Use of eucalyptol and trans-2-hexenal in repelling sericothrips variabilis

Abstract

This invention belongs to the field of agricultural pest repellency technology, specifically involving the application of eucalyptol and / or trans-2-hexenal in repelling soybean thrips. Addressing the problems of increased pesticide resistance and excessive pesticide residues in existing chemical control methods for soybean thrips, this invention, for the first time, clearly demonstrates that eucalyptol from Sichuan pepper volatiles and trans-2-hexenal from tomato volatiles possess significant repellent activity against soybean thrips, and determines their effective concentration range. Eucalyptol at a volume ratio concentration of 10... ‑2 and 10 ‑3 v / v has a significant evasion effect, at 10 ‑1 It exhibits a highly significant repellent effect at a v / v ratio; trans-2-hexenal at a volume ratio concentration of 10 ‑1 It exhibits a highly significant repellent effect at v / v. This invention provides a new technological means for the green repellent of soybean thrips, helping to reduce the use of chemical pesticides, lower the risk of pesticide residues, and ensure the safe production of leguminous crops.

Description

Technical Field

[0001] This invention relates to the field of agricultural pest repellency technology, specifically to the application of eucalyptol and trans-2-hexenal in repelling soybean thrips. Background Technology

[0002] Bean-sized thrips Megalurothrips usitatus As a global agricultural pest with a wide host range, cowpea is a major pest of leguminous crops, especially cowpeas. This pest can damage cowpeas throughout their entire growth cycle, causing flower drop and blackening of pods. Without effective control measures, it can cause significant economic losses to cowpeas. Currently, chemical control is still the primary method for controlling this pest. However, the long-term and frequent use of various chemical agents has led to increasing resistance and easily results in excessive pesticide residues in cowpeas. Therefore, exploring safe, sustainable, and green control technologies has become an urgent need in the field of cowpea pest control.

[0003] Plant-derived volatiles, as important informational chemicals for insect behavior regulation, have shown potential in the green control of pests. Related studies have shown that certain volatile components released by non-host plants have repellent effects on pests; for example, extracts from plants such as black pepper and cinnamon have been reported to exhibit repellent activity against other thrips species. (Red Sichuan pepper...) Zanthoxylum bungeanum With tomatoes Solanum lycopersicum As common plants, thrips have complex volatile compositions. However, regarding bean thrips, whether its volatiles contain highly effective repellent components, what those components are, and their effective concentrations are still lacking in systematic research and clear conclusions. This makes it impossible to develop plant-derived repellent products with clear and stable effects based on these two plants.

[0004] Therefore, the following problems still exist in the relevant technologies: For the specific pest *Thrips spp.*, the key repellent active ingredients in the volatiles of Sichuan pepper and tomato have not yet been identified and confirmed; their effective repellent concentration range is also unclear; and there is a lack of reliable data to support the conversion of these active ingredients into repellent formulations that can be used for practical control. This hinders the development of efficient and practical green control technologies for *Thrips spp.* based on these two plant resources. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides the application of eucalyptol and / or trans-2-hexenal in repelling soybean thrips, and based on this, provides a dedicated plant-derived repellent.

[0006] The first object of the present invention is to provide the use of eucalyptol and / or trans-2-hexenal in repelling soybean thrips.

[0007] Preferably, when eucalyptol is used, its volume concentration is 10. -3v / v to 10 -1 v / v; more preferably 10 - 1 v / v.

[0008] Preferably, when trans-2-hexenal is used, its volume ratio concentration is 10. -1 v / v.

[0009] A second objective of this invention is to provide a repellent for repelling soybean thrips, wherein the eucalyptol in the repellent has a volume fraction of 10. -3 v / v to 10 -1 v / v; and / or the volume fraction of the trans-2-hexenal is 10. -1 v / v.

[0010] Preferably, the repellent can be any one of a solution, emulsion, aerosol, or sustained-release formulation. Beneficial effects

[0011] This invention is the first to clearly demonstrate that eucalyptol in the volatiles of Sichuan pepper and trans-2-hexenal in the volatiles of tomato possess significant repellent activity against soybean thrips, and the effective concentrations of both have been determined, providing a new technical means for the green repellency of soybean thrips. This invention helps reduce the use of chemical pesticides, lowers the risk of pesticide residues, and promotes the safe production of crops such as cowpeas. Attached Figure Description

[0012] Figure 1 This is the behavioral response of the bean thrips to Sichuan peppercorns and tomatoes.

[0013] Figure 2 This is a total ion chromatogram of the volatile components of Sichuan pepper.

[0014] Figure 3 This is a total ion chromatogram of volatile components in tomatoes.

[0015] Figure 4 Behavioral response of soybean thrips to linalyl acetate.

[0016] Figure 5 Behavioral response of soybean thrips to eucalyptol.

[0017] Figure 6 Behavioral response of soybean thrips to trans-2-hexenal.

[0018] Figure 7 Behavioral response of soybean thrips to safflower.

[0019] Figure 8 Behavioral response of soybean thrips to β-caryophyllene.

[0020] Figure 9 Comparison of the field repellency effects of compound solutions with different ratios on soybean thrips. Detailed Implementation

[0021] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments, but the scope of protection of the present invention is not limited thereto. Example 1

[0022] This invention uses headspace solid-phase microextraction (HS-SPME) combined with gas chromatography-mass spectrometry (GC-MS) to collect and identify volatiles from Sichuan pepper and tomato leaves, employs a Y-type olfactometer for indoor behavioral response determination, and conducts field repellency tests to screen and verify key volatile components with significant repellency activity against soybean thrips.

[0023] I. Materials and Methods

[0024] 1. Test materials

[0025] (1) Test insect source

[0026] The bean thrips was collected from a cowpea experimental field in Lingao Village, Yazhou District, Sanya City, Hainan Province. It was brought back to the laboratory and subcultured in an artificial incubator with fresh broad beans. The culture conditions were: temperature (25±1)℃, relative humidity (55±5)%, photoperiod L:D=16:8, and the broad beans were replaced every 3 days. Female adults within 3 days of emergence were selected for the experiment.

[0027] (2) Test plants

[0028] The tomato seedlings were purchased from Shandong Shouhe Seed Industry Co., Ltd., and the variety is strawberry tomato. The Sichuan pepper powder was purchased from Anhui Yunshan Semi-Fish Technology Co., Ltd.

[0029] (3) Test instruments

[0030] Shimadzu TQ8040NX gas chromatograph-mass spectrometer, Shimadzu Corporation, Japan; solid-phase microextraction fiber and handle, Supelco, USA; electric thermostatic water bath, Beijing Changfeng Instrument Co., Ltd.; electronic analytical balance, Shanghai Lichen Bangxi Instrument Technology Co., Ltd.; atmospheric sampler, Beijing Kean Labor Protection New Technology Co., Ltd.; stereomicroscope, Leica Instruments GmbH, Germany.

[0031] (4) Test reagents

[0032] Eucalyptol and trans-2-hexenal were purchased from Macklin, β-caryophyllene and n-hexane were purchased from Sigma, linalyl acetate was purchased from Beijing Solarbio Science & Technology Co., Ltd., and juniperene was purchased from Shanghai Yuanye Biotechnology Co., Ltd. The purity of all reagents supplied was ≥98%.

[0033] 2. Determination of the selective behavior of soybean thrips towards Sichuan pepper and tomato

[0034] The behavioral responses of bean thrips to Sichuan pepper and tomato were determined using a Y-shaped olfactory instrument. The apparatus was connected in the following order: atmospheric sampler (exit), activated carbon drying tower, gas washing bottle (distilled water), glass rotor flowmeter, flavor source bottle, and Y-shaped tube. The airflow velocity of the atmospheric sampler was set to 300 ml / min, and the gas flow rate was controlled to 150 ml / min using the glass rotor flowmeter. The straight arm and side arm of the Y-shaped glass tube were both 15 cm long, with an inner diameter of 1 cm and an included angle of 75° between the two arms. Odorless silicone tubing was used for all connections. The Y-shaped tube was covered with a black light-blocking cloth, and an 8W strip light source was installed above the center to ensure that the light intensity received by both arms of the Y-shaped tube was consistent. The experiment was conducted in an environment with a temperature of 25±1℃ and a humidity of 70±5%.

[0035] Tomato seedlings or 20.0g of Sichuan pepper powder were placed in one flavor source bottle, while the control flavor source bottle was left empty. Female bean thrips, 1-3 days old, were selected and starved for 2 hours. A single thrips was introduced into the straight arm of a Y-tube. If the thrips crawled more than 1 / 3 of the way along one arm and remained there for more than 30 seconds, it was considered that the thrips had chosen that arm. If the thrips did not choose within 5 minutes, the thrips were discarded, and another thrips was used for the next test. 80 thrips were tested per treatment (number of responding thrips). Every 5 thrips tested, the positions of the two arms were reversed; every 10 thrips tested, the Y-tube was replaced. After each treatment, all test materials were rinsed sequentially with clean water and distilled water, then rinsed with anhydrous ethanol. After drying, the glassware was placed in a 180℃ electric thermostatic drying oven for 120 minutes to eliminate residual odors and prevent them from affecting subsequent treatments.

[0036] 3. Extraction and identification of volatile components from Sichuan pepper and tomatoes

[0037] (1) Headspace solid-phase microextraction for extraction of volatile components

[0038] The extraction head was pre-aged at 250℃ for 1 hour. 0.05 g of Sichuan pepper powder and 0.8 g of ground tomato leaf powder were placed in 20 ml headspace vials, and the pre-aged extraction head was inserted. Extraction was carried out in a 60℃ hot water bath for 30 min. After removing the extraction head, it was immediately inserted into the GCMS injection port for analysis and injection.

[0039] (2) Identification of volatile informational compounds

[0040] Chromatographic column: SH-I-5SiL MS (30m×0.25mm×0.25μm); The temperature program for Sichuan pepper was: initial temperature 65℃, increased to 90℃ at 25℃ / min and held for 1 min, increased to 105℃ at 1℃ / min and held for 2 min, then increased to 160℃ at 3℃ and held for 1 min, and finally increased to 250℃ at 30℃ / min and held for 2 min, with a split ratio of 20:1 and a column flow rate of 1.5 ml / min; The temperature program for tomato was: initial temperature 50℃ and held for 1 min, then increased to 185℃ at 6℃ / min, then increased to 240℃ at 20℃ / min, with splitless injection and a column flow rate of 1.00 ml / min; Injector temperature: 250℃; Carrier gas: Helium. Mass spectrometry conditions: Ion source: EI, ionization voltage 70 eV, ion source temperature 230℃, mass spectrometry detection range: 50~550 m / z.

[0041] 4. Behavioral response of soybean thrips to volatile informational compounds

[0042] Volatile compounds with a relative content greater than 10% in the leaves of Sichuan pepper and tomato were selected for behavioral reaction experiments on soybean thrips. The test compounds were diluted to 10% using n-hexane as a solvent. -1 10 -2 10 -3 10 -4 Four concentration gradients (v / v) were used. A Y-type olfactory instrument was employed. A filter paper disc (2cm × 2cm) containing 100μl of the test compound solution was placed in one of the odorant bottles, while a filter paper disc containing 100μl of n-hexane was placed in the other control odorant bottle. Solutions were prepared fresh and used immediately. Each concentration treatment involved 50 bean thrips.

[0043] 5. Field experiment to repel soybean thrips

[0044] To test the field repellency effects of compounds and their different formulations on soybean thrips, a field trial was conducted in January 2026 in a cowpea field in Tianyang Village, Lingao Town, Yazhou District, Sanya City, Hainan Province. Compounds showing good repellency in the indoor behavioral trials were used in the field experiment. 200 μl of the test compound solution was added to a blank lure, and a control lure was added with n-hexane solvent. The lures were then attached to the center of a 20*25 cm blue sticky insect board. Each treatment was randomly assigned to an independent row of cowpeas, approximately 20 m long. Within the same row, a sticky insect board with the corresponding treatment lure was suspended every 5 m along the row, at a height of 1.2 m above the ground. A row of cowpeas was used as an isolation strip between different treatment rows. Each treatment was repeated three times. After 2 days, the sticky insect boards were retrieved and counted under a stereomicroscope. The formulation components and ratios are shown in Table 1.

[0045] Table 1. Components and proportions of the initial screening formulation of soybean thrips repellent.

[0046]

[0047] 6. Data Processing and Analysis

[0048] SPSS 26.0 was used for statistical analysis. The chi-square test (χ²) was used to analyze the significance of differences in behavioral response data. One-way ANOVA was performed on the field trial data, and Tukey's method was used for multiple comparisons. p < 0.05 was considered statistically significant. Repellency rate = (1 - number of insects using the selected chemical standard odor source / total number of test insects) × 100%.

[0049] Volatile compounds were searched and identified using the NIST17-1, NIST17-2, and NIST17-S mass spectrometry libraries, and the relative content of each component was calculated using the peak area normalization method.

[0050] II. Results

[0051] 1. Behavioral response of bean thrips to Sichuan pepper and tomatoes

[0052] The results are as follows Figure 1 As shown, in the study of the selection behavior of soybean thrips towards Sichuan pepper and tomato, both Sichuan pepper and tomato exhibited highly significant repellent effects on soybean thrips. Specifically, the repellency rate of Sichuan pepper against soybean thrips was 93.75%, which was highly significant compared to the control (p=0.000, χ²=61.250), while the repellency rate of tomato against soybean thrips was 68.75%, which was also highly significant compared to the control (p=0.000, χ²=11.250).

[0053] 2. Identification of volatile components in Sichuan peppercorns and tomatoes

[0054] Figure 2 The total ion chromatogram of volatile components of Sichuan pepper is shown. Through mass spectrometry library comparison analysis, a total of 79 volatile components were identified, mainly alkenes, alcohols, esters and a small number of other compounds. Among them, 18 compounds with a matching degree greater than 80% and a relative content greater than 1% were identified (Table 2), accounting for 83.69% of the total volatile components of Sichuan pepper. Two compounds with a relative content greater than 10% were identified, namely linalyl acetate (26.82%) and eucalyptol (10.41%).

[0055] Table 2. Main volatile components of Sichuan pepper.

[0056]

[0057] Figure 3The total ion chromatogram of volatile components in tomato leaves was obtained. Through mass spectrometry library comparison and analysis, a total of 59 volatile components were identified, mainly alkenes, alcohols, aldehydes and a small number of other compounds. Among them, 11 compounds with a complexity greater than 80% and a relative content greater than 1% were identified (Table 3), accounting for 89.86% of the total volatile components in tomato leaves. Three compounds with a relative content greater than 10% were identified: trans-2-hexenal (37.18%), juniperene (19.38%) and β-caryophyllene (10.23%).

[0058] Table 3. Main volatile components in tomatoes

[0059]

[0060] 3. Behavioral response of soybean thrips to volatile informational compounds

[0061] Compounds with a relative content greater than 10% and a matching degree greater than 80% in Sichuan pepper and tomatoes were used in a bean thrips behavior reaction experiment. The compounds in the volatiles of Sichuan pepper that met this condition were linalyl acetate and eucalyptol. The experimental results showed that a volume ratio concentration of 10... -3 Linaloyl acetate at v / v concentrations significantly attracted soybean thrips (p=0.011), while other concentrations had no significant effect on the selective behavior of soybean thrips. Figure 4 ); 10 -2 10 -3 (v / v) eucalyptol had a significant repellent effect on soybean thrips (p=0.048, p=0.024), 10 -1 The v / v concentration of eucalyptol showed a highly significant repellent effect (p=0.002). Figure 5 This indicates that eucalyptol exhibits a dose-response effect; the repellency effect of the compound increases with increasing treatment concentration. The compounds with a relative content greater than 10% in the volatiles of tomato leaves are trans-2-hexenal, sapinene, and β-caryophyllene. Experimental results show that 10 -1 v / v trans-2-hexenal had a highly significant repellent effect on soybean thrips (p=0.000), while other concentrations showed no significant repellent effect. Figure 6 ); 10 -2 10 -3 (v / v) sapinene had a significant repellent effect on soybean thrips (p=0.011, p=0.024), 10 -1 v / v of sapinene showed a highly significant repellent effect (p=0.000). Figure 7 This indicates that sapinene also has a dose-response effect on soybean thrips; 10- 4β-caryophyllene v / v had a significant attraction effect on soybean thrips (p=0.024), meaning that β-caryophyllene only showed an attraction effect at low concentrations, while it had no significant attraction or repellency effect at higher concentrations. Figure 8 ).

[0062] Based on cost considerations, given the extremely high cost of halogenene among the three volatile compounds, the lower-cost trans-2-hexenal and eucalyptol were chosen as the subsequent test targets.

[0063] 4. Field experiment to repel bean thrips

[0064] Field trial results showed that the average number of thrips per sticky insect board was 161 in the control group (CK). The average number of thrips per sticky insect board for formulations A, B, C, D, E, F, and G were 81, 54, 63, 43, 76, 43, and 43, respectively, all lower than the CK. Data analysis revealed that the number of insects on sticky insect boards treated with individual compound solutions F and G was significantly lower than the CK (p=0.029, p=0.029), and the number of insects on the board treated with formulation D was also significantly lower than the CK (p=0.029). All three formulations showed good repellency effects, but there was no significant difference among them. The number of insects on the boards treated with formulations A, B, C, and E was not significantly different from the CK (p=0.243, p=0.057, p=0.095, p=0.196), as shown in the results. Figure 9 As shown in the figure. This indicates that under field conditions, eucalyptol and trans-2-hexenal, whether used alone or in a 2:1 ratio, can significantly repel soybean thrips. Other tested ratios of the same compound did not show significant effects in this experiment.

[0065] 5. Formulation and Field Application Methods

[0066] A 10% eucalyptol emulsifiable concentrate was prepared by mixing 10% eucalyptol, 5% Tween 80, and 85% ethanol by volume. A 200 μL / mL solution of this repellent was then sprayed evenly onto the plant surface at a rate of 1 mL per plant. The number of bean thrips was monitored before application and on days 1, 2, and 3 after application.

[0067] Without departing from the spirit and scope of this invention, based on the disclosure of this invention, many other variations and modifications conforming to the principles of this invention can be directly determined or derived. Therefore, the scope of protection of this invention should be understood and recognized as covering all such variations and modifications.

Claims

1. The application of eucalyptol in repelling soybean thrips, characterized in that, The volume concentration of eucalyptol is 10. - 3 v / v to 10 -1 v / v.

2. The application according to claim 1, characterized in that, The volume concentration of eucalyptol is 10. -1 v / v.

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