CN111937883A - Lycium barbarum fruit fly sex pheromone and extraction method and application thereof - Google Patents
Lycium barbarum fruit fly sex pheromone and extraction method and application thereof Download PDFInfo
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- CN111937883A CN111937883A CN202010848966.6A CN202010848966A CN111937883A CN 111937883 A CN111937883 A CN 111937883A CN 202010848966 A CN202010848966 A CN 202010848966A CN 111937883 A CN111937883 A CN 111937883A
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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
- A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
- A01N43/34—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one nitrogen atom as the only ring hetero atom
- A01N43/36—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one nitrogen atom as the only ring hetero atom five-membered rings
- A01N43/38—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one nitrogen atom as the only ring hetero atom five-membered rings condensed with carbocyclic rings
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01M—CATCHING, TRAPPING OR SCARING OF ANIMALS; APPARATUS FOR THE DESTRUCTION OF NOXIOUS ANIMALS OR NOXIOUS PLANTS
- A01M1/00—Stationary means for catching or killing insects
- A01M1/02—Stationary means for catching or killing insects with devices or substances, e.g. food, pheronones attracting the insects
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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
- A01N31/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic oxygen or sulfur compounds
- A01N31/02—Acyclic compounds
Abstract
The invention discloses a medlar fruit fly sex pheromone and an extraction method and application thereof, belonging to the field of biological control, the invention utilizes an extraction method to extract medlar fruit fly sex pheromone substances, and utilizes methods of electrophysiological technology, modern analytical chemistry, biological assay and the like to identify sex pheromone components released by males; the invention shows that the feeler potential reaction of female fruit flies of medlar to male insect extract is obviously higher than that of other treatments, which shows that the female fruit flies of medlar are induced by sex pheromone released by the male insects; 2 species of matters of the female trypetid which are not mated have GC-EAD activity on the male trypetid extract, namely 2-nonanol and indole, and compared with a gas chromatogram of the male and female trypetid extract, the 2 species of matters exist in the male trypetid only; the field trapping experiment shows that the combination of 2-nonanol and indole has a more remarkable effect of attracting the medlars than a single component, and the 2-sex pheromone substance has an effect of attracting both female and male medlars of the medlars.
Description
Technical Field
The invention relates to the field of biological control, in particular to a medlar fruit fly sex pheromone and an extraction method and application thereof.
Background
The medlar is a characteristic economic crop with homology of medicine and food in northwest China, is rich in medlar polysaccharide, amino acid and other nutrient substances, and is extremely easy to be damaged by diseases and insects. In an organic medlar plantation, the fruit flies of medlar are one of the main pests.
The fruit fly of Lycium barbarum, Neoceratitis asiatica (Becker), belongs to the family of Pacific Trypetidae (Trypetidae), and its larva exclusively feeds on fruit of Lycium barbarum, so it is named as Lycium barbarum fruit fly. The damage rate of the medlar is generally 22-55%, and reaches 80% when the damage rate is serious. Therefore, the fruit flies of the Chinese wolfberry become the first insect pests in Chinese wolfberry production, and the prevention and control situation is severe. The prevention and control method of the fruit fly of medlar comprises agricultural physical prevention and control, chemical prevention and biological prevention and control and the like. The agricultural physical control comprises maggot fruit picking and deinsectization, winter cultivation and pupa killing, film covering and smoldering disinsection and the like, has good effect, but wastes time and labor; the chemical control mainly comprises the steps of killing the Chinese wolfberry fruit fly imagoes by using medicaments in the initial emergence stage and the full emergence stage and killing the Chinese wolfberry fruit fly imagoes by using soil in the pupal stage, and the large-area use of the insecticides can cause environmental pollution, influence the health of people and livestock, improve pesticide residues, the drug resistance of pests and the like; the biological control method mainly uses biological agents, and the effect is not ideal. Because the larvae of the fruit flies of the Chinese wolfberry live in the fruits of the Chinese wolfberry, proper treatment measures are difficult to take in the larval stage, and the imagoes live freely and need to find mates in a Chinese wolfberry orchard for mating and spawning, the prevention and control work of the imagoes before spawning is particularly important. The sex pheromone which is safe, efficient and strong in specificity is researched and developed, male insects can be attracted, the mating of adults is interfered, and the male insects can be trapped and killed, so that the sex pheromone becomes an important part for comprehensive control of the fruit flies of the Chinese wolfberry, and can be used for monitoring the insect condition of the fruit flies of the Chinese wolfberry.
However, no effective sex pheromone of the medlars has been developed so far.
Disclosure of Invention
The invention aims to provide a sex pheromone for fruit flies of Chinese wolfberry and an extraction method and application thereof, which are used for solving the problems in the prior art.
In order to achieve the purpose, the invention provides the following scheme:
the invention provides a medlar fruit fly sex pheromone, which mainly comprises 2-nonanol and indole as active ingredients.
Further, the mass ratio of the 2-nonanol to the indole is 1: 1.
The invention also provides an extraction method of the lycium barbarum fruit fly sex pheromone, wherein the lycium barbarum fruit fly sex pheromone is extracted from lycium barbarum fruit fly male adults 3-8 days after eclosion.
Further, the extraction method comprises the following steps: soaking 3-8 days after eclosion male adult fruit fly of fructus Lycii in dichloromethane, adding anhydrous sodium sulfate, and filtering with diatomaceous earth to obtain filtrate.
Further, the male adult fruit fly medlar is 5 days after eclosion.
Further, the using amount ratio of the dichloromethane, the anhydrous sodium sulfate and the diatomite is 1 mL: 0.1 g: 0.1 g.
Further, the soaking time is at least 24 h.
The invention also provides application of the sex pheromone of the fruit flies of Chinese wolfberry as a sex attractant in monitoring, controlling and preventing the field occurrence condition of the fruit flies of Chinese wolfberry.
The invention discloses the following technical effects:
the invention utilizes the extraction method to extract the sexual pheromone substance of the fruit fly of medlar, and utilizes the methods of electrophysiological technology, modern analytical chemistry, biological assay and the like to identify the sexual pheromone component released by the male insect; and field trapping effect evaluation is carried out, and the medlar fruit fly sex attractant with good female trapping effect, low cost and popularization value is developed. The results show that the feeler potential reaction of the female fruit fly with medlar to the male insect extract is obviously higher than that of other treatments, which indicates that the female fruit fly with medlar is attracted by sex pheromone released by the male insect; 2 species of matters of the female trypetid which are not mated have GC-EAD activity on the male trypetid extract, namely 2-nonanol and indole, and compared with a gas chromatogram of the male and female trypetid extract, the 2 species of matters exist in the male trypetid only; the field trapping experiment shows that the combination of 2-nonanol and indole has a more remarkable effect of attracting the medlars than a single component, and the 2-sex pheromone substance has an effect of attracting both female and male medlars of the medlars.
The extraction method of the sex pheromone of the fruit fly with medlar has the advantages of simple and convenient operation, small insect quantity, short preparation period and high result reliability.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.
FIG. 1 is the EAG response of an unmatched female and male Lycium barbarum to a crude adult extract;
FIG. 2 is a GC-EAD reaction of unmatched female Lycium barbarum on male extract;
FIG. 3 is an ion flow diagram of extracts of male and female Lycium barbarum flies;
FIG. 4 is an ion fragment diagram of two sex pheromone substances of fruit fly, in which, A.2-nonanol; B. indole;
FIG. 5 shows the field trapping effect of sex attractant on Bactrocera lycii Rehd.
Detailed Description
Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The specification and examples are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including, but not limited to.
EXAMPLE 1 materials and methods
1.1 extraction of sex pheromone of fruit fly of Lycium barbarum
Newly emerged (<24h) adult females and males were placed in different cages each day, providing water and 10% honey water.
Collecting sex pheromone by leaching: and (3) selecting 10 female insects and 10 male insects which are not mated respectively to be placed into a 1.5ml sample bottle after the adults eclosion day 5 (the 5 th day is observed to be a mating peak period), adding 1ml dichloromethane for soaking, simultaneously adding 0.1g anhydrous sodium sulfate for drying, filtering an extracting solution by using 0.1g of diatomite after 24 hours, and storing the collected sample at a low temperature for later use.
1.2 assay of the Activity of the extract EAG of the fruit fly of Lycium barbarum
The EAG reaction of the unmatched female and male adult Lycium Chinense Mill on different extracts was tested respectively.
The EAG is specifically operated as follows: two were loaded with Ringer solution (750mg NaCl,35mg KCl,29mg CaCl)2·2H2O,100mL H2O) glass capillary connected to platinum wire electrode, recording electrode connected to PRG-3 probe, and output electric signal to data collecting and processing device IDAC-4: (
Germany) and then on a computer using the EAG 2000 software (b)
Germany) for identification and recordation. Under the binocular stereovision mirror, the head of the fruit fly with medlar is cut off, one glass electrode is connected with the neck incision of the fruit fly with medlar, and the other glass electrode is connected with the feeler end of the fruit fly with medlar. Measuring 10 μ L of the extract to be measured by a pipette, uniformly dripping the extract on a filter paper strip (5cm × 0.5cm), volatilizing in air for 15s, placing the extract in a glass dropper, and connecting the end of the dropper with a gas stimulation control device by a silicon hose after 20 s. Stimulating with 500mL/min continuous airflow for 0.2s, wherein the interval between two times of stimulation is not less than 2 min. Dichloromethane was used as a control. Each treatment was repeated 5 times. The absolute value of EAG is the reaction minus the EAG reaction of the control.
1.3 testing of the GC-EAD response of unmatched females to Male extracts
Selecting 5-day-old unmated female insects, quickly cutting off the head by using a scalpel, connecting the cut end of the head with a reference electrode, and connecting the tip of an antenna with a recording electrode. The electrodes were connected to a signal amplifier via silver-chloride plated wires, and the signal was amplified via a probe (PRG-3,
Germany) monitoring, using GC-EAD software
Germany) records and analyzes the data.
The GC-EAD analysis chromatographic column is an HP-5ms capillary column, and the sample detection procedure is as follows: the injection port temperature was 270 deg.C, the initial temperature was 50 deg.C (2 min hold), ramped up to 160 deg.C at a rate of 10 deg.C/min, then ramped up to 280 deg.C at a rate of 15 deg.C/min hold for 14min, for a total of 35 min. The detector is a flame ion detector and the temperature is 280 ℃. The amount of sample was 3. mu.L.
1.4 GC-MS identification of effective components of fruit fly sex pheromone of Chinese wolfberry
The instrument is an Agilent Technologies HP6890GC/5973MS Gas Chromatography (GC) -Mass Spectrometry (MS) spectrometer.
Gas phase (GC) conditions: HP-5MS quartz capillary column (30 mm. times.0.25 μm); column temperature: the initial temperature is 50 ℃, the temperature is kept for 2min, the temperature is increased to 160 ℃ at the speed of 10 ℃/min, the temperature is increased to 280 ℃ at the speed of 15 ℃/min, and the temperature is kept for 9 min; for a total of 30 min. The column flow rate was 1.0 ml/min; the temperature of a sample inlet is 250 ℃; the front column pressure is 100 kPa; the sample injection amount is 2.0 mu L; the split ratio is 10: 1; the carrier gas was high purity helium.
Mass Spectrometry (MS) conditions: an ionization mode EI; electron energy 70 eV; the transmission line temperature is 250 ℃; the ion source temperature is 230 ℃; the temperature of the quadrupole rods is 150 ℃; the mass range is 35-500; the method comprises the steps of adopting a Nist14 standard library and a mass spectrogram of a standard compound to qualitatively search the components of the extract by a computer, and then carrying out quantitative analysis on each component according to a peak area normalization method and an internal standard method.
1.5 determination of the field attraction effect of the sexual attractant for the fruit flies of Chinese wolfberry
Respectively preparing the identified 2 fruit fly sex pheromone substances of the medlar into N, I (single-label) and NI (mixed label) solutions of 1 mu g/mu L (normal hexane is used as a solvent), then adding 100 mu L of the solutions or normal hexane (contrast) into a groove of a rubber stopper, and storing the lure with the sex pheromone substances into a sealed sample bottle after the rubber stopper absorbs the added solutions.
Fixing the lure core added with the sex pheromone at the central position of the yellow sticky trap, and bending the sticky trap into a cylindrical shape. In an organic medlar garden (about 100 mu) in Dulan county in Qinghai province, in order to avoid the influences of illumination, wind direction and the like as much as possible, a coiled cylindrical pest sticking plate is vertically hung on a medlar tree at a position 1-1.5 m away from the ground (the distance is more than 5 m). And (3) arranging different treatment sticky boards by adopting a random block design, and counting and recording the number of female and male medlars on the sticky boards after 2 d.
1.6 data analysis
Differences between antennal potential response values and field trap experimental data multiple comparative analyses were performed using Turkey-test of one-way analysis of variance ANOVA.
Example 2 results and analysis
2.1 EAG reaction of Lycium barbarum to its extract
1) The EAG response of the non-mated female Chinese wolfberry fruit fly to the extracts of the female and male adults is obviously higher than that of the non-mated male Chinese wolfberry fruit fly to the extracts of the female and male adults (P < 0.05). 2) The antennal potential response of the female un-mated medfly to the male insect extract is significantly higher than the antennal potential response to the female insect extract (P < 0.05). 3) The response of the antennal potential of the unmated female to the male worm extract and the male worm abdominal extract is obviously higher than that of the male worm cephalothorax extract, but the response of the antennal potential of the male worm extract and the male worm abdominal extract is not obviously different. 4) The antennal potential responses of the unmated females to the male extract and the male headspace volatiles were not significantly different, as shown in figure 1.
2.2 GC-EAD reaction of Lycium barbarum on the extract
The GC-EAD reaction of the non-mated female fruit fly to the non-mated male insect extract detects 2 substances with GC-EAD activity, namely 2-Nonanol (2-Nonanol) and Indole (Indole), as shown in figure 2 and figure 4, the gas phase diagram of the female fruit fly and male insect extract further verifies that 2-Nonanol and Indole 2 substances are only present in the male insect extract, the 2 substances are not found in the female insect extract, as shown in figure 3, the temperature raising program is 50 ℃ (keeping for 2min), the temperature is raised to 160 ℃ at 10 ℃/min, and then the temperature is raised to 280 ℃ (keeping for 9min at 15 ℃/min).
2.3 field attraction effect of sex attractant on fruit fly of Lycium barbarum
The sex attractant respectively prepared by the combination of 2-nonanol and indole and the combination of the lure core prepared from the single component and the yellow armyworm plate in the field has the attraction effect on the medlars, the attraction effect of the combination of 2-nonanol and indole on the medlars is obviously higher than that of the single component of 2-nonanol and indole and a control, and the combination of 2-nonanol and lure can attract hundreds of medlars. Control and attractant treatments were able to attract female and male flies, as shown in figure 5.
The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.
Claims (8)
1. The wolfberry fruit fly sex pheromone is characterized in that the main active ingredients are 2-nonanol and indole.
2. The medlare fruit fly sex pheromone according to claim 1, wherein the mass ratio of the 2-nonanol to the indole is 1: 1.
3. The method for extracting sex pheromone of medlars according to claim 1 or 2, wherein the sex pheromone of medlars is extracted from male adults of medlars 3-8 days after eclosion.
4. The extraction method according to claim 1, wherein the extraction method is: soaking 3-8 days after eclosion male adult fruit fly of fructus Lycii in dichloromethane, adding anhydrous sodium sulfate, and filtering with diatomaceous earth to obtain filtrate.
5. The extraction method according to claim 3 or 4, wherein the male adult fruit fly lycium barbarum is 5 days after emergence.
6. The extraction method according to claim 4, wherein the dichloromethane, the anhydrous sodium sulfate and the diatomite are used in a ratio of 1 mL: 0.1 g: 0.1 g.
7. The extraction process according to claim 4, characterized in that the soaking time is at least 24 h.
8. Use of the sexual pheromone of medlars according to claim 1 or 2 as a sex attractant for monitoring and controlling the occurrence in the field of medlars.
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Cited By (1)
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| CN117397681A (en) * | 2023-12-15 | 2024-01-16 | 中国医学科学院药用植物研究所 | Barbary wolfberry fruit sex pheromone and application thereof |
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| CN117397681A (en) * | 2023-12-15 | 2024-01-16 | 中国医学科学院药用植物研究所 | Barbary wolfberry fruit sex pheromone and application thereof |
| CN117397681B (en) * | 2023-12-15 | 2024-02-27 | 中国医学科学院药用植物研究所 | Barbary wolfberry fruit sex pheromone and application thereof |
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