Bacillus thuringiensis, bacterial agent, composite insecticide and application thereof

Abstract

The application discloses a bacillus thuringiensis, a bacterial agent, a composite insecticide and application thereof, and belongs to the field of microorganisms and fermentation engineering. A bacillus thuringiensis BT001 is isolated from a diseased noctuid dead insect. The bacillus thuringiensis BT001 has high toxicity, a wide insecticidal spectrum, certain salt tolerance, heavy metal ion tolerance, alkali tolerance, high temperature tolerance and ultraviolet tolerance. The bacillus thuringiensis BT001 can tolerate various pesticide insecticides and can be used in combination with the pesticide insecticides to improve the insecticidal effect. The bacillus thuringiensis BT001 also has a growth promoting effect. Therefore, the bacillus thuringiensis BT001 can be used for preventing and treating agricultural insect pests and has important application prospects.

Description

Technical Field

[0001] This invention relates to the fields of microbiology and fermentation engineering, and particularly to the field of agricultural microbial control technology, specifically to a strain of Bacillus thuringiensis, a microbial agent, a compound insecticide, and their applications. Background Technology

[0002] In agricultural production, the damage caused by pests to crops can be divided into two categories based on the location of occurrence: above-ground and underground damage. Different types of pests have significantly different modes of damage and impacts, seriously threatening the safe production and stable yield of crops worldwide.

[0003] Lepidoptera pests are the main above-ground groups that damage crops, among which the fall armyworm (… Spodoptera frugiperda ), Spodoptera litura ( Spodoptera litura ) and Asian corn borer ( Ostrinia furnacalis Guené e Typical examples include the fall armyworm and the beet armyworm. Both are characterized by their omnivorous diet, wide host range, high reproductive rate, and ability to cause serious damage. They can infest various crops such as corn, soybeans, peanuts, and potatoes, with damage occurring during the larval stage. Taking the beet armyworm as an example, newly hatched larvae often congregate to feed on the leaf mesophyll. After the second instar, they disperse and gnaw on the lower epidermis and mesophyll, leaving only the upper epidermis. In the third instar, their appetite increases, causing the leaves to become mottled. After the fourth instar, they enter a voracious feeding stage, and in severe cases, they can completely destroy leaves, flowers, and fruits. The corn borer mainly damages gramineous crops such as corn and sorghum. Its larvae bore into the stems and rachis, damaging the internal tissues of the plant, easily leading to lodging and insufficient grain filling, thus causing severe yield reduction.

[0004] Plant parasitic nematodes are a major underground group that harms crops. These pests are important soil-borne harmful organisms, such as the southern root-knot nematode (…). Meloidogyne incognita This is particularly typical, and with the development of the greenhouse vegetable industry, its harm has become increasingly serious. The infection process of nematodes follows a clear pattern: under suitable conditions, the egg sacs hatch into second-instar larvae. The larvae swim in the soil to find host roots, and invade the interior by piercing tender tissues such as root tips with their stylets. They migrate to the vicinity of the vascular bundles to establish feeding sites. During this process, enzymes, effector proteins, and plant hormone analogs secreted by the nematode's salivary glands interfere with the plant's physiological metabolism, inducing root cells to form giant cells as a long-term nutrient source. This leads to root diseases such as root knots, rot, and necrosis, damaging the root structure and function, affecting the absorption and transport of water and nutrients, and ultimately causing the above-ground parts of the affected plants to show slow growth, yellowing, and stunting. In severe cases, the entire plant dies, causing significant yield losses.

[0005] Currently, the control of lepidopteran pests and nematodes generally adopts a comprehensive strategy with chemical control as the core and biological and physical control as supplementary measures. For chemical control, the focus for lepidopteran pests is on controlling the egg stage and early larval stages, commonly using 20% ​​chlorantraniliprole suspension at a dilution of 2000 times or 6% lambda-cyhalothrin suspension at a dilution of 2000 times. When applying pesticides, focus on the undersides of leaves, young stems, and heart leaves. For soil-borne nematodes, soil treatment can be carried out before sowing using furrow or hole application, or root irrigation can be used during the crop growing season. Commonly used pesticides include 5% abamectin microemulsion at a dilution of 1500 times or 41.7% fluopyram suspension at a dilution of 3000 times. For biological control, lepidopteran pests can be controlled in stages: during the egg stage, release parasitic natural enemies such as Trichogramma pyrenoidosa and Trichogramma purpureus; during the larval stage, release predatory natural enemies such as ladybugs and ladybugs. For nematodes, biocontrol agents such as *Paecilomyces lilacinus* and *Verticillium chrysogenum* are mainly used. These agents disrupt the egg and female structure of nematodes through spore germination and hyphal parasitism, thereby inhibiting their population growth. In terms of physical control, for chemotactic adult lepidopteran noctuid moths, sugar-vinegar solutions can be used to trap and kill them, or sex pheromones can be combined with sticky traps to capture males, reducing mating rates and population numbers. For nematodes, methods such as sunlight disinfection, high-temperature fumigation, hot water treatment of soil, or soaking of seedlings are commonly used to directly kill or weaken their activity, effectively reducing the nematode population in the field.

[0006] However, the aforementioned control measures face numerous bottlenecks: chemical pesticides easily lead to pesticide resistance in pests and pose a risk of pesticide residues, threatening food safety and the ecological environment; physical control is costly and difficult to implement on a large scale; biological control suffers from difficulties in the colonization of natural enemy insects and slow effectiveness. Against this backdrop, microbial pesticides have become the most feasible breakthrough in green pest control. Of particular note is Bacillus thuringiensis (Bt). Bacillus thuringiensis Bt (Bacillus thuringiensis) is a widely used microbial resource that has shown good results not only in the control of lepidopteran pests but also in the control of nematodes. Through the ecological regulation model of "using bacteria to control pests," it has become a core pillar of the green plant protection system, possessing environmental safety, targeted targeting, and sustainability, representing the scientific direction of modern agricultural pest control. However, with the use of Bt preparations, problems have emerged such as weak toxicity, narrow insecticidal spectrum, and unstable efficacy. Therefore, there is an urgent need to screen for new Bacillus thuringiensis species with strong toxicity, broad insecticidal spectrum, environmental tolerance, and high stability. Summary of the Invention

[0007] To address the problems existing in the prior art, the purpose of this invention is to provide a strain of Bacillus thuringiensis, a fungal agent, a compound insecticide, and their applications. The Bacillus thuringiensis strain of this invention has the advantages of high toxicity, a broad insecticidal spectrum, and strong environmental tolerance; it is also insensitive to many chemical agents and can be used in combination with chemical agents, exhibiting significant control effects against lepidopteran pests and nematodes.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] In a first aspect, the present invention provides a strain of Bacillus thuringiensis (Bt). Bacillus thuringiensis Bacillus thuringiensis BT001 was deposited on October 27, 2025, at the China Center for Type Culture Collection (CCTCC, Wuhan University, Wuhan, China), with accession number CCTCC NO: M 20252346 and taxonomic name: Bacillus thuringiensis BT001. Bacillus thuringiensis BT001.

[0010] The Bacillus thuringiensis of the present invention ( Bacillus thuringiensis Compared with existing reports of Bacillus thuringiensis, the main characteristics of BT001 are:

[0011] (1) High toxicity: The toxicity effect on the second instar larvae of the beet armyworm and the first- and second instar larvae of the fall armyworm can reach 100%;

[0012] (2) Broad insecticidal spectrum: It has insecticidal effects on above-ground pests such as beet armyworm, fall armyworm, and corn borer, as well as underground pests such as root-knot nematode.

[0013] (3) Stable efficacy: Bacillus thuringiensis BT001 has UVC resistance, is not sensitive to a variety of commonly used insecticides, does not produce antagonistic reactions, and has strong environmental tolerance.

[0014] (4) Synergistic effect: When Bacillus thuringiensis BT001 is used in combination with chemical agents such as matrine, diflubenzuron and thiazophos, its control effect can be synergistically improved, achieving a control effect of 1+1>2.

[0015] (5) Promote growth: It can increase the fresh weight of plants and the dry weight of roots, and promote healthy plant growth.

[0016] In summary, the Bacillus thuringiensis BT001 of this invention integrates multiple excellent properties and is a biocontrol bacterium with great application prospects.

[0017] A second aspect of the present invention provides a microbial agent containing the aforementioned Bacillus thuringiensis (Bt). Bacillus thuringiensis )BT001.

[0018] Preferably, the bacterial agent contains Bacillus thuringiensis (Bt). Bacillus thuringiensis BT001 exists in one or more of the following forms: cultured live bacteria, fermentation broth, bacterial suspension, and spore liquid.

[0019] Furthermore, the fermentation broth refers to the liquid produced after inoculating the microbial strain into a culture medium and culturing it for a period of time.

[0020] In some preferred embodiments of the present invention, the fermentation broth is prepared by the following method:

[0021] Bacillus thuringiensis ( Bacillus thuringiensis BT001 was inoculated into glucose-yeast medium and cultured at 37 ℃ and 180 r / min for 48 h with shaking.

[0022] More preferably, the glucose-yeast culture medium comprises: 5 g / L glucose, 5 g / L yeast extract, 5 g / L peptone, 1 g / L dipotassium hydrogen phosphate, 0.2 g / L magnesium sulfate, and 0.02 g / L manganese sulfate.

[0023] The bacterial suspension refers to the bacterial precipitate obtained by resuspending the precipitate after centrifugation of the fermentation broth.

[0024] The spore solution mentioned refers to the spores collected during the fermentation and culture of the strain and prepared with sterile water.

[0025] In a third aspect, the present invention provides the above-mentioned Bacillus thuringiensis (Bt). Bacillus thuringiensis Application of BT001 or fungicides in the preparation of insecticides.

[0026] In the above applications, the pests controlled by the insecticide include: beet armyworm, fall armyworm, corn borer and / or southern root-knot nematode.

[0027] A fourth aspect of the present invention provides a compound insecticide, said compound insecticide being composed of Bacillus thuringiensis (Bt). Bacillus thuringiensis BT001 is a compound of pesticide compounds;

[0028] The pesticide compound is selected from one or more of matrine, diflubenzuron, abamectin, thiazophos, and chlorantraniliprole.

[0029] Preferably, the compound insecticide is any one of the following (1)-(3):

[0030] (1) Bacillus thuringiensis-matrine compound insecticide: Bacillus thuringiensis ( Bacillus thuringiensis BT001 fermentation broth and matrine dilution were mixed at a volume ratio of 1:1.

[0031] (2) Bacillus thuringiensis-diflubenzuron compound insecticide: Bacillus thuringiensis ( Bacillus thuringiensis BT001 fermentation broth and dilute diflubenzuron solution were mixed at a volume ratio of 1:1.

[0032] (3) Bacillus thuringiensis-thiazophosphorus compound insecticide: Bacillus thuringiensis ( Bacillus thuringiensis It is a compound of BT001 fermentation broth and thiazophosphorus granules.

[0033] More preferably, in the above-mentioned Bacillus thuringiensis-matrine compound insecticide, Bacillus thuringiensis (Bt) Bacillus thuringiensis The spore concentration in the BT001 fermentation broth is 1×10⁻⁶. 6 CFU / mL - 1×10 7 CFU / mL; the concentration of matrine dilution is 5 mg / L-10 mg / L.

[0034] More preferably, in the above-mentioned Bacillus thuringiensis-diflubenzuron compound insecticide, Bacillus thuringiensis (Bt) Bacillus thuringiensis The spore concentration in the BT001 fermentation broth is 1×10⁻⁶. 7 CFU / mL; the concentration of the dilute diuretic was 250 mg / L.

[0035] More preferably, in the above-mentioned Bacillus thuringiensis-thiazophosphorus compound insecticide, Bacillus thuringiensis (Bt) Bacillus thuringiensis The viable cell count of BT001 fermentation broth was 5 × 10⁻⁶. 7 CFU / mL; Bacillus thuringiensis ( Bacillus thuringiensis BT001 fermentation broth and 10% thiazophosphorus granules were mixed at a ratio of 200mL:0.2g.

[0036] In a fifth aspect, the present invention provides the application of the above-mentioned compound insecticide in the control of plant pests; said plant pests are caused by the beet armyworm, fall armyworm, corn borer and / or southern root-knot nematode.

[0037] The beneficial effects of this invention are:

[0038] (1) This invention isolated a strain of Bacillus thuringiensis from dead diseased moths. Bacillus thuringiensis BT001, the Bacillus thuringiensis BT001 of the present invention is highly toxic and has a broad insecticidal spectrum. It has certain resistance to salt, heavy metal ions, alkali and high temperature, and also has a certain resistance to ultraviolet light. Moreover, it can withstand a variety of pesticides and insecticides.

[0039] (2) The Bacillus thuringiensis of the present invention ( Bacillus thuringiensis BT001 can be used in combination with pesticides and insecticides to synergistically improve insecticidal effects; the Bacillus thuringiensis BT001 of this invention also has a growth-promoting effect. Therefore, the Bacillus thuringiensis BT001 of this invention can be used for the control of agricultural pests and has broad application prospects. Attached Figure Description

[0040] Figure 1Diseased and dead cutworms; left is the beet armyworm, right is the fall armyworm.

[0041] Figure 2 : Colonies isolated from dead insects; In the figure, A is a typical colony morphology of suspected Bacillus thuringiensis; B is a microscopic image of the colony, showing typical parasporal crystals next to the spores.

[0042] Figure 3 Single colony of Bacillus thuringiensis.

[0043] Figure 4 Microscopic image of a single colony of Bacillus thuringiensis, showing typical parasporal crystals alongside the spores.

[0044] Figure 5 The toxicity of Bacillus thuringiensis BT001 against second instar larvae of the beet armyworm.

[0045] Figure 6 The results of susceptibility tests on four strains of Bacillus thuringiensis to chemical pesticides are shown. A represents the susceptibility test results of strain BT001 to eight chemical agents; B represents the susceptibility test results of strain NBL-B0207 to eight chemical agents; C represents the susceptibility test results of strain NBL-B0249 to eight chemical agents; and D represents the susceptibility test results of strain NBL-BS33 to eight chemical agents. In the figure, 1. imidacloprid; 2. diflubenzuron; 3. abamectin; 4. lambda-cyhalothrin; 5. azadirachtin; 6. matrine; 7. chlorantraniliprole; and 8. thiazophos are circled in red to indicate antagonistic reactions.

[0046] Figure 7 Results of ultraviolet light sensitivity test on four strains of Bacillus thuringiensis.

[0047] Figure 8 Microscopic images of Bacillus thuringiensis BT001 fermented on different culture media for 48 hours.

[0048] Figure 9 Microscopic image of Bacillus thuringiensis BT001; cultured in glucose-yeast medium for 48 hours (left), transferred to outdoor environment to induce spore shedding (middle), and finally completed shedding (right).

[0049] Figure 10 The control effect of Bacillus thuringiensis BT001 on the first instar larvae of fall armyworm on Chinese cabbage; in the figure, B is an enlarged view of the circled part in A.

[0050] Figure 11 The control effect of Bacillus thuringiensis BT001 on second instar larvae of the beet armyworm on Chinese cabbage. Detailed Implementation

[0051] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0052] To enable those skilled in the art to more clearly understand the technical solution of this application, the technical solution of this application will be described in detail below with reference to specific embodiments. Specific experimental conditions not specified in the embodiments are generally based on conventional conditions or conditions recommended by the reagent company; reagents, consumables, etc., used in the following embodiments, unless otherwise specified, can be obtained commercially. Wherein:

[0053] Nutrient broth (NB) medium: 10 g peptone, 3 g beef extract, 5 g sodium chloride, 1 L distilled water, pH natural, sterilized at 121 ℃ for 20 min.

[0054] NA medium: 10 g peptone, 3 g beef extract, 5 g sodium chloride, 20 g agar, 1 L distilled water, pH at rest, sterilized at 121 °C for 20 min.

[0055] LB medium: 10 g tryptone, 5 g yeast extract, 10 g sodium chloride, 1 L distilled water, pH 7, sterilized at 121 °C for 20 min.

[0056] Glucose-yeast medium: 5 g glucose, 5 g yeast extract, 5 g peptone, 1 g dipotassium hydrogen phosphate, 0.2 g magnesium sulfate, 0.02 g manganese sulfate, 1 L distilled water, pH 7. Glucose was sterilized separately at 115 °C for 15 min, and the remaining components were sterilized at 121 °C for 20 min. The sterilized glucose was then added to the sterilized remaining components under aseptic conditions.

[0057] Economical fermentation medium: 20 g soybean meal powder, 10 g corn starch, 1 g dipotassium hydrogen phosphate, 2 g calcium carbonate, 1 L distilled water, pH 7, sterilized at 121 ℃ for 20 min.

[0058] Bacillus thuringiensis NBL-B0207, NBL-B0249, and NBL-BS33 were all provided by the strain resource bank of the Science and Technology Innovation Center of Shandong Bilan Biotechnology Co., Ltd.

[0059] Imidacloprid, diflubenzuron, abamectin, lambda-cyhalothrin, azadirachtin, matrine, chlorantraniliprole, and thiazophos are all commercially available products.

[0060] Example 1: Isolation and Identification of Strains

[0061] 1. Isolation of bacterial strains:

[0062] Biocontrol bacteria were isolated and screened from dead diseased noctuid moths raised in the laboratory. The surface of the dead insects turned black (as shown in the image). Figure 1 As shown in the image, the infected dead insects were first disinfected with 5% sodium hypochlorite for 3 minutes, then with 75% alcohol for 30 seconds, followed by rinsing with sterile water for 5 minutes, 4 minutes, and 3 minutes respectively. Finally, the cleaned insects were placed in a sterile mortar, and a small amount of sterile water was added to cover the insects. The mortar was then ground thoroughly. After thorough grinding, 1 mL of the ground insects was placed in 99 mL of 0.9% physiological saline and shaken in a shaker at 30°C and 180 rpm for 1 hour. The shake flask was then removed and serially diluted with 0.9% physiological saline (to 10). -3 10 -4 Finally, take 100 μL of the diluted solution and spread it evenly on NA medium plates, and incubate at 37 ℃ upside down for 3 days.

[0063] The screening results of biocontrol bacteria in dead diseased cutworms are as follows: Figure 2 As shown, 10 -4 Multiple single colonies appeared on the plates coated with the diluted solution, including single colonies similar to Bacillus thuringiensis. Microscopic examination of the single colonies revealed parasporal crystals next to the spores. It was preliminarily inferred that the bacterium was Bacillus thuringiensis, named BT001, and further identification work was carried out.

[0064] 2. Identification of the strain:

[0065] 2.1 Colony morphology identification

[0066] Single colonies of strain BT001 were streaked onto NA agar plates. The plates were then inverted and incubated at 37°C for 24 hours. After incubation, grayish-white to milky-white, opaque colonies with a dry, rough surface were formed. The colony edges were irregularly serrated or diffused, with a diameter of 2-5 mm. The colonies were relatively hard and easily picked up. Figure 3 ).

[0067] 2.2 Identification of bacterial cell and spore morphology

[0068] Gram staining revealed that strain BT001 was Gram-positive (G). + Large bacilli, arranged in short chains or as single cells, with a cell size of approximately 1.0-1.2 μm × 3-5 μm. During the vegetative growth phase, the ends of the cells are blunt and rounded, and the cytoplasm is uniformly stained. After entering the sporulation phase, a colorless and transparent area (spore precursor) appears in the center of the cell. After culturing at 37°C for 48 hours, the spores begin to detach. The spores are elliptical, terminal or nearly terminal, and slightly smaller in diameter than the cell. After staining with crystal violet, the spores are observed to be hollow under an optical microscope, surrounded by purple rhomboid parasporal crystals. Figure 4 ).

[0069] 2.3 Molecular biological identification

[0070] The strain BT001 was identified by sequencing based on its 16S rRNA gene sequence. The genome of strain BT001 was extracted using universal primers for the 16S rRNA gene sequence (16S-1 sequence: 5'-AGAGTTTGATCCTGGCTCAG-3') (16S-2 sequence: 5'-CGGCTACCTTGTTACGAC-3'). The genome was sent to Sangon Biotech Co., Ltd. for sequencing. The 16S rRNA gene sequence of strain BT001 is shown in SEQ ID NO.1.

[0071] Analysis using Ezbiocloud revealed that strain BT001 belongs to the genus Bacillus (Bacillus). Bacillus ), which is related to Bacillus thuringiensis (Bacillus thuringiensis) SXFB3 (99.93%), Bacillus thuringiensis ( Bacillus thuringiensis B62 (99.86%) and Bacillus thuringiensis ( Bacillus thuringiensis ZG608 (99.79%) shares the highest 16S rRNA gene sequence similarity.

[0072] In summary, based on the results of colony morphology identification, cell and spore morphology identification, and molecular biological identification, strain BT001 was identified as Bacillus thuringiensis (Bt). Bacillus thuringiensis ).

[0073] Example 2: Comparative Test of the Virulence of Biocontrol Bacteria

[0074] 1. Test Methods

[0075] Bacillus thuringiensis BT001 and Bacillus thuringiensis NBL-B0207, NBL-B0249, and NBL-BS33, screened in Example 1, were used as test biocontrol bacteria for a comparative toxicity test to examine their toxicity against second instar larvae of the beet armyworm (Spodoptera litura). The characteristics of first to sixth instar larvae of the beet armyworm are shown in Table 1.

[0076] Table 1: Characteristics of 1st-6th instar larvae of the beet armyworm

[0077]

[0078] Fermentation broth preparation: Single colonies of the four Bacillus thuringiensis strains were picked and inoculated into nutrient broth (NB) medium. The cultures were incubated at 37 ℃ and 180 r / min with shaking for 48 h. The culture was terminated after microscopic examination showed that 80% of the spores had detached. The viable cell count of the fermentation broth for each of the four Bacillus thuringiensis strains was adjusted to 5 × 10⁴ cells / mL using sterile water. 7 CFU / mL.

[0079] The feeding method involved mixing 3 mL of fermentation liquid into every 10 g of artificial feed and stirring thoroughly to ensure the feed fully absorbed the fermentation liquid before feeding. Second-instar larvae of the beet armyworm were used as the test insect source. The toxicity test was conducted in disposable plastic petri dishes. The specific experimental protocol is as follows:

[0080] The experiment included six treatments: Treatment 1: Bacillus thuringiensis BT001 fermentation broth mixed into the feed; Treatment 2: Bacillus thuringiensis NBL-B0207 fermentation broth mixed into the feed; Treatment 3: Bacillus thuringiensis NBL-B0249 fermentation broth mixed into the feed; Treatment 4: Bacillus thuringiensis NBL-BS33 fermentation broth mixed into the feed; Treatment 5: An equal volume of 1000-fold diluted abamectin (5% active ingredient) mixed into the feed as a positive control; Treatment 6: An equal volume of NB medium mixed into the feed as a negative control. Each treatment consisted of 20 larvae, replicated three times. After treatment, the larvae were reared at 26±1℃, relative humidity 60-70%, and photoperiod 16:8 (L:D) for 7 days. The number of larval deaths was recorded, and the mortality rate was calculated using the following formula:

[0081] Mortality rate = (Number of dead insects / Total number of tested insects) × 100%

[0082] 2. Test Results:

[0083] The results of the four Bacillus thuringiensis strains on the toxicity of second-instar larvae of the beet armyworm are shown in Table 2.

[0084] Table 2: Virulence effects of four Bacillus thuringiensis strains on second-instar larvae of the beet armyworm.

[0085]

[0086] The results showed that the mortality rate of larvae in the negative control was 0%, while the mortality rate of larvae in the positive control was 100%. Strain BT001 exhibited a high toxicity against second-instar larvae of the beet armyworm; after 5 days of treatment with strain BT001, all larvae in the culture dish died. Figure 5 The mortality rate was as high as 100% in one strain; however, the mortality rate of larvae treated with the other three Bacillus thuringiensis strains NBL-B0207, NBL-B0249, and NBL-BS33 was less than 70%. Therefore, through in vitro toxicity tests on four Bacillus thuringiensis strains, it was preliminarily determined that strain BT001 had the best toxicity effect on noctuid moth larvae and can be further explored as a highly effective biocontrol strain.

[0087] Example 3: Sensitivity test of Bacillus thuringiensis to chemical pesticides

[0088] 1. Test Methods

[0089] Bacillus thuringiensis BT001 and Bacillus thuringiensis NBL-B0207, NBL-B0249, and NBL-BS33, screened in Example 1, were used as test biocontrol bacteria. Fermentation broths of four Bacillus thuringiensis strains were prepared according to the method in Example 2. The viable cell counts of the fermentation broths of the four Bacillus thuringiensis strains were adjusted to 5 × 10⁴ cells / mL using sterile water. 7 CFU / mL.

[0090] The pesticides tested were: 1. Imidacloprid (70% active ingredient content), 2. Diflubenzuron (25% active ingredient content), 3. Abamectin (5% active ingredient content), 4. Lambda-cyhalothrin (10% active ingredient content), 5. Azadirachtin (0.5% active ingredient content), 6. Matrine (0.5% active ingredient content), 7. Chlorantraniliprole (20% active ingredient content), and 8. Thiazolylphosphonate (3% active ingredient content). All pesticides were diluted 1000 times for testing.

[0091] First, 100 μL of fermentation broth from each of the four Bacillus thuringiensis strains was evenly spread onto NA medium plates. Double-layered sterile filter paper was evenly distributed on the spread medium, and 5 μL of pesticide was dropped onto each filter paper. This process was repeated three times. The plates were then incubated at 37°C for 24 h. The presence or absence of a clear zone on the plates was then observed to determine whether an antagonistic reaction occurred.

[0092] 2. Test Results

[0093] Strain BT001 is resistant to all eight chemical agents used (1. imidacloprid; 2. diflubenzuron; 3. abamectin; 4. lambda-cyhalothrin; 5. azadirachtin; 6. matrine; 7. chlorantraniliprole; 8. thiazophos). Figure 6 As shown in A), there is no antagonistic reaction, and it can be compounded with these 8 pesticides to form products or mixed for field application on crops.

[0094] Strain NBL-B0207 is partially intolerant to the eight chemical agents used (1. imidacloprid; 2. diflubenzuron; 3. abamectin; 4. lambda-cyhalothrin; 5. azadirachtin; 6. matrine; 7. chlorantraniliprole; 8. thiazophos). Figure 6 As shown in B), it has antagonistic reactions with 1. imidacloprid, 3. abamectin, and 7. chlorantraniliprole. It is not suitable to be compounded with these three pesticides to form products, and it is not recommended to mix them together when applying them in the field.

[0095] NBL-B0249 is partially intolerant to the eight chemical agents used (1. imidacloprid; 2. diflubenzuron; 3. abamectin; 4. lambda-cyhalothrin; 5. azadirachtin; 6. matrine; 7. chlorantraniliprole; 8. thiazophos). Figure 6(As shown in C), it has antagonistic reactions with 1. imidacloprid, 2. diflubenzuron, and 7. chlorantraniliprole, and is not suitable for compounding with these three pesticides to form products. It is not recommended to mix them together when applying them in the field.

[0096] NBL-BS33 is partially intolerant to some of the eight chemical agents used (1. imidacloprid; 2. diflubenzuron; 3. abamectin; 4. lambda-cyhalothrin; 5. azadirachtin; 6. matrine; 7. chlorantraniliprole; 8. thiazophos). Figure 6 As shown in D), it has antagonistic reactions with 1. imidacloprid, 2. diflubenzuron, 4. lambda-cyhalothrin, and 7. chlorantraniliprole. It is not suitable to be compounded with these four pesticides to form products, and it is not recommended to mix them together when applying them in the field.

[0097] Example 4: Tolerance test of Bacillus thuringiensis

[0098] 1. Test Methods

[0099] Bacillus thuringiensis BT001 and Bacillus thuringiensis NBL-B0207, NBL-B0249, and NBL-BS33, screened in Example 1, were used as test biocontrol bacteria. Fermentation broths of four Bacillus thuringiensis strains were prepared according to the method in Example 2. The fermentation broths were incubated in an 80°C water bath for 30 min, and then diluted with sterile water to obtain spore solutions with a final spore count of 550 CFU / mL. These spore solutions were used as the treatment solutions for the experiments described later.

[0100] 1.1 Experiments with different NaCl contents

[0101] Four strains of Bacillus thuringiensis spores (0.2 mL each) were spread onto NaCl agar plates containing 0% (control), 1%, 5%, 10%, 15%, and 20% NaCl, respectively. After removing error cells and dead cells, each plate contained approximately 100 viable spores, resulting in six treatment groups with three replicates per group. The plates were incubated at 37°C for 24 hours, and the growth of the strains was observed. The survival rate (survival rate = average colony count in each salinity treatment group / average colony count in the control group × 100%) was calculated by counting the colonies on each plate to assess the effect of salt stress on the growth of each strain.

[0102] 1.2 Cadmium ions with different contents (Cd) 2+ )test

[0103] Spray 0.2 mL of spores from each of the four Bacillus thuringiensis strains onto Cd. 2+Six treatment groups were established using NA medium plates with concentrations of 0 g / L (control), 0.1 g / L, 0.2 g / L, 0.4 g / L, 0.8 g / L, and 1.0 g / L. After removing error cells and dead cells, approximately 100 viable spores were observed on each plate. Each treatment group had three replicates. The plates were incubated at 37°C for 24 hours, and the growth of the bacterial strains was observed. The survival rate was calculated by counting the number of colonies on each plate (survival rate = Cd / spore concentration). 2+ The average colony count of the concentration treatment group / the average colony count of the control group (×100%) was used to evaluate different Cd concentrations. 2+ Inhibitory effect of concentration on the growth of each strain.

[0104] 1.3 Different pH Tests

[0105] 0.2 mL of spore suspension from each of the four Bacillus thuringiensis strains was spread onto NA agar plates at pH values ​​of 5, 7 (control), 9, and 12, respectively. Error cells and dead cells were removed. Each plate contained approximately 100 viable spores, resulting in four treatment groups with three replicates per treatment group. The plates were incubated at 37°C for 24 hours, and the growth of the strains was observed. The inhibitory effect of different pH values ​​on the growth of each strain was evaluated by counting the number of colonies on each plate and calculating the survival rate (survival rate = average colony count in each pH treatment group / average colony count in the control group × 100%).

[0106] 1.4 Tests at different temperatures

[0107] 0.2 mL of spore suspension from each of the four Bacillus thuringiensis strains was spread onto NA agar plates. Error cells and dead cells were removed, resulting in approximately 100 viable spores per plate. Three temperature gradients were used for treatment: 90℃, 80℃, and 70℃, with treatment times of 5 minutes and 10 minutes at each temperature. A control group of untreated spore suspensions was also included. A total of seven treatment groups were established, with three replicates for each group. After water bath treatment at the corresponding temperature, the spore suspensions were immediately spread onto NA plates. All plates were incubated at 37℃ for 24 hours. Colony counts were performed, and the survival rate was calculated (survival rate = average colony count in each heat-treated group / average colony count in the control group × 100%) to evaluate the effect of different temperature-time combinations on the survival rate of each strain.

[0108] 1.5 Ultraviolet Irradiation for Different Durations

[0109] Two mL of spore suspension from each of the four Bacillus thuringiensis strains was spread onto NA agar plates, removing any errors or dead cells. Each plate contained approximately 1000 viable spores. The plates were then irradiated vertically at a distance of 30 cm from a 30 W low-pressure mercury lamp (254 nm wavelength) for 0 min (control), 1 min, 5 min, 15 min, 30 min, and 60 min, for a total of six treatment groups. Each treatment group was replicated in triplicate. The plates were then incubated at 37 °C for 24 h, and the growth of the strains was observed.

[0110] 2. Test Results

[0111] 2.1 Tests with different NaCl contents

[0112] The survival rates of four Bacillus thuringiensis strains treated with different amounts of NaCl are shown in Table 3.

[0113] Table 3: Survival rates (%) of four test bacteria treated with different NaCl concentrations

[0114]

[0115] Based on the salt tolerance test data, all four Bacillus thuringiensis strains exhibited high survival rates within a salt concentration range of 1% to 5%, with a regular decreasing trend as the salt concentration increased. Significant differences in salt tolerance were observed among the strains, with BT001 showing the strongest salt tolerance. Starting from 10% NaCl, except for BT001 which showed a low survival rate (4.27%), the other three strains failed to grow at all. The results indicate that strain BT001 exhibits relatively superior salt tolerance.

[0116] 2.2 Different Cd contents 2+ test

[0117] Different Cd contents 2+ The survival rates of the four Bacillus thuringiensis strains are shown in Table 4.

[0118] Table 4: Cd content at different levels 2+ Survival rate (%) of the four test strains.

[0119]

[0120] Based on the effects of four Bacillus thuringiensis strains on cadmium ions (Cd) 2+ The results of the tolerance test showed that the survival rate of each strain increased with Cd. 2+ The concentration increases and then decreases significantly. This is true in the range of 0.1–0.2 g / L Cd. 2+Within the specified concentration range, all strains maintained a high survival rate (68.65%–93.04%), demonstrating a certain degree of tolerance. However, when the concentration increased to 0.4 g / L, the survival rate dropped sharply to 33.66%–54.97%. At a concentration of 0.8 g / L, the survival rate generally fell below 13%, indicating that this concentration severely inhibited the growth of the strains. Up to 1.0 g / L Cd... 2+ Under these conditions, the survival rate of all strains was zero, indicating that this concentration completely inhibited their growth. Among them, strain BT001 showed the best survival rate at concentrations of 0.1-0.8 g / L Cd. 2+ They all exhibited the highest tolerance at the specified concentrations, demonstrating relatively stronger potential for heavy metal resistance.

[0121] 2.3 Tests at different pH levels

[0122] The survival rates of the four Bacillus thuringiensis strains treated with different pH values ​​are shown in Table 5.

[0123] Table 5: Survival rate (%) of the four test strains under different pH treatments

[0124]

[0125] Based on the tolerance tests of four Bacillus thuringiensis strains under different alkaline pH conditions, all four strains showed high survival rates (85.43%–96.08%) at pH 9. When the environment reached the strongly alkaline pH 12, only strain BT001 retained trace growth (3.26%), while the other strains almost failed to survive, indicating that pH 12 was close to or reached their lethal threshold. Overall, strain BT001 showed relatively strong tolerance to both acidic and alkaline environments.

[0126] 2.4 Tests at different temperatures

[0127] The survival rates of the four Bacillus thuringiensis strains treated at different temperatures are shown in Table 6.

[0128] Table 6: Survival rates (%) of four test bacteria treated at different temperatures

[0129]

[0130] Based on the results of four Bacillus thuringiensis strains under different heat stress treatments, short-term high-temperature (90°C) treatment significantly reduced the survival rate of all four strains, while prolonged treatment time further exacerbated the heat-induced lethal effect, especially at 80°C and 90°C where time sensitivity was more pronounced. In contrast, heat treatment at 70°C had little impact on the growth of all strains. Under harsh high-temperature conditions (e.g., 90°C treatment for 10 minutes), strain BT001 exhibited the strongest heat tolerance and the highest survival rate (43.09%), suggesting that its spore structure may have better thermal stability.

[0131] 2.5 Ultraviolet Irradiation for Different Durations

[0132] The growth status of the four Bacillus thuringiensis strains is as follows: Figure 7 As shown, the four rows from top to bottom represent strains BT001, NBL-B0207, NBL-B0249, and NBL-BS33. Strain BT001 showed complete colony growth on plates after 0-5 minutes of 254nm UV irradiation, with no significant difference among the strains. After 15, 30, and 60 minutes of UV irradiation, some strains still survived. The number of surviving colonies on plates from strain BT001 was significantly higher than the other three Bacillus thuringiensis strains (NBL-B0207, NBL-B0249, and NBL-BS33), indicating stronger UVC resistance. This result confirms that BT001 possesses excellent DNA damage repair capabilities and cell structure tolerance. Although the stress mechanisms of natural sunlight (including UVA / UVB) differ from those of UVC, this intrinsic resistance suggests that this strain may have superior survival potential under natural conditions.

[0133] In summary, Bacillus thuringiensis BT001 not only exhibits the strongest toxicity but also demonstrates good tolerance to various pesticides, while also possessing certain resistance to heat, salinity, heavy metals, and UVC stress. Therefore, Bacillus thuringiensis BT001 was selected as a biocontrol bacterium for subsequent experiments, and it was biodeposited at the China Center for Type Culture Collection (CCTCC) using patent procedures, with accession number CCTCC NO: M 20252346.

[0134] Example 5: Optimization of fermentation medium for Bacillus thuringiensis BT001

[0135] 1. Test Methods

[0136] Single colonies of strain BT001 were picked using an inoculation loop and inoculated into three media: LB medium, glucose-yeast medium, and economical fermentation medium. The cultures were incubated at 37°C with shaking at 180 rpm. Samples were taken at 24 h and 48 h, and sporulation and spore shedding were examined under a microscope using crystal violet staining. If the spore shedding rate was >90%, the fermentation broth was subjected to serial dilution plating for viable cell and spore counts, with three replicates to ensure data reliability.

[0137] 2. Test Results

[0138] The results are as follows Figures 8-9 As shown, the results indicate that the culture medium type significantly affects the sporulation capacity and spore shedding rate of strain BT001. In LB medium, strain BT001 showed no sporulation after 48 h; in the economical fermentation medium, sporulation began after 24 h, but no spores shed after 48 h or even after 2 days of static incubation. In contrast, the glucose-yeast medium performed best, with normal sporulation and a spore shedding rate as high as 90% after 2 days of static incubation. Corresponding counting results confirmed that the viable cell count in the glucose-yeast medium after 48 h was 2.12 × 10⁻⁶. 8 CFU / mL) and spore count (1.50 × 10⁻⁶) 8 The CFU / mL ratio was significantly higher than that of other culture media, highlighting its comprehensive advantages in promoting efficient culture, sporulation, and spore shedding of strain BT001.

[0139] Example 6: In vitro toxicity test of Bacillus thuringiensis BT001 against fall armyworm larvae of different instars

[0140] 1. Test Methods

[0141] Based on the preferred results of Example 5, the fermentation broth of strain BT001 was prepared using glucose-yeast medium (37℃, 180r / min shaking culture for 48h), and the viable cell count in the fermentation broth was adjusted to 5×10⁻⁶. 7 CFU / mL; for later use.

[0142] The characteristics of fall armyworm larvae from the 1st to 6th instar are shown in Table 7:

[0143] Table 7: Characteristics of Fall Armyworm Larvae (1st-6th Instars)

[0144]

[0145] Note: The key identification features of the fall armyworm are the inverted Y-shaped markings on its head and the four black dots arranged in a square at the end of its abdomen.

[0146] Two methods are used to treat fall armyworm larvae:

[0147] Spraying treatment involves using a spray bottle to evenly spray the fermentation liquid onto the surface of the insects, ensuring that a liquid film is formed without dripping and that there is no standing water in the rearing box.

[0148] The mixing process is the same as in Example 2.

[0149] The experiment was conducted with 20 larvae per treatment for 1st and 2nd instar larvae, and 12 larvae per treatment for 3rd, 4th, 5th, and 6th instar larvae. Each treatment was replicated three times. A negative control was included for each instar, consisting of an equal volume of pure water. After treatment, all larvae were kept at 26±1℃, relative humidity of 60-70%, and a photoperiod of 16:8 (L:D). They were observed for 7 days, and the number of deaths in the treatment group and the negative control group was recorded and the mortality rate was calculated. The corrected mortality rate was then calculated using a formula.

[0150] Corrected mortality rate (%) = (treatment mortality rate - negative control mortality rate) / (1 - negative control mortality rate) × 100%.

[0151] 2. Test Results

[0152] The toxic effects of different treatment methods of fermentation broth on fall armyworm larvae are shown in Table 8.

[0153] Table 8: Corrected mortality rate (%) of fall armyworm larvae at different instars under different treatments of fermentation broth from strain BT001

[0154]

[0155] The experimental results showed that the fermentation broth of strain BT001 cultured for 48 h was toxic to fall armyworm larvae from the 1st to the 6th instar, and the toxic effect weakened with increasing larval age. The toxic effect was particularly significant on larvae below the 3rd instar, with a corrected mortality rate of ≥94% for larvae from the 1st to the 3rd instar under both treatments.

[0156] Example 7: In vitro toxicity test of Bacillus thuringiensis BT001 against larvae of different instars of Spodoptera litura.

[0157] 1. Test Methods

[0158] Based on the preferred results of Example 5, the fermentation broth of strain BT001 was prepared using glucose-yeast medium (37℃, 180r / min shaking culture for 48h), and the viable cell count in the fermentation broth was adjusted to 5×10⁻⁶. 7 CFU / mL, for later use.

[0159] Two methods, spraying and feeding, were used to treat larvae of different instars of the beet armyworm, with the specific treatment methods being the same as in Example 6. The experiment was conducted with 20 larvae per treatment for 1st and 2nd instar larvae, and 12 larvae per treatment for 3rd, 4th, 5th, and 6th instar larvae. Each treatment was repeated three times. A negative control was provided for each instar, consisting of an equal volume of pure water. After treatment, all larvae were kept at 26±1℃, relative humidity 60-70%, and photoperiod 16:8 (L:D) for 7 days. The number of deaths in the treatment and control groups was recorded, and the mortality rate was calculated. The corrected mortality rate was then calculated using a formula.

[0160] Corrected mortality rate (%) = (treatment mortality rate - negative control mortality rate) / (1 - negative control mortality rate) × 100%.

[0161] 2. Test Results

[0162] The toxic effects of different treatment methods of fermentation broth on the larvae of the beet armyworm are shown in Table 9.

[0163] Table 9: Corrected mortality rate (%) of Spodoptera litura larvae at different instars after different treatments of fermentation broth from strain BT001

[0164]

[0165] The experimental results showed that the fermentation broth of strain BT001, cultured for 48 h, exhibited toxic activity against 1st to 6th instar larvae of the beet armyworm. Under both treatments, the toxicity rate of strain BT001 against 1st to 3rd instar larvae was ≥91%, with the toxicity decreasing with increasing instar. However, the mortality rate of 6th instar larvae in the mixed feeding treatment was abnormally high. It is speculated that this may be because the beet armyworm enters a voracious feeding phase from the 5th to the 6th instar, rapidly increasing in size and significantly increasing its food intake, leading to a correspondingly higher dose of BT001 fermentation broth ingested, thus causing a sudden increase in mortality.

[0166] Example 8: Toxicity determination of Bacillus thuringiensis BT001 and its compound matrine against fall armyworm

[0167] 1. Test Methods

[0168] 1.1 Preparation of the test fermentation broth:

[0169] Based on the preferred results of Example 5, the fermentation broth of strain BT001 was prepared using glucose-yeast medium (37℃, 180 r / min shaking culture for 48 h). 5 mL of the fermentation broth was first taken to determine the spore concentration, which was found to be 1.5 × 10⁻⁶. 8 CFU / mL, for later use.

[0170] 1.2 Processing Group Settings

[0171] The treatment groups are as follows:

[0172] Treatment group 1 (T1): This group consists of different dilutions of strain BT001, specifically, fermentation broth of strain BT001 diluted with pure water to obtain four spore concentrations: 1×10⁻⁶. 5 CFU / mL, 1×10 6 CFU / mL, 1×10 7 CFU / mL, 1×10 8 CFU / mL. Used to calculate the LC50 of the fermentation broth of strain BT001. 50 .

[0173] Treatment Group 2 (T2): Different dilution groups of 0.5% matrine single agent, that is, 0.5% matrine was diluted with pure water to three concentrations, namely 500, 1000, and 1500 times, respectively, with the corresponding final concentrations of 10 mg / L. -1 5 mg•L -1 3.33 mg•L -1 Used to calculate the LC50 of matrine. 50 .

[0174] Based on the compatibility of Bacillus thuringiensis and matrine between treatment groups 1 and 2, and the results of the toxicity test of the two agents against fall armyworm larvae (LC50), 50 Then, set the ratio of the compound drug group, specifically: first, dilute 0.5% matrine with pure water to the corresponding final concentration of 10 mg / L. -1 5 mg•L -1 Next, take an equal volume of BT001 fermentation broth and mix it according to the required BT001 spore concentration in each of the following treatments. All mixtures are in a 1:1 ratio. Mix well before use.

[0175] Treatment group 3 (T3): 50 mL spore count 1×10 7 Fermentation broth of strain BT001 with CFU / mL and 50mL of a solution containing 10mg•L -1 A mixture of matrine;

[0176] Treatment group 4 (T4): 50 mL spore count 1×10 7 Fermentation broth of strain BT001 with CFU / mL and 50mL of a solution containing 5mg•L -1 A mixture of matrine;

[0177] Treatment group 5 (T5): 50 mL spore count 1×10 6 Fermentation broth of strain BT001 with CFU / mL and 50mL of a solution containing 10mg•L -1 Matrine mixture;

[0178] Treatment group 6 (T6): 50 mL of the fermentation broth of strain BT001 with a spore count of 1×10 6 CFU / mL was mixed with 50 mL of matrine at a concentration of 5 mg•L -1 .

[0179] 1.3 Virulence determination method

[0180] The poisoning test was carried out by the method of mixing food for feeding. The 3rd instar Spodoptera frugiperda larvae were used as the test insect source. 3 mL of the above-mentioned treatment solution was added to every 10 g of artificial feed, and it was stirred evenly. After the feed completely absorbed the treatment solution, it was fed. The feed mixed with the medicament was evenly placed in a 12-well plate, and then the 3rd instar Spodoptera frugiperda larvae were put in. There were 30 test insects in each treatment, and it was repeated three times. It was raised under the conditions of 26±1°C, relative humidity of 60 - 70%, and photoperiod of 16:8 (L:D). After the treatment, it was continuously observed for 7 days, and the mortality rate was recorded.

[0181] The LC 50 , 95% confidence interval, and virulence regression equation of the single medicament and the mixed treatment solution were calculated by SPSS software, and the co-toxicity coefficient (CTC) was calculated according to the Sun-Johnson method;

[0182] Virulence index (TI) = standard medicament LC 50 / test medicament LC 50 [[ID=2尽]]×100 (the standard medicament refers to the fermentation broth of strain BT001, and the test medicament is 0.5% matrine);

[0183] Actual measured virulence index of the mixture (ATI) = standard medicament LC 50 / mixed medicament LC 50 ×100 (the mixed medicament refers to T3 - T6);

[0184] Theoretical virulence index of the mixture (TTI) = virulence index of medicament A × percentage content of medicament A in the mixture + virulence index of medicament B × percentage content of medicament B in the mixture;

[0185] Co-toxicity coefficient (CTC) = (actual measured virulence index of the mixed medicament (ATI)) / (theoretical virulence index of the mixed medicament (TTI)) × 100;

[0186] When the co-toxicity coefficient (CTC) of the compound agent > 120, it shows a synergistic effect; when CTC ≤ 80, it shows an antagonistic effect; when 80 < CTC < 120, it shows an additive effect.

[0187] 2. Test results

[0188] The test results are shown in Table 10.

[0189] Table 10: Virulence determination of strain BT001 and its compound with matrine against Spodoptera frugiperda

[0190]

[0191] Based on the criteria for judging synergistic toxicity, the results of this study on the combination of Bacillus thuringiensis BT001 and matrine showed that fermentation broths of strain BT001 with different spore concentrations and matrine concentrations all exhibited a strong synergistic effect. Among these, the spore count was 1×10⁻⁶. 7 Fermentation broth of strain BT001 with CFU / mL and a concentration of 5 mg•L -1 The combination of matrine showed the most prominent synergistic effect, with a co-toxicity coefficient as high as 500.76. Even the T5 combination, which had the weakest synergistic effect, had a co-toxicity coefficient of 293.07, which was far higher than the threshold of synergistic effect, and also showed a significant synergistic effect.

[0192] Example 9: Toxicity determination of Bacillus thuringiensis BT001 and its compound diflubenzuron against Spodoptera litura.

[0193] 1.1 Preparation of the test fermentation broth:

[0194] Based on the preferred results of Example 5, the fermentation broth of strain BT001 was prepared using glucose-yeast medium (37℃, 180 r / min shaking culture for 48 h). 5 mL of the fermentation broth was first taken to determine the spore concentration, which was found to be 1.5 × 10⁻⁶. 8 CFU / mL, for later use.

[0195] 1.2 Processing Group Settings

[0196] The treatment groups are as follows:

[0197] Treatment group 1 (T1): This group consisted of different dilutions of strain BT001, specifically, fermentation broth of strain BT001 diluted with pure water to obtain four spore concentrations: 1×10⁻⁶. 5 CFU / mL, 1×10 6 CFU / mL, 1×10 7 CFU / mL, 1×10 8 CFU / mL. Used to calculate the LC50 of the fermentation broth of strain BT001. 50 .

[0198] Treatment Group 2 (T2): Different dilution groups of 25% diflubenzuron single agent, i.e., 25% diflubenzuron diluted with pure water to three concentrations, diluted 1000, 1500, and 2000 times respectively, with the corresponding final concentrations being 250 mg / L. -1 166.7 mg•L -1 125 mg•L -1 Used to calculate the median lethal concentration (LC50) of diflubenzuron.50 .

[0199] Based on the compatibility of Bacillus thuringiensis and diflubenzuron in treatment groups 1 and 2, and the results of the toxicity tests of the two agents against Spodoptera litura larvae (LC50), 50 Then, set the ratio of the compound drug group, specifically: first, dilute 25% diflubenzuron with pure water to the corresponding final concentration of 250 mg / L. -1 166.7 mg•L -1 Next, take an equal volume of BT001 fermentation broth and mix it according to the required BT001 spore concentration in each of the following treatments. All mixtures are in a 1:1 ratio. Mix well before use.

[0200] Treatment group 3 (T3): 50 mL spore count 1×10 7 Fermentation broth of strain BT001 with CFU / mL and 50 mL of a solution containing 250 mg•L -1 Mixed with diflubenzuron;

[0201] Treatment group 4 (T4): 50 mL spore count 1×10 7 The fermentation broth of strain BT001 with CFU / mL was mixed with 50 mL of a solution containing 166.7 mg•L. -1 Mixed with diflubenzuron;

[0202] Treatment group 5 (T5): 50 mL spore count 1×10 6 Fermentation broth of strain BT001 with CFU / mL and 50 mL of a solution containing 250 mg•L -1 Mixed with diflubenzuron;

[0203] Treatment group 6 (T6): 50 mL spore count 1×10 6 The fermentation broth of strain BT001 with CFU / mL was mixed with 50 mL of a solution containing 166.7 mg•L. -1 A mixture of diflubenzuron and chlorfluazuron.

[0204] 1.3 Methods for determining toxicity

[0205] Third-instar larvae of the fall armyworm were treated according to the toxicity determination method for the fall armyworm in Example 8.

[0206] 2. Test Results

[0207] According to the criteria for judging the co-toxicity coefficient, the results of the combined treatment of Spodoptera litura with the fermentation broth of strain BT001 and the dilute diflubenzuron solution showed that there was a significant difference compared with the effect of the mixture in Example 8 on the treatment of Spodoptera litura, with three situations: synergistic, additive, and antagonistic (Table 11).

[0208] Table 11: Toxicity determination of strain BT001 and its compound diflubenzuron against Spodoptera litura.

[0209]

[0210] As shown in Table 11, the co-toxicity coefficient of group T3 was 123.20, indicating a strong synergistic effect. The co-toxicity coefficient of group T4 was 82.64, indicating an additive effect. The co-toxicity coefficients of groups T5 and T6 were both <80, indicating antagonistic effects.

[0211] Therefore, the combined effect of Bacillus thuringiensis and diflubenzuron is highly dependent on their concentration ratio. A high concentration of BT001 fermentation broth combined with a high concentration of diflubenzuron dilution produces the best effect, resulting in a synergistic effect; conversely, a low concentration of BT001 fermentation broth combined with a high concentration of diflubenzuron dilution produces an antagonistic effect, reducing efficacy.

[0212] Example 10: Verification of the toxicity of strain BT001 against fall armyworm in a pot experiment.

[0213] 1. Test Methods

[0214] Based on the preferred results of Example 5, the fermentation broth of strain BT001 was prepared using glucose-yeast medium (37℃, 180r / min shaking culture for 48h), and the viable cell count in the fermentation broth was adjusted to 5×10⁻⁶. 7 CFU / mL, for later use.

[0215] Treatment of potted Chinese cabbage: 10 cabbages were planted in each pot. After reaching a height of 15 cm, the cabbages were inoculated with fall armyworm larvae. 10 fall armyworm larvae were inoculated in each pot. Two treatments were set up for each larval instar, and each treatment was repeated three times. Treatment group 1 (T1): control group with water; Treatment group 2 (T2): sprayed with fermentation liquid of strain BT001. When spraying, the liquid should evenly cover the cabbage leaves and form a film on the leaves without dripping. The plants were observed for 7 days after treatment.

[0216] Measure the total leaf area: Before releasing the larvae, randomly select control plants with the same growth as the test plants and measure the total area of ​​their healthy leaves as the initial average leaf area.

[0217] Measure the remaining area after damage: After the experiment, all damaged leaves were removed and scanned with a leaf area meter to obtain the remaining healthy leaf area.

[0218] Leaf area was measured using a portable leaf area meter LI-3000C. Experimental data were processed using Excel, and SPSS software was used for further statistical analysis and significance analysis.

[0219] Table 12: Pest Classification

[0220]

[0221] Single plant damage index =

[0222] Victim Index of Each Treatment Group =

[0223] Corrective efficacy (%) = ×100%

[0224] Damaged leaf area (%) = ×100%

[0225] 2. Test Results

[0226] The control effect was investigated 7 days later. Calculations were performed based on the pest classification in Table 12 and the formula below. The results are shown in Table 13 and... Figure 10 (The control effect on the first-instar fall armyworm is shown in the figure.)

[0227] Table 13: Control effect of inoculating Chinese cabbage with 1st and 2nd instar larvae of fall armyworm

[0228]

[0229] Strain BT001 showed significant control effects against first and second instar fall armyworm larvae released from Chinese cabbage. Treatment of first instar fall armyworm larvae inoculated on Chinese cabbage resulted in a 100% corrected control efficacy after 48 hours, while treatment of second instar fall armyworm larvae inoculated on Chinese cabbage resulted in a 75.68% corrected control efficacy after 48 hours. Pot experiments confirmed that strain BT001 exhibited certain control effects against both first and second instar fall armyworm larvae.

[0230] Example 11: Verification of the toxicity of strain BT001 and compound abamectin against Spodoptera litura in pot experiments

[0231] 1. Test Methods

[0232] Based on the preferred results of Example 5, the fermentation broth of strain BT001 was prepared using glucose-yeast medium (37℃, 180r / min shaking culture for 48h), and the viable cell count in the fermentation broth was adjusted to 5×10⁻⁶. 7 CFU / mL, for later use.

[0233] The insecticidal mechanism of abamectin is primarily stomach poison with secondary contact killing. It stimulates the insect's neuromuscular system to release γ-aminobutyric acid, thereby inhibiting nerve conduction in insects. Larvae exhibit paralysis symptoms upon contact with abamectin, becoming inactive and refusing to feed, and die after 2-4 days.

[0234] Treatment of potted Chinese cabbage: Ten cabbages were planted in each pot, and inoculation with insects began after they reached a height of 15 cm. First and second instar larvae of the beet armyworm were inoculated into the potted Chinese cabbage, with 10 larvae inoculated per pot. Four treatments were set up for each instar, and each treatment was repeated three times. Treatment group 1 (T1): control group with water; Treatment 2 (T2): sprayed with fermentation broth of strain BT001; Treatment 3 (T3): sprayed with diluted abamectin solution (5% abamectin diluted 1000 times); Treatment 4 (T4): sprayed with a mixture of fermentation broth of strain BT001 and diluted abamectin solution (5% abamectin diluted 1000 times) in a 1:1 volume ratio. During spraying, the treatment solution was required to evenly cover the Chinese cabbage leaves, forming a film on the leaves without dripping. Observation continued for 7 days after treatment.

[0235] The pest classification standards, damage index, and corrected control efficacy calculations are the same as in Example 10.

[0236] The Q-value was calculated using the Jin Zhengjun method to evaluate the interaction between the fermentation broth of strain BT001 and the diluted abamectin solution.

[0237] Q = Ea + b / (Ea + Eb - Ea × Eb)

[0238] Where Ea+b represents the inhibition rate of combined drug use; Ea and Eb represent the inhibition rates of drug A and drug B used alone, respectively.

[0239] In the formula, the numerator represents the "measured merging effect", the denominator is the "expected merging effect", and Q is the ratio of the two. Q < 0.85 indicates antagonism, 0.85 ≤ Q < 1.15 indicates addition, and Q ≥ 1.15 indicates synergy.

[0240] 2. Test Results

[0241] The control effect was investigated 7 days later, and the results are shown in Table 14 and 15. Figure 11 (The control effect on the second instar beet armyworm is shown in the figure.)

[0242] Table 14: Control effect of inoculating Chinese cabbage with 1st and 2nd instar larvae of the beet armyworm.

[0243]

[0244] Strain BT001 showed significant control effects against first and second instar beet armyworm larvae released from Chinese cabbage. The control efficacy of BT001 fermentation broth against first and second instar larvae was 71.01% and 62.00%, respectively, slightly lower than that against the chemical pesticide abamectin. When BT001 fermentation broth was combined with diluted abamectin, the control efficacy against first and second instar beet armyworm larvae in potted plants was ≥80%, with calculated Q values ​​>0.85, indicating an additive effect. This demonstrates the control advantage of combining Bacillus thuringiensis BT001 with the chemical pesticide abamectin.

[0245] Example 12: Virulence test and growth-promoting function determination of strain BT001 against southern root-knot nematodes infecting tomatoes

[0246] 1. Experimental Materials and Methods

[0247] 1.1 Method for determining the control efficacy of Bacillus thuringiensis BT001 against southern root-knot nematodes

[0248] Based on the preferred results of Example 5, the fermentation broth of strain BT001 was prepared using glucose-yeast medium (37℃, 180r / min shaking culture for 48h), and the viable cell count in the fermentation broth was adjusted to 5×10⁻⁶. 7 CFU / mL, for later use.

[0249] Tomato seedlings were raised in plug trays. Tomato seedlings of uniform size and height of 10 cm were selected for transplanting. Each pot was filled with a mixture of substrate and diseased soil from the Ningyang base greenhouse at a ratio of 1:1. Four treatment groups were set up, with 10 replicates for each treatment group.

[0250] The four treatments were as follows: Treatment 1 (T1): 200 mL of water was used as the control group (CK); Treatment 2 (T2): 200 mL of Bacillus thuringiensis BT001 fermentation broth was used; Treatment 3 (T3): 200 mL of water + 0.2 g of 10% thiazophosphorus granules were used (thiazophosphorus granules were mixed into the soil in the flowerpot before transplanting the tomato seedlings, and then water was used); Treatment 4 (T4): 200 mL of Bacillus thuringiensis fermentation broth + 0.2 g of 10% thiazophosphorus granules were used (thiazophosphorus granules were mixed into the soil in the flowerpot before transplanting the tomato seedlings, and then fermentation broth was used). The light and temperature were kept consistent after each treatment. After 15 days, each treatment solution was applied again, and the observation continued for another 30 days, for a total of 45 days.

[0251] Forty-five days after potting, 100 g soil samples were taken from each treatment and passed through three layers of sieves (200 mesh, 300 mesh, and 500 mesh, respectively). The insect population density was examined under a microscope, and the insect population reduction rate was calculated compared with the control group. The degree of infection of diseased plants and roots was investigated, and the disease index and control effect were calculated.

[0252] Nematode reduction rate (%) = ×100%

[0253] Disease Index (DI) = ×100

[0254] Prevention and control effect (%) = ×100%

[0255] Experimental data were processed using Excel, and SPSS software was used for further statistical analysis and significance analysis of differences.

[0256] Table 15: Grading Standards for Nematode Diseases

[0257]

[0258] 1.2 Determination of the growth-promoting function of Bacillus thuringiensis BT001 on tomato plants

[0259] At harvest, wash the roots with clean water, air dry at room temperature, and then use an electronic balance to determine the fresh weight of the tomato plants. After measuring the fresh weight, dry them in an oven at 65℃ until constant weight, and then measure the dry weight of the roots.

[0260] Experimental data were processed using Excel, and SPSS software was used for further statistical analysis and significance analysis of differences.

[0261] 2. Results

[0262] 2.1 Results of efficacy test of Bacillus thuringiensis BT001 against southern root-knot nematodes

[0263] The results are shown in Table 16. Both Bacillus thuringiensis BT001 and the chemical agent thiazophos showed significant effects in controlling southern root-knot nematodes. Compared with thiazophos alone (T3 group), the fermentation broth of strain BT001 alone (T2 group) showed similar nematode reduction rates (60.08% and 67.12%) and control effects (29.63% and 38.89%), with thiazophos being slightly better. When the fermentation broth of strain BT001 was used in combination with thiazophos (T4 group), all indicators were significantly improved, achieving the best control effect. The nematode reduction rate reached 77.46%, the disease index dropped to 30.00, and the control effect reached 66.67%. The calculated Q value was 1.17, indicating that the combined use of the two had a synergistic effect on southern root-knot nematodes (the determination method is the same as in Example 11).

[0264] Table 16: Control of Southern Root-Knot Nematodes by Bacillus thuringiensis BT001 and its Compound Chemical Agent Thiazolphosphine

[0265]

[0266] 2.2 Results of the growth-promoting effect of Bacillus thuringiensis BT001 and its compound chemical thiazophos on tomato plants

[0267] According to the data in Table 17, the fermentation broth of Bacillus thuringiensis BT001 and its combined treatment with the chemical pesticide thiazophos showed a significant growth-promoting effect on tomato plants.

[0268] Table 17: Growth-promoting effects of Bacillus thuringiensis BT001 and its compound chemical thiazophos on tomato plants

[0269]

[0270] Note: Different lowercase letters in the same column's subscript indicate significant differences. P <0.05)

[0271] Compared with the water control group, groups T2-T4 all effectively increased plant fresh weight and root dry weight. Groups T2 and T3 showed significant growth-promoting effects. Notably, group T4 exhibited the most pronounced growth-promoting effect, with both plant fresh weight and root dry weight reaching their maximum values, and the root dry weight significantly higher than other treatment groups. Therefore, the combined use of Bacillus thuringiensis BT001 and the chemical pesticide thiazophos not only effectively controls southern root-knot nematodes but also further promotes healthy crop growth.

[0272] Example 13: Verification of the field toxicity of strain BT001 and its compound chlorantraniliprole against corn borer.

[0273] 1. Test Methods

[0274] Based on the preferred results of Example 5, the fermentation broth of strain BT001 was prepared using glucose-yeast medium (37℃, 180 r / min shaking culture for 48 h). 5 mL of the fermentation broth was first taken to determine the spore concentration, which was found to be 1.5 × 10⁻⁶. 8 The fermentation broth was diluted with pure water to achieve spore concentrations of 1×10⁻⁶ CFU / mL. 8 CFU / mL, 1×10 7 Prepare fermentation broth at CFU / mL for later use.

[0275] Different treatments were set up in the field to verify the effectiveness of the insecticide against corn borer larvae. The experiment included six treatment groups: Treatment 1 (T1): negative control group (sprayed with glucose-yeast medium); Treatment 2 (T2): sprayed with BT001 spores at a concentration of 1×10⁻⁶. 8 Fermentation broth group with CFU / mL; Treatment 3 (T3): Spraying with strain BT001 spores at a concentration of 1×10⁻⁶. 7  Fermentation broth group with CFU / mL; Treatment 4 (T4): Spray with 20% chlorantraniliprole diluted solution (1000 times dilution); Treatment 5 (T5): Spray with strain BT001 mixed at a volume ratio of 1:1 (spore concentration of 1×10⁻⁶). 8 Fermentation broth (CFU / mL) and 20% chlorantraniliprole diluted 1000 times; Treatment 6 (T6): Spray with strain BT001 (spore concentration 1×10⁻⁶ CFU / mL) mixed at a volume ratio of 1:1. 7 Fermentation broth (CFU / mL) and 20% chlorantraniliprole diluted 1000 times.

[0276] Six plots were set up in the field, each 40m × 40m wide, and sprayed with one of the six treatment solutions. Each treatment solution was sprayed evenly over the leaves, ensuring a uniform film formation without dripping. Fourteen days after treatment, a five-point sampling method was used to investigate each plot. At each point, within a 1m × 1m quadrat, larval mortality and stem borer damage were recorded, with five plants surveyed at each point.

[0277] Reduction rate of damaged plants (%) = (Bottled stems of control group - Bottled stems of treatment group) / Bottled stems of control group × 100%

[0278] Insect population reduction rate (%) = (Insect population before pesticide application - Insect population after pesticide application) / Insect population before pesticide application × 100%

[0279] 2. Test Results

[0280] The results are shown in Table 18.

[0281] Table 18: Verification of the field toxicity of Bacillus thuringiensis and its compound chlorantraniliprole against corn borer larvae

[0282]

[0283] Note: Different lowercase letters in the same column's subscript indicate significant differences. P <0.05)

[0284] According to the field toxicity data provided in Table 18, the combined treatment of Bacillus thuringiensis (BT001) and chlorantraniliprole showed a certain control effect on corn borer larvae, among which the high concentration combination group (T5 group) performed better in multiple indicators.

[0285] Regarding the reduction rate of damaged plants, the T5 treatment group reached 78.95%, higher than all other treatment groups. In terms of the number of live insects, the T5 group had 0.32 live insects per plant, significantly lower than the insect populations in all other treatment groups, indicating that the combined treatment had a stronger suppressive effect on larval populations. Overall, the T5 group performed best in all indicators, demonstrating that the combined treatment can more effectively control the damage caused by corn borer larvae.

[0286] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A compound insecticide, characterized in that, The compound insecticide is composed of Bacillus thuringiensis (Bacillus thuringiensis). Bacillus thuringiensis BT001 is a compound of pesticide compounds; The Bacillus thuringiensis ( Bacillus thuringiensis The accession number for BT001 is CCTCC NO: M20252346; The compound insecticide is any one of the following (1)-(3): (1) Bacillus thuringiensis-matrine compound insecticide: Bacillus thuringiensis ( Bacillus thuringiensis BT001 fermentation broth and matrine dilution were mixed at a volume ratio of 1:

1. (2) Bacillus thuringiensis-diflubenzuron compound insecticide: Bacillus thuringiensis ( Bacillus thuringiensis BT001 fermentation broth and dilute diflubenzuron solution were mixed at a volume ratio of 1:

1. (3) Bacillus thuringiensis-thiazophosphorus compound insecticide: Bacillus thuringiensis ( Bacillus thuringiensis BT001 fermentation broth is a compound of thiazophosphorus granules; In the Bacillus thuringiensis-matrine compound insecticide, Bacillus thuringiensis (Bt) Bacillus thuringiensis The spore concentration in the BT001 fermentation broth is 1×10⁻⁶. 6 CFU / mL - 1×10 7 CFU / mL; the concentration of matrine dilution is 5 mg / L-10 mg / L; In the Bacillus thuringiensis-diflubenzuron compound insecticide, Bacillus thuringiensis (Bt) Bacillus thuringiensis The spore concentration in the BT001 fermentation broth is 1×10⁻⁶. 7 CFU / mL; the concentration of the diluent for diflubenzuron is 250 mg / L; In the Bacillus thuringiensis-thiazophos compound insecticide, Bacillus thuringiensis (Bt) Bacillus thuringiensis The viable cell count of the fermentation broth of BT001 was 5 × 10⁻⁶. 7 CFU / mL; Bacillus thuringiensis ( Bacillus thuringiensis The fermentation broth of BT001 was mixed with 10% thiazophosphorus granules at a ratio of 200mL:0.2g.

2. The compound insecticide according to claim 1, characterized in that, Bacillus thuringiensis (Bt) Bacillus thuringiensis The BT001 fermentation broth was prepared by the following method: Bacillus thuringiensis ( Bacillus thuringiensis BT001 was inoculated into glucose-yeast medium and cultured at 37 ℃ with shaking at 180 r / min for 48 h; The glucose-yeast culture medium consists of the following components: glucose 5 g / L, yeast extract 5 g / L, peptone 5 g / L, dipotassium hydrogen phosphate 1 g / L, magnesium sulfate 0.2 g / L, and manganese sulfate 0.02 g / L.

3. The application of the compound insecticide according to claim 1 or 2 in the control of plant pests, characterized in that, The plant pests mentioned are caused by the beet armyworm, the fall armyworm, or the southern root-knot nematode.

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