阅读说明：本技术 垫状棱子芹在杀螨杀虫方面的用途及其生物防治药剂和制备方法 (Application of Plectranthus Amboinicus in killing mite and pest, and biological control agent and preparation method thereof ) 是由 刘志龙 朱丽 落桑 刘奇志 于 2021-01-05 设计创作，主要内容包括：本发明“垫状棱子芹在杀螨杀虫方面的用途及其生物防治药剂和制备方法”属于生物防治领域。本发明涉及垫状棱子芹在杀螨杀虫方面的用途,以及基于垫状棱子芹精油的生物防治药剂。本发明的生物防治药剂可高效杀灭害螨及害虫,同时无毒无害、安全可靠、成本低廉。(The invention relates to application of pimpinella pulcherrima in the aspects of mite killing and insect killing, a biological control medicament and a preparation method thereof, and belongs to the field of biological control. The invention relates to application of pimpinella pulcherrima to mite and pest killing, and a biological control medicament based on pimpinella pulcherrima essential oil. The biological control medicament can efficiently kill harmful mites and pests, and is non-toxic, harmless, safe, reliable and low in cost.)

1. Use of Plectranthus Amboinicus in killing mite and pest is provided.

2. Use of basil according to claim 1, for combating mites and pests, where the mites are selected from the group consisting of pest mites; the insects are selected from agricultural pests, or, storage pests;

preferably, the pest mites are selected from tetranychus urticae; the agricultural pests are selected from frankliniella occidentalis, or the storage pests are selected from booklice.

3. Use of basil according to claim 1 or 2, for combating acarids and insects, wherein the acarids and insects are selected from contact or, alternatively, smoke.

4. Use of Plectranthus Amboinicus according to any of claims 1-3 for killing mites and insects, wherein Plectranthus Amboinicus essential oil is used for killing mites and insects.

5. Use of basil according to claim 4, for combating acarids and pests, characterized in that said basil essential oil is extracted from basil; preferably from the whole plant of Pimpinella brachycarpa.

6. Use of Plectranthus Amboinicus in preparing biocontrol agent is provided.

7. A biological control medicament is characterized in that the active ingredient of the medicament is the essential oil of the pimpinella pulchra.

8. The acaricide according to claim 7, characterized in that the biological control agents are selected from acaricides, and/or insecticides;

preferably, the acaricidal and insecticidal active ingredients of the basil essential oil comprise: alpha-pinene, beta-myrcene, linalool, alpha-terpineol and gamma-elemene.

9. A method for preparing biological control agent is characterized in that cushion-shaped pimpinella chinensis is boiled or distilled, condensed, extracted and solvent evaporated to obtain the biological control agent.

10. The method for preparing a biocontrol agent as claimed in claim 9, wherein said boiling with water is carried out for 8-12 hours, preferably 10 hours;

preferably, the distillation refers to steam distillation for 8-12h, preferably 10 h;

preferably, the condensation refers to condensation after the water boiling or distillation to obtain an oil-water mixture;

preferably, the extraction refers to extracting the oil-water mixture with n-hexane;

preferably, the temperature of the water bath for extraction is 38-45 ℃, preferably 40 ℃;

preferably, the mixture obtained by extraction is dried and then n-hexane is removed by evaporation;

preferably, the drying refers to drying with anhydrous sodium sulfate;

preferably, the evaporation refers to evaporation with a rotary evaporator.

Technical Field

The invention belongs to the field of biological control, and particularly relates to application of pimpinella pulcherrima to mite and pest killing, a biological control medicament of the pimpinella pulcherrima and a preparation method of the biological control medicament.

Background

Tetranychus urticae (Tetranychus urticae) is a worldwide pest mite and belongs to the class of Arachnida. The host plants of the tetranychus urticae are very wide, more than 800 plants of more than 50 families, and the common plants comprise various crops and nearly hundreds of weeds, such as strawberries, eggplants, cucumbers, tomatoes, hot peppers, soybeans, cowpeas, kidney beans, sorghum, watermelons, cucumbers, peaches, pears, apples, apricots, plums, cherries, geraniums, poinsettia, cotton and the like. The two-spotted spider mites and the nymph mites can all be harmed, the initial harmful mites mostly gather on two sides of the vein of the dorsum of the leaves, two sides of the vein of the front of the affected leaves show green loss, the leaves quickly lose green and become brown along with the increase of the number of the mites and the aggravation of the damage degree, the leaves gradually become hard and brittle, and finally become withered and yellow and fall off. The pesticide for preventing and controlling the tetranychus urticae mainly depends on applying a chemosynthetic acaricide, and because of long-term use, the tetranychus urticae has higher resistance to various common acaricides such as abamectin, pyridaben, fenpyroximate, thiamite and the like at present, and the recommended dose is basically ineffective. Meanwhile, the use of chemical agents also has the killing effect on non-target organisms, destroys the ecological balance and causes the pest mites to be rampant. The discovery of high-efficiency and environment-friendly acaricides is urgently needed.

Frankliniella occidentalis (Frankliniella occidentalis) belongs to Thysanoptera thrips, is a worldwide dangerous pest, has wide host range and is a food impurity, host plants comprise more than 500 important economic crops and food crops such as potatoes, eggplants, hot peppers, cucumbers, flowers, bananas, mangoes and the like, and the host types of Frankliniella occidentalis are continuously increased along with the continuous diffusion and spread of Frankliniella occidentalis. The Frankliniella occidentalis eats stems, leaves, flowers and fruits of plants by a rasping-sucking mouthpart, so that petals are discolored, leaves are shrunk, the stems and the fruits form scars, and finally, the plants are withered, and various plant viruses including tomato spotted wilt virus are spread, so that the agricultural production is seriously threatened. The chemical prevention and control method is still a main prevention and control means because the western flower thrips are small in size and secret in harm activities, parthenogenesis can be carried out, the reproductive capacity is strong, the population growth is rapid, but the long-term unreasonable use of chemical pesticides causes that the western flower thrips generate different degrees of drug resistance to insecticides such as organophosphorus, carbamate, pyrethroid and spinosad, so that the prevention and control difficulty is increased, and the pressure on the environment is increased. The discovery of green nuisanceless pesticide effective on frankliniella occidentalis is urgently needed in the production.

The louse prey (lipopeleis bostrychophilus) is an important storage pest belonging to the class of the insecta, order of rodentia. The psylla convolvulus can live among grains, plants, under the bark of trees, on lichens and old trees, is parthenogenesis, has tiny individuals, short development period, long service life of adults, poor appetite, strong hunger resistance and extremely high propagation speed, can cause serious economic loss when occurring in large quantity, has great harm to stored materials such as grains, foods, feeds, medicinal materials and the like, and can bring threat to the health of human beings because excrement adhered to the body of the psylla convolvulus can carry and propagate germs and cause allergy and has extremely high mobility. In actual production, storage class pest uses chemical fumigant prevention and cure more, and long-term single use hydrogen phosphide fumigant leads to the book louse of growing to the resistance to drug of hydrogen phosphide reinforcing, and easily causes the pest rampant again, can cause harm to human health even, seeks novel safe environmental protection's insecticide substitute and is the problem that needs solve urgently.

Pleurospermum hemsleyanum (Pleurospermum heidinii Diels) is a plant of genus Pleurospermum of family Umbelliferae, and is a unique plant in China. Perennial rosette-like herbs, 4-5 cm high and 10-15 cm in diameter. The root of the cushion-shaped prismatic celery is thick, strong, conical and straight. Thick and short stem, fleshy, 1-1.5 cm in diameter, with chestnut brown residual sheath at the base. The leaves are close to the flesh, the basal leaves are narrow and long and oval, the length is 2-5 cm, and the second time feather clefts; one pinna recovering piece 5-7 pairs, nearly without handle, small lobe inverted egg shape or spoon shape, length 1-2.5 mm; the petiole is 2-4 cm long and flat, and the base part of the petiole is 4 mm wide; the petiole is 2-4 cm long and flat, and the base is 4 mm wide. The diameter of the terminal growing umbrella-shaped inflorescence is 5-10 cm, most of bracts are generated, and the shape of leaves is the same; most of the umbrella spokes are small bracts 8-12, and the umbrella spokes are inverted egg-shaped or inverted needle-shaped, have the length of 4-8 mm and are not cracked or feather-cracked. The calyx teeth are nearly triangular; the petals are light red or white and are nearly round. The fruit has an oval shape or a wide oval shape, the length of the fruit is 4-5 mm, the fruit has dense small bubble-shaped protrusions, the fruit has wide wings and microwave-shaped folds, each edge groove has 1 oil pipe, and the synthetic surface has 2 oil pipes. The flowering period is 7-8 months, and the fruit period is 8-9 months. Counties such as eastern Tibet, West fruit Rou, Zhiduo, etc. of Qinghai. Growing on hilly grassland with elevation of about 5000 meters.

The application of the pimpinella pulcherrima to killing mites and insects has not been reported in the field.

Disclosure of Invention

The invention is based on the blank in the field, finds the effect of the pimpinella plementosa on killing tetranychus urticae, agricultural pest thrips occidentalis and storage pest psyllid, and provides the application of the pimpinella plementosa in the aspects of mite killing and insect killing in the field of biological control.

The technical scheme of the invention is as follows:

use of Plectranthus Amboinicus in killing mite and pest is provided.

The insects are selected from agricultural pests or storage pests; the mite is a pest mite;

preferably, the pest mites are tetranychus urticae;

preferably, the agricultural pest is frankliniella occidentalis, and the storage pest is booklice.

The acaricidal and insecticidal mode is selected from contact killing or smoking killing.

The essential oil of the pimpinella pulcherrima is adopted to kill mites and insects;

preferably, the basil essential oil is extracted from the whole basil grass. The whole plant refers to a whole plant of the cushion-shaped pimpinella includes roots and overground parts.

Use of Plectranthus Amboinicus in preparing biocontrol agent is provided.

A biological control medicament is characterized in that the active ingredient of the medicament is the essential oil of the pimpinella pulchra.

The biological control agent is selected from acaricides, and/or insecticides;

preferably, the acaricidal and insecticidal active ingredients of the basil essential oil comprise: alpha-pinene, beta-myrcene, linalool, alpha-terpineol and gamma-elemene.

A method for preparing biological control agent is characterized in that cushion-shaped pimpinella chinensis is boiled in water or distilled, condensed and extracted to obtain the biological control agent.

The water boiling refers to boiling treatment with water for 8-12h, preferably 10 h;

preferably, the condensation refers to condensation after the water boiling or distillation to obtain an oil-water mixture;

preferably, the distillation refers to steam distillation for 8-12h, preferably 10 h;

preferably, the extraction refers to extracting the oil-water mixture with n-hexane;

preferably, the temperature of the water bath for extraction is 38-45 ℃, preferably 40 ℃;

preferably, the mixture obtained by extraction is dried and then n-hexane is removed by evaporation;

preferably, the drying refers to drying with anhydrous sodium sulfate;

preferably, the evaporation refers to evaporation with a rotary evaporator.

The invention discovers the insecticidal and acaricidal effects of the basil chinensis and verifies the outstanding effects of the basil chinensis in the field of biological control, so the invention requests to protect the application of the basil chinensis in killing mites and insects, the application of the basil chinensis in preparing an acaricidal product, the application of the basil chinensis essential oil in controlling agricultural pests, the application of the basil chinensis essential oil in controlling storage pests and the preparation of the basil chinensis essential oil by taking the traditional Chinese medicinal material basil chinensis as a raw material, the basil chinensis essential oil is prepared by adopting the steps of boiling/distilling, condensing, extracting, dehydrating and evaporating a solvent, and the usage of the basil chinensis essential oil in an acaricidal and insecticidal medicament is characterized in that the medicinal active ingredient of the medicament is the basil chinensis essential oil. Meanwhile, the invention also provides a biological control medicament based on the pimpinella plenata essential oil, which can efficiently kill harmful mites and pests, is nontoxic and harmless, does not produce pollution, and is a novel insecticide/acaricide which is safe, reliable, low in cost, environment-friendly and efficient.

Detailed Description

The present invention will be described in further detail with reference to the following specific examples, which should not be construed as limiting the scope of the present invention.

Sources of biological material

The source of the test insects is as follows: the frankliniella occidentalis, tetranychus urticae and the ticks are artificially bred.

The cushion-shaped entire Pimpinella chinensis is collected from the soil cloth of Nanmu forest county, Xizang Rika.

The reagents used in the experimental examples of the present invention, the test reagents not specifically described, were all reagents conventional in the art or obtained by methods conventional in the art, and were commercially available as analytical reagents. Unless otherwise specified, the experimental procedures carried out in the experimental examples are all those routine in the art.

Group 1 example, New use of Pimpinella matsudana for biocontrol

The group of embodiments provides the use of the pimpinella pulcherrima in the aspects of mite killing and insect killing.

In a particular embodiment, the insect is selected from a pest, and the mite is a pest mite

In more specific embodiments, the pests are selected from agricultural pests or storage pests; the pest mites are tetranychus urticae;

in a preferred embodiment, the agricultural pest is selected from thrips occidentalis;

in a preferred embodiment, the stored pests are selected from the group consisting of psyllids.

In some embodiments, the control refers to killing;

in other embodiments, the mode of action on tetranychus urticae and thrips occidentalis is poisoning and the mode of action on psyllid is fumigation.

In a further embodiment, the essential oil of the pimpinella pulcherrima is adopted to kill mites and insects;

preferably, the basil essential oil is prepared from basil serving as a raw material by a distillation method.

Group 2 examples biocontrol pharmaceutical use of Pleurospermum scoparium

The present group of embodiments provides the use of basil apiacea in the preparation of a biological control medicament.

In particular embodiments, the biocontrol agents include, but are not limited to: insecticides, acaricides.

More specifically, the biocontrol agents may be formulated into dosage forms conventional in the art, including but not limited to: powders, wettable powders, drops, suspension concentrates, emulsifiable concentrates, sprays, aerosols, baits, mosquito-repellent incense, sticking agents, smoke agents, insecticidal coatings and the like. Group 3 examples of biocontrol agents of the invention

The present group of embodiments provides a biological control agent. All embodiments of this group share the following common features: the active ingredient of the biological control medicament is basil essential oil.

In particular embodiments, the dosage form of the biocontrol agent is selected from a miticide or an insecticide;

preferably, the active ingredients of the basil essential oil comprise: alpha-pinene, beta-myrcene, linalool, alpha-terpineol and gamma-elemene.

In further embodiments, the biocontrol agent further comprises a pesticide art-acceptable adjuvant common in the art, including but not limited to: emulsifiers, organic solvents, fillers, wetting agents, diluents, and the like.

Group 4 examples preparation of the biocontrol agents of the present invention

The present group of embodiments provides a method of preparing a biological control agent. All embodiments of this group share the following common features: the preparation method comprises the following steps: decocting or distilling, condensing, and extracting herba Pleurospermi Japonici.

In a particular embodiment, the distillation is steam distillation for 8-12h, preferably 10h, to extract substantially the volatile components of the plant material.

The boiling with water is carried out for 8-12h, preferably 10h, so as to boil essential oil components from plants completely.

Preferably, the extraction refers to extraction with n-hexane;

more specifically, using a 2L separatory funnel, n-hexane: and (3) obtaining a distillate of 1:1, and reserving an n-hexane layer in which the essential oil is dissolved after layering.

Preferably, the temperature of the extraction water bath is 38-45 ℃, preferably 40 ℃, and n-hexane can be quickly evaporated, condensed and recycled at the temperature.

Preferably, the mixture obtained by extraction is dried and then n-hexane is removed by evaporation;

preferably, the drying refers to drying with anhydrous sodium sulfate;

preferably, the evaporation refers to evaporation with a rotary evaporator.

Preferably, the specific gravity of the essential oil is 0.835 g/mL.

Experimental example 1 preparation of essential oil of Pleurospermum Amarum

3.5kg of cushion-shaped pimpinella is collected from soil cloth in Nanmu forest county of Xizang Rika, and is added with village, the mixture is treated by a water boiling method or a steam distillation method for 8h-12h, the obtained oil-water mixture is condensed and recovered, then is extracted by n-hexane, is dried by adding anhydrous sodium sulfate, and then is removed by a rotary evaporator, so that the cushion-shaped pimpinella essential oil is obtained. Wherein the water bath temperature is 40 ℃.

The yield of the basil essential oil is 0.10% (w/w), namely 0.1g of basil essential oil can be extracted from 100g of basil.

Experimental example 2 identification of essential oil component of Pleurospermum Amarum

Identifying essential oil components of the basil celery cushion by adopting a GC-MS detection method:

a detection instrument: a gas chromatograph (Agilent6890N) and a mass spectrometer (Agilent 5973N). .

Detection conditions are as follows: HP-5MS quartz capillary column with 30mm × 0.25mm × 0.25 μm has initial column temperature of 60 deg.C, holding for 1min, programmed heating to 180 deg.C at rate of 12 deg.C/min, holding for 0.5min, programmed heating to 280 deg.C at rate of 25 deg.C/min, holding for 18min, vaporizer temperature of 280 deg.C, helium as carrier gas, column flow of 1.0mL/min, and split ratio of 1: 10.

The ionization mode EI of the mass spectrum has the electron bombardment energy of 70eV, the scanning mass range of 20-550m/z and the scanning time of 2 s.

1 mu L of basil essential oil (100 times of acetone diluent) is taken for sample injection, analysis and identification are carried out by a gas chromatography-mass spectrometer, a Nist05 standard mass spectrum gallery is searched by an HP-MSD chemical workstation, and 26 peaks are detected and identified by combining related documents.

The ingredient identification results are shown in table 1 below:

TABLE 1 GC-MS determination of essential oil chemical composition of Pleurospermum pulchrum

The reason why the total percentage of all the components identified in table 1 above is less than 100% is that the unknown components still exist in the essential oil of Pimpinella brachycarpa which may be new compounds and cannot be identified by the existing GC-MS detection method.

Experimental example 3 acaricidal Effect of the biological control agent of the present invention

Tetranychus urticae-poisoning: LD₅₀＝20.36mg/mL(19.03，21.78)

The killing effect verification of the basidiolus chinensis essential oil is carried out on tetranychus urticae by adopting a leaf disc method, and the specific operation is as follows: coating polytetrafluoroethylene on the inner wall of the culture plate, and drying by electric air blowing; heating water and agar at a ratio of 100:1.5 in a microwave oven, mixing, pouring into 12-well cell culture plate (2-3 ml per well), and cooling to solidify; taking fresh kidney bean leaves without pesticide, and punching the leaves by using a puncher with the diameter of 19mm to prepare a circular leaf disc; dissolving a certain amount of basil essential oil in absolute ethyl alcohol (the amount is not more than 2% of the liquid medicine system), adding dimethyl sulfoxide (not more than 1% of the liquid medicine system) for assisting dissolution to prepare a mother solution, and diluting the mother solution to a required concentration by using distilled water to obtain the liquid medicine system; soaking the leaf disc in the agent for 10s, taking out, air drying, placing the kidney bean leaves soaked with the liquid medicine on a culture plate with agar poured therein with the back side of the kidney bean leaves upward, and inoculating 10 tetranychus urticae into each hole to form mites; firmly sticking the porous surface of the culture plate by rice paper, and putting the culture plate into an incubator at the temperature of 26-28 ℃, the relative humidity of 65-75% and the photoperiod L: D of 16: 8; observing and recording death number under the stereoscope after 24h, determining that the person who touches with the brush pen is alive and the person who does not touch is dead, and calculating LD₅₀。

Mortality (%) ═ death/test insect count 100

Corrected mortality (%) - (treatment-control mortality)/(1-control mortality) × 100

semi-Lethal Dose (LD)₅₀): refers to the dose required to cause death in 50% of the subjects tested. Calculated by using Probit software.

The results are shown in table 2 below:

TABLE 2 determination of acaricidal Activity of Plectranthus Amboinicus essential oil by leaf disc method

LD₅₀20.36mg/mL with a 95% confidence interval of (19.03, 21.78) and y-8.45 +6.46 x. The meaning of this semilethal dose is: the drug solution system (including distilled water, solvent-alcohol and cosolvent-dimethyl sulfoxide) contains 20.36mg of essential oil per ml, and half of the tested individuals die after treatment at such concentration.

Experimental example 4 agricultural Pest-killing Effect of the biological control agent of the present invention

Frankliniella occidentalis-poisoning: LD₅₀＝8.57mg/mL(7.84，9.38)

Frankliniella occidentalis-poisoning: dissolving a certain amount of cushion-shaped primula denticulata essential oil with anhydrous ethanol (the amount is not more than 1% of the liquid medicine system), adding tween 80 (not more than 0.5% of the liquid medicine system) for assisting dissolution, preparing a mother solution, and diluting with distilled water to the required concentration to obtain the liquid medicine system for later use; putting 1mL of liquid medicine into a glass scintillation bottle with the volume of 22mL, uniformly rolling for a plurality of circles to form a medicine film, pouring out the liquid medicine, and airing the scintillation bottle for later use. Taking fresh hyacinth beans without pesticide, preparing the hyacinth beans into a long section of 1cm square, immersing the hyacinth beans into the pesticide for 10s, taking out the hyacinth beans, airing the hyacinth beans, and placing the hyacinth beans in a scintillation bottle; setting 5 repetitions for each treatment, repeatedly inoculating second-instar nymphs of 10 Frankliniella occidentalis, and sealing the bottle mouth with a 150-mesh insect-proof net; and (5) placing the worm body at room temperature, observing the result after 24h, and stirring the worm body with a writing brush, wherein the worm body is recorded as dead after being immobilized.

Mortality (%) ═ death/test insect count 100

Corrected mortality (%) - (treatment-control mortality)/(1-control mortality) × 100

semi-Lethal Dose (LD)₅₀): refers to the dose required to cause death in 50% of the subjects tested. Calculated by using Probit software.

The results are shown in Table 3:

TABLE 3 determination of Pepper disc insecticidal Activity of Plectranthus Amboinicus essential oil

LD₅₀8.57mg/mL, 95% confidence interval (7.84, 9.38), y-4.47 +4.79 x. The meaning of this semilethal dose is: the liquid medicine system (including distilled water, solvent-alcohol and cosolvent-Tween) contains 8.57mg of essential oil per ml, and half of the tested individuals die after treatment at such concentration.

Experimental example 5 insecticidal Effect of the biological control agent of the present invention on storage pests

Psyllid-fumigation: LC (liquid Crystal)₅₀＝0.80mg/L(0.73，0.87)

Fumigating activity of the basil essential oil on the psyllium convolvulus: picking 10 test insects by a brush pen, putting the test insects into a small glass bottle (the death condition of the test insects is easy to observe) with the diameter of 0.5cm and the height of 1.6cm, coating polytetrafluoroethylene on the inner wall of a bottle mouth to prevent the test insects from escaping, putting the small glass bottle into a large ground glass bottle (290mL) with the height of 12cm and the diameter of 4cm, taking acetone as a solvent to dilute the medicament into an equal ratio series, sucking 10 microliter of medicament diluent (6 concentrations) by a pipette gun and dripping the diluent on a filter paper strip (the length is 7.5cm, the width is 1.5cm), adhering the filter paper strip into the ground bottle cap by using solid glue, quickly screwing the ground bottle cap, and winding a sealing film for one circle for sealing. After 24 hours, the death of the test insects was observed and recorded. Each concentration was repeated 5 times, each 10 replicates with no drug added to acetone as a blank. After 24 hours, observing, pouring the test insects in the small glass bottle on a piece of white paper, and stirring the bodies with a writing brush to ensure that the bodies die after being immobilized.

The mortality rate (%). is 100 x dead number/test number.

Corrected mortality (%) -100 × (treatment-control mortality)/(1-control mortality).

Semi-lethal dose (LC)₅₀) I.e. the dose that caused 50% of the mortality of the psyllids, the semi-lethal dose was calculated using the Probit software package.

The results are shown in Table 4:

TABLE 4 determination of fumigating Activity of essential oil of Pleurospermum odoratum by closed space method

LC₅₀0.80mg/L with 95% confidence interval (0.73, 0.87) and y 0.50+5.01 x. The meaning of this semilethal dose is: each liter of the closed space contains 0.80mg of essential oil, and half of the tested individuals die after being treated at the concentration.