Plant antibacterial composition for preventing and controlling saffron root rot, preparation method and application thereof

Abstract

The present invention belongs to the technical field of pesticides, and relates to a plant antibacterial composition for preventing and treating saffron root rot, a preparation method and an application thereof. The composition comprises: 5% to 55% of plant volatile oil, 1% to 20% of plant extract, 0.1% to 20% of emulsifier, 0.01% to 5% of auxiliary emulsifier, and purified water. The preparation method comprises: (1) extracting volatile oils from cinnamon and prickly ash together as an oil phase; (2) extracting liquorice, scutellaria baicalensis and coptis chinensis together with alcohol, and concentrating the alcohol extract under reduced pressure as an aqueous phase; (3) combining the oil phase with the aqueous phase; (4) adding an emulsifier and an auxiliary emulsifier, and stirring to obtain an emulsion. The present invention also provides an application thereof in preventing and treating saffron root rot. The composition has a significant inhibitory effect on the pathogenic bacteria of saffron root rot. Compared with chemical pesticides, it is safe and pollution-free to humans, animals and the environment, has no toxic residues after use, and is conducive to popularization and application.

Description

Technical Field

[0001] The invention relates to the technical field of pesticides, and in particular to a plant antibacterial composition for preventing and treating saffron root rot, a preparation method and application thereof. Background Art

[0002] Crocus sativus L Crocus sativus The dried stigma of L., a triploid plant, can only be propagated through bulbs. In production, too many bulb buds will disperse the nutrient transport, and individual bulbs will not be used to expand into mature bulbs. Therefore, bud removal before planting is a routine operation for field rejuvenation of saffron bulbs. Year-round asexual reproduction, the wounds caused by bud removal allow diseases to invade and accumulate year by year. In years with high incidence of diseases, the incidence of stem rot in some production areas can reach 40%, causing a sharp drop in saffron production, which has largely restricted the development of the saffron industry.

[0003] Among the currently known medicinal plant diseases, fungal diseases account for up to 80%, and fungal diseases are also the main diseases that cause saffron bulb rot. So far, the pathogens that have been publicly reported to cause saffron bulb rot include Aspergillus sp., Fusarium sp., Penicillium sp., Anthracnose sp., and Alternaria sp., among which Aspergillus brasiliensis, Fusarium oxysporum, and Penicillium citrinum are the most common pathogens of saffron root rot.

[0004] The sources of botanical antibacterial agents are very wide. At present, there are about 20,000 medicinal plants with antibacterial effects in the world. my country's "Chinese Medicine Dictionary" includes 4,773 traditional Chinese herbal medicines, and their efficacy and safety are relatively clear. Among them, there are at least 2,000 plants with antibacterial and antiseptic effects, many of which are recognized as safe and non-toxic substances.

[0005] At present, the development of new plant-derived antibacterial agents from plants has become a hot topic in current research. A large number of studies have shown that a variety of Chinese herbal medicines have a broad spectrum of antibacterial effects and can inhibit the growth of a variety of common pathogens and molds. There are many types of active ingredients with antibacterial or bactericidal activity in plants, including volatile oils, alkaloids, flavonoids, phenolic alcohols, quinones, saponins, glycosides, etc., which almost involve all kinds of plant components. With the rapid development of analytical chemistry, structural chemistry and analytical instruments and the increasing improvement of experimental methods, many antibacterial active ingredients in plant extracts have been separated, purified and structurally identified. These active ingredients show certain antibacterial effects in vitro and in vivo, and the antibacterial mechanism is very complex and diverse.

[0006] However, there is no report on the use of plant-derived antibacterial agents to inhibit the pathogenic bacteria of saffron root rot. Based on this, the present invention discloses a plant antibacterial composition for preventing and controlling saffron root rot, a preparation method and an application thereof. Summary of the invention

[0007] The purpose of the present invention is to overcome the deficiencies of the prior art, meet practical needs, and provide a plant antibacterial composition for preventing and treating saffron root rot, a preparation method and application thereof.

[0008] In order to achieve the purpose of the present invention, the technical solution adopted by the present invention is:

[0009] Disclosed is a plant antibacterial composition for preventing and treating saffron root rot. The composition comprises, by weight percentage, 5%-55% of plant volatile oil, 1%-20% of plant extract, 0.1%-20% of emulsifier, 0.01%-5% of auxiliary emulsifier, and the balance of purified water.

[0010] The plant volatile oil comprises extracts of the following components by weight: 3-15 parts of cinnamon and 1-5 parts of prickly ash.

[0011] The plant extract comprises extracts of the following components by weight: 5-15 parts of licorice, 2-8 parts of scutellaria baicalensis, and 1-3 parts of coptis chinensis.

[0012] The emulsifier is one or two or a mixture of more than two of sodium lauryl sulfate, sodium lauryl sulfonate, fatty alcohol polyoxyethylene ether, fatty acid sorbitan 60, polysorbate 80, gum arabic, and glyceryl monostearate.

[0013] The auxiliary emulsifier is one or two or a mixture of more than two of ethylene glycol, ethanol, propylene glycol, glycerol and polyglycerol ester.

[0014] Furthermore, the present invention also discloses a method for preparing a plant antibacterial composition for preventing and treating saffron root rot, comprising the following steps:

[0015] The cinnamon and pepper are removed from impurities, crushed, sieved, weighed and mixed according to proportion, and extracted with supercritical CO2 fluid to obtain a mixed vegetable oil containing cinnamon volatile oil and pepper volatile oil;

[0016] The liquorice, scutellaria baicalensis and coptis chinensis are impurity-removed, crushed, sieved and mixed, and refluxed with an ethanol solution for extraction, and the ethanol is removed under reduced pressure to obtain a plant extract containing effective ingredients of liquorice, scutellaria baicalensis and coptis chinensis;

[0017] The mixed vegetable oil obtained in step (1) is used as the oil phase, and the plant extract obtained in step (2) is used as the water phase, and purified water, an emulsifier, and an emulsifier are added, and stirred at high speed to obtain colostrum;

[0018] The colostrum obtained in step (3) is added to a high-pressure homogenizer for homogenization to obtain a uniform emulsion.

[0019] In step (1), the conditions of supercritical CO2 fluid extraction are: extraction temperature 25~45°C, extraction time 1~4 hours, extraction pressure 25~40 MPa.

[0020] In step (2), the concentration of the ethanol solution is 20% to 80%, and the reflux extraction time is 0.5 to 8 hours.

[0021] In step (3), the stirring speed of the high-speed stirring is 2000-20000 rpm, and the stirring time is 0.5-4 hours.

[0022] In step (4), the homogenization pressure is 100-1500 MPa, the number of homogenizations is 2-20 times, and the homogenization time is 30-120 seconds each time.

[0023] Furthermore, the present invention also discloses an application of a plant antibacterial composition for preventing and treating saffron root rot, characterized in that the plant antibacterial composition for preventing and treating saffron root rot is applied to prevent and treat saffron root rot, and is used to inhibit Penicillium citrinum, Aspergillus brasiliensis, and Fusarium oxysporum.

[0024] The beneficial effects of the present invention are:

[0025] (1) The plant antibacterial composition for preventing and controlling saffron root rot of the present invention is a pure plant preparation. Compared with traditional chemical pesticides, it is safe and pollution-free to humans, animals and the environment, has no toxic residue after use, and is conducive to promotion and application.

[0026] (2) The plant antibacterial composition for preventing and controlling saffron root rot of the present invention has a synergistic inhibitory effect on multiple pathogenic bacteria of saffron root rot, and the inhibition rate is more than 85%.

[0027] (3) The plant antibacterial composition for preventing and controlling saffron root rot of the present invention adopts an emulsion preparation process, is physically and chemically stable, does not deteriorate during long-term storage, and can be directly diluted with water when used, so it is convenient to use. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is a comparison chart of the plant antibacterial composition for preventing and controlling saffron root rot prepared in Example 3 and the antibacterial results of carbendazim on the pathogenic bacteria of saffron root rot.

[0029] Figure 2 This is a comparison chart of the plant antibacterial composition for preventing and controlling saffron root rot prepared in Example 3 and the field effects of carbendazim on the pathogenic bacteria of saffron root rot. Implementation

[0030] The present invention is further described below in conjunction with the accompanying drawings and embodiments:

[0031] Example 1: A plant antibacterial composition for preventing and controlling saffron root rot, comprising the following components in weight percentage: 30% plant volatile oil, 5% plant extract, 10% emulsifier (glyceryl monostearate), 2% co-emulsifier (propylene glycol), and the rest is purified water.

[0032] The above-mentioned plant volatile oil is extracted from the following substances in parts by weight, including: 3 parts of cinnamon and 1 part of pepper.

[0033] The plant extract is extracted from the following substances in parts by weight, including: 5 parts of licorice, 2 parts of scutellaria baicalensis, and 1 part of coptis chinensis.

[0034] The method for preparing the plant antibacterial composition for preventing and treating saffron root rot comprises the following steps:

[0035] Cinnamon and Zanthoxylum bungeanum were removed from impurities, crushed, passed through a 40-mesh sieve, weighed and mixed according to proportion, and extracted with supercritical CO2 fluid. The extraction conditions were: extraction temperature 40-45 ℃, extraction time 2 hours, extraction pressure 35 MPa, to obtain a mixed vegetable oil containing cinnamon volatile oil and Zanthoxylum bungeanum volatile oil.

[0036] (2) Remove impurities from licorice, scutellaria baicalensis, and coptis chinensis, crush them, pass them through a 40-mesh sieve, weigh them according to proportion, mix them, perform reflux extraction with a 60% ethanol solution for 4 hours, and remove the ethanol under reduced pressure to obtain a plant extract containing the effective ingredients of licorice, scutellaria baicalensis, and coptis chinensis.

[0037] (3) The mixed vegetable oil prepared in step (1) is used as the oil phase, the plant extract prepared in step (2) is used as the water phase, an appropriate amount of purified water, 10% glyceryl monostearate and 2% propylene glycol are added, and the mixture is stirred at a high speed of 10,000 rpm for 1 hour to obtain colostrum.

[0038] (4) Add the colostrum obtained in step (3) into a high-pressure homogenizer at a homogenization pressure of 800 MPa, 15 times of homogenization, and 60 seconds of homogenization each time to obtain colostrum.

[0039] Example 2, a plant antibacterial composition for preventing and controlling saffron root rot, comprising the following components in weight percentage: 45% plant volatile oil, 15% plant extract, 5% emulsifier (polysorbate 80), 5% co-emulsifier (glycerol), and the rest is purified water.

[0040] The plant volatile oil is extracted from the following substances in parts by weight, including: 10 parts of cinnamon and 5 parts of pepper.

[0041] The plant extract is extracted from the following substances in parts by weight, including: 10 parts of licorice, 6 parts of scutellaria baicalensis, and 2 parts of coptis chinensis.

[0042] The method for preparing the plant antibacterial composition for preventing and treating saffron root rot comprises the following steps:

[0043] (1) Cinnamon and Zanthoxylum bungeanum were removed from impurities, crushed, passed through a 40-mesh sieve, weighed and mixed according to proportion, and extracted with supercritical CO2 fluid under the following conditions: extraction temperature of 30-35 °C, extraction time of 6 h, and extraction pressure of 40 MPa to obtain a mixed vegetable oil containing cinnamon volatile oil and Zanthoxylum bungeanum volatile oil.

[0044] (2) Remove impurities from licorice, scutellaria baicalensis and coptis chinensis, crush them, pass them through a 40-mesh sieve, weigh them according to proportion, mix them, reflux extract them with a 60% ethanol solution for 6 hours, and remove the ethanol under reduced pressure to obtain a plant extract containing the effective ingredients of licorice, scutellaria baicalensis and coptis chinensis.

[0045] (3) The mixed vegetable oil prepared in step (1) is used as the oil phase, the plant extract prepared in step (2) is used as the water phase, an appropriate amount of purified water, 5% polysorbate 80 and 5% propylene glycol are added, and the mixture is stirred at a high speed of 15,000 rpm for 2 hours to obtain colostrum.

[0046] (4) Add the colostrum obtained in step (3) into a high-pressure homogenizer at a homogenization pressure of 1000 MPa, a homogenization number of 20 times, and a homogenization time of 80 seconds each time to obtain colostrum.

[0047] Example 3, a plant antibacterial composition for preventing and controlling saffron root rot, comprising the following components in weight percentage: 55% plant volatile oil, 20% plant extract, 10% emulsifier (glyceryl monostearate), 5% co-emulsifier (propylene glycol), and the rest is purified water.

[0048] The above-mentioned plant volatile oil is extracted from the following substances in parts by weight, including: 15 parts of cinnamon and 2 parts of pepper.

[0049] The plant extract is extracted from the following substances in parts by weight, including: 10 parts of licorice, 8 parts of scutellaria baicalensis, and 3 parts of coptis chinensis.

[0050] The method for preparing the plant antibacterial composition for preventing and treating saffron root rot comprises the following steps:

[0051] Cinnamon and Zanthoxylum bungeanum were removed from impurities, crushed, passed through a 40-mesh sieve, weighed and mixed according to proportion, and extracted with supercritical CO2 fluid. The extraction conditions were: extraction temperature 40-45 ℃, extraction time 4 hours, extraction pressure 35 MPa, to obtain a mixed vegetable oil containing cinnamon volatile oil and Zanthoxylum bungeanum volatile oil.

[0052] (2) Remove impurities from licorice, scutellaria baicalensis and coptis chinensis, crush them, pass them through a 40-mesh sieve, weigh them according to proportion, mix them, reflux extract them with 80% ethanol solution for 4 hours, remove the ethanol under reduced pressure, and obtain a plant extract containing the effective ingredients of licorice, scutellaria baicalensis and coptis chinensis.

[0053] (3) The mixed vegetable oil prepared in step (1) is used as the oil phase, the plant extract prepared in step (2) is used as the water phase, an appropriate amount of purified water, 10% glyceryl monostearate and 5% propylene glycol are added, and the mixture is stirred at a high speed of 12,000 rpm for 1.5 hours to obtain colostrum.

[0054] (4) Add the colostrum obtained in step (3) into a high-pressure homogenizer at a homogenization pressure of 1500 MPa, 15 homogenization times, and a homogenization time of 120 seconds each time to obtain colostrum.

[0055] Example 4, this example is the determination of the antibacterial activity of the above examples 1-3 against Aspergillus brasiliensis, Fusarium oxysporum and Penicillium citrinum, as follows:

[0056] Test pathogens: 3 strains of pathogenic fungi, including Penicillium citrinum ( Penicillium citrinum )、Aspergillus brasiliensis( Aspergillus brasiliensis ) and Fusarium oxysporum ( Fusarium oxysporum ), isolated from saffron bulb rot diseased plants of Jiande Flower Farmers Saffron Professional Cooperative in Zhejiang Province.

[0057] Activation of bacterial strains and preparation of culture medium: Add potato dextrose agar medium (PDA medium) into a conical flask, seal it and put it into a high-pressure steam sterilizer, sterilize it at 121 ℃ for 30 min, cool it to 40-50 ℃, mix it thoroughly and pour it into a sterile plastic culture dish; take the saffron root rot pathogens Aspergillus brasiliensis, Fusarium oxysporum and Penicillium citrinum stored in the test tube, inoculate it into the culture dish, and culture it in a 28 ℃ electric constant temperature incubator for 5 days.

[0058] The plant antibacterial compositions for preventing and controlling saffron root rot prepared in Examples 1-3 were taken respectively, filtered with a 0.22 um sterile filter membrane, and each plant antibacterial composition for preventing and controlling saffron root rot was diluted to a drug-containing plate with a concentration of 20 mg / ml (equivalent to the crude drug concentration) using PDA medium, and the PDA medium without the plant antibacterial composition was used as a blank control, and the 20 mg / ml was used as a control group. A fungal cake with a diameter of 6 mm was placed in the center of each plate, and cultured in an electric constant temperature incubator at 28°C for 5 days. The colony diameter was measured by the cross method, and the inhibition rate of the plant antibacterial composition for preventing and controlling saffron root rot prepared in Examples 1-3 on the three pathogens was calculated, and the formula is as follows:

[0059]

[0060] The results of the plant antibacterial composition for preventing and controlling saffron root rot and the inhibition of carbendazim on the pathogenic bacteria of saffron root rot prepared in Examples 1-3 are shown in Table 1. The results of the plant antibacterial composition for preventing and controlling saffron root rot and the inhibition zone of carbendazim on the pathogenic bacteria of saffron root rot prepared in Example 3 are shown in Table Figure 1 .

[0061] Table 1 Inhibition rate of Examples 1-3 on saffron root rot pathogens

[0062] Group Brazilian Aspergillus inhibition rate (%) Inhibition rate of Fusarium oxysporum (%) Penicillium citrinum inhibition rate (%) Example 1 92.4 89.6 95.7 Example 2 95.1 87.8 91.3 Example 3 93.7 93.5 90.9 Carbendazim control group 82.5 76.9 84.2

[0063] The results showed that the plant antibacterial compositions for preventing and controlling saffron root rot of Examples 1, 2 and 3 had significant inhibitory activity against the pathogens of saffron root rot, Aspergillus brasiliensis, Fusarium oxysporum and Penicillium citrinum, all higher than 85%, and significantly higher than the carbendazim control group.

[0064] Example 5, this example is the determination of the toxicity values ​​of Aspergillus brasiliensis, Fusarium oxysporum and Penicillium citrinum in Examples 1-3, as follows:

[0065] Test pathogens: 3 strains of pathogenic fungi, including Penicillium citrinum ( Penicillium citrinum )、Aspergillus brasiliensis( Aspergillus brasiliensis ) and Fusarium oxysporum ( Fusarium oxysporum ), isolated from saffron bulb rot diseased plants of Jiande Flower Farmers Saffron Professional Cooperative in Zhejiang Province.

[0066] Activation of strains and preparation of culture medium: Add potato dextrose agar medium (PDA medium) to a conical flask, seal it and place it in a high-pressure steam sterilizer, sterilize it at 121 ℃ for 30 min; cool it to 40-50 ℃, mix it thoroughly and pour it into a sterile plastic culture dish. Take the saffron root rot pathogens Aspergillus brasiliensis, Fusarium oxysporum and Penicillium citrinum stored in a test tube, inoculate it into a culture dish, and culture it in a 28 ℃ electric constant temperature incubator for 5 days.

[0067] The plant antibacterial compositions for preventing and controlling saffron root rot prepared in Examples 1-3 were respectively taken and filtered with a 0.22 um sterile filter membrane. The plant antibacterial compositions for preventing and controlling saffron root rot were diluted to 6 concentrations of 40, 20, 10, 5, 2.5, and 1.25 mg / ml with PDA medium. The inhibition rate of each concentration on pathogenic fungi was determined. Probit regression analysis using SPSS 26.0 software was performed, with the logarithm of the concentration as the independent variable x and the probability value of the inhibition rate as the dependent variable y. A toxicity regression equation was established, and the EC was calculated by the toxicity regression equation. 50 Toxicity value. EC 50The smaller the value, the stronger the toxicity of the plant to the pathogenic bacteria. The average EC values ​​of the plant antibacterial composition for preventing and controlling saffron root rot and carbendazim prepared in Examples 1-3 against the pathogenic bacteria of saffron root rot are 50 The toxicity results are shown in Table 2.

[0068] Table 2 Average EC of Examples 1-3 against saffron root rot pathogens 50 Toxicity value

[0069] The results showed that the plant antibacterial compositions for preventing and controlling saffron root rot of Examples 1, 2 and 3 had higher toxicity values ​​against saffron root rot pathogens Aspergillus brasiliensis, Fusarium oxysporum and Penicillium citrinum, and their toxicity values ​​were better than those of the carbendazim control group.

[0070] Example 6: This example is the field application of the plant antibacterial composition for preventing and treating saffron root rot prepared in Example 3, as follows:

[0071] Use sterile water to prepare a concentration of 1×10 5 Healthy saffron bulbs were soaked in a spore suspension of Aspergillus brasiliensis, Fusarium oxysporum and Penicillium citrinum at 3 CFU / mL and inoculated for 2 h.

[0072] After the infection, the plants were planted in soil, with 1,000 saffron bulbs planted in each group and three replicates for each treatment.

[0073] A certain concentration of the plant antibacterial composition for preventing and controlling saffron root rot prepared in Example 3 was sprayed on the soil planted with saffron bulbs, and the drug was administered once on the 5th day, the 25th day, and the 55th day, respectively. The saffron bulbs sprayed with carbendazim were used as a positive control, and the saffron bulbs sprayed with purified water were used as a blank control.

[0074] 100 bulbs were randomly selected on day 60 to observe the incidence of saffron bulbs and count the incidence of saffron bulb surface. The surface pictures of saffron bulbs in each group are shown in Figure 2 The results showed that only a small amount of bacterial plaque appeared in the saffron bulbs of Example 3, which was significantly better than the model group and the carbendazim group, indicating that the present invention can have a significant inhibitory effect on the pathogenic bacteria of saffron root rot.

[0075] The embodiments of the present invention disclose preferred embodiments, but are not limited thereto. A person skilled in the art can easily understand the spirit of the present invention based on the above embodiments and make different extensions and changes. However, as long as they do not deviate from the spirit of the present invention, they are all within the protection scope of the present invention.

Claims

1. A method for preparing a plant antibacterial composition for preventing and treating saffron root rot, which is used to prepare a plant antibacterial composition for preventing and treating saffron root rot, characterized in that: The plant antibacterial composition for preventing and treating saffron root rot comprises, by weight percentage: 5% to 55% of plant volatile oil, 1% to 20% of plant extract, 5% to 20% of emulsifier, 0.01% to 5% of auxiliary emulsifier, and the balance is purified water; The plant volatile oil comprises extracts of the following components by weight: 3-15 parts of cinnamon bark and 1-5 parts of prickly ash; The plant extract comprises extracts of the following components by weight: 5-15 parts of licorice, 2-8 parts of scutellaria, and 1-3 parts of coptis; The emulsifier is one or two or a mixture of more than two of sodium lauryl sulfate, sodium lauryl sulfonate, fatty alcohol polyoxyethylene ether, fatty acid sorbitan 60, polysorbate 80, gum arabic, and glyceryl monostearate; The auxiliary emulsifier is one or two or a mixture of more than two of ethylene glycol, ethanol, propylene glycol, glycerol, and polyglycerol ester; The preparation method comprises the following steps: (1) removing impurities from cinnamon and Sichuan pepper, crushing, sieving, weighing and mixing according to a proportion, and extracting with supercritical CO2 fluid to obtain a mixed vegetable oil containing cinnamon volatile oil and Sichuan pepper volatile oil; (2) removing impurities from licorice, scutellaria baicalensis and coptis chinensis, crushing, sieving and mixing them, refluxing and extracting them with an ethanol solution, and removing the ethanol under reduced pressure to obtain a plant extract containing effective ingredients of licorice, scutellaria baicalensis and coptis chinensis; (3) using the mixed vegetable oil obtained in step (1) as the oil phase and the plant extract obtained in step (2) as the water phase, adding purified water, an emulsifier, and an emulsifier co-agent, and stirring at high speed to obtain colostrum; (4) adding the colostrum obtained in step (3) into a high-pressure homogenizer for homogenization to obtain a uniform emulsion; Among them, the homogenization pressure is 100~1500 MPa, the number of homogenization times is 2~20 times, and the homogenization time is 30~120 seconds each time.

2. The preparation method according to claim 1, characterized in that: In step (1), the conditions of supercritical CO2 fluid extraction are: extraction temperature 25~45°C, extraction time 1~4 hours, extraction pressure 25~40 MPa.

3. The preparation method according to claim 1, characterized in that: In step (2), the concentration of the ethanol solution is 20% to 80%, and the reflux extraction time is 0.5 to 8 hours.

4. The preparation method according to claim 1, characterized in that: In step (3), the stirring speed of the high-speed stirring is 2000-20000 rpm, and the stirring time is 0.5-4 hours.

5. An application of a plant antibacterial composition for preventing and treating saffron root rot, characterized in that: The plant antibacterial composition for preventing and controlling saffron root rot is prepared by the preparation method according to any one of claims 1 to 4. The plant antibacterial composition for preventing and controlling saffron root rot is used to prevent and control saffron root rot, and is used to inhibit Penicillium citrinum, Aspergillus brasiliensis, and Fusarium oxysporum.

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