Application of limonin as pesticide synergist in corn pest control

Abstract

The application relates to application of limonin as a pesticide synergist in corn pest control, and the characteristics that low-concentration limonin has a significant synergistic effect on chemical pesticides. 0.5-4 mu g / mL limonin is mixed with 5% chlorantraniliprole and 25% thiamethoxam for use, the lethal concentration (LC50) of the mixture to corn borer, corn aphid and Sitobion avenae can be significantly reduced, and the insecticidal effect of chlorantraniliprole on corn borer and thiamethoxam on aphids is improved. Field efficacy tests show that 4 mu g / mL limonin mixed with 5% chlorantraniliprole not only significantly improves the field control effect of the pesticide on lepidopterous pests such as corn borer, but also reduces the actual field use amount of the chemical pesticide, that is, the purpose of reducing the pesticide and improving the synergistic effect is achieved, and the method has a positive effect on comprehensive field pest control.

Description

Technical Field

[0001] The present invention relates to the technical field of pesticide synergistic adjuvants. Specifically, the present invention relates to the application of limonin as a pesticide synergistic adjuvant in the prevention and control of corn pests. Background Art

[0002] For corn pests such as lepidopteran pests like Ostrinia furnacalis and sucking pests like Rhopalosiphum maidis, the current prevention and control mainly rely on chemical pesticides such as chlorantraniliprole and thiamethoxam. However, long-term and single use of chemical pesticides is likely to cause drug resistance on the one hand, and on the other hand, to ensure the control effect, it is necessary to apply sufficient liquid medicine, and focus on spraying the back of leaves, young leaves, tender buds, tender shoots, etc. of crops, which requires a large amount of liquid medicine.

[0003] Therefore, finding effective pesticide synergistic adjuvants is of great significance in enhancing the control effect of chemical pesticides against corn pests while reducing the dosage of pesticides.

[0004] Limonin (Chinese name: evodialactone), is a kind of plant secondary metabolite with special structures and functions. It is a tetranor product of triterpenoids and widely exists in plants of Rutaceae and Meliaceae. More than 300 structural analogs have been isolated so far. Limonin has the highest content in citrus and is an important substance causing bitterness in citrus. Research shows that some limonin compounds have biological activities such as anti-cancer, antibacterial, and anti-inflammatory, such as having antifeedant activity against insects. For example, limonin, nomilin, ichangensin, obacunone, azadirachtin, salannin, and gedunin, etc. For example, limonin and other limonin compounds have certain antifeedant activities against lepidopteran insects such as Spodoptera litura, Spodoptera frugiperda, Heliothis virescens, and Helicoverpa armigera. In addition, research also found that 10 mg / mL of limonin extracted from pomelo peel also has strong insecticidal activity against Aphis nerii, Icerya purchasi, and Eurydema gebleri, etc. Three limonin compounds (fraxinellone, obacunone, evodialactone) in the bark of Dictamnus dasycarpus have certain inhibitory effects on killing Bursaphelenchus xylophilus. The above research shows that higher concentrations of limonin have certain insecticidal and repellent activities and can be directly used for pest control, but there is no research report on using it as a pesticide synergistic adjuvant to indirectly improve the insecticidal activity of chemical pesticides. Summary of the Invention

[0005] Based on this, the present invention provides an application of limonin as a pesticide synergistic adjuvant in the prevention and control of corn pests. By using the characteristic that low-concentration limonin has a significant synergistic effect on chemical pesticides, the mixture with chemical pesticides not only significantly improves the field control effect of the pesticides against corn pests, but also reduces the actual field dosage of chemical pesticides, that is, it achieves the purpose of reducing pesticides and increasing efficiency, and has a positive effect on the comprehensive prevention and control of field pests.

[0006] Specifically, this invention relates to the application of limonene as a pesticide synergist in the control of corn pests.

[0007] The preferred concentration of limonene is 0.5~4µg / mL, and the optimal concentration of limonene is 2~4µg / mL.

[0008] The chemical pesticides described in this invention include thiamethoxam, chlorantraniliprole, flonicamid, thiamethoxam, imidacloprid, pirimicarb, deltamethrin, cypermethrin, malathion, abamectin, fenitrothion, etc.

[0009] The corn pests described in this invention include lepidopteran pests and piercing-sucking pests. Lepidopteran pests include the corn borer and the fall armyworm, while piercing-sucking pests include the corn aphid and the cereal constrictor aphid.

[0010] The preferred pesticides of this invention are 5% chlorantraniliprole or 25% thiamethoxam.

[0011] Furthermore, the preferred concentration of limonene in this invention is 1~4 µg / mL, and the concentration of 5% chlorantraniliprole is 0.006~0.025 µg / mL.

[0012] Another preferred concentration of limonene in this invention is 0.5~2µg / mL, and the concentration of 25% thiamethoxam is 0.572~1.938µg / mL.

[0013] When this invention is used in the field, the preferred concentration of limonene is 4 µg / mL, and the dosage of 5% chlorantraniliprole is 15 mL / acre.

[0014] This invention investigated the toxicity of limonene on four insect cell lines, including TnH5. The results showed that Hi5 cells treated with limonene at concentrations ranging from 1.25 ng / μL to 20 ng / μL exhibited numerous vacuoles; HaEpi cells showed extensive mitochondrial damage; while Sf9 and Ha cell lines showed no significant phenotypic differences. The effect of limonene treatment on the cell membrane integrity of the HaEpi cell line was examined using an inverted fluorescence microscope. No changes were found in the FDA fluorescence intensity or the proportion of cells stained with PI, indicating that limonene treatment of HaEpi cells did not disrupt cell membrane integrity within the test period of 0–6 hours. Laser confocal microscopy (LSCM) combined with DiBAC4(3) was used to detect the membrane potential (MP) of HaEpi cells. It was found that limonene treatment of HaEpi cells resulted in both depolarization and repolarization of the transient membrane potential. At a lower concentration (2 μg / mL), limonene treatment caused a certain degree of depolarization in the transient MP, while 10 μg / mL limonene treatment for 20 s produced significant depolarization. However, the degree of depolarization decreased with prolonged treatment time, tending to be consistent with the control. Continuous limonene treatment for 2-6 h revealed that after treatment with 2 μg / mL limonene, the fluorescence intensity of the cell membrane potential continuously decreased, indicating a very strong repolarization. In contrast, after treatment with 10 μg / mL limonene, the fluorescence intensity of the cell membrane potential first increased and then decreased, indicating a trend of initial depolarization followed by repolarization. These results suggest that the effect of limonene on changes in cell membrane potential may be related to ion channels.

[0015] The bioactivity of limonene in two lepidopteran pests (corn borer) and two piercing-sucking pests (corn aphid) was identified. At a concentration of 100 µg / mL, the survival rate of corn borer (86.67±13.33%) was not significantly different from the control group (100.00±0.00%) (P>0.05). At a limonene concentration of 25 µg / mL, the survival rate of fall armyworm (84.72±2.78%) was not significantly different from the control group (87.50±2.41%) (P>0.05). Even at a high concentration of 200 µg / mL, the survival rates of corn aphid and cereal aphid remained as high as (66.67±2.72)% and (85.00±5.53)%, respectively, which were not significantly different from the control groups (80.00±7.20% and 71.67±11.34%). The above results indicate that limonene has low insecticidal effect on corn borer at concentrations below 100 µg / mL, low insecticidal effect on fall armyworm at concentrations below 25 µg / mL, and low insecticidal effect on corn aphid and cereal constrictor aphid at concentrations below 200 µg / mL.

[0016] Indoor toxicity assays revealed that 1 µg / mL and 4 µg / mL limonene mixed with 5% chlorantraniliprole significantly enhanced the insecticidal effect against corn borer, with synergistic ratios of 2.75 and 3.67, respectively. Furthermore, 0.5 µg / mL and 2 µg / mL limonene mixed with 25% thiamethoxam significantly reduced the median lethal concentration (LC50) of thiamethoxam against corn aphid (synergistic ratios of 2.673 and 1.784, respectively) and cereal aphid (synergistic ratios of 1.08 and 16.60, respectively), and significantly enhanced the insecticidal effect of thiamethoxam against both corn aphid and cereal aphid.

[0017] Field efficacy trials showed that the combination of 5% chlorantraniliprole (15 mL / mu) and 4 µg / mL limonene exhibited better control efficacy against lepidopteran pests 15 days after application. Compared to the treatment with 5% chlorantraniliprole alone at 30 mL / mu, the combined treatment reduced the viable insect rate and average number of damaged plants by 18.60% and 19.05%, respectively. These results indicate that the combination of 5% chlorantraniliprole and 4 µg / mL limonene enhances the field control efficacy of chlorantraniliprole against lepidopteran pests in maize. This demonstrates that 4 µg / mL limonene significantly improves the control effect of the pesticide against lepidopteran pests in maize fields while simultaneously reducing the amount of pesticide required in the field. Attached Figure Description

[0018] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the present invention and, together with the description, serve to explain the principles of the invention. To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, those skilled in the art can obtain other drawings based on these drawings without any creative effort.

[0019] Figure 1 The results are the toxicity assays of limonene on four cell lines: Hi5, Sf9, Ha, and HaEpi (after 4 hours of treatment).

[0020] Figure 2 This is a graph showing the change in FDA fluorescence intensity over time after treatment with limonene;

[0021] Figure 3 This is a graph showing the effect of limonene on the transient dynamic changes of MP FI in HaEpi cells;

[0022] Figure 4 This is a graph showing the effect of limonene on the sustained effect of MP in HaEpi cells;

[0023] Figure 5 This is a graph showing the results of the toxicity test of limonene on the corn borer;

[0024] Figure 6 This is a graph showing the results of limonene toxicity testing on the fall armyworm.

[0025] Figure 7 This is a graph showing the results of indoor toxicity testing of limonene against corn aphids;

[0026] Figure 8 This is a graph showing the results of indoor toxicity testing of limonene against the cereal aphid.

[0027] Figure 9 This is a diagram showing the indoor control effect of limonene combined with chlorantraniliprole on corn borers. Detailed Implementation

[0028] The present invention will be further illustrated below through embodiments. It should be understood that the methods described in the embodiments of the present invention are merely for illustrating the present invention and are not intended to limit the present invention. Simple modifications to the present invention under the premise of the present invention's concept are all within the scope of protection claimed by the present invention.

[0029] 1. Test reagents

[0030] 1.1 Test reagents: Limonene (Shanghai Yuanye Biotechnology Co., Ltd., CAS#1180-71-8, purity 98%), Dimethyl sulfoxide (DMSO) (Sinopharm Chemical Reagent Co., Ltd.), 5% Chlorantraniliprole (FMC Corporation, USA), Thiamethoxam 25% water dispersible granules (Shandong Bainong Sida Biotechnology Co., Ltd.)

[0031] Sf-900ⅡSFM medium (GIBCO, USA), fetal bovine serum (GIBCO, USA), double antibiotics [streptomycin and penicillin (100×)] (GIBCO, USA), ethanol (analytical grade, Beijing Lanyi Company), fluorescein acetate (FDA) (purity > 98%, SIGMA, USA), propidium iodide (PI) (purity > 97%, SIGMA, USA), specific membrane potential fluorescent probe DiBAC4(3) (purity > 95%, SIGMA, USA).

[0032] 1.2 Insect Cell Line Culture and Consumables: The TnH5 cell line was derived from the ovarian cell line of *Spodoptera litura* (BTI-Tn-5B1-4 cell line, also known as the Hi5 cell line); the SF9 cell line was derived from the ovarian tissue of female *Spodoptera litura* pupae; the Ha cell line was derived from the embryonic cell line of *Helicoverpa armigera* (Ha-EM-5, abbreviated as Ha-EM); and the HaEpi cell line was derived from the epidermal cells of *Helicoverpa armigera*. All were provided by Professor Liu Kaiyu of Central China Normal University. The culture medium for Hi5, SF9, and HaEpi insect cells was Grace insect medium (purchased from Invitrogen). 8% fetal bovine serum (Gibco) and a final concentration of 1% penicillin and streptomycin were added to the Hi5 and SF9 cell lines before use. 10% fetal bovine serum (Gibco) and a final concentration of 1% penicillin and streptomycin were added to the HaEpi cell line before use. Ha cell line culture medium was Sf-900™ II SFM (purchased from Thermofisher), supplemented with 3% fetal bovine serum (Gibco) and a final concentration of 1% penicillin-dextrose antibody. 6-well cell culture plates were purchased from Shanghai Beyotime Biotechnology Co., Ltd. 15 mm confocal glass dishes were purchased from Zhejiang Shuohua Life Science Research Co., Ltd. Cellvis 18-well chambered confocal glass dishes were purchased from Hangzhou Xinyou Biotechnology Co., Ltd.

[0033] 1.3 Test Insects: The corn borer was collected in July 2023 from a cornfield in Qinglong Village, Xueshan Town, Enyang District, Bazhong City, Sichuan Province (106°33′24″E, 31°55′9″N). The fall armyworm was collected on July 20, 2019, from a summer cornfield in Banqiao Village, Dafan Town, Tongshan County, Hubei Province (114°38′37″E, 29°38′45″N). Both were transferred indoors and reared using artificial feed. After adult emergence, they were fed a 10% honey water solution to supplement sugar and moisture. Indoor rearing conditions: temperature (26±1)℃, relative humidity 60%±10%, photoperiod L:D=16:8. The corn aphid and the cereal constrictor aphid were collected in June 2024 from a cornfield in Shangbamiao Town, Enyang District, Bazhong City, Sichuan Province (106°33′42″E, 31°51′28″N). The population was maintained indoors using barley seedlings for continuous rearing. Indoor rearing conditions were: temperature (24±2)℃, relative humidity 60%±10%, and photoperiod L:D=16:8.

[0034] 1.4 Test Instruments: SPX-250B constant temperature biochemical incubator (Shanghai Yuefeng Company); BSC-1360 IIA2 biosafety cabinet (Beijing Donglian Company); Evos FLAuto eyeless inverted microscope (Life Technologies, USA); MVS-83 autoclave (Panasonic, Japan); Synergy 2 multi-functional microplate reader (BioTek, USA); LSM980 laser confocal microscope (ZEISS, Germany); PHS-3C pH meter (Shanghai Leici Company); ARS-125L-180T ultrapure water system (Chongqing Yiyang Company).

[0035] 2. Test Methods

[0036] 2.1 Determination of the toxicity of limonene to four insect cell lines

[0037] The four insect cell lines exhibiting good growth were inoculated into 48-well plates and cultured overnight at 28 °C. The reagents were diluted with sterile water to 1 mg / mL, filtered through a 0.22 μm filter, and stored at 4 °C protected from light. Limonene was dissolved in DMSO to a concentration of 10 mg / mL, and then diluted with insect cell culture medium to concentrations of 1.25 ng / μL, 2.5 ng / μL, 5 ng / μL, 10 ng / μL, 20 ng / μL, and 40 ng / μL, with a medium containing 0.0025% DMSO serving as a control. The original culture medium in the wells was discarded, and the corresponding diluted compounds or solvents were added (at a rate of 2 min / well). After culturing for 4 h, 8 h, 12 h, 24 h, 48 h, and 72 h, the cells were observed and photographed using an inverted microscope without an eyepiece, with at least three fields of view per well. Cell growth was observed.

[0038] 2.2 Effects of limonene on cell membrane permeability in HaEpi insects

[0039] 2.2.1 Detection of cell membrane integrity using fluorescence microscopy

[0040] One day before the experiment, HaEpi cells were passaged into 48-well cell plates. Once the cells reached approximately 80% confluence, the culture medium was discarded. The treatment groups received culture media containing 2 μg / mL and 10 μg / mL limonene, respectively, while the control group received culture media containing 0.1% DMSO. The reactions were terminated at 0 h, 2 h, 4 h, and 6 h. After treatment, the culture medium was discarded, and the cells were added to Grace Insect Medium containing 20 μmol / L FDA and incubated at room temperature in the dark for 30 min. Then, 150 μL of basal medium containing 8 μg / mL PI was added, and the cells were incubated at room temperature in the dark for 20 min. The dyes were retained for analysis. The number of cells entering the HaEpi cells with FDA and PI and the changes in fluorescence intensity were recorded. The changes in intracellular fluorescence intensity after drug treatment of HaEpi cells were also detected. The experiment was repeated five times.

[0041] 2.2.2 Laser confocal microscopy (LSCM) combined with DiBAC4(3) to detect HaEpi cell membrane potential (MP)

[0042] 2.2.2.1 Effects of limonene on transient dynamic changes in MP in HaEpi cells

[0043] The treatment groups were the same as in 2.2.1. One day before the experiment, HaEpi cells were passaged into 15 mm confocal glass dishes. After reaching approximately 80% confluence, the culture medium was discarded, and 500 μL of a 2 μM specific fluorescent probe DiBAC4(3) was added to the 15 mm culture dish. The dish was then incubated at room temperature in the dark for 20 min, with the dye retained. The dish was placed on the stage of a laser confocal microscope for continuous dynamic scanning (excitation wavelength 488 nm, emission wavelength 540 nm). The baseline fluorescence intensity was measured after 1 min of continuous scanning. After the baseline stabilized, the cells in each treatment group were scanned every 20 s to detect the dynamic changes in the fluorescence intensity (FI) of cells within 300 s. The experiment was repeated in triplicate.

[0044] 2.2.2.2 The sustained effect of limonene on HaEpi cell membrane potential (MP) over 6 hours

[0045] The treatment group was the same as in 2.2.1. After treatment with reagents for 2, 4, and 6 h, the culture medium was discarded, and the fluorescent probe DiBAC4(3) was added and loaded in the dark for 20 min. The samples were then placed on the stage of a laser confocal microscope and the fluorescence scanning program was started. The excitation wavelengths were 488 nm and 540 nm; the scanning mode was time-scan, the scanning density (resolution) was 512 × 512; the scanning speed was fast scan; and the focal plane was adjusted to make the fluorescence image clear. The changes in MPFI of cells after limonene treatment for 2, 4, and 6 h were detected. The experiment was repeated in 3 replicates.

[0046] 2.2.2.3 Data Statistics and Analysis

[0047] The transient dynamic changes of MP in HaEpi cells were detected using ZEISSZEN 3.12-Lite software. Randomly selected cells were used for dynamic measurements. Real-time measurements of images and time were performed using laser confocal microscopy to analyze the changes in transient fluorescence intensity of MP in HaEpi cells treated with limonene. The effects of limonene on the fluorescence intensity changes of MP and FDA in HaEpi cells at different time points were analyzed using ImageJ software. At least five fields of view were randomly selected, with at least 20 cells randomly selected from each field. The fluorescence values ​​of control group cells and the fluorescence values ​​of MP in HaEpi cells after different limonene treatment times were analyzed. All data were processed using Excel and then analyzed using SPSS 17.0 software for one-way ANOVA.

[0048] 2.3 Indoor toxicity test of limonene as an insecticidal active substance against four corn pests

[0049] 2.3.1 Toxicity determination against corn borer and fall armyworm

[0050] The feed method was used. Limonene was dissolved in DMSO to prepare a 20 mg / mL stock solution, which was then added to artificial feed to achieve final concentrations of 10, 25, 50, 100, 200, and 400 µg / mL. An equal volume of dilute aqueous solution was added as a control.

[0051] Corn borer: Cut the above-mentioned artificial feed into pieces and place them in plastic boxes with a diameter of 55 cm and a height of 3 cm. Inoculate each box with 10 early third instar corn borer larvae. Cover the boxes with double layers of gauze to prevent the corn borers from escaping, and then place them in the rearing room described in 1.3. Set up 4 independent biological replicates for each concentration, and check the survival of corn borers at 3 or 5 days.

[0052] Fall armyworm: Cut the above-mentioned artificial feed into pieces and place them in a 24-well plate. Inoculate one second-instar fall armyworm larva into each well and place it in the rearing room described in 1.3. Set up three independent biological replicates for each concentration and check the survival of corn borers at 3 or 5 days.

[0053] 2.3.2 Toxicity determination against corn aphid and cereal tubercle aphid

[0054] The immersion method was used. First, limonin was dissolved in DMSO to prepare a 20 mg / mL stock solution, which was then diluted with distilled water to create four concentration gradients: 50, 100, 200, and 400 µg / mL. The distilled water solution served as a control. Fresh, uniformly growing barley seedlings (approximately 4 cm in height) were selected and immersed in the prepared solutions for 10 seconds each. After drying in a cool, shaded place, the seedlings were placed in plastic boxes (55 cm in diameter and 3 cm high) with 2% agar gel added and lined with filter paper to maintain moisture. Fifteen wingless aphids of uniform size were inoculated into each well. The plastic boxes were sealed to prevent aphid escape and then placed in an aphid culture chamber. Four independent biological replicates were established for each concentration, and mortality was assessed after 24 or 48 hours.

[0055] Data analysis: SPSS 22.0 software was used to perform LSD (Least Significant Difference) comparative analysis on the mortality rates under different concentrations of isolidin.

[0056] 2.4 Indoor bioassay of limonene as a pesticide synergist against corn pests

[0057] 2.4.1 Indoor bioassay of two aphid species using a mixture of limonene and thiamethoxam

[0058] Three treatments were set up for corn aphids: (1) 25% thiamethoxam (0.5, 2.5, 5, 15, 30, 60 µg / mL), (2) 25% thiamethoxam (0.5, 2.5, 5, 15, 30, 60 µg / mL) + 0.5 µg / mL limonene, and (3) 25% thiamethoxam (0.5, 2.5, 5, 15, 30, 60 µg / mL) + 2 µg / mL limonene.

[0059] Three treatments were set up for the rice constrictor aphid: (1) 25% thiamethoxam (0.05, 0.25, 1.25, 2.5, 5.0, 10.0 µg / mL), (2) 25% thiamethoxam (0.05, 0.25, 1.25, 2.5, 5.0, 10.0 µg / mL) + 0.5 µg / mL limonene, and (3) 25% thiamethoxam (0.05, 0.25, 1.25, 2.5, 5.0, 10.0 µg / mL) + 2 µg / mL limonene.

[0060] The immersion method was used, as described above. Four independent biological replicates were set up for each concentration, and mortality was investigated after 48 hours. The experimental data were analyzed using the PROBIT model in SPSS Statistics 22.0 software to calculate the toxicity regression equation, LC50, and 95% confidence interval for each treatment. Synergistic ratio = LC50 of insecticide against target pest / LC50 of insecticide (+synergist) against target pest.

[0061] 2.4.2 Maximum effective concentration of limonene compound pesticide

[0062] Three treatments were set up for the corn aphid: (1) 25% thiamethoxam (0.5, 2.5, 5, 15, 30, 60 µg / mL), (2) 25% thiamethoxam (0.5, 2.5, 5, 15, 30, 60 µg / mL) + 4 µg / mL limonene, (3) 25% thiamethoxam (0.5, 2.5, 5, 15, 30, 60 µg / mL) + 8 µg / mL limonene, and (4) 25% thiamethoxam (0.5, 2.5, 5, 15, 30, 60 µg / mL) + 16 µg / mL limonene. The immersion method was used, and the operation was as shown above. The mortality rate was investigated after 48 h. The experimental data were analyzed using the PROBIT model in SPSS Statistics 22.0 software to calculate the toxicity regression equation, LC50, and 95% confidence interval for each treatment. Synergistic ratio = LC50 of insecticide to target pest / LC50 of insecticide (+synergist) to target pest.

[0063] 2.4.3 Indoor bioassay of a mixture of limonene and chlorantraniliprole in corn borer

[0064] Three treatments were set up: (1) 5% chlorantraniliprole (0.0004, 0.002, 0.01, 0.05, 0.2µg / mL), (2) 5% chlorantraniliprole (0.0004, 0.002, 0.01, 0.05, 0.2µg / mL) + 1µg / mL limonene, and (3) 5% chlorantraniliprole (0.0004, 0.002, 0.01, 0.05, 0.2µg / mL) + 4µg / mL limonene.

[0065] The feed method was used, and the operation was as shown above. Three independent biological replicates were set up for each concentration, and the larval survival was investigated after 5 days.

[0066] 2.5 Field control efficacy of limonene mixed with pesticides against lepidopteran pests such as corn borers

[0067] The experiment was conducted at the Ezhou Base of the Hubei Academy of Agricultural Sciences, using the Zhengdan 958 maize variety. It was carried out in July 2025, at the late whorl stage of maize. Two treatments were set up: Treatment 1: 5% chlorantraniliprole (30 mL / mu), and Treatment 2: 5% chlorantraniliprole (15 mL / mu) + limonene (final concentration 4 μg / mL). Each treatment plot was 1500 m², with a 5 m protective row between the two treatments, and no replicates were used. Unmanned aerial vehicle (UAV) spraying was employed, using 4 L of water per mu. Fifteen days after application, the occurrence and damage of lepidopteran pests on the entire maize plant were investigated, including corn borer, peach borer, and fall armyworm. Five random sampling points were collected for each treatment group and control group, with 50 maize plants surveyed at each point. The percentage of affected plants, the survival rate, and the relative control efficacy were calculated according to formulas (1), (2), and (3).

[0068] Damaged plant rate (%) = Number of damaged plants / Number of plants surveyed × 100 ‥‥‥‥‥‥‥‥‥‥‥ (1)

[0069] Survival rate (%) = Number of live larvae / Total number of insects surveyed × 100% ‥‥‥‥‥‥‥‥‥‥ (2)

[0070] Relative effect (%) = (CK-PT) / CK×100‥‥‥‥‥‥‥‥‥‥‥‥‥‥ (3)

[0071] In the formula:

[0072] CK – Percentage of affected plants in treatment 1;

[0073] PT – Percentage of affected plants in treatment 2.

[0074] 3. Test Results

[0075] 3.1 Determination of the toxicity of limonene to cells of four insect species

[0076] The results of the toxicity assay of limonene on the cells of four insect species are shown in the figure. Figure 1(Note: a-Hi5 cell control, b-Hi5 cells treated with 1.25 μg / mL limonene, c-Hi5 cells treated with 40 μg / mL limonene; d-Sf9 cell control, e-Sf9 cells treated with 1.25 μg / mL limonene, f-Sf9 cells treated with 40 μg / mL limonene; g-Ha cell control, h-Ha cells treated with 1.25 μg / mL limonene, i-Ha cells treated with 40 μg / mL limonene; j-HaEpi cell control, k-HaEpi cells treated with 1.25 μg / mL limonene, l-HaEpi cells treated with 40 μg / mL limonene). As shown in the figures, after limonene treatment, Hi5 cells exhibited a large number of vacuoles (approximately 40%) after 4 hours of treatment. Specifically, about 80% of cells treated with 1.25 ng / μL–20 ng / μL limonene for 8 hours produced vacuoles, significantly higher than the control group (approximately 20%). In HaEpi cells, mitochondrial damage was observed after 4 hours of treatment, with a damage rate of 50%–80%. After 12 hours of treatment, the damage rate reached almost 100%, higher than the 75% in the control group. However, after 48 hours of treatment, some mitochondrial repair was observed in limonene-treated cells, with a damage rate of only 25%–50%, lower than the 60% in the control group. There were no significant phenotypic differences between Sf9 and Ha cell lines after limonene treatment, although a small number of Ha cells showed mitochondrial damage.

[0077] The results of cytotoxicity assays showed that limonin had relatively low toxicity to the cells of the four insect species, but it was able to affect the mitochondrial homeostasis of the HaEpi cell line (epidermal cells).

[0078] 3.2 LSCM Detection of the Effect of Limonene Treatment on Cell Membrane Integrity of HaEpi Cell Line

[0079] Fluorescein diacetate (FDA) is an uncharged, lipid-soluble molecule that readily enters living cells. It is not fluorescent itself, but after entering the cell, it is hydrolyzed by non-specific esterases, releasing a fluorescein molecule that emits yellow-green fluorescence. The fluorescence intensity is positively correlated with cell viability. Prism inhibitor (PI) is a fluorescent probe that can intercalate into double-stranded DNA and RNA base pairs, emitting red light. It cannot penetrate intact cell membranes to enter the cell, but when the cell membrane is damaged or the cell is necrotic, it can enter the cell and intercalate into DNA and RNA, emitting orange-red fluorescence. Both dyes can effectively reflect changes in cell membrane integrity and cell viability.

[0080] The changes in intracellular fluorescence intensity of FDA and PI-labeled HaEpi cells over time after the addition of different concentrations of limonene are shown in the figure. Figure 2(Note: Data in the figure are mean ± standard error; the same lowercase letter on the column indicates that the difference between different concentrations at the same treatment time is not significant at the 0.05 level.) The figure shows that there were no significant differences in FDA fluorescence intensity between the CK group and the limonene 2 μg / mL and 10 μg / mL treatment groups at 2 h, 4 h, and 6 h (P<0.05).

[0081] Statistical analysis of the number of cells with PI entering HaEpi cells after limonene treatment (see Table 1) revealed that a small number of cells in each treatment showed PI staining, but there was no significant difference compared with the control group (P≥0.05). This indicates that the PI staining of individual cells in each treatment, including the control group, was due to damage caused by the procedure. The results show that limonene treatment of HaEpi cells did not disrupt the integrity of their cell membranes within the test period of 0–6 h.

[0082] Table 1. Percentage of PI-stained cells after limonene treatment (%)

[0083]

[0084] Note: The data in the figure are mean ± standard deviation. The same lowercase letter after the data in the same row indicates that there is no significant difference between different concentrations at the 0.05 level for the same treatment time.

[0085] 3.3 Effects of limonene treatment on HaEpi cell membrane potential

[0086] 3.3.1 Effects of limonene on transient dynamic changes in MP in HaEpi cells

[0087] DiBAC4(3) fluorescent probe is a lipophilic anionic fluorescent dye sensitive to cell membrane potential. It is non-fluorescent on its own, emitting fluorescence only after entering the cell and binding to proteins in the cytoplasm. Increased fluorescence intensity, i.e., increased membrane potential, indicates cell depolarization; conversely, decreased intracellular fluorescence intensity, i.e., decreased membrane potential, indicates cell hyperpolarization. The transient dynamic changes of limonene on MP in HaEpi cells are shown in [reference needed]. Figure 3(Note: AD: before treatment with 2 μg / mL limonene and at 20s, 60s, and 240s, respectively; EH: before treatment with 10 μg / mL limonene and at 20s, 60s, and 240s, respectively). After the instantaneous addition of 2 μg / mL limonene for 20s, the MP FI slightly increased from 106.85±7.39 to 113.61±5.94, and further increased to 125.43±6.97 after 240s, but there was no significant difference compared to the control group (P<0.05). After the instantaneous addition of 10 μg / mL limonene, the MPFI decreased from 74.20±3.33 to 45.56±4.86, a significant difference compared to the control group (P<0.05), and remained different for 120s. However, after 240s, it increased again to 71.40±5.78, but there was no significant difference compared to the control group. This indicates that HaEpi transient cell membrane potential exhibits depolarization and hyperpolarization when treated with limonene. Furthermore, treatment with a lower concentration (2 μg / mL) of limonene resulted in a certain degree of depolarization of transient MP, while treatment with 10 μg / mL of limonene for 20 s produced significant depolarization of MP. However, the degree of depolarization tended to decrease with treatment time, becoming consistent with the control.

[0088] 3.3.2 The sustained effect of limonene on the membrane potential (MP) of HaEpi cells

[0089] Changes in cell membrane potential and fluorescence intensity at different time points after treatment with limonene are shown in the figure. Figure 4 (Note: 0 μg / mL is the control treatment containing 0.1% DMSO solvent) As shown, the control group, which contains 0.1% DMSO, showed a decrease and then an increase in intracellular membrane potential fluorescence intensity within 2–6 h. However, after treatment with 2 μg / mL limonene, the cell membrane potential fluorescence intensity decreased significantly, and after treatment with 10 μg / mL limonene, the cell membrane potential fluorescence intensity increased and then decreased, indicating that the membrane potential showed a trend of depolarization followed by repolarization.

[0090] 3.4 Indoor toxicity test of limonene as a direct insecticidal active ingredient against four corn pests

[0091] 3.4.1 Determination of the toxicity of limonene to corn borer and fall armyworm

[0092] The results of indoor toxicity tests of limonene against corn borers are as follows: Figure 5As shown in the figure (Note: Data in the figure are mean ± standard error; different lowercase letters on the bars represent significant differences between different treatments (P<0.05) (LSD least significant difference method)): In the limonene treatment range of 10µg / mL to 100µg / mL, the survival rate was (86.67±13.33)% to (100.00±0.00)%, which was not significantly different from the survival rate of the control group (100.00±0.00%) (P>0.05). The results of the indoor toxicity determination of the fall armyworm are as follows... Figure 6 As shown in the figure (Note: Data in the figure are mean ± standard error; different lowercase letters on the bars represent significant differences between different treatments (P<0.05) (LSD least significant difference method)), when the limonene treatment concentration was 25 µg / mL, the survival rate was 84.72±2.78%, which was not significantly different from the control group (87.50±2.41%) (P>0.05). The results indicate that limonene has low insecticidal effect against corn borer at concentrations below 100 µg / mL and low insecticidal effect against fall armyworm at concentrations below 25 µg / mL.

[0093] 3.4.2 Determination of the toxicity of limonene to corn aphid and cereal tubercle aphid

[0094] The results of indoor toxicity tests of limonene against corn aphids are as follows: Figure 7 As shown in the figure (data are mean ± standard error, different lowercase letters on the columns represent significant differences between different treatments (P<0.05) (LSD least significant difference method)): under the treatment concentrations of limonene from 50µg / mL to 200µg / mL, the survival rate was (66.67±2.72)% to (78.33±4.19)%, which was not significantly different from the survival rate of the control group (80.00±7.20%) and other concentrations (P>0.05). The results of the indoor toxicity test of limonene against *Aphidius gracilis* are as follows. Figure 8 As shown in the figure (data are mean ± standard error, different lowercase letters on the bars represent significant differences between treatments (P<0.05) (LSD least significant difference method)), the survival rate under the highest limonene concentration of 400 µg / mL was (88.33±1.67)%, which was not significantly different from the mortality rate of the control group (71.67±11.34%) and other limonene concentration treatments (P>0.05). The results indicate that limonene itself has low insecticidal efficacy against corn aphids and cereal aphids.

[0095] 3.5 Indoor toxicity test of limonene as a pesticide synergist against three corn pests

[0096] 3.5.1 Effects of a mixture of low concentrations of limonene and thiamethoxam on two aphid species

[0097] Indoor bioassay results showed that the median lethal concentration (LC50) of 25% thiamethoxam water-dispersible granules against corn aphids was 1.179 (0.572–1.938) µg / mL. When 0.5 µg / mL and 2 µg / mL limonene were added, the LC50 values ​​were 0.441 (0.028–1.280) and 0.661 (0.002–2.456), respectively, with synergistic ratios of 2.673 and 1.784, respectively. This indicates that the combination with limonene can significantly improve the insecticidal effect of thiamethoxam against corn aphids (Table 2).

[0098] Table 2. Insecticidal effect of low concentration limonene mixed with thiamethoxam on corn aphids.

[0099]

[0100] Indoor bioassay results showed that the median lethal concentration (LC50) of 25% thiamethoxam water-dispersible granules against *Aphidius gracilis* was 0.249 (0.122–0.417) µg / mL. When 0.5 µg / mL and 2 µg / mL limonene were added, the LC50 values ​​were 0.207 (0.039–0.552) and 0.015 (0.001–0.055), respectively, with synergistic effects of 1.083 and 16.600, respectively. This indicates that the combination with limonene can significantly improve the insecticidal effect of thiamethoxam against *Aphidius gracilis* (Table 3).

[0101] Table 3. Insecticidal effect of low concentration limonene mixed with thiamethoxam on *Aphidius gracilis*.

[0102]

[0103] 3.5.2 Effects of a mixture of high concentrations of limonene and thiamethoxam on corn aphids

[0104] To further clarify the optimal ratio of limonin in compound pesticides, the synergistic effect of three limonin concentrations (4 µg / mL, 8 µg / mL, and 16 µg / mL) on thiamethoxam was analyzed under indoor conditions. The results showed that the synergistic ratios for the 4 µg / mL, 8 µg / mL, and 16 µg / mL limonin treatments were 1.731, 0.943, and 0.694, respectively. This indicates that higher concentrations of limonin as a synergistic adjuvant do not necessarily lead to better efficacy (see Table 4).

[0105] Table 4. Insecticidal effect of high concentration limonene combined with thiamethoxam on corn aphids.

[0106]

[0107] 3.5.3 Indoor control efficacy of limonene and chlorantraniliprole combination against corn borer

[0108] Results of 5-day indoor bioassays Figure 9 The results showed that the median lethal concentration (LC50) of 5% chlorantraniliprole against corn borer was 0.011 (0.006–0.025) µg / mL. When 1 µg / mL and 4 µg / mL limonene (LM) were added, the LC50 values ​​were 0.004 (0.002–0.010) and 0.003 (0.003–0.007) µg / mL, respectively, with synergistic effects of 2.75 and 3.67. The results indicated that the mixture of 1 µg / mL and 4 µg / mL limonene could significantly improve the insecticidal effect of 5% chlorantraniliprole against corn borer (Table 5).

[0109] Table 5. Insecticidal effect of limonene combined with chlorantraniliprole on corn borers.

[0110]

[0111] 3.6 The synergistic effect of limonene as an adjuvant on the control of lepidopteran pests in corn fields

[0112] The field efficacy results of chlorantraniliprole mixed with limonene against maize lepidopteran pests showed that 15 days after application, the survival rate and average number of damaged plants in treatment 1 (chlorantraniliprole 30 mL / mu) were lower than those in treatment 2 (chlorantraniliprole 15 mL / mu + limonene 4 µg / mL). In particular, the survival rate and average number of damaged plants in treatment 1 were reduced by 18.60% and 19.05% respectively compared with treatment 2, indicating that 4 µg / mL limonene can improve the field control efficacy of chlorantraniliprole against maize lepidopteran pests (Table 6).

[0113] Table 6. Field efficacy of limonene and chlorantraniliprole mixtures against lepidopteran pests of maize.

[0114]

[0115] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. Application of limonene as a pesticide synergist in the control of corn pests, wherein the corn pests are corn borer, corn aphid, and cereal aphid, and the pesticide is 5% chlorantraniliprole or 25% thiamethoxam; when the pesticide is 5% chlorantraniliprole, the concentration of limonene is 1~4µg / mL, and the concentration of 5% chlorantraniliprole is 0.006~0.025µg / mL; when the pesticide is 25% thiamethoxam, the concentration of limonene is 0.5~2µg / mL, and the concentration of 25% thiamethoxam is 0.572~1.938µg / mL.

2. The application of limonene as a pesticide synergist in the control of corn pests according to claim 1, characterized in that, When used in the field, the concentration of limonene is 4 µg / mL, and the dosage of 5% chlorantraniliprole is 15 mL / acre.

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