substances such as fungicides has been controversial. Excessive use may lead to residues of certain chemical substances, 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 drug resistance, which is also one of the reasons unacceptable to consumers. Some chemical control methods require coatings, and certain reagents will form a thin film on the surface of rough fruits and vegetables, thus 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 postharvest fruits. Summary of the Invention

[0005] To solve the deficiencies of the prior art, the purpose of the present invention is to provide a product for preventing and controlling Rhizopus soft rot, its application, and a method for preserving fruits and vegetables. The product for preventing and controlling Rhizopus soft rot provided by the present invention controls the pathogenic bacteria of fruit Rhizopus soft rot Rhizopus stolonifer while reducing the usage amount of antibacterial agents, has a synergistic effect on the prevention and control of fruit soft rot, and when used in foods, can also ensure the safety of fruits and vegetables, providing a new solution for the future prevention and control of fruit Rhizopus soft rot.

[0006] To achieve the above purpose, the technical solution of the present invention is as follows:

[0007] In the first aspect of the present invention, a product for preventing and controlling Rhizopus soft rot is provided. The product is composed of any one of oregano essential oil, 2,3-butanedione, nerol, and clove essential oil and methyl salicylate (MeSA);

[0008] The Rhizopus soft rot is caused by Rhizopus stolonifer ( Rhizopus stolonifer ).

[0009] In some embodiments of the present invention, the product is composed 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 median effective concentrations.

[0010] In some embodiments of the present invention, the product is composed 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 median effective concentrations.

[0011] In some embodiments of the present invention, the methyl salicylate and 2,3 - butanedione are compounded at a ratio of 1 / 4 - 1 / 2:1 / 4 - 1 / 2 of their respective median effective concentrations.

[0012] In some embodiments of the present invention, the methyl salicylate and 2,3 - butanedione are compounded at a ratio of 1 / 2:1 / 4 of their respective median effective concentrations.

[0013] In some embodiments of the present invention, the product is composed 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 median effective concentrations.

[0014] In some embodiments of the present invention, the product is composed of nerol and methyl salicylate, and the methyl salicylate and nerol are compounded at a ratio of 1 / 2:1 / 2 - 1 or 1 / 4:1 / 4 - 1 of their respective median effective concentrations.

[0015] In some embodiments of the present invention, the dosage form of the product includes any one of an emulsion in water, an emulsion, an oil agent, a suspension agent, or a solid preparation;

[0016] The solid preparation includes any one of a granule, a powder, a tablet, a capsule, and a seed coating agent.

[0017] The second aspect of the present invention provides an application of the above - mentioned product in preventing and controlling Rhizopus soft rot of fruits and vegetables;

[0018] The fruits and vegetables include any one or more of apples, pears, strawberries, figs, grapes, tomatoes, peaches, green peppers, and sweet potatoes.

[0019] In some embodiments of the present invention, the Rhizopus soft rot is caused by Rhizopus stolonifer ( Rhizopus stolonifer )

[0020] The third aspect of the present invention provides a method for preserving fruits and vegetables, including: fumigating the fruits and vegetables with the above - mentioned product;

[0021] The fruits and vegetables include any one or more of apples, pears, strawberries, figs, grapes, tomatoes, peaches, green peppers, and sweet potatoes.

[0022] The beneficial effects of the present invention are as follows:

[0023] The present invention provides a product for preventing and controlling Rhizopus soft rot, which is an antibacterial agent obtained by compounding methyl salicylate with oregano essential oil, 2,3-butanedione, nerol or clove essential oil. It can achieve a better preventive or therapeutic effect on Rhizopus soft rot while reducing the usage amount of each part. With a smaller dosage, it can achieve a better effect. Moreover, 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 and 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. Using the product of the present invention to prevent and control postharvest Rhizopus soft rot of fruits can not only reduce the usage amount of essential oil, but also improve the overall antibacterial effect, reduce the drug cost, increase the drug selection, facilitate operation, and enhance the postharvest storage resistance and edible safety of fruits and vegetables.

[0024] In particular, when methyl salicylate is compounded with 2,3-butanedione, it has a synergistic effect on inhibiting the growth of Rhizopus soft rot of fruits. When the two are compounded at their respective optimal concentrations, compared with the control group, the incidence rate of Rhizopus soft rot is significantly reduced. On the basis of reducing the usage amount of the two natural essential oils, it improves the overall antibacterial effect and fruit quality, reduces the drug cost, increases the drug selection, and enhances the postharvest storage resistance and edible safety of fruits and vegetables. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The accompanying drawings forming a part of the present invention are used to provide a further understanding of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation to the present invention.

[0026] Figure 1 Shows the inhibitory effect of different preservatives on Rhizopus stolonifer in Example 1 of the present invention;

[0027] Figure 2 Shows the inhibitory effect of different volatile compounds on Rhizopus stolonifer in Example 1 of the present invention;

[0028] Figure 3 Shows the in vivo antibacterial effect of different essential oil treatments on figs in Example 1 of the present invention;

[0029] Figure 4 Shows the lesion diameter and rot rate of figs treated with the compound of different concentrations of MeSA and oregano essential oil in Example 1 of the present invention;

[0030] Figure 5 Shows the lesion diameter and rot rate of figs treated with the compound of different concentrations of MeSA and clove essential oil in Example 1 of the present invention;

[0031] Figure 6Disease spot diameter and decay rate of fig treated with different concentrations of MeSA and 2,3-butanedione in Example 1 of the present invention;

[0032] Figure 7 Antibacterial effect in vivo of different essential oil treatments on peach in Example 1 of the present invention;

[0033] Figure 8 Disease spot diameter and decay rate of peach treated with different concentrations of MeSA and oregano essential oil in Example 1 of the present invention;

[0034] Figure 9 Disease spot diameter and decay rate of peach treated with different concentrations of MeSA and clove essential oil in Example 1 of the present invention;

[0035] Figure 10 Disease spot diameter and decay rate of peach treated with different concentrations of MeSA and 2,3-butanedione in Example 1 of the present invention;

[0036] Figure 11 Antibacterial effect in vivo of different essential oil treatments on tomato in Example 1 of the present invention;

[0037] Figure 12 Disease spot diameter and decay rate of tomato treated with different concentrations of MeSA and oregano essential oil in Example 1 of the present invention;

[0038] Figure 13 Disease spot diameter and decay rate of tomato treated with different concentrations of MeSA and clove essential oil in Example 1 of the present invention;

[0039] Figure 14 Disease spot diameter and decay rate of tomato treated with different concentrations of MeSA and 2,3-butanedione in Example 1 of the present invention;

[0040] Figure 15 Antibacterial effect in vivo of different essential oil treatments on green pepper in Example 1 of the present invention;

[0041] Figure 16 Disease spot diameter and decay rate of green pepper treated with different concentrations of MeSA and oregano essential oil in Example 1 of the present invention;

[0042] Figure 17 Disease spot diameter and decay rate of green pepper treated with different concentrations of MeSA and clove essential oil in Example 1 of the present invention;

[0043] Figure 18 Disease spot diameter and decay rate of green pepper treated with different concentrations of MeSA and 2,3-butanedione in Example 1 of the present invention.

[0044] Note: Figures 3 - 18Among them, 1 + 1 means the mixing of the original concentration of MeSA (166.8 μL / L) with the original concentrations 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 / 2 means the mixing of the original concentration of MeSA (166.8 μL / L) with 1 / 2 of the original concentrations 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 means the mixing of the original concentration of MeSA (166.8 μL / L) with 1 / 4 of the original concentrations of 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 means the mixing of 1 / 2 of the original concentration of MeSA (166.8 μL / L) with the original concentrations of 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 means the mixing of 1 / 2 of the original concentration of MeSA (166.8 μL / L) with 1 / 2 of the original concentrations of 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 means the mixing of 1 / 2 of the original concentration of MeSA (166.8 μL / L) with 1 / 4 of the original concentrations 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 means the mixing of 1 / 4 of the original concentration of MeSA (166.8 μL / L) with the original concentrations 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 means the mixing of 1 / 4 of the original concentration of MeSA (166.8 μL / L) with 1 / 2 of the original concentrations 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 means the mixing of 1 / 4 of the original concentration of MeSA (166.8 μL / L) with 1 / 4 of the original concentrations of oregano essential oil, clove essential oil or 2,3-butanedione (155.2 μL / L, 148 μL / L, 299.2 μL / L respectively). Detailed implementation method

[0045] The pathogen of Rhizopus soft rot grows relatively fast, and its penetration and colonization speeds are also fast. Currently, once fruits are infected by this pathogen, they are basically impossible to be completely eliminated. Once the fruits are infected, the disease will quickly spread to other fruits, resulting in significant losses. Some commonly used control methods at present are usually chemical methods, mainly including agricultural control, pesticides, chemical fungicides, etc. However, the use of chemical methods may lead to the residue of certain chemical substances. 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 Rhizopus stolonifer in postharvest fruits. Based on this, the present invention provides a product for preventing and controlling Rhizopus soft rot, its application, and a method for preserving fruits and vegetables.

[0046] In a typical embodiment of the present invention, a product for preventing and controlling Rhizopus soft rot is provided. The product is composed of any one of oregano essential oil, 2,3-butanedione, nerol, and clove essential oil and methyl salicylate.

[0047] The Rhizopus soft rot is caused by Rhizopus stolonifer ( Rhizopus stolonifer ).

[0048] Plant essential oils are a kind of natural volatile aromatic compounds and are secondary metabolites obtained from different organs of plants. Due to their biological properties, especially antifungal properties, they have the potential to be used as substitutes for many traditional antifungal agents in preserving fruits and vegetable products, and there are many studies on their application in fruit and vegetable preservation. In addition, as a way of using essential oils in fruit and vegetable preservation, fumigation can achieve a better antibacterial and fresh-keeping effect while keeping the fruit surface clean.

[0049] The present invention first determines the antibacterial effects of various bacteriostatic agents by measuring the mycelial inhibition rates of different food preservatives and volatile compounds on Rhizopus stolonifer Rhizopus stolonifer. Screening is carried out according to the maximum allowable use concentrations of different food preservatives and volatile compounds in foods. Further, Rhizopus stolonifer R can be inhibited according to 1 / 2 of the maximum allowable use concentrations of different food preservatives and volatile compounds. hizopus stolonifer is inhibited.

[0050] It should be noted that the screening is a commonly used screening method in the art. Specifically, it can be inoculating Rhizopus stolonifer Rhizopus stolonifer onto potato dextrose agar (PDA) media with and without food preservatives respectively for cultivation. After the control group fills the plate, the antibacterial effects are compared by measuring the colony diameters through the cross method. The screening method can also be: inoculating Rhizopus stolonifer Rhizopus stoloniferInoculate it into a potato dextrose agar (PDA) medium for cultivation. Use the fumigation method to add volatile organic compounds and sterile water respectively. After culturing until the control group fills the plate, measure the colony diameter by the cross method to compare the antibacterial effects.

[0051] In some embodiments of this implementation manner, the screening includes the following steps:

[0052] S1: Activation of the strain: Rhizopus stolonifer Rhizopus stolonifer The activation is carried out according to the ATCC guidelines. Place the isolated and purified strain in a 4°C refrigerator for storage. Each time it is used, pick a single colony stored for backup with an inoculation needle, and inoculate it into a sterilized PDA medium by the streaking method, and then place it in a constant temperature incubator and culture at 25°C for about 2 - 5 days for backup.

[0053] S2: Preparation of the medium: Potato dextrose agar (PDA) medium: Weigh a certain mass of PDA medium powder according to the required volume, add a certain amount of deionized water, heat it on an electric furnace, stir while heating until it reaches the boiling state, cool it, and then slowly dispense it into the prepared conical flasks, seal them with sealing films, and sterilize them at 121°C in an autoclave for 15 min for later use.

[0054] S3: Preparation of PDA medium containing food preservatives: After preparing the above PDA medium, weigh the corresponding concentrations of each food preservative and put them into a conical flask, and shake well. Pay attention to the properties of the food preservatives during this process. For products that are easily decomposed at high temperatures, they need to be put in after sterilization and cooling. For acidic food preservatives, the pH of the medium needs to be adjusted to be suitable for the acidic property to play. For ester food preservatives, small molecule co-solvents such as dimethyl sulfoxide (DMSO) can be added. Set negative controls and positive controls for each group of experiments according to the requirements, with three parallels in each group.

[0055] S4: Preparation of plates containing volatile compounds: After preparing the PDA medium, pour it into disposable sterile petri dishes. After transferring the Rhizopus stolonifer mycelial cake to the solidified medium, place a sterile filter paper disc with a diameter of 6 mm in the center of the petri dish lid, and drop 5 μL of volatile compounds on the filter paper disc. Set negative controls and positive controls for each group of experiments according to the requirements, with three parallels in each group.

[0056] S5: Determination of the antibacterial effects of different antibacterial agents: Use the cross method to measure the inhibition rate of mycelial growth (MGI) of different food preservatives and volatile compounds on Rhizopus stolonifer Rhizopus stolonifer mycelium.

[0057] A variety of food preservatives with antibacterial effects (ethylenediaminetetraacetic acid EDTA, disodium ethylenediaminetetraacetate EDTA disodium, 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, sodium metabisulfite or fumaric acid) and volatile compounds (ethanol, 2,3-butanedione, 2-phenylethanol, 4-propylphenol, clove essential oil, eugenol, dimethyl fumarate, sec-butylamine, thyme essential oil, peppermint essential oil, citronella essential oil, oregano essential oil, rosemary essential oil, nerol, (-)-carvone, (+)-carvone, wintergreen essential oil, methyl salicylate MeSA, tea tree essential oil, cinnamaldehyde, linalool, lemon essential oil, perilla essential oil or geraniol) were screened. Then the screened bacteriostatic agents were screened in the fruit body to select bacteriostatic agents with small amounts and excellent effects. They were compounded according to their respective bacteriostatic concentrations and different volume ratios, and the fruits were fumigated to accurately screen out those against Rhizopus soft rot Rhizopus stolonifer combinations that have an inhibitory effect on the growth of, that is, any one of oregano essential oil, 2,3-butanedione, nerol and clove essential oil combined with methyl salicylate. The screening method is simple and accurate, and the screened bacteriostatic agents are highly effective. The screened essential oils with inhibitory effects were compounded at different concentrations, which played a synergistic role in controlling Rhizopus stolonifer Rhizopus stolonifer while reducing the usage amount of bacteriostatic agents.

[0058] In some embodiments of this embodiment, the product is composed of oregano essential oil and methyl salicylate, and the methyl salicylate and oregano essential oil are compounded at 1 / 4 - 1:1 / 4 - 1 of their respective median effective concentrations.

[0059] It should be noted that the term median effective concentration (EC 50 ) refers to the drug concentration that can cause a certain effect in 50% of the test organisms in the toxicity experiment of exogenous substances. In a quantal response, it refers to the drug dose that can cause 50% of the maximum response intensity; in a qualitative response, it refers to the drug dose that only causes 50% of the experimental animals to show a positive reaction. The larger the median lethal dose and the smaller the median effective dose, the higher the safety of the drug. After detection, the EC 50 values of methyl salicylate, oregano essential oil, 2,3-butanedione, clove essential oil and nerol are 41.7 μL / L, 38.8 μL / L, 74.8 μL / L, 37.0 μL / L, 49.7 μL / L respectively.

[0060] In some embodiments of this embodiment, the product is composed of 2,3-butanedione and methyl salicylate, and methyl salicylate and 2,3-butanedione are compounded at a ratio of 1 / 4 - 1:1 / 4 - 1 of their respective median effective concentrations.

[0061] In some embodiments of this embodiment, methyl salicylate and 2,3-butanedione are compounded at a ratio of 1 / 4 - 1 / 2:1 / 4 - 1 / 2 of their respective median effective concentrations.

[0062] In some embodiments of this embodiment, methyl salicylate and 2,3-butanedione are compounded at a ratio of 1 / 2:1 / 4 of their respective median effective concentrations.

[0063] In some embodiments of this embodiment, the product is composed of clove essential oil and methyl salicylate, and methyl salicylate and clove essential oil are compounded at a ratio of 1 / 4 - 1:1 / 4 - 1 of their respective median effective concentrations.

[0064] In some embodiments of this embodiment, the product is composed of nerol and methyl salicylate, and methyl salicylate and nerol are compounded at a ratio of 1 / 2:1 / 2 - 1 or 1 / 4:1 / 4 - 1 of their respective median effective concentrations.

[0065] In some embodiments of this embodiment, the dosage form of the product includes but is not limited to any one of water emulsion, emulsion, oil agent, suspending agent or solid preparation;

[0066] The solid preparation includes but is not limited to any one of granule, powder, tablet, capsule and seed coating agent.

[0067] The second typical embodiment of the present invention provides an application of the above product in the prevention and control of root rot of fruits and vegetables;

[0068] The fruits and vegetables include any one or more of apple, pear, strawberry, fig, grape, tomato, peach, green pepper, sweet potato.

[0069] In some embodiments of this embodiment, the root rot is caused by Rhizopus stolonifer ).

[0070] The third typical embodiment of the present invention provides a method for preserving fruits and vegetables, including: fumigating the fruits and vegetables with the above product;

[0071] The fruits and vegetables include any one or more of apple, pear, strawberry, fig, grape, tomato, peach, green pepper, sweet potato.

[0072] The product provided by the present invention can be used in the field of disease prevention and control of fruits and vegetables, and it is a product that can reduce the usage amount while having an inhibitory effect onRhizopus stolonifer A biological fungicide with synergistic effect on prevention and control. Among them, when MeSA and 2,3-butanedione are used alone, they both have a significant inhibitory effect on the growth of Rhizopus stolonifer Rhizopus stolonifer When they are compounded, they have a synergistic effect on inhibiting the occurrence of bacterial soft rot; when MeSA and 2,3-butanedione are compounded according to their respective EC 50 at a ratio of 1 / 2:1 / 4, the occurrence of Rhizopus soft rot can be basically inhibited. In the present invention, MeSA and 2,3-butanedione with different sources and chemical structures are compounded and used, which has a synergistic effect on inhibiting the occurrence of Rhizopus soft rot. It can not only reduce the usage amount of the two bacteriostatic agents, but also improve the overall bacteriostatic effect, and enhance the postharvest storage tolerance and food safety of fruits and vegetables.

[0073] 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.

[0074] Example 1

[0075] I. Screening of binary bacteriostatic agent compound combinations with synergistic effect on inhibiting Rhizopus soft rot

[0076] 1. Soft rot pathogen Rhizopus stolonifer In vitro primary screening of bacteriostatic agents

[0077] Pick the single colony of Rhizopus stolonifer that has been cultured on the plate, and inoculate it into the sterilized PDA medium by the streaking method, and then put it into a constant temperature incubator and culture it at 25°C for about 2-5 days for standby. According to the maximum concentration for food obtained by consulting the literature (Table 1), taking the maximum concentration as Max, the inhibitory effect on Rhizopus stolonifer was measured at Max and 1 / 2Max concentrations, that is, the colony diameter was measured by the cross method, and the mycelial growth inhibition rate (MGI) was calculated. For the concentration of natural essential oils not specified in the national standard, in combination with consulting the literature, it was initially selected to drop 5 μL of plant essential oil on the filter paper disc to measure the inhibitory effect on Rhizopus stolonifer Rhizopus stolonifer The calculation formula of MGI is as follows: Rhizopus stolonifer The calculation formula of MGI is as follows:

[0078] Table 1 Maximum usage concentration (Max) of each food preservative

[0079]

[0080] 2. Determination of mycelial growth inhibition rate

[0081] Under aseptic conditions, pour the melted medium onto the plate. After transferring the Rhizopus stolonifer cake to the solidified medium, place a sterile filter paper disk with a diameter of 6 mm in the center of the petri dish lid, and add volatile compounds dropwise onto the filter paper disk at concentrations of 25, 50, 75, 100, 150, and 200 μL / L, respectively. Use the plate without adding the agent on the filter paper disk as the control. Seal the plate and place it at 25 °C for 2 - 5 d, then observe the inhibition results.

[0082] 3. Calculation of EC 50

[0083] Convert the concentration of the bacteriostatic agent in the PDA medium to logarithm, and respectively look up the corresponding probability values in the biostatistical probability value conversion table according to the mycelial growth inhibition rate. Use the logarithm of the concentration as the abscissa and the probability value obtained from the table lookup as the ordinate, and linearly regress to obtain the bacteriostatic toxicity equations of MeSA, oregano essential oil, 2,3 - butanedione, clove essential oil, and nerol to Rhizopus stolonifer Rhizopus stolonifer , and calculate the median effective concentration (EC 50 ) of each agent against Rhizopus stolonifer according to the toxicity equation.

[0084] 4. Evaluation of synergistic effect

[0085] 4.1 Design of compounding scheme

[0086] Compound MeSA with oregano essential oil, 2,3 - butanedione, clove essential oil, and nerol respectively, and find out the combinations that have a synergistic effect on the mycelial growth of Rhizopus stolonifer when compounded with MeSA. Assume that the EC 50 of MeSA is set as a, and the EC 50 of the other 4 bacteriostatic agents is set as b, and set the compounding ratios as combinations of 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. After compounding according to each ratio respectively for treatment, there are 3 parallels for each treatment, and measure the colony diameter by the cross - method after 2 - 5 d.

[0087] 4.2 Evaluation of synergistic effect

[0088] According to the method described in "1. Preliminary screening of bacteriostatic agents for soft rot pathogens Rhizopus stolonifer in vitro", measure the actual mycelial growth inhibition rate of the compounding combinations, and use the co - toxicity factor method to evaluate whether the compounding combinations have a synergistic effect.

[0089]

[0090] In the above formula:

[0091] F: Co - toxicity factor

[0092] ​M: Actual inhibition rate of the mixed bacteriostatic agent (%)

[0093] m: Theoretical inhibition rate of the mixed bacteriostatic agent (%)

[0094] 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 bacteriostatic agent compounding 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 bacteriostatic agents have a synergistic effect; when F ≤ -20, it indicates that the compounding of the two has an antagonistic effect; when F is between -20 and 20, it indicates that the two mixed bacteriostatic agents have an additive effect.

[0095] 5. In vivo bacteriostatic effect of the bacteriostatic agent

[0096] 5.1 Fruit selection and cleaning

[0097] Select figs, peaches, tomatoes, and green peppers with uniform size, consistent maturity, no mechanical damage, and no pests and diseases. After soaking them in 0.5% NaClO solution for 30 s, rinse the figs, peaches, tomatoes, and green peppers with clean water and air-dry them naturally for later use.

[0098] 5.2 Determination of bacteriostatic effect

[0099] Use sterile steel nails to punch holes in the epidermis of figs, peaches, and tomatoes respectively. For green peppers, use a sterile punch with a diameter of 1 cm to gently press on the center part of its surface to leave a circular mark, and then use a sterile blade to scrape off a thin layer of the green pepper epidermis according to the circular mark to form a wound, but do not pierce the epidermis to avoid the solution flowing into the cavity of the green pepper. Take 10 μL Rhizopus stolonifer Use a pipette gun to inject it into the hole or wound and air-dry it at room temperature. Put the fruits into a plastic basket (340 mm × 240 mm × 100 mm) and cover it with a polyethylene bag. Drop essential oils with different concentration ratios (four times the compound concentration in 4.1) on sterile filter paper, fix the sterile filter paper above the bag, use a wet gauze to keep the humidity in the basket at 90 - 95%, and then seal it. Then place the fruits in a 25°C appearance room to observe the disease situation. After 4 d, measure the diameter of the lesion by the cross method and count its incidence rate. The experiment is repeated three times.

[0100]

[0101] 6. Data processing

[0102] Use SPSS 25.0 to analyze the data, and Duncan multiple comparison is used to analyze the significant differences between values (P < 0.05). All charts are drawn using origin 2018 software.

[0103] 7. Experimental Results

[0104] 7.1 Rhizopus soft rot pathogen Rhizopus stolonifer Primary screening of bacteriostatic agents in vitro

[0105] Through literature review and preliminary experiments, antibacterial experiments were carried out for each concentration in Table 1 for each food preservative. According to the results ( Figure 1 ), it can be seen that benzoic acid, sodium sulfite, sodium nitrite, nisin, sodium bisulfite, and lysozyme have no inhibitory effect on Rhizopus stolonifer , while EDTA, disodium EDTA, natamycin, diphenyl ether, ethoxyquin, sodium diacetate, potassium sorbate, and ε-polylysine have a significant inhibitory effect on Rhizopus stolonifer , and β-naphthol and ε-polylysine can completely inhibit the growth of Rhizopus stolonifer at the selected concentrations.

[0106] For volatile compounds, 5 μL was used for fumigation treatment. As can be seen from Figure 2 , ethanol, 2-phenylethanol, rosemary essential oil, and perilla leaf essential oil have no inhibitory effect on Rhizopus stolonifer , while peppermint essential oil, citronella essential oil, nerol, (-)-carvone, tea tree essential oil, and linalool have a significant inhibitory effect on Rhizopus stolonifer , and 2,3-butanedione, 4-propylphenol, clove essential oil, eugenol, thyme essential oil, oregano essential oil, wintergreen essential oil, and methyl salicylate can completely inhibit the growth of Rhizopus stolonifer .

[0107] Considering the safety and actual price factors of food preservatives and the flavor influence of volatile compounds, MeSA, 2,3-butanedione, oregano essential oil, clove essential oil, and nerol were selected for fumigation treatment for subsequent experiments.

[0108] 7.2 Inhibitory effect of plant single essential oils on Rhizopus stolonifer mycelial growth and EC 50

[0109] The inhibitory effect of plant single essential oils on Rhizopus stolonifer mycelial growth is shown in Table 2. MeSA, oregano essential oil, clove essential oil, 2,3-butanedione, and nerol all showed varying degrees of inhibitory effect on Rhizopus stolonifer mycelial growth. When the concentrations of MeSA, oregano essential oil, clove essential oil, and nerol were 75, 100, 75, and 100 μL / L respectively, the mycelial inhibition rate could reach over 90%. Generally speaking, the antibacterial effects of these five plant essential oils are closely related to the concentration and increase with the increase of the concentration.

[0110] The concentrations of each plant essential oil in Table 2 were transformed into logarithms, and the mycelial growth inhibition rate was transformed into probit values for linear regression to obtain the results in Table 3. 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 antibacterial rate increases significantly. The correlations between the concentrations of five plant essential oils, namely MeSA, oregano essential oil, clove essential oil, 2,3-butanedione, and nerol, and the antibacterial effect are all relatively high, and the correlation coefficients are 0.9934, 0.9981, 0.9957, 0.9988, and 0.9975 respectively. The EC 50 values of MeSA, oregano essential oil, and clove essential oil are relatively close, being 41.7 μL / L, 38.8 μL / L, and 37.0 μL / L respectively, while the EC 50 values of 2,3-butanedione and nerol are higher, being 74.8 μL / L and 49.7 μL / L respectively. According to the EC 50 , the antibacterial strength of each plant essential oil against Rhizopus stolonifer is clove essential oil > oregano essential oil > MeSA > nerol > 2,3-butanedione.

[0111] Table 2 Mycelial inhibition rate and EC 50

[0112]

[0113] of plant essential oils against Rhizopus stolonifer 50

[0114]

[0115] 7.3. Synergistic effect of plant essential oil combinations on Rhizopus stolonifer mycelial growth

[0116] On the basis of measuring the inhibitory effects of each plant essential oil on Rhizopus stolonifer, the co-toxicity factor method was used to measure the synergistic effects of the combined essential oils. It can be seen from Table 4 that when the concentration combination of MeSA and nerol is 41.7 + 12.425 μL / L, the actual antibacterial rate of the two against the growth of Rhizopus stolonifer is significantly lower than the theoretical antibacterial rate, showing an antagonistic effect. However, the actual antibacterial rates of the combined combinations of MeSA with oregano essential oil, 2,3-butanedione, and clove essential oil are significantly higher than the theoretical antibacterial rates.

[0117] When the concentration combination of MeSA and 2,3-butanedione is 10.425 + 74.8 μL / L, the co-toxicity factor is between -20 and 20, indicating that the two mixed bacteriostatic agents have an additive effect. While for other concentration combinations, the co-toxicity factors are all greater than 20, indicating that the two mixed bacteriostatic agents have a synergistic effect at this time. When the concentration combination of MeSA and 2,3-butanedione is 20.85 + 18.7 μL / L, the co-toxicity factor is 165.15, which is significantly higher than that of other treatment groups. When the concentration combination of MeSA and 2,3-butanedione is 41.7 + 37.4 μL / L, the actual bacteriostatic rate has reached 100%, and it has achieved the effect of reducing dosage and increasing efficiency.

[0118] Table 4 Co-toxicity factors of essential oil compound combinations against Rhizopus stolonifer

[0119]

[0120] 7.4. Control effect of essential oil compound on fruit root rot caused by Rhizopus

[0121] Combined with the evaluation results of the synergistic effect, the compound combinations of MeSA with oregano essential oil, 2,3-butanedione, and clove essential oil were selected to measure the fruit rot rate and lesion diameter. From Figures 3 - 18 It can be seen that compared with the control group, the compound combinations 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 lesion diameter of the inoculated damaged fruits.

[0122] Overall, the effects of the compound combinations of MeSA with oregano essential oil and clove essential oil are similar, and the greater the concentration, the lower the incidence and the smaller the lesion diameter. In contrast, the compound combination of MeSA and 2,3-butanedione has the best effect. From Figures 3 - 6 It can be seen that for fig fruits, after injury inoculation, the incidence of the control group fruits reached 100%, while when the compound combination of MeSA and 2,3-butanedione was 1 / 2 + 1 / 4, the incidence decreased to 11.11%. Similarly, for tomato fruits, the incidence of the control group fruits reached 100%, and when the compound combination of MeSA and 2,3-butanedione was 1 / 2 + 1 / 4, the incidence decreased to 8.33%. Therefore, the compound combination of MeSA and 2,3-butanedione can significantly reduce the incidence and lesion diameter of fruit root rot caused by Rhizopus, and when the compound combination is 1 / 2 + 1 / 4, the incidence can be reduced by more than 80%, achieving the effect of reducing dosage and increasing efficiency.

[0123] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

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

2. The product according to claim 1, wherein The methyl salicylate and 2,3-butanedione are compounded at a ratio of 1 / 2:1 / 4 of their respective half-maximal effect concentrations.

3. The product according to claim 1, characterized in that, The dosage form of the product includes any one of emulsion, oil agent, suspending agent or solid preparation.

4. The product according to claim 1, wherein, The dosage form of the product is water emulsion.

5. The product according to claim 3, wherein, The solid preparation includes any one of granule, powder, tablet, capsule and seed coating agent.

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

7. The application according to claim 6, characterized in that, The fruits and vegetables include any one or more of apple, pear, strawberry, fig, grape, tomato, peach, green pepper, sweet potato.

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

9. A method for preserving fruits and vegetables, characterized in that, including: Fumigating the fruits and vegetables with the product according to any one of claims 1 - 5.

10. The method for preserving fruits and vegetables according to claim 9, wherein, The fruits and vegetables include any one or more of apple, pear, strawberry, fig, grape, tomato, peach, green pepper, sweet potato.

Images