Application of eugenol in preventing and treating schizaphis graminum

Abstract

This invention belongs to the field of plant protection, specifically relating to the application of eugenol in the control of wheat aphid. The research of this invention demonstrates the repellent, contact-killing, and fumigation effects of eugenol on the agricultural pest wheat aphid. The effects of eugenol on the growth and development of the agricultural pest wheat aphid were assessed using sex life table software, evaluating key parameters of wheat aphid growth and development and population changes at sublethal concentrations. The results showed that eugenol treatment can adversely affect the growth and development of wheat aphids, such as reducing survival and reproductive rates, and decreasing population size. Furthermore, eugenol has a certain synergistic effect with commonly used insecticides. Therefore, this invention has application value in the control of wheat aphids.

Description

Technical Field

[0001] This invention belongs to the field of plant protection, specifically relating to the application of eugenol in the control of wheat aphid. Background Technology

[0002] wheat aphid S. avenae The wheat aphid (Fabricius) belongs to the order Hemiptera and the family Aphidoidea. It is the dominant species of wheat aphid in my country, causing damage in all wheat-growing areas. It primarily feeds on wheat sap using its piercing-sucking mouthparts, causing wheat grains to shrivel, reducing grain weight and number, and also transmitting various plant viruses.

[0003] Currently, the control of wheat aphids mainly relies on chemical control. Chemical control is simple to operate and effective, and the most widely accepted chemical insecticides on the market mainly involve organophosphates, neonicotinoids, and pyrethroids. However, long-term use of single-type pesticides has led to varying degrees of resistance in aphids. Furthermore, while broad-spectrum chemical pesticides kill the target pests, they can also harm their natural enemies (such as ladybugs, lacewings, and parasitic wasps) and pollinators such as bees, thus disrupting the ecological balance.

[0004] Plant essential oils, as natural plant secondary metabolites, possess repellent, stomach poison, contact poisoning, attractant, antifeedant, and growth-inhibiting effects on pests, making them a preferred lead compound for new pesticide development. Compared to other chemical pesticides, plant essential oils are easily degraded, leave low residues, are less likely to induce resistance in pests, and have high safety for non-target organisms. The combined use of plant essential oils and chemical pesticides can also increase the toxicity of chemical insecticides. The main synergistic mechanism involves disrupting the insect's body surface structure to increase pesticide penetration; some plant essential oils and their active ingredients can inhibit the activity of enzymes related to pesticide resistance within pests, thereby weakening their detoxification ability. As synergists for chemical pesticides, plant essential oils can effectively reduce pesticide dosage, delay resistance development, and mitigate harm to the environment and non-target organisms, demonstrating significant economic and ecological benefits.

[0005] Traditionally, the control of wheat aphids has primarily relied on chemical control. However, long-term use or overuse of the same type of chemical pesticides has led to aphid resistance. Increased resistance inevitably leads to higher production costs. Meanwhile, chemical pesticides have high residue levels and are difficult to degrade, polluting the air and soil, and harming various non-target organisms, including plants, animals, beneficial soil microorganisms, and insects. Plant essential oils, on the other hand, are green, pollution-free, and highly safe, and are widely used in the food and cosmetics industries. Essential oils also have multi-target action, making it less likely for pests to develop resistance. Scientifically combining essential oils with existing chemical pesticides holds promise for improving pesticide penetration and effectiveness by disrupting the insect's body wall and inhibiting detoxification enzymes, providing a new pathway for reducing pesticide use while increasing efficiency. Summary of the Invention

[0006] In response to the current situation of high toxicity, high residue of chemical pesticides and the increasing resistance of pests, this invention develops a highly efficient, low-toxicity, safe and pollution-free plant-derived pesticide.

[0007] This invention clarifies the mechanism of action of eugenol on the major agricultural pest, the wheat aphid, and observes the effects of eugenol on the growth and development of the wheat aphid and its synergistic effect on commonly used insecticides.

[0008] This invention demonstrates the application of eugenol in the repellent, contact, and fumigation effects on the agricultural pest, the wheat aphid. The effects of eugenol on the growth and development of the wheat aphid were assessed using sex life table software, evaluating key parameters and population changes at sublethal concentrations. The results showed that eugenol treatment negatively impacted the growth and development of the wheat aphid, reducing survival and reproductive rates, and decreasing population size. The effects of eugenol on the body surface of the wheat aphid were studied using scanning electron microscopy, revealing that eugenol caused dehydration and shrinkage of the aphid's body surface, while also disrupting its structure. Eugenol also inhibited neurotrophic enzymes, detoxifying enzymes, and protective enzymes within the wheat aphid. Furthermore, eugenol exhibited a certain synergistic effect with commonly used insecticides.

[0009] Therefore, eugenol has the potential to be developed into a green pesticide. Eugenol has certain repellent, fumigation, and contact-killing effects on aphids; however, sublethal concentrations of eugenol treatment with wheat aphids lead to a decrease in their reproductive and survival rates, and a reduction in population size. Simultaneously, eugenol damages the waxy layer on the insect's body surface, causing increased dehydration and death. Eugenol also has a certain synergistic effect with commonly used insecticides. This opens up new possibilities for the integrated management of aphids.

[0010] Furthermore, the present invention is achieved through the following technical solution: First, this invention provides the application of eugenol in the control of wheat aphid.

[0011] The application described herein preferably aims to control wheat aphids through fumigation, repellency, or contact killing.

[0012] The preferred application involves preparing eugenol into an essential oil microemulsion, specifically using Tween 80 as a co-solvent; or dissolving eugenol in an acetone solution.

[0013] Meanwhile, this invention also provides the application of eugenol in combination with abamectin, thiamethoxam, flonicamid, or lambda-cypermethrin in the control of wheat aphid.

[0014] This invention also provides a compound pesticide for controlling wheat aphids, which is composed of eugenol and abamectin, thiamethoxam, flonicamid or cypermethrin.

[0015] The present invention also provides a method for preparing the compound agent described above. The method involves dissolving abamectin, flonicamid, thiamethoxam, or lambda-cyhalothrin technical grade in acetone to prepare a stock solution, which is then stored in the dark. Tween 80 is mixed with water and continuously stirred with a magnetic stirrer until the solution is completely homogeneous. The insecticide is then diluted with the prepared Tween solution to an insecticide solution of 1 to 625 mg / L. Add eugenol to the Tween solution to a final concentration of 0.01-0.10%, and stir continuously with a magnetic stirrer until the solution is completely homogeneous to obtain a Tween solution containing eugenol. This solution can then be used to dilute the insecticide solution.

[0016] The present invention also provides a method for controlling wheat aphids, wherein the method uses eugenol or the compound agent described above to control wheat aphids.

[0017] Furthermore, it can be applied to the plant environment through fumigation or spraying. For agricultural pests (excluding stored-grain pests), it can be applied appropriately in the field or used as a synergist in combination with other chemical insecticides.

[0018] The method for controlling wheat aphids is preferably applied at the early stage of wheat aphid infestation.

[0019] In this invention, the treatment method used to investigate the effects of eugenol on the fumigation, repellency, and growth and development of the wheat aphid is to prepare an essential oil microemulsion using 0.1% Tween 80 as a co-solvent under the action of a magnetic stirrer.

[0020] In this invention, the method used to treat the contact toxicity of eugenol against wheat aphid is to dissolve eugenol in an acetone solution.

[0021] In this invention, the main observation and photography equipment used for observing the effects of eugenol on the body surface of the wheat aphid is a scanning electron microscope (Hitachi Regulus 8100 SEM).

[0022] In this invention, the effect of eugenol on the growth and development of the wheat aphid is mainly analyzed by using sexual life table software to examine relevant parameters. Attached Figure Description

[0023] Figure 1 This shows the population prediction results of wheat aphid at different stages after eugenol treatment.

[0024] Figure 2 This is a scanning electron microscope image.

[0025] Figure 3 This shows the changes in the activity of key enzymes in the wheat aphid after eugenol treatment. Detailed Implementation

[0026] I. Experimental Materials and Methods 1. Test aphids The wheat aphid, collected in 2024 from the Hefei Academy of Agricultural Sciences in Anhui (31°49′21.30″N, 117°13′18.25″E), was continuously reared for multiple generations in insect rearing cages in a Beijing greenhouse and preserved as a breeding stock. The temperature was 20±1℃, the humidity was 60%~80%, and the light:dark ratio was 16 h : 8 h.

[0027] 2. Aphid breeding The wheat used for feeding is the aphid-susceptible variety Zhongmai 175.

[0028] 3. Test reagents Eugenol (99%) was purchased from Anhui Zesheng Anaiji Reagent Company. 4. Experimental design for repellency effect The repellency effect was determined in a closed environment using a Y-shaped olfactometer. Plant essential oils were diluted with 0.1% Tween 80 aqueous solution to concentrations of 6, 3, 1.5, 0.75, and 0.375 μL / mL, with the 0.1% Tween 80 aqueous solution serving as a control. The Y-shaped tube of the olfactometer had a main arm length of 10 cm, an inner diameter of 2.5 cm, and two side arms of 15 cm each, with an angle of 75° between the sidewalls. Before the experiment, the Y-shaped olfactometer was thoroughly cleaned with 75% anhydrous ethanol and dried to remove residual odors and other impurities. The sampler was connected to the Y-tube, and the atmospheric sampler was turned on, with the flow rate adjusted to 100 mL / min. Filter paper was cut into uniform shapes, and 200 μL of the control and treatment solutions were respectively placed on opposite sides of the Y-tube using a pipette. Untreated wheat seedlings were then placed for the aphids to feed on. Each experiment required the release of 30 adult wheat aphids, and the repellency rate was recorded 24 hours after the start of the experiment. Three biological replicates were set up for each concentration of each reagent. The entire experiment was conducted in the dark.

[0029] The avoidance activity is graded according to the avoidance rate: Grade 0, 0.01% to 0.1%; Grade I, 0.1% to 20%; Grade II, 20.01% to 40%; Grade III, 40.1% to 60%; Grade IV, 60.1% to 80%; Grade V, 80.1% to 100%.

[0030] 5. Experimental protocol for fumigation Fumigation activity was determined using a wide-mouth bottle sealed fumigation method. 0.1% Tween 80 was used as a co-solvent and control. Five concentrations of essential oil (10, 5, 2.5, 1.25, and 0.625 μL / mL) were prepared in a preliminary experiment. Wingless aphids (≥25 individuals) were collected in a wide-mouth bottle, and 50 μL of essential oil was pipetted onto a 2 cm × 2 cm filter paper, which was then suspended inside the bottle. Each treatment was repeated in triplicate, and mortality was recorded after 24 h.

[0031] 6. Test protocol for contact killing effect Contact toxicity was determined using the film-film method. Acetone was used as a co-solvent and control. Eugenol was prepared into six concentrations (1, 0.5, 0.25, 0.125, and 0.0625 μL / mL) in a preliminary experiment. 150 μL of the essential oil was added to a glass tube (1.5 cm in diameter and 6 cm in height) using a micropipette. The tube was placed on a roller mixer for two hours to allow the acetone to evaporate and the essential oil to form a uniform film adsorbed within the tube. The entire experiment was conducted in a fume hood. Each treatment was repeated in triplicate. Wingless aphids (≥25 individuals) were collected from each glass tube, and mortality was recorded after 3 hours.

[0032] 7. Life Table Experimental Protocol Eugenol was prepared into a microemulsion using a 0.1% Tween 80 aqueous solution with a magnetic stirrer. The microemulsion was then diluted to five concentration gradients: 6.000, 3.000, 1.500, 0.750, and 0.375 μL / mL. A 0.1% Tween 80 aqueous solution was used as a control. The toxicity was determined by the insect and leaf immersion method to identify the sublethal concentration.

[0033] Based on the toxicity test results, the sublethal concentration (LC50) was determined. 30 The optimal experimental concentration was determined by preparing eugenol with a concentration of 0.1% Tween 80 aqueous solution at an LC50 concentration. 30An emulsifier was used to raise first-instar aphids to adulthood. The adults were treated with the essential oil emulsifier, and first-instar nymphs of these adults were raised individually in 130 dishes. Wheat hydroponic seedlings (3-4 cm in length) were immersed in the emulsifier for 1 minute, then dried for the aphids to feed on. Observations were made daily, and the treated seedlings were replaced every two days with freshly treated seedlings. Molting and maturation times of nymphs were recorded. After maturity, the number of aphids laid was recorded, and nymphs were removed until the adults died. A sex life table for specific ages was established to calculate the developmental duration, survival rate, and number of aphids laid per female of the wheat aphid. The experiment was set up with three groups: a control group and a eugenol-treated group. The age-instar sex life table proposed by Chi and Liu was used to calculate the data. Based on data calculated from the sex life table and ignoring factors such as disease, predation, and parasitism, the population dynamics of wheat aphid (130 individuals / group) 60 days after eugenol disturbance were predicted using the TIMING-MSChart.

[0034] 8. Scanning Electron Microscopy Experimental Protocol Eugenol was prepared into a concentration of LC using a 0.1% Tween 80 aqueous solution. 50 Emulsifier: 3-5 wingless adult aphids treated for 24 h were placed in centrifuge tubes. The control group consisted of wingless adult aphids treated with 0.1% Tween 80 aqueous solution. 3% glutaraldehyde fixative was added to each centrifuge tube, and the samples were fixed at 4°C for 24 h. The fixed samples were washed with 0.1 mol / L PBS buffer (pH 7.2) for 15 min, repeated 3 times. The samples were then dehydrated in gradients of 10%, 30%, 50%, 70%, 80%, 90%, and 95% ethanol for 8 min at room temperature, followed by dehydration in 100% ethanol for 30 min. Ethanol was then replaced in three steps using mixtures of ethanol and tert-butanol in different ratios (3:1, 1:1, and 1:3, v / v), each step for 15 min, followed by replacement with 100% tert-butanol for 40 min. After drying, the samples were attached to the sample stage with conductive tape, sputtered with gold, and observed under a scanning electron microscope.

[0035] 9. Enzyme activity assay protocol Eugenol was prepared into a concentration of LC using a 0.1% Tween 80 aqueous solution. 30 LC 50 With LC 80 For emulsifier, 0.02 g of adult aphids of uniform size were collected after 8, 12, and 24 hours of treatment and placed in 1.5 mL centrifuge tubes. 180 mL of PBS buffer was added, and the aphids were thoroughly ground in liquid nitrogen at 5000 rpm / min. The supernatant was used as the enzyme solution to be tested. The enzyme activities of acetylcholinesterase (AChE), carboxylesterase (CarE), and catalase (CAT) were measured according to the relevant instructions of the enzyme-linked immunosorbent assay kit from Beijing Solarbio Science & Technology Co., Ltd.

[0036] 10. Synergistic effect experiment The toxicity of four commonly used insecticides (avermectin, flonicamid, thiamethoxam, and lambda-cyhalothrin) to the wheat aphid population was determined using the leaf-dip method. The technical grade insecticides were dissolved in acetone to prepare a stock solution, which was then stored in the dark. Tween 80 was then mixed with water at a ratio of 1:1000 and stirred continuously with a magnetic stirrer for at least 1 hour until the solution was completely homogeneous. The prepared Tween solutions were used to dilute the insecticides to concentrations of 1, 5, 25, 125, and 625 mg / L, with Tween 80 serving as a control. The preparation method for the synergistic solution is as follows: Tween 80 is mixed with water at a ratio of 1:1000, and eugenol is added to bring the final concentration of eugenol to 0.1%. The mixture is stirred continuously with a magnetic stirrer for at least 1 hour until the solution is completely homogeneous. The insecticide (1, 5, 25, 125, 625 mg / L) is diluted with the prepared Tween solution containing 0.1% eugenol. Wheat leaves infested with wingless aphids are immersed in the test solution for 5-10 seconds. The treated wheat leaves are then placed in plastic petri dishes lined with a layer of moist filter paper. Each concentration is repeated three times, ensuring that each petri dish contains at least 20 aphids. The synergistic effect is determined using the equivalent dose comparison method: R = LC of the single agent. 50 LC of the mixture 50 R>1 indicates synergistic effect, R<1 indicates antagonistic effect, and R~1 indicates no synergistic effect.

[0037] II. Experimental Results 1. Repellent effect results showed that the repellent effect of eugenol on wheat aphids gradually decreased with decreasing concentration. At eugenol concentrations of 6 and 3 μL / mL, the repellency rates were 86.7% and 64.4%, respectively, classified as Grade V and IV, with no significant difference between these two concentrations. At eugenol concentrations of 1.5 and 0.75 μL / mL, the repellency rates under sealed conditions were 31.1% and 15.6%, respectively, classified as Grade II and I. At a concentration of 0.375 μL / mL, there was no repellent effect under sealed conditions, indicating an attraction effect.

[0038] Table 1: Repellent effect of eugenol on wheat aphid in a closed environment (mean ± standard error)

[0039] Note that different lowercase letters indicate significant differences in repellent effects between different concentrations of the same essential oil (P<0.05). 2. Fumigation results showed that eugenol has fumigation toxicity against the wheat aphid. The toxicity regression line of fumigation was analyzed, and the LC50 of eugenol against the wheat aphid was determined. 50 It was 7.0419 uL / mL.

[0040] Table 2: Fumigation toxicity equation of eugenol against wheat aphid

[0041] The contact toxicity test results showed that eugenol has a contact toxicity effect on the wheat aphid, and the LC-15 toxicity test curve showed that... 50 It is 0.2455 uL / mL.

[0042] Table 3: Equation of contact toxicity of eugenol against wheat aphid

[0043] 3. Effects of eugenol on growth and development factors related to the wheat aphid Eugenol-treated wheat aphids were able to complete their life cycle, with the nymphal lifespan significantly prolonged compared to the control group. Adult lifespan, reproductive period, aphid production, and the pre-spawning phase were significantly shortened. The pre-spawning phase was significantly prolonged.

[0044] Table 4: Effects of eugenol on the developmental stages of the wheat aphid

[0045] Note: Data in the table are mean ± standard error; different letters after the data in the same row indicate significant differences (P < 0.05, paired repeated sampling). Compared with the control group, the intrinsic growth rate (r) of the wheat aphid treated with eugenol decreased by 0.08, the net growth rate (R0) decreased by 9.56, and the weekly growth rate (λ) decreased by 0.08, all of which were statistically significant (P<0.05). Furthermore, compared with the control group, the average generation cycle of the eugenol-treated group was significantly shortened by 1.26 days (P<0.05). The wheat aphid has paid a certain fitness cost to adapt to the eugenol environment.

[0046] Table 5: Effects of eugenol and eugenol treatment on life table parameters of wheat aphid populations

[0047] Note: Data in the table are mean ± standard error; different letters after the data in the same row indicate significant differences (P < 0.05, paired repeated sampling). Population projections for the wheat aphid were made based on age-instar sex life table data. The results showed that eugenol treatment effectively suppressed the population growth of the wheat aphid (see [link to relevant documentation]). Figure 1 The population growth rate of wheat aphids treated with eugenol was slow, with a predicted population size of approximately 1,614 individuals at 60 days, compared to approximately 80,000 individuals in the control group at 60 days.

[0048] 4. Changes in the body surface of the wheat aphid after eugenol treatment Depend on Figure 2It is known that eugenol treatment causes the wheat aphid to lose water and shrink, and the waxy layer on its body surface is damaged.

[0049] 5. Effects of eugenol on the activity of key enzymes in the wheat aphid Depend on Figure 3 It can be seen that, after treatment with different concentrations and for different durations, the enzyme activity of key enzymes related to resistance in wheat aphids decreased compared with the control group.

[0050] 6. Synergistic effect of eugenol on commonly used chemical insecticides

[0051] Eugenol has a certain synergistic effect on commonly used insecticides. As shown in Table 6, the synergistic ratios of eugenol to abamectin and thiamethoxam are 1.34 and 1.42, respectively; and to flonicamid and cypermethrin are 3.31 and 3.10, respectively.

[0052] Table 6: Synergistic effect of eugenol on chemical insecticides In summary, eugenol exhibits multiple mechanisms of action against the wheat aphid, and can be used in production as a repellent, insecticide, and synergist. Eugenol can influence the behavior and feeding of wheat aphids; simultaneously, it can enhance the penetration and effectiveness of chemical insecticides by damaging the aphid's body wall and inhibiting detoxification enzymes, providing a new pathway for achieving reduced pesticide use and increased efficiency.

Claims

1. Application of eugenol in the control of wheat aphid.

2. The application as described in claim 1, characterized in that, The purpose of controlling wheat aphids is to use fumigation, repellency, or contact killing.

3. The application as described in claim 2, characterized in that, Eugenol can be formulated into an essential oil microemulsion, specifically using Tween 80 as a co-solvent; or eugenol can be dissolved in an acetone solution.

4. Application of eugenol in combination with abamectin, thiamethoxam, flonicamid or high-efficiency cypermethrin in the control of wheat aphid.

5. A compound pesticide for controlling wheat aphids, characterized in that, It is composed of eugenol and abamectin, thiamethoxam, flonicamid or high-efficiency cypermethrin.

6. The method for preparing the compound drug as described in claim 5, characterized in that, Dissolve abamectin, flonicamid, thiamethoxam, or lambda-cyhalothrin technical in acetone to prepare a stock solution and store it away from light; mix Tween 80 with water and stir continuously with a magnetic stirrer until the solution is completely homogeneous; dilute the insecticide with the prepared Tween solution to an insecticide solution of 1 to 625 mg / L. Add eugenol to the Tween solution to a final concentration of 0.01-0.10%, and stir continuously with a magnetic stirrer until the solution is completely homogeneous to obtain a Tween solution containing eugenol. This solution can then be used to dilute the insecticide solution.

7. A method for controlling wheat aphids, characterized in that, It uses eugenol, or the compound agent as described in claim 5, to control wheat aphids.

8. The method as described in claim 7, characterized in that, It is applied to the plant environment through fumigation or spraying.

9. The method as described in claim 8, characterized in that, Apply the treatment at the early stage of the wheat aphid infestation.

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