CN109452320B - Application of clove volatile oil - Google Patents
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- CN109452320B CN109452320B CN201811359510.2A CN201811359510A CN109452320B CN 109452320 B CN109452320 B CN 109452320B CN 201811359510 A CN201811359510 A CN 201811359510A CN 109452320 B CN109452320 B CN 109452320B
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
- A01N65/08—Magnoliopsida [dicotyledons]
- A01N65/28—Myrtaceae [Myrtle family], e.g. teatree or clove
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Abstract
The invention discloses an application of clove bud volatile oil; namely the application of the compound in preventing and treating root rot of traditional Chinese medicinal materials or crops in agriculture or pathogenic bacteria causing diseases on the overground parts of the traditional Chinese medicinal materials or the crops; the experimental result shows that the clove mother volatile oil can inhibit the growth of 4 main pathogenic bacteria of fusarium oxysporum, fusarium solani and pythium and the hypha of the damaged Neurospora species, which cause the occurrence of root rot of panax notoginseng, and can completely inhibit 3 pathogenic bacteria of diseases on the overground part of panax notoginseng from inhibiting the hypha growth of botrytis cinerea, colletotrichum gloeosporioides and rhizoctonia solani; the mother clove volatile oil has the bacteriostatic effect equivalent to that of the chemical pesticide hymexazol, and the compounding of the mother clove and the hymexazol has a synergistic effect on a plurality of strains; because the volatile oil is volatile and has the characteristic of fragrance and dirt avoidance, the chemical fertilizer or pesticide developed by the volatile oil has the advantages of safety, effectiveness, low residue and the like.
Description
Technical Field
The invention relates to application of clove volatile oil, in particular to the prevention and treatment effect of clove volatile oil on root rot of traditional Chinese medicines or crops and various diseases of overground parts of the traditional Chinese medicines or the crops, and belongs to the technical field of biological pesticides.
Background
Notoginseng (radix Notoginseng)Panax notoginseng(Burk.) F.H.Chen) also named as pseudo-ginseng, is a perennial herb of Panax of Araliaceae, has the effects of removing blood stasis, stopping bleeding, relieving swelling and relieving pain, is a unique valuable cultivated medicinal plant in China, has a main production area in southwest areas of China, and has a cultivation history of more than 400 years from China to China in Wenshan state of Yunnan province. The root, stem, leaf and flower of pseudo-ginseng can be used as medicines, the medicine application range is very wide, the social demand is increasing continuously, the disease problem is getting more serious with the rapid increase of the planting area of pseudo-ginseng, and the disease problem becomes a main obstacle for pseudo-ginseng production. Most of the Chinese herbal medicine cultivation has land-based nature and is influenced by factors such as geological soil characteristics, climatic conditions, planting tradition and the like, so that a large amount of continuous cropping exists inevitably, the yield and the quality are reduced, the pesticide effect is reduced, and the problem to be solved in agricultural production is urgently solved.
At present, the prevention and treatment of diseases of the panax notoginseng mainly depend on various chemical agents, and although chemical pesticides are effective, the input cost of farmers is greatly increased, the soil is seriously polluted, and the quality of medicinal materials is reduced. In modern agricultural production, chemical pesticides are widely applied to prevention and control of plant diseases and insect pests, and are the main means for prevention and control of plant diseases at present. However, it has a fatal disadvantage that there is a problem of "3R" (residue, resistance and regeneration) when a chemical bactericide amount, an insecticide is used, and a high residue of an agricultural chemical causes serious environmental pollution. For example, hymexazol is a novel pesticide having unique effects on the prevention and control of diseases, but it is irritating to the skin and eyes and causes hepatotoxicity.
The plant volatile oil is not limited to traditional medicines, foods, tobacco, alcohol and tobacco, but also can be used for plant protection (insect killing, bacteriostasis, weeding and the like), which undoubtedly opens up a new way for the utilization of the plant ecosystem. The plant volatile oil is applied to the field of plant protection as a plant source pesticide, and is consistent with the suggested concept of pollution-free pesticides. In this sense, plant essential oils may also be classified as third generation pesticides.
Fructus Caryophylli is the nearly mature fruit of Eugenia caryophyllata (medicinal Eugenia caryophyllata) of Myrtaceae, and the dried fruit is oval or elliptical; the mother clove is pungent in flavor and warm in nature; can be used for treating sudden heart qi pain, stomach cold, emesis, toothache, halitosis, coldness of women, and infantile hernia.
At present, no literature disclosure related to the technical scheme of the invention is found.
Disclosure of Invention
The invention aims to provide a new application of clove bud volatile oil, namely an application of clove bud volatile oil in preventing and treating root rot of traditional Chinese medicinal materials or crops.
The clove volatile oil can also be applied to preventing and treating pathogenic bacteria causing diseases on overground parts of traditional Chinese medicinal materials or crops; the pathogenic bacteria is botrytis cinerea (Botrytis cinerea) Colletotrichum gloeosporioides (B) ((B))Colletotrichum gloeosporioides) Rhizoctonia solani (A), (B), (C), (B), (C), (B), (C)Rhizoctonia solani) And the like.
The invention relates to an application of clove volatile oil, which is used for preparing a preparation for preventing and treating root rot of traditional Chinese medicinal materials or crops or pathogenic bacteria causing diseases of overground parts of the traditional Chinese medicinal materials or the crops, namely the application of the clove volatile oil serving as an active ingredient in preparing the preparation for preventing and treating the pathogenic bacteria.
The ingredients (or effective ingredients) of the pathogenic bacteria prevention and treatment preparation are clove volatile oil, and one or more traditional Chinese medicines and auxiliary materials acceptable in crop disease prevention and treatment preparations can be added, or the pathogenic bacteria prevention and treatment preparation is compounded with other active ingredients to play a synergistic antibacterial role.
The clove volatile oil can be prepared into pesticides of various formulations or fertilizers of various forms by the prior art equipment.
The mother clove volatile oil is prepared by extracting through a conventional steam distillation method and drying the volatile oil through anhydrous sodium sulfate, and the preparation method is simple, low in cost of required reagents and beneficial to mass production.
The clove volatile oil can reduce the plant morbidity and disease index by inhibiting the spore germination and hypha growth of pathogenic bacteria; the synergistic effect of the pesticide composition and the chemical pesticide is realized, so that the purpose of reducing the application of the chemical pesticide is achieved.
The clove volatile oil is used as a natural bactericidal active substance, the activity of the clove volatile oil disappears or is decomposed by microorganisms after use, the clove volatile oil has no pollution to the environment and is safe to people and livestock; because the clove volatile oil is a plant source extract, the active ingredients of the clove volatile oil are complex and diverse, and the clove volatile oil is an action mechanism with multiple ingredients and multiple targets for pathogenic bacteria, so that target organisms are not easy to generate drug resistance to one or more ingredients, and a lasting prevention and treatment effect is achieved. Compared with the chemical synthetic pesticide, the active ingredients of the clove volatile oil are volatile and easy to degrade, and are bacteriostatic but not bactericidal, so that the harm to other beneficial microorganisms is not easy to cause, the soil micro-ecological structure is improved, the synergistic evolution of plants and microorganisms is promoted, the ecological environment is protected, and the quality of traditional Chinese medicinal materials or crops is improved.
Preparing 50mg/mL of clove volatile oil solution, wherein the solvent is aqueous solution containing DMSO with the volume concentration of 1% and Tween 80 with the volume concentration of 0.1%, and testing the clove volatile oil on 4 main pathogenic bacteria fusarium oxysporum occurring in the root rot of panax notoginsengBacteria (A), (B)Fusarium oxysporum) Fusarium solani (F.sp.), (Fusarium solani) Pythium, Pythium (II)Pythium aphanidermatum) And destruction of Neurospora (Cylindrocarpon destructans) And 3 pathogenic bacteria of botrytis cinerea (A) causing overground part diseases of panax notoginsengBotrytis cinerea) Colletotrichum gloeosporioides (B) ((B))Colletotrichum gloeosporioides) Rhizoctonia solani (A), (B), (C), (B), (C), (B), (C)Rhizoctonia solani) The effect of hyphal growth;
the invention opens up a new method for reducing the application of chemical pesticides, is beneficial to the research of science and technology workers on the action mechanism of the volatile oil, and has wider prospect in agricultural production; the clove mother volatile oil for reducing the occurrence of pseudo-ginseng diseases has the characteristics of low investment, simplicity in operation, high efficiency, volatility, low toxicity and low residue. Under the large background of the vigorous development of organic agriculture in China, the clove hair oil is an ideal chemical agent substitute for overcoming the diseases of pseudo-ginseng, and has important significance for the sustainable development of agriculture.
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FIG. 1 shows the effect of the clove bud volatile oil on the growth of Fusarium oxysporum (a), Fusarium solani (b), Neurospora destructor (c), Pythium vulgare (d), Botrytis cinerea (e), colletotrichum gloeosporioides (f) and Rhizoctonia solani (g) (colony diameter results);
FIG. 2 shows the effect of the clove bud volatile oil on the growth of Fusarium oxysporum, Fusarium solani, Neurospora destructor, Pythium, Botrytis cinerea, Colletotrichum gloeosporum and Rhizoctonia solani colonies (bacteriostasis rate).
Detailed Description
The present invention is further illustrated by the following figures and examples, but the scope of the present invention is not limited to the above description, and the examples are conventional methods unless otherwise specified, and reagents used are conventional commercially available reagents or reagents formulated according to conventional methods unless otherwise specified.
Example 1: preparation of clove volatile oil
(1) Adding water 8 times the mass of crushed flos Caryophylli powder into 1kg of crushed flos Caryophylli powder, soaking for 2h, extracting volatile oil from flos Caryophylli powder by steam in a heating distillation mode for 6h, condensing and collecting distillate;
(2) drying the collected volatile oil with anhydrous sodium sulfate, removing water, placing into brown sample bottle, sealing, and storing at-20 deg.C.
Example 2: activation of pathogenic bacteria
(1) Pathogenic bacteria isolated from root and aerial parts of Notoginseng radix, and purified and identified as Fusarium oxysporum (F.), (Fusarium oxysporum) Fusarium solani (F.sp.), (Fusarium solani) Pythium, Pythium (II)Pythium aphanidermatum) Destroying Neurospora (Cylindrocarpon destructans) Botrytis cinerea (A), (B), (C), (B), (C), (B), (C), (B), (C), (B), (C), (B), (C)Botrytis cinerea) Colletotrichum gloeosporioides (B) ((B))Colletotrichum gloeosporioides) And Rhizoctonia solani (F.), (Rhizoctonia solani) The strain is inoculated on a PDA culture medium for activation, and after 4 times of inoculation, a strain with vigorous growth is taken for standby;
(2) potato dextrose agar medium (PDA): 200g of potatoes, 20g of glucose, 20g of agar and 1000 mL of distilled water.
Example 3: GC-MS (gas chromatography-Mass spectrometer) determination of chemical components of clove bud volatile oil
(1) GC conditions were as follows: the instrument model is as follows: agilenggt Technologies 7890B-5977B; a chromatographic column: HP-5MS 30m × 250 μm × 0.25 μm;
(2) EI source ionization source temperature: 230 ℃; quadrupole temperature: 150 ℃; scanning range: 30-500 m/z; sample inlet temperature: 285 ℃; the split ratio is 10: 1; sample introduction amount: 1 mul; electron energy: 70 eV; column oven temperature program: keeping the temperature at 50 ℃ for 4min, heating to 120 ℃ at 5 ℃/min, heating to 180 ℃ at 1 ℃/min, and heating to 280 ℃ at 10 ℃/min and keeping the temperature for 16 min; wherein the RI value is obtained by calculation according to the retention time of the continuous carbon (C9-C25) of the n-alkane, the retention time and the mass spectrum of the compound are compared with a NIST 17.L database to determine the final compound, and the detection result is shown in the following table;
in the table: a, retention time; b is relative peak area; plus or minus relative deviation; and c, keeping index.
Example 4: determining the influence of the clove bud volatile oil on the hypha growth of the 7 pathogenic bacteria in example 2
(1) Under the aseptic operation condition, 15 mL of PDA culture medium is poured into each culture dish, after cooling and solidification, the pathogenic bacteria cultured for 7d on the PDA culture medium in the embodiment 2 are taken, a puncher with the diameter of 5mm is used for punching a bacterial block along the edge of a bacterial colony, and the bacterial block is inoculated in the center of the culture medium;
(2) placing 4 aseptic oxford cups at equal distance of 25 mm from the fungus block;
(3) dissolving the dried fructus caryophylli volatile oil obtained in the above example 1 in an aqueous solution containing DMSO with a volume concentration of 1% and Tween 80 with a volume concentration of 0.1% to obtain 50mg/mL fructus caryophylli volatile oil liquid;
(4) adding 200 μ L of the 50mg/mL mother clove volatile oil (previously sterilized by filtration with 0.22 μm organic filter) obtained in step (3) into each Oxford cup, culturing at 28 deg.C in a microbial incubator, and controlling with 1% volume concentration of DMSO and 0.1% volume concentration of Tween 80 aqueous solution and hymexazol, wherein each treatment is set to 5 times;
(5) after culturing for 7 days in the incubator, the colony diameter was measured by the "cross method".
The results are shown in figures 1 and 2, and it can be seen from figures 1 and 2 that the clove bud volatile oil has obvious inhibition effect on hypha growth of 7 pathogenic bacteria, and has the same effect as that of the chemical agent hymexazol, wherein the inhibition effect on fusarium solani is obviously better than that of hymexazol.
Example 5: MIC (minimum inhibitory concentration) determination method for clove volatile oil
(1) Example 1 the dried volatile oil of clove mother is dissolved in the water solution containing DMSO with the volume concentration of 1% and Tween 80 with the volume concentration of 0.1%, a plurality of concentration gradient liquids with the concentration range of 150-0.15mg/mL are prepared by a double dilution method, and the positive drugs hymexazol, the monomer compound eugenol, caryophyllene and eugenol acetate are prepared into the concentration gradient with the concentration range of 2.5-0.005mg/mL by the same method;
(2) the pathogen of example 2, which grew for 7 days, was washed with 20 mL 1/4PDA liquid medium (no agar added) to make the spore concentration 1X 104Spore suspension per mL;
(3) adding 50 μ L of flos Caryophylli volatile oil sterilized by 0.22 μm microporous membrane filtration and 150 μ L of spore suspension prepared in step (2) into 96-well plate;
(4) the negative control is 50 μ L of aqueous solution containing DMSO at a volume concentration of 1% and Tween 80 at a volume concentration of 0.1% and 150 μ L of spore suspension prepared in step (2); the positive control is 150 mu L of spore suspension prepared in the step (2);
(5) culturing a 96-well plate at a constant temperature of 28 ℃ for 36h, and then checking the growth condition of fungi in the hole; the absorbance of each well was measured at 595nm using a microplate reader (Thermo 1510), and the concentration of the agent corresponding to the well with absorbance of 0 was the MIC.
The results are given in the following table;
note: different letters in the same row indicate significant differences between treatments (P < 0.05).
Example 6: determination of interaction between clove volatile oil and hymexazol
(1) Preparing the MIC of the clove mother volatile oil and hymexazol obtained in the embodiment 5 into 8 times of MIC at the highest concentration, and then preparing 8 dilution gradients by a double dilution method;
(2) different concentrations of the clove bud volatile oil (25 muL) and different concentrations of hymexazol (25 muL) are added in a 96-well plate in combination, and then spore suspension (150 muL) prepared in the step (2) of the example 5 is added in each well;
(3) 150 μ L of the spore suspension prepared in the step (2) of example 5 and an aqueous solution containing DMSO at a volume concentration of 1% and Tween 80 at a volume concentration of 0.1% were negative controls;
(4) storing the 96-well plate at a constant temperature of 28 ℃ for 36h, and carrying out color comparison at 595nm by using an enzyme-labeling instrument (Thermo 1510) to detect the growth condition of fungi;
(5) MIC assay for combination: taking the concentration of a drug corresponding to growth of a fungaltfree pore as the MIC of the combined application of the mother clove volatile oil and hymexazol;
(6) and (3) calculating an FIC: FIC of volatile oil = MIC of volatile oil in combination with hymexazol/MIC of volatile oil, FIC of hymexazol = MIC of volatile oil in combination with hymexazol/MIC of hymexazol;
FICI = FIC of volatile oil + FIC of hymexazol, where FICI no greater than 0.5 is synergistic effect, FICI no greater than 0.5 is additive effect, FICI no greater than 1 is irrelevant effect, FICI >4 is antagonistic effect; the results are given in the following table;
Claims (1)
1. application of flos Caryophylli volatile oil in preventing and treating Notoginseng radix root rot disease with the pathogenic bacteria of StachybotrysCylindrocarpon destructans）；
The preparation method of the clove bud volatile oil comprises the following steps:
(1) adding water with the mass being 8 times of that of 1kg of crushed clove bud powder, soaking for 2h, extracting volatile oil in clove bud by using water vapor in a heating distillation mode for 6h, condensing and collecting distillate;
(2) drying the collected volatile oil with anhydrous sodium sulfate, and removing water.
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| CN115039797A (en) * | 2022-03-24 | 2022-09-13 | 福建省农业科学院亚热带农业研究所（福建省农业科学院蔗麻研究中心） | Clove essential oil antibacterial mixed solution and application thereof |
| CN115843792A (en) * | 2022-12-12 | 2023-03-28 | 云南中医药大学 | Preparation method of bacteriostatic active agent for enhancing fennel volatile oil |
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| CN100488359C (en) * | 2007-04-26 | 2009-05-20 | 江苏省农业科学院 | Eugenol micro-emulsion |
| CN101940206A (en) * | 2010-09-30 | 2011-01-12 | 江苏天容集团股份有限公司 | Application of eugenol in preventing and controlling plant diseases and bactericide |
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