Volatile substance with repellent activity to adults of prodenia litura in soybean

Abstract

The invention discloses a volatile substance with repelling activity to adults of prodenia litura in soybeans. The volatile substance is prepared from the following components: trans-2-hexenal or a composition of nonanal, leaf alcohol and hexanal. The trans-2-hexenal has a repelling effect on male adults of prodenia litura; the nonanal, leaf alcohol and hexanal composition has a repelling effect on female adults of prodenia litura. When the volatile substance is used as the prodenia litura imago repellent, the solvent is at least one of dichloromethane, paraffin oil or normal hexane; and the solute concentration of the repellent is 1-100 [mu] g / [mu] L. The volatile substances can be adsorbed, collected and extracted through the soybean volatile matter collecting device. According to the application, it is found for the first time that trans-2-hexenal in the soybean volatile matter has a repelling effect on male adults of prodenia litura, and the nonanal, leaf alcohol and hexanal composition has a repelling effect on female adults of prodenia litura, can be further used for research and development of plant source pest repelling agents, and plays a positive role in green prevention and control of prodenia litura.

Description

Technical Field

[0001] The present invention belongs to the fields of biopesticides and plant protection, and relates to the screening and application of key phytogenic compounds capable of repelling adult Spodoptera litura, and more specifically to volatile substances in soybeans that have repellent activity against adult Spodoptera litura. Background Art

[0002] Spodoptera litura belongs to the order Lepidoptera and the family Noctuidae, and harms nearly 400 crops in 110 families (such as Cucurbitaceae, Gramineae, Compositae, Cruciferae, etc.), and has characteristics such as voracious feeding and strong migration ability. At present, chemical control is mainly used to control the damage of this insect, but this has induced problems such as drug resistance, environmental pollution, and pesticide residues in agricultural products, and there is an urgent need to develop ecological prevention and control measures. Patent CN113142202B provides a method for controlling noctuid pests (Helicoverpa assulta, Spodoptera litura) in field crops, including steps such as the preparation of an oviposition repellent, the preparation of an oviposition promoter, and the setting of a monitoring device for the oviposition behavior of Lepidoptera Noctuidae insects. According to the oviposition habits of Lepidoptera fruit-boring Noctuidae insects, this invention uses an oviposition repellent to regulate the oviposition behavior of female adults, reduces the oviposition amount of gravid female adults on target hosts, so as to achieve the purpose of reducing the population density in the field, reducing the occurrence of fruit-boring and diseased fruits, and improving the technology of behavioral regulation of noctuid insects.

[0003] Using plant volatile substances to repel target pests, so that the pests stay away from the main cultivated crops and reduce the damage of pests, is a safe and effective method. Generally, volatile secondary metabolites derived from plants have simple structures, are easy to synthesize, and have advantages such as good environmental compatibility and safety for non-target organisms, which is one of the research and development directions of new environmentally friendly biopesticides. Patent CN105994424A provides a method for preparing a phytogenic insect repellent, which is mainly composed of a refined extract of tomato plants, a refined extract of coriander plants, and a surfactant OP. The mass percentages of the above material components are respectively: 30 - 50%, 10 - 20%, 1 - 2%. The main active ingredients of the tomato refined extract are n-hexanol and (Z)-3-hexenyl acetate; the active ingredients of the coriander refined extract are nonanal and trans-2-hexenal. The preparation method is to mix the synthesized refined extract of tomato plants, the refined extract of coriander plants, and the surfactant OP in proportion, and then add an appropriate amount of water and mix and stir to form a solvent. This insect repellent has characteristics such as being green and environmentally friendly, residue-free, easy to degrade, low cost, and convenient to use, and the repellent effect on moth larvae, aphids, etc. can reach more than 85%. However, this patent only targets the larvae of several pests and does not involve the effect on adults.

[0004] Existing research has shown that intercropping soybeans with green vegetables can significantly reduce the population of Spodoptera litura (Wan Nianfeng, Jiang Jiexian, Ji Xiangyun. Effects of intercropping soybeans or taro with green vegetables on controlling Spodoptera litura and the diversity of predatory natural enemies under protected conditions [J]. Chinese Journal of Eco-Agriculture, 2012, 20(2): 236-241), and it has been locally applied in Shanghai, Jiangsu, Zhejiang and other places. However, existing research has only verified the regulatory effect of soybeans at the production level, and has not yet clarified its key repellent components and action mechanisms, which limits the further optimization and popularization of this technology. Summary of the Invention

[0005] Based on the above-mentioned disadvantages in the existing technology, the purpose of this application is to provide volatile substances in soybeans that have repellent activity against adult Spodoptera litura. The inventors screened key phytogenic compounds with repellent activity against adult Spodoptera litura through gas chromatography-electroantennogram detection (GC-EAD), electroantennogram (EAG), and behavioral experiments, providing a theoretical basis for the development of repellents for adult Spodoptera litura and the control of Spodoptera litura.

[0006] One of the technical solutions of the present invention provides the application of trans-2-hexenal, nonanal + leaf alcohol + hexanal composition in controlling adult Spodoptera litura.

[0007] Furthermore, the trans-2-hexenal has a repellent effect on male adult Spodoptera litura; the nonanal + leaf alcohol + hexanal composition has a repellent effect on female adult Spodoptera litura.

[0008] Another technical solution of the present invention provides a repellent for male adult Spodoptera litura, wherein the solute component of the repellent for male adult Spodoptera litura is trans-2-hexenal; the concentration of the solute in the repellent for male adult Spodoptera litura is (1-100) μg / μL.

[0009] Furthermore, the solvent of the repellent for male adult Spodoptera litura is at least any one of dichloromethane, paraffin oil or n-hexane.

[0010] Furthermore, the repellent for male adult Spodoptera litura is obtained by an artificial preparation method or by collecting and extracting from natural plants.

[0011] Another technical solution of the present invention provides a repellent for female adult Spodoptera litura, wherein the solute component of the repellent for female adult Spodoptera litura is nonanal + leaf alcohol + hexanal composition; the ratio of nonanal, leaf alcohol, and hexanal in the repellent for female adult Spodoptera litura is 1:1:1; the concentration of the solute in the repellent for female adult Spodoptera litura is (1-100) μg / μL.

[0012] Furthermore, the solvent of the Spodoptera litura female adult repellent is at least one of dichloromethane, paraffin oil or n-hexane; the Spodoptera litura female adult repellent is obtained by an artificial preparation method or by collecting and extracting from natural plants.

[0013] The fourth technical solution of the present invention provides a method for extracting volatile substances with repellent activity against Spodoptera litura adults from soybeans.

[0014] Furthermore, this method is based on a collection device for soybean volatiles, and the structure of the device includes:

[0015] An air sampler (1), an activated carbon filter bottle (2), a humidifying conical flask (3), a first flowmeter (4), a glass cover (5), an adsorption tube (6), a second flowmeter (7); the above structures are sequentially connected in order through pipelines, and the second flowmeter (7) is connected to the air sampler (1), and the whole device forms a circulating and closed passage. The glass cover (5) is used to place plants; Porapak-Q adsorbent is placed in the adsorption tube (6) for collecting plant volatile samples.

[0016] Furthermore, based on the above collection device, the steps of the method include:

[0017] (1) Preparation before testing: Rinse the glass cover and dry it. After sealing the bottle mouth of the glass cover, continuously introduce air; wrap the upper mouth of the pot for planting soybeans with tin foil and expose the soybean plants to eliminate the influence of volatiles from the soil and the flower pot.

[0018] (2) Collection of volatiles: Turn on the air sampler, control the air flow rate at the air outlet and the air inlet to be the same. At this time, the Porapak-Q adsorbent in the adsorption tube continuously adsorbs the volatiles of the soybean plants. After continuously pumping air for a period of time, use a solvent to elute the Porapak-Q adsorbent adsorbed with volatiles. The obtained eluate contains volatile substances with repellent activity against Spodoptera litura adults, which can be further purified or compounded according to a ratio to be used as the Spodoptera litura male adult repellent and / or the Spodoptera litura female adult repellent.

[0019] The rinsing of the glass cover in step (1) means: rinsing the glass cover successively with distilled water, absolute ethanol, and acetone; the drying means: baking at 180 - 220 °C for 1 - 3 h, preferably baking in an oven at 200 °C for 2 h; the flow rate of continuously introducing air is 150 - 400 mL / min, preferably 200 mL / min.

[0020] In step (2), the air flow rates of both the air outlet and the air inlet are 480 - 520 mL / min, preferably 500 mL / min; the duration of continuous air extraction is 10 - 15 hours, preferably 12 hours; the eluent / solvent used for elution is any one of dichloromethane, paraffin oil, or n - hexane, preferably dichloromethane; the volume of the eluent / solvent is 1 - 5 mL, preferably 1.5 mL.

[0021] The present invention has the following advantages compared with the prior art:

[0022] (1) The present invention first discovers six volatile compounds (hexanal, trans - 2 - hexenal, leaf alcohol, leaf acetate, nonanal, and isophorone) in the volatile components of soybeans that have a behavioral response to adult Spodoptera litura. Adult Spodoptera litura has an electroantennogram response to these six chemical substances and shows a strong dose - dependence. Among them, trans - 2 - hexenal has a repellent effect on male adult Spodoptera litura; the composition of nonanal + leaf alcohol + hexanal has a repellent effect on female adult Spodoptera litura, and the ratio of nonanal, leaf alcohol, and hexanal is 1:1:1.

[0023] (2) The present invention discovers for the first time that trans - 2 - hexenal in soybean volatiles has a repellent effect on male adult Spodoptera litura, and the composition composed of nonanal, leaf alcohol, and hexanal has a repellent effect on female adult Spodoptera litura. This lays a foundation for the next - step research and development of plant - derived pest repellent products to control adult Spodoptera litura. Description of the Drawings

[0024] Figure 1 is a schematic structural diagram of the volatile collection of the present invention;

[0025] Description of the Reference Numerals in the Drawings

[0026] 1 - Atmospheric sampler; 2 - Activated carbon filter bottle; 3 - Humidifying conical flask; 4 - First flowmeter; 5 - Glass cover; 6 - Adsorption tube; 7 - Second flowmeter;

[0027] Figure 2 is the GC - EAD response curve of female adult Spodoptera litura to the volatiles of soybean flowering stage; where: 1: hexanal; 2: trans - 2 - hexenal; 3: leaf alcohol; 4: leaf acetate; 5: nonanal; 6: isophorone;

[0028] Figure 3 is the GC - EAD response curve of male adult Spodoptera litura to the volatiles of soybean flowering stage; where: 1: hexanal; 2: trans - 2 - hexenal; 3: leaf alcohol; 4: leaf acetate; 5: nonanal; 6: isophorone;

[0029] Figure 4It is the mass spectrometry of the volatiles during the soybean flowering period and the standard substances; among which: (a): hexanal; (b): trans-2-hexenal; (c): leaf alcohol; (d): leaf acetate; (e): nonanal; (f): isophorone;

[0030] Note: Figure 4 It is divided into 1 summary figure and 6 enlarged figures;

[0031] Figure 5 It is the electroantennogram-dose response of female Spodoptera litura adults to 6 standard volatiles (CK in the figure is the control group); Note: A: hexanal; B: trans-2-hexenal; C: leaf alcohol; D: leaf acetate; E: nonanal; F: isophorone. Different lowercase letters indicate significant differences (Tukey’s multiple range test, P<0.05);

[0032] Figure 6 It is the electroantennogram-dose response of male Spodoptera litura adults to 6 standard volatiles (CK in the figure is the control group); Note: A: hexanal; B: trans-2-hexenal; C: leaf alcohol; D: leaf acetate; E: nonanal; F: isophorone; Different lowercase letters indicate significant differences (Tukey’s multiple range test, P<0.05);

[0033] Figure 7 It is the device diagram for the determination of the behavioral selection reaction of the present invention; among which, 1, 6: atmospheric sampler; 2, 7: flowmeter; 3, 8: activated carbon filter bottle; 4, 9: humidifying conical flask; 5, 10: odor bottle; 11: Y-tube;

[0034] Figure 8 It is the behavioral selection of female Spodoptera litura adults for 6 standard volatile substances; Note: ** indicates significant difference of P<0.01 (x 2 -test);

[0035] Figure 9 It is the behavioral selection of male Spodoptera litura adults for 6 standard volatile substances; Note: * and ** respectively indicate significant differences of P<0.05 and P<0.01 (x 2 -test);

[0036] Figure 10 It is the behavioral selection of female Spodoptera litura adults for 4 volatile mixtures; Note: ** indicates significant difference of P<0.01 (χ 2 -test). Detailed implementation mode

[0037] To enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be described in detail below in conjunction with specific embodiments. It should be noted that the following embodiments will help those skilled in the art to further understand the present invention, but do not limit the present invention in any form. It should be pointed out that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made. These all belong to the protection scope of the present invention.

[0038] There are no special restrictions on the sources of all raw materials of the present invention, and those purchased on the market or prepared according to conventional methods well-known to those skilled in the art are all acceptable.

[0039] Example 1 A volatile collection device

[0040] As Figure 1 shown, the present invention uses the dynamic headspace sampling method of the adsorption tube 6 containing Porapak-Q adsorbent to extract the volatiles during the flowering period of soybeans, and uses GC-EAD and GC-MS to test and identify the components of the volatiles.

[0041] The structure of the volatile collection device includes: placing the flowering soybeans in the glass cover 5, connecting the upper switch (air outlet) of the glass cover 5 to the adsorption tube 6, placing Porapak-Q adsorbent in the adsorption tube 6, and connecting the adsorption tube 6 to the second flowmeter 7 and the air sampler 1 through a connecting tube; the bottom switch (air inlet) of the glass cover 5 is connected to the air delivery system, and is sequentially connected to the first flowmeter 4, the humidifying conical flask 3, the activated carbon filtering flask 2 and the air sampler 1 through a connecting tube.

[0042] The model of the air sampler 1 is QC-1B, which is produced by Beijing Ke'an Labor Protection Technology New Technology Co., Ltd. The dosage of the Porapak-Q adsorbent is 100 mg, the pore size of the adsorbent is 80-100 mesh, and the production company is Waters Associates, USA; the adsorption tube 6 is rinsed with 3 mL of dichloromethane (HPLC grade, purchased from Shanghai Anpu Experimental Technology Co., Ltd.) before adsorbing volatiles, using N2 with a flow rate of 70 mL / min as the protective gas, and then pre-treated by heating at 200 °C for 2 h.

[0043] Example 2 Headspace collection of soybeans volatiles during the flowering period

[0044] This example collects the volatiles of soybeans in the flowering period based on the device provided in Example 1. The specific process includes the following steps:

[0045] Step 1. Preparation before testing:

[0046] The glass cover was rinsed successively with distilled water, absolute ethanol, and acetone, and then placed in an oven at 200 °C for 2 h for pretreatment. After sealing the mouth of the glass cover, air was continuously introduced at an air flow rate of 200 mL / min; the upper opening of the pot for planting soybeans was wrapped with tin foil, leaving the soybean plants exposed, to eliminate the influence of volatiles from the soil and the flower pot (such as benzene, toluene, formaldehyde, etc.);

[0047] Step 2. Volatile collection:

[0048] The potted plant wrapped with tin foil was placed in the glass cover pretreated in Step 1. The upper switch (air outlet) of the glass cover was connected to the adsorption tube 6, and the adsorption tube 6 was connected to the second flowmeter 7 and the air sampler 1 through a connecting tube; the bottom switch (air inlet) of the glass cover was connected to the air delivery system, and was successively connected to the first flowmeter 4, the humidifying conical flask 3, the activated carbon filtering flask 2, and the air sampler 1 through a connecting tube; the air sampler 1 was turned on, and the air flow rates at the air outlet and the air inlet were both controlled at 500 mL / min. At this time, the Porapak-Q adsorbent in the adsorption tube continuously adsorbed the volatiles of the soybean plants. The pumping time lasted for 12 hours, and then the Porapak-Q adsorbent adsorbed with volatiles was eluted with 1.5 mL of dichloromethane and stored frozen at -20 °C.

[0049] Example 3 Determining the components in soybean volatiles that have an electrophysiological response to Spodoptera litura adults

[0050] (1) Determining the components that have an electrophysiological response to Spodoptera litura adults through a gas chromatography-electroantennogram detection system (GC-EAD)

[0051] In this example, the gas chromatography-electrophysiological antennal detecting system (GC-EAD) was produced by Syntech in the Netherlands. The chromatographic column was an HP-5MS capillary column (length × inner diameter × film thickness = 30 m × 0.25 mm × 0.25 μm). The dichloromethane eluate of the volatiles during the soybean flowering stage was injected without splitting, and the injection volume was 3 μL each time. The column temperature program: the initial value was maintained at 50 °C for 2 min, then heated at a rate of 4 °C / min to 120 °C and maintained for 2 min, and then heated at a rate of 20 °C / min to 250 °C and maintained for 4 min, running for a total of 32 min. The solvent delay was 3 min. The detector temperature was 280 °C, the injection port temperature was 250 °C, the carrier gas was high-purity nitrogen, and the flow rate was 25 mL / min. A Y-type separation device (Agilent Technologies, USA) was added to the chromatographic column. The Y-type separation device separated the volatiles coming out of the gas chromatography injection tube at a separation ratio of 1:1 by volume. Half of the test substances were sent to the antenna in the electroantennogram apparatus to measure the activity, and the other half of the test substances were sent to the FID detection of the gas chromatography.

[0052] Select adult Spodoptera litura with good activity at 2-3 days old. Under a dissecting microscope, quickly cut off the antennae from the base with a scalpel, and cut off a little of the tip of the antennae. Remove the reference electrode of the glass electrode, suck the base of the antennae by relying on the surface tension of the physiological saline, and then carefully fix the reference electrode. At the same time, connect the end of the antennae to the measuring electrode under a stereomicroscope. The connected antennae were 1 cm away from the gas flow glass tube. After the above antennae were connected well and the baseline was stable, inject 3 μL of the test volatiles into the chromatographic column; the whole experiment was repeated 4 times. The indoor temperature during the test was 23-27 °C, and the relative humidity was 60-80%.

[0053] (2) Detect the volatile components by gas chromatography-mass spectrometry (GC-MS)

[0054] In this example, a gas chromatography-mass spectrometry (GC-MS, model 7890B-5977B) produced by Agilent was used to identify the volatiles during the soybean flowering stage. The purchased soybean volatile standard (purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.) was used as a control and was also verified by GC-MS. The gas chromatography was equipped with an HP-5MS capillary column (length × inner diameter × film thickness = 30 m × 0.25 mm × 0.25 μm), the split ratio was 20:1, the carrier gas was helium, the column flow rate was 1 mL / min, the injection volume was 1 μL, and the injection port temperature was 250 °C. The temperature program: the initial temperature was 50 °C, after maintaining for 2 min, it was heated to 120 °C at a rate of 4 °C / min and maintained for 2 min, and then heated to 250 °C at a rate of 20 °C / min and maintained for 4 min. The solvent delay was 3 min.

[0055] Mass spectrometry conditions: The ion source type is EI, the ion source temperature is 230 °C, the source electron energy is 70 eV, the quadrupole temperature is 150 °C, and the mass spectrometry scanning range is 29 - 600 m / z. Compound identification is carried out by comparing the mass spectrometry data with the NIST20.L standard spectral library.

[0056] Figure 2 、 Figure 3 are the GC-EAD response curves of female and male adults of Spodoptera litura to the volatile compounds during the soybean flowering stage. The test results show that female and male adults of Spodoptera litura have a potential response to 6 compound components in the soybean volatiles. There are receptors on the antennae of Spodoptera litura adults that can sense chemical substances. When the adults of Spodoptera litura come into contact with these 6 compounds in the soybean volatiles, the neurons in the receptors will produce changes in electrical signals, which can be recorded by instruments and manifested as fluctuations in potential. The potential response is often closely related to the behavioral response of insects. Generally speaking, when the adults of Spodoptera litura have an obvious potential response to a certain compound, it may indicate that they will respond behaviorally to this compound, that is, they may show an avoidance or attraction effect.

[0057] Figure 4 are the mass spectrometry diagrams of the volatile compounds during the soybean flowering stage and the standard products. After GC-MS comparison, the 6 compounds are respectively identified as hexanal, trans-2-hexenal, leaf alcohol, leaf acetate, nonanal, and isophorone.

[0058] Example 4 Antenna potential-dose response of Spodoptera litura adults to 6 standard volatile compounds

[0059] To further verify the electrophysiological activity of the 6 EAD active compounds, the antenna potential-dose response of the antennae of female and male adults of Spodoptera litura to each standard volatile compound was detected.

[0060] In this example, an electroantennogram (EAG) apparatus (produced by Syntech, Netherlands) was used to measure the EAG electrophysiological responses of adult Spodoptera litura to soybean volatiles standards at different concentrations. Before the experiment started, the EAG apparatus was ventilated for 2 hours. Adult Spodoptera litura that emerged 2 - 3 days ago were selected. Under a dissection microscope, the antennae were quickly cut off from the base with a scalpel, and a little of the tip of the antenna was cut off. The reference electrode of the glass electrode was removed, and the base of the antenna was sucked by relying on the surface tension of physiological saline. Then the reference electrode was carefully fixed. At the same time, under a stereomicroscope, the end of the antenna was connected to the measuring electrode. The connected antenna was 1 cm away from the air flow glass tube. The Y - Ampl value of the EAG apparatus was selected as 0.5 mV, the time - baseline value was 15 s, the stimulating air flow in the EAG was 300 mL per minute, the continuous ventilation air flow was 600 mL per minute, and the time interval between each measurement was 30 s. The volatile standards hexanal, (E) - 2 - hexenal, leaf alcohol, ethyl cis - 3 - hexenoate, nonanal, and isophorone at concentrations of 1 μg / μL, 10 μg / μL, and 100 μg / μL were used as the experimental group, and the control group (CK) was paraffin oil solvent.

[0061] The preparation method of the above - mentioned volatile standards was as follows: A certain amount of the standard was weighed with an analytical balance and placed in a 2 - mL gas chromatography sample vial, and dissolved with liquid paraffin (purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.) to prepare a stock solution of the standard at a concentration of 100 μg / μL. Then, the volatile standards at concentrations of 10 μg / μL and 1 μg / μL were prepared by the gradient series concentration dilution method. The standards were stored in chromatography sample vials at 4°C and equilibrated at room temperature for 30 minutes before use.

[0062] 10 μL of the volatile standard was evenly dropped on a 3 cm×0.5 cm rectangular filter paper strip to measure the EAG response value. Each standard concentration was tested once on the same antenna from low to high, and the control group of liquid paraffin oil was tested twice before and after. A total of 6 antennae were continuously tested. The indoor environmental temperature was 25 ± 2°C, and the relative humidity was 70 ± 10%. The EAGPro software produced by Syntech, Netherlands was used to collect data. The Tukey’s multiple range test in one - way analysis of variance (ANOVA) was used to test the significance between the EAG response values. The statistical significance was set as P < 0.05. The F - statistic obtained from one - way analysis of variance is the ratio of the mean square between groups to the mean square within groups, and its calculation formula is: F = MS 组间 / MS 组内 . Among them, the mean square between groups MS 组间 is obtained by dividing the sum of squares between groups by the degrees of freedom between groups, and the mean square within groups MS 组内 is obtained by dividing the sum of squares within groups by the degrees of freedom within groups. The larger the F value, the more significant the difference between groups is relative to the difference within groups.

[0063] Figure 5 is the electroantennogram-dose response of female adult Spodoptera litura to 6 standard soybean volatiles. The experimental results showed that: the antennae of female adult Spodoptera litura had significant responses to hexanal (F = 53.43; P < 0.001; Figure 5 A), (E)-2-hexenal (F = 95.8; P < 0.001; Figure 5 B), cis-3-hexen-1-ol (F = 65.73; P < 0.001; Figure 5 C), cis-3-hexen-1-yl acetate (F = 49.89; P < 0.001; Figure 5 D), nonanal (F = 312.7; P < 0.001; Figure 5 E) and isophorone (F = 112.9; P < 0.001; Figure 5 F). The response degrees of female adult Spodoptera litura to different concentrations of soybean volatiles were different. Through the electroantennogram-dose response curve, it was found that 6 compounds from 1 μg / μL to 100 μg / μL could significantly stimulate the electroantennogram response of females, and 100 μg / μL could stimulate the electroantennogram response to the greatest extent.

[0064] Figure 6 is the electroantennogram-dose response of male adult Spodoptera litura to 6 standard soybean volatiles. The experimental results showed that: the antennae of male adult Spodoptera litura had significant responses to hexanal (F = 28.39; P < 0.001; Figure 6 A), (E)-2-hexenal (F = 44.44; P < 0.001; Figure 6 B), cis-3-hexen-1-ol (F = 141.2; P < 0.001; Figure 6 C), cis-3-hexen-1-yl acetate (F = 80.31; P < 0.001; Figure 6 D), nonanal (F = 108.2; P < 0.001; Figure 6 E) and isophorone (F = 179.9; P < 0.001; Figure 6 F). The response degrees of male adult Spodoptera litura to different concentrations of soybean volatiles were different. Through the electroantennogram-dose response curve, it was found that hexanal started to trigger a significant EAG response in male antennae at a dose of 100 μg / μL, while the other 5 standard soybean volatiles could significantly stimulate the electroantennogram response of males from 1 μg / μL to 100 μg / μL. Among them, 100 μg / μL could stimulate the electroantennogram response to the greatest extent.

[0065] Example 5 Determining the Components with Behavioral Responses to Female Adult Spodoptera litura

[0066] The behavioral response device used in this example is as Figure 7As shown in the figure, connect the device air sampler 1 and 6, flowmeter 2 and 7, drying tower 3 and 8 filled with activated carbon, humidifying device 4 and 9, odor bottle 5 and 10, and Y-shaped test tube 11 in sequence with a connecting tube. The air flow on both arms of the Y-shaped test tube 11 is maintained at 500 mL / min. Before the experiment, all instruments should be cleaned 3 times with distilled water and alcohol respectively to reduce errors. Select female Spodoptera litura adults at 2-3 days old for behavioral test determination.

[0067] In this embodiment, a Y-shaped test tube is used to verify the behavioral responses of 6 volatile standard substances to female Spodoptera litura adults. The specific process is as follows: Take the outlet of the air sampler as the air flow source, set the air flow rates of both arms of the olfactometer to 500 mL / min, and ventilate in advance for 5 min to ensure that there are no other impurity gases in the Y-shaped tube. Use 10 μg / μL volatile standard substances hexanal, trans-2-hexenal, leaf alcohol, leaf acetate, nonanal, and isophorone as the experimental group, and liquid paraffin as the control group. The preparation of the volatile standard substances is the same as that for the above-mentioned electroantennogram response determination.

[0068] During the test, take 10 μL of the volatile standard substance and liquid paraffin respectively and drop them onto a 3 cm × 0.5 cm rectangular filter paper strip, put them into the odor bottle to form an odor source, and connect them to the two side arms of the Y-shaped tube through a connecting tube. Release a single adult at the main arm opening of the Y-shaped tube, and the reaction time is 5 min. When the insect enters more than 1 / 2 of the side arm and stays for at least 30 s, record it as the selection result. If no selection is made within 5 min, stop the behavioral determination of this insect and do not record it. There are 30 female Spodoptera litura adults in each group. Each insect under the treatment of the same concentration compound is only tested once. The indoor environmental temperature is 25 ± 2 °C, and the relative humidity is 70 ± 10%.

[0069] Calculation formulas for the attraction rate and repulsion rate of female Spodoptera litura adults to the odor source:

[0070] Test selection rate = number of insects selecting the test odor source / total number of insects successfully selected; control selection rate = number of insects selecting the control odor source / total number of insects successfully selected. The significant differences in the Y-shaped tube behavioral response data were compared by the chi-square test (χ 2 )). The statistical significance was set at P < 0.05.

[0071] Figure 8 are the behavioral selection results of female Spodoptera litura adults for 6 volatile standard substances. The experimental results show that: nonanal (χ 2 = 72.000, df = 1, P < 0.001), leaf alcohol (χ 2 = 8.000, df = 1, P = 0.005), hexanal (χ 2= 8.000, df = 1, P = 0.005) had a significant attracting effect on female adults. The attracting rates of nonanal, leaf alcohol, and hexanal were 80%, 60%, and 60% respectively. In addition, these six substances did not have a significant repellent effect on female adults of Spodoptera litura.

[0072] Example 6 Determining the Components with Behavioral Responses to Male Adults of Spodoptera litura

[0073] The methods, steps, devices, etc. of this example were the same as those of Example 5, except that the female adults of Spodoptera litura were replaced with male adults of Spodoptera litura.

[0074] The experimental results were as Figure 9 shown. The experimental results found that trans-2-hexenal (χ 2 = 3.92, df = 1, P < 0.05) had a significant repellent effect on male adults, and its repellent rate was 56.67%.

[0075] Example 7 Determining the Repellent Effect of the Volatile Mixture with Behavioral Responses to Female Adults of Spodoptera litura

[0076] The above examples determined three soybean volatile components with behavioral responses to female adults of Spodoptera litura. In this example, through artificial preparation, the repellent effect of the mixture of these three components on female adults of Spodoptera litura was studied.

[0077] Take nonanal solution and hexanal solution with a concentration of 10 μg / μL each to prepare mixture 1 with a volume ratio of nonanal to hexanal of 1:1 for later use;

[0078] Take nonanal solution and leaf alcohol solution with a concentration of 10 μg / μL each to prepare mixture 2 with a volume ratio of nonanal to leaf alcohol of 1:1 for later use;

[0079] Take hexanal solution and leaf alcohol solution with a concentration of 10 μg / μL each to prepare mixture 3 with a volume ratio of hexanal to leaf alcohol of 1:1 for later use;

[0080] Take nonanal solution, leaf alcohol solution, and hexanal solution with a concentration of 10 μg / μL each to prepare mixture 4 with a volume ratio of nonanal, leaf alcohol, and hexanal of 1:1:1 for later use.

[0081] The behavioral response device used in this example was as Figure 7 shown. Connect the device air sampler 1, 6 → flowmeter 2, 7 → drying tower 3 filled with activated carbon, 8 → humidifying device 4, 9 → odor bottle 5, 10 → Y-shaped test tube 11 in sequence with a connecting tube. The air flow on both sides was maintained at 500 mL / min. Before the experiment, all instruments should be cleaned 3 times with distilled water and alcohol respectively to reduce errors. Female adults of Spodoptera litura aged 2 - 3 days were selected for behavioral test determination.

[0082] In this embodiment, a Y-shaped test tube was used to verify the attracting effect of a mixture of 4 volatiles on female adult Spodoptera litura. The specific process was as follows: Taking the outlet of an air sampler as the air flow source, the air flow rates of both arms of the olfactometer were set to 500 mL / min, and ventilation was carried out in advance for 5 min to ensure that there were no other impurity gases in the Y-shaped tube. Using mixtures 1, 2, 3, and 4 as the experimental groups and liquid paraffin as the control group, the preparation of the volatile standards was the same as that for the above-mentioned electroantennogram response measurement. During the test, 10 μL of the mixture or liquid paraffin was respectively dropped onto a rectangular filter paper strip of 3 cm × 0.5 cm, placed into an odor bottle to form an odor source, and connected to the two side arms of the Y-shaped tube through a connecting tube. A single adult was released from the main arm opening of the Y-shaped tube, and the reaction time was 5 min. When the insect entered more than 1 / 2 of the side arm and stayed for at least 30 s, it was recorded as the selection result. If no selection was made within 5 min, the behavioral determination of this insect was stopped and not recorded. There were 30 female adult Spodoptera litura in each group, and each insect under the treatment of the same concentration compound was only tested once. The indoor environmental temperature was 25 ± 2 °C, and the relative humidity was 70 ± 10%.

[0083] Formulas for the attraction rate and repellency rate of female adult Spodoptera litura to the odor source: Test selection rate = number of insects selecting the test odor source / total number of insects successfully selecting; Control selection rate = number of insects selecting the control odor source / total number of insects successfully selecting. The significant differences in the Y-shaped tube behavioral response data were compared through a chi-square test (χ 2 ). The statistical significance was set at P < 0.05.

[0084] Figure 10 This is the behavioral selection result of female adult Spodoptera litura for a mixture of 4 volatiles. The experimental results showed that mixture 4 (10 μg / μL nonanal: 10 μg / μL leaf alcohol:: 10 μg / μL hexanal = 1:1:1, χ 2 = 13.520, df = 1, P < 0.001) had a significant repellent effect on female adults, and its repellency rate was 63.33%.

[0085] The above description of the embodiment is for the convenience of those of ordinary skill in the art to understand and use the invention. Those familiar with the technology in this field can obviously make various modifications to these embodiments easily and apply the general principles described here to other embodiments without creative labor. Therefore, the present invention is not limited to the above embodiments, and the improvements and modifications made by those skilled in the art according to the disclosure of the present invention without departing from the scope of the present invention should be within the protection scope of the present invention.

Claims

1. The use of trans-2-hexenal in preventing and controlling the adults of Spodoptera litura, characterized in that: The trans-2-hexenal has a repellent effect on male adults of Spodoptera litura.

2. The use of a nonanal+leaf alcohol+hexanal composition in controlling the adult Spodoptera litura, characterized in that: The combination of nonanal+leaf alcohol+hexanal has a repellent effect on female adults of Spodoptera litura.

3. A male adult Spodoptera litura repellent, characterized in that: The solute component of the Spodoptera litura male adult repellent is trans-2-hexenal.

4. A female adult Spodoptera litura repellent, characterized in that: The solute component of the female adult Spodoptera litura repellent is a composition of nonanal+leaf alcohol+hexanal.

5. The repellent according to claim 3 or 4, characterized in that: The concentration of the solute in the repellent is 1 to 100 μg / μL.

6. The repellent according to claim 3 or 4, characterized in that: The solvent of the repellent is at least any one of dichloromethane, paraffin oil or n-hexane.

7. A method for extracting volatile substances having repellent activity against adults of Spodoptera litura from soybeans, characterized in that: The method is based on a device for collecting soybean volatiles, the structure of which includes: An atmospheric sampler (1), an activated carbon filter bottle (2), a humidifying conical bottle (3), a first flow meter (4), a glass cover (5), an adsorption tube (6), and a second flow meter (7); the above structures are connected in sequence through pipelines, and the second flow meter (7) is connected to the atmospheric sampler (1), and the entire device forms a circulating and closed passage; plants are placed in the glass cover (5); and Porapak-Q adsorbent is placed in the adsorption tube (6); The extraction method is based on the above-mentioned collection device, and the steps include: (1) Preparation before the test: rinse the glass cover and dry it, seal the mouth of the glass cover, and continue to let in air; wrap the top of the soybean planting pot with tin foil and expose the soybean plant to eliminate the influence of volatile substances in the soil and pot; (2) Volatile collection: Turn on the atmospheric sampler and control the air flow rates at the outlet and the inlet to be the same. At this time, the Porapak-Q adsorbent in the adsorption tube continues to adsorb the volatiles of the soybean plants. After continuous extraction for a period of time, the Porapak-Q adsorbent adsorbed with the volatiles is eluted with a solvent. The resulting eluate contains volatile substances that have repellent activity against the adult Spodoptera litura.

8. The method for extracting volatile substances having repellent activity against adults of Spodoptera litura from soybeans according to claim 7, characterized in that: The washing of the glass cover in step (1) refers to: washing the glass cover with distilled water, anhydrous alcohol and acetone in sequence; the drying refers to: baking at 180-220° C. for 1-3 hours; and the flow rate of the continuous introduction of air is 150-400 mL / min.

9. The method for extracting volatile substances having repellent activity against adults of Spodoptera litura from soybeans according to claim 7, characterized in that: The air flow rates at the air outlet and the air inlet in step (2) are both 480-520 mL / min.

10. The method for extracting volatile substances having repellent activity against adults of Spodoptera litura from soybeans according to claim 7, characterized in that: The continuous pumping time in step (2) is 10 to 15 hours; the eluent / solvent used for elution is any one of dichloromethane, paraffin oil or n-hexane.

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