Application of politeal bead scale attractant

Abstract

The invention discloses an application of an attractant for poliplius perfoliatus. According to the invention, two compounds, namely methyl jasmonate and d-dihydrocarvone, which have remarkable attraction activity on male Lipoliplius popliacea adults are reversely screened out through methods such as a molecular docking technology, electrophysiology and insect behavioristics, wherein the two compounds are methyl jasmonate and d-dihydrocarvone. A field trapping effect evaluation result shows that the methyl jasmonate has an obvious trapping effect on Lipoliplius popliacea male adults. Therefore, the methyl jasmonate can be used as the attractant for the male imago of the politeal-bead scales, and the lure containing the attractant can be used for trapping the male imago of the politeal-bead scales, so that the dependence on chemical pesticides is reduced, and the green prevention and control requirements are met.

Description

Technical Field

[0001] The invention belongs to the field of biological pesticides and relates to biological trapping of insects, in particular to the application of a liquorice carmine scale attractant. Background Art

[0002] The licorice scale (Porphyrophora sophorae) is an important economic pest belonging to the order Hemiptera and family Margarodidae. First-instar nymphs parasitize licorice roots and suck root sap for a living. The root tissue of the affected plant is damaged, eroding and drying up, resulting in obstructed water and nutrient transport. Aboveground leaves turn yellow and fall off, ultimately leading to the death of the entire plant. The licorice scale reproduces rapidly, spreads rapidly, and lives underground for long periods of time. Once it occurs, it is extremely difficult to control. The degree of damage increases with the number of years the licorice is cultivated. Typically, the damage rate in 2-3 year-old cultivated licorice reaches 20-50%, even leading to complete crop failure. Despite the continuous development of control technologies for the licorice scale, existing methods still have certain limitations. Although conventional chemical control methods can quickly reduce the number of pests, they can lead to the development of pest resistance and environmental pollution, seriously affecting the competitiveness and sustainable development of the artificial licorice industry. The development of new control technologies is urgent.

[0003] Attraction and control technology is a viable green approach for the control of licorice scale insects. In nature, plants and animals communicate through volatile chemicals, which act as signaling molecules and play a key role in interbiotic interactions. Leveraging these distinctive odors to attract licorice scale insects and achieve effective control can reduce pesticide use and mitigate insecticide resistance. However, there is currently no effective attractant for adult licorice scale insects.

[0004] In order to solve the above problems, the present invention is proposed. Summary of the Invention

[0005] The purpose of the present invention is to overcome the deficiencies of the prior art and provide an application of an attractant for glycyrrhiza uralensis. The attractant provided by the present invention has a good attracting effect on male adults of glycyrrhiza uralensis, high attracting efficiency, and is environmentally friendly.

[0006] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0007] Application of methyl jasmonate in the preparation of attractant for male adults of Glycyrrhiza uralensis scale.

[0008] The invention discloses an application of an attractant containing methyl jasmonate in trapping male adults of the glycyrrhiza uralensis scale.

[0009] Preferably, the attractant core uses methyl jasmonate as an active ingredient and is made into an attractant core for trapping male adults of the licorice scale insect through a carrier acceptable in the field of insect trapping.

[0010] More preferably, the carrier is a natural desulfurized rubber stopper, and the method for preparing the lure comprises: first dissolving methyl jasmonate in an organic solvent to obtain a methyl jasmonate solution, then evenly dropping the methyl jasmonate solution onto the natural desulfurized rubber stopper to ensure uniform absorption, sealing and packaging with aluminum film, and storing at low temperature.

[0011] More preferably, the organic solvent is dichloromethane.

[0012] More preferably, the low temperature is 4°C.

[0013] Beneficial effects:

[0014] This study, using molecular docking, electrophysiology, and insect behavior, identified two compounds with significant attractant activity against adult male licorice scale insects: methyl jasmonate and d-dihydrocarvone. Field evaluation of the trapping efficacy demonstrated that methyl jasmonate exhibited a significant trapping effect on adult male licorice scale insects. Therefore, methyl jasmonate can be used as an attractant for adult male licorice scale insects. Lures containing this attractant can be used to trap adult male licorice scale insects, reducing reliance on chemical pesticides and meeting the requirements of environmentally friendly pest control. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is the result of PCR amplification of PsopOR1 and PsopORco genes in Glycyrrhiza uralensis;

[0016] Figure 2 The agarose gel electrophoresis diagram of the positive clone plasmid after purification and enzyme digestion purification;

[0017] Figure 3 Agarose gel electrophoresis of cRNA before and after purification;

[0018] Figure 4 This is a fluorescence detection image of Xenopus oocytes;

[0019] Figure 5Figure 3: Two-electrode voltage-clamp recordings of the responses of Xenopus oocytes expressing PsopOR1 / PsopORco to volatiles. A shows the current response of Xenopus oocytes expressing PsopOR1 / PsopORco to 0.1 mM solutions of different odorants. B shows the response spectrum of Xenopus oocytes expressing PsopOR1 / PsopORco, with response values represented by numbers ± standard deviations (n=5). C shows the stimulation of Xenopus oocytes expressing PsopOR1 / PsopORco with d-dihydrocarvone concentration gradients. D shows the stimulation of Xenopus oocytes expressing PsopOR1 / PsopORco with methyl cis-jasmonate concentration gradients. E shows the dose curves of the responses of Xenopus oocytes expressing PsopOR1 / PsopORco to methyl jasmonate and d-dihydrocarvone. Response values are normalized to the maximum value as 100, and error bars represent standard deviations (n=4).

[0020] Figure 6 The EAG responses of male adults of R. glycyrrhizae to different concentrations of methyl jasmonate and d-dihydrocarvone;

[0021] Figure 7 The results of Y olfactometer behavioral assays of male insects to sensitive compounds;

[0022] Figure 8 The figure shows the trapping effect of various attractants on male adults of Licorice Scale. DETAILED DESCRIPTION

[0023] The essential contents of the present invention are described in detail below with reference to the embodiments, but the protection scope of the present invention is not limited thereto.

[0024] Example 1: Design of an attractant targeting the olfactory system (PsopOR1 / PsopORco receptor) of male adults of Psopus licoricei

[0025] 1. Cloning and expression vector construction of PsopOR1 and PsopORco

[0026] Specific primers were designed for PsopOR1 and PsopORco and PCR amplified. The results were detected by electrophoresis and compared with the marker. Figure 1 As shown, the size is consistent with the expected nucleotide sequence, and the band is single and clearly visible, which can be used for gel recovery experiments.

[0027] pGEMHE-GFP / mCherry was digested with Xmal / EcoRI (NEB). After linearization and purification with NheI (NEB), the results were as follows: Figure 2 As shown in the figure, one band was obtained by electrophoresis, indicating that the enzyme digestion effect was good.

[0028] Using the positive clone plasmid as a template, cRNA (ORco-GFP; OR1-mCherry) was obtained by in vitro transcription using a T7 in vitro transcription kit. The cRNA electrophoresis band after ORco-GFP transcription was tailing, and it needed to be purified. The DNA template was digested and the cRNA was purified and desalted. The results were as follows: Figure 3 As shown, the main bands detected by electrophoresis were bright and had clear edges, and high-quality cRNAs with specific tags for the two target genes were obtained.

[0029] 2. Fluorescence detection of Xenopus oocytes

[0030] The purified cRNA was injected into the oocytes of African clawed frogs. Figure 4 As shown, protein expression was detected using a fluorescence confocal microscope. No fluorescence was observed under all four light sources in the control injected with clear water. Green fluorescence was observed in oocytes injected with ORco-GFP cRNA alone under a 488nm light source, no fluorescence under white light and 546nm light sources, and green fluorescence was observed under a mixed light source of 488nm and 546nm. Red fluorescence was observed in oocytes injected with OR1-mCherry cRNA alone under a 546nm light source, no fluorescence under white light and 512nm light sources, and red fluorescence was observed under a mixed light source of 512nm and 546nm. Green fluorescence was observed in oocytes injected with a mixture of ORco-GFP and OR1-mCherry cRNA under a 488nm light source, red fluorescence was observed under a 546nm light source, no fluorescence under white light, and yellow fluorescence was observed under a mixed light source of 512nm and 546nm. This indicates that PsopORco and PsopOR1 are both successfully expressed on the oocyte membrane.

[0031] 3. PsopOR1 function two-electrode voltage clamp detection

[0032] After PsopOR1 was co-expressed with PsopORco in Xenopus oocytes, ligands were screened using TEVC technology, and 27 odorants screened in previous studies were selected as candidate ligands. Figure 5 As shown, subject to 10 -4 After odor stimulation, oocytes expressing PsopORco, PsopOR1, or the control did not produce significant currents. However, oocytes co-expressing PsopOR1 / PsopORco showed significant current responses to methyl jasmonate and d-dihydrocarvone ( Figure 5 A and B in the figure). The concentration gradient experiment showed that methyl jasmonate -4 The strongest current response was induced when M Figure 5 D), and the dose-response curve showed a positive correlation ( Figure 5(E) Compared with d-dihydrocarvone, methyl jasmonate has a lower EC50 (half effective concentration), indicating that it binds to the receptor more efficiently.

[0033] 4. EAG verification

[0034] The above two compounds (methyl jasmonate and d-dihydrocarvone) were diluted to 0.01μg / μL, 0.1μg / μL, 1μg / μL, 10μg / μL, and 100μg / μL concentration gradients (solvent: dichloromethane), and the antennal EAG response under different concentration gradients was measured, and dichloromethane was used as a control. The results are shown in Figure 2. Figure 6 As shown in Figure 2, the antennal potential (EAG) response was strongest at 100 μg / μL of methyl jasmonate ( Figure 6 ), significantly higher than d-dihydrocarvone (p<0.05), indicating that male insects are more sensitive to methyl jasmonate. At low concentrations (0.01-1 μg / μL), methyl jasmonate still induced detectable EAG signals, while d-dihydrocarvone had a weaker response.

[0035] 5. Behavioral measurement

[0036] The above two compounds (methyl jasmonate and d-dihydrocarvone) were subjected to Y olfactometer behavioral assay, and the results were as follows: Figure 7 As shown, 100 μg / μL methyl jasmonate and d-dihydrocarvone had direct behavioral activity against the glycyrrhizic scale insect, with male insects showing significantly higher selectivity for methyl jasmonate than for the control (p < 0.01). At a concentration of 100 μg / μL, methyl jasmonate had a higher attraction rate than d-dihydrocarvone (male insects showed no significant difference in selectivity for d-dihydrocarvone compared to the control).

[0037] Using molecular docking techniques, electrophysiological methods (such as two-electrode voltage clamp assays), and insect behavioral studies, a reverse screening approach identified two compounds with significant attractant activity against adult male licorice scale insects: methyl jasmonate and d-dihydrocarvone. Methyl jasmonate (at a concentration of 100 μg / μL) exhibited superior attractant activity.

[0038] Example 2: Preparation of methyl jasmonate attractant

[0039] The molecular formula of methyl jasmonate is C 13 H 20 O3, CAS number 1211-29-6, is a colorless oily liquid with a strong floral, green, sweet and long-lasting aroma similar to that of Jasmine sambac absolute. Its melting point is N / A, boiling point is 110°C, and flash point is >230°F. It is soluble in ethanol and oils. It should be stored in a sealed and dry place at room temperature. Its structural formula is as follows.

[0040]

[0041] Dissolve methyl jasmonate in dichloromethane to obtain an attractant with a concentration of 100μg / μL. Take out the natural desulfurized blank rubber head bait cores from the package and put them into 100mL conical flasks in groups of 10. Add 1000μL of attractant to each conical flask, stirring with a glass rod while adding to ensure uniform absorption. A single bait core with a load of 10,000μg of active ingredient is made, which can maintain high-efficiency trapping for 5 days. Seal the bait core with 10 pieces of aluminum film per bag and store at 4°C.

[0042] Example 3: Field trapping effect evaluation

[0043] In mid-August 2024, the bait prepared in Example 2 was used to trap the licorice scale insects at the Gaoshawo licorice planting base in Yanchi County, Ningxia. The traps used commercial blank sticky insect boards (20 cm × 25 cm). One bait was placed in the center of each trap and hung at 2 / 3 of the height of the licorice plant. Ten trapping points were set and the trapping time was 5 days. At the same time, the solvent dichloromethane was used as a control to evaluate the killing effect of licorice scale insects on male adults in the field.

[0044] The experimental results are as follows Figure 8 As shown, the methyl jasmonate attractant produced significantly higher catches than the dichloromethane control, reaching 20.58 ± 8.31 insects per trap, approximately double the capture rate of the dichloromethane control. Furthermore, statistics on the number of other insects captured revealed that these were non-natural enemy flies, with a low capture rate, demonstrating the lure's strong specificity.

[0045] This study, using molecular docking, electrophysiology, and insect behavior, identified two compounds with significant attractant activity against adult male licorice scale insects: methyl jasmonate and d-dihydrocarvone. Field evaluation of the trapping efficacy demonstrated that methyl jasmonate exhibited a significant trapping effect on adult male licorice scale insects. Therefore, methyl jasmonate can be used as an attractant for adult male licorice scale insects. Lures containing this attractant can be used to trap adult male licorice scale insects, reducing reliance on chemical pesticides and meeting the requirements of environmentally friendly pest control.

[0046] The purpose of the above embodiments is to specifically introduce the essential content of the present invention, but those skilled in the art should know that the protection scope of the present invention should not be limited to this specific embodiment.

Claims

1. Application of methyl jasmonate in the preparation of attractants for male adults of Glycyrrhiza uralensis scale.

2. The use of an attractant containing methyl jasmonate in trapping male adults of the licorice scale insect.

3. The use according to claim 2, characterized in that: The lure core is made of methyl jasmonate as an active ingredient and a carrier acceptable in the field of insect trapping, and is used to trap male adults of the licorice scale insect.

4. The use according to claim 3, characterized in that: The carrier is a natural desulfurized rubber stopper.

5. The use according to claim 4, characterized in that The preparation method of the lure comprises: firstly dissolving methyl jasmonate in an organic solvent to obtain a methyl jasmonate solution, then evenly dropping the methyl jasmonate solution onto a natural desulfurized rubber stopper to ensure uniform absorption, sealing and packaging with an aluminum film, and storing at a low temperature.

6. The use according to claim 5, characterized in that: The organic solvent is dichloromethane.

7. The use according to claim 5, characterized in that: The low temperature is 4°C.

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