Application of ursolic acid lactone in prevention and treatment of spodoptera exigua

Abstract

The application provides application of ursolic acid lactone in prevention and treatment of Spodoptera exigua, and relates to the technical field of pest control. The ursolic acid lactone can produce significant antifeedant effect on Spodoptera exigua at a certain concentration, can inhibit the growth of Spodoptera exigua larvae to a certain extent, and can even cause the death of Spodoptera exigua larvae, and can be used as a new Spodoptera exigua control agent. The present application overcomes the shortcomings of the prior art, and the plant source extract component, i.e. ursolic acid lactone, can effectively control Spodoptera exigua, and the component is green and safe, reduces environmental pollution, and has a good prospect in the development of Spodoptera exigua control agents.

Description

Technical Field

[0001] This invention relates to the field of pest control technology, specifically to the application of ursolic acid lactone in the control of beet armyworm. Background Technology

[0002] The beet armyworm is one of the world's most widely distributed agricultural pests, with over 170 host species, including beets, corn, onions, peanuts, Chinese cabbage, and roses. Beet armyworm outbreaks are most prevalent in autumn due to the low rainfall and mild climate, which are ideal for its growth and reproduction. Therefore, the beet armyworm population in autumn accounts for 61% to 96% of the annual total. Its larvae primarily feed on leaves, tender stems, flower buds, and petals. Young larvae gnaw on the leaf tissue, creating transparent window spots, while older larvae voraciously feed on leaves, causing notches or holes. In severe cases, only the veins remain, and the larvae can also cause malformed flower buds that fail to open, directly impacting the quality of cut flowers.

[0003] Currently, the control of beet armyworm mainly relies on chemical pesticides. However, the long-term and large-scale use of chemical pesticides has led to pesticide resistance in beet armyworms and reduced efficacy of major insecticides, while also causing environmental pollution. Pesticide resistance has become a major challenge in beet armyworm control, necessitating the search for new, green control technologies to reduce reliance on chemical methods.

[0004] Dehydro(11,12)ursolic acid lactone (DUA) is a triterpenoid compound extracted from a specific plant, with the molecular formula C2. 30 H 46 Currently, research on ursolic acid lactone mainly focuses on its antibacterial and antiviral properties, without addressing its role in pest control. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides the application of ursolic acid lactone in the control of beet armyworm. Specifically, the use of ursolic acid lactone can effectively induce beet armyworm to refuse to eat and can limit the growth and development of beet armyworm larvae, thus achieving a good control effect.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] The application of ursolic acid lactone in the control of beet armyworm, wherein the ursolic acid lactone is used to limit the development of beet armyworm, kill beet armyworm, or inhibit beet armyworm feeding.

[0008] Preferably, the ursolic acid lactone has an effective inhibitory concentration (EC) of 100 mg / L in inhibiting the beet armyworm. 50 It is 0.015 mg / mL.

[0009] Preferably, the concentration of the ursolic acid lactone in inhibiting feeding by the beet armyworm is ≥0.1 mg / mL.

[0010] Preferably, the application method involves dissolving ursolic acid lactone in a solvent and then spraying it onto crops to control beet armyworm.

[0011] Preferably, the concentration of the ursolic acid lactone dissolved in the solvent is ≥0.01 mg / mL.

[0012] This invention provides the application of ursolic acid lactone in the control of beet armyworm, and its advantages compared with the prior art are:

[0013] In this invention, ursolic acid lactone exhibits a good antifeedant effect against beet armyworm at low concentrations, and the antifeedant rate increases with increasing concentration, demonstrating a clear dose-response relationship. Specifically, at a concentration of 0.1 mg / mL, the antifeedant rate against beet armyworm exceeds 60%, indicating a significant inhibitory effect. Furthermore, when beet armyworms consume food containing a certain concentration of ursolic acid lactone, their growth and development are significantly restricted, and they may even die, demonstrating the sustained sublethal effect of ursolic acid lactone on insect development. Moreover, ursolic acid lactone is a plant-derived extract, making it green and safe, and suitable for long-term control of beet armyworm. Attached Figure Description

[0014] Figure 1 This is a schematic diagram illustrating the effect of ursolic acid lactone of the present invention on the growth and development of beet armyworm;

[0015] Figure 2 This is a schematic diagram illustrating the effect of ursolic acid on the growth and development of the beet armyworm according to the present invention.

[0016] Figure 3 This is a schematic diagram illustrating the effect of different concentrations of ursolic acid lactone on the activity of acetylcholinesterase in beet armyworm.

[0017] Figure 4 This is a schematic diagram illustrating the effect of different concentrations of ursolic acid on the acetylcholinesterase activity in beet armyworm.

[0018] Figure 5 The present invention relates to ursolic acid lactone (DUA) EC. 50 Schematic diagram showing the effect of concentration on the activity of carboxylesterase in beet armyworm;

[0019] Figure 6 This invention relates to ursolic acid (UA) EC 50 Schematic diagram showing the effect of concentration on the activity of carboxylesterase in beet armyworm. Detailed Implementation

[0020] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. 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 are within the scope of protection of the present invention. Example:

[0021] Ursolic lactone (DUA) (purchased from Wuhan Tianzhi Biotechnology Co., Ltd.) was used as the experimental compound, and ursolic acid (UA) (Bailinwei) was used as the control compound.

[0022] 1. Effects of two compounds on the feeding behavior of the beet armyworm:

[0023] (1) The test was conducted under the following conditions: relative humidity of 70%, experimental temperature of (26±1)℃, and photoperiod of (L12:D12).

[0024] After cleaning and drying the cabbage leaves, use a hole punch to create leaf discs with a diameter of 3 cm. Then, dissolve the compound in a 50% ethanol aqueous solution to prepare a specific concentration, and use the leaf immersion method to test the feeding behavior of beet armyworm larvae.

[0025] (2) Beet armyworms were fed cabbage leaves, and then 3rd instar larvae of similar size and weight were selected. After 8 hours of starvation, a double-selection biological test was performed: a leaf dish (containing the test compound (T); a leaf dish soaked in deionized water (CK)) was placed on either side of each petri dish, and a larva was introduced into the center of each dish. One day later, the details of the larvae feeding on the leaves were recorded: a transparent grid paper (1 mm) was used to... 2 Lay the leaves flat on a table, place them on top of grid paper, and trace the holes eaten by beet armyworm larvae with a pencil, calculating the area. Divide the same drug solution into groups of 15, with 3 parallel groups. Calculate the feeding rejection rate by the area of ​​the eaten leaves.

[0026] Refusal rate = (Leaf area consumed by control group - Leaf area consumed by treatment group) / (Leaf area consumed by control group + Leaf area consumed by treatment group) × 100%;

[0027] The specific results are shown in Table 1 below:

[0028] Table 1

[0029]

[0030] A significant inhibitory effect on beet armyworm feeding was defined as a refusal rate of ≥60%. Ursolic acid lactone achieved a refusal rate of 68.05% at a concentration of 0.1 mg / mL, while ursolic acid, a known insecticidal ingredient, showed a refusal rate of less than 60% against beet armyworm at a higher concentration of 2.0 mg / mL. Therefore, ursolic acid lactone has a better refusal effect on beet armyworm feeding than ursolic acid.

[0031] 2. Determination of the effects of ursolic acid lactone and ursolic acid on the growth and development of beet armyworm larvae:

[0032] The leaf immersion method was used, in which cabbage leaves were completely immersed in ursolic acid and ursolic acid solutions of different concentration gradients for 30 seconds. After immersion, the leaves were removed and air-dried in a clean bench until no obvious droplets remained on the surface before being placed in finger tubes. Third-instar beet armyworm larvae of uniform physiological condition were selected and subjected to 8 hours of starvation. Their initial body weight was measured and recorded using an electronic balance. Thirty third-instar beet armyworm larvae were placed in finger tubes containing cabbage leaves immersed in different concentrations of the compounds, with parallel control experiments also conducted.

[0033] The tube opening was covered with double layers of sterile gauze and sealed with a rubber ring to ensure ventilation and prevent insect escape. Fresh treated leaf discs were replaced every 24 hours after treatment began, and insect excrement was removed simultaneously. The weight changes of surviving larvae were measured at fixed time points. Three biological replicates were set up for each group; the dose-time effects of different concentrations of ursolic acid lactone and ursolic acid on the growth and development of beet armyworm larvae were determined after 6 days of continuous rearing.

[0034] The results of the treatment with ursolic acid lactone are shown below. Figure 1 In treatment with 0.01 mg / mL ursolic acid lactone, the daily average body weight gain rate of larvae decreased, with an inhibition rate of 37.06% on day 4. Significant larval developmental retardation was observed. When the concentration was increased to 0.1 mg / mL, the biotoxicity of ursolic acid lactone significantly increased, with an inhibition rate of 44.44% on day 6, and the inhibition rate increased with increasing concentration. Exposure to ursolic acid lactone led to progressive metabolic disturbances in larvae. Beet armyworm larvae died during the experiment, with mortality accelerating with increasing concentration; dead individuals often exhibited typical metabolic poisoning symptoms such as body blackening and lethargy. Surviving beet armyworms also showed inhibited growth and development; these larvae pupated and subsequently emerged as smaller moths. This indicates that ursolic acid lactone has a sustained sublethal effect on insect growth and development.

[0035] The results of ursolic acid treatment for beet armyworm are shown in Figure 2The effects of feeding different concentrations of ursolic acid on the body weight of beet armyworms were investigated. As the concentration increased, the rate of weight gain decreased. At a ursolic acid concentration of 1.0 mg / mL, the growth rate of beet armyworms slowed over time, reaching a maximum inhibition rate of 41.21% on day six. At a ursolic acid concentration of 2.0 mg / mL, the growth rate significantly decreased over time, reaching an inhibition rate of 50.25% on day six. These results indicate that the resistance of beet armyworms increases with increasing treatment concentration. During the experiment, beet armyworm larvae died during the experiment, with the mortality rate increasing with increasing concentration. The surviving beet armyworms also exhibited inhibited growth and development. These larvae pupated and then emerged as small moths.

[0036] The above tests show that ursolic acid lactone can effectively inhibit the growth and development of beet armyworm at relatively low concentrations.

[0037] 3. Toxicity tests of ursolic acid lactone and ursolic acid against beet armyworm:

[0038] Following the experimental setup described above, different concentrations of ursolic acid lactone and ursolic acid were used to determine the EC50 effect on the beet armyworm. 50 The values ​​and results are shown in Table 2 below:

[0039] Table 2

[0040]

[0041] 4. Effects of ursolic acid lactone and ursolic acid on enzyme activity in beet armyworm larvae:

[0042] 4.1 Determination of acetylcholinesterase activity in beet armyworm larvae:

[0043] (1) Enzyme solution preparation

[0044] The compound was prepared at specific concentrations, and each concentration was fed to fourth-instar larvae of the beet armyworm using the leaf-dip method. Ethanol was used as a control. Treatments were conducted at 24 h and 48 h. The selected larvae were cleaned in distilled water and air-dried. Each larva was then added to 0.1 mol / L pH 7.8 phosphate buffer, with each treatment repeated three times. The mixture was centrifuged at 8000g for 10 min at 4°C. The supernatant was collected and placed on ice as the test sample.

[0045] (2) Assay of acetylcholinesterase activity

[0046] Preheat the microplate reader for at least 30 minutes. Add reagents according to the kit instructions, mix well, and incubate for 2 minutes. Measure the absorbance at 412 nm and record it as Assay A and Control A. During the assay, place the sample and working solution on ice to prevent denaturation and inactivation.

[0047] Calculate the AChE activity of the sample according to the formula in the instruction manual:

[0048] AChE enzyme activity (U / g) = [ΔA ÷ (ε × d) × V color development × 10] 9 ]÷(W×Vsample÷Vtotalsample×Vsupernatant÷Venzymecatalyst) ÷T=2255×ΔA÷W;

[0049] ΔA: A determination - A control; ε: TNB molar extinction coefficient, 13.6 × 10⁻⁶ 3 L / mol / cm; d: optical path length of the cuvette, 1 cm; Vdeveloped: total volume of the colorimetric reaction system, 0.2 mL = 2 × 102 -4 L; unit conversion factor, 1 mol = 1 × 10 9 nmol; Venzyme: total volume of enzyme reaction, 0.115 mL; Vsupernatant: volume of supernatant aspirated, 0.01 mL; Vtotal: volume of extract added, 1 mL; W: sample mass, g; Vsample: volume of sample added, 0.015 mL; T: reaction time, 5 min.

[0050] For details of the ursolic acid lactone treatment results, please refer to [link / reference]. Figure 3 The results showed that, compared with the control, the activity of AChE in the third instar larvae of the beet armyworm, treated with ursolic acid lactone for 24 h (T24), initially decreased and then increased with increasing concentration. When the concentration of ursolic acid lactone was 0.01 mg / mL, AChE activity was inhibited and decreased by 13.29% compared with the control, but the difference was not statistically significant. When the concentration of ursolic acid lactone was 0.05 mg / mL, AChE activity was increased compared with the control, with an increase of 3.80% in vivo. When the concentration of ursolic acid lactone was 0.1 mg / mL, AChE activity was inhibited compared with the control, but the difference was not statistically significant. After treatment with ursolic acid lactone for 48 h (T48) in the third instar larvae of the beet armyworm, AChE activity showed a trend of initial inhibition followed by activation. When the concentration of ursolic acid lactone was 0.01 mg / mL, AChE activity was inhibited compared to the control, with an in vivo AChE activity inhibition of 16.26%. When the concentration of ursolic acid lactone was 0.05 mg / mL, AChE activity was activated compared to the control, but the in vivo AChE activity increased by 5.65%. When the concentration of ursolic acid lactone was 0.1 mg / mL, AChE activity was activated compared to the control, with an in vivo AChE activity activation of 12.53%.

[0051] The changes in acetylcholinesterase activity in the bodies of third-instar larvae of the beet armyworm after feeding on leaves treated with different concentrations of ursolic acid are as follows: Figure 4The results showed that, compared with the control, in third-instar larvae of the beet armyworm, 24 h after drug administration (T24), AChE activity initially decreased and then increased with increasing ursolic acid concentration. At a ursolic acid concentration of 1.0 mg / mL, AChE activity was inhibited by 14.01% compared to the control; at 1.5 mg / mL, AChE activity was activated, increasing by 18.44%; at 2.0 mg / mL, there was no significant difference in AChE activity compared to the control. 48 h after drug administration (T48), AChE activity in third-instar larvae of the beet armyworm also showed a trend of initially decreasing and then increasing. When the ursolic acid concentration was 1.0 mg / mL, AChE activity was inhibited by 8.85% compared with the control; when the concentration was 1.5 mg / mL, AChE activity was activated compared with the control, and the in vivo AChE activity increased by 21.78%; when the concentration was 2.0 mg / mL, there was no significant difference in AChE activity compared with the control.

[0052] 4.2 Determination of carboxylesterase activity in beet armyworm larvae:

[0053] (1) Enzyme solution preparation

[0054] Using ethanol as a solvent, the compound was prepared to a concentration of EC. 50 Different concentrations of the pesticide were applied to the pronotum of 4th instar beet armyworm larvae, with 2 μL of each concentration as a control. After treatment, the larvae were placed in petri dishes containing fresh cabbage leaves. Live larvae were collected at 0 h, 2 h, 4 h, 6 h, 8 h, and 12 h after treatment to prepare the enzyme solution. The treated larvae were homogenized in 1 mL of 0.1 mol / L pH 7.8 phosphate buffer on ice. The supernatant was diluted 10-fold with distilled water and then processed according to the assay procedure. Each treatment was repeated three times, and centrifuged at 4000 g for 10 min at 4 ℃.

[0055] (2) Assay of carboxylesterase activity

[0056] Preheat the microplate reader for at least 30 minutes, add reagents according to the kit instructions, and measure the absorbance of the control group and treatment group at 450 nm for 10 seconds. Record these values ​​as A1 control and A1 measurement. Quickly place the microplate reader in a 37°C incubator for 5 minutes, and then quickly measure the absorbance at 5 minutes and 10 seconds. Record these values ​​as A2 control and A2 measurement.

[0057] Calculate the CarE activity of the sample according to the formula in the instruction manual:

[0058] CarE enzyme activity (U / g) = (ΔA test tube - ΔA blank tube) × V total ÷ 0.5 ÷ (W ÷ V total sample × V sample) ÷ T × F = 8 × (ΔA test tube - ΔA blank tube) × F;

[0059] Vsample total: Total volume of supernatant, 1 mL; Vsample: Volume of sample added, 0.01 mL; ΔAcontrol = A2 control - A1 control, ΔAdetermination = A2 determination - A1 determination; T: Reaction time, 5 min; W: Sample mass, g; Vreaction total: Total volume of reaction system, 0.2 mL; F: Dilution factor.

[0060] Use DUA's EC 50 After treatment with different concentrations of carboxylesterase (CarE) on third-instar larvae of the beet armyworm, the activity of their carboxylesterase exhibited a dynamic response. Figure 5 The larvae feed on EC containing DUA. 50 After pesticide application to leaves, CarE activity was significantly activated initially, then gradually decreased with prolonged exposure, but remained higher than the control group throughout the process. When beet armyworm larvae immediately fed on the pesticide-containing leaves, CarE activity was significantly activated compared to the control, increasing by 50.74% (t = 10.530, P < 0.002), indicating that the pesticide triggered a rapid response mechanism of the insect's detoxification enzyme system at the initial stage of pesticide contact. When DUA treatment lasted for 2 h and 4 h, CarE activity was activated compared to the control, with activation rates of 41.00% and 34.84%, respectively, but still lower than the activity immediately after treatment. This dynamic change may reflect the phased characteristics of CarE gene expression regulation in insects. The experimental results suggest that DUA may continuously stimulate CarE synthesis to cope with exogenous toxicity pressure, but the compensatory capacity of the enzymatic reaction system gradually weakens with prolonged exposure.

[0061] Use UA's EC 50 Changes in CarE activity in the bodies of 3rd instar larvae of the beet armyworm after treatment with different concentrations are as follows: Figure 6 As shown in the figure, the results indicated that the activity of CarE in the third instar larvae of the beet armyworm was activated with increasing time after drug treatment. When the beet armyworm larvae immediately fed on the drug-containing leaves, the CarE activity was activated compared to the control, with an increase of 14.76%; the maximum CarE activity was reached when the UA treatment time was 8 h, with an in vivo CarE activity of 39.71%.

[0062] In conclusion, ursolic acid lactone has a significantly stronger effect on carboxylesterase and acetylcholinesterase in beet armyworm than ursolic acid, indicating that ursolic acid lactone has a higher potential for controlling beet armyworm.

[0063] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. 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. Such 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 ursolic acid lactone in the control of beet armyworm, characterized in that, The ursolic acid lactone is used to limit the development of beet armyworm, kill beet armyworm, or inhibit beet armyworm feeding; the concentration of the ursolic acid lactone inhibiting beet armyworm feeding is ≥0.1 mg / mL.

2. The application according to claim 1, characterized in that: The ursolic acid lactone effectively inhibits the medium concentration EC of beet armyworm. 50 It is 0.015 mg / mL.

3. The application according to claim 1, characterized in that: The application method involves dissolving ursolic acid lactone in a solvent and then spraying it onto crops to control beet armyworm.

4. The application according to claim 3, characterized in that: The concentration of the ursolic acid lactone dissolved in the solvent is ≥0.01 mg / mL.

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