A complex microbial agent capable of secreting insect toxin and its application in biological control

Abstract

The application discloses a kind of composite microbial inoculum that can secrete insect toxin in the field of agricultural biotechnology and application thereof in biological control, the composite microbial inoculum is made of Bacillus thuringiensis subspecies israelensis and three kinds of microbial bodies of Xenorhabdus nematophila, with two specific synthesized compounds, i.e., benzamide derivative containing cyano and difluoromethoxy and acetamide derivative containing dichloromethoxy benzyl and difluoroacetyl, and various formulation aids are compounded.The preparation mainly includes respectively cultivating the three kinds of microorganisms and treating into bacterial suspension, then mixing, adding the two kinds of compound micro powder together with the aid and homogenizing.The composite microbial inoculum can effectively secrete or induce the production of insect toxin through the synergistic effect of microorganism and compound, significantly enhances the control effect on pests, has the advantages of wide insecticidal spectrum and good environmental compatibility, and is suitable for biological control in agricultural field.

Description

Technical Field

[0001] This invention relates to the field of agricultural biotechnology, specifically to a compound microbial agent that can secrete insect toxins and its application in biological control. Background Technology

[0002] For a long time, agricultural production has faced the severe challenge of pest infestation. Traditional chemical control methods rely on synthetic organic pesticides, which, while able to quickly suppress pest populations in the short term, have increasingly prominent and difficult-to-overcome inherent drawbacks. Extensive and unscientific use has led to a continuous rise in pesticide resistance among pest populations, rendering many once-effective products gradually ineffective. More seriously, the residues and spread of chemical pesticides in the environment pose a persistent threat to soil health, water safety, non-target organisms (including pollinating insects and natural enemies), and the diversity of agricultural ecosystems. Ultimately, through accumulation in the food chain, this impacts the quality and safety of agricultural products and human health. Society's urgent demand for green and safe agricultural products makes the development of environmentally friendly alternative control technologies an inevitable direction for sustainable agricultural development.

[0003] Against this backdrop, microbial-based biological control technologies have received widespread attention and achieved some application. For example, biocontrol agents prepared using certain insect-pathogenic bacteria, fungi, or viruses have demonstrated good specificity and environmental compatibility. However, existing microbial pesticides still face several significant bottlenecks in practical application. On the one hand, their insecticidal effect often relies on a single strain or a single toxin protein, resulting in a relatively narrow control spectrum. They are only effective against specific groups of pests and cannot meet the needs of controlling complex pest outbreaks. On the other hand, the field efficacy of live microbial agents is greatly affected by environmental factors (such as temperature, ultraviolet radiation, and rainfall), and their stability and persistence are often unsatisfactory, with large fluctuations in efficacy. Furthermore, compared with chemical pesticides, most biocontrol agents have a relatively slow rate of action, which is not conducive to rapid control of damage during pest outbreaks. More importantly, long-term use of biological products with a single mechanism of action also carries the potential risk of inducing pest resistance, which limits the longevity of their life cycle and the reliability of their application.

[0004] To overcome these limitations, researchers have begun to focus on the complex interactions between insects and microorganisms in nature, particularly symbiotic microbial systems that provide chemical defense for their insect hosts. These symbiotic bacteria can synthesize and secrete specific bioactive substances to help insects defend against predators. This discovery provides a disruptive approach to designing novel biological control strategies: by simulating or modifying these natural symbiotic defense systems, a synergistic system can be constructed by combining artificially designed functional compounds with carefully selected pathogens or beneficial microorganisms. However, achieving efficient and stable compounding of synthetic compounds and functional microorganisms, enabling them to co-colonize and synergistically act after application, and precisely produce or activate multiple toxic effects in situ or on the insect's body or surface, thereby simultaneously achieving rapid efficacy, broad-spectrum activity, sustained efficacy, and low resistance risk, remains a key challenge that current technologies have not effectively addressed. Most existing solutions involve simple compounding of microorganisms or simple mixing with known pesticides, lacking innovative design and deep integration from the perspective of the mechanism of action, and failing to truly build an efficient, stable, and intelligent "compound-microorganism" synergistic platform. This invention aims to propose a completely new solution based on a deep understanding of this technological gap. Summary of the Invention

[0005] The purpose of this invention is to provide a compound microbial agent that can secrete insect toxins and its application in biological control, which solves the technical problems of existing biological pesticides, such as narrow insecticidal spectrum, low efficacy, easy development of resistance by pests, and environmental pollution and residues caused by chemical pesticides.

[0006] The present invention achieves the above objectives through the following technical solutions:

[0007] A compound microbial agent capable of secreting insect toxins, characterized in that the preparation steps of the compound microbial agent include:

[0008] S1. Inoculate Bacillus thuringiensis subsp. Israel and nematode-pathogenic Bacillus into LB medium and culture with shaking at 25-30℃ to obtain cultures;

[0009] S2. Centrifuge the three cultures separately at 3-5℃ to collect the bacterial cells; wash the bacterial cells with sterile physiological saline, and finally resuspend the bacterial cells in phosphate buffer containing trehalose to obtain a bacterial suspension; mix the bacterial suspensions under sterile conditions to obtain a compound bacterial solution.

[0010] S3. Grind 4-cyano-2-(difluoromethoxy)benzamide and N-(3,5-dichloro-2-methoxybenzyl)-2,2-difluoroacetamide into micro powders respectively, and add them to the composite bacterial solution under sterile conditions; at the same time, add alkyl naphthalene sulfonate, alkyl polysaccharide and magnesium aluminum silicate; mix under stirring to obtain bacterial agent slurry; dispense the bacterial agent slurry into liquid bacterial agent.

[0011] In this invention, the process of preparing a bacterial agent by combining 4-cyano-2-(difluoromethoxy)benzamide and N-(3,5-dichloro-2-methoxybenzyl)-2,2-difluoroacetamide with three functional microorganisms embodies a sophisticated chemical-biological synergy and physical enhancement mechanism. Its mechanism of action is not a simple mixture of components, but a multi-level synergistic system. At the biological synergy level, the three selected bacteria constitute a functionally complementary symbiotic complex. Bacillus thuringiensis, as a classic pathogen, provides basic pathogenic effects and crystalline toxins against the midgut epithelial cells of insects; nematode-pathogenic bacteria excel at secreting various complex toxins and antibacterial substances, and can suppress host immunity. The combination of the two may form a microecological synergy during colonization, enhancing overall environmental adaptability, stress resistance, and the ability to continuously secrete active substances. At the chemical-biological interaction level, the addition of the two synthetic compounds introduces a key "chemical weapon" module. The first benzamide derivative, with its cyano and difluoromethoxybenzamide structures, can be recognized and metabolized by enzymes secreted by the complex microbial flora (especially), acting as a "precursor toxin." It is activated within the pest or at the microbial colonization site, potentially releasing toxic small molecules or producing new inhibitory substances. The second difluoroacetamide derivative, with its dichloro-substituted benzene ring and highly electronegative difluoroacetyl group, possesses excellent lipid solubility and membrane permeability. It can effectively interfere with the lipid barrier or cell membrane function of the insect epidermis, acting as a "penetration enhancer" and "metabolic disruptor," paving the way for the invasion and action of microorganisms and their toxins. At the physical formulation level, grinding the compounds into microparticles greatly increases their specific surface area, promoting their dispersion in the bacterial solution and their efficiency in contacting microbial cells and target pests. Various added adjuvants play roles in dispersion, wetting, thickening, and stabilization, ensuring the stability of the active microorganisms and compounds during formulation storage and ensuring uniform adhesion to the crop surface and effective contact with the target after application. In summary, this compound microbial agent constructs a novel biological control system that is highly efficient, stable, and has multiple mechanisms of action through a three-pronged approach: functional symbiosis among microbial species, precise interaction between synthetic compounds and microbial metabolism, and physical safeguards provided by formulation technology. This results in a significant improvement in control efficacy.

[0012] According to a preferred embodiment of the present invention, in step S1, the rotation speed of the shaking culture at 25-30°C is 180-220 rpm.

[0013] According to a preferred embodiment of the present invention, in step S2, the three cultures are centrifuged at 4°C for 10-15 minutes.

[0014] According to a preferred embodiment of the present invention, in step S3, the mixing time under stirring is 1-2 hours.

[0015] According to a preferred embodiment of the present invention, the preparation method of 4-cyano-2-(difluoromethoxy)benzamide includes: dissolving methyl 4-cyano-2-hydroxybenzoate in anhydrous N,N-dimethylformamide in a dry three-necked flask, adding sodium carbonate under nitrogen protection, and stirring at 0-2°C; introducing difluorochloromethane and reacting at 8-10°C to obtain a reaction solution; pouring the reaction solution into ice water, adjusting the pH to 6-7, filtering, washing with water, and drying to obtain a crude product; placing the crude product in a single-necked flask, adding methanol and ammonia solution, and stirring at room temperature; after the reaction is completed, distilling under reduced pressure to obtain a concentrate, pouring the concentrate into ice water, stirring, filtering, washing the filter cake successively with cold water and cold methanol, and drying in a vacuum drying oven at 45-55°C.

[0016] In this invention, the preparation of 4-cyano-2-(difluoromethoxy)benzamide follows a stepwise transformation mechanism from ester to ether to amide. The starting point for synthesis is methyl benzoate containing a phenolic hydroxyl group and a cyano group. In an anhydrous and air-isolated environment, sodium carbonate first undergoes a deprotonation reaction with the phenolic hydroxyl group in the starting material molecule, generating a highly reactive phenoxy anion. This anion, acting as a strong nucleophile, attacks the partially positively charged carbon atom in the subsequently introduced difluorochloromethane molecule under low-temperature conditions, resulting in a nucleophilic substitution reaction. The chlorine atom in the difluorochloromethane acts as a leaving group, successfully introducing a difluoromethoxy group in situ onto the hydroxyl group of the benzene ring, forming a methyl benzoate intermediate containing both a difluoromethoxy and a cyano group. Low-temperature control of this reaction step is crucial for suppressing side reactions and ensuring the efficient introduction of the difluoromethoxy group at the designated position. Subsequently, this ester intermediate undergoes ammonolysis in an alkaline environment provided by methanol and concentrated ammonia. The carbonyl carbon in the ester group is subjected to nucleophilic attack by the ammonia molecule, undergoing an addition-elimination process. The methoxy group departs in the form of methanol, ultimately converting the ester group into an amide group. The entire reaction pathway is clear: the key difluoromethoxy group is introduced through nucleophilic substitution by the phenoxy anion, and the final functional group transformation is completed through a classic ammonolysis reaction, yielding a structurally stable target benzamide derivative with potential biological activity.

[0017] According to a preferred embodiment of the present invention, the stirring time at 0-2°C is 30-60 min.

[0018] According to a preferred embodiment of the present invention, the method for preparing N-(3,5-dichloro-2-methoxybenzyl)-2,2-difluoroacetamide includes:

[0019] A1: 3,5-Dichloro-2-methoxybenzoic acid and thionyl chloride were mixed in anhydrous toluene, and N,N-dimethylformamide was added. The mixture was refluxed at 78-82 °C. The mixture was distilled under reduced pressure to obtain an acyl chloride intermediate. The acyl chloride intermediate was dissolved in anhydrous tetrahydrofuran and added dropwise to a methanol solution containing ammonia at 8-10 °C. The mixture was stirred at room temperature to obtain a reaction mixture. The reaction mixture was concentrated to obtain a concentrate. The concentrate was dissolved in ethyl acetate, washed with saturated sodium bicarbonate solution and brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain 3,5-dichloro-2-methoxybenzamide. 3,5-dichloro-2-methoxybenzamide was suspended in anhydrous tetrahydrofuran, cooled in an ice bath, and lithium aluminum hydride was added. The mixture was brought to room temperature and refluxed. Under ice bath cooling, water, sodium hydroxide solution, and water were added dropwise in sequence to quench the reaction. The mixture was filtered, the filter cake was washed with tetrahydrofuran, the filtrates were combined, and concentrated under reduced pressure to obtain 3,5-dichloro-2-methoxybenzylamine.

[0020] A2: Dissolve 3,5-dichloro-2-methoxybenzylamine in dichloromethane and cool in an ice bath to 0-2°C; add triethylamine and 2,2-difluoroacetic anhydride sequentially, stir at 0-2°C, then raise to room temperature and continue stirring to obtain a reaction solution; dilute the reaction solution with dichloromethane, wash sequentially with hydrochloric acid, saturated sodium bicarbonate solution and brine, dry the organic phase with anhydrous magnesium sulfate, filter, and concentrate under reduced pressure to obtain the crude product; purify the crude product by silica gel column chromatography.

[0021] In this invention, the preparation of N-(3,5-dichloro-2-methoxybenzyl)-2,2-difluoroacetamide embodies a tandem reaction mechanism from carboxylic acid to amide, followed by reduction to finally form a specific amide. The process begins with benzoic acid having dichloro substitution and a methoxy group. The first step is an acyl chloride reaction. Under the catalytic action of an amide compound, the sulfur atom of thionyl chloride attacks the carboxyl oxygen, forming a highly reactive acyl chloride via an intermediate, while simultaneously releasing sulfur dioxide and hydrogen chloride gas. This acyl chloride intermediate does not require separation; it encounters a methanol solution of ammonia at low temperature. Ammonia, acting as a nucleophile, rapidly attacks the carbonyl carbon of the acyl chloride, generating the corresponding benzamide. This is the first amide bond in the molecule's construction. Subsequently, the crucial step is the reduction of the benzamide using the strong reducing agent lithium aluminum hydride. Lithium aluminum hydride provides hydride anions, which first attack the carbonyl carbon of the amide, and through a series of intermediates, ultimately completely reduce the carbonyl group to a methylene group, thereby converting benzamide into benzylamine. This reaction step achieves a fundamental transformation of the functional group from amide to amine, and is the core of constructing the desired "benzylamine" skeleton in the target molecule. Finally, the obtained benzylamine undergoes an acylation reaction with difluoroacetic anhydride. In the presence of a basic organic base, the amino nitrogen atom of benzylamine acts as a nucleophilic center, attacking a carbonyl carbon in difluoroacetic anhydride to form a new amide bond, thereby obtaining the final target product. This route, through a precise design of "activation-amidation-deep reduction-reacylation," efficiently constructs a specific benzylamine amide structure linked to a difluoroacetyl group.

[0022] According to a preferred embodiment of the present invention, in step A1, the stirring reaction time at room temperature is 2-4 hours.

[0023] According to a preferred embodiment of the present invention, in step A2, the stirring time after raising the temperature to room temperature is 4-6 hours.

[0024] The present invention also provides the application of the compound bacterial agent that can secrete insect toxins in biological control.

[0025] The beneficial effects of this invention are as follows:

[0026] The compound microbial agent capable of secreting insect toxins provided by this invention represents a significant breakthrough and optimization in terms of mechanism of action, control effect, and overall benefits compared to existing single microbial or chemical pesticide products. Its technical advantages are specifically reflected in the following aspects:

[0027] First, this invention fundamentally expands the insecticidal spectrum and improves insecticidal efficiency and speed through a unique dual mechanism of "multi-strain synergy" and "chemical-biological synergy." Existing biological pesticides mostly rely on a single strain or its single toxin, resulting in a narrow target area and limited and unstable control effects. This invention innovatively combines *Bacillus thuringiensis* subsp. Israel and *Bacillus nematodes*. *Bacillus thuringiensis* subsp. Israel specifically produces a crystalline toxin that destroys the insect's gut; while the protein toxin complex produced by *Bacillus nematodes* has been proven effective against various pests and exhibits a synergistic effect with *Bacillus thuringiensis*, enabling it to target pest populations resistant to the latter. The synergistic effect of these three functionally complementary strains constitutes a three-dimensional attack network. This invention creatively introduces 4-cyano-2-(difluoromethoxy)benzamide and N-(3,5-dichloro-2-methoxybenzyl)-2,2-difluoroacetamide. These two compounds are not directly effective toxins, but rather act as "precursors" or "synergists." They can be taken up and metabolized by specific microorganisms in the compound microbial agent, or activated by the insect's enzyme system within the insect, thereby being converted in situ into more toxic substances or significantly enhancing the expression and secretion of the strain's own toxins. This design simulates and optimizes highly efficient symbiotic defense systems in nature, realizing a paradigm shift from "application-based activity" to "on-demand production of activity at the target site." This not only significantly improves the specificity and intensity of the toxic effect but also overcomes the defect that single-target action easily leads to resistance, providing a completely new solution for dealing with increasingly resistant agricultural pests.

[0028] Secondly, the compound microbial agent of this invention has demonstrated highly efficient, broad-spectrum, and long-lasting pest control effects in practical field applications, while minimizing negative impacts on the environment and non-target organisms. Compared to the widespread harm to the environment and non-target organisms caused by traditional chemical pesticides, and the shortcomings of some biological insecticides such as short-lasting effects and the need for frequent application, the microbial agent of this invention provides continuous protection through the successful colonization of microorganisms in the rhizosphere or leaf surface of crops. The microorganisms in the compound microbial community can continuously reproduce and metabolize to produce active substances, while the added small molecule compounds provide a long-lasting "ammunition" reserve, enabling a longer protection period with a single application. In addition, adjuvants such as alkyl naphthalene sulfonates and alkyl polysaccharides used in the formulation of the microbial agent further improve the adhesion, spread, and stability of the agent on the crop surface. In actual control cases, similar green control technology systems based on biocontrol bacteria such as Bacillus thuringiensis, combined with other ecological regulation methods, have successfully improved the comprehensive control efficacy of major pests such as potato beetles to a high level of over 95%, and significantly reduced the amount of chemical pesticides used. This indirectly confirms the enormous potential of multi-strategy synergistic technologies. This invention elevates this synergistic concept to the molecular and microbial community level, and is expected to produce excellent and stable control effects on a variety of important agricultural pests, including Lepidoptera and Coleoptera. Its speed of action is due to the rapid activation of chemical precursors and the synergistic attack of multiple toxins, and will be faster than traditional biological pesticides.

[0029] Finally, this invention offers significant environmental and ecological benefits and broad prospects for industrial application, aligning with the strategic direction of sustainable agricultural development. All active ingredients in this compound microbial agent are based on biologically derived or biodegradable chemical designs, preventing the formation of persistent residues in the environment like traditional chemical pesticides. The microbial components are highly safe for mammals, birds, and the natural enemies of pests, thus protecting the biodiversity and natural control forces of the farmland ecosystem. From an industrialization and commercial perspective, the strains used in this invention and the raw materials for compound synthesis are all conventional commercially available products, with a clear process route and the feasibility of large-scale production. Integrating active ingredients with different mechanisms of action into a single product simplifies the application process for farmers and reduces overall management costs. Faced with the current widespread occurrence of pesticide resistance and the surging market demand for green and high-quality agricultural products, this invention provides a highly efficient, safe, and environmentally friendly novel biological control tool. It not only effectively ensures crop yields but also serves as a key technological support for promoting the transformation of crop pest and disease control towards a fully green approach, holding significant positive implications for safeguarding national food security and agricultural product quality safety. Detailed Implementation

[0030] The following detailed embodiments are only used to further illustrate this application and should not be construed as limiting the scope of protection of this application. Those skilled in the art can make some non-essential improvements and adjustments to this application based on the above application content.

[0031] Example 1

[0032] Preparation of 4-cyano-2-(difluoromethoxy)benzamide: The preparation was carried out in a dry 500 mL three-necked round-bottom flask equipped with a magnetic stirrer, a low-temperature thermometer, a nitrogen inlet, and a reflux condenser (connected to a gas bubbler). First, 20.0 g of methyl 4-cyano-2-hydroxybenzoate was added to the flask. Then, 200 mL of anhydrous N,N-dimethylformamide, dried through a 4A molecular sieve, was injected using a syringe. The magnetic stirrer was turned on until the solid was completely dissolved. The reaction system was cooled in an ice-salt bath until the internal temperature stabilized at 0 °C. Under continuous nitrogen purging, 4.8 g of powdered sodium carbonate was weighed using an electronic balance. While maintaining 0 °C and vigorous stirring, the sodium carbonate was slowly added to the reaction solution in ten batches, with approximately 2 minutes between each batch, using a spatula to prevent violent exothermic reactions and foam overflow. After all the sodium carbonate was added, stirring was continued at 0 °C for 40 minutes, at which point the solution became a pale yellow suspension. Remove the ice-salt bath and transfer the reaction flask to a pre-cooled constant-temperature circulating water bath at 8°C. Pass dichlorofluoromethane gas from a cylinder sequentially through a concentrated sulfuric acid drying bottle and a gas flow meter, inserting the outlet tube below the surface of the reaction liquid. Control the gas flow rate to maintain stable bubbling in the reaction liquid, keeping the internal temperature at 8°C. Continuously purge the gas and allow the reaction to proceed for 3.0 hours. During the reaction, take samples every 30 minutes using a capillary tube for thin-layer chromatography (TLC) analysis (silica gel GF254 plate, developing solvent 3:1 petroleum ether / ethyl acetate, observed under a 254nm UV lamp) until the starting material spot (Rf≈0.5) essentially disappears, leaving only the product spot (Rf≈0.7). After the reaction is complete, remove the gas tube. Add 800g of crushed ice and 800mL of deionized water to another 2000mL plastic beaker and pre-cool to 3°C. Under vigorous stirring, the reaction solution was slowly added dropwise to the ice-water mixture using a large-pore glass dropping funnel, controlling the dropping rate to keep the mixing temperature below 20°C. After the addition was complete, a milky white suspension was obtained. While stirring, the pH of the mixture was slowly adjusted to 6.5 using 10% (v / v) dilute hydrochloric acid (confirmed using precise pH paper). At this point, a large amount of white flocculent solid precipitated. Stirring continued in the ice-water bath for 30 minutes to ensure complete precipitation. The mixture was then filtered using a Buchner funnel and slow-speed qualitative filter paper. The filter cake was washed with 3 × 100 mL of pre-cooled deionized water to thoroughly remove DMF and salts. The wet filter cake was transferred to a watch glass and dried in a vacuum drying oven at 45°C for 6 hours to obtain crude methyl 4-cyano-2-(difluoromethoxy)benzoate. All of this crude product was transferred to a dry 500 mL single-necked round-bottom flask. 100 mL of anhydrous methanol and 100 mL of 25% ammonia solution were added sequentially. A magnetic stir bar and condenser were assembled, and the mixture was stirred vigorously at 25°C for 24 hours. After the reaction was complete, the reaction flask was connected to a rotary evaporator, and vacuum distillation was performed at 50°C in a water bath to remove most of the methanol, yielding a viscous, oily concentrate.Add 200 mL of ice water to the concentrate and stir vigorously with a glass rod for 1 hour, during which white crystals continuously precipitate. Filter the solid using a Buchner funnel. Wash the filter cake successively with 50 mL of pre-cooled deionized water and 20 mL of pre-cooled anhydrous methanol. Transfer the filter cake to a watch glass and dry it in a vacuum drying oven at 50 °C to constant weight (approximately 8 hours) to obtain 4-cyano-2-(difluoromethoxy)benzamide.

[0033] Preparation of N-(3,5-dichloro-2-methoxybenzyl)-2,2-difluoroacetamide: Step A1.1 (acyl chloride): In a dry 250 mL round-bottom flask, add 20.0 g of 3,5-dichloro-2-methoxybenzoic acid. Add 100 mL of anhydrous toluene and stir to partially suspend the solid. Add 15.0 mL of freshly distilled thionyl chloride using a syringe. Finally, add 0.2 mL of N,N-dimethylformamide as a catalyst. Immediately assemble a reflux condenser (topped with a calcium chloride drying tube) and a magnetic stir bar onto the flask. Heat the reaction system in an oil bath at 80 °C under reflux. The solid dissolves rapidly, and the reaction solution turns pale yellow. Maintain reflux for 4.0 hours. After the reaction is complete, convert the reaction flask to a vacuum distillation apparatus. Under a 50°C water bath, excess thionyl chloride and most of the toluene were first evaporated under reduced pressure to obtain a pale yellow to light brown oily liquid, which is the intermediate 3,5-dichloro-2-methoxybenzoyl chloride. It was used directly in the next step without purification.

[0034] Step A1.2 (Amideation): Dissolve the oily acyl chloride intermediate obtained in the previous step in 80 mL of anhydrous tetrahydrofuran (THF) and transfer it to a 100 mL constant-pressure dropping funnel. Add 100 mL of a 7.0 M ammonia methanol solution pre-cooled to 8 °C to a 1000 mL three-necked flask and place the flask in an ice-water bath to keep it cool. Under vigorous stirring, slowly add the THF solution containing the acyl chloride to the ammonia solution dropwise, controlling the dropping rate to keep the internal reaction temperature below 10 °C. After the addition is complete (about 30 minutes), remove the ice bath and allow the reaction system to naturally warm to 25 °C, and continue stirring at this temperature for 3.0 hours. After the reaction is complete, connect the reaction flask to a rotary evaporator and concentrate under reduced pressure in a 40 °C water bath to remove most of the solvent, obtaining a white paste. Add 150 mL of ethyl acetate to this paste and stir until completely dissolved. The solution was transferred to a 500 mL separatory funnel and washed successively with 80 mL of saturated sodium bicarbonate solution and 80 mL of saturated sodium chloride solution. The organic phase was dried over anhydrous sodium sulfate for 30 minutes, filtered, and the filtrate was concentrated under reduced pressure to obtain 3,5-dichloro-2-methoxybenzamide, which can be used directly in the next step.

[0035] Step A1.3 (Reduction): In a dry 500mL three-necked flask, add 20.0g of the 3,5-dichloro-2-methoxybenzamide prepared in the previous step. Add 150mL of anhydrous THF and stir to form a suspension. Cool the flask to 0°C in an ice-salt bath. Under nitrogen protection, accurately weigh 5.7g of lithium aluminum hydride powder using an electronic balance. Under vigorous stirring and while maintaining 0°C, carefully add the lithium aluminum hydride to the suspension in five batches, with an interval of approximately 5 minutes between each batch. After all the addition is complete, remove the ice-salt bath and allow the reaction solution to naturally warm to 25°C while stirring for 30 minutes. Then, heat the reaction system to the reflux temperature of THF (approximately 66°C) and maintain reflux at this temperature for 6.0 hours. After the reaction is complete, cool the reaction flask back to 0°C in an ice-salt bath. Very important: Under vigorous stirring, slowly add the following quenchers dropwise sequentially using a constant-pressure dropping funnel: first, add 5.7 mL of deionized water (note: vigorous bubbling may occur initially), then add 5.7 mL of 15% (w / w) sodium hydroxide aqueous solution, and finally add 17.1 mL of deionized water. After the addition is complete, continue stirring at 0°C for 30 minutes to obtain a gray granular precipitate. Add approximately 10 g of anhydrous magnesium sulfate and stir for 10 minutes to absorb excess water. Filter through a Buchner funnel and a diatomaceous earth layer, and wash the filter cake with a large amount (approximately 200 mL) of anhydrous THF. Combine the filtrate and washings, and concentrate under reduced pressure using a rotary evaporator to obtain 3,5-dichloro-2-methoxybenzylamine.

[0036] Step A2 (acylation): In a dry 250 mL three-necked flask, add 10.0 g of the 3,5-dichloro-2-methoxybenzylamine prepared in the previous step. Add 80 mL of anhydrous dichloromethane and stir to dissolve. Cool the reaction flask to 0 °C in an ice-water bath. Add 5.6 mL of triethylamine and 6.2 mL of 2,2-difluoroacetic anhydride sequentially using a syringe. After the addition is complete, stir the reaction at 0 °C for 1.0 h. Then, remove the ice bath and allow the reaction system to warm naturally to 25 °C, and continue stirring at this temperature for 5.0 h. TLC monitoring (developing solvent: petroleum ether / ethyl acetate = 2:1) shows that the starting amine spot disappears. After the reaction is complete, transfer the reaction solution to a 500 mL separatory funnel and dilute with 100 mL of dichloromethane. Wash sequentially with 50 mL of 1.0 M hydrochloric acid, 50 mL of saturated sodium bicarbonate solution, and 50 mL of saturated sodium chloride solution. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a pale yellow oily crude product. The crude product was purified by silica gel column chromatography (silica gel specifications: 200-300 mesh, column diameter 5 cm, column height 30 cm; eluent: petroleum ether / ethyl acetate gradient elution, from 5:1 to 2:1). The fractions rich in the target product were collected (monitored by TLC), combined, and concentrated under reduced pressure to give N-(3,5-dichloro-2-methoxybenzyl)-2,2-difluoroacetamide.

[0037] Preparation of compound bacterial agents that secrete insect toxins:

[0038] Strain activation and expansion culture: One loop each of *Bacillus thuringiensis* subsp. *Israelica* (Bti) and *Xenorhabdus nematophila* were taken from -80℃ glycerol storage tubes and streaked onto two separate LB agar plates under sterile conditions. The plates were inverted and placed in a 30℃ incubator for 24 hours until single colonies formed. One morphologically typical and plump single colony was picked from each plate and aseptically inoculated into two 500mL Erlenmeyer flasks, each containing 100mL of sterile LB liquid medium. The flasks were placed in a shaker and cultured at 28℃ and 220rpm for 18 hours until the cultures reached late logarithmic growth (OD600≈1.8-2.0), yielding two homogeneous, turbid seed cultures.

[0039] Cell collection and washing: Transfer the two cultures to pre-chilled 50 mL sterile centrifuge tubes (approximately 40 mL per tube). Centrifuge at 4°C and 8000 × g for 12 minutes using a high-speed refrigerated centrifuge. Carefully discard the supernatant to obtain the cell pellet. To remove residual culture medium, add 20 mL of pre-chilled sterile physiological saline (0.85% NaCl, w / v) to the cell pellet in each centrifuge tube, vortex thoroughly to resuspend the cells, and then centrifuge again at 4°C and 8000 × g for 12 minutes. Repeat this washing process twice.

[0040] Preparation of composite bacterial suspensions: Washed bacterial cells were resuspended in 10 mL of 0.1 M phosphate-buffered saline (PBS, pH 7.0) containing 5% (w / v) trehalose. The viable cell concentration of each suspension was determined using the plate count method, and the concentrations of the three suspensions were uniformly adjusted to 1.0 × 10¹⁰ CFU / mL using the same trehalose-PBS buffer. In a sterile operating room, using sterilized pipettes and containers, 30.0 g (approximately 30 mL) of *B. ti* bacterial suspension and 20.0 g (approximately 20 mL) of *Bacillus nematodes* bacterial suspension were respectively poured into a sterilized 200 mL capped glass bottle. The mixture was stirred with a sterilized magnetic stir bar at a gentle speed (approximately 200 rpm) for 10 minutes to obtain the composite bacterial solution.

[0041] Mixing and Formulation: Accurately weigh 1.50 g of the previously prepared 4-cyano-2-(difluoromethoxy)benzamide and 1.00 g of N-(3,5-dichloro-2-methoxybenzyl)-2,2-difluoroacetamide. Place both in an agate mortar and grind for 30 minutes, then pass through a 400-mesh stainless steel sieve to obtain a uniformly mixed composite powder. Under continuous aseptic stirring (approximately 200 rpm) of the bacterial solution, slowly and in batches sprinkle the composite powder into the solution using a sterilized spatula. After adding all the powder, continue stirring for 20 minutes to ensure thorough dispersion. Subsequently, add 1.50 g of alkylnaphthalene sulfonate (dispersant), 1.00 g of alkyl polysaccharide glycoside (wetting agent), and 0.50 g of magnesium aluminum silicate (suspension stabilizer) to the mixture in sequence. Increase the stirring speed to 300 rpm and continue stirring at 25°C for 1.5 hours until a uniform, stable suspension without visible particles is formed.

[0042] Dispensing and Storage: Under aseptic conditions, dispense 40.0g of the prepared bacterial slurry into 50mL amber sterile glass reagent bottles. Immediately tighten and seal the bottles. Label the product with the name, batch number, and preparation date. Store the dispensed liquid bacterial agent in a cool, dark place at 4°C. Testing showed that after 90 days of storage under these conditions, the viable bacterial survival rate was greater than 90%, indicating good physical stability of the preparation.

[0043] Example 2

[0044] The specific implementation method is the same as in Example 1, except that the preparation of 4-cyano-2-(difluoromethoxy)benzamide is carried out in a dry 300mL three-necked round-bottom flask equipped with a magnetic stir bar, a low-temperature thermometer, a nitrogen inlet, and a reflux condenser (connected to a gas bubbler). First, 15.0g of methyl 4-cyano-2-hydroxybenzoate is added to the flask. Then, 150mL of anhydrous N,N-dimethylformamide (DMF), dried through a 4A molecular sieve, is injected using a syringe. Magnetic stirring is started to completely dissolve the solid. The reaction system is cooled in an ice bath until the internal temperature stabilizes at 1°C. Under continuous nitrogen purging, 3.6g of powdered sodium carbonate is weighed using an electronic balance. While maintaining 1°C and vigorous stirring, the sodium carbonate is slowly added to the reaction solution in eight batches, with approximately 2 minutes between each batch. After all the sodium carbonate has been added, stirring is continued for 50 minutes while maintaining 1°C. The reaction flask is then transferred to a pre-cooled constant-temperature circulating water bath at 9°C. Dichlorofluoromethane gas was passed through a concentrated sulfuric acid drying bottle and a gas flow meter, with the outlet tube inserted below the surface of the reaction solution. The gas flow rate was controlled to maintain the internal temperature of the reaction between 9-10°C. The gas was continuously introduced for 2.5 hours, during which the reaction progress was monitored by TLC (silica gel GF254 plate, developing solvent: petroleum ether / ethyl acetate, v / v). After the reaction was completed, the gas tube was removed. In another 1500 mL plastic beaker, 600 g of crushed ice and 600 mL of deionized water were added and pre-cooled to 5°C. Under vigorous stirring, the reaction solution was slowly added dropwise to this ice-water mixture using a large-pore glass dropping funnel. After the addition was complete, the pH of the mixture was slowly adjusted to 6.8 with 10% (v / v) dilute hydrochloric acid while stirring. The mixture was then filtered using a Buchner funnel and slow-speed qualitative filter paper. The filter cake was washed with 3 × 80 mL of pre-cooled deionized water. The wet filter cake was transferred to a watch glass and dried in a vacuum drying oven at 48°C for 6 hours to obtain 16.5 g of crude 4-cyano-2-(difluoromethoxy)benzoate intermediate as a white powder. The crude product was transferred entirely to a dry 250 mL single-necked round-bottom flask. 80 mL of anhydrous methanol and 80 mL of 25% (w / w) ammonia solution were added sequentially. A magnetic stir bar and condenser were attached, and the mixture was stirred vigorously at 28°C for 20 hours. After the reaction was complete, the flask was connected to a rotary evaporator, and most of the methanol was removed by vacuum distillation at a water bath temperature of 50°C. 150 mL of ice water was added to the resulting concentrate, and the mixture was stirred vigorously with a glass rod for 1 hour. The solid was collected by suction filtration using a Buchner funnel. The filter cake was washed sequentially with 40 mL of pre-cooled deionized water and 15 mL of pre-cooled anhydrous methanol. The filter cake was transferred to a petri dish and dried in a vacuum drying oven at 52°C to constant weight to obtain 4-cyano-2-(difluoromethoxy)benzamide.

[0045] Preparation of N-(3,5-dichloro-2-methoxybenzyl)-2,2-difluoroacetamide: Step A1.1 (acyl chloride): In a dry 250 mL round-bottom flask, add 15.0 g of 3,5-dichloro-2-methoxybenzoic acid. Add 75 mL of anhydrous toluene and stir. Add 11.0 mL of freshly distilled thionyl chloride using a syringe. Add 0.15 mL of N,N-dimethylformamide (DMF) as a catalyst. Assemble a reflux condenser and a magnetic stirrer. Heat the reaction system under reflux in an oil bath at 81 °C for 3.5 hours. After the reaction is complete, switch to a vacuum distillation apparatus and evaporate excess thionyl chloride and toluene in a water bath at 50 °C to obtain the acyl chloride intermediate.

[0046] Step A1.2 (amidation): Dissolve the acyl chloride intermediate obtained in the previous step in 60 mL of anhydrous tetrahydrofuran (THF) and transfer the solution to a constant-pressure dropping funnel. Add 75 mL of a 7.0 M ammonia methanol solution pre-cooled to 9 °C to a 500 mL three-necked flask and place the flask in an ice-water bath. Under vigorous stirring, slowly add the acyl chloride THF solution to the ammonia solution, controlling the internal temperature below 11 °C. After the addition is complete, remove the ice bath, raise the reaction system to 25 °C, and continue stirring at this temperature for 3.5 hours. After the reaction is complete, concentrate under reduced pressure to remove most of the solvent. Dissolve the paste in 120 mL of ethyl acetate. Transfer the solution to a separatory funnel and wash successively with 60 mL of saturated sodium bicarbonate solution and 60 mL of saturated sodium chloride solution. Dry the organic phase with anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure to give 15.8 g of a white solid, which is 3,5-dichloro-2-methoxybenzamide.

[0047] Step A1.3 (Reduction): In a dry 500 mL three-necked flask, add 14.0 g of the 3,5-dichloro-2-methoxybenzamide prepared in the previous step. Add 120 mL of anhydrous THF and stir to form a suspension. Cool the flask to 0°C in an ice bath. Under nitrogen protection, add 4.0 g of lithium aluminum hydride powder in four batches. After all the powder has been added, remove the ice bath and heat to 25°C and stir for 30 minutes. Then, heat to the THF reflux temperature and maintain reflux for 5.5 hours. After the reaction is complete, cool the reaction flask back to 0°C in an ice bath. Quench the reaction by slowly adding 4.0 mL of deionized water, 4.0 mL of 15% (w / w) sodium hydroxide aqueous solution, and 12.0 mL of deionized water dropwise while stirring vigorously. Add approximately 8 g of anhydrous magnesium sulfate and stir for 10 minutes. Filter through a Buchner funnel and a diatomaceous earth layer, and wash the filter cake with a large amount of anhydrous THF. The filtrates were combined and concentrated under reduced pressure to obtain 3,5-dichloro-2-methoxybenzylamine.

[0048] Step A2 (acylation): In a dry 250 mL three-necked flask, add 7.0 g of the 3,5-dichloro-2-methoxybenzylamine prepared in the previous step. Add 60 mL of anhydrous dichloromethane (DCM) and stir to dissolve. Cool the reaction flask to 1 °C in an ice-water bath. Add 3.9 mL of triethylamine and 4.3 mL (0.035 mol) of 2,2-difluoroacetic anhydride sequentially. After the addition is complete, stir the reaction mixture at 1 °C for 1.0 h. Then, remove the ice bath, raise the temperature of the reaction system to 25 °C, and continue stirring at this temperature for 4.5 h. After the reaction is complete, dilute the reaction solution with 80 mL of DCM and transfer it to a separatory funnel. Wash sequentially with 40 mL of 1.0 M hydrochloric acid, 40 mL of saturated sodium bicarbonate solution, and 40 mL of saturated sodium chloride solution. Dry the organic phase with anhydrous magnesium sulfate, filter, and concentrate the filtrate under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography (eluting with a gradient of petroleum ether and ethyl acetate) to give N-(3,5-dichloro-2-methoxybenzyl)-2,2-difluoroacetamide.

[0049] Preparation of the compound bacterial agent capable of secreting insect toxins: The bacterial strains were activated, cultured, and the cells collected and washed according to the aseptic method described in Example 1. The two types of washed bacterial cells were resuspended in 0.1M PBS (pH 7.0) containing 5% (w / v) trehalose, and the viable cell concentration was adjusted to 1.0 × 10¹⁰ CFU / mL. In an aseptic environment, 25.0 g of *Bacillus cereus* suspension and 15.0 g of *Bacillus nematodes* suspension were measured and poured into a sterilized 200 mL capped glass bottle. The mixture was gently stirred for 10 minutes to obtain 65.0 g of the compound bacterial solution. 1.00 g of 4-cyano-2-(difluoromethoxy)benzamide and 0.60 g of N-(3,5-dichloro-2-methoxybenzyl)-2,2-difluoroacetamide were accurately weighed, ground together, and passed through a 400-mesh sieve to obtain the compound micro-powder. While continuously aseptically stirring the compound bacterial solution, the micronized compound was slowly and gradually added to the solution, and stirring continued for 20 minutes after the addition was complete. Subsequently, 1.20 g of alkyl naphthalene sulfonate, 0.80 g of alkyl polysaccharide, and 0.40 g of magnesium aluminum silicate were added sequentially to the mixture. The stirring speed was increased to 250 rpm, and stirring was continued at 28°C for 2.0 hours to form a homogeneous slurry. The prepared bacterial agent slurry was aseptically dispensed into 50 mL amber sterile glass reagent bottles, 40.0 g per bottle, and the caps were tightened and sealed. The dispensed liquid bacterial agent was stored in a cold storage at 4°C, protected from light.

[0050] Example 3

[0051] The specific implementation method is the same as in Example 1, except that the preparation of 4-cyano-2-(difluoromethoxy)benzamide is carried out in a dry 300mL three-necked round-bottom flask equipped with a magnetic stir bar, a low-temperature thermometer, a nitrogen inlet, and a reflux condenser (connected to a gas bubbler). First, 12.0g of methyl 4-cyano-2-hydroxybenzoate is added to the flask. Then, 120mL of anhydrous N,N-dimethylformamide dried through a 4A molecular sieve is injected using a syringe. Magnetic stirring is turned on to completely dissolve the solid. The reaction system is cooled in an ice bath until the internal temperature stabilizes at 2°C. Under continuous nitrogen purging, 2.9g of powdered sodium carbonate is weighed using an electronic balance. While maintaining 2°C and vigorous stirring, the sodium carbonate is slowly added to the reaction solution in six batches, with an interval of approximately 2 minutes between each batch. After all the sodium carbonate has been added, stirring is continued for 60 minutes while maintaining 2°C. The reaction flask is then transferred to a constant-temperature circulating water bath pre-cooled to 10°C. Dichlorofluoromethane gas was passed through a concentrated sulfuric acid drying bottle and a gas flow meter, with the outlet tube inserted below the surface of the reaction solution. The gas flow rate was controlled to maintain the internal temperature of the reaction at approximately 10°C. The gas was continuously introduced for 3.0 hours, during which the reaction progress was monitored by TLC. After the reaction was completed, the gas tube was removed. In another 1000 mL plastic beaker, 500 g of crushed ice and 500 mL of deionized water were added and pre-cooled to 4°C. Under vigorous stirring, the reaction solution was slowly added dropwise to this ice-water mixture using a large-pore glass dropping funnel. After the addition was complete, the pH of the mixture was slowly adjusted to 7.0 using 10% (v / v) dilute hydrochloric acid while stirring. The mixture was then filtered using a Buchner funnel and slow-speed qualitative filter paper. The filter cake was washed with 3 × 60 mL of pre-cooled deionized water. The wet filter cake was transferred to a watch glass and dried in a vacuum drying oven at 50°C for 6 hours to obtain crude methyl 4-cyano-2-(difluoromethoxy)benzoate intermediate. The crude product was transferred entirely to a dry 250 mL single-necked round-bottom flask. 60 mL of anhydrous methanol and 60 mL of a 25% (w / w) ammonia solution were added sequentially. A magnetic stir bar and condenser were attached, and the mixture was stirred vigorously at 30 °C for 22 hours. After the reaction was complete, the flask was connected to a rotary evaporator, and most of the methanol was removed by vacuum distillation at a water bath temperature of 50 °C. 100 mL of ice water was added to the resulting concentrate, and the mixture was stirred vigorously with a glass rod for 1 hour. The solid was collected by suction filtration through a Buchner funnel. The filter cake was washed sequentially with 30 mL of pre-cooled deionized water and 10 mL of pre-cooled anhydrous methanol. The filter cake was transferred to a watch glass and dried to constant weight in a vacuum drying oven at 55 °C to obtain 4-cyano-2-(difluoromethoxy)benzamide.

[0052] Preparation of N-(3,5-dichloro-2-methoxybenzyl)-2,2-difluoroacetamide:

[0053] Step A1.1 (Acyl Chlorination): In a dry 250 mL round-bottom flask, add 12.0 g of 3,5-dichloro-2-methoxybenzoic acid. Add 60 mL of anhydrous toluene and stir. Add 9.0 mL of freshly distilled thionyl chloride using a syringe. Add 0.12 mL of N,N-dimethylformamide as a catalyst. Assemble a reflux condenser and a magnetic stir bar. Heat the reaction system under reflux in an oil bath at 82 °C for 4.0 hours. After the reaction is complete, switch to a vacuum distillation apparatus and evaporate excess thionyl chloride and toluene in a water bath at 50 °C to obtain the acyl chloride intermediate.

[0054] Step A1.2 (amidation): Dissolve the acyl chloride intermediate obtained in the previous step in 50 mL of anhydrous tetrahydrofuran and transfer it to a constant-pressure dropping funnel. Add 60 mL of a 7.0 M ammonia methanol solution pre-cooled to 10 °C to a 500 mL three-necked flask and place the flask in an ice-water bath. Under vigorous stirring, slowly add the acyl chloride THF solution to the ammonia solution, controlling the internal temperature below 12 °C. After the addition is complete, remove the ice bath, raise the reaction system to 25 °C, and continue stirring at this temperature for 4.0 hours. After the reaction is complete, concentrate under reduced pressure to remove most of the solvent. Dissolve the paste in 100 mL of ethyl acetate. Transfer the solution to a separatory funnel and wash successively with 50 mL of saturated sodium bicarbonate solution and 50 mL of saturated sodium chloride solution. Dry the organic phase with anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure to obtain 3,5-dichloro-2-methoxybenzamide.

[0055] Step A1.3 (Reduction): In a dry 500 mL three-necked flask, add 11.0 g of the 3,5-dichloro-2-methoxybenzamide prepared in the previous step. Add 100 mL of anhydrous THF and stir to form a suspension. Cool the flask to 0°C in an ice bath. Under nitrogen protection, add 3.1 g of lithium aluminum hydride powder in three batches. After all the powder has been added, remove the ice bath and heat to 25°C and stir for 30 minutes. Then, heat to the THF reflux temperature and maintain reflux for 7.0 hours. After the reaction is complete, cool the reaction flask back to 0°C in an ice bath. Quench the reaction by slowly adding 3.1 mL of deionized water, 3.1 mL of 15% (w / w) sodium hydroxide aqueous solution, and 9.3 mL of deionized water dropwise while stirring vigorously. Add approximately 5 g of anhydrous magnesium sulfate and stir for 10 minutes. Filter through a Buchner funnel and a diatomaceous earth layer, and wash the filter cake with a large amount of anhydrous THF. The filtrates were combined and concentrated under reduced pressure to obtain 3,5-dichloro-2-methoxybenzylamine.

[0056] Step A2 (acylation): In a dry 250 mL three-necked flask, add 5.0 g of the 3,5-dichloro-2-methoxybenzylamine prepared in the previous step. Add 50 mL of anhydrous dichloromethane and stir to dissolve. Cool the reaction flask to 2 °C in an ice-water bath. Add 2.8 mL of triethylamine and 3.0 mL of 2,2-difluoroacetic anhydride sequentially. After the addition is complete, stir the reaction at 2 °C for 1.0 h. Then, remove the ice bath, raise the temperature of the reaction system to 25 °C, and continue stirring at this temperature for 6.0 h. After the reaction is complete, dilute the reaction solution with 70 mL of LDCM and transfer it to a separatory funnel. Wash sequentially with 35 mL of 1.0 M hydrochloric acid, 35 mL of saturated sodium bicarbonate solution, and 35 mL of saturated sodium chloride solution. Dry the organic phase with anhydrous magnesium sulfate, filter, and concentrate the filtrate under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography (eluting with a gradient of petroleum ether and ethyl acetate) to give N-(3,5-dichloro-2-methoxybenzyl)-2,2-difluoroacetamide.

[0057] Preparation of the compound bacterial agent capable of secreting insect toxins: The bacterial strains were activated, cultured, and the cells collected and washed according to the aseptic method described in Example 1. The two types of washed bacterial cells were resuspended in 0.1M PBS (pH 7.0) containing 5% (w / v) trehalose, and the viable cell concentration was adjusted to 1.0 × 10¹⁰ CFU / mL. In an aseptic workbench, 20.0 g of *Bacillus cereus* suspension and 25.0 g of *Bacillus nematodes* suspension were measured and poured into a sterilized 200 mL capped glass bottle, and gently stirred for 10 minutes to obtain 75.0 g of the compound bacterial solution. 2.00 g of 4-cyano-2-(difluoromethoxy)benzamide and 1.50 g of N-(3,5-dichloro-2-methoxybenzyl)-2,2-difluoroacetamide were accurately weighed, ground together, and passed through a 400-mesh sieve to obtain the compound micro-powder. While continuously aseptically stirring the compound bacterial solution, the micronized compound was slowly and gradually added to the solution in batches, and stirring continued for 20 minutes after the addition was complete. Subsequently, 2.00 g of alkyl naphthalene sulfonate, 1.50 g of alkyl polysaccharide, and 1.00 g of magnesium aluminum silicate were added sequentially to the mixture. The stirring speed was increased to 350 rpm, and stirring was continued at 30°C for 1 hour to form a homogeneous slurry. The prepared bacterial slurry was aseptically dispensed into 50 mL amber sterile glass reagent bottles, 40.0 g per bottle, and the caps were tightened and sealed. The dispensed liquid bacterial agent was stored in a cold storage at 4°C, protected from light.

[0058] Comparative Example 1

[0059] The specific implementation method is the same as in Example 1, except that 4-cyano-2-(difluoromethoxy)benzamide and N-(3,5-dichloro-2-methoxybenzyl)-2,2-difluoroacetamide are not added. The preparation steps of the compound bacterial agent are as follows: 50.0 g of a compound bacterial solution composed of 30.0 g of Bacillus thuringiensis subsp. Israel suspension and 20.0 g of nematode pathogenic bacillus suspension is prepared according to the same method as in Example 1. 1.50 g of alkylnaphthalene sulfonate, 1.00 g of alkyl polysaccharide, and 0.50 g of magnesium aluminum silicate are directly added to this compound bacterial solution. The mixture is stirred at 300 rpm for 1.5 hours at 25°C, homogenized, and then dispensed and stored to obtain a control bacterial agent without modified compounds.

[0060] Comparative Example 2

[0061] The specific implementation method is the same as in Example 1, except that only 4-cyano-2-(difluoromethoxy)benzamide is added, and N-(3,5-dichloro-2-methoxybenzyl)-2,2-difluoroacetamide is not added. The preparation steps of the compound bacterial agent are as follows: 50.0g of compound bacterial solution is prepared according to the same method as in Example 1. 1.50g of 4-cyano-2-(difluoromethoxy)benzamide is weighed, ground into a fine powder, and added to the compound bacterial solution. Subsequently, 1.50g of alkylnaphthalene sulfonate, 1.00g of alkyl polysaccharide, and 0.50g of magnesium aluminum silicate are added sequentially. The mixture is stirred at 300rpm for 1.5 hours at 25°C, homogenized, and then dispensed and stored to obtain a control bacterial agent containing only one modified compound.

[0062] Comparative Example 3

[0063] The specific implementation method is the same as in Example 1, except that 4-cyano-2-(difluoromethoxy)benzamide and N-(3,5-dichloro-2-methoxybenzyl)-2,2-difluoroacetamide are replaced with compounds that are structurally similar but different. Benzamide and dichlorobenzamide are selected as substitutes. The preparation steps of the compound bacterial agent are as follows: 50.0g of compound bacterial solution is prepared according to the same method as in Example 1. 1.50g of benzamide and 1.00g of dichlorobenzamide are weighed, ground into micro powder, and added to the compound bacterial solution. Subsequently, 1.50g of alkylnaphthalene sulfonate, 1.00g of alkyl polysaccharide, and 0.50g of magnesium aluminum silicate are added sequentially. The mixture is stirred at 300rpm for 1.5 hours at 25°C, homogenized, and then dispensed and stored to obtain a control bacterial agent containing a common structural amide compound.

[0064] Performance testing

[0065] According to relevant standards, the compound bacterial agents capable of secreting insect toxins prepared in Examples 1-3 and Comparative Examples 1-3 were tested according to the following performance test methods:

[0066] All performance tests were conducted in a controlled laboratory environment to quantitatively evaluate the efficacy, stability, and safety of the compound microbial agent. The test insects were sensitive strains artificially reared under standard conditions: the diamondback moth (Plutellaxylostella) third instar larvae were represented by Lepidoptera, and the potato beetle (Leptinotarsadecemlineata) adults were represented by Coleoptera. Before testing, the insects were reared for at least two complete generations using clean host plant leaves in a rearing room at 25±1℃, 70%±5% relative humidity, and a photoperiod of L16:D8. Healthy, active individuals of uniform size were used for the experiment. The main tests consisted of the following three aspects:

[0067] Indoor toxicity and efficacy determination: The test samples (examples and comparative products) were diluted 500 times with sterile deionized water containing 0.05% (v / v) Tween-80. A standard Potter spray tower was used to quantitatively spray fresh, uniformly sized cabbage leaves (for diamondback moth) or potato leaves (for potato beetle) on both sides at a pressure of 0.7 kg / cm². Each leaf received approximately 1.5 µL / cm² of the pesticide solution. After the leaves were allowed to air dry for 30 minutes, they were placed in transparent plastic rearing boxes (15cm × 10cm × 5cm). Each rearing box served as a replicate, with 20 test insects in each box, along with an appropriate amount of untreated leaves as food. Leaves sprayed with an equal volume of sterile water containing 0.05% Tween-80 served as a blank control. Each treatment had four independent replicates. The treated rearing boxes were placed in an artificial climate chamber under the above-described standard environmental conditions. The mortality of the test insects was checked at 24h, 48h, and 72h after treatment. Insects were considered dead if they showed absolutely no coordinated movement when gently touched with a soft brush. The number of dead insects was recorded, and the corrected mortality rate at each time point was calculated using the Abbott formula.

[0068] Storage stability test: Samples were aliquoted into 10mL sterile centrifuge tubes (5mL per tube) and sealed. Accelerated storage tests were conducted at 4±1℃ in a refrigerator and 37±1℃ in a constant temperature incubator. Samples were taken at the start of storage (day 0) and after 30, 60, and 90 days. Viable cell count was determined using the plate count method: Samples were serially diluted 10-fold with 0.85% sterile physiological saline. Three suitable dilutions were selected, and 100µL of each was spread onto LB agar plates, with three replicates for each dilution. After incubating the plates upside down at 30℃ for 48 hours, colony-forming units were counted, and the number of viable cells per milliliter of sample was calculated. Simultaneously, the physical properties of the samples were visually observed and recorded, including changes in layering, precipitation, flocculation, clumping, or color.

[0069] Acute toxicity assays for non-target aquatic organisms: Daphnia magna and Daniorerio embryos were used as model organisms for evaluation. Daphnia magna acute activity inhibition test: Healthy larvae aged 6-24 hours were selected, and the test agent was prepared into a series of concentrations (100 mg / L, 50 mg / L, 25 mg / L, 12.5 mg / L, and 6.25 mg / L) using standard dilution water. Each concentration was replicated in four places, with five larvae in 20 mL of the test solution per replicate. Static exposure was performed at 20±1℃ under a photoperiod of L16:D8. Observations were conducted at 24 and 48 hours, and the number of Daphnia magna unable to swim spontaneously within 15 seconds was recorded, calculating the immobilization rate. Zebrafish embryo acute toxicity test: Normal zebrafish embryos were collected within 2 hours of fertilization, and the test agent was prepared into a series of dilutions with a maximum concentration of 100 mg / L using aquaculture water. Twenty embryos were placed in each concentration group into a 6-well plate, with 5 mL of reagent per well, and four replicates were performed. The plates were statically exposed at 28 ± 0.5 °C, with the reagent changed every 24 hours. At 96 hours post-exposure, the number of dead embryos (loss of pigmentation, cardiac arrest) and the number of deformities (such as scoliosis, pericardial edema, delayed hatching, etc.) were observed and recorded under a stereomicroscope.

[0070] Performance test results:

[0071] Table 1: Performance Test Results

[0072]

[0073] As can be seen from Table 1, Examples 1-3, compared with Comparative Examples 1-3, effectively solved the key problems of the prior art through their unique design.

[0074] First, addressing the issue of a narrow insecticidal spectrum, the examples demonstrated highly effective killing of both the diamondback moth (Lepidoptera) and the potato beetle (Coleoptera), with 72-hour corrected mortality rates consistently at high levels of 91.8-95.2% and 85.1-88.5%, respectively. This indicates that the present invention achieves broad-spectrum control of multiple pest groups through the synergy of a composite functional strain and a specific compound, overcoming the limitation of single-target microbial agents. Second, regarding the issue of low efficacy, the insecticidal efficacy of the examples significantly surpasses all comparative examples. In particular, compared to Comparative Example 1, which did not contain any synergistic compounds, Example 1 showed improved control efficacy. This is directly attributed to the two newly designed compounds acting as a "precursor toxin" and a "penetration enhancer," which, together with the microorganisms, constructed a synergistic system of "in-situ activation and multi-pathway attack," significantly improving the speed of action and the final control efficacy.

[0075] Regarding the issue of pests easily developing resistance, the complex synergistic mechanism of multiple species, multiple compounds, and multiple action sites provided by this invention makes it difficult for pests to develop comprehensive resistance through a single mutation pathway. When Comparative Example 2 contained only one compound, the control efficacy showed a significant decrease, while when Comparative Example 3 used a compound with a common structure, the control efficacy was even lower than that of the pure fungal agent group. This, in turn, confirms the irreplaceable role of the specific compound combination introduced by this invention in interfering with the development of resistance.

[0076] Finally, regarding environmental safety, all examples and comparative examples showed practically non-toxic acute toxicity to Daphnia magna, and the core components of the microbial agent are biodegradable microorganisms and easily metabolized organic molecules, fundamentally avoiding the high residue and pollution risks of traditional chemical pesticides. In summary, the embodiments of this invention successfully integrate the three major objectives of highly efficient broad-spectrum insecticide, resistance management, and environmental friendliness, systematically providing a solution to the bottlenecks of existing technologies.

[0077] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. A compound bacterial agent capable of secreting insect toxins, characterized in that, The preparation steps of the compound microbial agent include: S1. Bacillus thuringiensis subsp. Israel and nematode-pathogenic bacteria were inoculated into LB medium and cultured with shaking at 25-30℃ to obtain three cultures. S2. Centrifuge the three cultures separately at 3-5℃ to collect the bacterial cells; wash the bacterial cells with sterile physiological saline, and finally resuspend the bacterial cells in phosphate buffer containing trehalose to obtain a bacterial suspension; mix the bacterial suspensions under sterile conditions to obtain a compound bacterial solution. S3. Grind 4-cyano-2-(difluoromethoxy)benzamide and N-(3,5-dichloro-2-methoxybenzyl)-2,2-difluoroacetamide into micro powders respectively, and add them to the composite bacterial solution under sterile conditions; at the same time, add alkyl naphthalene sulfonate, alkyl polysaccharide glycoside, and magnesium aluminum silicate; mix under stirring to obtain bacterial agent slurry; dispense the bacterial agent slurry into liquid bacterial agents.

2. The compound bacterial agent capable of secreting insect toxins according to claim 1, characterized in that, In step S1, the shaking rotation speed is 180-220 rpm at 25-30℃.

3. The compound bacterial agent capable of secreting insect toxins according to claim 1, characterized in that, In step S2, the three cultures are centrifuged at 4°C for 10-15 minutes.

4. The compound bacterial agent capable of secreting insect toxins according to claim 1, characterized in that, In step S3, the mixing time under stirring is 1-2 hours.

5. The compound bacterial agent capable of secreting insect toxins according to claim 1, characterized in that, The preparation method of the 4-cyano-2-(difluoromethoxy)benzamide includes: dissolving methyl 4-cyano-2-hydroxybenzoate in anhydrous N,N-dimethylformamide in a dry three-necked flask, adding sodium carbonate under nitrogen protection, and stirring at 0-2°C; introducing difluorochloromethane and reacting at 8-10°C to obtain a reaction solution; pouring the reaction solution into ice water, adjusting the pH to 6-7, filtering, washing with water, and drying to obtain a crude product; placing the crude product in a single-necked flask, adding methanol and ammonia solution, and stirring at room temperature; after the reaction is completed, distilling under reduced pressure to obtain a concentrate, pouring the concentrate into ice water, stirring, filtering, washing the filter cake successively with cold water and cold methanol, and drying in a vacuum drying oven at 45-55°C.

6. The compound bacterial agent capable of secreting insect toxins according to claim 5, characterized in that, Stirring time at 0-2℃ is 30-60 minutes.

7. The compound bacterial agent capable of secreting insect toxins according to claim 1, characterized in that, The preparation method of the N-(3,5-dichloro-2-methoxybenzyl)-2,2-difluoroacetamide includes: A1: 3,5-Dichloro-2-methoxybenzoic acid and thionyl chloride were mixed in anhydrous toluene, and N,N-dimethylformamide was added. The mixture was refluxed at 78-82 °C. The mixture was distilled under reduced pressure to obtain an acyl chloride intermediate. The acyl chloride intermediate was dissolved in anhydrous tetrahydrofuran and added dropwise to a methanol solution containing ammonia at 8-10 °C. The mixture was stirred at room temperature to obtain a reaction mixture. The reaction mixture was concentrated to obtain a concentrate. The concentrate was dissolved in ethyl acetate, washed with saturated sodium bicarbonate solution and brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain 3,5-dichloro-2-methoxybenzamide. 3,5-dichloro-2-methoxybenzamide was suspended in anhydrous tetrahydrofuran, cooled in an ice bath, and lithium aluminum hydride was added. The mixture was brought to room temperature and refluxed. Under ice bath cooling, water, sodium hydroxide solution, and water were added dropwise in sequence to quench the reaction. The mixture was filtered, the filter cake was washed with tetrahydrofuran, the filtrates were combined, and concentrated under reduced pressure to obtain 3,5-dichloro-2-methoxybenzylamine. A2: Dissolve 3,5-dichloro-2-methoxybenzylamine in dichloromethane and cool in an ice bath to 0-2°C; add triethylamine and 2,2-difluoroacetic anhydride sequentially, stir at 0-2°C, then raise to room temperature and continue stirring to obtain a reaction solution; dilute the reaction solution with dichloromethane, wash sequentially with hydrochloric acid, saturated sodium bicarbonate solution and brine, dry the organic phase with anhydrous magnesium sulfate, filter, and concentrate under reduced pressure to obtain the crude product; purify the crude product by silica gel column chromatography.

8. The compound bacterial agent capable of secreting insect toxins according to claim 7, characterized in that, In step A1, the reaction time at room temperature with stirring is 2-4 hours.

9. The compound bacterial agent capable of secreting insect toxins according to claim 7, characterized in that, In step A2, the stirring time is 4-6 hours after the temperature is raised to room temperature.

10. The application of a compound microbial agent capable of secreting insect toxins according to any one of claims 1-9 in biological control.

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