Inhibitors for controlling spodoptera frugiperda and use thereof

Abstract

The application discloses an inhibitor for preventing and treating Spodoptera frugiperda and application thereof, relates to the technical field of insect prevention and treatment, and the inhibitor takes rapamycin or AZD-8055 as a main active ingredient, takes DMSO or water as a solvent, the rapamycin is a macrolide immunosuppressant produced by Streptomyces hygroscopicus, and the AZD-8055 is an ATP competitive TOR kinase inhibitor; the effective working concentration of the rapamycin is 0.1 muM-50 muM; the effective working concentration of the AZD-8055 is 0.1 muM-50 muM; the inhibitor makes the Sf9 cell of the ovary cell line of the Spodoptera frugiperda change in shape or die; the inhibitor provided by the application takes the rapamycin or the AZD-8055 as an active ingredient, targets the TOR signal path of insects, has no cross resistance with existing chemical pesticides and Bt protein, can effectively cope with resistant populations, and solves the problem of the drug resistance of the Spodoptera frugiperda.

Description

Technical Field

[0001] This invention relates to the field of insect control technology, specifically to inhibitors for controlling fall armyworm and their applications. Background Technology

[0002] Currently, the control of fall armyworm still relies mainly on emergency measures using chemical pesticides. However, the long-term and extensive use of chemical pesticides not only leads to environmental safety and pesticide residue problems, but also, according to the latest research, the fall armyworm has developed extensive resistance to many traditional chemical pesticides and Bt transgenic maize. The effectiveness of existing control agents is continuously declining, the pressure on control is increasing sharply, and it is difficult to meet the needs of sustainable control.

[0003] Therefore, there is an urgent need to develop novel, green pesticide formulations with unique mechanisms of action. Rapamycin (RAP) is a macrolide immunosuppressant produced by *Streptomyces hygroscopicus*, and is a specific inhibitor of the TOR protein. It forms a binary complex by specifically binding to the FKBP12 protein, which then binds to the FRB domain of the TOR protein and inhibits its activity. When the FKBP12 gene is mutated, rapamycin cannot bind to it, and the TOR protein is no longer inhibited by rapamycin. In plants, due to adaptive mutations in the FKBP12 gene, the ability to bind to rapamycin is lost, so rapamycin has no inhibitory effect on the plant's TOR protein; however, in fungi and mammals, rapamycin has the ability to inhibit TOR protein activity. Therefore, rapamycin can be used as a biological pesticide for plant diseases and pests without being toxic to the plants themselves. In yeast and mammals, rapamycin can only inhibit the activity of TORC1, but not TORC2. Therefore, although rapamycin has high specificity for inhibiting TOR, it cannot completely inhibit TOR activity. AZD-8055 is an ATP-competitive TOR kinase inhibitor. As a TOR inhibitor, AZD-8055 overcomes the limitations of the rapamycin-based inhibition pathway by binding to the ATP-binding sites in the TOR kinase structural regions required for the function of both TOR complexes, leading to complete inhibition of TOR activity. Therefore, RAP and AZD-8055 have the potential to be developed into green biopesticides. However, there are currently no reports on using RAP or AZD-8055 to inhibit the proliferation of fall armyworm ovarian cells and thus control the fall armyworm. How to utilize the properties of RAP and AZD-8055 to develop novel, highly efficient, and low-toxicity control methods against the fall armyworm is an urgent problem to be solved in this field. Summary of the Invention

[0004] The purpose of this invention is to provide an inhibitor for controlling fall armyworm and its application, in order to solve the problems of poor environmental safety, prominent resistance issues, and poor control efficacy of existing fall armyworm control agents.

[0005] To achieve the above objectives, the present invention provides the following technical solution: an inhibitor for controlling fall armyworm, wherein the inhibitor has rapamycin or AZD-8055 as the main active ingredient and DMSO or an aqueous solution containing DMSO as the solvent, wherein rapamycin is a macrolide immunosuppressant produced by Streptomyces hygroscopicus and AZD-8055 is an ATP-competitive TOR kinase inhibitor.

[0006] The effective working concentration of the rapamycin is 0.1 μM to 50 μM; the effective working concentration of the AZD-8055 is 0.1 μM to 50 μM.

[0007] The inhibitor caused morphological changes or death in the fall armyworm ovarian cell line Sf9; the morphological changes included cell swelling, cell shrinkage, and the appearance of apoptotic bodies.

[0008] This invention also discloses the application of inhibitors for controlling fall armyworm in the preparation of pesticides for controlling fall armyworm.

[0009] Furthermore, the pesticide is a pesticide that inhibits the proliferation of the fall armyworm ovarian cell line Sf9 and induces abnormal morphology or death of Sf9 cells.

[0010] Furthermore, the pesticide formulation is at least one of wettable powder, water-dispersible granules, suspension concentrate, microemulsion, and soluble concentrate.

[0011] Furthermore, the pesticide is applied by foliar spraying or soil treatment.

[0012] Furthermore, the pesticide uses rapamycin and / or AZD-8055 as active ingredients, or rapamycin and / or AZD-8055 are used in combination with other pesticide active ingredients.

[0013] Compared with existing technologies, the inhibitor for controlling fall armyworm provided by this invention and its application have the following beneficial effects:

[0014] (1) The inhibitor of the present invention uses rapamycin and AZD-8055 as active ingredients. Rapamycin has no inhibitory effect on plant TOR protein and is safe and harmless to crops. AZD-8055 has a specific target. Both have no environmental residual pollution risk and meet the requirements of green and pollution-free pesticides. This solves the problem of poor environmental safety of pesticides for controlling fall armyworm.

[0015] (2) The inhibitor of the present invention targets the TOR signaling pathway in insects and has no cross-resistance with existing chemical pesticides and Bt protein. It can effectively resist resistant populations and solve the problem of pesticide resistance in fall armyworm.

[0016] (3) The inhibitor of the present invention can significantly inhibit the proliferation of Sf9 cells and induce apoptosis at a low concentration of 0.1 μM, and can cause cell disintegration and death at a high concentration. The dose-effect is clear, the control efficiency is high, and the inhibitory effect is significant.

[0017] (4) RAP and AZD-8055 can be used alone or in combination, taking into account both suppression efficiency and usage cost. They can be adapted to different green and sustainable control scenarios for fall armyworm in fields, and their application is flexible. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in this invention. For those skilled in the art, other drawings can be obtained based on these drawings.

[0019] Figure 1 The effect of different concentrations of rapamycin (RAP) on the growth status of fall armyworm Sf9 cells is shown in the figure provided in the embodiments of the present invention.

[0020] Figure 2 The graph showing the effect of different concentrations of rapamycin (RAP) on the survival rate of fall armyworm Sf9 cells is provided in the embodiments of the present invention.

[0021] Figure 3 This is a diagram illustrating the effect of different concentrations of AZD-8055 on the growth status of Sf9 cells of fall armyworm, as provided in an embodiment of the present invention.

[0022] Figure 4 The graph shows the effect of different concentrations of AZD-8055 on the survival rate of Sf9 cells of fall armyworm, as provided in the embodiments of the present invention. Detailed Implementation

[0023] To enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings.

[0024] Example 1:

[0025] Please see Figures 1 to 4 This is an inhibitor used to control fall armyworm, with rapamycin (RAP) or AZD-8055 as the main active ingredients and DMSO as the solvent. Rapamycin is a macrolide immunosuppressant produced by Streptomyces hygroscopicus; AZD-8055 is an ATP-competitive TOR kinase inhibitor.

[0026] The effective working concentration of rapamycin is 0.1 μM to 50 μM;

[0027] The effective working concentration of AZD-8055 is 0.1 μM to 50 μM;

[0028] Normal Sf9 ovarian cell lines of fall armyworm exhibit uniform, round cell morphology with clear outlines, good intracellular light transmittance, and homogeneous structure. Rapamycin inhibits the TOR signaling pathway, thereby affecting TOR protein activity and specifically blocking protein synthesis in Sf9 cells. AZD-8055 can bind to the ATP-binding site in the TOR kinase structural region required for the function of the TORC1 and TORC2 complex, leading to complete inhibition of TOR activity. The inhibitor proposed in this invention for controlling fall armyworm can cause morphological changes or death in Sf9 cells. Apoptotic cells of the fall armyworm ovarian cell line Sf9 exhibit cell shrinkage, diverse morphologies (rod-shaped, oval, polygonal), accompanied by budding and bubbling phenomena, with blurred cell outlines. The appearance of apoptotic bodies is a typical characteristic, and cell swelling is also an important phenotype of apoptosis in this cell line.

[0029] Preparation of inhibitors:

[0030] (1) Reagents and materials:

[0031] Reagents: Rapamycin (RAP) and dimethyl sulfoxide (DMSO) were purchased from Solarbio; rapamycin (catalog number: HY-10219); AZD-8055 (catalog number: HY-10422) were purchased from MedChemexpress (MCE); Sf-900™ II SFM serum-free medium (catalog number: 11496015) and Nunc EasY Flask culture flasks (25cm) were used. 3 All reagents (item number 156367) were purchased from Thermo Fisher Scientific; 96-well cell culture plates were purchased from Corning Incorporated; other reagents without a specified source were domestically produced analytical grade products, commonly available in the market.

[0032] The test cell line was the fall armyworm ovary cell line Sf9, obtained from the USDA Insect Pathology Laboratory. The culture medium was Sf-900™ II SFM, and the cells were cultured in a cell culture incubator at 28°C and 4.5% CO2. The cells were passaged every 4–6 days, and cells in the logarithmic growth phase that were plump and round were selected for subsequent experiments.

[0033] (2) Preparation of inhibitor stock solution and working solution:

[0034] RAP: Prepare a high-concentration RAP stock solution using DMSO. After aseptic filtration, store the prepared stock solution at 4°C for later use. The specific steps are as follows: Accurately weigh an appropriate amount of RAP. Dissolve the RAP in a small amount of DMSO, then prepare a 10mM RAP stock solution using Sf-900™ II SFM culture medium. After sterilizing the RAP stock solution by filtration, dilute it with Sf-900™ II SFM basal culture medium to prepare a series of working solutions with varying mass concentrations (0.01μM, 0.1μM, 1μM, 10μM, 50μM), ensuring that the final DMSO content in each working solution is less than 0.1% (v / v). The solvent control is a culture medium containing an equal amount of DMSO.

[0035] A high-concentration AZD-8055 stock solution was prepared using DMSO. The prepared stock solution was aseptically filtered and stored at 4°C for later use. The specific steps are as follows: Accurately weigh an appropriate amount of AZD-8055. First, dissolve AZD-8055 in a small amount of DMSO. Then, prepare a 10mM AZD-8055 stock solution using Sf-900™ II SFM culture medium. After filtering and sterilizing the AZD-8055 stock solution, dilute it with Sf-900™ II SFM basal culture medium to prepare a series of RAP stock solution working solutions with mass concentration gradients (0.01μM, 0.1μM, 1μM, 10μM, 50μM), ensuring that the final DMSO content in each concentration working solution is less than 0.1% (v / v). The solvent control is a culture medium containing an equal amount of DMSO.

[0036] Example 2:

[0037] Inhibitor efficacy verification:

[0038] (1) Culture and treatment of Sf9 ovarian cell line from fall armyworm:

[0039] Cellular resuscitation:

[0040] Non-primary cultured cells are generally cryopreserved in liquid nitrogen. When culturing is required, the frozen cells need to be taken out of the liquid nitrogen and thawed: First, turn on the water bath and adjust the temperature to 37°C; take the desired cells out of the liquid nitrogen tank and immediately place them in the 37°C water bath to thaw them quickly; wipe the outer surface of the cryovials and culture medium bottles clean with medical alcohol and place them in a clean bench; add the cell suspension to a centrifuge tube and add 1 mL of culture medium, and gently pipette; centrifuge at 1500 rpm for 3 min; discard the supernatant, add 1 mL of culture medium, and pipette to mix well to suspend the cells; transfer the cell suspension to a 50 mL culture flask and add 5 mL of culture medium; place in a cell culture incubator for culture.

[0041] Cell culture:

[0042] First, sterilize the laminar flow hood for 30 minutes. Then, remove the cell culture flask from the incubator and observe the cell growth status under a 10× inverted microscope. A normal state is characterized by a monolayer, adherence to the wall, plumpness, roundness, and transparency. Tap the cell culture flask to allow the suspended cells to settle. Discard the old culture medium and add 3 ml of fresh Sf-900 Ⅲ SFM medium. Then, use a pipette to resuspend the cells and remove any foam. Divide the cells into 3 flasks (1 ml each), and add another 3 ml to each flask to make a final solution of 4-5 ml. Finally, place the aliquoted cells in a CO2 cell culture incubator at 28°C for 4-6 days. Once the Sf9 cells have covered the bottom of the cell culture flask, passage them again.

[0043] Cell cryopreservation:

[0044] The most crucial step in cell cryopreservation is to gradually and slowly lower the temperature to avoid ice crystal formation inside the cells and causing damage. Adherent cells are collected by pipetting and centrifugation, while suspension cells are collected by centrifugation. Approximately 10⁶ cells are added to 1 mL of cryopreservation solution and dispersed into a single-cell suspension using a pipette. The cell suspension is then added to cryovials. If using 1.8 mL cryovials, each cryovial should ideally contain no more than 1.5 mL of cell suspension. The cell names and cryopreservation dates are labeled on the cryovials. The cryovials are wrapped in a thick cloth or tissue paper and incubated at 4°C for 0.5 hours, then at -20°C for 2 hours, then at -40°C for 2 hours, and finally at -80°C overnight. The next day, the cells are placed in liquid nitrogen. The location of the cryovials is recorded, and a cryopreservation log is maintained.

[0045] Cytotoxicity assay:

[0046] Ovarian cell line Sf9 from fall armyworm in the logarithmic growth phase was adjusted to 1.0 × 10⁻⁶ cells. 3 Sf9 cell suspension was seeded at a density of 10 cells / mL into 96-well plates. After culturing the Sf9 cells for 12 hours and allowing them to fully adhere, the cell plate was inverted, the culture medium was poured out, and rapamycin solution was added. The rapamycin (RAP) treatment groups were treated with 0 μM (DMSO), 0.01 μM, 0.1 μM, 1 μM, 10 μM, and 50 μM, respectively. The negative control group was treated with Sf-900™ II SFM basal culture medium containing DMSO. Each concentration was used in 5 replicates, and the experiment was repeated 3 times. After 48 hours of dark treatment, the experimental results were analyzed, and the data were analyzed using SPSS software for variance analysis.

[0047] Ovarian cell lines Sf9 from fall armyworms in the logarithmic growth phase were used, and adjusted to approximately 1.0 × 10⁻⁶ cells. 3Sf9 cell suspension was seeded at a density of 10 cells / mL into 96-well plates. After culturing the Sf9 cells for 12 hours and allowing them to fully adhere, the cell plate was inverted, the culture medium was poured out, and AZD-8055 solution was added. The AZD-8055 treatment groups were treated with 0 μM (DMSO), 0.01 μM, 0.1 μM, 1 μM, 10 μM, and 50 μM, respectively. The negative control group was treated with Sf-900™ II SFM basal culture medium containing DMSO. Each concentration was used in 5 replicates, and the experiment was repeated 3 times. After 48 hours of dark treatment, the experimental results were analyzed, and the data were analyzed using SPSS software for variance analysis.

[0048] (2) Cell morphology observation

[0049] Forty-eight hours after drug treatment, the morphological changes of Sf9 cells in each group were observed and photographed under an inverted microscope (10× or 20× objective lens). Special attention was paid to cell size, shape, adherence, cell membrane integrity, and the presence or absence of apoptotic bodies.

[0050] Effects of different concentrations of rapamycin on the growth status of Sf9 cells:

[0051] Figure 1 The effects of different concentrations of rapamycin on the growth status of Sf9 cells were demonstrated, such as... Figure 1 As shown, Sf9 cells were treated with six concentrations of rapamycin solution, from low to high, for 48 hours. The results showed that five of the concentrations induced morphological changes in Sf9 cells, exhibiting a dose-response effect. The negative control group (0 μM RAP) showed Sf9 cells with regular morphology, mostly spherical, with clear cell boundaries, smooth surfaces, good adherence, and tight arrangement. After 48 hours of RAP treatment, at a concentration of 0.01 μM, cell morphology showed no significant change compared to the control group. When the RAP concentration reached 0.1 μM and above, Sf9 cell morphology changed significantly: some cells shrank and became rounded, some cells abnormally swelled, the cell surface became rough, the outline became indistinct, and cell budding and apoptotic bodies were visible. As the RAP concentration increased to 1 μM and 10 μM, the number of shrunken and dead cells increased significantly, and the cell density decreased. When the RAP concentration reached 50 μM, most cells disintegrated and died, cell debris increased, and adherent cells became extremely sparse. The above results indicate that the inhibitory and cytotoxic effects of RAP on Sf9 cells are significantly dose-dependent, validating the effectiveness of the working concentration range of rapamycin.

[0052] Results of rapamycin toxicity assay on Sf9 cells:

[0053] Figure 2The graph shows the effect of different concentrations of rapamycin (RAP) on the survival rate of Sf9 cells of fall armyworm. The horizontal axis represents the concentration of rapamycin in the culture medium, and the vertical axis represents the number of surviving Sf9 cells. Turkey's one-way ANOVA was used to compare the significance of differences. An asterisk indicates a significant difference at the 5% level (P < 0.05). Figure 2 As shown in Table 1, compared with the negative control DMSO treatment, the cell viability of the 0.01 μM RAP treatment group was 93.37%, with no significant difference (P>0.05). However, the cell viability of the 0.1 μM, 1 μM, 10 μM, and 50 μM RAP treatment groups decreased to 62.19%, 55.91%, 56.10%, and 28.14%, respectively. Statistical analysis showed that the cell viability of these concentration treatment groups was significantly different from the control group (P<0.05), indicating that RAP at concentrations of 0.1 μM and above can significantly inhibit the proliferation activity of Sf9 cells and has significant toxicity to Sf9 cells. This confirms the effectiveness of RAP at a working concentration of not less than 0.1 μM, and the inhibitory effect is concentration-dependent.

[0054] Table 1. Effects of different concentrations of RAP on Sf9 cell viability.

[0055]

[0056] Effects of different concentrations of AZD-8055 on the growth status of Sf9 cells:

[0057] Figure 3 The effects of different concentrations of AZD-8055 on the growth status of Sf9 cells were demonstrated, such as Figure 3 As shown, the negative control group cells without AZD-8055 treatment were mostly spherical with regular morphology, smooth surface, and tightly packed adherent cells with normal morphology. After 48 hours of AZD-8055 treatment, the cell morphology of the 0.01 μM AZD-8055 treatment group was similar to that of the control group. When the AZD-8055 concentration reached 0.1 μM, Sf9 cell morphology changed, with cells beginning to shrink and exhibiting a small amount of swelling. When the concentration increased to 1 μM, drastic changes occurred in cell morphology, with a large number of cells shrinking and dying, a significant decrease in cell density, and visible cell debris. At concentrations of 10 μM and 50 μM, almost all cells disintegrated, with very few adherent cells. These results indicate that AZD-8055 can induce morphological changes in Sf9 cells, and that the morphological disruption and killing effects of AZD-8055 on Sf9 cells are dose-dependent, with an effective concentration similar to that of RAP, validating the effectiveness of the working concentration range of AZD-8055.

[0058] Results of AZD-8055 cytotoxicity assay on Sf9 cells:

[0059] Figure 4 The graph shows the effect of different concentrations of AZD-8055 on the survival rate of Sf9 cells of fall armyworm. The horizontal axis represents the concentration of AZD-8055 in the culture medium, and the vertical axis represents the number of surviving Sf9 cells. Turkey's one-way ANOVA was used to compare the significance of differences. An asterisk indicates a significant difference at the 5% level (P < 0.05). Figure 4 As shown in Table 2, AZD-8055 exhibits significant toxicity to Sf9 cells. The cell viability rate in the 0.01 μM AZD-8055 treatment group was 97.13%, showing no significant difference compared to the control group treated with DMSO. The cell viability rates in the 0.1 μM, 1 μM, 10 μM, and 50 μM AZD-8055 treatment groups were 77.24%, 51.61%, 53.23%, and 25.45%, respectively. Compared to the control group, these concentrations significantly inhibited Sf9 cell activity (P < 0.05). This result is highly consistent with morphological observations, confirming that AZD-8055 can effectively inhibit the proliferation of fall armyworm ovarian cells at low concentrations (0.1 μM and above), and the inhibitory effect is concentration-dependent.

[0060] Table 2. Effects of different concentrations of AZD-8055 on Sf9 cell viability.

[0061]

[0062] In summary, this invention demonstrates that the TOR inhibitors rapamycin (RAP) and AZD-8055 can effectively inhibit the proliferation of the fall armyworm ovarian cell line Sf9. At concentrations of 0.1 μM and above, both significantly induced morphological changes (swelling, shrinkage, apoptosis, etc.) in Sf9 cells and reduced cell viability in a clearly dose-dependent manner. Therefore, inhibitors with effective concentrations of RAP and AZD-8055 can serve as novel, green pesticides for the control of fall armyworm, providing a new application approach for controlling this major invasive pest.

[0063] Example 3:

[0064] Application of inhibitors for controlling fall armyworm in the preparation of pesticides for controlling fall armyworm: This pesticide can inhibit the proliferation of the fall armyworm ovarian cell line Sf9, induce abnormal morphology or death of Sf9 cells, and uses rapamycin and / or AZD-8055 as active ingredients, or rapamycin and / or AZD-8055 in combination with other active pesticide ingredients. The pesticide formulation is selected from at least one of wettable powder, water-dispersible granules, suspension concentrate, microemulsion, and soluble concentrate, and is applied by foliar spraying or soil treatment.

[0065] The specific implementation method is as follows: First, reagents and cells are prepared. Rapamycin is a macrolide immunosuppressant produced by Streptomyces hygroscopicus, AZD-8055 is an ATP-competitive TOR kinase inhibitor, and dimethyl sulfoxide (DMSO) is used as the solvent. Sf-900™ IISFM medium, commonly used pesticide adjuvants, and the fall armyworm ovarian cell line Sf9 are also prepared. Sf9 cells are cultured in Sf-900™ IISFM medium in a conventional incubator at 28°C and 4.5% CO2. They are passaged every 4 to 6 days, and cells in the logarithmic growth phase with uniform morphology, round shape, and good adhesion are used for the experiment.

[0066] Subsequently, pesticide stock solutions were prepared by dissolving rapamycin and AZD-8055 separately in DMSO to prepare high-concentration stock solutions. After sterile filtration, the solutions were stored at 4°C in the dark. When using, the solutions were diluted with Sf-900™ IISFM medium or field water to ensure that the effective working concentration of rapamycin was 0.1 μM to 50 μM and the effective working concentration of AZD-8055 was 0.1 μM to 50 μM. When compounding, the two stock solutions were mixed in the required proportion to ensure that the total active ingredient concentration was within the effective range.

[0067] Next, different formulations of pesticides were prepared. For soluble formulations, DMSO was used as the solvent and anhydrous ethanol as the co-solvent. Rapamycin and / or AZD-8055 were dissolved, then diluted to the target concentration with culture medium or field water, and stirred until homogeneous to obtain a transparent and uniform soluble solution. For microemulsions, the active ingredient was dissolved in DMSO, Tween-80 was added as an emulsifier, propylene glycol as a stabilizer, and sterile water or field water was slowly added dropwise while stirring at high speed to form a transparent and stable microemulsion. For suspensions, rapamycin and / or AZD-8055 were pre-dissolved in DMSO, and lignin sulfonate was added as a dispersant and xanthan gum. Using glue as a thickener and ethylene glycol as an antifreeze, water is added and the mixture is dispersed by high-speed shearing and then milled until the particle size is uniform to obtain a suspension. When preparing a wettable powder, rapamycin and / or AZD-8055 are mixed with diatomaceous earth and kaolin as carriers, sodium dodecyl sulfate as a wetting agent, and naphthalene sulfonate formaldehyde condensate as a dispersant. The mixture is then ultra-finely pulverized and sieved to obtain a wettable powder. When preparing a water-dispersible granule, the active ingredient is mixed with soluble starch as a carrier, polyvinyl alcohol as a binder, and sodium carboxymethyl starch as a disintegrant. The mixture is then pulverized, granulated, dried, and sized to obtain a water-dispersible granule. All of the above dosage forms are diluted with water to the effective working concentration before use.

[0068] The different formulations of pesticides mentioned above were used to treat Sf9 cells and verify their application effects. Sf9 cells in the logarithmic growth phase were taken and their density adjusted to 1.0 × 10⁶ cells / year. 3Cells were seeded at a density of 1 / mL in 96-well plates and cultured for 12 hours until the cells were fully adhered. The original culture medium was discarded, and pesticide formulations containing different concentrations of rapamycin and / or AZD-8055 were added. Blank controls and DMSO solvent controls were also set up. Each treatment was performed in 5 replicates, and the results were repeated 3 times. After 48 hours of dark treatment, cell morphology was observed and the survival rate was detected.

[0069] The results showed that the cells in the blank control group and the solvent control group were regular round, with clear outlines, good light transmittance, and dense arrangement, without morphological abnormalities or cell death. After treatment with pesticides containing rapamycin and / or AZD-8055, the proliferation of Sf9 cells was significantly inhibited, and the cells showed morphological abnormalities such as swelling, shrinkage, budding, blistering, and blurred outlines. Apoptotic bodies were visible in the high-concentration treatment group, and the cells eventually disintegrated and died. The inhibitory effect was concentration-dependent, with a significant inhibitory effect produced at concentrations of 0.1 μM and above.

[0070] This pesticide can be applied in two ways. The first is foliar spraying, where the prepared soluble concentrate, microemulsion, suspension, wettable powder, or water-dispersible granule is diluted with water to an effective concentration and sprayed evenly on both sides of the leaves of crops such as corn and rice, with a focus on the heart leaves and tender leaves. The second is soil treatment, where the diluted pesticide is irrigated into the soil around the crop roots or applied with irrigation water to control the fall armyworm.

[0071] In addition, this pesticide can be used in combination with other pesticide active ingredients. Rapamycin and / or AZD-8055 can be combined with conventional pesticide active ingredients for controlling fall armyworm, such as chlorantraniliprole and abamectin, at the registered dosage to make soluble, microemulsion or suspension formulations, which can be applied by foliar spraying. The combination can have a synergistic effect, further enhancing the inhibitory effect on Sf9 cell proliferation and delaying the development of pest resistance.

[0072] The foregoing has only described certain exemplary embodiments of the present invention by way of illustration. Undoubtedly, those skilled in the art can modify the described embodiments in various ways without departing from the spirit and scope of the present invention. Therefore, the foregoing drawings and descriptions are illustrative in nature and should not be construed as limiting the scope of protection of the claims of the present invention.

Claims

1. The application of an inhibitor for controlling fall armyworm in the preparation of pesticides for controlling fall armyworm, characterized in that, The inhibitor uses AZD-8055 as the main active ingredient and DMSO or an aqueous solution containing DMSO as the solvent. AZD-8055 is an ATP-competitive TOR kinase inhibitor. The effective working concentration of AZD-8055 is 0.1 μM to 50 μM; The inhibitor caused morphological changes or death in the fall armyworm ovarian cell line Sf9; the morphological changes included cell swelling, cell shrinkage, or the appearance of apoptotic bodies.

2. The application of the inhibitor for controlling fall armyworm according to claim 1 in the preparation of pesticides for controlling fall armyworm, characterized in that, The pesticide in question is one that inhibits the proliferation of the fall armyworm ovarian cell line Sf9 and induces abnormal morphology or death of Sf9 cells.

3. The application of the inhibitor for controlling fall armyworm according to claim 1 in the preparation of pesticides for controlling fall armyworm, characterized in that, The pesticide is formulated as a wettable powder, water-dispersible granule, suspension concentrate, microemulsion, or soluble concentrate.

4. The application of the inhibitor for controlling fall armyworm according to claim 1 in the preparation of pesticides for controlling fall armyworm, characterized in that, The pesticide is applied by foliar spraying or soil treatment.

5. The application of the inhibitor for controlling fall armyworm according to claim 1 in the preparation of pesticides for controlling fall armyworm, characterized in that, The pesticide uses AZD-8055 as its active ingredient, or combines AZD-8055 with other active pesticide ingredients.

Images