A biocontrol agent containing bacillus toyonensis, a preparation method and application thereof

Abstract

The application discloses a biocontrol agent containing bacillus toyonensis and a preparation method and application thereof, and belongs to the technical field of microorganisms. The biocontrol agent comprises an active component and an auxiliary material, the active component is composed of bacillus toyonensis CGMCC No. 34320, flavobacterium breve CGMCC No. 1.10125 and one of low-toxicity chemical insecticidal synergists, imidacloprid, acetamiprid or thiamethoxam, and the mass ratio of the three is (3-5):(1-3):(0.5-1). The application has the three functions of killing adult insects, inhibiting reproduction and inducing crop stress resistance through the synergistic action of the two bacteria and the precise compound of the bacteria and the medicine, can efficiently prevent and control cotton aphids, inhibit cotton aphid reproduction and improve the stress resistance of cotton plants, has good storage stability and is suitable for large-scale application.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a biocontrol agent containing Bacillus oryzae, its preparation method, and its application. Background Technology

[0002] Cotton is an important economic crop, and cotton aphids are one of the most significant pests in cotton production. Cotton aphids use their piercing-sucking mouthparts to feed on the sap of tender leaves, stems, and bolls, causing leaf curling, stunted growth, and boll shedding. They also secrete honeydew, leading to sooty mold, severely impacting cotton yield and quality. For a long time, the control of cotton aphids has relied primarily on chemical insecticides, such as imidacloprid, acetamiprid, and thiamethoxam. However, the extensive and singular use of chemical pesticides has led to varying degrees of resistance in cotton aphids to multiple pesticides, resulting in a decline in control effectiveness year by year. Simultaneously, pesticide residues have adversely affected the farmland ecosystem, pollinating insects, and the health of humans and livestock.

[0003] Currently, cotton aphid control mainly relies on the single application of chemical pesticides. Long-term, high-frequency use has led to aphid resistance increasing year by year, resulting in a continuous decline in control efficacy. This also causes problems such as excessive pesticide residues, damage to soil microbiota, death of natural enemy insects, and environmental pollution. While single microbial inoculants are green, safe, residue-free, and less prone to resistance development, they suffer from technical drawbacks such as slow onset of action, poor rapid-acting properties, unstable efficacy in low-temperature and dry environments, and difficulty in completely eradicating aphids when used alone. These limitations make it difficult to meet the high-efficiency pest control requirements of large-scale cotton cultivation.

[0004] Microbial biocontrol agents have gradually become an important direction for green control of aphids due to their advantages such as being environmentally friendly, leaving no residue, and being less likely to induce resistance. However, existing biocontrol agents still have obvious shortcomings: First, the commonly used strains are mostly conventional strains such as Bacillus subtilis and Bacillus cereus, which have limited biocontrol activity. When used alone, their control efficacy is generally low, and they are slow to take effect and have a short duration of action. Second, existing compound microbial agents are mostly simple superpositions of strains, lacking a functional synergistic mechanism, resulting in weak synergistic effects. Third, the technology of compounding microorganisms with pesticides generally suffers from problems such as high dosage of chemical agents, easy inactivation of microorganisms, and obvious antagonistic effects. Moreover, most products only focus on insect control effects and cannot simultaneously improve crop resistance, resulting in single-function products.

[0005] For example, Chinese patent application CN202010846976.6 discloses a microbial insecticide for controlling aphids. This microbial insecticide is obtained through solid-state activation and culture of microbial strains, which include: *Neotrichum aphidii* 22-28%, *Acinetobacter baumannii* 15-20%, *Bacillus thuringiensis* 12-18%, *Bacillus amyloliquefaciens* 13-18%, *Bacillus subtilis* 10-15%, and *Serratia marcescens* 12-16%. The beneficial effects of this invention are: utilizing the symbiotic relationship between *Neotrichum aphidii* and *Acinetobacter baumannii*, and the synergistic effect among *Acinetobacter baumannii*, *Bacillus thuringiensis*, *Bacillus amyloliquefaciens*, *Bacillus subtilis*, and *Serratia marcescens*, *Neotrichum aphidii* helps *Acinetobacter baumannii*, *Bacillus thuringiensis*, *Bacillus amyloliquefaciens*, and *Bacillus subtilis* invade aphid cells and ultimately cause disease in aphids, thus improving the efficiency of insecticidal control.

[0006] However, the bacterial agent components described in this scheme are complex, containing bacteria and fungi of various genera, making the production process difficult to control. Furthermore, there may be complex interspecies competition or antagonistic effects between different strains, making it difficult to guarantee product stability.

[0007] Therefore, there is an urgent need to develop a new strain and strain combination, and to develop a novel biocontrol agent that overcomes the shortcomings of existing biocontrol agents, such as slow onset of action, unstable efficacy, and single function, by optimizing the preparation process and compounding system, so as to meet the needs of green pest control and sustainable development of cotton. Summary of the Invention

[0008] The purpose of this invention is to address the shortcomings of existing technologies by providing a biocontrol agent containing *Bacillus terrestris*, its preparation method, and its application. This invention uses a domestically preserved, highly active *Bacillus terrestris* as the core, combined with *Flavobacterium granulosum* to construct a synergistic dual-strain system, and further incorporates a low-toxicity chemical insecticide. Through functional complementarity and precise synergistic effects, it achieves multiple benefits, including efficient control of cotton aphids, inhibition of aphid reproduction, and enhancement of crop stress resistance. The composition is simple, stable, and has low production costs, making it suitable for industrial-scale application.

[0009] To achieve the above-mentioned technical objectives, the technical solution adopted by the present invention is as follows: A biocontrol agent containing Bacillus terrestris, comprising an active ingredient and agriculturally acceptable adjuvants, wherein the active ingredient comprises Bacillus terrestris (… Bacillus toyonensis ), auxiliary biocontrol strains and low-toxicity chemical insecticide synergists; the auxiliary biocontrol strain is Flavobacterium granulosum ( Flavobacterium granuli The low-toxicity chemical insecticide synergist is one of imidacloprid, acetamiprid, and thiamethoxam.

[0010] Preferably, the mass ratio of Bacillus oryzae, Flavobacterium granulosum, and low-toxicity chemical insecticide synergist in the active components is (3-5):(1-3):(0.5-1).

[0011] Preferably, the Bacillus terrestris has the accession number CGMCC No. 34320, the accession date is April 23, 2025, and the accession address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, China General Microbiological Culture Collection Center.

[0012] The strain of *Flavobacterium granulosum* has the strain number CGMCCNo.1.10125 and the original deposit date is July 1, 2009. This strain was purchased from the China General Microbiological Culture Collection Center and does not require biological preservation.

[0013] Preferably, the excipients include a carrier, a dispersant, a wetting agent, a binder, and a disintegrant, in a mass ratio of (50-65):(3-7):(1-3):(1-3):(3-7).

[0014] More preferably, the carrier is at least one of diatomaceous earth, kaolin, bentonite, and talc; the dispersant is at least one of sodium lignosulfonate, sodium methylene bisnaphthalene sulfonate, and soapberry powder; the wetting agent is at least one of sodium alkyl sulfate, sodium alkyl sulfonate, and fatty alcohol polyoxyethylene ether; the binder is at least one of polyvinyl alcohol, sodium carboxymethyl cellulose, and starch; and the disintegrant is at least one of sodium bicarbonate, citric acid, and urea.

[0015] Preferably, the mass ratio of the active ingredient to the agriculturally acceptable excipient is (20-40):(60-80).

[0016] A method for preparing a biocontrol agent containing Bacillus terrestris, comprising the following preparation steps: (1) Activation of strains: Bacillus oryzae and Flavobacterium granulosum were inoculated into the corresponding solid slant culture medium and activated by static incubation at constant temperature to obtain activated strains of the two strains; (2) Seed culture preparation: Each activated bacterial strain was inoculated into the corresponding liquid seed culture medium and cultured in a shaker to prepare Bacillus oryzae seed culture and Flavobacterium granulosum seed culture, respectively. (3) Fermentation culture: The two seed liquids were transferred to the fermentation culture medium of the fermenter according to the inoculation amount, and fermented by aeration and stirring to obtain Bacillus oryzae fermentation broth and Flavobacterium granulosum fermentation broth respectively. (4) Concentration and powdering: The bacterial cells of each fermentation broth were collected by centrifugation, washed with sterile water, and dried under low temperature and vacuum to prepare Bacillus oryzae powder and Flavobacterium granulosum powder, respectively. (5) Compounding of active components: Weigh out two kinds of bacterial powder and low-toxic chemical insecticide synergist according to the ratio, add them to a mixer and stir at low speed until uniform to obtain compound active components; (6) Formulation: The compound active components and excipients are mixed in proportion, and then crushed, granulated, sieved and granulated to prepare granular biocontrol agents.

[0017] Preferably, in step (1), the activation culture temperature of Bacillus orientalis is 28-32℃ and the culture time is 24-36h; the activation culture temperature of Flavobacterium granulosum is 26-30℃ and the culture time is 36-48h; in step (2), the shaking speed is 180-220r / min, the culture time of Bacillus orientalis is 18-24h, and the culture time of Flavobacterium granulosum is 24-30h; in step (3), the fermentation temperature is 28-30℃, the dissolved oxygen concentration is 20%-30%, the fermentation time is 48-72h, and the inoculum size is 5%-10%v / v.

[0018] Preferably, the culture medium compositions used for each stage of the two strains are as follows: Culture media for Bacillus subtilis: Solid slant culture medium: peptone 8-12 g / L, yeast extract 4-6 g / L, sodium chloride 8-12 g / L, agar 18-20 g / L, pH 7.0-7.2; Liquid seed culture medium: peptone 8-12 g / L, yeast extract 4-6 g / L, sodium chloride 8-12 g / L, pH 7.0-7.2; Fermentation medium: peptone 7-9 g / L, yeast extract 5-7 g / L, glucose 8-12 g / L, sodium chloride 4-6 g / L, dipotassium hydrogen phosphate 0.8-1.2 g / L, magnesium sulfate 0.1-0.3 g / L, pH 7.0-7.2; Culture media for Flavobacterium granulosum: Solid slant culture medium: peptone 8-12 g / L, beef extract 2-4 g / L, sodium chloride 4-6 g / L, agar 18-20 g / L, pH 7.2-7.4; Liquid seed culture medium: peptone 8-12 g / L, beef extract 2-4 g / L, sodium chloride 4-6 g / L, pH 7.2-7.4; Fermentation medium: peptone 7-9 g / L, beef extract 4-6 g / L, glucose 4-6 g / L, sodium chloride 4-6 g / L, dipotassium hydrogen phosphate 0.4-0.6 g / L, magnesium sulfate 0.05-0.15 g / L, pH 7.2-7.4; All the above culture media should be autoclaved at 121℃ for 20 min before use.

[0019] Preferably, the stirring speed in step (5) is 300-500 r / min and the stirring time is 10-20 min.

[0020] Application of a biocontrol agent containing Bacillus oryzae in controlling cotton aphids, inhibiting cotton aphid reproduction, and enhancing the stress resistance of cotton plants.

[0021] The general application method of the biocontrol agent containing Bacillus oryzae is as follows: At the early stage of cotton aphid infestation, the biocontrol agent is applied by soil application or foliar spraying. For soil application, the dosage is 150-300g per acre, followed by watering or shallow tilling to distribute the agent in the rhizosphere. For foliar spraying, the agent is diluted with water at a mass ratio of 1:600-1:800, soaked and filtered, and then evenly sprayed on cotton shoots, the back of leaves, and buds. Depending on the insect infestation, a second spray can be applied every 7-10 days.

[0022] The core innovation of this invention is based on the multifunctional bioactivity of Bacillus oryzae (CGMCC No. 34320), and a three-level synergistic enhancement system is constructed around this core: First, the core strain plays a leading role: Bacillus terrestris possesses triple activities of insecticidal, propagation-inhibiting, and stress-resistant properties. The core strain of this invention, Bacillus thuringiensis CGMCC No. 34320, is a novel strain independently isolated and preserved by the applicant. It is the functional core of the entire biocontrol system and possesses three biological activities: ① Adult insecticidal effect: This strain can secrete lipopeptides and surfactant metabolites, which can kill cotton aphid adults through both contact poisoning and stomach poisoning; ② Reproductive inhibition effect: The secondary metabolites of this strain can interfere with the development of the cotton aphid reproductive system, significantly reduce the number of eggs laid by adults and the egg hatching rate, prolong the nymph development period, and inhibit the growth of the insect population from the perspective of the intrinsic growth rate; ③ Crop stress resistance induction effect: This strain can stably colonize the rhizosphere and leaves of cotton, activate the plant defense signaling pathway mediated by salicylic acid and jasmonic acid, and induce a significant upregulation of the activities of defense enzymes such as phenylalanine ammonia-lyase (PAL), peroxidase (POD), and superoxide dismutase (SOD), thereby enhancing the cotton's drought resistance, disease resistance, and insect resistance.

[0023] Second, synergistic enhancement by two bacteria: Flavobacterium granulosum assists in enhancing the inhibition of proliferation and colonization. Based on the core strain, it is combined with Flavobacterium granulosum CGMCC No.1.10125. The two have no antagonistic effect and their functions are highly complementary, achieving a synergistic effect of 1+1>2: the metabolites of Flavobacterium granulosum can further interfere with the reproductive physiology of aphids, and strengthen the inhibitory effect on cotton aphid oviposition and hatching; at the same time, Flavobacterium granulosum can form a dense biofilm on the plant surface, providing a colonization microenvironment for Bacillus orientalis, improving the drought resistance of the compound bacterial community and prolonging the effect of the bacterial agent.

[0024] Third, precise synergy between fungicides and pesticides: rapid and effective supplementation of chemical insecticides reduces pesticide use while increasing efficacy. This treatment combines any one of imidacloprid, acetamiprid, or thiamethoxam with a dual-strain system. Through precise formulation, it avoids the antagonistic and inactivation problems inherent in traditional microbial pesticide combinations, achieving a perfect balance between rapid and long-lasting effects: chemical pesticides quickly kill large numbers of adult insects in the field, rapidly reducing the insect population and compensating for the slow onset of action of microbial agents; the core and auxiliary strains continuously exert long-lasting insect control, reproductive inhibition, and stress-inducing effects, controlling insect population rebound at its source while repairing physiological damage to plants caused by chemical pesticides. The synergistic effect of these three components significantly exceeds the simple sum of the effects of each individual component, ensuring effective control while significantly reducing pesticide residues and delaying the development of aphid resistance.

[0025] In summary, compared with the prior art, the present invention has the following outstanding substantive features and significant progress: (1) The core biocontrol strain of this invention is the self-isolated and preserved Bacillus oryzae CGMCC No.34320, which is a brand-new strain that has not been publicly reported in the prior art. It has triple activities of killing adult insects, inhibiting reproduction and inducing crop stress resistance. It is different from all conventional biocontrol strains with single functions, and fundamentally solves the problem of the lack of core multifunctional strains in the existing biocontrol technology.

[0026] (2) This invention is based on a core multifunctional strain and is specifically combined with Flavobacterium granulosum to achieve functional complementarity, rather than simply superimposing strains. After the two work together, the overall insecticidal and propagation-inhibiting effects are improved, while the colonization ability of the bacterial community is enhanced and the duration of effectiveness is extended. This effectively controls the rebound of insect populations at the population level and solves the problems of short duration of effectiveness and easy rebound of insect populations in traditional biocontrol agents.

[0027] (3) Precise combination of fungal agents and pesticides significantly reduces pesticide use and increases efficacy. This invention uses precise combination of chemical agents and a dual-strain system, avoiding the antagonistic and inactivation problems of traditional fungal agent combinations, and achieving a combination of rapid and long-lasting effects. The amount of chemical agents used is reduced compared to conventional amounts, which greatly reduces pesticide residues while ensuring efficient insect control, delaying the development of aphid resistance, and protecting the field ecology.

[0028] (4) The microbial agent of the present invention has three functions: pest control, reproduction inhibition and crop stress resistance enhancement. It can be used for multiple purposes, reduce the number of times the agent is used in the field and reduce planting costs. The components are simplified, the production process is easy to control, the product has good storage stability, and it is suitable for the field cotton planting mode. It has broad prospects for promotion and application. Attached Figure Description

[0029] Figure 1 A colony morphology diagram of Bacillus thomsonii with accession number CGMCC No. 34320; Figure 2 A staining characteristic image of Bacillus thomsonii with accession number CGMCC No. 34320; Figure 3The image shows the plate antagonistic effect between Bacillus truncatum (CGMCC No. 34320) and Flavobacterium granulosum (CGMCC No. 1.10125), where A represents Bacillus truncatum and B represents Flavobacterium granulosum. Detailed Implementation

[0030] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the embodiments will be described in detail below. The following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0031] Example 1 A biocontrol agent containing Bacillus terrestris, comprising an active ingredient and agriculturally acceptable adjuvants, wherein the active ingredient comprises Bacillus terrestris (… Bacillus toyonensis ), auxiliary biocontrol strains and low-toxicity chemical insecticide synergists; the auxiliary biocontrol strain is Flavobacterium granulosum ( Flavobacterium granuli The low-toxicity chemical insecticide synergist mentioned is imidacloprid.

[0032] The active ingredients contain Bacillus oryzae, Flavobacterium granulosum, and a low-toxicity chemical insecticide synergist in a mass ratio of 3:1:0.5.

[0033] The Bacillus tumefaciens described is deposited with accession number CGMCC No. 34320, on April 23, 2025, at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, China General Microbiological Culture Collection Center, Institute of Microbiology, Chinese Academy of Sciences.

[0034] The core Bacillus tsutchuensis used in this embodiment was isolated and screened from cotton aphids that died naturally in the cotton field. The specific isolation and screening steps are as follows: (1) Sample collection: Wingless adult cotton aphids that died naturally, had stiff bodies, and showed no obvious mold on their body surface were collected from cotton fields in the budding stage in Liaocheng, Shandong, the main cotton producing area. They were placed in sterile centrifuge tubes, refrigerated at 4℃ and brought back to the laboratory for later use.

[0035] (2) Surface disinfection of insects: Select intact dead aphids, place them in sterile petri dishes, soak them in 75% ethanol for 30 seconds, discard the ethanol, add 0.5% sodium hypochlorite solution and soak for 2 minutes for surface sterilization; then rinse them repeatedly with sterile physiological saline 3 times, take the last rinse solution and spread it on LB solid plates, and incubate at 30℃ for 48 hours. No colony growth was observed, which proved that the insect surface disinfection was thorough.

[0036] (3) Tissue homogenization and spore enrichment: Under aseptic conditions, the disinfected aphids were transferred to a sterile mortar, and 1 mL of sterile physiological saline was added and ground thoroughly until the tissue homogenate was formed. The homogenate was transferred to a sterile centrifuge tube and heated in an 80°C constant temperature water bath for 10 min to kill the vegetative cells of non-spore-forming bacteria and enrich spore-forming bacteria.

[0037] (4) Separation and purification: The heated homogenate was serially diluted 10-fold with sterile physiological saline, and 10-fold dilutions were selected. -3 10 -4 10 -5 Three dilutions, 100 μL each, were spread onto LB agar plates, with each dilution replicated three times. The plates were incubated at 30°C inverted for 24-48 h. After colonies grew on the plates, single colonies of different morphology, color, and size were picked and purified three times on LB agar plates using the streak method to obtain pure culture strains, which were then numbered and stored.

[0038] (5) Initial screening of biocontrol activity: The pure strains isolated were screened for cotton aphid toxicity and reproduction inhibition activity by leaf immersion method. Finally, a Bacillus strain with the highest toxicity against adult cotton aphids and the most significant reproduction inhibition effect was obtained and numbered respectively.

[0039] (6) Strain Identification and Preservation: Based on morphological observation, physiological and biochemical tests, and 16S rRNA gene sequence homology comparison, the colonies of this strain were found to be round, milky white, with a rough, wrinkled surface; the cells were rod-shaped, Gram-positive, and capable of forming endospores; the 16S rRNA sequence showed 99.2% homology with the Bacillus tsao-ko type strain, thus identifying it as Bacillus tsao-ko. This strain was deposited on April 23, 2025, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 34320, and serves as the core experimental strain for all subsequent examples. Colony and staining images are shown below. Figure 1-2 As shown.

[0040] The 16S rRNA sequence of the aforementioned Bacillus orientalis is shown in SEQ ID NO. 1.

[0041]

[0042] The strain of *Flavobacterium granulosum* has the strain number CGMCCNo.1.10125 and the original deposit date is July 1, 2009. This strain was purchased from the China General Microbiological Culture Collection Center and does not require biological preservation.

[0043] The excipients include a carrier, a dispersant, a wetting agent, a binder, and a disintegrant, in a mass ratio of 50:7:1:3:3.

[0044] The carrier is diatomaceous earth; the dispersant is sodium lignosulfonate; the wetting agent is sodium alkyl sulfate; the binder is polyvinyl alcohol; and the disintegrant is sodium bicarbonate.

[0045] The mass ratio of the active ingredient to agriculturally acceptable excipients is 20:80.

[0046] A method for preparing a biocontrol agent containing Bacillus terrestris, comprising the following preparation steps: (1) Activation of strains: Bacillus oryzae and Flavobacterium granulosum were inoculated into the corresponding solid slant culture medium and activated by static incubation at constant temperature to obtain activated strains of the two strains; (2) Seed culture preparation: Each activated bacterial strain was inoculated into the corresponding liquid seed culture medium and cultured in a shaker to prepare Bacillus oryzae seed culture and Flavobacterium granulosum seed culture, respectively. (3) Fermentation culture: The two seed liquids were transferred to the fermentation culture medium of the fermenter according to the inoculation amount, and fermented by aeration and stirring to obtain Bacillus oryzae fermentation broth and Flavobacterium granulosum fermentation broth respectively. (4) Concentration and powdering: The bacterial cells of each fermentation broth were collected by centrifugation, washed with sterile water, and dried under low temperature and vacuum to prepare Bacillus oryzae powder and Flavobacterium granulosum powder, respectively. (5) Compounding of active components: Weigh out two kinds of bacterial powder and low-toxic chemical insecticide synergist according to the ratio, add them to a mixer and stir at low speed until uniform to obtain compound active components; (6) Formulation: The compound active components and excipients are mixed according to the specified ratio, then pulverized, granulated, and sieved to prepare a granular biocontrol agent. The resulting biocontrol agent has an effective total viable count ≥1.5×10⁻⁶. 9 CFU / g.

[0047] In step (1), the activation culture temperature of Bacillus oryzae is 28-32℃ and the culture time is 24h; the activation culture temperature of Flavobacterium granulosum is 26-30℃ and the culture time is 36h; in step (2), the shaking speed is 180r / min, the culture time of Bacillus oryzae is 18h, and the culture time of Flavobacterium granulosum is 24h; in step (3), the fermentation temperature is 28-30℃, the dissolved oxygen concentration is 20%-30%, the fermentation time is 48h, and the inoculum size is 10%v / v.

[0048] The composition of the culture medium used for each stage of the two strains is as follows: Culture media for Bacillus subtilis: Solid slant culture medium: peptone 8-12 g / L, yeast extract 4-6 g / L, sodium chloride 8-12 g / L, agar 18-20 g / L, pH 7.0-7.2; Liquid seed culture medium: peptone 8-12 g / L, yeast extract 4-6 g / L, sodium chloride 8-12 g / L, pH 7.0-7.2; Fermentation medium: peptone 7-9 g / L, yeast extract 5-7 g / L, glucose 8-12 g / L, sodium chloride 4-6 g / L, dipotassium hydrogen phosphate 0.8-1.2 g / L, magnesium sulfate 0.1-0.3 g / L, pH 7.0-7.2; Culture media for Flavobacterium granulosum: Solid slant culture medium: peptone 8-12 g / L, beef extract 2-4 g / L, sodium chloride 4-6 g / L, agar 18-20 g / L, pH 7.2-7.4; Liquid seed culture medium: peptone 8-12 g / L, beef extract 2-4 g / L, sodium chloride 4-6 g / L, pH 7.2-7.4; Fermentation medium: peptone 7-9 g / L, beef extract 4-6 g / L, glucose 4-6 g / L, sodium chloride 4-6 g / L, dipotassium hydrogen phosphate 0.4-0.6 g / L, magnesium sulfate 0.05-0.15 g / L, pH 7.2-7.4; All the above culture media should be autoclaved at 121℃ for 20 min before use.

[0049] In step (5), the stirring speed is 300 r / min and the stirring time is 10 min.

[0050] Example 2 A biocontrol agent containing Bacillus terrestris, comprising an active ingredient and agriculturally acceptable adjuvants, wherein the active ingredient comprises Bacillus terrestris (… Bacillus toyonensis ), auxiliary biocontrol strains and low-toxicity chemical insecticide synergists; the auxiliary biocontrol strain is Flavobacterium granulosum ( Flavobacterium granuli The low-toxicity chemical insecticide synergist mentioned is acetamiprid.

[0051] The active ingredients contain Bacillus oryzae, Flavobacterium granulosum, and a low-toxicity chemical insecticide synergist in a mass ratio of 4:2:0.7.

[0052] The Bacillus tumefaciens described is deposited with accession number CGMCC No. 34320, on April 23, 2025, at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, China General Microbiological Culture Collection Center, Institute of Microbiology, Chinese Academy of Sciences.

[0053] The screening and isolation methods used for Bacillus terrestris were the same as in Example 1.

[0054] The strain of *Flavobacterium granulosum* has the strain number CGMCCNo.1.10125 and the original deposit date is July 1, 2009. This strain was purchased from the China General Microbiological Culture Collection Center and does not require biological preservation.

[0055] The excipients include a carrier, a dispersant, a wetting agent, a binder, and a disintegrant, in a mass ratio of 60:5:2:2:5.

[0056] The carrier is kaolin; the dispersant is sodium methylene bisnaphthalene sulfonate; the wetting agent is sodium alkyl sulfonate; the binder is sodium carboxymethyl cellulose; and the disintegrant is citric acid.

[0057] The mass ratio of the active ingredient to agriculturally acceptable excipients is 30:70.

[0058] A method for preparing a biocontrol agent containing Bacillus terrestris, comprising the following preparation steps: (1) Activation of strains: Bacillus oryzae and Flavobacterium granulosum were inoculated into the corresponding solid slant culture medium and activated by static incubation at constant temperature to obtain activated strains of the two strains; (2) Seed culture preparation: Each activated bacterial strain was inoculated into the corresponding liquid seed culture medium and cultured in a shaker to prepare Bacillus oryzae seed culture and Flavobacterium granulosum seed culture, respectively. (3) Fermentation culture: The two seed liquids were transferred to the fermentation culture medium of the fermenter according to the inoculation amount, and fermented by aeration and stirring to obtain Bacillus oryzae fermentation broth and Flavobacterium granulosum fermentation broth respectively. (4) Concentration and powdering: The bacterial cells of each fermentation broth were collected by centrifugation, washed with sterile water, and dried under low temperature and vacuum to prepare Bacillus oryzae powder and Flavobacterium granulosum powder, respectively. (5) Compounding of active components: Weigh out two kinds of bacterial powder and low-toxic chemical insecticide synergist according to the ratio, add them to a mixer and stir at low speed until uniform to obtain compound active components; (6) Formulation: The compound active components and excipients are mixed according to the specified ratio, then pulverized, granulated, and sieved to prepare a granular biocontrol agent. The resulting biocontrol agent has an effective total viable count ≥ 2.5 × 10⁻⁶. 9 CFU / g.

[0059] Step (1) The activation culture temperature of Bacillus oryzae is 28-32℃ and the culture time is 30h; the activation culture temperature of Flavobacterium granulosum is 26-30℃ and the culture time is 40h; in step (2), the shaking speed is 220r / min, the culture time of Bacillus oryzae is 21h, and the culture time of Flavobacterium granulosum is 26h; in step (3), the fermentation temperature is 28-30℃, the dissolved oxygen concentration is 20%-30%, the fermentation time is 60h, and the inoculum size is 7%v / v.

[0060] The culture medium composition used for each stage of the two strains is the same as in Example 1.

[0061] In step (5), the stirring speed is 400 r / min and the stirring time is 15 min.

[0062] Example 3 A biocontrol agent containing Bacillus terrestris, comprising an active ingredient and agriculturally acceptable adjuvants, wherein the active ingredient comprises Bacillus terrestris (… Bacillus toyonensis ), auxiliary biocontrol strains and low-toxicity chemical insecticide synergists; the auxiliary biocontrol strain is Flavobacterium granulosum ( Flavobacterium granuli The low-toxicity chemical insecticide synergist mentioned is thiamethoxam.

[0063] The active ingredients contain Bacillus oryzae, Flavobacterium granulosum, and a low-toxicity chemical insecticide synergist in a mass ratio of 5:3:1.

[0064] The Bacillus tumefaciens described is deposited with accession number CGMCC No. 34320, on April 23, 2025, at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, China General Microbiological Culture Collection Center, Institute of Microbiology, Chinese Academy of Sciences.

[0065] The screening and isolation methods used for Bacillus terrestris were the same as in Example 1.

[0066] The strain of *Flavobacterium granulosum* has the strain number CGMCCNo.1.10125 and the original deposit date is July 1, 2009. This strain was purchased from the China General Microbiological Culture Collection Center and does not require biological preservation.

[0067] The excipients include a carrier, a dispersant, a wetting agent, a binder, and a disintegrant, in a mass ratio of 65:3:3:1:7.

[0068] The carrier is bentonite; the dispersant is soapberry powder; the wetting agent is fatty alcohol polyoxyethylene ether; the binder is starch; and the disintegrant is urea.

[0069] The mass ratio of the active ingredient to agriculturally acceptable excipients is 40:60.

[0070] A method for preparing a biocontrol agent containing Bacillus terrestris, comprising the following preparation steps: (1) Activation of strains: Bacillus oryzae and Flavobacterium granulosum were inoculated into the corresponding solid slant culture medium and activated by static incubation at constant temperature to obtain activated strains of the two strains; (2) Seed culture preparation: Each activated bacterial strain was inoculated into the corresponding liquid seed culture medium and cultured in a shaker to prepare Bacillus oryzae seed culture and Flavobacterium granulosum seed culture, respectively. (3) Fermentation culture: The two seed liquids were transferred to the fermentation culture medium of the fermenter according to the inoculation amount, and fermented by aeration and stirring to obtain Bacillus oryzae fermentation broth and Flavobacterium granulosum fermentation broth respectively. (4) Concentration and powdering: The bacterial cells of each fermentation broth were collected by centrifugation, washed with sterile water, and dried under low temperature and vacuum to prepare Bacillus oryzae powder and Flavobacterium granulosum powder, respectively. (5) Compounding of active components: Weigh out two kinds of bacterial powder and low-toxic chemical insecticide synergist according to the ratio, add them to a mixer and stir at low speed until uniform to obtain compound active components; (6) Formulation: The compound active components and excipients are mixed according to the specified ratio, then pulverized, granulated, and sieved to prepare a granular biocontrol agent. The resulting biocontrol agent has an effective total viable count ≥ 4.0 × 10⁻⁶. 9 CFU / g.

[0071] In step (1), the activation culture temperature of Bacillus oryzae is 28-32℃ and the culture time is 36h; the activation culture temperature of Flavobacterium granulosum is 26-30℃ and the culture time is 48h; in step (2), the shaking speed is 220r / min, the culture time of Bacillus oryzae is 24h, and the culture time of Flavobacterium granulosum is 30h; in step (3), the fermentation temperature is 28-30℃, the dissolved oxygen concentration is 20%-30%, the fermentation time is 72h, and the inoculum size is 5%v / v.

[0072] The culture medium composition used for each stage of the two strains is the same as in Example 1.

[0073] In step (5), the stirring speed is 500 r / min and the stirring time is 20 min.

[0074] Comparative Example 1 This comparative example, except for the absence of *Bacillus toyosum* in the active ingredient, maintains the total mass of *Flavobacterium granulosum* equal to the total mass of the two microorganisms in Example 1. All other raw materials and process steps are the same as in Example 1. That is: A biocontrol agent comprising an active ingredient and agriculturally acceptable adjuvants, wherein the active ingredient comprises a supportive biocontrol strain and a low-toxicity chemical insecticide synergist; the supportive biocontrol strain is *Flavobacterium granulosum* (…). Flavobacteriumgranuli The low-toxicity chemical insecticide synergist mentioned is imidacloprid.

[0075] The active ingredient contains a 4:0.5 mass ratio of Flavobacterium granulosum to a low-toxicity chemical insecticide synergist.

[0076] The strain of *Flavobacterium granulosum* has the strain number CGMCCNo.1.10125 and the original deposit date is July 1, 2009. This strain was purchased from the China General Microbiological Culture Collection Center and does not require biological preservation.

[0077] A method for preparing a biocontrol agent includes the following preparation steps: (1) Activation of strains: Flavobacterium granulosum was inoculated onto solid slant culture medium and activated by static incubation at constant temperature to obtain activated strains; (2) Seed culture preparation: Each activated bacterial strain was inoculated into the corresponding liquid seed culture medium and cultured in a shaker to prepare the seed culture of Flavobacterium granulosum. (3) Fermentation culture: The seed liquid was transferred to the fermentation culture medium of the fermenter according to the inoculation amount, and fermentation culture was carried out by aeration and stirring to obtain the fermentation broth of Flavobacterium granulosum; (4) Concentration and powdering: The fermentation broth is centrifuged to collect the bacterial cells, washed with sterile water, and then dried under low temperature and vacuum to prepare fine-grained Flavobacterium powder. (5) Compounding of active components: Weigh the bacterial powder and low-toxic chemical insecticide synergist according to the ratio, add them to the mixer and stir at low speed until uniform to obtain the compound active components; (6) Formulation: The compound active components and excipients are mixed in proportion, and then crushed, granulated, sieved and granulated to prepare granular biocontrol agents.

[0078] Comparative Example 2 This comparative example, except for the absence of *Flavobacterium granulosum* in the active ingredient, maintains the total mass of *Bacillus toyota* equal to the total mass of the two microorganisms in Example 1. All other raw materials and process steps are the same as in Example 1. That is: A biocontrol agent containing Bacillus terrestris, comprising an active ingredient and agriculturally acceptable adjuvants, wherein the active ingredient comprises Bacillus terrestris (… Bacillus toyonensis (and low-toxicity chemical insecticide synergists; the low-toxicity chemical insecticide synergists are imidacloprid.)

[0079] The active ingredient contains Bacillus oryzae and a low-toxicity chemical insecticide synergist in a mass ratio of 4:0.5.

[0080] The Bacillus tumefaciens described was deposited on April 23, 2025, with accession number CGMCC No. 34320. The deposit address was No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, China General Microbiological Culture Collection Center. The screening and isolation methods were the same as in Example 1.

[0081] A method for preparing a biocontrol agent containing Bacillus terrestris, comprising the following preparation steps: (1) Activation of strains: Bacillus davidiana was inoculated into the corresponding solid slant culture medium and activated by static incubation at constant temperature to obtain activated strains; (2) Seed culture preparation: The activated bacterial strains were inoculated into the corresponding liquid seed culture medium and cultured in a shaker to prepare Bacillus tsutsugamushi seed culture; (3) Fermentation culture: The seed liquid was transferred to the fermentation culture medium in the fermenter according to the inoculation amount, and fermentation culture was carried out by aeration and stirring to obtain the fermentation broth of Bacillus davidianus. (4) Concentration and powdering: The bacterial cells of each fermentation broth are collected by centrifugation, washed with sterile water, and dried under low temperature and vacuum to prepare Bacillus tsori powder. (5) Compounding of active components: Weigh the bacterial powder and low-toxic chemical insecticide synergist according to the ratio, add them to the mixer and stir at low speed until uniform to obtain the compound active components; (6) Formulation: The compound active components and excipients are mixed in proportion, and then crushed, granulated, sieved and granulated to prepare granular biocontrol agents.

[0082] Comparative Example 3 This comparative example is identical to Example 1 in all raw materials and process steps except that the low-toxicity chemical insecticide synergist imidacloprid is not used in the active ingredient (an equal amount of starch is used instead). That is: A biocontrol agent containing Bacillus terrestris, comprising an active ingredient and agriculturally acceptable adjuvants, wherein the active ingredient comprises Bacillus terrestris (… Bacillus toyonensis ), auxiliary biocontrol strains and starch; the auxiliary biocontrol strain is Flavobacterium granulosum ( Flavobacterium granuli ).

[0083] The active components contain Bacillus oryzae, Flavobacterium granulosum, and starch in a mass ratio of 3:1:0.5.

[0084] The Bacillus tumefaciens described was deposited on April 23, 2025, with accession number CGMCC No. 34320. The deposit address was No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, China General Microbiological Culture Collection Center. The screening and isolation methods were the same as in Example 1.

[0085] The strain of *Flavobacterium granulosum* has the strain number CGMCCNo.1.10125 and the original deposit date is July 1, 2009. This strain was purchased from the China General Microbiological Culture Collection Center and does not require biological preservation.

[0086] Comparative Example 4 This comparative example is identical to Example 1 in all raw materials and process steps, except for the use of different types of Bacillus orientalis in the active ingredient. That is: A biocontrol agent containing Bacillus terrestris, comprising an active ingredient and agriculturally acceptable adjuvants, wherein the active ingredient comprises Bacillus terrestris (… Bacillus toyonensis ), auxiliary biocontrol strains and low-toxicity chemical insecticide synergists; the auxiliary biocontrol strain is Flavobacterium granulosum ( Flavobacterium granuli The low-toxicity chemical insecticide synergist mentioned is imidacloprid.

[0087] The active ingredients contain Bacillus oryzae, Flavobacterium granulosum, and a low-toxicity chemical insecticide synergist in a mass ratio of 3:1:0.5.

[0088] The Bacillus tumefaciens specimen, with accession number CGMCC No. 1.4698, was purchased from the China General Microbiological Culture Collection Center. No biopreservation is required.

[0089] The strain of *Flavobacterium granulosum* has the strain number CGMCCNo.1.10125 and the original deposit date is July 1, 2009. This strain was purchased from the China General Microbiological Culture Collection Center and does not require biological preservation.

[0090] Comparative Example 5 This comparative example is identical to Example 1 in all raw materials and process steps, except for the use of different species of Flavobacterium granulosum in the active ingredient. That is: A biocontrol agent containing Bacillus terrestris, comprising an active ingredient and agriculturally acceptable adjuvants, wherein the active ingredient comprises Bacillus terrestris (… Bacillus toyonensis ), auxiliary biocontrol strains and low-toxicity chemical insecticide synergists; the auxiliary biocontrol strain is Flavobacterium granulosum ( Flavobacterium granuli The low-toxicity chemical insecticide synergist mentioned is imidacloprid.

[0091] The active ingredients contain Bacillus oryzae, Flavobacterium granulosum, and a low-toxicity chemical insecticide synergist in a mass ratio of 3:1:0.5.

[0092] The Bacillus tumefaciens described was deposited on April 23, 2025, with accession number CGMCC No. 34320. The deposit address was No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, China General Microbiological Culture Collection Center. The screening and isolation methods were the same as in Example 1.

[0093] The strain of *Flavobacterium granulosum* has the strain number CGMCC No. 1.12067 and the original deposit date is January 10, 2012. This strain was purchased from the China General Microbiological Culture Collection Center and does not require biological preservation.

[0094] Comparative Example 6 A microbial insecticide prepared according to the formulation and method of Example 1 in prior art document CN202010846976.6 is used as a prior art comparison.

[0095] Performance testing Antagonistic effect test of strains: To ensure that there is no antagonistic effect between the compound strains and thus affect the biocontrol effect, the cross streak plate method was used to conduct an antagonistic test on Bacillus oryzae CGMCC No.34320 and Flavobacterium granulosum CGMCC No.1.10125.

[0096] The tested Bacillus subtilis and Flavobacterium granulosum were inoculated into LB liquid medium and cultured at 30°C with shaking at 180 rpm for 24 h until the logarithmic growth phase was reached. The bacterial suspension concentration was then adjusted to 1×10⁻⁶. 8 CFU / mL. *Bacillus oryzae* was inoculated into the center of an LB agar plate and incubated at 28°C for 24 h. Then, *Flavobacterium granulosum* was inoculated into the plate perpendicular to the first streak and incubated at 28°C for another 48 h. This was repeated twice, and the colony growth at and around the intersection of the two streaks was observed.

[0097] If both strains can grow normally at the crossover point without producing a clear inhibition zone, it is determined that there is no antagonistic effect; if a clear zone appears at the crossover point or the growth of one strain is significantly inhibited, it is determined that there is an antagonistic effect.

[0098] The results are as follows Figure 3 As shown, both strains grew well at the intersection of the two scribbled lines and in the surrounding area, with normal colony morphology and no transparent inhibition zone or growth inhibition was observed. This result indicates that there is no antagonistic effect between *Bacillus oryzae* CGMCC No. 34320 and *Flavobacterium granulosum* CGMCC No. 1.10125 selected in this invention, and they are suitable for combined use.

[0099] Indoor toxicity testing 1. Test insect source The cotton aphids (Aphis gossypii) used in the test were collected from cotton-growing areas in Liaocheng City, Shandong Province. They were reared for more than three generations using cotton seedlings in an artificial climate chamber. Healthy, active, wingless adult aphids of the same age were selected for the test. The rearing conditions were: temperature 25±1℃, relative humidity 70%±5%, and photoperiod L:D=16:8.

[0100] 2. Test reagents Examples 1-3 describe the biocontrol agents prepared; Comparative Examples 1-5 describe the control formulations prepared; Comparative Example 6 describes the microbial insecticide prepared according to existing technology; Comparative Example 7 describes the conventional control agent as 5% imidacloprid emulsifiable concentrate (commercially available, conventional field application concentration); a blank control of water was also included. All examples and comparative examples were diluted with sterile water at a mass ratio of 1:600, thoroughly shaken, and allowed to stand for 10 minutes. The supernatant suspension was then used as the test solution.

[0101] 3. Test Methods Indoor toxicity assays were performed using the leaf immersion method. Fresh, healthy cotton leaves (variety: Lumianyan 37, growth stage: 4-5 leaves) were collected and prepared into circular leaf discs with a diameter of 2 cm using a perforator. Each test pesticide solution was poured into a sterile petri dish, and the leaf disc was immersed in the solution for 10 seconds using sterile forceps, gently agitating it to ensure both sides of the leaf disc were fully moistened. The leaf discs were then removed, and excess solution was gently blotted off onto sterile filter paper. They were then allowed to air dry at room temperature for approximately 30 minutes until no watermarks remained on the surface.

[0102] Line the bottom of a 9cm diameter petri dish with a layer of absorbent cotton and add a suitable amount of sterile water to maintain moisture. Cover the absorbent cotton with a layer of qualitative filter paper. Place the dried leaf discs, back side up, on the filter paper, with 3 leaf discs in each dish. Gently pick up wingless adult aphids with a soft brush, placing 5 aphids on each leaf disc (i.e., 15 aphids per dish), allowing the aphids to crawl naturally to the back of the leaf discs to feed. Set up 3 petri dishes as replicates for each treatment (i.e., a total of 45 test insects per treatment), and the entire experiment was independently replicated 3 times (i.e., a total of 135 test insects per treatment).

[0103] The petri dishes were placed in an artificial climate chamber and cultured under the same conditions as the tested insect sources. The number of cotton aphid deaths was observed and recorded under a stereomicroscope at 24h, 48h, 72h, and 96h after treatment. Aphids that did not move at all when gently touched with a paintbrush were considered dead; those that only showed slight leg twitching but could not crawl normally were also considered dead.

[0104] 4. Calculation Method The adjusted mortality rate is calculated using the following formula (I): Corrected mortality rate (%) = (treatment group mortality rate - control group mortality rate) / (1 - control group mortality rate) × 100% (I) in, Mortality rate in the treatment group (%) = (Number of dead insects in the treatment group / Total number of insects tested in the treatment group) × 100%; Mortality rate of control group (%) = (Number of dead insects in control group / Total number of insects tested in control group) × 100%.

[0105] 5. Data Processing Each treatment was performed in three independent replicates, with 45 test insects per treatment (3 petri dishes) per replicate, for a total of 135 test insects per treatment across the three replicates. The final data are expressed as the arithmetic mean of the three replicates.

[0106] The corrected mortality rate results of indoor toxicity assays of each treatment against cotton aphids are shown in Table 1.

[0107] Table 1. Results of indoor toxicity tests of each treatment against cotton aphids (corrected mortality rate, %) As shown in Table 1, the biocontrol agents prepared in Examples 1-3 of this invention have significant control effects on cotton aphids, and the corrected mortality rate increases with the extension of treatment time. After 96 hours of treatment, the corrected mortality rate of Examples 1-3 all reached over 93%, with Example 3 showing the best effect at 95.23%, which is significantly better than Comparative Examples 1-6 and Comparative Example 7 (conventional chemical agents).

[0108] Specifically, Comparative Example 1 contained only *Flavobacterium granulosum* and imidacloprid, lacking the core strain *Bacillus terrestris*, and its 96-hour corrected mortality rate was only 60.15%, indicating that the synergistic effect of *Flavobacterium granulosum* alone combined with chemical insecticides is limited. Comparative Example 2 contained only *Bacillus terrestris* and imidacloprid, lacking the auxiliary strain *Flavobacterium granulosum*, and its 96-hour corrected mortality rate was 75.82%, higher than Comparative Example 1, but still significantly lower than the example, indicating that *Flavobacterium granulosum* plays an important auxiliary synergistic role in the *Bacillus terrestris* biocontrol system, possibly due to its role in promoting the growth of *Bacillus terrestris* on aphids. The colonization and synergistic effects of metabolites in the epidermis or intestines are related. Comparative Example 3 contained only a combination of Bacillus terrestris and Flavobacterium granulosum without the addition of low-toxicity chemical insecticide synergists. Its 96-hour corrected mortality rate was 56.34%, which, although demonstrating the biocontrol potential of the two bacteria, showed a slower onset of action and lower late-stage efficacy compared to the "bacterium + pesticide" synergistic model. This demonstrates that the introduction of chemical agents is crucial for improving both rapid onset and final efficacy. Comparative Examples 4 and 5 used Bacillus terrestris and Flavobacterium granulosum strains from other sources, respectively. Their efficacy was lower than that of the examples using the specific strains of this invention (CGMCC No. 34320 and CGMCC No. 1.10125), indicating that the specific strain combination screened in this invention has superior synergistic biocontrol activity. Comparative Example 6 is a prior art control, with a 96-hour corrected mortality rate of 70.25%, significantly lower than that of the examples of this invention, further confirming the superiority of the "Bacillus terrestris + Flavobacterium granulosum + chemical insecticide" ternary synergistic system of this invention.

[0109] Furthermore, it is noteworthy that conventional chemical agents (Comparative Example 7) exhibited high rapid efficacy at 24h and 48h (68.72% and 82.15%, respectively), but the efficacy growth stagnated or even slightly decreased at 72h and 96h (86.34% and 81.67%, respectively). This may be related to the aphids developing contact resistance, the short residual effect of the agent, or the loss of the agent from the leaf surface. In contrast, the embodiments of the present invention not only maintained an extremely high mortality rate in the mid-to-late stages (72h-96h) but also showed a continuous upward trend, indicating that the biocontrol agent combines the persistence of biological control with the rapid efficacy of chemical control, effectively overcoming the shortcomings of single control methods.

[0110] Field efficacy trials 1. Test location and time The experiment was conducted from June to August 2025 at a cotton planting base in Liaocheng City, Shandong Province. The experimental field was flat, with convenient irrigation and drainage, and the soil fertility was moderate and evenly distributed. The cotton variety used was Lumianyan 37, and conventional field management was employed. No other pesticides or fungicides were applied during the entire experimental period.

[0111] 2. Experimental Design The experiment included 11 treatments: Examples 1-3, Comparative Examples 1-7, and a blank control (water treatment). Each treatment was replicated three times, resulting in a total of 33 plots arranged in a randomized block design. Each plot had an area of ​​30 m². 2 (5m×6m), with 1m wide isolation rows between the small sections to prevent drug drift interference.

[0112] Foliar spraying was applied during the cotton budding stage (June 20th), at the initial stage of cotton aphid infestation (approximately 500 aphids per 100 plants on average). For all examples and comparative examples 1-6, the fungicide was diluted with water at a ratio of 1:600 ​​by weight, stirred thoroughly, and then evenly sprayed using a backpack sprayer onto both sides of the cotton plant stems and leaves, as well as the buds and bolls, ensuring the leaves were moistened but not dripping. Comparative example 7 (conventional chemical agent group) used 10% imidacloprid wettable powder, diluted with water according to the conventional field recommended dosage (2.5g / mu of active ingredient, 25g / mu of formulation, dilution ratio of 2000). The blank control was sprayed with an equal volume of water. The amount of pesticide solution used per plot was approximately 3L.

[0113] 3. Survey Methodology At 3, 7, 14, and 21 days after application, a five-point sampling method was used in each plot, with two cotton plants surveyed at each point (a total of 10 plants per plot). The total number of live cotton aphids (including wingless adults and nymphs, but excluding winged aphids) was counted on the upper, middle, and lower parts of each cotton plant. Each survey was conducted by the same team of technicians to ensure consistency.

[0114] 4. Calculation Method The insect population reduction rate and control effectiveness are calculated using the following formulas (II) and (III): Insect population reduction rate (%) = (Number of live insects before application - Number of live insects after application) / Number of live insects before application × 100% (II) Control efficacy (%) = (Pest population reduction rate in the treated area - Pest population reduction rate in the control area) / (1 - Pest population reduction rate in the blank control area) × 100% (III) 5. Data Processing All data from three replicate plots, with 10 cotton plants per plot, were included in the statistical analysis. The final data is expressed as the arithmetic mean of the three replicates.

[0115] The field control efficacy of each treatment against cotton aphids is shown in Table 2.

[0116] Table 2. Field control efficacy of each treatment against cotton aphids (%) Note: "—" indicates that the insect population in the blank control group grew naturally and the control effect was not calculated.

[0117] As shown in Table 2, Examples 1-3 of this invention exhibited excellent control effects against cotton aphids under field conditions. Three days after application, the control efficacy of the conventional chemical pesticide group (Comparative Example 7) was the highest (82.67%), slightly higher than that of the examples of this invention (78.26%-81.53%), demonstrating the rapid-acting nature of chemical pesticides. However, seven days after application, the control efficacy of the examples of this invention (86.53%-89.37%) was on par with that of the conventional chemical pesticide group (85.43%); fourteen days after application, the control efficacy of the examples of this invention (92.17%-94.28%) was significantly higher than that of the conventional chemical pesticide group (78.56%); and twenty-one days after application, the control efficacy of the conventional chemical pesticide group had decreased to 68.34%, while the examples of this invention maintained a high level of 89.64%-91.87%. This result indicates that while conventional chemical agents, when used alone, have good rapid effects, their duration of action is short. In contrast, this invention achieves a perfect combination of rapid and long-lasting effects through the synergistic combination of chemical agents and microbial agents, resulting in a significantly better control effect 14-21 days after application compared to conventional chemical agents alone.

[0118] Comparative Example 1 (without Bacillus oryzae) showed a control efficacy of only 58.42% 14 days after application, Comparative Example 2 (without Flavobacterium granulosum) showed 73.56%, and Comparative Example 3 (without chemical insecticide synergists) showed only 52.86%, all lower than Example 1 (92.17%). Comparative Example 6 (existing technology CN202010846976.6) showed a control efficacy of only 65.37% 14 days after application, decreasing to 58.91% after 21 days. This indicates that while the existing microbial agent combination has some residual effect, its rapid action is insufficient, and its field stability is poor, making it difficult to meet the control needs during cotton aphid outbreaks.

[0119] cotton aphid reproduction inhibition test 1. Test Methods Healthy, wingless adult aphids reared indoors were individually inoculated into petri dishes containing fresh cotton leaves. Using a soft brush, the test solutions from Examples 1-3 and Comparative Examples 1-7 were evenly applied to the leaf surface (the test solutions were prepared in the same manner as in indoor toxicity testing), with sterile water treatment serving as a control. Each treatment contained 30 aphids, with three replicates (i.e., a total of 90 aphids per treatment). The entire experiment was independently replicated three times (i.e., a total of 270 aphids per treatment). Twenty-four hours after inoculation, all adult aphids were examined under a stereomicroscope and removed, leaving only newly hatched nymphs on each leaf. From the date of removal of adult aphids, the number of newly hatched nymphs on each leaf was observed and recorded daily under a stereomicroscope for 10 consecutive days. Simultaneously, the developmental status of the nymphs was recorded daily, with the first molt of the nymph marking the entry into the second instar. The time from the first molt of the nymph to the entry into the second instar was calculated as the developmental period.

[0120] 2. Calculation Method Aphid production per female = Total number of nymphs produced by a single female aphid within 10 days.

[0121] The aphid suppression rate is calculated using the following formula (IV): Aphid suppression rate (%) = (Aphid production per female in control - Aphid production per female in treatment) / Aphid production per female in control × 100% (IV) The survival rate of newly hatched nymphs = (total number of nymphs surviving within 10 days / total number of aphids laid by female aphids) × 100%.

[0122] The survival inhibition rate of offspring is calculated using the following formula (V): Farrowing survival inhibition rate (%) = (Control survival rate - Treatment survival rate) / Control survival rate × 100% (V); Nymphal development period = the number of days from the first emergence of nymphs to the development of the second instar.

[0123] 3. Data Processing All data from each of the three replicates (30 insects per replicate) were included in the final statistics (a total of 270 insects per treatment). The final data are expressed as the arithmetic mean of the three replicates.

[0124] The results of the inhibitory effects of each treatment on cotton aphid reproduction are shown in Table 3.

[0125] Table 3. Inhibitory effect of each treatment on cotton aphid reproduction As shown in Table 3, Examples 1-3 of this invention have a significant inhibitory effect on cotton aphid reproduction. The number of aphids per female decreased from 41.67 in the control to 16.12-18.23, with an aphid production inhibition rate of 56.28%-61.34%; the survival rate of newly hatched nymphs decreased from 96.78% in the control to 48.23%-52.36%, with a pupal survival inhibition rate of 45.89%-50.16%; and the nymphal development period was extended from 6.18 days in the control to 8.72-9.15 days.

[0126] The conventional chemical pesticide group (Comparative Example 7) showed an aphid production inhibition rate of only 32.01%, a larval survival inhibition rate of only 29.00%, and a nymphal development period of only 6.52 days. This indicates that although conventional chemical pesticides have a good rapid killing effect on adults, their ability to inhibit the reproduction of cotton aphids is limited, and they cannot fundamentally control the continuous growth of aphid populations. In contrast, this invention utilizes the specific interference effect of the secondary metabolites of Bacillus oryzae CGMCC No. 34320 on the cotton aphid reproductive system, combined with the synergistic effect of Flavobacterium granulosum, to achieve deep inhibition of cotton aphid reproduction at the intrinsic growth rate level. Its aphid production inhibition rate (56.28%-61.34%) was 24.27-29.33 percentage points higher than that of the conventional chemical pesticide group (32.01%).

[0127] The aphid inhibition rate of Comparative Example 1 (without Bacillus toyota) was only 21.92%, Comparative Example 2 (without Flavobacterium granulosum) was 40.57%, and Comparative Example 6 (existing technology) was 25.96%, all of which were far lower than the 56.28% of Example 1, further verifying the superiority of the three-level synergistic system of the present invention.

[0128] Cotton plant stress resistance induction test 1. Test Methods Cotton (Lu Mian Yan 37 variety) was grown in pots in an artificial climate chamber. The pots were 25cm in diameter, with 2 seedlings per pot, and routine water and fertilizer management was applied. When the seedlings reached the 4-leaf stage, the roots were drenched with the test pesticide solutions from Examples 1-3 and Comparative Examples 1-7 (Comparative Example 7 used a conventional concentration of imidacloprid solution), 50mL per pot. An equal volume of water served as a control. Each treatment consisted of 10 pots, with 3 replicates (30 pots per treatment). The entire experiment was independently replicated 3 times (90 pots per treatment).

[0129] Seven days after treatment, between 9:00 and 10:00 AM, the third fully expanded leaf from the top of the main stem of each cotton plant in each treatment group was collected. Two leaves were collected from each pot, and leaves from the same treatment were mixed together as one sample. The samples were quickly frozen with liquid nitrogen and then transferred to a -80°C freezer for storage.

[0130] Enzyme extraction: Weigh 0.5g of frozen leaves, add 5mL of pre-cooled 50mmol / L phosphate buffer (pH 7.0, containing 1% polyvinylpyrrolidone), grind thoroughly in an ice bath to form a homogenate, centrifuge at 4℃ and 12000r / min for 20min, and take the supernatant as crude enzyme solution for the determination of the activities of the following three enzymes.

[0131] Phenylalanine ammonia-lyase (PAL) activity assay: Take 0.1 mL of crude enzyme solution, add 1.9 mL of 50 mmol / L borate buffer (pH 8.8, containing 0.02 mol / L L-phenylalanine), react in a water bath at 37℃ for 60 min, then add 0.2 mL of 6 mol / L hydrochloric acid to terminate the reaction, and measure the absorbance at a wavelength of 290 nm. OD value is calculated hourly. 290 A change of 0.01 corresponds to one unit of enzyme activity (U). PAL activity is expressed as U / g FW.

[0132] Peroxidase (POD) activity assay: The guaiacol method was used. 0.1 mL of crude enzyme solution was added to 2.9 mL of reaction solution (50 mmol / L phosphate buffer, pH 7.0, containing 0.2% guaiacol and 0.3% H2O2). The absorbance was measured at 470 nm, recorded every 30 seconds for 3 minutes. The OD activity was measured per minute. 470 A change of 0.01 corresponds to one unit of enzyme activity (U). POD activity is expressed as U / gFW.

[0133] Superoxide dismutase (SOD) activity assay: The nitroblue tetrazolium (NBT) photoreduction method was used. 0.1 mL of crude enzyme solution was added to 3 mL of reaction solution (50 mmol / L phosphate buffer, pH 7.8, containing 13 mmol / L methionine, 75 μmol / L NBT, 2 μmol / L riboflavin, and 0.1 mmol / L EDTA). The reaction was carried out under 4000 lx light for 20 min. A light-protected control was used to zero the reaction, and the absorbance was measured at 560 nm. The amount of enzyme required to inhibit 50% of NBT photoreduction was defined as one unit of enzyme activity (U). SOD activity was expressed as U / g FW.

[0134] 2. Data Processing All data from each treatment with 3 replicates, each with 10 pots, and 3 independent replicates were included in the final statistics (a total of 90 independent sample data per treatment). The final data are expressed as the arithmetic mean of the 3 replicates.

[0135] 3. Experimental Results The effects of each treatment on the activity of defense enzymes in cotton leaves are shown in Table 4.

[0136] Table 4. Effects of each treatment on the activity of defense enzymes in cotton leaves (U / g FW) As shown in Table 4, the activities of the three defense enzymes, PAL, POD, and SOD, in cotton leaves treated in Examples 1-3 of this invention were significantly higher than those in the blank control and each comparative ratio. In Example 1, the activities of PAL, POD, and SOD were 68.34 U / g FW, 126.45 U / g FW, and 89.56 U / g FW, respectively, representing increases of 109.2%, 116.8%, and 112.5% ​​compared to the blank control.

[0137] It is noteworthy that the activities of the three defense enzymes (PAL 35.12, POD 62.78, SOD 46.23) in the conventional chemical agent group (Comparative Example 7) were essentially the same as those in the blank control (PAL 32.67, POD 58.34, SOD 42.15), showing no significant improvement. This indicates that conventional chemical agents do not induce or enhance the stress resistance of cotton plants, and long-term use may even cause physiological damage to the plants due to chemical stress. In contrast, this invention, through the stable colonization of Bacillus oryzae CGMCC No. 34320 in the rhizosphere of cotton, effectively activates the salicylic acid and jasmonic acid-mediated plant defense signaling pathways, achieving a significant improvement in the stress resistance of cotton plants—a functional advantage not possessed by conventional chemical agents.

[0138] The activities of all three enzymes in Comparative Example 1 (excluding Bacillus toyosumi) were lower than those in Example 1, indicating that Bacillus toyosumi is the core strain that plays a role in inducing stress resistance. Although the enzyme activities in Comparative Example 2 (excluding Flavobacterium granulosum) were higher than those in Comparative Example 1, they were still lower than those in Example 1, indicating that the biofilm formed by Flavobacterium granulosum provided a better colonization microenvironment for Bacillus toyosumi, thereby enhancing the induction of defensive enzyme expression.

[0139] Storage stability test The granular biocontrol agents prepared in Examples 1-3 were stored in sealed containers protected from light at room temperature (25±2℃) and 4℃, respectively. Viable bacterial counts were determined at 0, 3, 6, and 12 months of storage.

[0140] Method for determining viable cell count: Accurately weigh 10.0 g of granule sample under each storage condition, add 90 mL of sterile physiological saline (containing 0.05% Tween-80), and place on a shaker at 200 r / min for 30 min to ensure thorough dispersion of bacteria. Perform 10-fold serial dilutions of the sample suspension with sterile physiological saline, select an appropriate dilution, and spread 100 μL onto LB agar plates. Repeat each dilution three times. Incubate at 30℃ for 48 h, then count the colonies and convert the count to the viable cell count per gram of sample (CFU / g).

[0141] The viable cell survival rate is calculated using the following formula (VI): Viable bacteria survival rate (%) = (number of viable bacteria at a certain storage time point / initial number of viable bacteria) × 100% (VI).

[0142] Three stored samples were processed as replicates, and the average of the counts from three plates for each sample was taken. The entire experiment was independently repeated three times, and the final data was expressed as the arithmetic mean of the three replicates.

[0143] The viable cell survival rates of the microbial agents under various storage conditions are shown in Table 5.

[0144] Table 5. Viable bacterial survival rate (%) of the microbial agent under different storage conditions As shown in Table 5, the granular biocontrol agent prepared by this invention exhibits good storage stability. After 12 months of storage at room temperature, the viable bacterial survival rate of Examples 1-3 remained at 82.37%-84.23%; after 12 months of storage at 4℃, the survival rate was even higher at 89.64%. This indicates that the excipient system selected in this invention has a good protective effect on the activity of the bacterial strains, and the product has a shelf life of more than 12 months, meeting the needs of industrial production and commercial application.

[0145] In summary, the above experimental results fully demonstrate that the biocontrol agent containing *Bacillus tumefaciens* provided by this invention not only exhibits significant insecticidal, reproductive-inhibiting, and stress-inducing triple activities against cotton aphids, but also, through the synergistic effect of the two bacteria and the precise combination of the agent and the fungus, achieves a significantly better overall control effect than single strains or conventional chemical agents. Furthermore, this agent possesses excellent storage stability, effectively ensuring its effectiveness in field application. Therefore, this invention has significant application value and broad market prospects in the field of green control of cotton aphids.

[0146] It should be noted that the above embodiments are merely some preferred embodiments of the present invention, and not all embodiments. Obviously, based on the above embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

Claims

1. A biocontrol agent containing Bacillus terrestris, characterized in that, It includes an active ingredient and agriculturally acceptable excipients, wherein the active ingredient includes Bacillus toyota (… Bacillus toyonensis ), auxiliary biocontrol strains and low-toxicity chemical insecticide synergists; the auxiliary biocontrol strain is Flavobacterium granulosum ( Flavobacterium granuli The low-toxicity chemical insecticide synergist is one of imidacloprid, acetamiprid, and thiamethoxam.

2. The biocontrol agent containing Bacillus terrestris according to claim 1, characterized in that, The active ingredients contain a mass ratio of Bacillus oryzae, Flavobacterium granulosum, and low-toxicity chemical insecticide synergist of (3-5):(1-3):(0.5-1).

3. The biocontrol agent containing Bacillus terrestris according to claim 1, characterized in that, The Bacillus terrestris strain has the accession number CGMCC No. 34320 and the accession date is April 23, 2025. It is deposited at the China General Microbiological Culture Collection Center; the Flavobacterium granulosum strain has the strain number CGMCC No. 1.10125 and was purchased from the China General Microbiological Culture Collection Center.

4. The biocontrol agent containing Bacillus terrestris according to claim 1, characterized in that, The excipients include a carrier, a dispersant, a wetting agent, a binder, and a disintegrant, with a mass ratio of (50-65):(3-7):(1-3):(1-3):(3-7).

5. The biocontrol agent containing Bacillus terrestris according to claim 4, characterized in that, The carrier is at least one of diatomaceous earth, kaolin, bentonite, and talc; the dispersant is at least one of sodium lignosulfonate, sodium methylene bisnaphthalene sulfonate, and soapberry powder; the wetting agent is at least one of sodium alkyl sulfate, sodium alkyl sulfonate, and fatty alcohol polyoxyethylene ether; the binder is at least one of polyvinyl alcohol, sodium carboxymethyl cellulose, and starch; and the disintegrant is at least one of sodium bicarbonate, citric acid, and urea.

6. The biocontrol agent containing Bacillus terrestris according to claim 1, characterized in that, The mass ratio of the active ingredient to agriculturally acceptable excipients is (20-40):(60-80).

7. A method for preparing a biocontrol agent containing *Bacillus orientalis* as described in any one of claims 1-6, characterized in that, The preparation steps include the following: (1) Activation of strains: Bacillus oryzae and Flavobacterium granulosum were inoculated into the corresponding solid slant culture medium and activated by static incubation at constant temperature to obtain activated strains of the two strains; (2) Seed culture preparation: Each activated bacterial strain was inoculated into the corresponding liquid seed culture medium and cultured in a shaker to prepare Bacillus oryzae seed culture and Flavobacterium granulosum seed culture, respectively. (3) Fermentation culture: The two seed liquids were transferred to the fermentation culture medium of the fermenter according to the inoculation amount, and fermented by aeration and stirring to obtain Bacillus oryzae fermentation broth and Flavobacterium granulosum fermentation broth respectively. (4) Concentration and powdering: The bacterial cells of each fermentation broth were collected by centrifugation, washed with sterile water, and dried under low temperature and vacuum to prepare Bacillus oryzae powder and Flavobacterium granulosum powder, respectively. (5) Compounding of active components: Weigh out two kinds of bacterial powder and low-toxic chemical insecticide synergist according to the ratio, add them to a mixer and stir at low speed until uniform to obtain compound active components; (6) Formulation: The compound active components and excipients are mixed in proportion, and then crushed, granulated, sieved and granulated to prepare granular biocontrol agents.

8. The method for preparing the biocontrol agent containing Bacillus oryzae according to claim 7, characterized in that, In step (1), the activation culture temperature of Bacillus orientalis is 28-32℃ and the culture time is 24-36h; the activation culture temperature of Flavobacterium granulosum is 26-30℃ and the culture time is 36-48h; in step (2), the shaking speed is 180-220r / min, the culture time of Bacillus orientalis is 18-24h, and the culture time of Flavobacterium granulosum is 24-30h; in step (3), the fermentation temperature is 28-30℃, the dissolved oxygen concentration is 20%-30%, the fermentation time is 48-72h, and the inoculum size is 5%-10%v / v.

9. The method for preparing the biocontrol agent containing Bacillus oryzae according to claim 7, characterized in that, In step (5), the stirring speed is 300-500 r / min and the stirring time is 10-20 min.

10. The application of a biocontrol agent containing Bacillus oryzae as described in any one of claims 1-6 in controlling cotton aphids, inhibiting cotton aphid reproduction, and enhancing the stress resistance of cotton plants.

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