Codling moth oviposition repellent and application thereof

Abstract

The invention relates to the technical field of agricultural pest repellent agents, in particular to a codling moth oviposition repellent agent and application thereof. The codling moth oviposition repellent comprises an active component, wherein the active component is selected from one or more of palmitic acid and linoleic acid. The repellent provided by the invention has a good oviposition repellent effect on female insects, and has a good development prospect and application value in the aspect of prevention and control of codling moths.

Description

Technical Field

[0001] This invention relates to the field of repellents for agricultural pests, and more specifically, to a repellent for the oviposition of the codling moth and its application. Background Technology

[0002] The codling moth (Cydiapomonella), belonging to the family Lepidoptera and the family Cydiapodidae, is one of the major invasive agricultural pests in my country. Originating in south-central Eurasia, it has rapidly spread to almost all apple-producing regions worldwide. Due to its overlapping generations, adaptability, resistance, and strong reproductive capacity, it is highly susceptible to outbreaks under suitable conditions. The larvae bore into the fruit, typically causing fruit damage exceeding 50%, and in severe cases reaching 70%–100%, resulting in significant agricultural economic losses. Currently, production relies mainly on chemical control for the codling moth, but the long-term and frequent use of insecticides has led to pesticide resistance, pesticide residues, environmental pollution, and harm to natural enemies. Therefore, there is an urgent need to develop new, environmentally friendly, and highly effective control technologies for the codling moth.

[0003] Insect oviposition selection is an adaptive strategy developed over a long period of evolution, playing a crucial role in the survival and reproduction of species. Utilizing insect oviposition preferences for pest control is a relatively new technology that has emerged in recent years. While significant progress has been made in the screening of attractants, the screening of repellents is becoming increasingly urgent in the control of agricultural pests. Substances that repel insect oviposition are called insect oviposition deterring pheromones (ODPs). Developing novel, green, and highly efficient pest behavior regulators based on these ODPs can effectively reduce pest damage, possessing high potential development value and broad application prospects.

[0004] The codling moth is selective in its oviposition sites in the field, typically avoiding laying eggs in areas where larvae cause damage. Therefore, developing an insect repellent using extracts from the excrement of codling moth larvae as the main component plays a crucial role in the green control of this pest. Currently, research reports on oviposition repellents for the codling moth are scarce. Summary of the Invention

[0005] This invention provides an oviposition repellent for codling moths and its application, aiming to provide an environmentally friendly and highly specific biological control method that can effectively control codling moths, with low control costs and environmental friendliness.

[0006] To achieve the above objectives, the present invention is implemented through the following technical solution:

[0007] In a first aspect, the present invention provides an oviposition repellent for the codling moth, comprising an active component selected from one or more palmitic acid and linoleic acid.

[0008] In a preferred embodiment, the palmitic acid and linoleic acid are derived from the excrement of codling moth larvae, and the active ingredient is an extract of codling moth larvae excrement.

[0009] In the preferred embodiment, the feces of codling moth larvae are impregnated with hexane, the liquid is collected, and the volume is adjusted with hexane to obtain an extract of codling moth larvae feces.

[0010] In a preferred embodiment, the soaking time is 0.8–1.2 h; 0.2–0.5 g of codling moth larvae excrement is extracted and diluted to a final volume to obtain 100 mL of codling moth larvae excrement extract.

[0011] In the preferred embodiment, 0.2g of codling moth larvae excrement is extracted and diluted to a final volume to obtain 100mL of codling moth larvae excrement extract.

[0012] In the preferred embodiment, the concentration of palmitic acid is 0.35 μg / μL and the concentration of linoleic acid is 1 μg / μL.

[0013] In a preferred embodiment, the repellent further includes a solvent, wherein the solvent is n-hexane.

[0014] In a second aspect, the present invention provides the application of the above-mentioned codling moth oviposition repellent in the control of codling moth.

[0015] In the preferred embodiment, the above application is used to repel the codling moth from laying eggs at the application site.

[0016] In the preferred embodiment, the active components palmitic acid and / or linoleic acid are used to repel the oviposition of mated female adults in the above application.

[0017] The beneficial effects of this invention are as follows:

[0018] This invention uses hexane as an extractant to extract codling moth larvae excrement extract from the excrement of codling moth larvae. This extract can be used to control codling moths. The active components have been identified as palmitic acid or linoleic acid. The codling moth larvae excrement extract effectively repels codling moth oviposition, showing good repellency towards female moths. The preparation method is simple and easy to implement, and the application method is convenient and straightforward. It has excellent development and application prospects and is valuable in the preparation of insecticides and the control of codling moths. Attached Figure Description

[0019] Figure 1 This is a schematic diagram of the experimental design scheme for the oviposition selection of the codling moth of the present invention;

[0020] Figure 2 This is the oviposition selection of female adult codling moths under treatment with extracts of codling moth larvae in different solvents according to Example 1 of the present invention;

[0021] Figure 3 This is the oviposition inhibition effect of insect excrement extracts in different solvents on adult female codling moths in Example 1 of the present invention;

[0022] Figure 4 This is Example 2 of the present invention, which describes the oviposition selection of female adult codling moths under different concentrations of n-hexane insect excrement extract treatment;

[0023] Figure 5 This is a TIC diagram of artificial feed for codling moths and n-hexane extract of insect excrement from Example 3 of the present invention;

[0024] Figure 6 This is Example 4 of the present invention, which describes the oviposition selection of female adult codling moths treated with palmitic acid and linoleic acid.

[0025] Figure 7 This is the EAG reaction of the codling moth of the apple tree to the hexane insect excrement extract and active ingredient in Example 5 of the present invention. Detailed Implementation

[0026] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0027] In this invention, unless otherwise stated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Unless otherwise specified, the experimental methods in the embodiments are conventional methods. Where specific conditions are not specified in the embodiments, they should be performed under conventional conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0028] Example 1: Effects of different solvents on larval excrement extracts on oviposition rate of codling moth

[0029] Excrement from 5-day-old codling moth larvae was collected daily, sealed, and stored at -18℃ for later use. 2.5g of larval excrement was added to 30mL of different solvents (acetone, n-hexane, methanol, dichloromethane, and ethanol), stirred, and soaked for 60min. The soaking solution was repeatedly extracted and diluted to 50mL with the same organic solvent, resulting in five extracts with a concentration of 2.5g / 50mL. An indoor oviposition experiment was conducted to test the effect of the five different solvent extracts on the oviposition rate of the codling moth. Newly emerged female moths that had mated for two nights were selected as experimental moths, and two treatment methods were applied respectively. Figure 1As shown, 2 mL of the extract was applied to the upper part (treatment 1) and lower part (treatment 2) of a disposable plastic cup (3 replicates each). The corresponding solvent was used as a control. After the solvent evaporated, 5 test moths were placed in the cup 2 hours before oviposition, and honey water was provided. The number of eggs in the treatment and control areas was counted after 24 hours. Each experiment was repeated 6 times. The oviposition avoidance rate was calculated as (egg count in the control area - egg count in the treatment area) / (egg count in the control area + egg count in the treatment area) × 100%, which was used to evaluate the oviposition avoidance activity of the extract. The oviposition inhibition index (ODI) was calculated as follows: ODI = (N... 对照 -N 处理 ) / (N 对照 +N 处理 ODI (Optimal Displacement Index) values ​​are used to evaluate the oviposition-repelling effect on adult insects; among which, N 处理 To handle the number of eggs in the treated area, N 对照 The number of eggs laid in the control area is shown. When ODI > 0, it indicates that the tested extract has a more significant attraction and repulsion effect on adult oviposition; when ODI < 0, it indicates that the tested extract has an attraction effect on adult oviposition; when ODI ≈ 0, it indicates that the tested extract has no activity.

[0030] The oviposition preferences of female codling moths were studied using indoor oviposition experiments under different solvent treatments with larval excrement extracts. Figure 2 The results showed that, regardless of whether the upper part (treatment 1) or the lower part (treatment 2) of the oviposition cup was treated, adult female codling moths showed no oviposition preference for ethanol-based insect excrement extract (treatment 1: P = 0.957; treatment 2: P = 0.500), acetone-based insect excrement extract (treatment 1: P = 0.522; treatment 2: P = 0.054), dichloromethane-based insect excrement extract (treatment 1: P = 0.380; treatment 2: P = 0.938), and methanol-based insect excrement extract (treatment 1: P = 0.970; treatment 2: P = 0.451). However, between the control and n-hexane-based insect excrement extract, the codling moths preferred to lay eggs in the control area (treatment 1: P = 0.0001; treatment 2: P = 0.0001). Note: Figure 2 In the figure, * indicates a significant difference (independent samples t-test, *p<0.05, **p<0.01, ***p<0.001).

[0031] The inhibitory effects of larval excrement extracts in different solvents on oviposition of female codling moths of the codling moth are shown in [reference needed]. Figure 3The results showed that acetone-based insect excrement extract (treatment 1: P = 0.938; treatment 2: P = 0.467) had some oviposition-attracting activity compared to the control, but it was not significant; ethanol-based insect excrement extract (treatment 2: P = 0.640), dichloromethane-based insect excrement extract (treatment 1: P = 0.287; treatment 2: P = 0.950), and methanol-based insect excrement extract (treatment 1: P = 0.696; treatment 2: P = 0.344) had some inhibitory activity compared to the control, but it was not significant; while n-hexane-based insect excrement extract had significant oviposition-inhibiting activity, with oviposition inhibition indices of 0.732 (P = 0.0002) and 0.584 (P = 0.0001) for treatment 1 and treatment 2, respectively. Note: Figure 3 In the figure, * indicates a significant difference (paired t-test, *p<0.05, **p<0.01, ***p<0.001).

[0032] The effects of different solvent-based larval excrement extracts on the oviposition rate of female codling moths are shown in Table 1. The oviposition rate of codling moths in the hexane-based larval excrement extract treatment area was 7.60 ± 1.13, significantly lower than that in the control area. The oviposition avoidance rate and oviposition inhibition index were 67.30% and 0.66, respectively, indicating that only the hexane-based larval excrement extract had a strong inhibitory effect on oviposition of female codling moths.

[0033] Table 1. Effects of codling moth larval excrement extract on adult oviposition.

[0034]

[0035] Note: Data in the table are mean ± standard error. * indicates that the treatment and control were significantly different after an independent samples t-test.

[0036] Example 2: Effects of different concentrations of n-hexane larval excrement extract on the oviposition rate of female codling moths.

[0037] Weigh out 1g, 2g, and 3g of codling moth larvae excrement, respectively. Place each sample of excrement in hexane, stir, and soak for 60 minutes. Dilute the soaked solution to 1L to prepare hexane-based larval excrement extracts at concentrations of 1g / L, 2g / L, and 3g / L. Select newly emerged female moths that have mated for two nights as test moths. Apply 2mL of the extract to the top and bottom of a disposable plastic cup. After complete evaporation, place five test moths in the cup two hours before oviposition, providing honey water. Count the oviposition rate in both the treatment and control areas after 24 hours.

[0038] Treatment with different concentrations of hexane-based insect excrement extracts all mitigated the oviposition selection of female adult codling moths. Figure 4Compared with the control, the number of eggs laid by adult female codling moths in the 1.0 g / L treatment area was less than that in the control, but there was no significant difference between the two (P = 0.0706); the number of eggs laid in the 2.0 g / L (P = 0.0353) and 3.0 g / L (P = 0.0231) treatment areas was significantly less than that in the control.

[0039] The oviposition rates of *Copterocarpus macrocarpus* treated with 1.0 g / L, 2.0 g / L, and 3.0 g / L hexane insect excrement extract were 13.67 ± 2.96, 8.67 ± 4.26, and 10.00 ± 2.08, respectively, with oviposition avoidance rates of 35.96%, 60.81%, and 54.14%, and oviposition inhibition indices of 0.35, 0.56, and 0.54, respectively. This indicates that the 2.0 g / L hexane insect excrement extract had the strongest inhibitory effect on oviposition of female *Copterocarpus macrocarpus* moths.

[0040] Table 2. Effects of different concentrations of n-hexane insect excrement extract on the oviposition rate of adult codling moths.

[0041]

[0042] Example 3: Identification of active ingredients in hexane-containing larval excrement extract

[0043] The sample to be tested in this embodiment is the hexane larval excrement extract prepared in Example 1 using hexane as the extractant and solvent. The preparation method of the artificial feed sample includes taking artificial feed of codling moths; the formula and preparation method of the artificial feed are described in patent application CN201910839547.3. The artificial feed sample is prepared according to the process in Example 1 for preparing the hexane larval excrement extract. Specifically, the detection process is as follows:

[0044] The GC-MS system used was an Agilent 7890B, with an HP-5 column (30 mm × 0.32 mm × 0.25 μm, Agilent), and nitrogen as the carrier gas (25 mL / min). The temperature program was as follows: initial temperature 40 °C, ramped at 3 °C / min to 80 °C with no retention, then ramped at 5 °C / min to 260 °C and held for 15 min, with a 1 μL injection volume, an injector temperature of 250 °C, and an FID detector temperature of 260 °C. The mass spectrometry analysis conditions were: electron impact (EI) ion source, electron energy 70 eV, nitrogen as carrier gas (1.0 mL / min), mass spectrometry ion source temperature of 250 °C, and an injection volume of 1 μL. The NIH / EPA / MS / DS computer spectral library was used for searching, and the temperature program was the same as for the GC-MS.

[0045] GC-MS results showed that the hexane-based insect excrement extract exhibited significant and strong peaks at retention times of 38.96 min and 42.35 min, while these peaks were absent in the artificial feed. This indicates that the substances corresponding to these two peaks are secreted by larvae into the excrement. Mass spectrometry identified these two peaks as palmitic acid and linoleic acid, respectively. Figure 5 ).

[0046] Example 4: Indoor activity determination of active ingredients in hexane-containing larval excrement extract

[0047] An oviposition avoidance test was conducted on the codling moth using an oviposition selection method. Newly emerged female moths that had mated for two nights were selected as test moths. 2 mL of the active ingredient linoleic acid (1 μg / μL) or palmitic acid (0.35 μg / μL) was applied to the upper and lower parts of a disposable plastic cup (3 replicates each). After the solvent evaporated, 5 test moths were placed in the cup 2 hours before oviposition, and honey water was provided. (See illustration) Figure 1 The number of eggs in the control group and the data were statistically analyzed after 24 hours. Each experiment was repeated 6 times.

[0048] like Figure 6 As shown, the results indicate that in the indoor experiment, the number of times the codling moth chose to lay eggs in the control group was significantly higher than the number of times it chose to lay eggs in the treatment group (P<0.05). This suggests that the female codling moth changes its choice of oviposition location after sensing the active ingredient.

[0049] Example 5: Antennae Potential Response Experiment (EAG)

[0050] Electrophysiological responses of male and female adult codling moths to insect excrement extracts and active ingredients were determined using EAG assays. The specific procedures were as follows: Using hexane as a control, the EAG responses of hexane-based insect excrement extract (2 g / L), linoleic acid (1 μg / μL), and palmitic acid (0.35 μg / μL) were tested. Codling moth antennae were cut off at the base, with 0.5–1 mm removed from both ends. The antennae were then fixed to electrodes with conductive adhesive, and the EAG probe was inserted. The instrument was adjusted until the baseline stabilized before measurement. 1 μL of the test substance was dropped onto filter paper. After the hexane evaporated, the sample was placed in a Pasteur tube. A filter paper strip with an equal volume of hexane was used as a control. Ten active antennae were tested for each substance (one antenna per insect). Each treatment was repeated three times, with a 30-second interval between stimulations. Control measurements were performed before and after each substance measurement. The relative EAG response value was calculated as: |EAG measurement value (mv)| - mean response value before and after the control measurements (mv).

[0051] like Figure 7As shown, the EAG response results indicated that the highest relative EAG response value (1.55 ± 0.08 mV) in 3-day-old female adults (mated) to hexane-based insect excrement extract was significantly higher than that in male adults and 1-day-old female adults (unmated) (F = 254.83, P < 0.001). Compared with hexane treatment, the EAG response of male adults to insect excrement extract was significantly reduced (t = 3.47, P = 0.0055), while the EAG response of 1-day-old female adults (unmated) showed no significant change (t = 1.46, P = 0.18). However, the relative EAG response of 3-day-old female adults (mated) to insect excrement extract was significantly increased (t = 15.64, P = 0.0001), indicating that mated female adults have a strong electrophysiological response to insect excrement extract. Figure 7 A). Male adults and 1-day-old unmated females showed the highest EAG relative response values ​​to palmitic acid, at 0.37±0.04 and 0.41±0.03 mv respectively, significantly higher than those to hexane and linoleic acid. 3-day-old mated female adults showed the highest EAG relative response value to linoleic acid, at 1.24±0.02 mv, significantly higher than the control, indicating that 3-day-old mated females have a strong electrophysiological response to the active ingredient linoleic acid. Figure 7 B~7D). Note: Figure 7 In the table, data are mean ± standard error; A: Different lowercase and uppercase letters on the column indicate significant differences in EAG response values ​​of different adults to hexane and hexane excrement extract, respectively (P < 0.05) (Tukey method); * indicates significant differences in EAG response values ​​between female and male adults to the same substance (independent samples t-test, *, P < 0.05; **, P < 0.01; ***, P < 0.001). BD: * indicates significant differences in EAG response values ​​between adult treatments and controls (independent samples t-test, *, P < 0.05; **, P < 0.01; ***, P < 0.001).

[0052] This invention discloses the application of larval excrement extract as a repellent in the control of the codling moth. The hexane extract of codling moth larvae provided by this invention exhibits good repellent effect against oviposition by female moths and can be used for the control of the codling moth. The hexane extract of larval excrement provided by this invention effectively repels oviposition by the codling moth and has promising development and application prospects, possessing application value in the preparation of insecticides and the control of the codling moth.

[0053] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A repellent for oviposition of the codling moth, characterized in that, It includes an active component, which is selected from one or more of palmitic acid and linoleic acid.

2. The codling moth oviposition repellent according to claim 1, characterized in that, The palmitic acid and linoleic acid are derived from the excrement of codling moth larvae, and the active ingredient is an extract of codling moth larvae excrement.

3. The codling moth oviposition repellent according to claim 2, characterized in that, After the excrement of codling moth larvae was impregnated with hexane, the liquid was collected and diluted to volume with hexane to obtain an extract of codling moth larvae excrement.

4. The codling moth oviposition repellent according to claim 3, characterized in that, The soaking time is 0.8–1.2 h; 0.2–0.5 g of codling moth larvae excrement is extracted and diluted to a final volume to obtain 100 mL of codling moth larvae excrement extract.

5. The codling moth oviposition repellent according to claim 4, characterized in that, Each 0.2g of codling moth larvae excrement was extracted and diluted to a final volume to obtain 100mL of codling moth larvae excrement extract.

6. The codling moth oviposition repellent according to claim 1, characterized in that, The concentration of palmitic acid was 0.35 μg / μL, and the concentration of linoleic acid was 1 μg / μL.

7. The oviposition repellent for the codling moth according to claim 1 or 2, characterized in that, The repellent also includes a solvent, wherein the solvent is n-hexane.

8. The application of the oviposition repellent of the codling moth according to any one of claims 1 to 7 in the control of the codling moth.

9. The application according to claim 8, characterized in that, Used to repel codling moths from laying eggs at the application site.

10. The application according to claim 9, characterized in that, The active components palmitic acid and / or linoleic acid are used to repel the oviposition of mated female adults.

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