Use of gamma-terpinene in preparing aphidius gifuensis attractant and screening method thereof

Abstract

The application discloses application of gamma-terpinene in preparation of an aphidius gifuensis attractant and a screening method thereof. The gamma-terpinene is prepared into a working solution with n-hexane and can affect the behavior selection of the aphidius gifuensis under the volatile odor background of wheat plants. The screening method takes 9 kinds of chemical sensing proteins and 15 kinds of odor binding proteins of the aphidius gifuensis as molecular targets, carries out molecular docking scoring on candidate volatiles of cnidium monnieri, and screens gamma-terpinene and beta-caryophyllene; 5 kinds of representative proteins are selected for binding conformation analysis, and a Y-type olfactometer is used for behavior verification, so that the gamma-terpinene is determined as a plant source attractant active ingredient of the aphidius gifuensis. The application can improve the pertinence of screening of the plant source attractant active ingredient of the aphidius gifuensis.

Description

Technical Field

[0001] This invention belongs to the field of green pest control technology in agriculture, specifically involving the application of γ-terpinene in the preparation of attractants for tobacco aphid parasitic wasps and its screening method. Background Technology

[0002] Wheat aphid ( Sitobion miscanthi Wheat aphids (Takahashi) are a significant pest in wheat production. They negatively impact wheat yield and quality by sucking food, transmitting plant viruses, and disrupting normal wheat growth and development. Long-term reliance on chemical pesticides for aphid control can easily lead to increased pesticide resistance and may adversely affect natural enemies such as the tobacco aphid wasp. Therefore, fully utilizing parasitic natural enemies to regulate wheat aphid populations is one of the important technical approaches to achieving green control of wheat aphids.

[0003] Tobacco aphid parasitic wasp ( Aphidius gifuensis *Aphidius cibarius* is an important parasitic natural enemy of wheat aphids, capable of locating suitable host habitats by sensing volatile chemical signals released by the host, host plant, and its habitat. Plant volatile components can participate in the long-distance orientation and short-distance host localization of *Aphidius cibarius*, and regulating the behavioral responses of *Aphidius cibarius* through plant volatile components has the potential value of reducing chemical pesticide use and improving the utilization efficiency of natural enemy insects.

[0004] Cnidium monnieri ( Cnidium monnieri (L.) Cusson can serve as a functional plant around wheat fields. Observations have shown that planting *Cnidium monnieri* around wheat fields helps increase the number of *Aphidius spp.* and enhance aphid parasitism. However, *Cnidium monnieri* is not a host plant for aphids, and *Aphidius spp.* is not the dominant parasitic wasp of the main aphids on *Cnidium monnieri*. Therefore, the attraction of *Cnidium monnieri* to *Aphidius spp.* may be related to the volatile components released by *Cnidium monnieri* itself.

[0005] Cnidium monnieri contains a wide variety of volatile components. Conducting insect olfactory behavior experiments on each component individually would be labor-intensive, inefficient, and lack specificity. Insect odor-binding proteins and chemoreceptor proteins are involved in the perception and recognition of exogenous volatile compounds. By constructing three-dimensional structural models of various chemoreceptor proteins and odor-binding proteins from the tobacco aphid wasp, molecular docking scoring of candidate volatiles from Cnidium monnieri was performed. Then, the candidate volatiles with the best scores were selected for olfactory behavior verification, which can improve the specificity of screening for plant-derived attractant active ingredients.

[0006] Therefore, it is necessary to establish a screening method that combines multi-target molecule docking scoring, representative protein binding conformation analysis, Y-type olfactometer behavior verification, and wheat plant volatile odor background verification to screen plant-derived attractant active ingredients from the volatile components of Cnidium monnieri. Summary of the Invention

[0007] To address the issues of unclear plant-derived attractant active ingredients for the tobacco aphid wasp in existing technologies and the low efficiency of conducting individual behavioral tests for screening, this paper provides the application of γ-terpinene in the preparation of attractants for the tobacco aphid wasp, and provides a method for screening plant-derived attractant active ingredients for the tobacco aphid wasp from the volatile components of Cnidium monnieri.

[0008] To address the aforementioned technical problems, the present invention provides the following technical solutions.

[0009] In a first aspect, the present invention provides the use of γ-terpinene in the preparation of an attractant for attracting the tobacco aphid wasp.

[0010] Furthermore, the attractant is used to attract tobacco aphid parasitic wasps in an environment containing the volatile odor of wheat plants; the background of the volatile odor of wheat plants refers to the odor environment containing the volatile components of wheat plants formed after the airflow passes through the wheat plant material.

[0011] Furthermore, the γ-terpinene is used at a working solution concentration of 0.01 to 10.0 μL / mL.

[0012] Furthermore, the working solution concentration of the γ-terpinene is 0.01 μL / mL, 0.1 μL / mL, 1.0 μL / mL, or 10.0 μL / mL.

[0013] Furthermore, the working solution concentration refers to the volume of γ-terpinene contained in each milliliter of γ-terpinene working solution. The γ-terpinene can be prepared into a working solution of the corresponding concentration using n-hexane.

[0014] Furthermore, the *Aphidius cirrhosa* mentioned is a female adult *Aphidius cirrhosa*.

[0015] Secondly, this invention provides a method for screening plant-derived attractant active ingredients of the tobacco aphid parasitic wasp from the volatile components of Cnidium monnieri, comprising the following steps: S1. Obtain the amino acid sequences of the chemosensory proteins AgifCSP1-AgifCSP9 and the odor binding proteins AgifOBP1-AgifOBP9, AgifOBP11-AgifOBP15 and AgifOBP17 of the tobacco aphid wasp, and construct the three-dimensional structural models of the chemosensory proteins and odor binding proteins. S2. Obtain the volatile components of Cnidium monnieri and establish a candidate volatiles library; S3. The candidate volatiles are molecularly docked with the chemosensory protein and the odor binding protein, respectively. The candidate volatiles are screened according to the binding energy obtained from the molecular docking to obtain the initial volatiles. S4. The attraction effect of the primary screening volatiles on the tobacco aphid wasp was determined using a Y-type olfactometer, and the influence of the primary screening volatiles on the behavioral selection of the tobacco aphid wasp was determined against the background of volatile odors from wheat plants. Based on the measurement results, γ-terpinene was determined to be the plant-derived active attractant of the tobacco aphid wasp.

[0016] Furthermore, the amino acid sequences of the chemosensory proteins AgifCSP1–AgifCSP9 and the odor-binding proteins AgifOBP1–AgifOBP9, AgifOBP11–AgifOBP15 and AgifOBP17 were obtained from the NCBI database and publicly available literature. The amino acid sequences of AgifOBP13 and AgifOBP14 are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively. The sequence identifiers and sources of each protein are shown in Table 1.

[0017] Further, in step S1, the amino acid sequences of the chemosensory protein and the odor-binding protein are used to predict the N-terminal signal peptide; for amino acid sequences predicted to contain an N-terminal signal peptide, the N-terminal signal peptide sequence is removed, and a three-dimensional protein structure model is constructed using AlphaFold2; for amino acid sequences for which no N-terminal signal peptide is predicted, a three-dimensional protein structure model is directly constructed using the corresponding amino acid sequence; in step S3, the molecular docking is performed using AutoDock Vina.

[0018] Further, in step S2, the candidate volatiles library is established based on publicly reported volatile components of Cnidium monnieri; the candidate volatiles include styrene, α-pinene, β-myrcene, D-limonene, trans-β-ocimene, γ-terpinene, β-caryophyllene, and cis-β-farnesene.

[0019] Further, in step S3, AutoDock Vina is used to perform molecular docking between the candidate volatiles and the 9 chemosensory proteins and 15 odor-binding proteins, respectively. γ-terpinene and β-caryophyllene are selected as the initial volatiles based on the molecular docking score.

[0020] Furthermore, each candidate volatile substance was independently docked with each odor-binding protein and chemoreceptor protein three times. The lowest binding energy of each docking was recorded, and the average value of the lowest binding energy was calculated. The average value was used as the docking score of the corresponding candidate volatile substance with the protein.

[0021] Furthermore, based on the molecular docking scores of γ-terpinene and β-caryophyllene with the aforementioned 9 chemosensory proteins and 15 odor-binding proteins, AgifCSP2, AgifCSP5, AgifOBP6, AgifOBP11, and AgifOBP15 were selected as representative proteins, and the binding conformation analysis of the protein-ligand complexes formed by the primary screening volatiles with each representative protein was performed.

[0022] Furthermore, the Y-type olfactometer test in step S4 was conducted under the conditions of a temperature of 25±1℃, relative humidity of 70±5%, airflow speed of 200 mL / min, and a dark room. The tested parasitic wasps were adult parasitic wasps that had emerged within 48 hours. They were starved for 2 hours before the test, and 5 parasitic wasps were released each time. A total of 25 individuals who made effective selections were obtained for each sex and each treatment.

[0023] Compared with the prior art, the present invention has at least the following beneficial effects.

[0024] (1) The present invention found through olfactory behavior experiments that γ-terpinene can induce the directional selection behavior of the tobacco aphid parasitic wasp and can be used as a plant-derived attractant active ingredient of the tobacco aphid parasitic wasp to prepare tobacco aphid parasitic wasp attractant.

[0025] (2) The present invention further found that γ-terpinene can still affect the behavioral selection of the tobacco aphid parasitic wasp in the background of volatile odor of wheat plants, indicating that γ-terpinene has potential value for the regulation of the behavior of the tobacco aphid parasitic wasp in wheat habitat.

[0026] (3) The γ-terpinene described in this invention has a regulatory effect on the behavioral selection of both female and male *Aphidius chinensis*, and its behavioral response shows certain concentration and sex differences. Female adult *Aphidius chinensis* undertake host search and parasitism behaviors; therefore, the influence of γ-terpinene on the behavior of female adult *Aphidius chinensis* is beneficial to improving its application value as an attractant active ingredient.

[0027] (4) This invention uses 9 chemosensory proteins and 15 odor-binding proteins of the tobacco aphid wasp as molecular screening targets to perform overall molecular docking scoring on the candidate volatiles of Cnidium monnieri. Based on the scoring results, AgifCSP2, AgifCSP5, AgifOBP6, AgifOBP11 and AgifOBP15 are selected as representative proteins for binding conformation analysis. Combined with Y-type olfactometer behavior verification and wheat plant volatile odor background verification, this invention can improve the targeting of the plant-derived attractant active ingredients of the tobacco aphid wasp and reduce the workload of conducting olfactory behavior tests on all candidate volatiles one by one. Attached Figure Description

[0028] Figure 1The binding energy heatmaps are obtained by molecular docking of candidate volatile compounds of Cnidium monnieri with 9 chemosensory proteins and 15 odor-binding proteins of the aphid parasitic wasp. The color scale represents the average value of the lowest binding energy obtained by molecular docking, and the unit is kcal / mol.

[0029] Figure 2 This diagram shows the molecular docking results of γ-terpinene with representative chemoreceptor proteins and odor-binding proteins of the tobacco aphid wasp. In the diagram, A, B, and E are the three-dimensional binding conformations of γ-terpinene with AgifCSP2, AgifCSP5, and AgifOBP6, respectively, and C, D, and F are the two-dimensional interaction diagrams of γ-terpinene with AgifCSP2, AgifCSP5, and AgifOBP6, respectively.

[0030] Figure 3 The diagram shows the molecular docking results of γ-terpinene with representative chemosensory and odor-binding proteins of the tobacco aphid wasp. In the diagram, A and B are the three-dimensional binding conformations of γ-terpinene with AgifOBP11 and AgifOBP15, respectively, and C and D are the two-dimensional interaction diagrams of γ-terpinene with AgifOBP11 and AgifOBP15, respectively.

[0031] Figure 4 The diagram shows the molecular docking results of β-caryophyllene with representative chemosensory and odor-binding proteins of the tobacco aphid wasp. In the diagram, A, B, and E are the three-dimensional binding conformations of β-caryophyllene with AgifCSP2, AgifCSP5, and AgifOBP6, respectively, and C, D, and F are the two-dimensional interaction diagrams of β-caryophyllene with AgifCSP2, AgifCSP5, and AgifOBP6, respectively.

[0032] Figure 5 The diagram shows the molecular docking results of β-caryophyllene with representative chemosensory and odor-binding proteins of the tobacco aphid wasp. In the diagram, A and B are the three-dimensional binding conformations of β-caryophyllene with AgifOBP11 and AgifOBP15, respectively, and C and D are the two-dimensional interaction diagrams of β-caryophyllene with AgifOBP11 and AgifOBP15, respectively.

[0033] Figure 6 A physical diagram of the experimental setup for selecting the behavior of the Y-type olfactory sensor.

[0034] Figure 7The graph shows the behavioral selection results of the aphid wasp on the scents of Cnidium monnieri, wheat, and a combination of Cnidium monnieri and wheat. In the graph, A represents the behavioral selection results of the aphid wasp on the scent of Cnidium monnieri and air; B represents the behavioral selection results of the aphid wasp on the combination of Cnidium monnieri and wheat and wheat scents; C represents the behavioral selection results of the aphid wasp on the scents of Cnidium monnieri and wheat and wheat scents; and D represents the behavioral selection results of the aphid wasp on the combination of Cnidium monnieri and wheat and the scent of Cnidium monnieri. ns indicates no significant difference between the treatment group and the control group; * indicates a significant difference between the treatment group and the control group; and *** indicates an extremely significant difference between the treatment group and the control group. The same applies below.

[0035] Figure 8 The figure shows the effects of different concentrations of γ-terpinene on the behavioral selection of *Aphidius nicotine*, where A represents the effects of different concentrations of γ-terpinene on the behavioral selection of female *Aphidius nicotine*, and B represents the effects of different concentrations of γ-terpinene on the behavioral selection of male *Aphidius nicotine*.

[0036] Figure 9 The figure shows the effects of different concentrations of β-caryophyllene on the behavioral selection of *Aphidius nicotineus*. In the figure, A represents the effects of different concentrations of β-caryophyllene on the behavioral selection of female *Aphidius nicotineus*, and B represents the effects of different concentrations of β-caryophyllene on the behavioral selection of male *Aphidius nicotineus*.

[0037] Figure 10 The figure shows the effect of γ-terpinene on the behavioral selection of *Aphidius chinensis* under the background of volatile odor of wheat plants. In the figure, A shows the behavioral selection results of female *Aphidius chinensis* under the treatment of wheat odor combined with γ-terpinene and wheat odor combined with n-hexane; B shows the behavioral selection results of female *Aphidius chinensis* under the treatment of wheat odor combined with γ-terpinene and γ-terpinene alone; C shows the behavioral selection results of male *Aphidius chinensis* under the treatment of wheat odor combined with γ-terpinene and wheat odor combined with n-hexane; and D shows the behavioral selection results of male *Aphidius chinensis* under the treatment of wheat odor combined with γ-terpinene and γ-terpinene alone.

[0038] Figure 11 The figure shows the effect of β-caryophyllene on the behavioral selection of *Aphidius chinensis* under the background of volatile odor of wheat plants. In the figure, A shows the behavioral selection results of female *Aphidius chinensis* under the treatment of wheat odor combined with β-caryophyllene and wheat odor combined with n-hexane; B shows the behavioral selection results of female *Aphidius chinensis* under the treatment of wheat odor combined with β-caryophyllene and β-caryophyllene alone; C shows the behavioral selection results of male *Aphidius chinensis* under the treatment of wheat odor combined with β-caryophyllene and wheat odor combined with n-hexane; and D shows the behavioral selection results of male *Aphidius chinensis* under the treatment of wheat odor combined with β-caryophyllene and β-caryophyllene alone. Detailed Implementation

[0039] The present invention will be further described below with reference to specific embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of protection of the present invention.

[0040] Unless otherwise stated, the reagents, instruments and materials used in the following examples are all available through conventional commercial channels, and the experimental methods used are all conventional methods in the art.

[0041] The purity of γ-terpinene standard was 95%, purchased from Shanghai Dibai Biotechnology Co., Ltd. The purity of β-caryophyllene standard was 80%, purchased from Shanghai Maclean Biochemical Technology Co., Ltd. Hexane was of chromatographic grade and purchased from Tianjin Kaitong Chemical Reagent Co., Ltd.

[0042] The tested *Aphidius spp.* wasp was obtained from Yunnan Green Biotechnology Co., Ltd., and was continuously reared under conditions of 25±1℃, relative humidity 70–75%, and a photoperiod of 8 h light / 16 h darkness. Adult *Aphidius spp.* wasp were fed a 10% honey solution after emergence.

[0043] The wheat variety used in the experiment was Jimai 22, which was used when it reached the 3-leaf stage; the Cnidium monnieri material used in the experiment was obtained from the experimental field of Shandong Agricultural University in Tai'an City, Shandong Province, and was used when it reached the flowering stage.

[0044] Example 1: Molecular docking of candidate volatiles from Cnidium monnieri with odor-related proteins from the tobacco aphid wasp 1.1 Establishment of the candidate volatile matter library A candidate volatiles library was established based on the publicly available information on the volatile components of Cnidium monnieri, compiled by Jiang et al., using the following literature: Jiang X, Zhao L, Segers A, Chang CY, Zhang XR, Ju Q, Ge F. Herbivore-induced plant volatiles (HIPVs) from companion plant could enhance predator recruitment and biocontrol of cereal aphids. Entomologia Generalis, 2025, 45(4): 1067–1077. DOI: 10.1127 / entomologia / 3224. The candidate volatiles included styrene, α-pinene, β-myrcene, D-limonene, trans-β-ocimene, γ-terpinene, β-caryophyllene, and cis-β-farnesene.

[0045] The three-dimensional structures of each candidate volatile were downloaded from the PubChem database. The candidate volatiles were then subjected to hydrogenation and energy minimization using Open Babel and converted to PDBQT format.

[0046] 1.2 Construction of three-dimensional protein structure models The amino acid sequences of the chemosensory proteins AgifCSP1–AgifCSP9, as well as the odor-binding proteins AgifOBP1–AgifOBP9, AgifOBP11–AgifOBP15, and AgifOBP17, were obtained from the NCBI database and public literature. The sequence identifiers and sources of each protein are shown in Table 1.

[0047] The amino acid sequences of AgifOBP13 and AgifOBP14 are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively, and are disclosed in the supplementary materials of the following literature: Jiang X, Qin Y, Jiang J, Xu Y, Francis F, Fan J, Chen J. Spatial Expression Analysis of Odorant Binding Proteins in Both Sexes of the Aphid Parasitoid Aphidius gifuensis and Their LigandBinding Properties. Frontiers in Physiology, 2022, 13:877133.

[0048] Table 1. Sequence identifiers and sources of chemoreceptor and odor-binding proteins from the tobacco aphid wasp.

[0049] AgifOBP13, SEQ ID NO: 1: MDKLIGLSLFFTLVSSSAIMEDLAIVRICNATDSVDISILNDYMLNHDFHTLENHQLRQLSCFLLCIYSEYNWMDHHGSFKIHNIKSWMHRAKLPTDHIEILLKRCITSELTDPCTRARHFTECFWSNHQGILNANHRHTLHSIIRKKDTE AgifOBP14, SEQ ID NO: 2: MQTKADIRRECRKQTGVAWDPLSKFKNGDFNENDPKLKCYLKCFMQKYGIFGDDSIYIDRVLRYLPYSMQKTSKNTLEKCNLIPSTDSCDKAFQLLKCYFKSQPEVIFLKLLYYFTV The amino acid sequences of the chemoreceptor and odor-binding proteins were used to predict N-terminal signal peptides. For amino acid sequences predicted to contain N-terminal signal peptides, the N-terminal signal peptide sequence was removed, and a three-dimensional protein structure model was constructed using AlphaFold2. For amino acid sequences where no N-terminal signal peptide was predicted, a three-dimensional protein structure model was directly constructed using the corresponding amino acid sequence. Three-dimensional structure models with high confidence were selected, checked and preprocessed using PyMOL, and saved in PDB format. The protein receptors were hydrogenated and charged using AutoDockTools, and the processed protein receptors were converted to PDBQT format.

[0050] 1.3 Molecular docking and determination of volatiles in the initial screening The docking center was determined based on the binding pocket of each protein, and a docking cassette was set to cover the protein binding cavity. Batch molecular docking was performed using AutoDock Vina. Unless otherwise specified, the other parameters were set to the software defaults. Styrene, α-pinene, β-myrcene, D-limonene, trans-β-ocimene, γ-terpinene, β-caryophyllene, and cis-β-farnesene were molecularly docked with AgifCSP1~AgifCSP9, AgifOBP1~AgifOBP9, AgifOBP11~AgifOBP15, and AgifOBP17, respectively.

[0051] Each candidate volatile was independently docked with each chemosensory protein or odor-binding protein three times. The lowest binding energy in the conformation obtained by each docking was recorded, and the average of the three lowest binding energies was calculated. The average value was used as the docking score of the corresponding candidate volatile and protein.

[0052] The molecular docking scores of each candidate volatile with 9 chemoreceptor proteins and 15 odor-binding proteins are as follows: Figure 1 As shown in the figure. The candidate volatiles were ranked based on their molecular docking scores with the 24 proteins. The results showed that γ-terpinene and β-caryophyllene had relatively low binding energies to various chemoreceptor and odor-binding proteins of the tobacco aphid wasp. Therefore, γ-terpinene and β-caryophyllene were selected as the initial screening volatiles for subsequent visualization analysis of representative protein binding conformations and verification of the olfactory behavior of the tobacco aphid wasp.

[0053] 1.4 Conformational analysis of the binding of representative proteins to primary screening volatiles Based on the molecular docking scores of γ-terpinene and β-caryophyllene with the above 24 proteins, the chemosensory proteins AgifCSP2 and AgifCSP5, as well as the odor binding proteins AgifOBP6, AgifOBP11 and AgifOBP15, were selected as representative proteins. The binding conformation of the protein-ligand complexes formed by the representative proteins with γ-terpinene and β-caryophyllene was visualized and analyzed.

[0054] PyMOL was used to analyze the three-dimensional binding sites of γ-terpinene and β-caryophyllene in the binding cavities of representative proteins. Discovery Studio was used to analyze the hydrophobic interactions, hydrogen bonding interactions, van der Waals interactions, and related amino acid residues between proteins and ligands.

[0055] The three-dimensional binding conformations and two-dimensional interaction results of γ-terpinene with AgifCSP2, AgifCSP5, AgifOBP6, AgifOBP11 and AgifOBP15 are as follows: Figure 2 and Figure 3 As shown; the three-dimensional binding conformations and two-dimensional interaction results of β-caryophyllene with AgifCSP2, AgifCSP5, AgifOBP6, AgifOBP11 and AgifOBP15 are as follows. Figure 4 and Figure 5 As shown.

[0056] The results showed that both γ-terpinene and β-caryophyllene could enter the binding cavity of the representative protein and form various non-covalent interactions with the amino acid residues within the binding cavity. The above binding conformation analysis results indicate that γ-terpinene and β-caryophyllene can form protein-ligand complexes with the representative protein, providing a screening basis for subsequent verification of the olfactory behavior of the tobacco aphid wasp.

[0057] Example 2: Effects of wheat, Cnidium monnieri and their compound odors on the behavioral selection of the tobacco aphid parasitic wasp. The effects of wheat odor, Cnidium monnieri odor, and Cnidium monnieri-wheat compound odor on the behavioral selection of the tobacco aphid parasitic wasp were determined using a Y-type olfactometer.

[0058] Set up the following test treatment: (1) Cnidium monnieri vs. air; (2) The aroma of Cnidium monnieri + wheat compound vs. wheat; (3) Cnidium monnieri vs. wheat; (4) The aroma of Cnidium monnieri + wheat compound vs. Cnidium monnieri.

[0059] 10 g of wheat was used in the wheat-containing scent source, 10 g of Cnidium monnieri was used in the Cnidium monnieri-containing scent source, and 10 g of Cnidium monnieri and 10 g of wheat were used in the Cnidium monnieri and wheat compound scent source, respectively. No plant material was placed in the air control scent source.

[0060] The tested aphid wasps were adults within 48 hours of emergence, and were starved for 2 hours before the experiment. The experimental conditions were: temperature 25±1℃, relative humidity 70±5%, dark room conditions, and airflow rate of 200 mL / min.

[0061] Five aphid wasps were released into the main arm of the Y-shaped olfactory instrument each time, and the observation time was 5 minutes. Aphid wasps that entered any selection arm for more than 5 cm and stayed for more than 10 seconds were recorded as having made a selection; those that did not meet the selection criteria within the specified time were recorded as having no response. A total of 25 individuals of each sex and each treatment made valid selections were obtained.

[0062] The number of *Aphidius nicotineus* selected in the treatment arm, the control arm, and the number of unresponsive individuals were recorded separately. The chi-square test was used to compare the number of *Aphidius nicotineus* selected in the treatment odor source and the control odor source. Unresponsive individuals were not included in the significance analysis of the number of selected individuals in the treatment group and the control group.

[0063] See results Figure 7 The results showed that female *Aphidius nicotineus* exhibited selective behavior toward the scent of *Cnidium monnieri* and the combined scent of *Cnidium monnieri* and wheat, while the response of male *Aphidius nicotineus* was relatively weak, indicating that the volatile components released by *Cnidium monnieri* can influence the behavioral selection of *Aphidius nicotineus*.

[0064] Example 3: The attraction effect of γ-terpinene on the tobacco aphid wasp Working solutions of γ-terpinene were prepared using n-hexane at concentrations of 10.0 μL / mL, 1.0 μL / mL, 0.1 μL / mL, and 0.01 μL / mL, respectively.

[0065] 10 μL of the γ-terpinene working solution was dropped onto a 1 cm × 2 cm filter paper, and the carrier was placed in the treatment odor source bottle. An equal volume of n-hexane was dropped onto the same carrier, and the carrier was placed in the control odor source bottle. Clean air flowed through the treatment and control odor source bottles respectively, and then entered the corresponding selection arm of the Y-type olfactory instrument.

[0066] The tested *Aphidius citrinum* wasps were adults that had emerged within 48 hours of their emergence and were starved for 2 hours before the experiment. The experimental conditions were: temperature 25±1℃, relative humidity 70±5%, dark chamber, and airflow rate 200 mL / min. Five *Aphidius citrinum* wasps were released each time, and the experiment was repeated for each sex and each concentration treatment until a total of 25 *Aphidius citrinum* wasps that made effective selections were obtained.

[0067] The number of female and male *Aphidius oryzae* individuals selected for the γ-terpinene treatment arm, the n-hexane control arm, and the number of unresponsive individuals were recorded and analyzed using the chi-square test.

[0068] The results showed that γ-terpinene exhibited concentration- and sex-specific behavioral selection effects on the tobacco aphid wasp; the specific significance of different concentration treatments is as follows: Figure 8 As shown.

[0069] The above results indicate that γ-terpinene can be used as a plant-derived attractant for the tobacco aphid wasp and can be used to prepare tobacco aphid wasp attractants.

[0070] Example 4: Effects of β-caryophyllene on the behavioral selection of the tobacco aphid parasitic wasp β-Caryophyllene was prepared into working solutions of 10.0 μL / mL, 1.0 μL / mL, 0.1 μL / mL and 0.01 μL / mL using n-hexane.

[0071] The treatment arm was set with β-caryophyllene working solution, and the control arm was set with an equal volume of n-hexane. The amount of working solution, the tested aphid parasitic wasps, the experimental conditions, the selection of judgment methods, and the statistical analysis methods were the same as in Example 3.

[0072] See results Figure 9 The results showed that, within the test concentration range of 0.01–10.0 μL / mL, there was no significant difference in the selection of β-caryophyllene-treated odor source and n-hexane control odor source between female and male aphid wasps.

[0073] Example 5: Effects of γ-terpinene on the behavioral selection of the tobacco aphid parasitic wasp under the background of volatile odors from wheat plants. To determine the effect of γ-terpinene on the behavioral selection of the tobacco aphid parasitic wasp against the background of volatile odors in wheat plants, the following experimental treatments were set up: (1) The combined odor of γ-terpinene and wheat scent, compared with the combined odor of n-hexane and wheat scent; (2) The combined odor of γ-terpinene and wheat odor, compared with the odor of γ-terpinene.

[0074] The working solution concentrations of γ-terpinene were 10.0 μL / mL, 1.0 μL / mL, 0.1 μL / mL, and 0.01 μL / mL, respectively. For the odor source treated with γ-terpinene, 10 μL of the corresponding concentration of γ-terpinene working solution was used; for the hexane control odor source, an equal volume of hexane was used; and for the odor source treated with wheat, 10 g of wheat was used.

[0075] The test wasps, experimental conditions, airflow speed, release quantity, selection criteria, and statistical analysis methods were the same as in Example 3.

[0076] The results showed that, against the backdrop of volatile odors from wheat plants, γ-terpinene exhibited concentration- and sex-specific behavioral selection effects on the tobacco aphid wasp, with some tested concentrations showing significant differences compared to the corresponding controls. Specific results are as follows: Figure 10 As shown.

[0077] The above results indicate that γ-terpinene can not only induce selective behavior in aphid parasitoids on its own, but also influence their behavioral selection in environments containing volatile wheat plant odors.

[0078] Example 6: Effects of β-caryophyllene on the behavioral selection of the tobacco aphid parasitic wasp under the background of volatile odors in wheat plants. Set up the following test treatment: (1) The combined odor of β-caryophyllene and wheat odor, compared with the combined odor of n-hexane and wheat odor; (2) The combined odor of β-caryophyllene and wheat odor, compared with the odor of β-caryophyllene.

[0079] The working solution concentrations of β-caryophyllene were 10.0 μL / mL, 1.0 μL / mL, 0.1 μL / mL, and 0.01 μL / mL, respectively. The working solution volume, the tested wheat, the tested tobacco aphid parasitic wasps, the experimental conditions, the selection and judgment methods, and the statistical analysis methods were the same as in Example 5.

[0080] See results Figure 11 The results showed that β-caryophyllene could influence the behavioral selection of the tobacco aphid wasp under certain test conditions against the background of volatile odors from wheat plants. Based on the combined results of molecular docking scores, representative protein binding conformation analysis, and olfactory behavior experiments, γ-terpinene was identified as the plant-derived attractant active ingredient of the tobacco aphid wasp.

Claims

1. Application of γ-terpinene in the preparation of attractants for attracting adult tobacco aphid parasitic wasps.

2. The application according to claim 1, characterized in that, The attractant is used to attract adult tobacco aphid wasps against a background of volatile odors from wheat plants.

3. The application according to claim 1, characterized in that, The γ-terpinene was prepared into a working solution using n-hexane as a solvent, wherein the volume concentration of γ-terpinene in the working solution was 0.01–10.0 μL / mL.

4. The application according to any one of claims 1 to 3, characterized in that, The adult tobacco aphid wasps mentioned are female adults.

5. A method for screening plant-derived attractant active ingredients of the tobacco aphid wasp from the volatile components of Cnidium monnieri, characterized in that, Includes the following steps: S1. Obtain the amino acid sequences of the chemosensory proteins AgifCSP1-AgifCSP9 and the odor-binding proteins AgifOBP1-AgifOBP9, AgifOBP11-AgifOBP15 and AgifOBP17 of the tobacco aphid wasp. The amino acid sequences of AgifOBP13 and AgifOBP14 are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively. Construct three-dimensional structural models of the chemosensory proteins and odor-binding proteins. S2. Obtain the volatile components of Cnidium monnieri and establish a candidate volatiles library; S3. The candidate volatiles are molecularly docked with the chemosensory protein and the odor binding protein, respectively. The candidate volatiles are screened according to the binding energy obtained from the molecular docking to obtain the initial volatiles. S4. The attraction effect of the primary screening volatiles on adult *Aphidius spp.* was determined using a Y-type olfactometer. The effect of the primary screening volatiles on the behavioral selection of adult *Aphidius spp.* was also determined against the background of volatile odors from wheat plants. Based on the measurement results, γ-terpinene was identified as the plant-derived active attractant of *Aphidius spp.* 6. The method according to claim 5, characterized in that, In step S2, the candidate volatiles include styrene, α-pinene, β-myrcene, D-limonene, trans-β-ocimene, γ-terpinene, β-caryophyllene, and cis-β-farnesene.

7. The method according to claim 5, characterized in that, In step S1, the amino acid sequences of the chemosensory protein and the odor-binding protein are used to predict the N-terminal signal peptide. For amino acid sequences predicted to contain an N-terminal signal peptide, the N-terminal signal peptide sequence is removed, and a three-dimensional protein structure model is constructed using AlphaFold2. For amino acid sequences for which no N-terminal signal peptide is predicted, a three-dimensional protein structure model is directly constructed using the corresponding amino acid sequence. In step S3, the molecular docking is performed using AutoDock Vina.

8. The method according to any one of claims 5 to 7, characterized in that, In step S3, γ-terpinene and β-caryophyllene were screened as primary volatiles based on the binding energy obtained from molecular docking, and AgifCSP2, AgifCSP5, AgifOBP6, AgifOBP11 and AgifOBP15 were selected as representative proteins. The binding conformations of the primary volatiles with each representative protein were analyzed.

9. The method according to claim 8, characterized in that, Working solutions of γ-terpinene and β-caryophyllene were prepared using n-hexane as a solvent, with volume concentrations of γ-terpinene or β-caryophyllene in the working solutions being 0.01 μL / mL, 0.1 μL / mL, 1.0 μL / mL, and 10.0 μL / mL, respectively. The tested parasitic wasps included female and male adults.

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