Tea wing bug aggregation pheromone as well as preparation method and application thereof

Abstract

The invention provides a tea wing bug aggregation pheromone. The tea wing bug aggregation pheromone takes methyl benzoylformate as an effective component. The invention further provides a preparation method and application of the tea wing bug aggregation pheromone. The tea wing bug aggregation pheromone has the advantages of being green, safe, long in validity period and free of negative effects on the yield and quality of fruit trees. The application frequency of chemical pesticides can be effectively reduced by using the tea wing bug aggregation pheromone, so that the pesticide application interval is prolonged, and the problems of pesticide residues and phytotoxicity of fruit trees are reduced.

Description

Technical Field

[0001] This application belongs to the field of agricultural pest control. Specifically, this application provides a tea-winged bug aggregation pheromone, its preparation method, and its application. Background Technology

[0002] The tea-winged bug (Halyomorpha halys) belongs to the family Pentatomidae in the order Hemiptera. It is also known as the wood stink bug or tea-winged bug. The tea-winged bug has a wide host range, infesting fruit trees such as apples, pears, peaches, cherries, apricots, crabapples, and hawthorns, as well as crops like soybeans, beans, and sugar beets. It also damages trees such as elms, smoke trees, and wolfberries. It sucks sap from branches, leaves, young shoots, flower buds, and fruits, with the most severe damage occurring to fruits. Damaged fruits become corky, resulting in deformed, bumpy fruits. Besides the direct damage caused by sucking sap, its most important function is the transmission of viruses during this process. For example, paulownia witches'-broom disease, a devastating disease of paulownia trees, is primarily spread by the tea-winged bug.

[0003] Currently, the main method for controlling the tea-winged bug is still the use of chemical pesticides. However, long-term use of chemical pesticides easily leads to pesticide resistance in the bugs, and the tea-winged bug exhibits a clear host migration phenomenon. Using chemical pesticides not only fails to achieve good control but also easily pollutes the environment and causes phytotoxicity. The use of chemical pesticides to control this pest is highly indiscriminate and inefficient. Finding green, safe, and effective control methods to reduce pesticide use is an urgent problem to be solved.

[0004] In northern my country, eggs laid by the tea-winged bug before mid-July can develop into adults that same year and mate to lay more eggs; however, eggs laid after mid-July cannot develop into sexually mature adults that year. Non-overwintering adults often appear in pairs, feeding on the same fruit, while overwintering adults seek suitable locations to hibernate, exhibiting a clear aggregation phenomenon. Preliminary investigations indicate that non-overwintering adults are mainly concentrated on the flowers of the smoke tree (Cotinus coggygria), while overwintering adults move to the leaves after the flowers wither in autumn and winter. This phenomenon suggests that the smoke tree, as an important host plant, may contain substances in its floral and leaf tissues that are of significant value to the feeding behavior and reproductive activities of adult bugs.

[0005] Insect aggregation pheromones are chemical substances produced by insects that induce aggregation behavior in both males and females of the same species. In most insects, aggregation pheromones are produced by males and affect both sexes. The main components of aggregation pheromones have been isolated and identified from various insects, primarily including those in the orders Coleoptera, Orthoptera, Hemiptera, Thysanoptera, and Blattodea.

[0006] Insect aggregation pheromones hold great promise for monitoring insect population dynamics and for green pest control. In monitoring insect population dynamics, aggregation pheromones can not only monitor the emergence and damage time of pests but also reflect the occurrence dynamics and distribution of pest populations, playing a crucial role in guiding pest control. In green pest control, aggregation pheromones offer advantages such as being environmentally friendly and pollution-free. By setting up aggregation pheromone traps in the field to capture pests, not only can pest population density be reduced, but the base population for the next generation can also be decreased. Therefore, how to provide an effective method for monitoring and controlling the tea-winged bug using aggregation pheromones in production is a technical problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0007] On the one hand, this application provides a tea-winged bug aggregation pheromone, characterized in that the tea-winged bug aggregation pheromone uses methyl benzoate as the active ingredient.

[0008] Furthermore, the aggregation pheromone of the tea-winged bug also contains n-hexane as a diluent.

[0009] Furthermore, the concentration of methyl benzoate in the aggregation pheromone of the tea-winged bug is 0.5 μg / μL-1 μg / μL.

[0010] On the other hand, this application provides a method for preparing the aggregation pheromone of the tea-winged bug, the method comprising diluting methyl benzoate with n-hexane.

[0011] On the other hand, this application provides the application of the tea-winged bug aggregation pheromone in the preparation of products for trapping tea-winged bugs.

[0012] Furthermore, the product for trapping the tea winged bug includes a slow-release bottle and a trap.

[0013] Furthermore, the sustained-release bottle includes a sustained-release carrier.

[0014] The slow-release carrier can be any type of carrier known in the art that can cooperate with methyl benzoate, including but not limited to one or more of silicone rubber, rubber, hollow fibers, microcapsules, polyethylene, and porous starch. Preferably, the slow-release carrier is polyethylene. The target insects are preferably overwintering adult females and / or non-overwintering adult males of the tea-winged bug.

[0015] On the other hand, this application provides a method for trapping overwintering adult female and male tea-winged bugs and / or non-overwintering adult male tea-winged bugs, wherein the method uses the aforementioned tea-winged bug aggregation pheromone.

[0016] On the other hand, this application provides a method for repelling non-overwintering female adult tea bugs, wherein the method uses the aforementioned tea bug aggregation pheromone.

[0017] On the other hand, this application provides a method for collecting the aggregation pheromone of the tea-winged bug. The method includes placing overwintering male adult tea-winged bugs in a gas collecting bottle, collecting the volatiles of the bugs with an adsorption column, and then eluting the adsorption column with n-hexane to obtain the aggregation pheromone of the tea-winged bug.

[0018] Furthermore, the overwintering male adults of the tea-winged bug were pre-reared: they were fed with smoke tree leaves for one month and placed in an incubator at a temperature of 18°C ​​and a photoperiod of 10L:14D to maintain their reproductive diapause state, so as to promote the synthesis and release of more aggregation pheromones, making them easier to detect.

[0019] Furthermore, the period for collecting volatile substances from the insect body was from 9:00 to 14:00, and the period for collecting gaseous volatile substances was 4 hours. During collection, the insect body was placed in an incubator with a temperature of 18°C ​​and a photoperiod of 10L:14D.

[0020] Compared with existing technologies, this invention has the following advantages and technical effects: The aggregation pheromone provided by this invention is specifically designed to control adult tea-winged bugs. Because adult tea-winged bugs exhibit significant host migration during the fruit tree's growth period, they not only harm fruit trees but also locust and elm trees. Chemical pesticide control is indiscriminate and inefficient. This invention, however, can concentrate and kill overwintering adults of the tea-winged bug, reducing the overwintering population in the current year and positively impacting the population size in the following year. Secondly, the indiscriminate and excessive use of chemical pesticides can lead to significant pesticide resistance in tea-winged bugs and adversely affect natural enemies in the field. The aggregation pheromone provided by this invention is a substance used by tea-winged bugs to transmit information within the population, thus preventing pesticide resistance. Furthermore, the attractant prepared using this pheromone can continuously interfere with the aggregation behavior of tea-winged bugs, reducing the insect population density in the field without affecting the number of other natural enemies. The aggregation pheromone provided by this invention has the advantages of being green and safe, having a long effective period, and having no negative impact on the yield and quality of fruit trees. Furthermore, the use of the aggregation pheromone in this invention reduces the frequency of chemical pesticide application, thereby lengthening the pesticide application interval and reducing pesticide residues and phytotoxicity problems in fruit trees. Attached Figure Description

[0021] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below.

[0022] Figure 1A The antennal potential response of overwintering male insects in Example 2 to 10 substances was measured, with each substance being 100 μg.

[0023] Figure 1B The antennal potential responses of overwintering female insects in Example 2 to 10 substances were measured, with each substance being 100 μg.

[0024] Figure 1C The antennal potential response of non-overwintering male insects in Example 2 to 10 substances was measured at a dosage of 100 μg.

[0025] Figure 1D The antennal potential response of non-overwintering male insects in Example 2 to 10 substances was measured at a dosage of 10 μg.

[0026] Figure 1E The antennal potential response of non-overwintering male insects to 10 substances in Example 2 was measured at a dosage of 1 μg.

[0027] Figure 1F The antennal potential response of the non-overwintering female insects in Example 2 to 10 substances was measured at a dosage of 100 μg.

[0028] Figure 1G The antennal potential response of the non-overwintering female insects in Example 2 to 10 substances was measured at a dosage of 10 μg.

[0029] Figure 1H The antennal potential response of the non-overwintering female insects in Example 2 to 10 substances was measured at a dosage of 1 μg.

[0030] Figure 2 In Example 2, a double-selection test was used to study the response of overwintering and non-overwintering male and female adults to methyl benzoate, with a dosage of 100 μg for both generations.

[0031] Figure 3 In Example 4, a trapping experiment was conducted to study the response of overwintering and non-overwintering adult males and females to methyl benzoate. The dosage for overwintering adults was 500 μg, and the dosage for non-overwintering adults was 1000 μg. Detailed Implementation

[0032] The following embodiments are provided to better understand the present invention, but are not limited thereto. These embodiments are for illustrative purposes only and do not limit the scope of protection of the present invention in any way.

[0033] Unless otherwise specified, the equipment and reagents used in each embodiment are all commercially available.

[0034] The test insects in this embodiment of the invention were collected from Fragrant Hills, Beijing. Adult tea-winged bugs that had gathered for overwintering were brought indoors to identify their sexes, and the volatile compounds were collected. After relieving reproductive diapause, non-overwintering adults were obtained through normal rearing conditions.

[0035] Example 1

[0036] A method for collecting aggregation pheromones from the tea-winged bug, utilizing a dynamic headspace method, specifically includes the following steps:

[0037] The adult tea-winged bugs used in the experiment need to be reared in advance: they are reared with smoke tree leaves for one month, and the overwintering adults are placed in an incubator with a temperature of 18℃ and a photoperiod of 10L:14D to ensure that they can maintain reproductive diapause; the non-overwintering adults are placed in an incubator with a temperature of 26℃ and a photoperiod of 16L:8D.

[0038] Overwintering and non-overwintering male adults of the tea-winged bug were starved for 24 hours and then placed in Erlenmeyer flasks. The flasks were connected to an atmospheric sampler using rubber tubing to collect volatiles. An empty Erlenmeyer flask (without any contents) was also connected to the sampler using rubber tubing to collect volatiles as a blank control. Air was extracted from the flasks using activated carbon at a rate of 300 mL / min for 4 hours. Volatiles collected in an adsorption tube filled with PoraPak Q (80-100 mesh, 200 mg adsorbent) were washed three times with n-hexane, and a total of 1.5 mL was collected for GC-MS identification. The GC-MS conditions were as follows: instrument model: Agilent 7890B / 7200; column: HP-5MS flexible quartz capillary column; injection port temperature: 250℃; temperature program: initial temperature 70℃, hold for 2 min, increase to 200℃ at 5℃ / min, hold for 5 min, then increase to 280℃ at 10℃ / min, hold for 10 min. The mass spectrometry conditions were: ionization source: EI, ion source temperature: 230℃, GC-MS transfer line temperature: 280℃, mass scan range: 40–500 amu, EI ionization energy: 70 eV. Volatile compounds were identified using the NIST mass spectrometry standard library. The results are shown in Table 1.

[0039] Table 1. GC-MS analysis of body surface components of overwintering male adults of the tea-winged bug.

[0040]

[0041]

[0042] Example 2

[0043] In conjunction with Example 1, ten compounds were selected for antennal potential response testing. Methyl benzoylformate, 4-ethyl-m-xylene, dodecanol, 3-phenylbutanol, butyl stearate, and n-tetane were derived from insect volatiles; trans-2-decenal was the alarm pheromone of the tea-winged bug; linalool oxide and linalool were derived from plant volatiles; and methyl (2E,4E,6Z)-decorienoic acid was the main component of the amber bug lure. Methyl (2E,4E,6Z)-decorienoic acid was purchased from Targetmol, and the others were purchased from McLean Biotech Co., Ltd.

[0044] Each compound was diluted with n-hexane to prepare solutions of 0.1, 1, and 10 μg / μL, and each solution was prepared fresh before use. 0.5 cm × 5 cm filter paper strips were prepared and folded into a "V" shape. 10 μL of each of the tested odors was added to the filter paper strip, which was then inserted into a Pasteur tube. During the experiment, the instrument was first turned on, and the metal and glass electrodes were installed. Two-thirds of the volume of 0.1 M KCl solution was poured into the glass electrode, and then the glass electrode was placed over the metal electrode. The airflow rate was adjusted to 10 mL / s, and the stimulation time was 2 s. Overwintering male and female adults of the tea-winged bug and non-overwintering male and female adults were selected as experimental subjects. The antennae were cut off along the base using surgical scissors, and the first segment of the flagellum was removed. Under a microscope, the reference electrode was connected to the base of the antennae, and the recording electrode was connected to the tip of the antennae. After successful connection, the instrument was observed. Once the baseline stabilized, the test odors were added and recorded. During testing, the end of the Pasteur tube was connected to the silicone tube, and the tip was placed in a small hole on the ventilation metal tube. Stimulation was performed by stepping on a foot pedal. An interval of at least 20 seconds was required between each stimulation to allow the antennae to recover. Hexane was used as a control and required two stimulations (first and last). The stimulation order of the 10 different odors was randomized, and different concentrations of the same odor were stimulated in ascending order of concentration. A 10×AC / DC headstage preamplifier was used to amplify the stimulation signal. Only one antenna was tested per adult insect. The results are shown in Figure 1. Although methyl benzoate only volatilizes specifically in overwintering male adults, it shows a significant antennal potential response in both overwintering and non-overwintering male and female adults.

[0045] Based on antennal electrophysiology, this study primarily verified the behavioral selection response of methyl benzoate to the tea-winged bug. The olfactory behavioral response of adult tea-winged bugs to volatile substances was measured using a Y-type olfactometer. 10 μL of the volatile substance to be tested was added to qualitative filter paper (50 mm × 10 mm), with 10 μL of n-hexane as the control. The air sampler was adjusted to a flow rate of 500 ml / min for both branches. Five minutes before the experiment, excess gas in both Erlenmeyer flasks was purged using the air sampler. During the test, one adult bug, starved for 12 hours, was placed at the end of the main branch. If the adult bug remained in either arm of the Y-tube for more than 1 minute, it was considered to have made a selection; if it remained in the main arm, it was considered to have no response. Each adult bug was tested only once, and the arms were swapped every 5 bugs tested to eliminate the influence of odor source location and light. Results are as follows: Figure 2As shown, methyl benzoate has a significant attraction effect on overwintering male and female adults. Non-overwintering male adults show a clear attraction to methyl benzoate, while non-overwintering female adults show a clear repulsion. This phenomenon may be because methyl benzoate promotes the aggregation of male and female adults for overwintering, increasing the overwintering survival rate; while females that have broken diapause (i.e., non-overwintering generation) need to complete mating to facilitate population reproduction, and therefore prefer to select males that have broken diapause and no longer volatilize methyl benzoate, thus exhibiting a clear repulsion to methyl benzoate.

[0046] Example 3

[0047] A method for preparing the aggregation pheromone of the tea-winged bug involves diluting a methyl benzoate standard with n-hexane to 0.5 μg / μL and 1 μg / μL, thereby obtaining the tea-winged bug aggregation pheromone inducer.

[0048] Example 4

[0049] The preparation of a slow-release bottle for trapping the tea winged bug includes the following steps: take the tea winged bug aggregation pheromone obtained in Example 3, and add 1000 μL of it using a polyethylene slow-release bottle as a slow-release carrier to obtain the tea winged bug aggregation pheromone slow-release bottle.

[0050] Four empty slow-release bottles were used. Two bottles contained the aggregation pheromone of the tea-winged bug, and the other two contained n-hexane as a control. The prepared slow-release bottles were placed inside the traps, and the four traps were placed at the four corners of a 1m × 1m × 1m rearing cage and secured. Slow-release bottles containing the same liquid were placed in diagonally opposite sections, while adjacent sections contained bottles containing different liquids. Overwintering and non-overwintering male and female adults were placed separately in the rearing cage. After 24 hours, the number of unselected adults in each trap and rearing cage was counted. The final results are as follows: Figure 3 As shown, the results are the same as in Example 2. Figure 2 The result.

Claims

1. A type of aggregation pheromone for the tea-winged bug, characterized in that, The aggregation pheromone of the tea-winged bug uses methyl benzoate as the active ingredient.

2. The aggregation pheromone of the tea-winged bug according to claim 1, wherein the aggregation pheromone further comprises n-hexane as a diluent; and the concentration of methyl benzoate in the aggregation pheromone is 0.5 μg / μL-1 μg / μL.

3. The method for preparing the aggregation pheromone of the tea-winged bug according to claim 1 or 2, characterized in that, The preparation method includes diluting methyl benzoate with n-hexane.

4. The application of the tea-winged bug aggregation pheromone according to claim 1 or 2 in the preparation of products for trapping tea-winged bugs.

5. The application according to claim 4, wherein the product for trapping the tea winged bug comprises a slow-release bottle and a trap; the slow-release bottle comprises a slow-release carrier.

6. A method for trapping overwintering adult female and male *Tetracentron sinense* and / or non-overwintering adult male *Tetracentron sinense*, characterized in that, The method uses the aggregation pheromone of the tea-winged bug according to claim 1 or 2 to trap overwintering female and male tea-winged bug adults and / or non-overwintering male tea-winged bug adults.

7. A method for repelling non-overwintering generation female adult tea-winged bugs, characterized in that, The method uses the aggregation pheromone of the tea-winged bug according to claim 1 or 2 to repel non-overwintering female tea-winged bug adults.

8. A method for collecting aggregation pheromones from the tea-winged bug, characterized in that, The method involves placing overwintering male adult tea bugs in a gas collecting bottle, collecting the insect's volatiles using an adsorption column, and then eluting the adsorption column with n-hexane to obtain the tea bug aggregation pheromone.

9. The method according to claim 8, wherein the overwintering male adult tea winged bug is pre-reared: it is fed with smoke tree leaves for one month and placed in an incubator at a temperature of 18°C ​​and a photoperiod of 10L:14D to maintain its reproductive diapause state, so as to promote its synthesis and release of more aggregation pheromones, thereby making it easier to detect.

10. The method according to claim 8 or 9, wherein the time period for collecting insect volatiles is 9:00-14:00, and the time for collecting gaseous volatiles is 4 hours; during collection, it is also placed in an incubator with a temperature of 18°C ​​and a photoperiod of 10L:14D.

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