A pesticide composition containing roberts green muscardine and rotenone and its application

Abstract

The present application relates to a pesticide composition containing Roberts green muscardine and rotenone and its application, in particular to the application of the pesticide composition in the prevention and treatment of Asian corn borer, belonging to the field of biological pesticides. The active ingredient of the pesticide composition includes Roberts green muscardine MrGX0603 pure spore powder and rotenone, and the Roberts green muscardine MrGX0603 has been preserved in Guangdong Microbial Culture Collection Center on July 1, 2026, with the preservation number of GDMCC No.68257. The pesticide composition is compounded by Roberts green muscardine MrGX0603 and rotenone in a specific mass ratio, has a significant synergistic effect on the prevention and treatment of Asian corn borer, wherein rotenone plays a quick-acting insecticidal role, quickly knocks down Asian corn borer larvae, Roberts green muscardine realizes sustained infection, colonization and pest control, effectively improves the overall control effect, and greatly reduces the use amount of single pesticide and the risk of resistance.

Description

Technical Field

[0001] This invention relates to a pesticide composition containing Metarhizium anisopliae and rotenone and its application, specifically to the application of the pesticide composition in the control of Asian corn borer, belonging to the field of biological pesticides. Background Technology

[0002] Metarhizium robertsii, belonging to the genus Metarhizium in the subphylum Deuteromycetes, is an important biocontrol microorganism. It infects pests through its body wall, multiplies within the insect, and produces toxins such as metamizole, ultimately leading to the pest's death. Metarhizium robertsii possesses advantages such as a wide host range, strong pathogenicity, continuous pest control through field colonization, and safety against non-target organisms, making it an excellent biocontrol strain for controlling Lepidoptera and Coleoptera pests. Rotenone is a natural isoflavone compound extracted from the roots and stems of plants in the genera *Derris* and *Tephrosia* of the legume family. As a classic plant-derived pesticide, its mechanism of action is to inhibit mitochondrial respiratory chain complex I in insects, blocking energy metabolism and leading to paralysis and death of pests. Rotenone has broad-spectrum insecticidal activity, effectively inhibiting insect respiration and exhibiting strong antifeedant, contact, and stomach poison effects. In addition, rotenone degrades rapidly in air and light, posing a low risk of residue, and is therefore considered an environmentally friendly biopesticide.

[0003] The Asian corn borer (Ostrinia furnacalis) is a major lepidopteran pest in corn production. Its larvae bore into the whorls, stalks, cobs, and kernels of corn, causing damage such as mosaic virus, stalk breakage, and ear rot. In severe cases, it can reduce corn yield by more than 30%, affecting both yield and quality. Currently, the control of the Asian corn borer still relies primarily on chemical pesticides. The long-term and excessive use of organophosphates, pyrethroids, and diamides has not only led to a continuous increase in pesticide resistance in the Asian corn borer and a decline in control effectiveness year by year, but has also caused a series of problems, including excessive pesticide residues, damage to the farmland ecological environment, and damage to non-target organisms (natural enemies and beneficial microorganisms), which contradicts the development concepts of green and ecological agriculture. Microbial pesticides for the control of the Asian corn borer, such as single-component formulations like Beauveria bassiana, Bacillus thuringiensis, and azadirachtin formulations, generally suffer from poor rapid action, unstable efficacy, and short-lasting effects, making it difficult to meet the needs of rapid pest control and long-term yield protection in the field. Regarding the control of corn borers using biopesticide compositions, such as Zhang Yunyue's journal article "Research on the Combined Control of Asian Corn Borers by Microbial Pesticides," the study utilized two fungal insecticides, Beauveria bassiana and Metarhizium anisopliae, in combination to control Asian corn borers, as well as a combination of fungal insecticides and Bt. The results showed that the biopesticide compositions had a slow effect on corn borers, and the control efficacy was not ideal. Patent CN 118844471 A describes a combination of Verticillium chrysosporium and rotenone for the control of corn borers, but only records a co-toxicity coefficient of 120.767-172.159 against third-instar larvae of the corn borer; it does not record the field control efficacy of this composition against corn borers.

[0004] To date, there are no domestic literature reports on the use of a combination of Metarhizium anisopliae and rotenone for the control of Asian corn borer. Therefore, developing a novel biopesticide formulation combining Metarhizium anisopliae and rotenone could overcome the shortcomings of single biopesticides, single chemical pesticides, and microbial pesticide combinations, providing a new option for the green control of Asian corn borer and playing a significant role in promoting green agriculture and ensuring safe corn production. Summary of the Invention

[0005] To overcome the shortcomings of existing technologies, the present invention aims to provide a pesticide composition containing Metarhizium anisopliae and rotenone and its application. By combining the entomopathogenic fungus (Metarhizium anisopliae) with the plant-derived pesticide (rotenone), a synergistic control effect of "rapid-acting + sustained-effect" can be achieved. Specifically, the plant-derived pesticide quickly knocks down pests, exerting a rapid insecticidal effect; while the entomopathogenic fungus continuously infects and colonizes, achieving long-term pest control. Simultaneously, it can reduce the dosage of single pesticides and decrease the risk of resistance development. This addresses the shortcomings of single biological pesticides, single chemical pesticides, and microbial pesticide combinations, providing a new option for the green control of Asian corn borers and is of great significance for promoting green agricultural development and ensuring safe corn production.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: On the one hand, the present invention provides a pesticide composition containing Metarhizium robertsii and rotenone. The active ingredients of the pesticide composition include pure spores of Metarhizium robertsii MrGX0603 and rotenone. Metarhizium robertsii MrGX0603 has been deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC) on July 1, 2026, with accession number GDMCC No. 68257. The deposit address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Academy of Sciences, Postcode: 510070.

[0007] In a preferred embodiment of the pesticide composition of the present invention, the mass ratio of Metarhizium anisopliae MrGX0603 to rotenone in the pesticide composition is (1~35):(1~3), and the total mass of Metarhizium anisopliae and rotenone accounts for 3% to 4% of the total mass of the pesticide composition.

[0008] In a preferred embodiment of the pesticide composition of the present invention, the mass ratio of Metarhizium anisopliae MrGX0603 to rotenone in the pesticide composition is 2.5:1, and the total mass of Metarhizium anisopliae and rotenone accounts for 3.5% of the total mass of the pesticide composition.

[0009] In a preferred embodiment of the pesticide composition of the present invention, the Metarhizium anisopliae MrGX0603 pure spore powder is a high-purity conidial powder prepared by liquid-solid two-phase fermentation, with a spore content ≥3.5×10⁻⁶. 10 Spores / g, spore germination rate ≥90%, drying loss ≤3.0%, meeting the quality requirements for pesticide formulation processing.

[0010] In a preferred embodiment of the pesticide composition of the present invention, the rotenone is a high-purity product extracted naturally with a purity of ≥95%, which can be purchased commercially.

[0011] On the other hand, the present invention provides a dispersible oil suspension, wherein the effective component of the dispersible oil suspension includes the active component of the pesticide composition described in the present invention.

[0012] As a preferred embodiment of the dispersible oil suspension of the present invention, the dispersible oil suspension further includes pesticide adjuvants, which include surfactants, thickeners, ultraviolet protectants and oil-based components; the surfactants include emulsifiers, aqueous dispersants and non-aqueous dispersants.

[0013] As a preferred embodiment of the dispersible oil suspension of the present invention, the dispersible oil suspension comprises, by mass percentage, 2%–3% pure spore powder of Metarhizium anisopliae, 0.8%–1.2% rotenone, 12%–18% surfactant, 2%–4% thickener, 0.8%–1.2% UV protectant, and oil base to make up to 100%.

[0014] As a preferred embodiment of the dispersible oil suspension of the present invention, the dispersible oil suspension comprises, by mass percentage, 2.5% Metarhizium anisopliae pure spore powder, 1% rotenone, 10% emulsifier, 3% non-aqueous dispersant, 2% aqueous dispersant, 3% thickener, 1% UV protectant, and oil base to make up to 100%.

[0015] In a preferred embodiment of the dispersible oil suspension of the present invention, the emulsifier includes TENSIOFIX 96DB10 and / or Atlas G-1086; the non-aqueous dispersant includes Atlox-4914; the aqueous dispersant includes Atlas G-5002L; the thickener is selected from at least one of A200, silica, and ATLOX RHEOSTRUX 100; the UV protectant includes ZnO; and the oil base is selected from at least one of sunflower oil, corn oil, soybean oil, peanut oil, and blended vegetable oils.

[0016] In a preferred embodiment of the dispersible oil suspension agent of the present invention, the emulsifier includes TENSIOFIX 96DB10 and Atlas G-1086; the thickener includes A200, silica and ATLOX RHEOSTRUX100; and the oil base is sunflower seed oil.

[0017] In another aspect, the present invention provides a method for preparing the dispersible oil suspension, the method comprising the following steps: (1) Pretreatment: Add a thickener to a small amount of oil base, heat to ensure that the thickener is completely dissolved, and obtain a uniform oil phase matrix; (2) Mixing and dispersing: Emulsifier, dispersant, the other two thickeners, UV protectant and rotenone are added to the oil base in sequence and placed in a high-speed stirrer to mix the components evenly to obtain a preliminary mixture; (3) Sand milling: Transfer the preliminary mixture into a sand mill, adjust the speed of the sand mill to 2500 r / min, and mill for 1 hour to refine the particles in the mixture to a particle size ≤5μm, so as to ensure the emulsification and dispersibility of the formulation; (4) Final preparation: Filter the mixture after sand milling to remove impurities, then add pure spore powder of Metarhizium anisopliae MrGX0603 and the pretreated oil phase matrix, place it in a high-speed homogenizer, and homogenize at 3000 r / min for 30 min to make the spores evenly dispersed in the formulation, thus obtaining the dispersible oil suspension product.

[0018] On the other hand, the present invention provides the application of the pesticide composition or the dispersible oil suspension in the control of Asian corn borer, which can be used for green control of Asian corn borer larvae in corn fields, and is especially suitable for pest control in the production of organic corn and green food corn.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) The pesticide composition provided by the present invention combines entomopathogenic fungi (Metarhizium anisopliae MrGX0603) with plant-derived pesticides (rotenone). Within a specific mass ratio range of (1~35): (1~3), it has a significant synergistic effect on the control of Asian corn borers, achieving a synergistic control effect of "rapid effect + sustained effect". That is, the plant-derived pesticide quickly knocks down the pests and exerts a rapid insecticidal effect; the entomopathogenic fungi continuously infect and colonize, achieving long-term pest control, effectively improving the overall control effect, while significantly reducing the amount of single agent used and reducing the risk of resistance.

[0020] (2) The dispersible oil suspension agent protected by this invention uses sunflower seed oil as the oil base and does not use organic solvents, thus avoiding the harm of organic solvents to the environment, pesticide applicators and non-target organisms. It also has excellent stability at room temperature and cold storage, and all quality indicators meet the relevant standards for dispersible oil suspension agents. At the same time, it can effectively protect the activity of Roberts green metamorphosum MrGX0603 spores, improve the field UV resistance and survival rate of spores, prolong the effective period of the formulation, and expand the types of biological pesticide formulations.

[0021] (3) The pesticide composition and dispersible oil suspension protected by this invention are safe for corn crops, have no phytotoxicity after field application, and are environmentally friendly and easily degradable. They make up for the shortcomings of single biological pesticides, single chemical pesticides and microbial pesticide compositions, and provide a new option for green control of Asian corn borer. They are of great significance for promoting green agricultural development and ensuring safe corn production. Attached Figure Description

[0022] Figure 1 The morphology of Asian corn borer larvae infected with Metarhizium anisopliae MrGX0603 after being kept moist is shown. Figure 1 In the diagram, A represents the growth of mycelium in a localized area of ​​the insect body. Figure 1 In the diagram, B represents mycelium covering the insect body. Figure 1 In this context, C represents the sporulation of mycelium on the insect body.

[0023] Figure 2 The dispersion states of pure spores of Metarhizium anisopliae in different vegetable oil bases after standing for 1 hour are shown. From left to right, they are sunflower seed oil, corn oil, peanut oil, soybean oil, and blended vegetable oil.

[0024] Figure 3 The emulsified dispersion states of different surfactants and sunflower seed oil mixtures in water. Figure 3 The first to third rows from top to bottom in the middle are state diagrams for 1 h, 24 h, and 48 h of resting, respectively.

[0025] Figure 4 The mixtures of different thickeners and sunflower seed oil, after standing for 24 hours, are as follows: from left to right: silica, xanthan gum, sodium carboxymethyl cellulose, ATLOX RHEOSTRUX 100, A200, 305 thickener, magnesium aluminum silicate, bentonite, organobentonite, Tensiofix 869, fumed silica, and CK.

[0026] Figure 5 The effect of UV irradiation time on spore germination rate under different amounts of UV protectant treatment.

[0027] Figure 6 The finished product is a 3.5% Metarhizium anisopliae-rotenone dispersible oil suspension prepared according to the present invention.

[0028] Figure 7 The field control efficacy of Metarhizium anisopliae and rotenone dispersible oil suspension against Asian corn borer in maize fields was studied. Figure 7 A represents the plant growth in the 3.5% Metarhizium anisopliae·rotenone OD treatment group. Figure 7 B represents the growth of plants in the control group. Figure 7 C and D represent the dead corn insects in the treatment groups after pesticide application. Figure 7 E represents corn borers wrapped in mycelium on plants in the treatment group. Detailed Implementation

[0029] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0030] Unless otherwise specified, the experimental methods used in these examples are conventional methods, and the materials, reagents, and strains used are all commercially available. Specifically, *Metarhizium anisopliae* MrGX0603 is a highly pathogenic strain (GDMCC No. 68257) obtained through screening and can be acquired using conventional isolation and culture methods; rotenone purity is ≥95%; and all adjuvants are commercially available pesticide-grade products.

[0031] Example 1: This example illustrates the screening of the active ingredients in the pesticide composition described in this invention. 1. Bioactivity determination of Metarhizium anisopliae MrGX0603 against Asian corn borer The Asian corn borer tested were second-instar larvae artificially reared indoors. The rearing conditions were a temperature of 27±1℃, a relative humidity of 70±5%, and a photoperiod of 16L:8D. During the rearing period, they were fed artificial feed, which was changed every two days. Healthy larvae with consistent vitality were selected for live testing.

[0032] Metarhizium anisopliae MrGX0603, deposited by the laboratory of South China Agricultural University at the Guangdong Provincial Microbial Culture Collection Center (GDMCC) on July 1, 2026, with accession number GDMCC No. 68257, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Academy of Sciences, Postcode: 510070. MrGX0603 was inoculated onto PDA plates and incubated at 28℃ for 14 days. After full sporulation, spores were scraped from the fully sporulated plates using a spatula and collected in 20 mL centrifuge tubes. 10 mL of sterile 0.1% Tween-80 solution was added, and the mixture was thoroughly shaken and filtered through double-layered sterilized filter paper to prepare 1×10⁻⁶ centrifuge tubes. 8 A spore suspension at spores / mL is prepared for later use.

[0033] Bioactivity was determined according to the People's Republic of China Agricultural Industry Standard NY / T 1154.6-2006 "Guidelines for Indoor Bioassay Testing of Pesticides - Part 6: Insecticidal Activity Test - Immersion Method". Healthy, uniformly grown second-instar Asian corn borer larvae were selected with a brush and transferred to sterilized 2 mL centrifuge tubes. 1 mL of the tested bacterial strain spore suspension was added, the tubes were quickly sealed, and the tubes were inverted 10 times. The tubes were then transferred to disposable petri dishes lined with filter paper and placed in an artificial climate incubator at 27±1℃, 70±5% relative humidity, and a photoperiod of 16 L:8 D. Ten larvae were used per group, with three replicates. The Asian corn borer larvae treated with immersion were fed artificial feed, with the feed changed every two days. Larval mortality was observed at 1, 3, 5, 7, 9, and 11 days after treatment. The number of dead larvae was recorded, and the corrected mortality rate was calculated. The calculation formula is as follows:

[0034] Experimental results: Metarhizium anisopliae MrGX0603 showed good activity, 1×10 8 After 7 days of treatment with a spore suspension at a concentration of spores / mL, the corrected mortality rate exceeded 60%; after 11 days of treatment, the corrected mortality rate was 78.57%, exceeding 70%. Virulence regression analysis showed that the virulence regression equation for *Metarhizium anisopliae* MrGX0603 after 11 days of treatment was y=0.031+0.719x, with a correlation coefficient of 0.9751. The median lethal concentration (LC50) against 2-year-old Asian corn borers was... 50 It is 8.21×10 6 Spores / mL, 95% confidence interval is 3.18 × 10⁻⁶ 6 ~17.26×10 6 Spores / mL. The surface of infected and dead larvae is gradually covered with green mycelium, forming a layer resembling... Figure 1 The bioactivity of Metarhizium anisopliae MrGX0603 against the Asian corn borer is shown in Table 1.

[0035] Table 1 Bioactivity of MrGX0603 against Asian corn borer

[0036] The data in the table represent the corrected mortality rates of corn borer larvae after treatment with different concentrations of MrGX0603 spore suspensions. The data are the mean ± standard error of three replicates, where CK is the mortality rate of the tested corn borers.

[0037] 2. Determination of the toxicity of rotenone to the Asian corn borer The rearing conditions for the tested Asian corn borer larvae were consistent with those for the above-mentioned insect pathogenic fungal bioactivity assays. Healthy second-instar larvae with consistent vigor were selected for toxicity assays.

[0038] A stock solution of rotenone with a concentration of 30,000 mg / L was prepared using dimethyl sulfoxide as a solvent and stored at 4°C for later use. During the experiment, the stock solutions were diluted to working concentrations of 50 mg / L and 250 mg / L using 0.1% Tween-80 solution. Asian corn borers were treated with the above-described method, and larval mortality was observed at 1, 3, and 5 days after treatment. The number of dead larvae was recorded, and the mortality rate and corrected mortality rate were calculated.

[0039] Experimental Results: After treatment of Asian corn borer larvae with different concentrations of rotenone, the corrected mortality rate increased significantly with increasing rotenone concentration and treatment time. Specifically, the corrected mortality rate was 35.90% after 6 days of treatment at a concentration of 50 mg / L, 58.97% after 6 days at a concentration of 100 mg / L, and reached 82.05% after 6 days at a concentration of 200 mg / L. The toxicity determination results of different concentrations of rotenone against the Asian corn borer are shown in Table 2. Toxicity regression analysis yielded the following regression equation for 6 days of treatment: y = 1.979 + 1.656x, with a correlation coefficient of 0.9839. The median lethal concentration (LC50) was... 50 The concentration was 66.60 mg / L, with a 95% confidence interval of 48.67–86.06 mg / L.

[0040] Table 2. Toxicity of rotenone to Asian corn borer

[0041] The toxicity of rotenone at different concentrations to second-instar larvae of the Asian corn borer was determined by the immersion method. Ten larvae were treated per larva. The data in the table are the corrected mean mortality rate ± standard error of four replicates. CK is the mortality rate of the tested Asian corn borers after immersion treatment in 0.1% Tween-80 solution.

[0042] 3. Safety determination of rotenone against Metarhizium anisopliae MrGX0603 Rotenone was diluted to 500 mg / L using 0.1% Tween-80 solution and filtered through a disposable syringe filter with a pore size of 0.22 μm to ensure sterility. Sterilized PDA was heated to dissolve and cooled to approximately 45°C. Sterile rotenone solution was then added to the PDA in the specified proportions and mixed thoroughly to prepare toxin-containing plates with final concentrations of 25 mg / L, 66 mg / L, and 150 mg / L. *Metarhizium anisopliae* (MrGX0603) mycelial pellets were punched and transferred to the center of the toxin-containing plates using an inoculation needle. PDA without rotenone was used as a control. Each treatment was repeated three times. After inoculation, the plates were incubated at 28°C. Colony diameter was measured using the cross-sectional method at 7 and 14 days.

[0043] The toxic medium plate was prepared using the above method, with a concentration of approximately 1×10⁻⁶. 6 A spore suspension of 100 μL was evenly spread onto a plate in a clean bench. After 24 h, the number of germinating spores was counted under an optical microscope. Germination was considered to have occurred when the spores swelled to twice their original size or extended beyond the germ tube. The spore germination rate was calculated using the following formula:

[0044] Experimental results: Under low, medium, and high concentrations of rotenone, the spore germination rate of strain MrGX0603 showed no significant difference from the control group, with germination rates all exceeding 93%. The results indicate that at a lethal concentration of 66 mg / L, rotenone did not significantly affect the mycelial growth and spore germination of strain MrGX0603. The effects of rotenone on the mycelial growth and spore germination rate of MrGX0603 are shown in Table 3.

[0045] Table 3. Effects of rotenone on mycelial growth and spore germination rate of entomopathogenic fungi

[0046] The data in the table show the colony diameter and spore germination rate of strain MrGX0603 under different rotenone concentrations. Colony diameter data are the mean ± standard error of three replicates, and spore germination rate data are the mean ± standard error of four replicates. Different lowercase letters in the same column indicate significant differences at the p < 0.05 level as determined by the DMRT test.

[0047] 4. Determination of the combined toxicity of Metarhizium anisopliae MrGX0603 and rotenone against the Asian corn borer. Metarhizium anisopliae MrGX0603 and rotenone were dissolved separately in 0.1% Tween-80 solution and adjusted to their respective LC ratios. 50 Concentration, for later use. The prepared rotenone solution and spore suspension were mixed thoroughly at mass ratios of 1:0, 3:1, 1.15:1, 1:1.5, 1:2.5, 1:3.5, 1:5.5, 1:8, 1:14, 1:35, and 0:1, respectively. A 0.1% Tween-80 solution was used as a control. The total volume of the prepared solution for each treatment was 10 mL, and the mixture was repeated three times. Asian corn borers were treated according to the method described above. Larval mortality was observed and recorded at 1, 3, 5, and 7 days after treatment. The mortality rate and corrected mortality rate were calculated using the formula. Referring to the method of Yin Fei et al., the synergistic virulence index (CF value) of *Metarhizium anisopliae* MrGX0603 and rotenone was calculated using the formula.

[0048]

[0049] Judgment criteria: CF value > 20 indicates synergistic effect; 0 ≤ CF value ≤ 20 indicates additive effect; CF value < 0 indicates antagonistic effect.

[0050] Experimental results: Rotenone and MrGX0603 in LC 50The combined toxicity results of rotenone and MrGX0603 at different mass ratios for infiltration treatment of Asian corn borer are shown in Table 4. After 7 days of treatment, when the mass ratios of rotenone and MrGX0603 were 3:1, 1.15:1, and 1:1.5, the corrected mortality rates were 56.67%, 56.67%, and 60.00%, respectively, and the synergistic toxicity indices (CF values) were -5.56, -5.56, and 0, respectively, showing an additive effect. When the mass ratio was 1:2.5, the corrected mortality rate was 73.33%, and the CF value was 22.22, showing a synergistic effect. The CF values ​​of the other mass ratio combinations were all less than -20, showing an antagonistic effect. Therefore, rotenone and MrGX0603 were combined at a mass ratio of 1:2.5, in which the concentration of rotenone was 39.96 mg / L and the concentration of Metarhizium anisopliae MrGX0603 was 3.28 × 10⁻⁶. 6 Spores / mL.

[0051] Table 4. Co-toxicity of different ratios of rotenone and MrGX0603 against Asian corn borer.

[0052] The data in the table represent the lethality and synergistic toxicity index of rotenone and MrGX0603 after 7 days of mixing in different proportions on corn borers. The corrected mortality rate is the mean ± standard error of three replicates. CK represents the mortality rate of the tested corn borers.

[0053] Example 2: Preparation and quality testing of pure spores of Metarhizium anisopliae MrGX0603 (1) Liquid seed culture: MrGX0603 mycelium cake was punched with a puncher and inoculated into insect powder PDB medium (200g potato, 20g glucose, 5g insect powder, 1L deionized water). The culture was shaken at 28℃ and 180 r / min for 48 h to obtain seed liquid. (2) Solid fermentation: 300 g of rice was put into a polypropylene plastic bag and sterilized by moist heat at 121℃ for 15 min. After cooling, the seed liquid was inoculated at a rate of 20% (m:m). The mixture was mixed evenly and cultured at 28℃ and 80%-90% relative humidity for 7-14 days until the fungus produced full spores. (3) Spore collection: Spread out the rice covered with spores and use an electric vibrating sieve to separate the spore powder from the rice; (4) Drying and storage: Dry using a vacuum freeze dryer for 5-7 hours. After passing quality inspection, store in a sealed refrigerator at 4℃.

[0054] The quality indicators of pure spore powder are shown in Table 5, including a spore germination rate of 92.00%, a drying weight loss of 2.94%, and a spore content of 3.53 × 10⁻⁶. 10Spores / g. Based on the results in Table 4, the mass ratio of the active ingredient rotenone to MrGX0603 was determined to be 1:2.5 (m:m).

[0055] Table 5 Quality Indicators of Pure Spore Pollen of Metarhizium anisopliae MrGX0603 from Roberts

[0056] The data in the table are the mean ± standard error of three replicates.

[0057] Example 3: Development of a 3.5% Metarhizium anisopliae·Rotenone Dispersible Oil Suspension 1. Screening of pharmaceutical adjuvants The screening of adjuvants for dispersible oil suspensions was based on the core principle of "not affecting the activity of Metarhizium anisopliae spores and improving the emulsification, dispersibility, and stability of the formulation." Oil-based agents, surfactants, thickeners, and UV protectants were screened separately, as detailed below: (1) Oil base screening: The tested oil bases included sunflower oil, corn oil, peanut oil, soybean oil, and blended vegetable oil, a total of 5 types, all of which were commercially available food-grade vegetable oils. 0.25 g of pure spore powder was weighed into a 10 mL sterile centrifuge tube, and 5 mL of each different oil base was added. The tubes were shaken well, and each treatment was repeated 3 times. All samples were stored at room temperature and their dispersion was initially observed. The spore germination rate was measured after 7 days, 15 days, and 30 days of storage to test the safety of the 5 oil bases to the spores. Pure spore powder from the same batch without oil bases was used as a control group.

[0058] Experimental Results: During the one-month storage period, the spore germination rate of the treated groups was significantly higher than that of the control group. Furthermore, the spores in the treated groups maintained high germination activity even with prolonged storage, indicating that all five tested plant oils effectively maintained spore viability and had a significant protective effect. In the three measurements, the spore germination rate stored in sunflower seed oil was the highest (see Table 6). Therefore, sunflower seed oil was selected as the optimal oil base for subsequent experiments in this invention's dispersible oil suspension. The dispersion states of *Metarhizium anisopliae* pure spores in different plant oil bases are shown below. Figure 2 As shown, Figure 2 The images show the dispersion of pure spores of Metarhizium anisopliae in different vegetable oil bases after standing for 1 hour. From left to right, they are sunflower oil, corn oil, peanut oil, soybean oil, and blended vegetable oil.

[0059] Table 6 Effect of oil base on spore germination rate of MrGX0603

[0060] The data in the table are the mean ± standard error of three replicates. Different lowercase letters after the data in the same column indicate significant differences according to the DMRT test (p < 0.05).

[0061] Surfactant screening: A total of 17 surfactants were tested, and their details are shown in Table 7.

[0062] Table 7 Surfactant Information

[0063] ① Safety test of surfactants against Metarhizium anisopliae: 10 mL of sterile water was added to 15 mL centrifuge tubes, and 100 μL of surfactant was added dropwise to prepare 17 kinds of aqueous solutions containing surfactants. Sterile water without surfactant was used as a control. 50 mg of pure spore powder was added under sterile conditions, and the mixture was shaken thoroughly. Each treatment was repeated 3 times. After storage at room temperature for 3, 7, 15 and 30 days, the spore germination rate was measured.

[0064] Experimental results: The spore germination rate of the seven surfactant treatment groups (Atlas G-5002L, NP-10, Atlox-4914, TENSIOFIX 96DB10, Atlas G-1086, dispersant 5040, and agricultural emulsion 600#) was not significantly different from that of the control group, and all remained above 71%. This indicates that these seven surfactants have good safety for Metarhizium anisopliae MrGX0603. The effects of each surfactant on the spore germination rate of MrGX0603 are shown in Table 8.

[0065] ② Evaluation of the emulsifying and dispersing properties of surfactants: Surfactants were added to sunflower seed oil to prepare sunflower seed oil containing 15% surfactant. The dispersibility of the surfactant in the oil was observed during the preparation process. The above mixed oil was shaken well, and 1 mL was added to 9 mL of water. The mixture was shaken well, and the emulsifying properties of the surfactant in water were observed. The emulsifying and dispersing stability of the sample was observed and recorded after standing for 1 h, 24 h, and 48 h.

[0066] Experimental Results: During the functional screening of single-agent agents, the dispersibility of surfactants in sunflower seed oil containing 15% surfactant was observed in Table 9. The 17 tested surfactants showed good dispersibility in sunflower seed oil, all at grade three or higher. The emulsification and dispersion of the sunflower seed oils containing different surfactants were added to water and stirred. The results are also shown in Table 9. Except for EL40, Atlox-4916, and Agricultural Emulsion 600#, which were rated grade five, the remaining surfactants were rated grade three and could be dispersed after stirring. Except for dispersant 5040, which had no emulsifying function, the others formed a milky white liquid after being added to water, indicating good emulsifying ability. The states of the mixtures after standing in water for 1 h, 24 h, and 48 h are shown in Table 9. Figure 3As shown, surfactants Atlox-4914, TENSIOFIX 96DB10, Atlas G-1086, and Atlas G-1096 maintained good emulsifying properties and did not separate after standing for 1 h, 24 h, and 48 h.

[0067] Based on the above safety and functionality test results, NP-10 and Agricultural Emulsion 600# showed inferior emulsifying effects compared to Atlox-4914, TENSIOFIX 96DB10, Atlas G-1086, and Atlas G-1096. Furthermore, the spore germination rate of the Atlas G-1096 treatment group was significantly lower than the control group after 30 days of storage, thus it was excluded. After standing for 48 hours, TENSIOFIX 96DB10 and Atlas G-1086 showed the best emulsifying effects, followed by Atlox-4914. Atlox-4914 can also be used as a non-aqueous dispersant. Atlas G-5002L and Dispersant 5040 function as aqueous dispersants. After standing for 48 hours, their dispersibility was observed, and Atlas G-5002L performed better than Dispersant 5040. Therefore, the dispersible oil suspension of this invention ultimately selected TENSIOFIX 96DB10 and Atlas G-1086 as emulsifiers, Atlox-4914 as a non-aqueous dispersant, and Atlas G-5002L as an aqueous dispersant.

[0068] Table 8 Effect of surfactants on the germination rate of MrGX0603 spores

[0069] The data in the table are the mean ± standard error of three replicates. Different lowercase letters after the data in the same column indicate significant differences according to the DMRT test (p < 0.05).

[0070] Table 9 Evaluation of the emulsifying and dispersing properties of surfactants

[0071] Emulsification and dispersion effect rating standards: Level 1: Rapid, automatic, and uniform dispersion; with slight agitation, it forms a fluorescent blue or pale white transparent emulsion; Level 2: Automatic and uniform dispersion; with slight agitation, it forms a blue or pale milky white translucent emulsion; Level 3: White cloud-like or filamentous dispersion; with slight agitation, it forms a pale blue or milky white opaque emulsion; Level 4: White granular sedimentation; after agitation, it forms an opaque emulsion; Level 5: It settles as an oil; agitation does not completely emulsify it; and it quickly separates into layers after agitation stops.

[0072] (3) Thickener screening The tested thickeners included A200, magnesium aluminum silicate, silica, fumed silica, xanthan gum, organobentonite, Tensiofix 869, ATLOX RHEOSTRUX 100, bentonite, sodium carboxymethyl cellulose, and 305 thickener, totaling 11 types.

[0073] ① Functional determination of thickeners Add 4% thickener to sunflower seed oil, shake well, and let stand at room temperature for 24 hours. Observe the suspension height and bottom formation. The less oil separation, the better the suspension.

[0074] Experimental Results: Thickener 305, when added to sunflower seed oil, still separated into layers after shaking, and was therefore excluded. Observation of mixtures of different thickeners and sunflower seed oil after standing for 24 hours revealed that A200 and fumed silica were soluble in the oil, while the others all showed separation. Among them, ATLOX RHEOSTRUX 100 recovered to a dispersed state the fastest after shaking, followed by Tensiofix 869, silica, and sodium carboxymethyl cellulose. Bentonite, organobentonite, magnesium aluminum silicate, and xanthan gum showed a bottoming phenomenon.

[0075] ② Safety test of thickener against Metarhizium anisopliae Add 10 mL of sterile water to 15 mL centrifuge tubes, and add thickeners to prepare 2% (m / m) aqueous solutions containing different thickeners. Use sterile water without thickener as a control. Under sterile conditions, add 50 mg of pure spore powder, shake thoroughly to mix evenly, and repeat each treatment three times. After storing at room temperature for 5 and 10 days, determine the spore germination rate.

[0076] Experimental Results: After storage for 5 and 10 days, respectively, there were no significant differences in spore germination rates between the treatment groups using thickeners A200, ATLOX RHEOSTRUX 100, xanthan gum, sodium carboxymethyl cellulose, silica, and fumed silica and the control group. The spore germination rates all exceeded 90%, indicating relative safety for the spores. The spore germination rates of the other thickener treatment groups were lower, indicating some inhibitory effect on the fungus, and these were excluded. The effects of each thickener on the spore germination rate of MrGX0603 are shown in Table 10.

[0077] Based on the functional assay results of the single agent, xanthan gum exhibited a bottoming-out phenomenon and was therefore not used for subsequent experiments. Since A200 is a different commercial product of fumed silica, only one of the tested thickeners, A200 or fumed silica, needs to be retained. Given that the spore germination rate of the A200 treatment group was higher than that of the fumed silica treatment group after 5 and 10 days of storage, A200 was retained. Therefore, the final selected thickeners for the dispersible oil suspension of this invention are A200, ATLOX RHEOSTRUX 100, silica, and / or sodium carboxymethyl cellulose. The effects of different thickeners on the stability of the dispersible oil suspension are as follows: Figure 4 As shown.

[0078] Table 10 Effect of thickener on the germination rate of MrGX0603 spores

[0079] The data in the table are the mean ± standard error of three replicates. Different lowercase letters after the data in the same column indicate significant differences according to the DMRT test (p < 0.05).

[0080] (4) Screening of UV protectants The tested UV protectants included ascorbic acid, sodium carboxymethyl starch, TiO2, fluorescent whitening agent, and ZnO, totaling five types.

[0081] ① Safety test of UV protectant against Metarhizium anisopliae Take 10 mL of sunflower seed oil, add 50 mg of UV protectant, then add 50 mg of pure spore powder, shake thoroughly to mix evenly, with no UV protectant as the control. Each treatment is repeated 3 times. After storage at room temperature for 7 and 15 days, the spore germination rate is measured.

[0082] ②Determination of UV protection performance 50 mg of pure spore powder of Metarhizium anisopliae was thoroughly mixed with 25 mg of UV protectant and placed in a petri dish lined with filter paper. After irradiation with a 254 nm UV lamp at a distance of 40 cm for 4 and 8 h, the spore germination rate was measured. Pure spore powder without UV protectant was used as a control.

[0083] Experimental Results: After 15 days of storage at room temperature, except for the fluorescent whitening agent, the spore germination rates of the other four UV protectant treatment groups were not significantly different from the control group, indicating high safety for *Metarhizium anisopliae* MrGX0603. After 4 hours of UV irradiation, the spores treated with TiO2, ZnO, fluorescent whitening agent, and sodium carboxymethyl starch maintained good germination rates, significantly higher than the control group, while the spore germination rate of the ascorbic acid treatment group was only 64.33%. After 8 hours of UV irradiation, only the spore germination rates of the TiO2 and ZnO treatment groups were significantly better than the other treatment groups and the control group, indicating that these two UV protectants have good protective performance for *MrGX0603*, while the protective performance of ascorbic acid is weak, with only 18% of spores germinating. The spore germination rates of *MrGX0603* under different conditions in UV protectants are shown in Table 11.

[0084] Considering the spore germination rate under both room temperature storage and ultraviolet irradiation conditions, ZnO was ultimately selected as the best performing ultraviolet protectant for the dispersible oil suspension of this invention.

[0085] Table 11 Spore germination rate of MrGX0603 under different conditions in UV protectant

[0086] The data in the table are the mean ± standard error of three replicates. Different lowercase letters after the data in the same column indicate significant differences according to the DMRT test (p < 0.05).

[0087] 2. Formulation optimization and preparation of dispersible oil suspensions (1) Re-screening of surfactants Based on the initial screening results of surfactants, a secondary screening was conducted on surfactants with different functions and good effects, using appropriate combinations. The dosage of surfactants was tested according to the recommended dosages of each adjuvant; the recommended dosage for emulsifiers was 5%-10%, and for dispersants, it was 1%-3%. Different dosages of emulsifiers (TENSIOFIX 96DB10, Atlas G-1086), non-aqueous dispersants (Atlox-4914), and aqueous dispersants (Atlas G-5002L) were combined, and 3% thickener and an appropriate amount of sunflower seed oil were added and milled. Then, 1% pure spore powder was added for homogenization to obtain small-scale formulations. These small-scale formulations were then stored at 4℃, 25℃, and 54℃ for 14 days, at room temperature, and at high temperature, respectively. The flow and oil separation of the small-scale formulations were observed and recorded.

[0088] Experimental Results: Under normal temperature storage conditions, all nine treatments exhibited good stability. Under hot storage conditions, treatment group 5 (5% TENSIOFIX 96DB10 + 5% Atlas G-1086 + 3% Atlox-4914 + 1% Atlas G-5002L) and treatment group 7 (10% TENSIOFIX 96DB10 + 3% Atlox-4914 + 2% Atlas G-5002L) showed good stability. Under cold storage conditions, treatment groups 2, 4, and 7 exhibited "excellent" oil separation grades. Based on the above results, treatment groups 5 and 7 were selected as backup formulations for surfactants.

[0089] Table 12 Effects of different amounts of surfactant compounded

[0090] The evaluation criteria are as follows: Excellent grade: no oil separation or only a small amount of oil separation, with an oil separation rate of <5%; Good grade: oil separation rate of 5%-10%; Poor grade: oil separation rate of >10%.

[0091] (2) Secondary screening of thickeners Based on the initial screening results of thickeners, the thickeners with better performance were further screened by dosage combinations, with the total amount of thickener not exceeding 5%. Different amounts of A200, silica, sodium carboxymethyl cellulose, and ATLOX RHEOSTRUX 100 were combined, and 20% surfactant and an appropriate amount of sunflower seed oil were added and sand-milled for later use. Then, different combinations of thickeners and 1% pure spore powder were added and homogenized to obtain small-scale formulations. The formulations were stored at 4℃, 25℃, and 54℃ for 14 days, and the flow and oil separation of the samples were observed and recorded.

[0092] Experimental Results: Treatments 1-5 all showed "poor" oil separation ratings regardless of whether they were stored at room temperature, in hot conditions, or in cold conditions. Treatment 6 (1% A200 + 1% silica + 1% ATLOX RHEOSTRUX 100) achieved excellent or good oil separation under all three storage conditions, and maintained good fluidity throughout. Treatments 7, 8, and 9 all showed acceptable oil separation, with treatments 8 and 9 being superior to treatment 7. Therefore, treatment 6 was selected as the preferred thickener system, while treatment 9 (1.5% A200 + 1% silica + 1% sodium carboxymethyl cellulose + 0.5% ATLOX RHEOSTRUX 100), a different combination from treatment 6, was selected as an alternative (Table 13).

[0093] Table 13 Effects of different amounts of thickener in compound formulation

[0094] The evaluation criteria are as follows: Excellent grade: no oil separation or only a small amount of oil separation, with an oil separation rate of <5%; Good grade: oil separation rate of 5%-10%; Poor grade: oil separation rate of >10%.

[0095] (3) Screening of UV protectant dosage Based on the initial screening results of UV protectants, ZnO, a UV protectant with high safety and good protective performance, was screened for dosage. ZnO was mixed with pure spore powder at dosages of 1%, 2%, and 3%, and irradiated with a 254 nm UV lamp at a distance of 40 cm for 4 h and 8 h, respectively. The spore germination rate was then measured.

[0096] Experimental results: When the UV protectant ZnO was added to pure spore powder at a concentration of 1%-3%, the spore germination rate after 4 h and 8 h of UV irradiation was as follows: Figure 5 As shown, after 4 hours of UV irradiation, the spore germination rate of the three treatment groups with different addition amounts was not significantly different from that of the control group. After 8 hours of UV irradiation, the spore germination rate of the treatment groups was significantly higher than that of the control group, but there was no significant difference among the three treatment groups. This indicates that the addition amount of ZnO at 1%-3% can provide good protection for spores. When the addition amount increases from 1% to 3%, the improvement in spore protection effect is not significant. Therefore, the dosage of UV protectant was determined to be 1%.

[0097] (4) Final formula optimization The two surfactant formulations and two thickener formulations obtained from the screening were combined, and the amount of aqueous dispersant (Atlas G-5002L) was increased to the maximum recommended amount (3%), resulting in 7 treatments. The remaining components were then added to prepare formulation samples, which were stored at 4℃ and 25℃ for 14 days, respectively. The flow and oil separation of the samples were observed and recorded.

[0098] Experimental Results: Tables 14-15 show that Treatment 5 had an oil separation rate of 12.5% ​​under hot storage conditions, which was considered unqualified. The oil separation rates of the remaining combinations were all below 10% under normal temperature storage, hot storage, and cold storage conditions, achieving an evaluation grade of "good" or above. Among them, Treatment 2 (5% TENSIOFIX 96DB10 + 5% Atlas G-1086 + 3% Atlox-4914 + 2% Atlas G-5002L as surfactants, 1% A200 + 1% silica + 1% ATLOX RHEOSTRUX 100 as thickeners) performed best, showing virtually no oil separation under normal temperature and cold storage conditions, and an oil separation rate of 4.91% under hot storage conditions, achieving an "excellent" evaluation grade, with good fluidity in all conditions. Therefore, the formulation of Treatment 2 was determined as the final formulation.

[0099] Based on the above adjuvant screening results, the addition amount of each component was optimized using the suspension rate, emulsification and dispersibility, storage stability, and spore activity of the formulation as evaluation indicators. Finally, the optimal formulation (mass percentage) of 3.5% Metarhizium anisopliae·rotenone dispersible oil suspension was determined as follows: 2.5% pure spore powder of Metarhizium anisopliae MrGX0603, 1% rotenone, 5% TENSIOFIX96DB10, 5% Atlas G-1086, 3% Atlox-4914, 2% Atlas G-5002L, 1% A200, 1% silica, 1% ATLOX RHEOSTRUX 100, 1% ZnO, and 77.5% sunflower seed oil.

[0100] Table 14 Different combinations of surfactants and thickeners

[0101] Table 15 Oil separation of samples after adding different combinations of surfactants and thickeners

[0102] (5) Preparation of dispersible oil suspension The dispersible oil suspension was prepared using a media milling method. The specific preparation process is as follows: ① Pretreatment: Slowly add a thickener (ATLOX RHEOSTRUX 100) to the sunflower seed oil and heat it to ensure that the thickener is completely dissolved to obtain a uniform oil phase matrix.

[0103] ② Mixing and dispersing: Emulsifier, dispersant, the other two thickeners (A200, white carbon black), UV protectant, and rotenone are added to the oil base in sequence and placed in a high-speed stirrer to mix the components evenly and obtain a preliminary mixture; ③ Sand milling: Transfer the preliminary mixture into a sand mill, adjust the speed of the sand mill to 2500 r / min, and mill for 1 hour to refine the particles in the mixture to a particle size ≤5 μm, ensuring the emulsification and dispersibility of the formulation.

[0104] ④ Final Preparation: Filter the milled mixture to remove impurities, then add pure spore powder of *Metarhizium anisopliae* MrGX0603 and the pretreated oil phase matrix. Place the mixture in a high-speed homogenizer and homogenize at 3000 r / min for 30 min to ensure uniform dispersion of spores in the formulation, thus obtaining the 3.5% *Metarhizium anisopliae*·rotenone dispersible oil suspension. Figure 6 As shown, the finished product is a uniform, light green, oily liquid with no sedimentation, no layering, and good fluidity.

[0105] 3. Preparation quality testing The quality of the prepared 3.5% Metarhizium anisopliae·rotenone dispersible oil suspension was tested in accordance with relevant national and industry standards for dispersible oil suspensions. The test indicators included suspension rate, oil separation rate, residual amount after pouring, residual amount after washing, and spore germination rate. Each indicator was repeated three times, and the data were statistically analyzed using SPSS 22 software.

[0106] The detection method is as follows: (1) Suspension rate determination: Refer to the method in GB / T 35670-2017 "Pesticide Water Dispersible Granules" with slight adjustments, dilute the formulation, place it in a stoppered graduated cylinder, let it stand for 30 min, and determine the suspension rate.

[0107] (2) Determination of oil separation rate: The preparation was placed in a sealed container and stored at room temperature (25±2℃) and cold storage (0±2℃) for 14 days respectively. The layering of the preparation was observed and the oil separation rate was calculated.

[0108] (3) Determination of residual amount after pouring and residual amount after washing: Refer to the relevant requirements in GB / T 28137-2011 "Determination of persistent foaming property of pesticides" to determine the residual amount after pouring and residual amount after washing of the formulation.

[0109] (4) Spore germination rate determination: Take a small amount of the preparation, dilute it with 0.1% Tween-80 solution, inoculate it into PDA medium, and incubate at 28℃ for 24 h to determine the spore germination rate.

[0110] Experimental Results: The quality test results of 3.5% Metarhizium anisopliae·rotenone dispersible oil suspension are shown in Table 16. The suspension rate of the formulation was 94.41±0.52%, the oil separation rate after 14 days of storage at room temperature was 4.35±0.72%, and there was basically no oil separation after 14 days of cold storage (oil separation rate <1%). The residual amount after pouring was 4.42±0.25%, the residual amount after washing was 0.74±0.16%, and the spore germination rate was 90.33±1.12%. All indicators met the relevant standard requirements for dispersible oil suspensions, indicating that the formulation is of stable quality and can be used for field application. The germination rate of Metarhizium anisopliae spores in the formulation under different storage conditions is shown in Table 17.

[0111] Table 16 Relevant Indicators of Metarhizium anisopliae·Rotenone Dispersible Oil Suspension

[0112] The data in the table are the mean ± standard error of three replicates.

[0113] Table 17 Spore germination rate of Metarhizium anisopliae in OD of Metarhizium anisopliae·rotenone

[0114] The data in the table are the mean ± standard error of three replicates.

[0115] Example 4: In-vitro bioactivity assay of the formulation A 2.5% Metarhizium anisopliae dispersible oil suspension was prepared as a control formulation, with the adjuvant system being consistent with the formulation of the present invention, and the oil base being supplemented to make up the balance; a 20 mg / L rotenone solution was also prepared as a control.

[0116] The 3.5% Metarhizium anisopliae·rotenone dispersible oil suspension of the present invention was diluted with water at 200 times, 500 times, and 800 times, respectively; at the same time, 2.5% Metarhizium anisopliae dispersible oil suspension diluted 500 times, rotenone solution at 20 mg / L, and water were set up as blank control groups.

[0117] Following the indoor bioassay method for pesticides, healthy, uniform second-instar larvae of the Asian corn borer were selected. Each treatment was replicated three times, with 10-20 larvae per replicate. Larval mortality was assessed at 1, 3, 5, and 7 days after application, and the corrected mortality rate and stunted larvae rate were calculated.

[0118] The experimental results (see Table 18) show that the corrected mortality rates after 7 days of treatment with the formulation of this invention diluted 200 times, 500 times, and 800 times were 91.11%, 80.56%, and 43.33%, respectively, all significantly higher than those of Metarhizium anisopliae single agent (15.87%) and Rotenone single agent (20.00%). The stunted insect rate in each treatment ranged from 22.54% to 48.89%, significantly higher than that of Metarhizium anisopliae single agent (7.94%), demonstrating both rapid and sustained insecticidal effects, and the synergistic effect of the compound formulation was significant.

[0119] Table 18 Activity of Metarhizium anisopliae·Rotenone OD formulation against Asian corn borer

[0120] The data in the table are the mean ± standard error of three replicates. Different lowercase letters after the data in the same column indicate significant differences according to the DMRT test (p < 0.05). CK is the mortality rate of the tested corn borer.

[0121] Comparative Example 1: Indoor Bioactivity Determination of Metarhizium anisopliae MaGX19S02 Combined with Rotenone This comparative example uses the *Metarhizium anisopliae* MaGX19S02, as described in the prior published patent CN121817212A, as the active strain. The remaining formulation, preparation process, bioassay conditions, test insects, dilution gradient, survey time points, and number of repetitions are completely consistent with Examples 3 and 4 of this invention, only the strain is replaced. This is used to compare the indoor bioactivity of different combinations of *Metarhizium anisopliae* and rotenone with *Aspergillus simonii* in *Aspergillus corn borer*.

[0122] Formulation preparation: The total mass percentage of active ingredients was maintained at 3.5%, and the mass ratio of bacteria to drug, MaGX19S02:rotenone, was 2.5:1. The addition amounts of emulsifier, dispersant, thickener, UV protectant ZnO, and sunflower seed oil, as well as the sand milling and homogenization processes, were completely in accordance with the optimal formulation in Example 3 to obtain a 3.5% MaGX19S02·rotenone dispersible oil suspension.

[0123] Bioassay setup: Test insect source: Second-instar Asian corn borer larvae were reared indoors at 27±1 ℃ and 70±5% relative humidity. The bioassay procedure using the immersion method was the same as in Example 4.

[0124] Experimental treatments: 3.5% MaGX19S02・rotenone OD diluted 200, 500, and 800 times; control group (CK) was prepared with water. Each treatment was replicated three times, with 10-20 larvae per replicate. Mortality was calculated and corrected at 1, 3, 5, and 7 days after application, and the synergistic toxicity index (CF) was calculated.

[0125] Table 19 Activity of 3.5% MaGX19S02·rotenone dispersible oil suspension against Asian corn borer

[0126] The data in the table are the mean ± standard error of three replicates; different lowercase letters after the data in the same column indicate significant differences at the p < 0.05 level as determined by the DMRT test.

[0127] Table 19 shows that the insecticidal activity of MaGX19S02 combined with rotenone against the Asian corn borer was low. The highest concentration (200-fold dilution) treatment showed a corrected mortality rate of only 38.89% after 7 days, significantly lower than the 91.11% of the MrGX0603 combined formulation at the same concentration in this invention. The synergistic toxicity index calculation results showed that the CF values ​​of each treatment were between 0 and 20, indicating only an additive effect, and no synergistic enhancement was observed. These results indicate that not any Metarhizium anisopliae strain combined with rotenone can produce a synergistic effect; the Metarhizium anisopliae MrGX0603 selected in this invention achieves synergistic enhancement with rotenone at a specific ratio.

[0128] Example 5: Potted Plant Efficacy Test Select potted corn plants with uniform growth and 5 leaves, and artificially inoculate them with newly hatched Asian corn borer larvae, 10 larvae per pot. After the larvae have established themselves, apply pesticides.

[0129] Experimental setup: 200x, 400x, and 800x dilutions of 3.5% Metarhizium anisopliae·rotenone dispersible oil suspension; control groups included 500x dilution of 2.5% rotenone emulsifiable concentrate, 500x dilution of 8 billion spores / mL Metarhizium anisopliae CQMa421 dispersible oil suspension, 200x dilution of 2.5% Metarhizium anisopliae Roberts dispersible oil suspension, and a water blank control. Each treatment was repeated 5 times, sprayed evenly until the leaves were moist but not dripping.

[0130] Seven days after application, the number of surviving insects was investigated, and the insect population reduction rate and control effect were calculated.

[0131] Pot experiment results (see Table 20) showed that the 200-fold dilution of the formulation of this invention achieved a control efficacy of 87.80%, significantly better than the single-agent controls; the 400-fold dilution achieved a control efficacy of 78.05%, significantly higher than the single-agent rotenone emulsifiable concentrate (70.73%) and Metarhizium anisopliae CQMa421 (60.98%), and significantly better than 2.5% Metarhizium anisopliae dispersible oil suspension (34.15%); the 800-fold dilution achieved a control efficacy of 68.29%. The formulation was safe for maize plants, and no phytotoxicity was observed during the experiment.

[0132] Table 20. Control efficacy of Metarhizium anisopliae·rotenone OD formulation against Asian corn borer.

[0133] The data in the table are the mean ± standard error of 5 replicates. Different lowercase letters after the data in the same column indicate significant differences according to the DMRT test (p < 0.05). Example 6: Field efficacy trial The field trial was conducted at the Qilin North Farm experimental field of South China Agricultural University, where the maize growth was uniform and the natural occurrence of Asian corn borer was even.

[0134] Experimental treatments included: 200, 400, and 800-fold dilutions of 3.5% *Metarhizium anisopliae*·rotenone dispersible oil suspension; control agents included 500-fold dilution of 2.5% rotenone emulsifiable concentrate, 500-fold dilution of 8 billion spores / mL *Metarhizium anisopliae* CQMa421 dispersible oil suspension, 200-fold dilution of 2.5% *Metarhizium anisopliae* Roberts dispersible oil suspension, and a water blank control. Each plot was 4 m². 2 The study employed a randomized block design with three replicates per treatment. Conventional spraying was conducted using an electric sprayer, with a water consumption of 40 L per acre. A comprehensive survey of the insect population was conducted before application. The number of live insects was assessed 3 and 7 days after application, and the percentage of damaged corn plants was assessed 21 days after application. The insect population reduction rate, control efficacy, and percentage of damaged plants were calculated.

[0135] Field results (see Table 21) show that: 7 days after application, the 3.5% Metarhizium anisopliae·rotenone OD formulation of this invention, diluted 200 times, achieved a control efficacy of 61.74% on the 7th day, significantly better than the control efficacy of rotenone emulsifiable concentrate (46.80%) and 2.5% Metarhizium anisopliae dispersible oil suspension (25.72%), and higher than the control efficacy of CQMa421 single agent (54.53%); the control efficacy at 400 times dilution was 52.73%, also better than the 8 billion spores / mL Metarhizium anisopliae CQMa421 dispersible oil suspension; at 800 times dilution, the control efficacy was 42.60% on the 7th day after application. Although the short-term control effect was slightly lower than that of rotenone single agent, the difference was not significant. Moreover, the control efficacy of rotenone single agent is highly dependent on the application dose. At low doses, it degrades faster in the field due to photolysis and has a shorter duration of effect. It also has inherent defects such as high risk of drug resistance and high non-target toxicity. The compound formulation of this invention maintains stable insecticidal activity even at low doses, while also possessing a long-lasting effect, resulting in superior overall control performance. 21 days after application, the percentage of damaged corn plants treated with 200-fold and 400-fold dilutions (5.80%–6.05%) was significantly lower than the blank control (20.64%) and the single-agent treatment of Metarhizium anisopliae (14.18%), demonstrating outstanding sustained efficacy. Field control effects are as follows... Figure 7 As shown, no phytotoxicity was observed in corn during the experiment.

[0136] Table 21 Field control efficacy of 3.5% Metarhizium anisopliae·rotenone OD formulation against Asian corn borer.

[0137] The data in the table are the mean ± standard error of three replicates. Different lowercase letters after the data in the same column indicate significant differences according to the DMRT test (p < 0.05).

[0138] In summary, the pesticide composition provided by this invention, through the combination of entomopathogenic fungi (Metarhizium anisopliae MrGX0603) and plant-derived pesticides (rotenone), exhibits a significant synergistic effect in the control of Asian corn borers within a specific mass ratio range of (1~35):(1~3). It can achieve a synergistic control effect of "rapid effect + sustained effect," that is, the plant-derived pesticide quickly knocks down the pests and exerts a rapid insecticidal effect; while the entomopathogenic fungi continuously infect and colonize, achieving long-term pest control, effectively improving the overall control effect. At the same time, it significantly reduces the amount of single agent used and reduces the risk of resistance development, providing a new option for the green control of Asian corn borers. This is of great significance for promoting the development of green agriculture and ensuring the safe production of corn. Furthermore, the dispersible oil suspension protected by this invention uses sunflower seed oil as the oil base and does not use organic solvents, thus avoiding the harm of organic solvents to the environment, pesticide applicators, and non-target organisms. It also exhibits excellent stability at room temperature and during cold storage, and all quality indicators meet the relevant standards for dispersible oil suspensions. At the same time, it can effectively protect the spore activity of Metarhizium anisopliae MrGX0603, improve the spore's field UV resistance and survival rate, extend the formulation's duration of action, and expand the types of biological pesticide formulations.

[0139] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A pesticide composition containing Metarhizium anisopliae and rotenone, characterized in that, The active ingredients of the pesticide composition include pure spores of Metarhizium anisopliae MrGX0603 and rotenone. Metarhizium anisopliae MrGX0603 has been deposited at the Guangdong Provincial Center for Microbial Culture Collection on July 1, 2026, with accession number GDMCC No. 68257.

2. The pesticide composition according to claim 1, characterized in that, In the pesticide composition, the mass ratio of the pure spore powder of Metarhizium anisopliae MrGX0603 to rotenone is (1~35):(1~3), and the total mass of the pure spore powder of Metarhizium anisopliae MrGX0603 and rotenone accounts for 3% to 4% of the total mass of the pesticide composition.

3. The pesticide composition according to claim 2, characterized in that, In the pesticide composition, the mass ratio of the pure spore powder of Metarhizium anisopliae MrGX0603 to rotenone is 2.5:1, and the total mass of the pure spore powder of Metarhizium anisopliae MrGX0603 and rotenone accounts for 3.5% of the total mass of the pesticide composition.

4. A dispersible oil suspension, characterized in that, The effective components of the dispersible oil suspension include the active components of the pesticide composition according to any one of claims 1 to 3; the dispersible oil suspension further includes pesticide adjuvants, which include surfactants, thickeners, ultraviolet protectants and oil-based components; the surfactants include emulsifiers, aqueous dispersants and non-aqueous dispersants.

5. The dispersible oil suspension agent as described in claim 4, characterized in that, The dispersible oil suspension comprises, by weight percentage, 2%–3% pure spore powder of Metarhizium anisopliae, 0.8%–1.2% rotenone, 12%–18% surfactant, 2%–4% thickener, 0.8%–1.2% UV protectant, and oil base to make up to 100%.

6. The dispersible oil suspension agent as described in claim 5, characterized in that, The dispersible oil suspension comprises, by weight percentage, 2.5% pure spore powder of Metarhizium anisopliae, 1% rotenone, 10% emulsifier, 3% non-aqueous dispersant, 2% aqueous dispersant, 3% thickener, 1% UV protectant, and oil base to make up to 100%.

7. The dispersible oil suspension agent as described in claim 6, characterized in that, The emulsifier includes TENSIOFIX96DB10 and / or Atlas G-1086; the non-aqueous dispersant includes Atlox-4914; the aqueous dispersant includes Atlas G-5002L; the thickener is selected from at least one of A200, silica, and ATLOX RHEOSTRUX 100; the UV protectant includes ZnO; and the oil base is selected from at least one of sunflower oil, corn oil, soybean oil, peanut oil, and blended vegetable oils.

8. The dispersible oil suspension agent as described in claim 7, characterized in that, The emulsifiers include TENSIOFIX 96DB10 and Atlas G-1086; the thickeners include A200, silica, and ATLOX RHEOSTRUX 100; and the oil base is sunflower seed oil.

9. The method for preparing the dispersible oil suspension according to any one of claims 4 to 8, characterized in that, The preparation method includes the following steps: (1) Pretreatment: Add a thickener to a small amount of oil base, heat to ensure that the thickener is completely dissolved, and obtain a uniform oil phase matrix; (2) Mixing and dispersing: Emulsifier, dispersant, the other two thickeners, UV protectant and rotenone are added to the oil base in sequence and placed in a high-speed stirrer to mix the components evenly to obtain a preliminary mixture; (3) Sand milling: Transfer the preliminary mixture into a sand mill, adjust the speed of the sand mill, and mill for a period of time to refine the particles in the mixture to a particle size ≤5μm; (4) Final preparation: Filter the mixture after sand milling to remove impurities, then add pure spore powder of Metarhizium anisopliae MrGX0603 and the pretreated oil phase matrix, and place it in a high-speed homogenizer for homogenization to obtain the finished product of the dispersible oil suspension.

10. The application of the pesticide composition according to any one of claims 1 to 3 or the dispersible oil suspension according to any one of claims 4 to 8 in the control of Asian corn borer.

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