Microbial pesticide for preventing and treating crop pests and preparation method thereof

Abstract

The invention relates to the field of microbial pesticides, and particularly discloses a microbial pesticide for preventing and treating crop pests and a preparation method thereof. The preparation method of the microbial pesticide for preventing and treating crop pests comprises the following steps: S1, extracting polysaccharide; s2, extracting insecticidal protein; s3, loading: dispersing insecticidal protein, Bt 4D19 polysaccharide, 2-methylimidazole and zinc acetate in a weight ratio of (8-12): (5-10): 500: (50-55) in water, stirring for reaction, filtering to obtain a precipitate, washing and drying to obtain a polysaccharide-protein-metal composite organic framework; s4, pesticide preparation. The microbial pesticide provided by the invention has high insecticidal efficacy, strong stability and good liquid medicine adhesion ability, has an obvious control effect on corn borers and cotton bollworms, has a survival rate of not higher than 10%, is applied after heating heat treatment at 55 DEG C, has a survival rate of not higher than 18%, and is applied after light treatment, and has a survival rate of not higher than 22%.

Description

Technical Field

[0001] The present application relates to the technical field of microbial pesticides, and in particular to a microbial pesticide for controlling crop pests and a preparation method thereof. Background Art

[0002] Microbial pesticides refer to biogenic pesticides with microbial live bodies as active ingredients for controlling pests, diseases, weeds, rats and other harmful organisms. They have strong selectivity, are safe for humans, livestock, crops and the natural environment, do not harm natural enemies, are not prone to generating resistance, can overcome the pollution of chemical pesticides to the ecological environment and reduce the pesticide residues in agricultural and sideline products, improve the quality of agricultural and sideline products, and effectively promote the growth of rural economy and the increase of farmers' income. Among them, Bacillus thuringiensis is a microbial low-toxic insecticide with excellent control effect, mainly acting through stomach poisoning, and has a better effect when used in the low-instar stage of pests. It mainly has a good control effect on the larvae of some Lepidoptera pests, and can be used to control Pieris rapae, Parnara guttata, Geometridae, Dendrolimus punctatus, Helicoverpa assulta, Ostrinia furnacalis, Helicoverpa armigera, Cnaphalocrocis medinalis, Psychidae, Agrotis ypsilon, etc. Its insecticidal principle is that after being ingested by pests, Bacillus thuringiensis parasitizes in the midgut of the host, grows and reproduces in the appropriate alkaline environment in the intestine. The crystal toxin is hydrolyzed by proteases in the insect intestine to form smaller subunits with toxicity, and they act on the midgut epithelial cells of the insect body, causing intestinal paralysis, perforation, insect body paralysis and cessation of feeding. Subsequently, Bacillus thuringiensis enters the hemocoel to reproduce, causing septicemia and resulting in the death of the insect body. Bacillus thuringiensis mainly comes from soil and is widely distributed in soil, insect corpses, plant surfaces, stored materials or warehouse dust, sewage and other environments around the world. Precisely because the main insecticidal components in microbial pesticides all come from the natural environment, using such pesticides can not only achieve good insecticidal effects, but also have no negative impact on the natural environment. However, precisely because of this, microbial pesticides (especially taking Bacillus thuringiensis pesticides as an example) have the defect of poor stability. During the field application process, they are easily affected by environmental factors, the control effect is unstable, and the insecticidal efficacy will be greatly reduced after being exposed to light and heat. Therefore, some researchers believe that it should be compounded with a metal skeleton to improve the long-term control effect of microbial pesticides. However, this will lead to insufficient pesticide efficacy and cannot meet the insecticidal needs of users. Summary of the Invention

[0003] In order to solve the above technical problems, the present application provides a microbial pesticide for controlling crop pests and a preparation method thereof.

[0004] First aspect, the present application provides a preparation method of a microbial pesticide for controlling crop pests, including the following steps: S1. Extracting polysaccharide: Taking Bacillus thuringiensis 4D19 strain, culturing, collecting the bacterial liquid, collecting the supernatant, performing alcohol precipitation to obtain the crude polysaccharide, digesting the residual protein, desalting, performing ion exchange chromatography and gel filtration chromatography to obtain Bacillus thuringiensis 4D19 polysaccharide; S2. Extracting insecticidal protein: Taking Bacillus thuringiensis HD73 strain, performing strain activation, culturing, washing, lysing the cells to obtain the supernatant, adding acetic acid to extract the protein, and washing the obtained solid substance to obtain the insecticidal protein; S3. Loading: Dispersing the insecticidal protein, Bacillus thuringiensis 4D19 polysaccharide, 2-methylimidazole and zinc acetate with a weight ratio of (8-12):(5-10):500:(50-55) in water, stirring and reacting, filtering to obtain the precipitate, washing and drying to obtain the polysaccharide-protein-metal composite organic framework; S4. Pesticide formulation: Blending the polysaccharide-protein-metal composite organic framework, dispersant, oily transfer agent and water with a weight ratio of (20-25):(2.5-3):(0.2-0.4):100 to obtain the microbial pesticide.

[0005] By adopting the above technical solution, the present application extracts polysaccharide and insecticidal protein from Bacillus thuringiensis 4D19 strain and Bacillus thuringiensis HD73 strain respectively, and loads them all into the metal-organic framework. Bacillus thuringiensis 4D19 polysaccharide can significantly improve the insecticidal efficacy of the insecticidal protein, and the two can also be evenly distributed in the metal-organic framework and improve the thermal stability and light stability of the overall microbial pesticide, thereby improving the effective period of the microbial pesticide. And the present application also adds a dispersant, which can significantly improve the uniform dispersion of the polysaccharide-protein-metal composite organic framework, avoid poor insecticidal effects in some areas caused by uneven dispersion, and also adds an oily transfer agent, which can effectively reduce the droplet drift phenomenon in the use of the microbial pesticide, promote the adhesion of the liquid medicine and improve the insecticidal efficacy.

[0006] The present application strictly controls the weight ratio of the insecticidal protein, Bacillus thuringiensis 4D19 polysaccharide and 2-methylimidazole, and at the same time improves the insecticidal efficacy and stability of the insecticide; it also strictly controls the weight ratio of the polysaccharide-protein-metal composite organic framework, dispersant, oily transfer agent and water, and while ensuring the efficacy, it improves the adhesion of the liquid medicine and the internal dispersion uniformity of the microbial pesticide as much as possible during use.

[0007] In summary, the microbial pesticide of the present application combines high insecticidal efficacy, strong stability, and good liquid attachment ability. Experimental tests have proven that 4 days after application, its control effects on the Asian corn borer and the cotton bollworm are obvious, and the survival rates are not higher than 10%. After heating treatment at 55°C and then applying the pesticide, the survival rates of the Asian corn borer and the cotton bollworm can still be not higher than 18%. After light treatment and then applying the pesticide, the survival rates of the Asian corn borer and the cotton bollworm can still be not higher than 22%. In the unmanned aerial vehicle spray application test, the droplet coverage rate is not lower than 25%.

[0008] Preferably, in the step S3, the weight ratio of the insecticidal protein, Bt 4D19 polysaccharide, and 2-methylimidazole is 10:8:500.

[0009] By adopting the above technical solution, the present application strictly controls the weight ratio, which can further improve the loading efficiency and stability of the insecticidal protein and the polysaccharide in the metal-organic framework, enhance the overall insecticidal efficacy of the microbial pesticide, optimize the synergistic effect between the polysaccharide and the insecticidal protein, significantly improve the insecticidal efficacy of the insecticidal components, and effectively extend the effective period of the microbial pesticide.

[0010] Preferably, in the step S3, the weight ratio of 2-methylimidazole to zinc acetate is 500:53.

[0011] By adopting the above technical solution, the present application strictly controls the weight ratio of the two, which can precisely control the formation conditions of the metal-organic framework, thereby optimizing the structural stability of the polysaccharide-protein-metal composite organic framework. This ratio helps to improve the loading capacity of the composite organic framework for the insecticidal protein and Bt 4D19 polysaccharide, further enhance the thermal stability and light stability of the microbial pesticide, extend its effective period, and ensure that the control effects on pests such as the Asian corn borer and the cotton bollworm in practical applications are not affected by environmental factors.

[0012] Preferably, in the step S4, the dispersant includes copper quinolate and sodium lignosulfonate.

[0013] By adopting the above technical solution, the present application can significantly improve the uniform dispersion of the polysaccharide-protein-metal composite organic framework in water, avoid poor insecticidal effects in some areas caused by uneven dispersion, and thus improve the overall control effect of the microbial pesticide.

[0014] Preferably, the weight ratio of copper quinolate to sodium lignosulfonate is (2-3):1.

[0015] By adopting the above technical solution, the ratio of this application optimizes the performance of the dispersant, significantly improves the uniform dispersion of the polysaccharide-protein-metal composite organic framework in water, and avoids the problem of poor insecticidal effect in some areas caused by uneven dispersion. Thus, the dispersant under this ratio enhances the overall stability of the microbial pesticide, ensuring the uniform distribution and continuous release of the drug efficacy during use.

[0016] Preferably, in the step S4, the oily transfer agent includes one or more of methyl linseed oil, soybean oil, methyl soyate, methyl rice bran oil, methyl coconut oil, and soybean oil fatty acid.

[0017] Preferably, the oily transfer agent includes methyl coconut oil and soybean oil.

[0018] Preferably, the weight ratio of methyl coconut oil to soybean oil is 10:(2 - 3).

[0019] By adopting the above technical solution, the methyl coconut oil and soybean oil of this application are compounded according to a specific weight ratio, which can significantly improve the liquid attachment ability of the microbial pesticide, effectively reduce the phenomenon of droplet drift, and thus enhance the insecticidal efficacy. Specifically, this weight ratio optimizes the synergistic effect between the two oily transfer agents, making the pesticide cover the crop surface more evenly during application and enhancing the control effect on pests.

[0020] Preferably, in the step S4, the weight ratio of the polysaccharide-protein-metal composite organic framework, the dispersant, the oily transfer agent, and water is 22.5:2.7:0.3:100.

[0021] By adopting the above technical solution, this application strictly controls the weight ratio, enabling the microbial pesticide to further optimize the dispersion uniformity and attachment ability of the liquid medicine while ensuring high insecticidal efficacy. The polysaccharide-protein-metal composite organic framework provides the core insecticidal component and enhances the thermal stability and light stability; the reasonable ratio of the dispersants quinoline copper and sodium lignosulfonate ensures the uniform distribution of the active ingredients and avoids insufficient local drug efficacy; the oily transfer agents methyl coconut oil and soybean oil are proportionally combined, effectively reducing droplet drift and promoting the attachment of the liquid medicine to the crop surface, thereby improving the overall insecticidal effect.

[0022] In a second aspect, this application provides a microbial pesticide prepared by the method for preparing a microbial pesticide for controlling crop pests as described above.

[0023] In summary, this application has the following beneficial technical effects: The microbial pesticide of the present application can significantly improve the insecticidal effect, and has obvious control effects on crop pests such as corn borers and cotton bollworms. The survival rates after 4 days of applying the pesticide are all not higher than 10%. After heat treatment at 55°C and then applying the pesticide, the survival rates of corn borers and cotton bollworms can still be not higher than 18%, indicating its excellent thermal stability. After light treatment and then applying the pesticide, the survival rates of corn borers and cotton bollworms can still be not higher than 22%, reflecting good light stability. In addition, due to the addition of a dispersant, the uniform dispersion of the polysaccharide-protein-metal composite organic framework can be significantly improved, avoiding poor insecticidal effects in some areas caused by uneven dispersion. The addition of an oily transfer agent can effectively reduce the phenomenon of droplet drift, promote the adhesion of the liquid medicine, and improve the insecticidal efficacy. In the unmanned aerial vehicle spraying test, the droplet coverage rate is not less than 25%. These effects jointly ensure that the microbial pesticide has high insecticidal efficacy, strong stability and good liquid medicine adhesion ability. Detailed implementation manners

[0024] Material sources Unless otherwise specified, the raw materials used in the present application are all commercially available products, specifically: The lysis solution is prepared by mixing sodium carbonate, disodium ethylenediaminetetraacetate, β-mercaptoethanol and deionized water with a weight ratio of 5.3:16.81:30:800, adjusting the pH to 9.5 - 10 with 1M NaOH, making the volume up to 1L, and filtering and sterilizing with a 0.22μm filter membrane.

[0025] The Bt 4D19 bacillus strain and the Bt HD73 bacillus strain are both provided by the Institute of Plant Protection, Chinese Academy of Agricultural Sciences.

[0026] The present application will be further described in detail below with reference to examples and comparative examples.

[0027] Example 1.1 A preparation method of a microbial pesticide for controlling crop pests, comprising the following steps: S1. Extraction of polysaccharides: streak the Bacillus 4D19 strain on LB solid medium, culture at 30°C until a single colony grows, pick a single colony and place it in liquid LB medium, culture it at 30°C and 220r / min for 12h, transfer it to LB liquid medium according to 1% inoculum, culture it in a shaker at 30°C and 220rpm for 30h, observe that the bacteria are vegetative without lysis, add 1mol / L NaOH solution to adjust the pH of the bacterial solution to 8-9, treat it at 12000rpm for 30min, collect the supernatant, add 5% trichloroacetic acid solution with one tenth volume of the supernatant, place it at room temperature for 2h to denature the protein, collect the supernatant with 12000rpm for 30min, adjust the pH to 6-7, add 95% anhydrous ethanol with three times volume of the supernatant, precipitate polysaccharides at 4°C overnight, treat it at 12000rpm for 30min, discard the supernatant to obtain the precipitate, and obtain Bt 4D19 polysaccharide; S2. Extraction of insecticidal protein: Bacillus HD73 strain was revived in 1 / 2LB solid culture medium and cultured at 30°C for 24h. Then, a single colony was picked and transferred to 1 / 2LB liquid culture medium. The culture was activated by shaking culture at 30°C and 200rpm for 16h. The activated bacterial liquid was inoculated into 1 / 2LB liquid culture medium at a ratio of 1:100 (v / v) for expansion culture. The culture was stopped when most of the bacteria were lysed under microscopic examination at 30°C and 200rpm. The fermentation liquid was centrifuged at 4°C and 8000rpm for 10min. The supernatant was discarded and the precipitate was retained with an appropriate amount of pre-cooled 1mol / L The NaCl solution was fully mixed, centrifuged at 4°C and 8000rpm for 10min, the precipitate was washed twice with pre-cooled sterile water again, centrifuged under the same conditions, the lysis solution was added to the culture precipitate at a volume ratio of 100:3, stirred evenly, incubated on ice at 100rpm for 1h, centrifuged at 4°C and 10000rpm for 10min to collect the supernatant, repeated the above operation once, added an appropriate amount of HAc to the supernatant, adjusted the solution pH to 4.5-5.0, placed the solution in ice water for at least 1h, centrifuged at 4°C and 13000rpm for 15min, the precipitate was fully washed 3 times with pre-cooled sterile water to remove the remaining HAc, and the precipitate was freeze-dried to obtain the insecticidal protein; S3. Load: Disperse the Bt 4D19 polysaccharide obtained in step S1 and the insecticidal protein obtained in step S2 in a 1.6 mol / L 2-methylimidazole solution, ensuring that the weight ratio of the insecticidal protein, Bt 4D19 polysaccharide, and 2-methylimidazole is 8:10:500. Invert and mix well until completely dissolved. Then add a 0.04 mol / L zinc acetate solution, ensuring that the weight ratio of 2-methylimidazole to zinc acetate is 500:50. Let it stand at room temperature for 30 min, then centrifuge at 8000 rpm for 10 min, filter to obtain a precipitate, wash the precipitate 3 times with distilled water, and dry to obtain a polysaccharide-protein-metal composite organic framework; S4. Pesticide formulation: Blend 20 parts of the polysaccharide-protein-metal composite organic framework obtained in step S3, 3 parts of a dispersant (quinoline copper and sodium lignosulfonate in a 1:1 ratio), 0.2 parts of an oil-based transfer agent (methyl linolenate, soybean oil, and methyl soyate in a 1:1:1 ratio), and 100 parts of water to obtain a microbial pesticide.

[0028] Example 1.2 A method for preparing a microbial pesticide for controlling crop pests, comprising the following steps: S1. Extract polysaccharide: Streak the Bt 4D19 bacillus strain on an LB solid medium and culture it at 30 °C until single colonies grow. Pick a single colony into a liquid LB medium and culture it at 30 °C and 220 r / min for 12 h. Transfer it to an LB liquid medium at an inoculation amount of 1% and place it in a shaker at 30 °C and 220 rpm for 30 h. When it is observed that the bacteria are in the vegetative state and there is no lysis, add a 1 mol / L NaOH solution to adjust the pH of the bacterial solution to 8 - 9. After treating it at 12000 rpm for 30 min, collect the supernatant. Add a 5% trichloroacetic acid solution that is one-tenth of the volume of the supernatant and let it stand at room temperature for 2 h to denature the protein. After treating it at 12000 rpm for 30 min, collect the supernatant, adjust the pH to 6 - 7, add 95% anhydrous ethanol that is three times the volume of the supernatant, precipitate the polysaccharide overnight at 4 °C, and after treating it at 12000 rpm for 30 min, discard the supernatant to obtain a precipitate, thus obtaining Bt 4D19 polysaccharide; S2. Extract insecticidal protein: Resuscitate the Bt HD73 bacillus strain using 1 / 2LB solid medium, culture it at 30 °C for 24 h. Then pick a single colony and transfer it to 1 / 2LB liquid medium, and culture it with shaking at 30 °C and 200 rpm for 16 h for activation. The activated bacterial solution is inoculated into 1 / 2LB liquid medium at a ratio of 1:100 (v / v) for scale-up culture, and the culture is stopped when most of the bacteria are lysed under microscopy after culturing with shaking at 30 °C and 200 rpm. The fermentation broth is centrifuged at 4 °C and 8000 rpm for 10 min, the supernatant is discarded, and the precipitate is mixed well with an appropriate amount of pre-cooled 1 mol / L NaCl solution, and then centrifuged at 4 °C and 8000 rpm for 10 min. The precipitate is washed twice with pre-cooled sterile water and centrifuged under the same conditions. Add lysis buffer to the culture precipitate according to a volume ratio of 100:3, stir evenly, incubate with shaking at 100 rpm on ice for 1 h, and centrifuge at 4 °C and 10000 rpm for 10 min to collect the supernatant. Repeat the above operation once, add an appropriate amount of HAc to the supernatant, adjust the pH of the solution to 4.5 - 5.0, place the solution in ice water and let it stand for at least 1 h, centrifuge at 4 °C and 13000 rpm for 15 min, and wash the precipitate thoroughly with pre-cooled sterile water 3 times to remove the remaining HAc. The precipitate is freeze-dried to obtain the insecticidal protein; S3. Loading: Disperse the Bt 4D19 polysaccharide obtained in step S1 and the insecticidal protein obtained in step S2 in a 1.6 mol / L 2-methylimidazole solution, ensuring that the weight ratio of the insecticidal protein, Bt 4D19 polysaccharide, and 2-methylimidazole is 12:5:500. Invert and mix well until completely dissolved, then add a 0.04 mol / L zinc acetate solution, ensuring that the weight ratio of 2-methylimidazole to zinc acetate is 500:55. Let it stand at room temperature for 30 min and then centrifuge at 8000 rpm for 10 min. Filter to obtain the precipitate, wash the precipitate 3 times with distilled water, and dry to obtain the polysaccharide-protein-metal composite organic framework; S4. Pesticide formulation: Mix 25 parts of the polysaccharide-protein-metal composite organic framework obtained in step S3, 2.5 parts of a dispersant (quinoline copper and sodium lignosulfonate at a ratio of 1.5:1), 0.4 parts of an oil-based transfer agent (methyl linolenate, methyl rice bran oil, methyl coconut oil, and soybean oil fatty acid at a ratio of 1:1:1), and 100 parts of water to obtain the microbial pesticide.

[0029] Example 1.3 A preparation method of a microbial pesticide for controlling crop pests, which is different from Example 1.1 in that: in step S3, the weight ratio of the insecticidal protein, Bt 4D19 polysaccharide and 2-methylimidazole is 10:8:500, and the weight ratio of 2-methylimidazole to zinc acetate is 500:53, and the rest are the same as in Example 1.1.

[0030] Example 1.4 A preparation method of a microbial pesticide for controlling crop pests, which is different from Example 1.3 in that: in step S4, the weight ratio of the polysaccharide-protein-metal composite organic framework, dispersant, oily transfer agent and water is 22.5:2.7:0.3:100, and the rest are the same as in Example 1.3.

[0031] Example 2.1 A preparation method of a microbial pesticide for controlling crop pests, which is different from Example 1.4 in that: in step S4, the weight ratio of quinoline copper to sodium lignosulfonate is 2:1, and the rest are the same as in Example 1.4.

[0032] Example 2.2 A preparation method of a microbial pesticide for controlling crop pests, which is different from Example 1.4 in that: in step S4, the weight ratio of quinoline copper to sodium lignosulfonate is 3:1, and the rest are the same as in Example 1.4.

[0033] Example 3.1 A preparation method of a microbial pesticide for controlling crop pests, which is different from Example 1.4 in that: in step S4, the oily transfer agent is a coconut oil methyl ester and soybean oil with a ratio of 10:5, and the rest are the same as in Example 1.4.

[0034] Example 3.2 A preparation method of a microbial pesticide for controlling crop pests, which is different from Example 1.4 in that: in step S4, the oily transfer agent is a coconut oil methyl ester and soybean oil with a ratio of 10:1, and the rest are the same as in Example 1.4.

[0035] Example 3.3 A preparation method of a microbial pesticide for controlling crop pests, which is different from Example 1.4 in that: in step S4, the oily transfer agent is a coconut oil methyl ester and soybean oil with a ratio of 10:2, and the rest are the same as in Example 1.4.

[0036] Example 3.4 A preparation method of a microbial pesticide for controlling crop pests, which is different from Example 1.4 in that: in step S4, the oily transfer agent is a coconut oil methyl ester and soybean oil with a ratio of 10:3, and the rest are the same as in Example 1.4.

[0037] Comparative Example 1 It is different from Example 1.4 in that: step S1 is removed, and the weight ratio of the insecticidal protein to 2-methylimidazole in step S3 is 18:500, and the rest are the same as in Example 1.4.

[0038] Comparative Example 2 It is different from Example 1.4 in that: the Bt HD73 bacillus strain is used in step S1, and the Bt4D19 bacillus strain is used in step S2, and the rest are the same as in Example 1.4.

[0039] Comparative Example 3 It is different from Example 1.4 in that: in step S3, the weight ratio of the insecticidal protein, Bt 4D19 polysaccharide and 2-methylimidazole is 5:15:500, and the rest are the same as in Example 1.1.

[0040] Comparative Example 4 It is different from Example 1.4 in that: in step S3, the weight ratio of 2-methylimidazole to zinc acetate is 500:60, and the rest are the same as in Example 1.1.

[0041] Comparative Example 5 It is different from Example 1.4 in that: in step S4, the weight ratio of the polysaccharide-protein-metal composite organic framework, dispersant, oil-based transfer agent and water is 30:2:0.5:100, and the rest are the same as in Example 1.4.

[0042] Comparative Example 6 It is different from Example 1.4 in that: step S1 is removed, and the weight ratio of the insecticidal protein to 2-methylimidazole in step S3 is 40:500, and the rest are the same as in Example 1.4.

[0043] Performance Detection 1. The microbial pesticides obtained in the examples and comparative examples were respectively added to the insect feed, and the concentration was prepared to be 300 ug / g. The feed was evenly divided and filled into 24-well plates. 2 one-day-old test insect larvae (1 corn borer and 1 cotton bollworm) were inoculated into each well. The test was repeated 5 times and placed in an incubator at a temperature of 26±1°C, and the 4-day insect survival rate was recorded (denoted as survival rate a / %). 2. The microbial pesticides obtained in the examples and comparative examples were sealed and placed in a 55°C constant temperature metal bath for 6 hours for heat storage. Subsequently, the above operations were repeated, and they were respectively added to the insect feed, and the concentration was prepared to be 300 ug / g. The feed was evenly divided and filled into 24-well plates. 2 one-day-old test insect larvae (1 corn borer and 1 cotton bollworm) were inoculated into each well. The test was repeated 5 times and placed in an incubator at a temperature of 26±1°C, and the 4-day insect survival rate was recorded (denoted as survival rate b / %). 3. Spread the microbial pesticides obtained in the examples and comparative examples evenly in petri dishes. After air-drying, irradiate with 100 W ultraviolet light for 6 h. Then repeat the above operation, add them to the insect feed respectively, and prepare a concentration of 300 μg / g. Divide the feed evenly into 24-well plates. Inoculate 2 one-day-old test insect larvae (1 Asian corn borer and 1 cotton bollworm) into each well. Repeat the test 5 times. Place them in an incubator at a temperature of 26 ± 1 °C, and record the survival rate of insects on the 4th day (recorded as survival rate c / %). Data analysis: 4. Use a drone to spray the microbial pesticides obtained in the examples and comparative examples. The environmental temperature is 25 - 31 °C, the wind is east wind level 1, and there is no precipitation during the spraying period. Before spraying, arrange 3 droplet information sampling points on the flight path of the plant protection drone, and arrange 3 sampling points 6 m away from the flight path. After the spray liquid is mixed evenly, add it to the drone. The drone flies and sprays at a speed of 5 m / s, a height of 3.5 m, and a flow rate of 2 L / min. After the water-sensitive paper dries, recover it and analyze the droplet coverage rate.

[0044] Table 1 Performance Detection Table As can be seen from Table 1, after the microbial pesticides of Examples 1.1 - 1.2 of the present application are used to control the Asian corn borer, its survival rate can be reduced to 8.5 - 8.6%, and after being used to control the cotton bollworm, its survival rate can be reduced to 9.1%. After the heat-treated microbial pesticide is used to control the Asian corn borer, its survival rate can be reduced to 10.1 - 10.2%, and after being used to control the cotton bollworm, its survival rate can be reduced to 17.3 - 17.6%. After spraying, the coverage rate can reach 25.7 - 25.8%.

[0045] In Comparative Example 1, the Bt 4D19 polysaccharide was removed from the present application, and the results showed that the survival rate of pests increased greatly, proving that the Bt 4D19 polysaccharide of the present application can significantly improve the insecticidal efficacy of the insecticidal protein. It can be evenly distributed in the metal-organic framework and improve the thermal stability and light stability of the overall microbial pesticide, thereby improving the effective period of the microbial pesticide.

[0046] In Comparative Example 2, the strains for extracting the Bt 4D19 polysaccharide and the strains for the insecticidal protein in the present application were changed, and the results showed that the survival rate of pests increased greatly, proving that the polysaccharide and the insecticidal protein were respectively extracted from the Bt 4D19 bacillus strain and the Bt HD73 bacillus strain in the present application and were all loaded in the metal-organic framework, and could exert a good cooperative insecticidal effect.

[0047] In Comparative Examples 3-5, the present application changed the ratios of different substances. The results showed that the survival rate of pests increased significantly or the stability of the microbial pesticide was impaired. This proves that the weight ratio of the present application, while ensuring the efficacy of the pesticide, maximally improves the adhesion of the microbial pesticide solution during use and the degree of uniform dispersion inside, thus enhancing the efficacy and stability of the insecticide.

[0048] In Comparative Example 6, the Bt 4D19 polysaccharide in the present application was removed and the dosage of the insecticidal protein was increased. However, the results still showed that the survival rate of pests increased significantly, proving that the Bt 4D19 polysaccharide in the present application can significantly enhance the insecticidal efficacy of the insecticidal protein.

[0049] The embodiments of this specific implementation manner are all preferred embodiments of the present application, and do not limit the protection scope of the present application accordingly. Therefore, all equivalent changes made according to the structure, shape, and principle of the present application should be covered within the protection scope of the present application.

Claims

1. A method for preparing a microbial pesticide for controlling crop pests, characterized in that: The following steps are involved: S1. Extracting polysaccharides: taking Bacillus 4D19 strain, culturing, collecting bacterial liquid, collecting supernatant, ethanol precipitation, obtaining crude polysaccharides, digesting residual protein, desalting, ion exchange chromatography, gel filtration chromatography, obtaining Bt 4D19 polysaccharides; S2. Extracting insecticidal protein: taking Bacillus HD73 strain, activating the strain, culturing, washing, lysing the cells to obtain supernatant, adding acetic acid to extract protein, and washing the obtained solid matter to obtain insecticidal protein; S3, loading: dispersing insecticidal protein, Bt 4D19 polysaccharide, 2-methylimidazole and zinc acetate in a weight ratio of (8-12): (5-10): 500: (50-55) in water, stirring to react, filtering to obtain a precipitate, washing, and drying to obtain a polysaccharide-protein-metal composite organic framework; S4. Pesticide preparation: Mix a polysaccharide-protein-metal composite organic framework, a dispersant, an oily transfer agent and water in a weight ratio of (20-25): (2.5-3): (0.2-0.4): 100 to obtain a microbial pesticide.

2. The method for preparing a microbial pesticide for controlling crop pests according to claim 1, characterized in that: In step S3, the weight ratio of the insecticidal protein, Bt 4D19 polysaccharide and 2-methylimidazole is 10:8:

500.

3. The method for preparing a microbial pesticide for controlling crop pests according to claim 1, characterized in that: In step S3, the weight ratio of 2-methylimidazole to zinc acetate is 500:

53.

4. The method for preparing a microbial pesticide for controlling crop pests according to claim 1, characterized in that: In the step S4, the dispersant includes copper quinoline and sodium lignin sulfonate.

5. The method for preparing a microbial pesticide for controlling crop pests according to claim 4, characterized in that: The weight ratio of the copper quinoline to sodium lignin sulfonate is (2-3):

1.

6. The method for preparing a microbial pesticide for controlling crop pests according to claim 1, characterized in that: In step S4, the oily transfer agent includes one or more of linseed oil methyl ester, soybean oil, soybean oil methyl ester, rice bran oil methyl ester, coconut oil methyl ester and soybean oil fatty acid.

7. The method for preparing a microbial pesticide for controlling crop pests according to claim 6, characterized in that: The oily delivery agents include coconut methyl ester and soybean oil.

8. The method for preparing a microbial pesticide for controlling crop pests according to claim 7, characterized in that: The weight ratio of the coconut oil methyl ester to soybean oil is 10:(2-3).

9. The method for preparing a microbial pesticide for controlling crop pests according to claim 1, characterized in that: In the step S4, the weight ratio of the polysaccharide-protein-metal composite organic framework, the dispersant, the oily transfer agent and water is 22.5:2.7:0.3:

100.

10. The microbial pesticide prepared by the method for preparing a microbial pesticide for controlling crop pests according to any one of claims 1 to 9.

Images