Corn rhizosphere microbial complex microbial inoculant for regulating and controlling taxis of spodoptera frugiperda

Abstract

The invention relates to the technical field of agricultural microorganisms, and discloses a corn rhizosphere microbial complex microbial inoculant for regulating and controlling the taxis of spodoptera frugiperda, which comprises the following components in percentage by mass: 15-35% of fermentation carbon source mixed liquor, 5-25% of complex microbial fermentation product, and 50-80% of carrier and auxiliary material component, wherein the fermentation carbon source mixed solution is composed of molasses accounting for 18-28% of the total mass of the fermentation carbon source mixed solution, crude glycerin accounting for 8-22% of the total mass of the fermentation carbon source mixed solution, cellulase hydrolysate accounting for 38-48% of the total mass of the fermentation carbon source mixed solution, corn starch processing byproduct liquid accounting for 10-15% of the total mass of the fermentation carbon source mixed solution, and the balance deionized water; multiple types of carbon sources are provided through fermentation carbon source mixed liquid, the compound microbial fermentation product is subjected to two-stage culture mixing and then is used for parallel fermentation, and a carrier and auxiliary material components are used for fixing and forming, so that the compound microbial fermentation product keeps stable coexistence and metabolism synergy in the preparation and use processes; therefore, the pest behavior resistance evolution capability is enhanced in the application.

Description

Technical Field

[0001] This application relates to the field of agricultural microbial technology, specifically to a maize rhizosphere microbial compound agent for regulating the tropism of fall armyworm. Background Technology

[0002] The fall armyworm (PMO) is a migratory lepidopteran pest with a wide feeding range, primarily damaging gramineous crops such as maize. Its larvae feed on leaves, flowers, and ears, significantly impacting maize yield and quality. Due to its complex behavioral characteristics, including phototaxis and chemotaxis, the fall armyworm easily forms swarms in the field, leading to inconsistent effectiveness of conventional chemical control and the potential for inducing resistance evolution. As maize is a major host of the fall armyworm, regulating its tropism and suppressing its feeding and oviposition habits through the rhizosphere ecosystem has become an important research direction for control strategies. Applying microbial agents that can regulate the pest's tropism in the maize rhizosphere can achieve ecological behavioral disturbance and control effects, thereby reducing reliance on chemical pesticides and maintaining the stability of the crop ecosystem.

[0003] Existing microbial compound agents are typically prepared by culturing and mixing multiple strains with antagonistic, growth-promoting, or nitrogen-fixing abilities separately, and then using an inert carrier. However, during use, a single carbon source system is generally used for carbon source supply and fermentation, which can easily lead to insufficient stability among the microbial communities, metabolic imbalance in the fermentation system, and difficulty in ensuring that the preparation forms a sustained and effective synergistic effect during application. Summary of the Invention

[0004] This application provides a maize rhizosphere microbial compound agent for regulating the tropism of fall armyworm, which solves the problems of insufficient stability among microbial communities, metabolic imbalance in the fermentation system, and difficulty in ensuring the formation of a continuous and effective synergistic effect of the agent during application.

[0005] In a first aspect, this application provides a maize rhizosphere microbial compound agent for regulating the tropism of fall armyworm, comprising the following components by mass percentage: 15-35% fermentation carbon source mixture, 5-25% compound microbial fermentation product, and 50-80% carrier and excipient components; The fermentation carbon source mixture consists of molasses (18–28% by mass), crude glycerol (8–22% by mass), cellulase hydrolysate (38–48% by mass), and corn starch processing by-product liquid (10–15% by mass), with the remainder being deionized water. The complex microbial fermentation products were obtained by mixing the following strains through two-stage culture: Bacillus subtilis CGMCC No. 1.1470 accounts for 30–60% of the total mass of the complex microbial fermentation products; Pseudomonas fluorescens CGMCC No. 1.1803 accounts for 10–40% of the total mass of the complex microbial fermentation products; Trichoderma harzianum CGMCC No.3.18371 accounts for 10–30% of the total mass of the fermentation products from the complex microbial fermentation process; Candida tropicalis CGMCC No.2.1082 accounts for 5–20% of the total mass of the complex microbial fermentation products; The carrier and excipient components consist of 40–55% bentonite, 10–15% sodium humate, 4–5% sodium alginate, 8–10% maltodextrin, and 0.3–0.5% silica aerogel, accounting for 40–55% of the total mass of the carrier and excipient components.

[0006] By employing the above technical solution, molasses provides monosaccharides and oligosaccharides that are easily utilized by microorganisms, crude glycerol provides a slow-release carbon source to maintain the stability of substrate concentration during fermentation, cellulase hydrolysate contains a variety of soluble sugars to meet the metabolic needs of different strains, and corn starch processing by-products contain soluble sugars and nitrogen sources to supplement nutrients. This allows for the formation of multiple carbon source types and the maintenance of material balance during fermentation. The composite microbial fermentation product consists of Bacillus subtilis CGMCC No.1.1470, Pseudomonas fluorescens CGMCC No.1.1803, Trichodermaharzianum CGMCC No.3.18371, and Candida tropicalis CGMCC No.2.1082. Each strain decomposes and utilizes carbon sources through different metabolic pathways. Bacterial strains can rapidly proliferate and produce metabolites, fungal strains can form hyphal structures to enhance system stability, and yeast can participate in redox regulation. The four strains, when mixed and cultured, form a metabolically complementary microbial community system. The carrier and excipients consist of bentonite, sodium humate, sodium alginate, maltodextrin, and silica aerogel. Bentonite provides the specific surface area for microbial adsorption; sodium humate has a multifunctional structure to enhance adsorption and binding; sodium alginate forms a gel structure to immobilize microorganisms; maltodextrin improves film-forming properties and flowability during drying; and silica aerogel forms a porous structure to maintain particle morphology and internal porosity. This ensures high activity of the microorganisms during drying and storage, resulting in stable fermentation, controllable product moisture content, and high microbial survival rate. Ultimately, this enhances resistance evolution against pests and improves the utilization rate of low-cost renewable carbon sources.

[0007] Preferably, the cellulase hydrolysate is a liquid obtained by hydrolyzing corn stalks as raw material with 1.0–2.0% cellulase (based on dry corn stalk mass) at pH 4.8–5.2 and temperature 45–55°C for 24–48 hours. By employing the above technical solution, cellulase hydrolysate utilizes the catalytic action of cellulase on cellulose and hemicellulose in corn stalks to convert macromolecular carbohydrates into soluble monosaccharides, oligosaccharides, and soluble organic acids. Using corn stalks as raw material releases natural soluble carbon sources and some nutrients during hydrolysis, and the resulting liquid contains various carbon source components that can be utilized by strains in the fermentation products of complex microorganisms. By controlling the amount of cellulase added, pH, temperature, and hydrolysis time, a hydrolysate with a balanced soluble sugar content can be obtained while maintaining stable enzyme activity. This allows the liquid to form multiple types of carbon source structures with molasses, crude glycerol, and corn starch processing byproducts in the fermentation carbon source mixture. This maintains a continuous carbon source supply during fermentation and reduces the need for external carbon sources, thereby improving the utilization rate of low-cost renewable carbon sources.

[0008] Preferably, the corn starch processing by-product liquid is a corn extract produced during the wet corn starch production process, and the total soluble sugar content of the corn extract is 15–25 g / L; By employing the above-mentioned technical solution, the corn extract forms a homogeneous liquid during the corn soaking and separation process through the dissolution of soluble sugars, amino nitrogen, and trace organic acids. The liquid contains soluble sugars, short-chain polysaccharides, and some amino acids. When used as a component of the fermentation carbon source mixture, this liquid provides directly usable carbon sources and some organic nitrogen components within the system. Together with molasses, crude glycerol, and cellulase hydrolysate, it forms a multiphase nutrient system where carbon and nitrogen sources coexist, thereby improving substrate absorption and metabolic balance during the fermentation stage. The synergistic use of this corn extract with other components reduces the need for external nitrogen sources and enables the reuse of waste by-products, thus achieving the beneficial effect of improving the utilization rate of low-cost renewable carbon sources.

[0009] Secondly, this application provides a method for preparing a maize rhizosphere microbial compound agent to regulate the tropism of fall armyworm, comprising the following steps: S1. Prepare the fermentation carbon source mixture; S2. Preparation of composite microbial fermentation products; S3. Introduce the fermentation products of the composite microorganisms into the fermentation carbon source mixture for parallel fermentation; S4. Concentrate the fermentation broth after fermentation is complete; S5. Prepare the carrier and excipient components, and then mix the concentrated fermentation broth with the carrier and excipient components for adsorption. S6. Dry and granulate the adsorption mixture; By employing the above technical solution, a fermentation carbon source mixture is prepared. By mixing molasses, crude glycerol, cellulase hydrolysate, and corn starch processing by-products in a specific ratio, multiple types of usable carbon sources can be provided during the fermentation stage, maintaining the stability of substrate supply. A composite microbial fermentation product is prepared. Through two-stage cultivation of multiple strains, bacteria, fungi, and yeast form a complementary system at the nutritional and metabolic levels, providing a metabolically active composite microbial community for subsequent parallel fermentation. The composite microbial fermentation product is inoculated into the fermentation carbon source mixture for parallel fermentation. Through the synchronous metabolism of multiple strains, stepwise utilization of the carbon source and metabolic balance among microorganisms in the fermentation system are achieved, thereby maintaining the stability of the fermentation process. The fermentation broth after fermentation is concentrated to remove excess water and enrich metabolites, ensuring that the concentration of active substances and microorganisms in the fermentation broth meets the requirements for formulation. A carrier and excipient components are prepared, and the concentrated fermentation broth is mixed with the carrier and excipient components for adsorption. The porous structure and gel properties of the carrier and excipient components allow for uniform distribution of microorganisms during adsorption, forming a stable wet mixture. The adsorbed mixture is dried and granulated. Through drying immobilization and particle size control, a solid formulation with good flowability and structural integrity is obtained. The sequential arrangement of the above steps and the coordination of each step can maintain cell activity, control moisture content, and maintain uniform dispersion of materials during the preparation process, thereby achieving the beneficial effects of stable fermentation process, stable product yield, and good microbial activity.

[0010] Preferably, in step S1, the specific steps for preparing the fermentation carbon source mixture are as follows: Add molasses, crude glycerol, cellulase hydrolysate, corn starch processing by-product liquid and deionized water to a stirrer and stir at a speed of 200–400 rpm for 20–40 min. By employing the above technical solution, molasses, crude glycerol, cellulase hydrolysate, corn starch processing by-product liquid, and deionized water are added to a stirrer and stirred until homogeneous. Stirring ensures thorough dispersion of the liquid raw materials in the system. The soluble sugars in the molasses and crude glycerol form a mixed system containing both fast-acting and slow-release carbon sources. The oligosaccharides in the cellulase hydrolysate and the soluble sugars and amino compounds in the corn starch processing by-product liquid are uniformly distributed in the solution under stirring. This prevents substrate utilization imbalances caused by uneven local carbon source concentrations during the initial fermentation stage. The homogeneous fermentation carbon source mixture formed after stirring has a stable material composition and high system homogeneity. The above operations ensure a stable carbon source supply and fermentation system during fermentation, thereby improving the stability of the fermentation process and the material conversion efficiency during preparation.

[0011] Preferably, in step S2, the specific steps for preparing the composite microbial fermentation product are as follows: First, the primary seed cultures of the four strains were cultured for 18–20 h at a temperature of 28–32°C and a pH of 6.4–6.8. The primary seed culture that has been cultured is inoculated into the secondary culture medium and cultured for 36–48 h at a temperature of 26–30°C and a dissolved oxygen of 20–40%. Finally, mix the four strains that have been cultured thoroughly. The specific steps for preparing the composite microbial fermentation product using the above technical solution are as follows: First, the primary seed culture of four strains is cultured to the logarithmic growth phase under set temperature and pH conditions to obtain physiologically stable seed culture. Then, the cultured primary seed culture is inoculated into secondary culture medium for further culture to achieve cell proliferation and accumulation of extracellular metabolites. Finally, the four strains are mixed evenly after culture to obtain the composite microbial fermentation product. This step controls the cell growth stage through graded culture, allowing Bacillus subtilis CGMCC No.1.1470, Pseudomonas fluorescens CGMCC No.1.1803, Trichoderma harzianum CGMCC No.3.18371, and Candidatropicalis CGMCC No.2.1082 to reach stable metabolic states, thereby avoiding growth competition among strains in the initial culture stage. This achieves the coexistence and balance of the composite microbial community during fermentation, thereby enhancing the evolutionary ability of pest resistance behavior.

[0012] Preferably, in step S4, the concentration process is as follows: After the fermentation broth is finished, the solids are removed by centrifugation and then concentrated by membrane filtration until the volume of the fermentation broth is reduced to 1 / 3–1 / 5 of the original volume. By employing the above technical solution, centrifugation removes solids from the fermentation broth, eliminating interference from residual culture medium particles and bacterial fragments in the subsequent concentration process and preventing clogging of the membrane filtration system. Membrane filtration, driven by pressure, achieves liquid-phase permeation and macromolecular retention separation, removing excess water without damaging the bacterial cell and its metabolite structure, thereby increasing the concentration of effective components in the fermentation broth. The combined use of centrifugation and membrane filtration reduces the system's heat load while simultaneously reducing liquid volume and enriching effective components. The treated concentrated fermentation broth has a high content of active components and suitable viscosity, allowing for direct use in the mixing and adsorption of carrier and excipient components. This ensures stable material moisture content and concentration during subsequent adsorption and drying stages, achieving stable bacterial activity and controlled moisture content in the final product during formulation.

[0013] Preferably, the specific steps for preparing the carrier and excipient components are as follows: Dissolve sodium alginate and maltodextrin in deionized water at 40–60°C, and stir at 300–500 rpm for 15–30 minutes to form a uniform sol. Add bentonite and sodium humate to the sol and continue stirring for 20–40 minutes until it is uniformly gelatinized; Then, silica aerogel was added for adsorption and dispersion, and the water content of the system was adjusted to 30–40%. By employing the above technical solution, sodium alginate and maltodextrin are dissolved to form a sol with a certain viscosity and film-forming properties, providing a basic network structure for the carrier system. The addition of bentonite increases the specific surface area and physical adsorption capacity of the system, while the multifunctional structure of sodium humate enhances the ionic bonding and dispersion stability between materials. The addition of silica aerogel creates a porous support structure, improving internal pore connectivity and uniform liquid phase distribution. Through stirring, the various carrier and auxiliary components are fully dispersed in the system and achieve structural composite formation, resulting in a homogeneous carrier matrix with suitable adhesion and controllable water content. This ensures uniform bonding and structural stability during mixing and adsorption with the fermentation broth, thereby improving the adhesion stability of microorganisms and the consistency of the carrier system formation.

[0014] Preferably, in step S5, the specific steps of mixed adsorption are as follows: The fermentation broth is mixed with the carrier and excipients, and stirred at room temperature for 15–45 minutes to form a wet mixture; By employing the above technical solution, the concentrated fermentation broth is directly mixed with the prepared carrier and excipient components, allowing the microorganisms and their metabolites in the fermentation broth to be uniformly distributed within the carrier matrix during stirring. The stirring operation ensures full contact between the gel network formed by sodium alginate and the porous structures of bentonite, sodium humate, and silica aerogel, thereby enhancing the adsorption and immobilization effect of microorganisms on the carrier surface and in the pores. Liquid components in the fermentation broth can partially enter the carrier structure, forming a stable binding layer between the microorganisms and the carrier. This achieves uniform binding of the liquid fermentation products with the solid carrier system, preventing cell aggregation or sedimentation before drying, ensuring material dispersibility and carrier binding strength during subsequent drying, and thus improving the stability of cell immobilization and the uniformity of the formulation structure.

[0015] Preferably, in step S6, the specific steps of drying and granulation are as follows: The wet mixture is subjected to fluidized bed drying, wherein the fluidized bed drying temperature is 50–55°C and the time is 45–60 min; The dried wet mixture is sieved and granulated, wherein the particle size after sieving and granulation is 150–220 μm. By employing the above technical solution, fluidized bed drying suspends the wet mixture under controlled airflow, ensuring uniform heating and gradual evaporation of moisture, thus avoiding the impact of localized overheating on bacterial activity. During the drying process, the gel film formed by sodium alginate and maltodextrin solidifies under fluidized conditions, while bentonite and sodium humate maintain the stability of the particle structure. The porous structure of silica aerogel promotes airflow penetration, improving drying efficiency. The dried material is then sieved and granulated to remove excessively large or small particles, resulting in a formulation with a uniform particle size distribution. This results in a dense particle surface structure with good dispersibility, ensuring the flowability and stability of the particles during storage and application. By maintaining bacterial activity, a solid formulation with stable moisture content, uniform particle size, and intact physical structure is obtained, thereby improving formulation stability and product consistency.

[0016] This application provides a compound microbial agent for the rhizosphere of maize used to regulate the tropism of the fall armyworm. It has the following beneficial effects: 1. This application provides multiple types of carbon sources through a fermentation carbon source mixture. The composite microbial fermentation products are mixed after two-stage culture and used for parallel fermentation. The carrier and excipient components are used for fixation and shaping, so that the composite microbial fermentation products maintain stable coexistence and metabolic synergy during formulation and use, thereby enhancing the ability of pest resistance evolution in application.

[0017] 2. The fermentation carbon source mixture of this application consists of molasses, crude glycerol, cellulase hydrolysate and corn starch processing by-product liquid. The cellulase hydrolysate and corn extract are used to provide soluble components that can be directly utilized. Through mixing and homogenization and parallel fermentation, continuous supply and step-by-step utilization are achieved, thereby improving the utilization rate of low-cost renewable carbon sources.

[0018] 3. This application forms a porous and gel structure that can be used to fix composite microbial fermentation products by dissolving, gelatinizing and adsorbing the carrier and excipient components. Combined with the mixed adsorption in S5 and the drying and granulation in S6, formulation particles with structural integrity and good flowability are obtained, thereby maintaining the microbial activity and uniform dispersion of materials during storage and application. Attached Figure Description

[0019] Figure 1 This is a flowchart illustrating the preparation method of a maize rhizosphere microbial compound agent for regulating the tropism of fall armyworm, as described in this application. Detailed Implementation

[0020] The present application will be further described in detail below with reference to embodiments and comparative examples.

[0021] Example 1 A maize rhizosphere microbial compound agent for regulating the tropism of fall armyworm, comprising the following components by mass percentage: 15% fermentation carbon source mixture, 5% compound microbial fermentation product, and 80% carrier and excipient components; The fermentation carbon source mixture consists of molasses (18% by mass), crude glycerol (8% by mass), cellulase hydrolysate (38% by mass), and corn starch processing by-product liquid (10% by mass), with the remainder being deionized water. The compound microbial fermentation product was obtained by mixing the following strains through two-stage culture: Bacillus subtilis CGMCC No. 1.1470 accounted for 30% of the total mass of the compound microbial fermentation product; Pseudomonas fluorescens CGMCC No. 1.1803 accounted for 40% of the total mass of the compound microbial fermentation product; Trichoderma harzianum CGMCC No. 3.18371 accounted for 10% of the total mass of the compound microbial fermentation product; and Candida tropicalis CGMCC No. 2.1082 accounted for 20% of the total mass of the compound microbial fermentation product. The carrier and excipient components consist of 40% bentonite, 10% sodium humate, 4% sodium alginate, 8% maltodextrin and 0.3% silica aerogel, with the remainder being agriculturally acceptable inert fillers. The cellulase hydrolysate is a liquid obtained by hydrolyzing corn stalks with 1.0% cellulase (based on dry corn stalk mass) at pH 4.8 and 45°C for 24 hours. The corn starch processing by-product liquor is the corn extract produced during the wet corn starch production process, and the total soluble sugar content of the corn extract is 15 g / L. The preparation method of this compound microbial agent includes the following steps: S1, preparing a fermentation carbon source mixture: The specific steps for preparing the fermentation carbon source mixture are as follows: add molasses, crude glycerol, cellulase hydrolysate, corn starch processing by-product liquid and deionized water into a stirrer and stir at a speed of 200 rpm for 20 min. S2. Preparation of composite microbial fermentation products: The specific steps for preparing the compound microbial fermentation product are as follows: First, the primary seed culture of the four strains is cultured at 28°C and pH 6.4 for 18 hours; the cultured primary seed culture is then inoculated into the secondary culture medium and cultured at 26°C and dissolved oxygen 20% for 36 hours; finally, the four cultured strains are mixed evenly. S3. Introduce the fermentation products of the composite microorganisms into the fermentation carbon source mixture for parallel fermentation; S4. Concentrate the fermentation broth after fermentation is complete: The specific steps of the concentration process are as follows: After centrifuging to remove solids, the fermentation broth after the culture is completed is concentrated by membrane filtration until the volume of the fermentation broth is reduced to 1 / 5 of the original volume. S5. Prepare the carrier and excipient components, then mix the concentrated fermentation broth with the carrier and excipient components for adsorption: The specific steps for preparing the carrier and excipient components are as follows: Sodium alginate and maltodextrin are dissolved in deionized water at 40°C and stirred at 300 rpm for 15 minutes to form a uniform sol; bentonite and sodium humate are added to the sol and stirred for another 20 minutes until uniform gelatinization is achieved. Then, silica aerogel was added for adsorption and dispersion, and the water content of the system was adjusted to 30%. The specific steps of mixed adsorption are as follows: the fermentation broth is mixed with the carrier and excipient components, and stirred at room temperature for 15 minutes to form a wet mixture; S6. Drying and granulating the adsorption mixture: The specific steps for drying and granulation are as follows: The wet mixture was dried in a fluidized bed at a temperature of 50°C for 45 minutes. The dried wet mixture is sieved and granulated, with a particle size of 150 μm after sieving and granulation.

[0022] Example 2 A maize rhizosphere microbial compound agent for regulating the tropism of fall armyworm, comprising the following components by mass percentage: 25% fermentation carbon source mixture, 15% compound microbial fermentation product, and 60% carrier and excipient components; The fermentation carbon source mixture consists of molasses (23% by mass), crude glycerol (15% by mass), cellulase hydrolysate (43% by mass), and corn starch processing by-product liquid (12.5% ​​by mass), with the remainder being deionized water. The compound microbial fermentation product was obtained by mixing the following strains through two-stage culture: Bacillus subtilis CGMCC No. 1.1470 accounted for 45% of the total mass of the compound microbial fermentation product; Pseudomonas fluorescens CGMCC No. 1.1803 accounted for 25% of the total mass of the compound microbial fermentation product; Trichoderma harzianum CGMCC No. 3.18371 accounted for 20% of the total mass of the compound microbial fermentation product; and Candida tropicalis CGMCC No. 2.1082 accounted for 10% of the total mass of the compound microbial fermentation product. The carrier and excipient components consist of bentonite (47.5% by mass), sodium humate (12.5% ​​by mass), sodium alginate (4.5% by mass), maltodextrin (9% by mass), and silica aerogel (0.4% by mass), with the remainder being agriculturally acceptable inert fillers. The cellulase hydrolysate is a liquid obtained by hydrolyzing corn stalks with 1.5% cellulase (based on dry corn stalk mass) at pH 5.0 and 50°C for 36 hours. The corn starch processing by-product liquor is the corn extract produced during the wet corn starch production process, and the total soluble sugar content of the corn extract is 20 g / L. The preparation method of this compound microbial agent includes the following steps: S1, preparing a fermentation carbon source mixture: The specific steps for preparing the fermentation carbon source mixture are as follows: add molasses, crude glycerol, cellulase hydrolysate, corn starch processing by-product liquid and deionized water into a stirrer and stir at a speed of 300 rpm for 30 min. S2. Preparation of composite microbial fermentation products: The specific steps for preparing the compound microbial fermentation product are as follows: First, the primary seed culture of the four strains is cultured at 30°C and pH 6.6 for 19 hours; the cultured primary seed culture is then inoculated into the secondary culture medium and cultured at 28°C and dissolved oxygen 30% for 42 hours; finally, the four cultured strains are mixed evenly. S3. Introduce the fermentation products of the composite microorganisms into the fermentation carbon source mixture for parallel fermentation; S4. Concentrate the fermentation broth after fermentation is complete: The specific steps of the concentration process are as follows: After centrifuging to remove solids, the fermentation broth after the culture is completed is concentrated by membrane filtration until the volume of the fermentation broth is reduced to 1 / 4 of the original volume. S5. Prepare the carrier and excipient components, then mix the concentrated fermentation broth with the carrier and excipient components for adsorption: The specific steps for preparing the carrier and excipient components are as follows: Sodium alginate and maltodextrin are dissolved in 50°C deionized water and stirred at 400 rpm for 22.5 min to form a uniform sol. Add bentonite and sodium humate to the sol and continue stirring for 30 minutes until homogeneous gelatinization; then add silica aerogel for adsorption and dispersion, and adjust the water content of the system to 35%. The specific steps of mixed adsorption are as follows: the fermentation broth is mixed with the carrier and excipient components, and stirred at room temperature for 30 minutes to form a wet mixture; S6. Drying and granulating the adsorption mixture: The specific steps for drying and granulation are as follows: the wet mixture is dried in a fluidized bed at a temperature of 52.5°C for 52.5 min. The dried wet mixture is sieved and granulated, with a particle size of 185 μm after sieving and granulation.

[0023] Example 3 A maize rhizosphere microbial compound agent for regulating the tropism of fall armyworm, comprising the following components by mass percentage: 35% fermentation carbon source mixture, 25% compound microbial fermentation product, and 50% carrier and excipient components; The fermentation carbon source mixture consists of molasses (28% by mass), crude glycerol (22% by mass), cellulase hydrolysate (38% by mass), and corn starch processing by-product liquid (12% by mass), with the remainder being deionized water (28% + 22% + 38% + 12% = 100%). The compound microbial fermentation product was obtained by mixing the following strains through two-stage culture: Bacillus subtilis CGMCC No. 1.1470 accounted for 60% of the total mass of the compound microbial fermentation product; Pseudomonas fluorescens CGMCC No. 1.1803 accounted for 10% of the total mass of the compound microbial fermentation product; Trichoderma harzianum CGMCC No. 3.18371 accounted for 30% of the total mass of the compound microbial fermentation product; and Candida tropicalis CGMCC No. 2.1082 accounted for 5% of the total mass of the compound microbial fermentation product. The carrier and excipient components consist of bentonite (55% by mass), sodium humate (15%), sodium alginate (5%), maltodextrin (10%), and silica aerogel (0.5%), with the remainder being agriculturally acceptable inert fillers. The cellulase hydrolysate is a liquid obtained by hydrolyzing corn stalks with 2.0% cellulase (based on dry corn stalk mass) at pH 5.2 and temperature 55°C for 48 hours. The corn starch processing by-product liquor is the corn extract produced during the wet corn starch production process, and the total soluble sugar content of the corn extract is 25 g / L. The preparation method of this compound microbial agent includes the following steps: S1, preparing a fermentation carbon source mixture: The specific steps for preparing the fermentation carbon source mixture are as follows: add molasses, crude glycerol, cellulase hydrolysate, corn starch processing by-product liquid and deionized water into a stirrer and stir at a speed of 400 rpm for 40 min. S2. Preparation of composite microbial fermentation products: The specific steps for preparing the compound microbial fermentation product are as follows: First, the primary seed culture of the four strains is cultured at 32°C and pH 6.8 for 20 hours; the cultured primary seed culture is then inoculated into the secondary culture medium and cultured at 30°C and dissolved oxygen 40% for 48 hours; finally, the four cultured strains are mixed evenly. S3. Inoculate the fermentation products of the composite microorganisms into the fermentation carbon source mixture for parallel fermentation; S4. Concentrate the fermentation broth after fermentation. The specific steps of the concentration process are as follows: After centrifuging to remove solids, the fermentation broth after the culture is completed is concentrated by membrane filtration until the volume of the fermentation broth is reduced to 1 / 3 of the original volume. S5. Prepare the carrier and excipient components, then mix the concentrated fermentation broth with the carrier and excipient components for adsorption: The specific steps for preparing the carrier and excipient components are as follows: Sodium alginate and maltodextrin are dissolved in deionized water at 60°C and stirred at 500 rpm for 30 minutes to form a uniform sol. Add bentonite and sodium humate to the sol and continue stirring for 40 minutes until it is uniformly gelatinized; Then, silica aerogel was added for adsorption and dispersion, and the water content of the system was adjusted to 40%. The specific steps of mixed adsorption are as follows: the fermentation broth is mixed with the carrier and excipient components and stirred at room temperature for 45 minutes to form a wet mixture; S6. Drying and granulating the adsorption mixture: The specific steps of drying and granulation are as follows: the wet mixture is dried in a fluidized bed at a temperature of 55°C for 60 minutes; the dried wet mixture is then sieved and granulated to a particle size of 220 μm.

[0024] Comparative Example 1 The only difference from Example 1 is that the corn starch processing by-product liquid in the fermentation carbon source mixture is replaced with soybean meal enzymatic hydrolysate.

[0025] Comparative Example 2 The only difference from Example 1 is that the molasses content in the fermentation carbon source mixture is increased to 30%.

[0026] Comparative Example 3 The only difference from Example 1 is that the content of Pseudomonas fluorescens CGMCC No.1.1803 in the compound microbial fermentation product is increased to 45%.

[0027] Comparative Example 4 The only difference from Example 1 is that the fermentation was changed from parallel fermentation to sequential fermentation.

[0028] Comparative Example 5 The only difference from Example 1 is that the silica aerogel content in the carrier and excipient components is increased to 1.0%.

[0029] Comparative Example 6 The only difference from Example 1 is that the concentration step is omitted, and the fermentation broth is directly introduced into S5 to mix and adsorb with the carrier and excipient components.

[0030] Comparative Example 7 The only difference from Example 1 is that the conditions for preparing cellulase hydrolysate are changed to pH 6.0, 60°C, enzyme addition amount of 0.5%, and hydrolysis for 12 hours.

[0031] Comparative Example 8 The only difference from Example 1 is that no sieving and granulation were performed.

[0032] I. Test on the evolutionary ability of behavioral resistance to fall armyworm Experimental Content: Compound microbial agents from Examples 1-3 and Comparative Examples 1-8 were selected. Zhengdan 958 maize seeds were used as the test crop. The uniform seedling conditions were 16 hours / day light, 25±2℃ temperature, and 60±5% humidity, cultivating the seedlings to the 3-leaf, 1-heart stage. The test insects were fall armyworm egg masses without resistance, with an initial population of 500 individuals per group. Instruments used included a Y-type olfactometer with a 20 cm arm length and 3 cm inner diameter, an artificial climate chamber, a stereomicroscope, and an insect behavior recorder. The experimental methods followed the "Standard for Test Methods of Insect Behavior" GB / T38583-2020 and the "Guidelines for Pesticide Resistance Risk Assessment" NY / T1859.1-2010. Each test microbial agent was evenly mixed into the soil of 10 kg seedling pots at a dosage of 10 g / pot. Each group was replicated in triplicate, with 10 pots of maize per replicate. When maize reached the 5-leaf stage, maize roots and rhizosphere soil from each treatment group were placed in the Y-shaped olfactometer arm, while soil from the blank control group was placed in the control arm. Activated carbon-filtered air was introduced at a flow rate of 50 ml / min. Third-instar nymphs, starved for 4 hours, were placed at the base of the olfactometer at a rate of 30 per group. Insect selection behavior was observed for 120 minutes. The avoidance rate was calculated as: Avoidance rate = (Number of nymphs choosing the control arm - Number of nymphs choosing the test arm) / Total number of nymphs × 100%. Subsequently, maize from each treatment group was transferred to an artificial climate chamber, and third-instar nymphs were introduced at a density of 20 nymphs per chamber. Each generation was reared until pupation and emergence, after which egg masses were collected to hatch the next generation. This rearing process was repeated for 10 generations, with the avoidance rate measurement repeated every two generations. The avoidance rate retention rate was calculated as: Avoidance rate retention rate = (Nth generation avoidance rate / First generation avoidance rate) × 100%. The LC50 of the 10th generation was determined using the stomach poison method. The resistance index was calculated as follows: Resistance index = LC50 of the 10th generation of the treatment group / LC50 of the initial population. The avoidance rate, retention rate and resistance index data of each generation were recorded in real time.

[0033] II. Low-cost renewable carbon source utilization rate testing experiment Experimental Content: The compound microbial agents of Examples 1-3 and Comparative Examples 1-8 were selected. The carbon source materials were corn straw crushed to 20 mesh with a cellulose content of 38.5%, corn starch processing by-product liquid with a total soluble sugar content of 20 g / L, molasses with a total sugar content of 80°Bx, and industrial-grade crude glycerol with a purity of 95%. The detection reagents included DNS reagent, Solarbio's cellulase kit and hemicellulase kit. The instruments used included an Agilent 1260 high-performance liquid chromatograph, a UV spectrophotometer, a centrifuge with a speed of 8000 rpm, and a constant temperature incubator. The experimental method referred to "Method for Determination of Microbial Carbon Source Utilization" QB / T4872-2015. Fermentation carbon source mixtures were prepared according to the carbon source ratio of each sample, and the corresponding compound microbial fermentation products were inoculated. Fermentation was carried out according to the preparation method. Comparative Example 4 used a sequential fermentation method, with three replicates for each group. After fermentation, samples were taken, and the total sugar content in the mixture before and after fermentation was determined using the DNS method. The total sugar utilization rate was calculated as: Total sugar utilization rate = (Total sugar content before fermentation - Total sugar content after fermentation) / Total sugar content before fermentation × 100%. Cellulase activity in the fermentation broth was determined using a cellulase kit, and residual cellulose content was determined using high-performance liquid chromatography (HPLC). The cellulose degradation rate was calculated as: Cellulose degradation rate = (Initial cellulose content - Residual cellulose content) / Initial cellulose content × 100%. The consumption rates of glucose and fructose in the corn starch by-product broth were determined using HPLC; this consumption rate is the corn starch by-product utilization rate. Simultaneously, the total viable cell count and total carbon source input cost were statistically analyzed. The carbon source cost-effectiveness was calculated as: Carbon source cost-effectiveness = Total viable cell count in fermentation broth / Total carbon source input cost, in CFU / yuan. All test data were recorded and compared.

[0034] III. Testing of Microbial Active Product Yield and Finished Product Yield Experimental Procedure: The compound bacterial agents from Examples 1-3 and Comparative Examples 1-8 were selected. Detection reagents included the antimicrobial peptide ELISA kit and chitinase activity assay kit from Shanghai Enzyme-Linked Biotechnology Co., Ltd. Instruments used included a freeze dryer, a Malvern Mastersizer 3000 laser particle size analyzer, a high-performance liquid chromatograph, and a colony counter. Experimental methods followed the standards GB / T20287-2006 "Standard for Testing the Activity of Microbial Preparations" and GB / T6284-2016 "General Method for Determination of Moisture in Chemical Products". Three parallel samples were selected for each group, and concentrated fermentation broth and finished formulations were obtained according to the corresponding preparation processes. The concentration of antimicrobial peptides in the concentrated broth was determined using the antimicrobial peptide ELISA kit. The antimicrobial peptide yield was calculated as: Antimicrobial peptide yield = Antimicrobial peptide concentration in concentrated broth × Concentration factor, in mg / L. Chitinase activity was determined using the DNS method. The chitinase yield was calculated as: Chitinase yield = Total enzyme activity in fermentation broth / Dry weight of microorganisms in fermentation broth, in U / g. The viable cell counts of the concentrated fermentation broth and the finished formulation were measured separately. The viable cell retention rate was calculated as follows: Viable cell retention rate = Viable cell count of formulation × Volume of concentrated broth / (Viable cell count of concentrated broth × Mass of formulation) × 100%. The particle size of the formulation was measured using a laser particle size analyzer, and the percentage of particles in the 150-220 micrometer range was statistically analyzed; this percentage represents the particle pass rate. The yield rate was calculated as follows: Yield rate = Actual qualified formulation mass / Theoretical formulation mass × 100%. The moisture content of the formulation was measured according to GB / T6284-2016 standard, and the data for each indicator were systematically recorded and compared horizontally.

[0035] Table 1. Test on the evolution of behavioral resistance to fall armyworm.

[0036] Table 2. Low-cost renewable carbon source utilization rate test

[0037] Table 3. Tests for the yield of microbial active products and the finished product yield of formulations

[0038] Based on the test data results of the above embodiments and comparative examples, the following conclusions are drawn: 1. Based on Examples 1-3 and Comparative Example 1, and combined with the test results, it can be seen that corn starch processing by-product liquid, as an important component of low-cost renewable carbon source, forms a carbon source system adapted to microbial metabolism with molasses, crude glycerol, and cellulase hydrolysate. This system can simultaneously improve carbon source utilization and microbial synergistic metabolic efficiency. If it is replaced with soybean meal enzymatic hydrolysate, the compatibility between the carbon source and the metabolic needs of the strain decreases. This not only reduces the total sugar utilization and carbon source cost-effectiveness, but also weakens the repellency effect and resistance evolution ability of the compound microbial agent against fall armyworm. At the same time, it leads to a decrease in the yield of active products such as antimicrobial peptides and chitinase.

[0039] 2. Based on Examples 1-3 and Comparative Example 2, and combined with the test results, it can be seen that controlling the proportion of molasses in the fermentation carbon source mixture at 18%-28% is the key to ensuring the balance of the carbon source system. Molasses, in synergy with other carbon sources, can provide balanced nutrition for the strains. If the proportion of molasses is increased to 30%, which exceeds the reasonable range, it will lead to an imbalance in carbon source concentration, inhibit the metabolic activity of strains such as Pseudomonas fluorescens, not only reduce the cellulose degradation rate and by-product utilization rate, but also decrease the repellency retention rate and increase the resistance index, while weakening the yield of active products and the retention rate of viable bacteria in the formulation.

[0040] 3. Combining Examples 1-3 and Comparative Example 3 with the test results, it can be seen that the 10%-40% proportion of Pseudomonas fluorescens CGMCC No.1.1803 in the compound microbial fermentation product is the core to achieve synergistic effects among strains. It forms an insect-resistant metabolic network with strains such as Bacillus subtilis. If its proportion is increased to 45%, which is beyond the reasonable range, it will lead to an imbalance in competition among strains, inhibit the activity of strains such as Trichodermaharzianum, not only reducing the repellency effect and resistance evolution ability, but also affecting the carbon source metabolism efficiency and active product synthesis, resulting in a significant decrease in the overall performance of the compound microbial agent.

[0041] 4. Combining Examples 1-3 and Comparative Example 4 with the test results, it can be seen that parallel fermentation is the key to ensuring the synchronous growth of the four strains and the synergistic accumulation of metabolites. It can provide a consistent growth environment for the strains and promote the synergistic generation of insect-resistant active substances and carbon source metabolic enzymes. If sequential fermentation is changed, the asynchronous growth of the strains will lead to an imbalance of metabolites, which will not only reduce the total sugar utilization rate and cellulose degradation rate, but also reduce the repulsion rate and the yield of active products, while increasing the risk of resistance evolution of fall armyworm.

[0042] 5. Based on Examples 1-3 and Comparative Example 5, and combined with the test results, it can be seen that the proportion of silica aerogel in the carrier and excipient components of 0.3%-0.5% is the key to optimizing the adsorption performance and permeability of the carrier. It can improve the adsorption stability of the carrier on the fermentation broth and protect the activity of microorganisms. If its proportion is increased to 1.0%, which is beyond the reasonable range, it will lead to a decrease in the permeability of the carrier and a densification of the adsorption structure. This will not only reduce the retention rate of viable bacteria and the qualified rate of particles, but also reduce the yield of the finished product. At the same time, it will affect the colonization efficiency of the compound microbial agent in the rhizosphere environment and weaken the persistence of the repulsion effect.

[0043] 6. Combining Examples 1-3 and Comparative Example 6 with the test results, it can be seen that the fermentation broth concentration treatment is an important step to increase the concentration of microbial active products and optimize the adsorption efficiency of the carrier. The concentration treatment can increase the unit content of active substances such as antimicrobial peptides and chitinase, and promote the full binding with the carrier. If the concentration treatment is omitted, the fermentation broth concentration will be too low, resulting in the dispersion of active products. This will not only reduce the yield of active products and the cost-effectiveness of carbon sources, but also reduce the retention rate of viable bacteria and the yield of finished products. At the same time, it will weaken the repellency effect of the compound microbial agent on fall armyworm and the resistance evolution ability.

[0044] 7. Based on Examples 1-3 and Comparative Example 7 and the test results, it can be seen that the preparation conditions of cellulase hydrolysate are the key to ensuring efficient degradation of cellulose. They can maximize the activation of cellulase activity and provide sufficient available carbon source for microorganisms. If the conditions are deviated from, the cellulose degradation rate will decrease significantly. Insufficient carbon source supply will lead to limited microbial metabolism, which will not only reduce carbon source utilization and cost-effectiveness, but also cause a simultaneous decrease in repulsion effect, yield of active products and finished product yield.

[0045] 8. Based on Examples 1-3 and Comparative Example 8, and combined with the test results, it can be seen that the sieving and granulation process ensures the uniformity of the formulation particles, improves the application effect and stability. Uniform particle size can ensure the synchronous release of microorganisms and active products adsorbed by the carrier. If sieving and granulation are not performed, the uneven particle size of the formulation particles will lead to differences in adsorption stability, which will not only reduce the particle qualification rate and finished product rate, but also cause uneven colonization of microorganisms in the rhizosphere environment, weaken the consistency of the repulsion effect and the resistance evolution ability, and at the same time affect the effective release efficiency of the active product.

[0046] Although embodiments of this application have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of this application, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A compound microbial agent for regulating the tropism of fall armyworm in maize rhizosphere, characterized in that, It comprises the following components by mass percentage: 15–35% fermentation carbon source mixture, 5–25% complex microbial fermentation products, and 50–80% carrier and auxiliary materials; The fermentation carbon source mixture consists of molasses (18–28% by mass), crude glycerol (8–22% by mass), cellulase hydrolysate (38–48% by mass), and corn starch processing by-product liquid (10–15% by mass), with the remainder being deionized water. The complex microbial fermentation products were obtained by mixing the following strains through two-stage culture: Bacillus subtilis CGMCC No. 1.1470 accounts for 30–60% of the total mass of the complex microbial fermentation products; Pseudomonas fluorescens CGMCC No. 1.1803 accounts for 10–40% of the total mass of the complex microbial fermentation products; Trichoderma harzianum CGMCC No.3.18371 accounts for 10–30% of the total mass of the fermentation products from the complex microbial fermentation process; Candida tropicalis CGMCC No.2.1082 accounts for 5–20% of the total mass of the complex microbial fermentation products; The carrier and excipient components consist of 40–55% bentonite, 10–15% sodium humate, 4–5% sodium alginate, 8–10% maltodextrin, and 0.3–0.5% silica aerogel, accounting for 40–55% of the total mass of the carrier and excipient components.

2. The maize rhizosphere microbial compound agent for regulating the tropism of fall armyworm according to claim 1, characterized in that, The cellulase hydrolysate is a liquid obtained by hydrolyzing corn stalks with 1.0–2.0% cellulase (based on dry corn stalk mass) at pH 4.8–5.2 and temperature 45–55°C for 24–48 hours.

3. The maize rhizosphere microbial compound agent for regulating the tropism of fall armyworm according to claim 1, characterized in that, The corn starch processing by-product liquid is a corn extract produced during the wet corn starch production process, and the total soluble sugar content of the corn extract is 15–25 g / L.

4. A method for preparing a maize rhizosphere microbial compound inoculant to regulate the tropism of fall armyworm, characterized in that, The preparation of a maize rhizosphere microbial compound agent for regulating the attraction of fall armyworm as described in any one of claims 1-3 includes the following steps: S1. Prepare the fermentation carbon source mixture; S2. Preparation of composite microbial fermentation products; S3. Introduce the fermentation products of the composite microorganisms into the fermentation carbon source mixture for parallel fermentation; S4. Concentrate the fermentation broth after fermentation is complete; S5. Prepare the carrier and excipient components, and then mix the concentrated fermentation broth with the carrier and excipient components for adsorption. S6. Dry and granulate the adsorption mixture.

5. The method for preparing a maize rhizosphere microbial compound agent for regulating the tropism of fall armyworm according to claim 4, characterized in that, The specific steps for preparing the fermentation carbon source mixture in step S1 are as follows: Add molasses, crude glycerol, cellulase hydrolysate, corn starch processing by-product liquid and deionized water to a stirrer and stir at a speed of 200–400 rpm for 20–40 min.

6. The method for preparing a maize rhizosphere microbial compound agent for regulating the tropism of fall armyworm according to claim 4, characterized in that, In step S2, the specific steps for preparing the composite microbial fermentation product are as follows: First, the primary seed cultures of the four strains were cultured for 18–20 h at a temperature of 28–32°C and a pH of 6.4–6.

8. The primary seed culture that has been cultured is inoculated into the secondary culture medium and cultured for 36–48 h at a temperature of 26–30°C and a dissolved oxygen of 20–40%. Finally, mix the four strains that have been cultured thoroughly.

7. The method for preparing a maize rhizosphere microbial compound agent for regulating the tropism of fall armyworm according to claim 4, characterized in that, In step S4, the concentration process is as follows: After the fermentation broth is finished, the solids are removed by centrifugation, and then the broth is concentrated by membrane filtration until the volume of the fermentation broth is reduced to 1 / 3–1 / 5 of the original volume.

8. The method for preparing a maize rhizosphere microbial compound agent for regulating the tropism of fall armyworm according to claim 4, characterized in that, The specific steps for preparing the carrier and excipient components are as follows: Dissolve sodium alginate and maltodextrin in deionized water at 40–60°C, and stir at 300–500 rpm for 15–30 minutes to form a uniform sol. Add bentonite and sodium humate to the sol and continue stirring for 20–40 minutes until it is uniformly gelatinized; Then silica aerogel was added for adsorption and dispersion, and the water content of the system was adjusted to 30–40%.

9. The method for preparing a maize rhizosphere microbial compound agent for regulating the tropism of fall armyworm according to claim 4, characterized in that, The specific steps of the mixed adsorption in step S5 are as follows: The fermentation broth is mixed with the carrier and excipients and stirred at room temperature for 15–45 minutes to form a wet mixture.

10. The method for preparing a maize rhizosphere microbial compound agent for regulating the tropism of fall armyworm according to claim 4, characterized in that, In step S6, the specific steps for drying and granulation are as follows: The wet mixture is subjected to fluidized bed drying, wherein the fluidized bed drying temperature is 50–55°C and the time is 45–60 min; The dried wet mixture is sieved and granulated, wherein the particle size after sieving and granulation is 150–220 μm.

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