CN109247347B - Application of fingered citron volatile oil - Google Patents
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- CN109247347B CN109247347B CN201811359515.5A CN201811359515A CN109247347B CN 109247347 B CN109247347 B CN 109247347B CN 201811359515 A CN201811359515 A CN 201811359515A CN 109247347 B CN109247347 B CN 109247347B
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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
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- 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]
- A01N65/36—Rutaceae [Rue family], e.g. lime, orange, lemon, corktree or pricklyash
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- C09K17/00—Soil-conditioning materials or soil-stabilising materials
- C09K17/14—Soil-conditioning materials or soil-stabilising materials containing organic compounds only
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- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
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Abstract
The invention discloses a new application of fingered citron volatile oil, namely the application of the fingered citron volatile oil in preparing fertilizers or pesticides for preventing and treating root rot of traditional Chinese medicinal materials or crops; the experimental result shows that the fingered citron volatile oil can inhibit the growth of 3 main pathogenic bacteria of fusarium oxysporum, fusarium solani and mycelium destroying the stylosanthes causing the occurrence of the root rot of panax notoginseng; the fingered citron volatile oil and the hymexazol are compounded to have the addition and synergistic effect on the strains; the volatile oil of the fingered citron is added into the continuous cropping soil, so that the morbidity and the disease index of the root rot of the panax notoginseng can be reduced; 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 a new application of fingered citron volatile oil, in particular to a prevention and treatment effect of the fingered citron volatile oil on root rot of traditional Chinese medicines or 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, and the disease problem is getting more serious with the rapid increase of the planting area of pseudo-ginseng, which becomes a main obstacle for the production of pseudo-ginseng. 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.
Finger citron (Citrus medica L.var.sarcodactylisSwingle) is a rutaceae plant, evergreen shrub or small tree, commonly used as a traditional Chinese medicine, or as an ornamental plant due to its fruity character.
At present, no report on the application of the fingered citron volatile oil in the prevention and control of crop diseases and insect pests is found.
Disclosure of Invention
The invention aims to provide a new application of fingered citron volatile oil, which is an application in the field of biological pesticides, namely an application in preparing fertilizers or pesticides for preventing and treating root rot of traditional Chinese medicinal materials or crops.
The invention relates to application of fingered citron volatile oil, which is used for preparing a preparation for preventing and treating root rot of traditional Chinese medicinal materials or crops, namely the application of the fingered citron volatile oil serving as an active ingredient in preparing the preparation for preventing and treating pathogenic bacteria.
The ingredients (or effective ingredients) of the preparation for preventing and treating pathogenic bacteria are fingered citron volatile oil, and one or more traditional Chinese medicines and auxiliary materials acceptable in preparations for preventing and treating crop diseases can be added, or the preparation is compounded with other active ingredients to play a synergistic antibacterial role.
The fingered citron volatile oil can be prepared into pesticides of various formulations or fertilizers of various forms by the prior art equipment.
In the invention, the inhibition effect of the fingered citron volatile oil on spore germination and hypha growth is determined, the MIC values of the fingered citron volatile oil and hymexazol are in the same order of magnitude, and the antibacterial effect is equivalent. The fingered citron volatile oil and the chemical pesticide hymexazol are compounded, and the antibacterial effect on a plurality of bacterial strains is found to have the additive and synergistic effect; the purpose of reducing the application of chemical pesticides is achieved through the synergistic effect of the fingered citron volatile oil and the chemical pesticides.
The fingered citron volatile oil is prepared by extracting the volatile oil 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 volatile oil of the fingered citron is used as a natural bactericidal active substance, the activity of the volatile oil disappears or is decomposed by microorganisms after the volatile oil is used, the volatile oil has no pollution to the environment and is safe to people and livestock; the volatile oil of the fingered citron is a plant source extract, the active ingredients of the volatile oil are complex and various, and the 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 volatile oil of the fingered citron are volatile and easy to degrade, and the volatile oil of the fingered citron is bacteriostatic but not bactericidal, so that the volatile oil of the fingered citron is not easy to cause harm to other beneficial microorganisms, improves the micro-ecological structure of soil, promotes the co-evolution of plants and microorganisms, protects the ecological environment, and improves the quality of traditional Chinese medicinal materials or crops.
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 fingered citron volatile oil for reducing the occurrence of pseudo-ginseng diseases has the characteristics of low investment, simple operation, high efficiency, easy volatilization, low toxicity and low residue; under the large background of the vigorous development of organic agriculture in China, the fingered citron 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 bergamot volatile oil and other compound pairs of the present inventionFusarium oxysporum (F.), (Fusarium oxysporum) Fusarium solani (F.sp.), (Fusarium solani) And destruction of Neurospora (Cylindrocarpon destructans) Effect of colony growth results (colony diameter); wherein a is gamma-terpinene; b is d-limonene; c is gamma-terpinene + d-limonene; d is volatile oil of fructus Citri Sarcodactylis; e is a negative control (containing 1% volume concentration of DMSO and 0.1% volume concentration of Tween 80 in water); f is flutriafol; g is hymexazol;
FIG. 2 shows the volatile oil of finger citron and other compounds of the present invention to Fusarium oxysporum (F.oxysporum) ((R))Fusarium oxysporum) Fusarium solani (F.sp.), (Fusarium solani) And destruction of Neurospora (Cylindrocarpon destructans) The effect of colony growth (bacteriostatic rate);
FIG. 3 shows the disease incidence and disease index of Panax notoginseng; a is radix Notoginseng planted in continuous cropping soil; and B is the notoginseng planted in the continuous cropping soil containing 0.3mg/g of the volatile oil of the fingered citron.
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 fingered citron volatile oil
(1) Adding water with the mass 8 times of that of the powder into 300g of crushed fingered citron powder, soaking for 2h, extracting volatile oil in the flos caryophylli by using water vapor in a heating distillation mode for 6h, condensing and collecting distillate;
(2) drying the extracted volatile oil with anhydrous sodium sulfate, removing water, placing into brown sample bottle, sealing, and storing at-20 deg.C for use.
Example 2: activation of pathogenic bacteria
(1) Separating root rot pathogenic bacteria of Notoginseng radix, and purifying and identifying as Fusarium oxysporum (F.) (Fusarium oxysporum) Fusarium solani (F.sp.), (Fusarium solani) And destruction of Neurospora (Cylindrocarpon destructans），Inoculating the strain on a PDA culture medium for activation, and taking a strain which grows vigorously for later use after inoculating for 4 times;
(2) potato dextrose agar medium (PDA): 200 g of potato, 20 g of glucose, 20 g of agar and 1000 mL of distilled water.
Example 3: GC-MS (gas chromatography-Mass spectrometer) determination of chemical components of fingered citron volatile oil
(1) GC conditions were as follows: the instrument model is as follows: agilenggt Technologies 7890B-5977B; a chromatographic column: HP-5MS 30 m × 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;
a: a retention time; b: relative peak area ± relative deviation; c: the index is retained.
Example 4: the influence of the volatile oil of the fingered citron on the growth of hypha of 3 pathogenic bacteria in example 2 is determined
(1) Under the aseptic operation condition, 15mL 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 25mm from the fungus block;
(3) dissolving the fingered citron volatile oil dried in the embodiment 1 in an aqueous solution containing DMSO with the volume concentration of 1% and Tween 80 with the volume concentration of 0.1% to prepare the fingered citron volatile oil with the concentration of 50 mg/mL;
(4) adding 200 μ L of the 50mg/mL fingered citron volatile oil liquid (previously sterilized by filtration with 0.22 μm organic filter) obtained in the step (3) into each Oxford cup, culturing in a microbial incubator at a constant temperature of 28 ℃, and setting 5 times of repetition for each treatment by taking the aqueous solution containing 1% volume concentration of DMSO and 0.1% volume concentration of Tween 80 without adding volatile oil and hymexazol as controls;
(5) after culturing for 7 days in an incubator, measuring the diameter of a bacterial colony by adopting a cross method;
the results are shown in figures 1 and 2, and it can be seen from figures 1 and 2 that the clove volatile oil has obvious inhibition effect on the hypha growth of 3 pathogenic bacteria, and has the same effect as the hymexazol which is a chemical agent, wherein the inhibition effect on fusarium solani and the fusarium oxysporum is better than the hymexazol.
Example 5: fingered citron volatile oil MIC determination
(1) Dissolving the volatile oil of the fingered citron in the example 1 in an aqueous solution containing DMSO with the volume concentration of 1% and Tween 80 with the volume concentration of 0.1%, configuring a plurality of concentration gradient liquids with the concentration range of 75-0.15mg/mL by a double dilution method, and configuring positive drugs hymexazol, monomeric compound d-limonene and gamma-terpinene into concentration gradients with the concentration range of 2.5-0.005 mg/mL by the same method;
(2) the pathogen of example 2, which grew for 7 days, was washed with 20 mL1/4PDA broth (no agar added) to make the spore concentration 1X 104Spore suspension per mL;
(3) adding 50 μ L of 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. mu.L of an aqueous solution containing DMSO at a volume concentration of 1% and Tween 80 at a volume concentration of 0.1% and 150. mu.L of the 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 results are given in the following table;
note: different letters in the same row indicate significant differences between treatments (P < 0.05).
Example 6: interaction determination of fingered citron volatile oil and hymexazol
(1) Preparing the MIC of the fingered citron volatile oil and the hymexazol obtained in the example 5 into 8 times of MIC at the highest concentration, and then preparing 8 dilution gradients by adopting a double dilution method;
(2) adding the fingered citron volatile oil (25 mu L) with different concentrations and the hymexazol (25 mu L) with different concentrations into a 96-well plate in a combined manner, and then adding the spore suspension (150 mu L) prepared in the step (2) in the example 5 into 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 the medicine corresponding to the growth of the fungus-free pores as the MIC of the combination of the fingered citron volatile oil and the 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;
(7) FICI = FIC of volatile oil + FIC of hymexazol, where FICI no greater than 0.5 is synergistic, FICI no greater than 0.5 is additive, FICI no greater than 1 is irrelevant, FICI >4 is antagonistic, and the results are given in the following table;
example 7: experiment for inhibiting panax notoginseng in-vitro morbidity by fingered citron volatile oil
Mixing the volatile oil of fructus Citri Sarcodactylis at a ratio of 0.3mg/g into continuous cropping soil (continuous cropping soil for more than three years), standing for one week, mixing the volatile oil and the volatile oil, and directly transplanting the healthy Notoginseng radix seedling into the continuous cropping soil; meanwhile, the healthy panax notoginseng seedlings planted in the continuous cropping soil of the panax notoginseng continuously planted for more than three years without adding the volatile oil of the fingered citron are used as a control;
planting 80 pseudo-ginseng plants in each group, and grading according to the withering degree of the plants after 30 days of planting: 0 is no disease symptom; 1 is partial leaf wilting; 2 all leaves showed signs of wilting; 3, the stem shows wilting symptom; 4, both leaves and stems showed wilting symptoms.
The incidence and disease index are calculated according to the following formula:
incidence rate = (number of diseased plants/total number of plants) × 100%;
disease index = 100 × ∑ (number of diseased plants at each stage × corresponding grade)/(number of investigated total plants × highest representative value);
the experimental results are shown in the following table and figure 3, and it can be seen from the figure that the volatile oil is added into the continuous cropping soil, the morbidity and disease index of the root rot of panax notoginseng can be reduced, and the volatile oil is safe and effective due to the characteristics of volatility and aroma for avoiding dirt;
Claims (1)
1. application of fingered citron volatile oil in preparation of fertilizer or pesticide for preventing and treating crop root rot, wherein pathogenic bacteria of the root rot is fusarium solani (F.) (Fusarium solani) And destruction of Neurospora (Cylindrocarpon destructans）；
The fingered citron volatile oil is prepared by adding water with the mass being 8 times of that of powder into 300g of crushed fingered citron powder, soaking for 2 hours, extracting the volatile oil in the fingered citron by using water vapor in a heating distillation mode for 6 hours, condensing and collecting distillate; drying the extracted volatile oil with anhydrous sodium sulfate, removing water, placing into brown sample bottle, sealing, and storing at-20 deg.C for use.
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