Aggregation pheromone of scirtothrips dorsalis, luring core and application thereof

Abstract

The present application belongs to the technical field of plant protection agents, and provides a gathering pheromone of Scirtothrips kauwae, a lure core and application thereof. The gathering pheromone of Scirtothrips kauwae provided by the present application is composed of only active ingredient (R)-2-methyl butyric acid-(R)-lavandulyl ester. The present application verifies the attraction of (R)-2-methyl butyric acid-(R)-lavandulyl ester to male and female adult Scirtothrips kauwae through indoor Y-type olfactometer test and field attraction test, and determines the component of the gathering pheromone of Scirtothrips kauwae, thereby providing technical support for effective prevention and control of Scirtothrips kauwae.

Description

Technical Field

[0001] This invention relates to the field of plant protectant technology, and in particular to an aggregation pheromone, lure, and application of the horn-toothed thrips. Background Technology

[0002] Odontothrips loti (Haliday), belonging to the genus Odontothrips in the family Thripidae of the suborder Thysnoptera, suborder Terebrantia, is a significant pest of alfalfa. Odontothrips can damage alfalfa at any stage of its growth and development. During the seedling and branching stages, it primarily damages the heart leaves and young leaves, causing leaf wrinkling and whitening of the infested areas, resulting in white spots—a typical symptom. During the budding and flowering stages, Odontothrips begin to damage the flower organs. Females lay eggs in the flower buds, and after hatching, they feed on petals and pollen, damaging the stigma, preventing the alfalfa from flowering, and even causing flower and pod drop, leading to the withering of the entire plant. Severe infestations of Odontothrips can cause poor plant growth and development, stunted growth, and even the withering of the entire alfalfa field during the seedling and branching stages, seriously threatening the quality and yield of alfalfa.

[0003] Currently, the main methods for controlling *Thrips hornedilum* include chemical control, agricultural control, and biological control, with chemical control being the most commonly used. However, applying chemical pesticides during the seedling and branching stages of alfalfa can affect its normal growth and development; the flowering period of alfalfa coincides with the harvest period, and spraying pesticides at this time can lead to excessive pesticide residues, threatening livestock safety. Furthermore, long-term application of pesticides can increase the resistance of *Thrips hornedilum*, reducing the effectiveness of control. Therefore, there is an urgent need to develop new, efficient, and green control technologies. Summary of the Invention

[0004] This invention provides an aggregation pheromone, a decoy, and their applications for the horned thrips, solving problems such as environmental pollution and drug resistance associated with chemical control, and achieving green control of the horned thrips.

[0005] In a first aspect, the present invention provides an aggregation pheromone of the bullhorn-toothed thrips.

[0006] The aggregation pheromone of *Thrips hornbillii* provided by this invention is composed of (R)-2-methylbutyric acid-(R)-lavandulyl[(R)-2-Methylbutanoate].

[0007] This invention utilizes a Y-type olfactometer to measure the olfactory responses of male and female *Thrips hornbillii* to their own odor sources. Volatile compounds from both male and female *Thrips hornbillii* are extracted using headspace solid-phase microextraction (HS-SPME), and the components of the volatile compounds are separated and identified using gas chromatography-mass spectrometry (GC-MS). An aggregation pheromone, (R)-2-methylbutyric acid-(R)-lavender ester, which has a significant attractant effect on both male and female *Thrips hornbillii*, has been developed, providing an effective method for the green control of *Thrips hornbillii*.

[0008] Although the aggregation pheromone components of five thrips species have been isolated and identified, the main components of the aggregation pheromone of western flower thrips (Frankliniella occidentalis) and flower thrips (F. intonsa) are neryl-(S)-2-methylbutanoate and (R)-lavandulylacetate; the main component of the aggregation pheromone of palm thrips (Thrips palmi) is (R)-lavandulyl3-methyl-3-butenoate; the main components of the aggregation pheromone of silk thrips (Megalurothrips sjostedti) are (R)-lavandulyl3-methylbutanoate and (R)-lavandulol; and the main components of the aggregation pheromone of common thrips (Megalurothrips) are... The main component of the aggregation pheromone of *Usitatus* is farnesyl acetate ((E,E)-farnesyl acetate). However, there are no reports on the aggregation pheromone of *Thymus hornulatus*. Even within the Thripidae family, the aggregation pheromones of different thrips species in different genera vary greatly. Based on the known aggregation pheromones of five thrips species, it is impossible to determine the components of the aggregation pheromone of *Thymus hornulatus* reported in this invention. Moreover, although the procedure of using headspace solid-phase microextraction combined with gas chromatography-mass spectrometry (GC-MS) to separate and identify the components of volatiles is common, the GC-MS spectrum cannot directly indicate the components of the aggregation pheromone, and there is no known database for reference regarding thrips volatiles. Therefore, the final determination of the aggregation pheromone of *Thymus hornulatus* is inseparable from the inventor's creative work.

[0009] Secondly, this invention provides the application of (R)-2-methylbutyric acid-(R)-lavender ester, the only active ingredient, as the aggregation pheromone of *Thrips hornflower*, in attracting *Thrips hornflower*.

[0010] In some embodiments of the present invention, the dosage of (R)-2-methylbutyric acid-(R)-lavender ester used to attract male *Thrips hornbillae* is 1-10 ng, and the dosage of (R)-2-methylbutyric acid-(R)-lavender ester used to attract female *Thrips hornbillae* is 1-10 ng.

[0011] More preferably, the dosage of (R)-2-methylbutyric acid-(R)-lavender ester for attracting male *Thrips hornbillae* is 10 ng, and the dosage of (R)-2-methylbutyric acid-(R)-lavender ester for attracting female *Thrips hornbillae* is 10 ng.

[0012] Thirdly, the present invention provides a lure for attracting horned thrips.

[0013] The lure provided by the present invention includes a sustained-release carrier and an aggregation pheromone placed within the sustained-release carrier, wherein the aggregation pheromone is composed of (R)-2-methylbutyric acid-(R)-lavender ester, which is the only active ingredient.

[0014] In some embodiments of the present invention, the amount of (R)-2-methylbutyric acid-(R)-lavender ester in a single sustained-release carrier is 40 to 80 μg.

[0015] The single sustained-release carrier uses a standard commercially available specification, with a diameter of 1 cm and a length of 1.5 cm.

[0016] More preferably, the amount of (R)-2-methylbutyric acid-(R)-lavender ester in a single sustained-release carrier is 80 μg.

[0017] In some embodiments of the present invention, the sustained-release carrier is one or more selected from rubber, polyethylene, hollow fiber, microcapsules, paraffin oil, and porous starch. The rubber can be natural rubber or synthetic rubber, preferably in the form of a rubber stopper; the polyethylene is preferably in the form of a polyethylene tube.

[0018] Fourthly, the present invention provides a method for preparing the above-mentioned lure for attracting horned thrips.

[0019] The method for preparing the lure provided by the present invention includes: rinsing and soaking the sustained-release carrier with n-hexane, drying it, and then adding the aggregation pheromone to the sustained-release carrier.

[0020] The order of hexane rinsing and soaking is not critical, as long as it removes impurities from the slow-release carrier. Drying can be done by baking, and after drying, it needs to be cooled to room temperature before adding the aggregation pheromone. The lure core is preferably prepared and used immediately.

[0021] Fifthly, the present invention provides the application of the above-mentioned lure core in the green control of horn-toothed thrips.

[0022] This invention provides an aggregation pheromone, lure, and application of the horned thrips. Volatile substances from male and female horned thrips were extracted using headspace solid-phase microextraction and analyzed by GC-MS. The resulting aggregation pheromone, composed of (R)-2-methylbutyric acid-(R)-lavender ester, exhibits a significant attraction effect on both male and female horned thrips, providing technical support for the effective control of this pest. Attached Figure Description

[0023] Figure 1 The olfactory selection behavior of male and female adult *Thrips hornbillii* towards male odor sources (***: P<0.001; **: P<0.01; *: P<0.05; NS: no significant difference; chi-square test).

[0024] Figure 2 The olfactory selection behavior of male and female adult *Thrips hornbillii* towards the odor source of female insects (***: P<0.001; **: P<0.01; *: P<0.05; NS: no significant difference; chi-square test).

[0025] Figure 3 This is the total ion current chromatogram (TIC) of the gaseous volatiles from male and female adult thrips (A: gaseous volatiles from female adults; B: gaseous volatiles from male adults).

[0026] Figure 4 This is a comparison of the mass spectrum of the informational compound released by the male *Thrips hornbill* with the standard mass spectrum of the NIST20s library. A: Mass spectrum of the informational compound released by the male *Thrips hornbill*; B: Mass spectrum of 2-methylbutyrate lavender ester.

[0027] Figure 5 This is the total ion chromatogram of co-injection of male thrips horn-toothed thrips and 2-methylbutyrate lavender ester chemical standard;

[0028] Figure 6 Comparison of total ion flux diagrams of lavender alcohol produced by hydrolysis of male thrips hornflower thrips extract with (R)-lavender alcohol and (S)-lavender alcohol;

[0029] Figure 7This is a comparison of the total ion flux diagrams of the volatiles of the male thrips hornflower and (R)-2-methylbutyric acid-(R)-lavender ester and (S)-2-methylbutyric acid-(R)-lavender ester;

[0030] Figure 8 The olfactory selection behavior of female (A) and male (B) *Thrips hornbillii* to different doses of (R)-2-methylbutyric acid-(R)-lavender ester (♂: male; ***: P<0.001; **: P<0.01; *: P<0.05, chi-square test);

[0031] Figure 9 The field trapping effect of different doses of (R)-2-methylbutyric acid-(R)-lavender ester on the sexual adult thrips of the horn-toothed flower thrips. Detailed Implementation

[0032] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0033] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0034] In the examples, the test thrips were :

[0035] The adult population of *Thrips hornbillii* was collected in August 2021 from the alfalfa base of the Grassland Research Institute in Hohhot, Inner Mongolia Autonomous Region. They were reared indoors in cages made of 300-mesh mesh using fresh alfalfa plants. The rearing conditions were: temperature 25±1℃, relative humidity 60±10%, and photoperiod 14:10h.

[0036] During the experiment, healthy adult insects aged 1-3 days are generally selected as the odor source and collected from the insect rearing cage using a homemade insect suction tube; the test insects are healthy adult insects that have emerged within 1 day and are picked out from the insect rearing cage.

[0037] The data analysis method in this embodiment is as follows: :

[0038] Data processing was performed using SPSS 22.0 software. Data from olfactory measurements were analyzed using the chi-square test. It was assumed that the ratio of thrips selected from both sides of the Y-shaped tube odor source was 1:1; individuals that did not make a selection were not included in the statistics. Field data were compared using a generalized linear model (Poisson distribution) to compare the average number of thrips attracted per white board under different treatments; multiple comparisons were performed using the LSD method.

[0039] Example: Isolation, identification and application of aggregation pheromones from *Thrips hornbillae*

[0040] 1. Behavioral selection test

[0041] This experiment used a Y-type olfactometer to determine the behavioral responses of male and female adult *Thrips hornbillae* to volatile substances released by the thrips. The Y-type olfactometer mainly consists of a Y-tube, a Teflon flexible tube, a scent source bottle, a glass rotor flowmeter, activated carbon, and an air pump. The experimental procedure involves turning on the air pump, allowing air to flow through the activated carbon and a three-way connector to form two clean airflows. These airflows then pass through the glass rotor flowmeter and the scent source bottle, respectively, carrying the gases from the two scent source bottles into the side arms of the Y-tube, where they finally meet on the main arm. The Y-tube is typically made of transparent glass, with an adaptation arm and test arm length of 6 cm and an inner diameter of 5 mm. The laboratory environment was maintained at 25±2℃ and 50±10% humidity.

[0042] The experiment set up two groups of odor sources: "male insect-blank control" and "female insect-blank control," to test the olfactory selection behavior of male and female insects. Clean, odorless glass gas washing bottles were sealed at both ends with nylon mesh. A filter paper with a few drops of distilled water was placed inside the bottle to keep it moist. Fifty healthy male or female insects were placed in each bottle as odor sources. The blank control bottle contained only a filter paper with a few drops of distilled water. After connecting the odor source bottles to the apparatus, the gas pump was turned on, and the flow rate of the test arm was adjusted to 80 mL / min. The odor sources were illuminated with a cold light source (11,000-12,000 lx) for 30 minutes before the experiment began. During the test, a soft brush was used to pick up one adult insect to be tested and placed it at the opening of the Y-shaped adaptation arm for observation. When the insect passed through half of a test arm of the Y-shaped tube and remained there for more than 30 seconds, it was considered to have made a selection; if the insect did not make a selection within 5 minutes of entering the adaptation arm, it was considered not to have selected. For every 5 thrips tested, the Y-tube was rotated 180°; for every 10 thrips tested, the Y-tube was replaced, and the positions of the flavor source bottles on both sides of the device and the Teflon tubing connecting the flavor source bottles to the Y-tube at the rear end were simultaneously reversed. Each group of experiments tested 20 thrips each time, for a total of 5 tests.

[0043] After the experiment, all parts of the device were cleaned in an ultrasonic cleaner with water for 10 minutes, then removed and rinsed repeatedly with anhydrous ethanol. Finally, they were placed in an oven at 100°C for 2 hours.

[0044] As a result, the olfactory behavioral responses of both male and female adult *Thrips hornbill* to the odor source from male insects were as follows: Figure 1As shown. The male *Thrips hornbill* species exhibited a highly significant selectivity for the male odor source; 62 males chose the male odor source, while 36 chose the blank control (χ²). 2 =6.898, P=0.009). The selection behavior of females was similar to that of males; females exhibited a highly significant selectivity towards male odor sources, with 67 females choosing male odor sources and 31 choosing the blank control (χ² = 6.898, P = 0.009). 2 =13.224, P=0.00028). Therefore, the odorant of the male *Thrips hornbill* has a significant attractant effect on both male and female *Thrips hornbill* of the same species.

[0045] The olfactory behavioral responses of male and female adult *Thrips hornbill* to odor sources from female insects are as follows: Figure 2 As shown. Male *Thrips hornbill* do not show selectivity for the odor source from females; 46 males selected the odor source from females, while 52 selected the blank control (χ²). 2 =0.367, P=0.544). The selection behavior of females was similar to that of males; females did not select for the odor source of females, with 43 females choosing the odor source and 57 choosing the blank control (χ² = 0.367, P = 0.544). 2 =0.3165, P=0.5737). The results showed that the odorant of the female *Thrips hornbillae* had no attraction effect on the male and female adults of *Thrips hornbillae*.

[0046] The above experimental results indicate that male *Thrips hornbillii* adults can release a volatile informational compound that induces significant positive directional behavior in both male and female adults, while females cannot produce an informational compound with the same effect. Functionally, this informational compound is an aggregation pheromone for *Thrips hornbillii*.

[0047] 2. Extraction of volatiles from male and female adult thrips (Horse Horn Thrips)

[0048] Volatile compounds from male and female *Thrips hornblight* were extracted using headspace solid-phase microextraction (HS-SPME). Before extraction, the extraction tip (100 μm, Polydimethylsiloxane Coating, Supelco) of the manual SPME sampler was activated at 200 °C for 15-20 min at the injection port of a gas chromatography-mass spectrometry (GC-MS, SHIMADZU GC-MS-QP2010plus). Forty female or male *Thrips hornblight* larvae within one day of emergence were transferred to 1.5 mL transparent vials and irradiated with a cold light source (11,000-12,000 lx) at a distance of 2 cm from the vials. The SPME extraction tip was inserted into the vial to extract the volatile compounds released by the females or males for 4-5 h. Subsequently, the informational compounds released by *Thrips hornblight* were analyzed by GC-MS. GC-MS operating conditions: nonpolar column Rxi-5ms (30m×0.25mm×0.25μm); splitless injection; programmed temperature rise (40℃, hold for 2 min, increase to 150℃ at 10℃ / min, hold for 0 min, increase to 180℃ at 1℃ / min, hold for 0 min, increase to 200℃ at 10℃ / min, hold for 1 min); injection port temperature 200℃; carrier gas He (>99.999%); ion source EI, ion source temperature 230℃; quadrupole temperature 150℃; electron energy 70eV; scan mass range 35-500 amu.

[0049] 3. Identification of the chemical composition and structural analysis of aggregated pheromones

[0050] The total ion chromatograms of volatiles from male and female adult thrips were analyzed to determine the elution positions of the target compounds. Then, the NIST20s library built into the instrument was searched using the target peak mass spectrometer. The most matching result was selected based on the degree of match between the search and the mass spectrum peak type and characteristic ion peak information. At the same time, the mass spectrum information was compared with the NIST online database for preliminary qualitative analysis.

[0051] Based on preliminary qualitative results, the compound 2-methylbutyric acid lavender ester was synthesized as follows: A clean 25 mL reaction flask was prepared, and 2 mL of dichloromethane was added dropwise. 1 mmol of lavender alcohol and 1 mmol of 2-methylbutyric acid were weighed and added to the reaction flask, followed by 0.05 mmol of a novel, highly efficient catalyst—4-dimethylaminopyridine (DMAP). The reaction flask was placed in an ice bath, and 1.2 mmol of dicyclohexylcarbodiimide (DCC) was slowly added. A magnet was placed in the flask, and the mixture was stirred using a magnetic stirrer. After 30 min, the mixture was stirred at room temperature for 24 h. After the reaction was complete, the reaction byproducts were filtered using a vacuum funnel, and the precipitate was washed 2-3 times with petroleum ether. The filtrate was washed successively with appropriate amounts of saturated sodium bicarbonate aqueous solution, dilute hydrochloric acid, and water, dried over magnesium sulfate, and filtered. The resulting filtrate was concentrated using a rotary evaporator and purified by column chromatography to obtain the pure product.

[0052] The components of 2-methylbutyrate lavender ester and compounds released by male insects were compared and confirmed using a chiral CYCLOSIL-B column (30m × 0.25mm × 0.25μm). The total ion flow rates of male insect volatiles, 2-methylbutyrate lavender ester, and co-injection of both were compared. The GC temperature program was as follows: 55℃, hold for 2 min, increase to 150℃ at 10℃ / min, hold for 1 min, increase to 160℃ at 1℃ / min, hold for 0 min. The co-injection method was as follows: the synthesized 2-methylbutyrate lavender ester was diluted to a standard solution of 0.01 ng / μL with chromatographically pure n-hexane. Then, the volatiles released by the insects and the chemical standard were extracted sequentially using the same extraction device and co-injected. The extraction time for insect gaseous volatiles was 2 h, and the extraction conditions for the chemical standard were 0.01 ng / μL, 1 μL, 30 min. Analyze the total ion chromatogram (TIC) of the mixture of insect volatiles and chemical standards.

[0053] The gas chromatographic separation results of the volatile substances from male and female adult *Thrips hornbill* are shown below. Figure 3 A distinct chromatographic peak was observed in the TIC chromatogram of the volatiles released by the male insect, with an elution time of 18.167 min. Figure 3 (B) The above results indicate that male *Thrips hornbill* can release a large amount of a signaling compound. Conversely, this component is not present in the volatile gases emitted by females. Figure 3 (A)

[0054] Using the NIST20s mass spectral library built into the mass spectrometer (MS), the mass spectra of informational compounds released by male adult *Thrips hornbill* were analyzed. Figure 4In step A), substance identification was performed. Based on the ionic fragments m / z 154, m / z 136, m / z 121, m / z 93, m / z 69, and m / z 41 in the figure, the compound was determined to be a monoterpene structure. Ionic fragments m / z 85 and m / z 57 are C4H9CO, which forms a 5-carbon saturated fatty acid. + and C4H9 + Therefore, it is speculated that the aggregation pheromone component of the horn-flowered thrips is likely composed of monoterpene alcohol C. 10 H 18 O and saturated five-carbon fatty acid C5H 10 The ester compounds formed by O2 esterification reaction have the molecular formula C. 15 H 26 O2, with a relative molecular mass of 238. Further analysis based on the search matching degree and the degree of agreement between the mass spectrometry peak type and characteristic ion peaks preliminarily identified the target peak as 2-methylbutyrate lavender ester (…). Figure 4 (Medium B). Further qualitative analysis was performed using total ion chromatograms and mass spectra of co-injection of the chemical standard 2-methylbutyrate lavender ester and the volatiles of male thrips horntooth, on chiral chromatography. The results showed that after co-injection of the chemical standard and the volatiles of the male insect, there was only one major chromatographic peak, with an elution time of 19.767 min, indicating that they are the same substance ( ). Figure 5 Therefore, it can be determined that the volatile component released by the male horn-toothed thrips is 2-methylbutyrate lavender ester.

[0055] 4. Determination of the chirality of aggregated pheromone components

[0056] Since 2-methylbutyric acid lavender ester has two chiral groups, it could be one of four chiral structures ((R)-2-methylbutyric acid-(R)-lavender ester, (R)-2-methylbutyric acid-(S)-lavender ester, (S)-2-methylbutyric acid-(R)-lavender ester, (S)-2-methylbutyric acid-(S)-lavender ester). This invention first uses a hydrolysis method to determine the chirality of the lavender alcohol group by hydrolyzing the natural aggregation pheromone component extract of *Thrips hornwortii* to obtain lavender alcohol, and then uses chiral column separation to determine the chirality of the other chiral group.

[0057] First, (S)-lavenderol (retention time 18.558 min) and (R)-lavenderol (retention time 18.833 min) were separated on a chiral CYCLOSIL-B column using a temperature program (55℃, hold for 1 min, increase to 160℃ at 5℃ / min, hold for 0 min, increase to 200℃ at 10℃ / min, hold for 0 min). The aggregation pheromone component of male *Thrips hornbillii* was extracted using a solution extraction method. Approximately 1000 males were soaked in n-hexane for 24 h, concentrated with nitrogen, and then dissolved in 300 μl of ethanol. Two drops of 1N NaOH solution (1 mol / L) were added, and the mixture was reacted in a water bath at 35℃ for 30 min. The solution was then neutralized with 1N HCl (1 mol / L). The product was extracted with n-hexane. The n-hexane solution was dried over anhydrous MgSO4. After concentration under nitrogen flow, lavender alcohol, generated from the hydrolysis of the natural aggregated pheromone component, was compared with (S)-lavender alcohol and (R)-lavender alcohol using a chiral chromatographic column CYCLOSIL-B to determine the chirality of the lavender alcohol group. The corresponding chiral lavender alcohols were then esterified with (R)-2-methylbutyric acid and (S)-2-methylbutyric acid, respectively, to generate (R)-2-methylbutyric acid-(R)-lavender ester and (S)-2-methylbutyric acid-(R)-lavender ester. A temperature program (100℃, hold for 1 min, increase to 170℃ at 10℃ / min, hold for 0 min, increase to 180℃ at 1℃ / min, hold for 0 min) was applied to the chiral chromatographic column CYCLOSIL-B. On a LOSIL-B, (R)-2-methylbutyric acid-(R)-lavender ester (retention time 11.244 min) and (S)-2-methylbutyric acid-(R)-lavender ester (retention time 11.333 min) were separated. The aggregation pheromone component released by the male insect was compared with (R)-2-methylbutyric acid-(R)-lavender ester and (S)-2-methylbutyric acid-(R)-lavender ester on a chiral chromatographic column to determine the final chirality.

[0058] The results showed that, through comparison of the total ion flux diagrams of lavender alcohol produced by hydrolysis of the male thrips hornbill thrips extract with those of (R)-lavender alcohol and (S)-lavender alcohol, the retention times of lavender alcohol and (R)-lavender alcohol produced by hydrolysis of the male thrips extract were the same, both being 18.833 min. Figure 6Therefore, the chirality of the lavenderol group in the aggregation pheromone component released by male *Thrips hornbillii* was determined to be (R)-lavenderol. (R)-lavenderol was esterified with (R)-2-methylbutyric acid and (S)-2-methylbutyric acid, respectively, to generate (R)-2-methylbutyric acid-(R)-lavender ester and (S)-2-methylbutyric acid-(R)-lavender ester. The aggregation pheromone component released by the male was compared with that of (R)-2-methylbutyric acid-(R)-lavender ester and (S)-2-methylbutyric acid-(R)-lavender ester by GC-MS. It was found that the retention time of the aggregation pheromone component released by the male was the same as that of (R)-2-methylbutyric acid-(R)-lavender ester, which was 11.242 min. Figure 7 Therefore, the aggregation pheromone component of *Thrips hornflower* was determined to be (R)-2-methylbutyric acid-(R)-lavender ester.

[0059] 5. Indoor induction activity assay of artificially synthesized aggregation pheromone components

[0060] The attractant activity of different doses of the synthetic aggregation pheromone component (R)-2-methylbutyric acid-(R)-lavender ester on adult male and female *Thrips hornbillii* was determined using a Y-type olfactometer. Four doses were used: 0.1 ng, 1 ng, 10 ng, and 100 ng. Before behavioral selection testing, the synthetic aggregation pheromone component was diluted with n-hexane to concentrations of 0.01 ng / μL, 0.1 ng / μL, 1 ng / μL, and 10 ng / μL. 10 μL of the prepared solution was added dropwise to filter paper and quickly placed into the flavor source bottle, representing doses of 0.1 ng, 1 ng, 10 ng, and 100 ng. For each dose treatment, a flavor source bottle containing filter paper with 10 μL of n-hexane was used as a control. Before the experiment, the control and treated flavor source bottles were assembled on the apparatus, allowed to stand for 10 min, and then purged with airflow for 5 min. The measurement method was the same as above. Each test involved 20 worms, and each dose treatment was repeated 5 times.

[0061] As a result, the olfactory selection behaviors of female and male *Thrips hornbillii* to different doses of (R)-2-methylbutyric acid-(R)-lavender ester were as follows: Figure 8 As shown in Figures A and B. When the dose of (R)-2-methylbutyric acid-(R)-lavender ester was 10 ng, female insects exhibited extremely significant selectivity for (R)-2-methylbutyric acid-(R)-lavender ester (χ²). 2 =4.000, P=0.046)( Figure 8 (A). When the dosage of (R)-2-methylbutyric acid-(R)-lavender ester was 1 and 10 ng, male insects exhibited extremely significant selectivity for (R)-2-methylbutyric acid-(R)-lavender ester (1 ng: χ²). 2 =4.84, P=0.028; 10ng: χ2 =17.62, P<0.0001)( Figure 8 (B). This shows that (R)-2-methylbutyric acid-(R)-lavender ester has an attractive effect on both male and female adult thrips.

[0062] 6. Field trapping experiment

[0063] The lure used in this invention is made by adding a specific dose of aggregation pheromone component to a green rubber stopper carrier. Before preparing the lure, the green rubber stopper, which serves as the carrier, is first rinsed with n-hexane (chromatographic grade) to remove surface impurities, then soaked in n-hexane for at least 24 hours to clean internal impurities, and then dried in an oven at 80°C for at least 2 hours. The dried and cooled rubber stopper is then sealed and stored in a glass bottle. The lure is prepared and used immediately; the artificially synthesized aggregation pheromone component is first diluted to different concentrations and then added to the green rubber stopper carrier using a pipette.

[0064] The artificially synthesized aggregation pheromone component (R)-2-methylbutyric acid-(R)-lavender ester in the green rubber stopper carrier was treated with four doses: 0 μg (blank control), 40 μg, 60 ng, and 80 μg.

[0065] The experiment was conducted in September 2023 at the experimental base of the Grassland Research Institute of the Chinese Academy of Agricultural Sciences in Hohhot, Inner Mongolia (40°34'52"N", 111°47'12"E), covering an area of ​​approximately 3 mu (about 0.2 hectares). The weather during the experiment was sunny or partly cloudy, with temperatures ranging from 15 to 28°C. Different doses and proportions of attractants were fixed in the center of white insect-attracting boards (25cm x 20cm). To minimize environmental interference, alfalfa was removed from both ends of the field, and the boards were positioned at least 2 meters from both ends of the field edge. The distance between boards in the same row was also at least 2 meters. To avoid uneven insect population distribution and the influence of different treatments on the experimental results, the different treatments were randomly arranged, with each treatment surrounded by other treatments. Each treatment had eight replicates. The number of female and male insects on the white boards of different treatments was collected and analyzed after 24 hours.

[0066] Field trapping effect Figure 9 As shown, compared with a blank whiteboard, attractants containing 40, 60, and 80 μg of (R)-2-methylbutyric acid-(R)-lavender ester significantly increased the attraction of the whiteboard to female, male, and adult thrips (female: χ²) 2 =115.76, df=3, P<0.0001; Male insect: χ 2 =309.57, df=3, P<0.0001; Total female and male: χ 2=408.29, df=3, P<0.0001). Among them, 80μg had the most obvious attraction effect, attracting 3.64 times, 6.19 times, and 4.86 times more females, males, and the total number of females and males than the blank white board, respectively.

[0067] In summary, this invention has demonstrated through behavioral selection experiments that male *Thrips hornbillae* can release a pheromone compound that attracts both male and female adults. Furthermore, the volatiles of male *Thrips hornbillae* were extracted using headspace solid-phase microextraction, and GC-MS analysis identified a compound as (R)-2-methylbutyric acid-(R)-lavender ester. Further indoor Y-type olfactometer experiments and field attraction experiments verified that this compound has an attractant effect on both male and female *Thrips hornbillae* adults, clarifying that this compound is an aggregation pheromone component of *Thrips hornbillae*.

[0068] It should be noted that the endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0069] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "specific implementation," or "some specific implementations," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0070] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. The application of aggregation pheromones from *Thrips hornbillii* in attracting *Thrips hornbillii*, characterized in that... The aggregation pheromone of the bullhorn thrips is composed of (R)-2-methylbutyric acid-(R)-lavender ester; The dosage of (R)-2-methylbutyric acid-(R)-lavender ester used to attract male *Thrips hornbillae* is 1-10 ng, and the dosage of (R)-2-methylbutyric acid-(R)-lavender ester used to attract female *Thrips hornbillae* is 1-10 ng.

2. The application according to claim 1, characterized in that, The dosage of (R)-2-methylbutyric acid-(R)-lavender ester used to attract male *Thrips hornbillae* was 10 ng, and the dosage of (R)-2-methylbutyric acid-(R)-lavender ester used to attract female *Thrips hornbillae* was 10 ng.

3. A lure for attracting thrips, characterized in that, The lure core includes a slow-release carrier and the aggregation pheromone of claim 1 disposed within the slow-release carrier; The amount of (R)-2-methylbutyric acid-(R)-lavender ester in a single sustained-release carrier is 40~80 μg.

4. The lure for attracting *Thrips hornedius* according to claim 3, characterized in that, The amount of (R)-2-methylbutyric acid-(R)-lavender ester in a single sustained-release carrier is 80 μg.

5. The lure for attracting *Thrips hornedius* according to claim 3, characterized in that, The sustained-release carrier is one or more of the following: rubber, polyethylene, hollow fiber, microcapsule, paraffin oil, and porous starch.

6. The method for preparing the lure for attracting *Thrips hornedius* according to any one of claims 3-5, characterized in that, include: After rinsing and soaking the sustained-release carrier with n-hexane and drying it, the aggregation pheromone is added to the sustained-release carrier.

7. The application of the lure according to any one of claims 3-5 in the green control of horn-toothed thrips.

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