CN112485414A - Determination method and application of volatile active compound with effect of repelling dichocrocis punctiferalis - Google Patents
Determination method and application of volatile active compound with effect of repelling dichocrocis punctiferalis Download PDFInfo
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Abstract
The invention discloses a method for determining a volatile active compound with a function of repelling dichocrocis punctiferalis, and an application thereof, wherein the method comprises the following steps: step 1, collecting sunflower disc volatile matters in a flowering phase and a mature phase respectively by using a headspace dynamic gas sampling method, analyzing the collected volatile matters by using a gas chromatography-mass spectrometer, and identifying the types of the sunflower disc volatile matters in the two phases respectively; step 2, comparing and analyzing the sunflower disc volatile matters in the flowering period and the mature period, and selecting 12 volatile compounds which are respectively unique in the two periods and have large content change between the two periods to perform behavior reaction test on the mated female moths of the dichocrocis punctiferalis; and 3, testing the behavioral response of the mated female moths of the dichocrocis punctiferalis to the selected 12 compounds by using a Y-type olfactometer, and determining that the beta-phellandrene in the female moths can be used as a repellent of the dichocrocis punctiferalis when the concentration is 0.01 (V/V). The method accurately measures the volatile active compounds with the effect of repelling dichocrocis punctiferalis.
Description
Technical Field
The invention relates to a plant-derived repellent, in particular to a determination method and application of a volatile active compound with a repelling effect on dichocrocis punctiferalis.
Background
The dichocrocis punctiferalis Guen bee belongs to Lepidoptera and Cnaphalocrocis medinalis, is damaged by the fact that larvae eat fruits such as Chinese chestnuts, peaches, sunflowers and the like, and is an important boring pest which is difficult to control. In recent years, with the adjustment of agricultural industrial structure in China, the harm of dichocrocis punctiferalis is becoming more serious. For example, in Huang-Huai-Hai-Xia corn producing area and Xian mountain summer corn, the population quantity of the dichocrocis punctiferalis on the corn ear is rapidly increased, so that the damage degree of the dichocrocis punctiferalis exceeds that of Asian corn borers (Ostrinia furnacalis), and the dichocrocis punctiferalis becomes a main pest in the ear stage of the local summer corn. And because the prevention and the treatment of the dichocrocis punctiferalis mainly depend on chemical pesticides at the present stage, the problems of drug resistance generation of pests, ecological environment pollution and the like are caused. Therefore, the problem that an environment-friendly, economic and effective green prevention and control technology is sought to realize the effective field control of dichocrocis punctiferalis is urgently needed to be solved.
In recent years, the regulation and control of insect behaviors by utilizing host plant volatile compounds to design specific attractants or repellents has developed into an important measure for green prevention and control of pests. At present, sex pheromones or host plant volatile compounds are applied to design attractants to carry out field control on dichocrocis punctiferalis.
However, so far, reports on the formula of sex pheromone attractant of dichocrocis punctiferalis and the use of host plant volatile matters as an attractant have been developed internationally, but no report on dichocrocis punctiferalis repellent has been found.
Disclosure of Invention
Based on the problems in the prior art, the invention aims to provide a method for determining a volatile active compound with a function of repelling dichocrocis punctiferalis, and an application thereof, and can solve the problem that the dichocrocis punctiferalis cannot be better prevented and controlled in the field by using a push-pull strategy because no medicament capable of repelling dichocrocis punctiferalis exists at present.
The purpose of the invention is realized by the following technical scheme:
the embodiment of the invention provides a method for measuring a volatile active compound with a function of repelling dichocrocis punctiferalis, which comprises the following steps:
step 1, collecting sunflower disc volatile matters in a flowering phase and a mature phase by using a headspace dynamic gas sampling method, and identifying the collected volatile matters by using a gas chromatography-mass spectrometer to determine the types of the sunflower disc volatile matters;
and 2, comparing and analyzing the volatile components of the sunflower discs in the two periods, and screening twelve compounds which are specific in the two periods and have large content change between the two periods: D-Limonene (D-Limonene), Diethyl phthalate (Diethyl phthalate), n-Pentadecane (Pentadecane), camphene (camphene), beta-Phellandrene (beta-Phellandrene), Terpinolene (Terpinolene), terpinene (gamma-terpinene), alpha-Phellandrene (alpha-Phellandrene), Eucalyptol (Eucalyptol), (1S) - (-) -beta-Pinene ((1S) - (-) -beta-Pinene) and Limonene (Limonene) as subsequent test samples having activity on peach borers;
and 3, respectively measuring twelve compounds selected as test samples through a Y-type olfactometer behavior test of the dichocrocis punctiferalis, and determining that the beta-phellandrene has a repellent effect on the dichocrocis punctiferalis when the concentration of the beta-phellandrene is 0.01(V/V) and can be used as a repellent of the dichocrocis punctiferalis.
The embodiment of the invention also provides application of the beta-phellandrene with the volume concentration of 0.01(V/V) as a repellent for repelling female moths of dichocrocis punctiferalis.
According to the technical scheme provided by the invention, the determination method and the application of the volatile active compound having the effect of repelling dichocrocis punctiferalis have the following beneficial effects:
the method accurately determines that the beta-phellandrene has good repelling effect on female moths of dichocrocis punctiferalis through the volatile components of the sunflower disc, can be used as a plant source repellent of dichocrocis punctiferalis, is used for field control of the dichocrocis punctiferalis, and has an important effect on population control of the dichocrocis punctiferalis in fields.
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In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the description of the embodiments will be briefly introduced 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 based on these drawings without creative efforts.
FIG. 1 is a diagram showing the behavior reaction test result of the dichocrocis punctiferalis Y-type olfactometer in the determination method provided by the embodiment of the present invention, wherein the volume concentration of the compounds is 0.01(V/V),*indicating that the difference between the test compound and the control is significant,**indicating that the difference between the test compound and the control is very significant.
Detailed Description
The technical solutions in the embodiments of the present invention are clearly and completely described below with reference to the specific contents of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments of the present invention without making any creative effort, shall fall within the protection scope of the present invention. Details which are not described in detail in the embodiments of the invention belong to the prior art which is known to the person skilled in the art.
The embodiment of the invention provides a method for measuring a volatile active compound with a function of repelling dichocrocis punctiferalis, which is characterized by comprising the following steps:
step 1, collecting sunflower disc volatile matters in a flowering phase and a mature phase respectively by using a headspace dynamic gas sampling method, and identifying the types of the collected volatile matters by using a gas chromatography-mass spectrometer;
and 2, selecting the volatiles (12 in total) with characteristics of the sunflower volatiles in the flowering phase and the mature phase and large content change between the volatiles for the sexual borer behavior reaction test, wherein the 12 volatiles are respectively D-Limonene (D-Limonene), Diethyl phthalate (Diethyl phthalate), n-Pentadecane (Pentadecane), camphene (camphene), beta-Phellandrene (beta-Phellandrene), Terpinolene (Terpinolene), terpinene (gamma-Terphin), alpha-Phellandrene (alpha-Phellandrene), Eucalyptol (Eucalyptol), (1S) - (-) -beta-Pinene ((1S) - (-) -beta-Pinene) and Limonene (limone).
And 3, testing the behavioral response of mated female moths of the dichocrocis punctiferalis to the 12 compounds screened in the step 2 by using a Y-type olfactometer, determining that the beta-phellandrene has a remarkable repelling effect on the dichocrocis punctiferalis when the concentration of the beta-phellandrene is 0.01(V/V), and using the beta-phellandrene as a dichocrocis punctiferalis repellent.
In the step 1 of the method, the volatile matters of the sunflower discs in the flowering phase and the mature phase are respectively collected by a headspace dynamic gas sampling method:
collecting sunflower disc volatile matters in a flowering phase and a mature phase respectively by adopting a mode of combining dynamic headspace adsorption and solvent elution, wherein an adsorbent used in the dynamic headspace adsorption is activated for 2 hours at 170 ℃ before being used, and then is rinsed and dried by 3mL each of 99.5% methanol, 99.9% anhydrous ether and 99.9% chromatographically pure hexane;
after the collection is finished, the volatile matters adsorbed by the adsorbent are eluted by 350 mu L of chromatographically pure hexane, then 200 mu L of eluent is absorbed and placed in a 2mL chromatographic bottle, 1 mu L of n-nonyl acetate with the concentration of 0.48 mg/mu L is added as an internal standard, the internal standard is shaken and shaken uniformly by a vortex instrument, and the mixture is placed in a refrigerated cabinet at the temperature of 18 ℃ below zero for storage, thus completing the collection of the sunflower disc volatile matters.
In the above determination method, the collecting of the daily sunflower disc volatiles by combining dynamic headspace adsorption and solvent elution is specifically as follows:
air is pumped out by an air sampling instrument and the air flow is monitored, the air flow is kept within the range of 450-600mL/min, the air sequentially enters a distilled water bottle and an active carbon filter bottle, the air passes through a turkey bag provided with a sunflower disc along a Teflon tube, the other end of the bag opening is connected to an adsorption column provided with 50mg of adsorbent, after the adsorbent adsorbs and collects volatile matters of the sunflower disc, the residual air monitors the air flow at an air outlet end through a panel type gas flowmeter and is kept within the range of 350-450mL/min, seamless connection is carried out between different components through a clean latex tube, and the whole device continuously extracts for 4 hours.
In the step 1 of the method, identifying the types of the collected volatile matters by adopting a gas chromatography-mass spectrometer:
and qualitatively and quantitatively analyzing the collected volatile matters by using a gas chromatography-mass spectrometer under the following analysis conditions: the capillary column is DB-5; helium is taken as carrier gas, and the flow rate is 26 cm/s; the sample introduction amount is 1 mu L, and the sample introduction is not divided; the sample injection temperature is 250 ℃; the temperature programming is as follows: keeping at 37 deg.C for 6 min; then heating from 37 ℃ to 70 ℃ at the heating rate of 2 ℃/min, and keeping for 5 min; then heating from 70 ℃ to 20 ℃ at the heating rate of 5 ℃/min, and keeping for 5 min; balancing for 0.5min, wherein the temperature of a gas spectrum-mass spectrum interface is 280 ℃, the temperature of a mass spectrum ion source is 230 ℃, the temperature of a quadrupole rod is 150 ℃, the ionization energy is 70eV, and the scanning range is 30-300 m/z;
performing qualitative analysis on the extracted volatile components by comparing with mass spectrograms in an NIST atlas;
part of the compounds were further verified by retention time and mass spectra with the purchased standard compounds;
converting the peak area of the compound and the peak area of an internal standard compound to obtain the relative content and the absolute content of each component;
according to the qualitative and quantitative analysis of the volatile matter, the composition of the sunflower disc volatile matter in the flowering phase and the mature phase is obtained respectively. Volatile matters (12 types in total) which are unique to each of the two periods and have large content change between the two periods are selected by comparing and analyzing the volatile matter components of the two periods, and the behavior reaction test of the dichocrocis punctiferalis female moths is carried out. The 12 compounds are D-Limonene (D-Limonene), Diethyl phthalate (Diethyl phthalate), n-Pentadecane (Pentadecane), camphene (camphene), beta-Phellandrene (beta-Phellandrene), Terpinolene (Terpinolene), terpinene (gamma-terpinene), alpha-Phellandrene (alpha-Phellandrene), Eucalyptol (Eucalyptol), (1S) - (-) -beta-Pinene ((1S) - (-) -beta-Pinene) and Limonene (Limonene), respectively. Whether the 12 volatile compounds have biological activity or not is investigated through subsequent behavioral response tests.
Referring to fig. 1, in step 3 of the method, the behavior reaction test of the dichocrocis punctiferalis Y-type olfactometer is as follows:
taking 10 mu L of a prepared sample to be detected by using a pipette, dripping the prepared sample to be detected on a W-shaped filter paper sheet, randomly putting the sample into a glass cap at the front end of one side arm of a Y-shaped olfactometer, putting the W-shaped filter paper sheet dropwise added with 10 mu L of liquid paraffin into the other glass cap as a contrast, and allowing clean air to flow in the arm for 10min at the flow rate of 300 mL/min;
introducing a pre-prepared mated female borer of dichocrocis punctiferalis into a main arm of the Y-type olfactometer, recording the selection behavior of the dichocrocis punctiferalis female moth, testing one mated dichocrocis punctiferalis female moth each time, testing 50 mated dichocrocis punctiferalis female moths in each test sample, selecting when the dichocrocis punctiferalis female moths climb over one third of a certain side arm and last for more than 30s, and marking as non-selection if no reaction occurs within 2 min.
The embodiment of the invention provides application of beta-phellandrene with volume concentration of 0.01(V/V) as a repellent with a repellent effect on female moths of dichocrocis punctiferalis.
The method accurately measures that the beta-phellandrene (0.01V/V) has good repelling effect on female moths of dichocrocis punctiferalis through the volatile components in the sunflower discs, can be used as a botanical dichocrocis punctiferalis repellent, is used for field control of the moths, and has an important effect on population control of the dichocrocis punctiferalis in fields.
The embodiments of the present invention are described in further detail below.
The embodiment of the invention provides a method for measuring a volatile active compound with a function of repelling dichocrocis punctiferalis, which comprises the following steps:
step 1) firstly, respectively collecting sunflower disc volatile matters in a flowering phase and a mature phase by using a headspace dynamic gas sampling method, and respectively carrying out qualitative and quantitative analysis on the collected sunflower disc volatile matters in the two phases by using a gas chromatography-mass spectrometry (GC-MS) combined instrument; then comparing the difference of the volatile matter components of the sunflower in the flowering phase and the mature phase, selecting the volatile matters (12 types in total) which are respectively unique in the two phases and have large content change between the two phases for test determination, and finally determining the volatile matter components of the sunflower disc which have the effect of repelling the dichocrocis punctiferalis according to a behavior test of a Y-type olfactometer;
and 2) determining the components of the botanical repellent of the dichocrocis punctiferalis and the appropriate concentration of beta-phellandrene (0.01V/V), wherein the beta-phellandrene is a standard substance with the concentration of more than 97%, and the product has high purity and does not contain other impurities.
The method comprises the following specific steps:
step 1, extracting plant volatile odor compounds from sunflower discs:
the experiment collects sunflower discs volatile odor compounds: each collection was set to 3 replicates, one set of controls.
The dynamic headspace adsorption method can capture the volatile matters of the animals and plants, can perform qualitative and quantitative analysis on trace compounds, can truly reflect the chemical components and the proportion thereof released by the tested animals and plants according to the determination result, is convenient to carry and is suitable for field operation, so the collection of the sunflower disc volatile matters in the experiment is performed by adopting a method combining the dynamic headspace adsorption and solvent elution. Wherein, the adsorbent is activated for 2h at 170 ℃ before use, and then is rinsed and dried by 3mL each of 99.5 percent methanol, 99.9 percent anhydrous ether and 99.9 percent chromatographically pure hexane. The specific process is as follows: air is pumped out by an air sampling instrument and the gas flow is monitored (the air flow is kept within the range of 600mL/min of 450-. The different parts are connected seamlessly by using a clean latex tube, and the whole device is extracted continuously for 4 hours.
After the collection, the volatile matter adsorbed by the adsorbent is immediately eluted by 350 mu L of chromatographically pure hexane, then 200 mu L of eluent is absorbed and placed in a 2mL chromatographic bottle, 1 mu L of n-nonyl acetate with the concentration of 0.48 mg/mu L is added as an internal standard, the internal standard is shaken and shaken uniformly by a vortex instrument, and the mixture is stored in a refrigerated cabinet at the temperature of 18 ℃ below zero for standby.
Step 2. identification of the compound:
the volatile matter was qualitatively and quantitatively determined using a gas chromatography-mass spectrometer (GC-MS) instrument. The analysis conditions were as follows: the capillary column was DB-5(Polyethylene Glycol 20000,60 m.times.0.25 mm.times.0.15 μm, Agilent, USA). Helium was used as a carrier gas, and the flow rate was 26 cm/s. The sample volume is 1 mu L, and the sample introduction is not carried out by shunting. The injection temperature was 250 ℃. The temperature programming is as follows: keeping at 37 deg.C for 6 min; then heating from 37 ℃ to 70 ℃ at the heating rate of 2 ℃/min, and keeping for 5 min; then the temperature is raised from 70 ℃ to 20 ℃ at the temperature raising rate of 5 ℃/min and is kept for 5 min. The balance is 0.5min, the interface temperature of gas spectrum/mass spectrum is 280 ℃, the ion source temperature of mass spectrum is 230 ℃, the temperature of quadrupole is 150 ℃, the ionization energy is 70eV, and the scanning range is 30-300 m/z. The extracted volatile components were characterized by comparison with mass spectra in the NIST gallery. Some compounds were further verified by retention time and mass spectra with the purchased standard compounds. And converting by comparing the peak area of the compound with the peak area of the internal standard compound to obtain the relative content and the absolute content of each component.
See table 1, step 3. comparative analysis of flowering and maturity sunflower volatiles, screening for test compounds:
volatile matters which are specific to the two periods and volatile matters with large content change between the two periods (12 types in total) are selected by comparing and analyzing the volatile matter components of the two periods, and the behavior reaction test of the dichocrocis punctiferalis female moths is carried out. The 12 compounds are D-Limonene (D-Limonene), Diethyl phthalate (Diethyl phthalate), n-Pentadecane (Pentadecane), camphene (camphene), beta-Phellandrene (beta-Phellandrene), Terpinolene (Terpinolene), terpinene (gamma-terpinene), alpha-Phellandrene (alpha-Phellandrene), Eucalyptol (Eucalyptol), (1S) - (-) -beta-Pinene ((1S) - (-) -beta-Pinene) and Limonene (Limonene), respectively.
Referring to fig. 1, step 4. dichocrocis punctiferalis mated female moths were subjected to Y-type olfactometer behavioral response testing: a liquid transfer gun is used for taking 10 mu L of a prepared sample to be detected and dripping the prepared sample to be detected on a W-shaped filter paper sheet, the sample to be detected is randomly placed in a glass cap at the front end of one side arm, a W-shaped filter paper sheet dropwise added with 10 mu L of liquid paraffin is placed in the other glass cap to serve as a contrast, clean air is allowed to flow in the arm for 10min, and the flow rate is 300 mL/min. Introducing one end of a mated female moth of the dichocrocis punctiferalis into a main arm of a Y-type olfactometer, and observing the selection behavior of the female moth. Each time, 1 female moth was tested, and a total of 50 mated female moths of dichocrocis punctiferalis were tested per sample. When the female moth of the dichocrocis punctiferalis climbs one third of a certain side arm for more than 30 seconds, the selection is regarded as being selected, and the non-reaction within 2min is regarded as being not selected.
The results and analysis were as follows:
(1) identification of sunflower volatile components:
and analyzing and identifying the main compound components of the sunflower volatile matter by using a gas chromatography-mass spectrometer (GC-MS).
The peak emergence time of the sunflower disc volatile matter in the flowering phase is mostly distributed in three periods of 15-25min, 30-35min and 39-45min, and 49 compounds (containing 50 internal standards in total, see the following table 1) are detected in total; the peak emergence time of sunflower volatile matters in the mature period is mostly and intensively distributed in two stages of 10-16min and 18-23min, and 45 compounds (containing 46 internal standards in total, see the following table 1) are detected in total. By comparing and analyzing the volatile matter types of the two periods, volatile matters (12 types in total) which are respectively unique to the two periods and have large content change between the two periods are selected: D-Limonene (D-Limonene), Diethyl phthalate (Diethyl phthalate), n-Pentadecane (pentadecene), camphene (camphene), beta-Phellandrene (beta-Phellandrene), Terpinolene (Terpinolene), terpinene (gamma-terpinene), alpha-Phellandrene (alpha-Phellandrene), Eucalyptol (Eucalyptol), (1S) - (-) -beta-Pinene ((1S) - (-) -beta-Pinene) and Limonene (limone), and these volatiles were used as candidates for the subsequent behavioral experiments.
(2) Screening of active compounds:
and (3) measuring the behavior reaction of the dichocrocis punctiferalis adult to a single compound by using a Y-type olfactometer, and screening out a compound component with repellent activity to the dichocrocis punctiferalis female moth.
The Y-type olfactometer behavior test is carried out on the mated female moths of the dichocrocis punctiferalis, and the beta-phellandrene is found to have obvious repellent effect on the mated female moths at the concentration of 0.01 (V/V).
According to the data, the beta-phellandrene measured from the volatile active compound extracted from the dichocrocis punctiferalis host plant-sunflower disc has an obvious repellent effect on the dichocrocis punctiferalis female moth when the concentration is 0.01 (V/V). Can be applied to the field population control of dichocrocis punctiferalis, and has the advantages of economy, effectiveness, no environmental pollution and the like.
In conclusion, the detection method successfully screens and obtains the repellent components suitable for the dichocrocis punctiferalis repellent, can be used for preventing and treating the dichocrocis punctiferalis adults, and solves the problems that the prevention and treatment effect of the agents in the larval stage is not ideal, and the environmental pollution is caused by chemical pesticides.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are included in the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.
Claims (6)
1. A method for measuring a volatile active compound having a repelling effect on dichocrocis punctiferalis is characterized by comprising the following steps:
step 1, collecting sunflower disc volatile matters in a flowering phase and a mature phase by using a headspace dynamic gas sampling method, and identifying the collected volatile matters by using a gas chromatography-mass spectrometer to determine the types of the sunflower disc volatile matters;
and 2, comparing and analyzing the volatile components of the sunflower discs in the two periods, and screening twelve compounds which are specific to the two periods and have large content change between the two periods: d-limonene, diethyl phthalate, n-pentadecane, camphene, beta-phellandrene, terpinolene, terpinene, alpha-phellandrene, eucalyptol, (1S) - (-) -beta-pinene and limonene as subsequent test samples with activity on dichocrocis punctiferalis;
and 3, respectively measuring twelve compounds selected as test samples through a Y-type olfactometer behavior test of the dichocrocis punctiferalis, and determining that the beta-phellandrene has a repellent effect on the dichocrocis punctiferalis when the concentration of the beta-phellandrene is 0.01V/V and can be used as a repellent of the dichocrocis punctiferalis.
2. The method for determining the volatile active compound with the effect of repelling dichocrocis punctiferalis according to claim 1, wherein in the step 1, the volatile matters collected from sunflower discs by using a headspace dynamic gas sampling method are as follows:
collecting sunflower disc volatile matters in a flowering phase and a mature phase respectively by adopting a mode of combining dynamic headspace adsorption and solvent elution, wherein an adsorbent used in the dynamic headspace adsorption is activated for 2 hours at 170 ℃ before being used, and then is rinsed and dried by 3mL each of 99.5% methanol, 99.9% anhydrous ether and 99.9% chromatographically pure hexane;
after the collection is finished, the volatile matters adsorbed by the adsorbent are eluted by 350 mu L of chromatographically pure hexane, then 200 mu L of eluent is absorbed and placed in a 2mL chromatographic bottle, 1 mu L of n-nonyl acetate with the concentration of 0.48 mg/mu L is added as an internal standard, the internal standard is shaken and shaken uniformly by a vortex instrument, and the mixture is placed in a refrigerated cabinet at the temperature of 18 ℃ below zero for storage, thus completing the collection of the volatile matters of the sunflower discs.
3. The method for determining the volatile active compounds with the effect of repelling dichocrocis punctiferalis as claimed in claim 2, wherein the manner of collecting sunflower disc volatiles by combining dynamic headspace adsorption and solvent elution is specifically as follows:
air is pumped out by an air sampling instrument and the air flow is monitored, the air flow is kept within the range of 450-600mL/min, the air sequentially enters a distilled water bottle and an active carbon filter bottle, the air passes through a turkey bag provided with a sunflower disc along a Teflon tube, the other end of the bag opening is connected to an adsorption column provided with 50mg of adsorbent, after the volatile matter of the sunflower disc is trapped by the adsorbent, the residual air monitors the air flow at an air outlet end through a panel type flowmeter and is kept within the range of 350-450mL/min, seamless connection is carried out between different components through a clean latex tube, and the whole device continuously extracts for 4 hours.
4. The method for determining the volatile active compounds having the effect of repelling dichocrocis punctiferalis as claimed in any one of claims 1 to 3, wherein in step 1 of the method, the volatile matters collected are analyzed by gas chromatography-mass spectrometry as follows:
and (3) carrying out qualitative and quantitative analysis on the collected volatile matters by adopting a gas chromatography-mass spectrometry combined method, wherein the analysis conditions are as follows: the capillary column is DB-5; helium is taken as carrier gas, and the flow rate is 26 cm/s; the sample introduction amount is 1 mu L, and the sample introduction is not divided; the sample injection temperature is 250 ℃; the temperature programming is as follows: keeping at 37 deg.C for 6 min; then heating from 37 ℃ to 70 ℃ at the heating rate of 2 ℃/min, and keeping for 5 min; then heating from 70 ℃ to 20 ℃ at the heating rate of 5 ℃/min, and keeping for 5 min; balancing for 0.5min, wherein the temperature of a gas spectrum-mass spectrum interface is 280 ℃, the temperature of a mass spectrum ion source is 230 ℃, the temperature of a quadrupole rod is 150 ℃, the ionization energy is 70eV, and the scanning range is 30-300 m/z;
the extracted volatile components are characterized by comparison with mass spectrograms in an NIST atlas;
part of the compounds were further verified by retention time and mass spectra with the purchased standard compounds;
converting the peak area of the compound with the peak area of an internal standard compound to obtain the quantification of each component;
through comparative analysis of the volatile components of the sunflower discs in the flowering phase and the mature phase, 12 volatile compounds which are respectively unique in the two phases and have large content change between the two phases are screened out, and the volatile compounds comprise: d-limonene, diethyl phthalate, n-pentadecane, camphene, beta-phellandrene, terpinolene, terpinene, alpha-phellandrene, eucalyptol, (1S) - (-) -beta-pinene and limonene as test samples for subsequent determination.
5. The method for determining the volatile active compound having the effect of repelling dichocrocis punctiferalis according to any one of claims 1 to 3, wherein in step 3 of the method, the behavior of dichocrocis punctiferalis Y-type olfactometer is measured as follows:
taking 10 mu L of a prepared sample to be detected by using a pipette, dripping the prepared sample to be detected on a W-shaped filter paper sheet, randomly putting the sample into a glass cap at the front end of one side arm of a Y-shaped olfactometer, putting the W-shaped filter paper sheet dropwise added with 10 mu L of liquid paraffin into the other glass cap as a contrast, and allowing clean air to flow in the arm for 10min at the flow rate of 300 mL/min;
introducing a pre-prepared mated female borer of dichocrocis punctiferalis into a main arm of the Y-type olfactometer, recording the selection behavior of the dichocrocis punctiferalis female moth, testing one mated dichocrocis punctiferalis female moth each time, testing 50 mated dichocrocis punctiferalis female moths in each test sample, selecting when the dichocrocis punctiferalis female moths climb over one third of a certain side arm and last for more than 30s, and marking as non-selection if no reaction occurs within 2 min.
6. The application of beta-phellandrene with the volume concentration of 0.01V/V as a repellent for repelling female moths of dichocrocis punctiferalis.
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