阅读说明：本技术 花椒簕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 fructus Zanthoxyli Acanthopanax trifoliatus in killing mite and pest, and its biological control agent and preparation method ) 是由 刘志龙 赵燚 刘奇志 于 2020-12-14 设计创作，主要内容包括：本发明“花椒簕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。本发明涉及花椒簕在杀螨杀虫方面的用途,以及基于花椒簕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农业害虫,同时无毒无害、安全可靠、成本低廉。(The invention relates to application of pricklyash trifoliate acanthopanax in mite and pest killing, a biological control medicament and a preparation method thereof, and belongs to the field of biological control. The invention relates to application of acanthopanax trifoliatus in mite killing and insect killing, and a biological control medicament based on acanthopanax trifoliatus essential oil. The biological control medicament can efficiently kill harmful mites and agricultural pests, and is non-toxic, harmless, safe, reliable and low in cost.)

1. The application of the acanthopanax trifoliatus in the aspects of killing mites and insects.

2. The use of the trifoliate pricklyash fruit in the aspect of killing mites and pests as claimed in claim 1, wherein the mites are selected from pest mites, and the pests are selected from agricultural pests;

preferably, the pest mites are tetranychus urticae;

preferably, the agricultural pest is selected from thrips occidentalis.

3. The use of the trifoliate pricklyash fruit in the aspect of killing mites and insects as claimed in claim 1 or 2, wherein the modes of killing mites and insects are contact killing and/or spraying.

4. The use of the trifoliate acanthopanax root in the aspects of mite killing and insect killing according to any one of claims 1 to 3, characterized in that the trifoliate acanthopanax root essential oil is used for killing mites and insects;

preferably, the zanthoxylum trifoliate acanthopanax essential oil is extracted from stem and leaf parts of the zanthoxylum trifoliate acanthopanax trifoliate.

5. The application of the acanthopanax trifoliatus in preparing the biocontrol medicament.

6. A biological control medicament is characterized in that the active component of the biological control medicament is pricklyash trifoliate acanthopanax essential oil.

7. The acaricide according to claim 6, characterized in that the biological control agents are selected from acaricides, and/or insecticides;

preferably, the acaricidal and insecticidal active ingredients of the pricklyash trifoliate acanthopanax essential oil comprise: 1,8-cineole, linalool, camphor, eugenol and beta-caryophyllene.

8. A method for preparing biological control medicament is characterized in that the biological control medicament is prepared by boiling stem and leaf parts of pricklyash trifoliate acanthopanax with water, condensing and extracting.

9. The method for preparing a biocontrol agent as claimed in claim 8, wherein said boiling with water is carried out for 4-8 hours, preferably 6 hours;

preferably, the distillation refers to steam distillation for 4 to 8 hours, preferably 6 hours;

preferably, the extraction refers to extraction 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 pricklyash peel trifoliate acanthopanax in mite and pest killing, a biological control medicament and a preparation method thereof.

Background

Tetranychus urticae (Tetranychus urticae) Is a kind of organism seriously harming crops, and has wide host range, small size, fast reproduction, high hidden property and high resistance to drug, and tetranychus urticae is mainly usedThe feed is taken from the back of the leaves of the crops, so the feed is a pest which is difficult to control. At present, the main measure for preventing and controlling tetranychus urticae is to apply acaricide, and the resistance of tetranychus urticae is gradually enhanced under the long-term medicament selection pressure; meanwhile, the use of chemical agents can kill and kill non-target organisms while preventing and treating mites, destroy ecological balance and cause the mites to be rampant.

Frankliniella occidentalis (C. Et D. Don.) (Frankliniella occidentalis) The insect belongs to thrips of Thysanoptera, has a wide host range, and known more than 500 host plants damage economic fruits such as peaches, plums, apples, strawberries, grapes and vegetables such as tomatoes, lettuce, hot peppers and beans, thus seriously threatening agricultural production. In addition, the western flower thrips is an agricultural pest which is difficult to control because the pest is small in size, the pest is hidden in movement and has certain drug resistance. Therefore, the demand for developing efficient, low-toxicity, low-residue, environmentally friendly pesticides is continuously increasing; in the comprehensive control of pests, botanical insecticides are favored.

Fructus Zanthoxyli Acanthopancis (fructus Zanthoxyli)Zanthoxylum scandens) Belonging to genus Zanthoxylum of family Rutaceae, is vine-shaped shrub. The branches and the stems have short barbs, the compound leaves with odd feathers are mutually grown or the upper parts of the leaf shafts are grown, and the leaf shafts have short barbs; poly umbellate panicles, axillary or terminal; the infructescence and the stalk have no hair, or have the skin thinned and slightly soft hair, the top end has short mango tip, and the oil gland point is not obvious; the flowering period is 3-5 months, and the fruit period is 7-8 months. The trifoliate pricklyash fruit is distributed in the south of Yangtze river and often grows in hillside shrubs.

The field has not reported the mite-killing and insect-killing application of the acanthopanax trifoliatus.

Disclosure of Invention

The invention is based on the blank in the field, finds the effect of the trifoliate pricklyash fruit in killing tetranychus urticae koch and agricultural pest thrips occidentalis, and provides the application of the trifoliate pricklyash fruit in killing mites and insects in the field of biological control.

The technical scheme of the invention is as follows:

the application of the acanthopanax trifoliatus in the aspects of killing mites and insects.

The mites are selected from pest mites, and the pests are selected from agricultural pests;

preferably, the pest mites are tetranychus urticae;

preferably, the agricultural pest is selected from thrips occidentalis.

The acaricidal and insecticidal mode is contact killing and/or spraying.

The pricklyash peel and acanthopanax trifoliatus essential oil is adopted to kill mites and insects;

preferably, the zanthoxylum trifoliate acanthopanax essential oil is extracted from stem and leaf parts of the zanthoxylum trifoliate acanthopanax trifoliate.

The application of the acanthopanax trifoliatus in preparing the biocontrol medicament.

A biological control medicament is characterized in that the active component of the biological control medicament is pricklyash trifoliate acanthopanax essential oil.

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

preferably, the acaricidal and insecticidal active ingredients of the pricklyash trifoliate acanthopanax essential oil comprise: 1,8-Cineole (1, 8-Cineole), Linalool (Linalool), Camphor (Camphor), Eugenol (Eugenol), beta-Caryophyllene (beta-Caryophyllene).

A method for preparing biological control medicament is characterized in that the biological control medicament is prepared by boiling stem and leaf parts of pricklyash trifoliate acanthopanax with water, condensing and extracting.

The water boiling refers to boiling treatment with water for 4-8h, preferably 6 h;

preferably, the distillation refers to steam distillation for 4 to 8 hours, preferably 6 hours;

preferably, the extraction refers to extraction 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 discloses an insecticidal and acaricidal effect of Acanthopanax trifoliatus pioneer, verifies the outstanding effect of Acanthopanax trifoliatus in the field of biological control, extracts Acanthopanax trifoliatus essential oil and identifies active ingredients with the functions of killing mites and killing insects in the essential oil. The invention discloses application of fructus Zanthoxyli Acanthopancis in killing mite and pest, application of fructus Zanthoxyli Acanthopancis in preparing mite-killing product, application of fructus Zanthoxyli Acanthopancis essential oil in preventing and treating agricultural pest, and preparation of fructus Zanthoxyli Acanthopancis essential oil by decocting in water, distilling, condensing, extracting, dehydrating, and evaporating solvent. The acanthopanax trifoliatus grows in large quantities of hillside shrubs, has strong climbing ability and can be obtained in large quantities, and the acanthopanax trifoliatus essential oil is used for killing mites and insects, thereby conforming to the concepts of environmental protection and changing waste into valuables.

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 and tetranychus urticae is artificially bred.

The acanthopanax trifoliatus is a wild plant, is collected in West Xiu district of Anshun city in Guizhou province in 6 months in 2020, and is identified by Liu Quanju professor of Living university, Beijing, university and Life sciences.

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 application of Zanthoxylum trifoliatus for biological control

The group of embodiments provide the application of the acanthopanax trifoliatus in the aspects of mite killing and insect killing.

In particular embodiments, the pest is selected from a pest mite or an agricultural pest;

in more specific embodiments, the pest mite is tetranychus urticae;

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

In some embodiments, the control refers to killing;

in other embodiments, the acaricidal and insecticidal means is contact killing, and/or spraying.

In a further embodiment, the pricklyash trifoliate acanthopanax essential oil is used for killing mites and insects;

preferably, the zanthoxylum trifoliate acanthopanax essential oil is extracted from stem and leaf parts of the zanthoxylum trifoliate acanthopanax trifoliate.

Group 2 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 component of the biological control medicament is pricklyash peel and trifoliate acanthopanax root essential oil.

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

preferably, the insecticidal and acaricidal active ingredients of the acanthopanax trifoliatus essential oil comprise 1,8-Cineole (1, 8-Cineole), Linalool (Linalool), Camphor (Camphor), Eugenol (Eugenol) and beta-Caryophyllene (beta-caryophylelene).

Group 3 example, Process for the preparation of the biological control Agents of the 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: the stem and leaf part of the prickly ash trifoliate acanthopanax is boiled in water or distilled, condensed and extracted to obtain the prickly ash trifoliate acanthopanax.

In specific embodiments, the distillation refers to steam distillation for 4-8h, 6h, to sufficiently extract volatile components from the plant material.

The boiling with water means boiling with water for 4-8h, preferably 6h, so that the essential oil components can be boiled out from the fresh plant as completely as possible.

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

more specifically, using a 2L separatory funnel, n-hexane: and (3) distilling off =1:1, and after layering, keeping an n-hexane layer of dissolved essential oil.

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 0.89 g/mL.

Experimental example 1 preparation of Zanthoxylum trifoliatus essential oil

Collecting stem and leaf parts of fructus Zanthoxyli Acanthopancis, decocting with water for 6h, condensing and recovering the obtained oil-water mixture, extracting with n-hexane, drying with anhydrous sodium sulfate, and removing n-hexane with rotary evaporator to obtain fructus Zanthoxyli Acanthopancis essential oil. Wherein the water bath temperature is 40 ℃.

The yield of the zanthoxylum Ilicifolia essential oil is 0.20 percent (w/w), namely 0.3g of the zanthoxylum Ilicifolia essential oil can be extracted from 100g of fresh zanthoxylum Ilicifolia plants.

Experimental example 2 identification of the essential oil components in Acanthopanax trifoliatus

Identifying the components of the zanthoxylum trifoliate essential oil by adopting a GC-MS detection method:

a detection instrument: a gas chromatograph (Agilent 6890N) and a mass spectrometer (Agilent 5973N). .

Detection conditions are as follows: HP-5MS30mm × 0.25mm × 0.25 μm quartz capillary column, initial column temperature 60 deg.C, keeping for 1min, programming to 180 deg.C at a rate of 12 deg.C/min, keeping for 0.5min, programming to 280 deg.C at a rate of 25 deg.C/min, keeping for 18min, vaporizer temperature 280 deg.C, carrier gas is helium, column flow is 1.0mL/min, and split ratio is 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 pricklyash trifoliate plant 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 atlas is retrieved by an HP-MSD chemical workstation, and 35 peaks are detected and identified by combining related documents.

The ingredient identification results are shown in table 1 below:

TABLE 1

The reason why the total percentage of all the components identified in the above table 1 is less than 100% is that unknown components still exist in the zanthoxylum trifolium essential oil, and the unknown components are possibly 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-touch out: LD₅₀ =8.52 mg/mL（6.90, 10.68）

The killing effect verification of the pricklyash peel and trifolium acanthopanax essential oil is carried out on tetranychus urticae by adopting a leaf disc method, and the specific operation is as follows: taking fresh hyacinth bean leaves without pesticide, punching the leaves to prepare a circular leaf disc with the diameter of 3 cm; dissolving 30 microliters of prickly ash trifoliate acanthopanax essential oil in absolute ethyl alcohol (the amount of the absolute ethyl alcohol is not more than 1% of the liquid medicine system), adding dimethyl sulfoxide (the amount of the dimethyl sulfoxide is not more than 1% of the liquid medicine system) to assist in dissolving, preparing mother liquor, and diluting the mother liquor to a required concentration by using distilled water to obtain the liquid medicine system; the inner wall of the 12-hole plate is coated with polytetrafluoroethylene to prevent mites from escaping, and 1mL of 1% agar water is added for keeping the leaf discs moist; soaking the leaf disk in the medicament for 10s, taking out, drying, and placing in a 12-hole plate; each treatment is provided with 5 repetitions, and 10 active mite-forming heads are inoculated into each repetition; the obtained product is placed in an illumination incubator, the temperature is 25 ℃, the humidity is 80%, the light// dark =16h//8h, the result is observed under a stereoscope after 24h, the mite body is stirred by a writing brush, and the death is recorded when the mite body is still.

Mortality (%) = number of deaths/number of test insects 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:

LD₅₀=8.52 mg/mL, 95% confidence interval (6.90, 10.68), y = -7.62+1.90 x. The meaning of this semilethal dose is: per ml of medicinal liquid system (including distilled water, solvent-alcohol and cosolvent)Agent-dimethylsulfoxide) contained 8.52mg of essential oil, and half of the subjects died when treated at this concentration.

It is known in the art that only 5 to 6 effective concentrations are required in toxicological tests, and at this concentration, the individual having the insect die, and the concentrations of 100% and 0% mortality cannot be counted as effective concentrations, so that the semi-lethal dose for killing mites and insects was determined in the experimental examples 3 and 4 of the present invention based on the 5 series concentrations listed in tables 2 and 3, respectively.

Experimental example 4 insecticidal Effect of the biological control agent of the present invention

Frankliniella occidentalis-touch-kill, and/or, spray: dissolving 20 microliters of prickly ash trifoliate acanthopanax essential oil in absolute ethyl alcohol (the amount of the absolute ethyl alcohol is not more than 1% of the liquid medicine system), adding dimethyl sulfoxide (the amount of the dimethyl sulfoxide is not more than 1% of the liquid medicine system) to assist in dissolving, preparing mother liquor, and diluting the mother liquor to a required concentration by using distilled water 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 2cm long sections, 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 (%) = number of deaths/number of test insects 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:

LD₅₀=7.64 mg/mL, 95% confidence interval (6.88, 8.43), y =0.28+4.26 x. The meaning of this semilethal dose is: liquid medicine per mlThe system (including distilled water, solvent-alcohol and cosolvent-dimethyl sulfoxide) contains 7.64 mg of essential oil, and half of the tested individuals die after treatment at such concentration.