Application of extracellular polysaccharide or extracellular polysaccharide-containing extract in improving sunlight or ultraviolet degradation resistance of insecticidal protein

Abstract

The invention relates to application of extracellular polysaccharide or an extracellular polysaccharide-containing extract in improving sunlight or ultraviolet degradation resistance of insecticidal protein. The invention finds that the extracellular polysaccharide-containing extract extracted from the bacillus thuringiensis G033A can obviously reduce the degradation degree of the bacillus thuringiensis insecticidal protein after being irradiated by ultraviolet or sunlight, and the higher the extract content is, the stronger the insecticidal protein protection capability is; the pesticide effect of the G033A wettable powder can still be well exerted after the G033A wettable powder is irradiated by sunlight, and the lasting period of the G033A wettable powder is prolonged.

Description

Technical Field

[0001] The invention relates to the field of biocides, and in particular to the application of extracellular polysaccharides or extracts containing extracellular polysaccharides in improving the resistance of insecticidal proteins to sunlight or ultraviolet degradation. Background Art

[0002] Bacillus thuringiensis ( Bacillus thuringiensis , Bt) wettable powder can be stored for 1 to 2 years under light-proof conditions. However, spores and insecticidal crystal proteins are easily inactivated and degraded under ultraviolet conditions, which affects the product's long-lasting effect and field control effect. There are three types of ultraviolet rays in natural sunlight: weak effect wavelength UVA (wavelength 320 to 400 nm), strong effect wavelength UVB (wavelength 290 to 320 nm) and super strong effect wavelength UVC (wavelength 190 to 280 nm). UVC is almost absorbed by the atmospheric ozone layer and rarely reaches the ground, so it is mainly UVA and UVB that cause the degradation of Bt insecticidal crystal proteins.

[0003] In order to protect Bt from the external environment and prolong its field persistence, enhancing Bt's anti-ultraviolet ability is a very critical factor. The current methods mainly include the following aspects: 1) adding adjuvants, such as adding biochar to improve the anti-ultraviolet ability of Cry1Ac protein; 2) changing the dosage form, such as in CN202111025625X, the Bt preparation is prepared into granules.

[0004] Many strains of Bacillus thuringiensis produce exopolysaccharides, and the uses of their exopolysaccharides need to be further developed.

[0005] When preparing Bacillus thuringiensis preparations, the cells produced by liquid fermentation are generally collected and further prepared into preparations. In this process, the fermentation supernatant after collecting the cells is discarded. Since the extracellular polysaccharides are secreted into the fermentation supernatant during the fermentation process, they are also regarded as waste and discarded, which greatly wastes the extracellular polysaccharides. How to more effectively utilize the extracellular polysaccharides produced by Bacillus thuringiensis is an urgent problem to be solved. Summary of the invention

[0006] One of the present inventions provides the use of exopolysaccharide or an extract containing the exopolysaccharide in improving the resistance of insecticidal proteins to sunlight or ultraviolet degradation.

[0007] In a specific embodiment, the wavelength of the ultraviolet light is 345 nm to 395 nm.

[0008] In a specific embodiment, the wavelength of the ultraviolet light is 365 nm to 380 nm.

[0009] In a specific embodiment, the UV intensity of the sunlight is above 690 μW / cm2; the average UV intensity at 12:00 is 4542 μW / cm2, and the highest UV intensity is 5140 μW / cm2.

[0010] In a specific embodiment, the exopolysaccharide or the extract is extracted from Bacillus thuringiensis G033A.

[0011] In a specific embodiment, it is characterized in that the insecticidal protein is expressed by Bacillus thuringiensis G033A.

[0012] In a specific embodiment, the extract is obtained by the following steps: 1) adjusting the pH value of the fermentation broth of Bacillus thuringiensis G033A to 8 to 9, then performing a first centrifugation, and collecting a first supernatant after the first centrifugation; 2) adding trichloroacetic acid or an aqueous solution of trichloroacetic acid to the first supernatant, thereby treating the first supernatant with the trichloroacetic acid to obtain a first treated liquid, and then performing a second centrifugation to collect a second supernatant after the second centrifugation; 3) After adjusting the pH value of the second supernatant to 6 to 7, ethanol is added to the second supernatant to obtain a second treatment liquid, the second treatment liquid is precipitated, and then a third centrifugation is performed, and the precipitate after the third centrifugation is collected, and the precipitate is the wet extract containing extracellular polysaccharides.

[0013] In a specific embodiment, the culture medium for culturing the Bacillus thuringiensis G033A is one of an industrial culture medium, an LB culture medium, a sugar-producing culture medium with glucose as a substrate, and a sugar-producing culture medium with sucrose as a substrate.

[0014] In a specific embodiment, in step 2), the mass / volume content of the trichloroacetic acid in the first supernatant is 0.4% to 0.5%.

[0015] In a specific embodiment, in step 3), the volume of ethanol added to the second supernatant is 3 to 3.5 times the volume of the second supernatant.

[0016] In a specific embodiment, in step 2), the first treatment solution is placed at 25 to 35° C. for 1.5 to 3 hours.

[0017] In a specific embodiment, in step 3), the second treatment solution is placed at 2 to 8° C. for 12 to 20 hours.

[0018] In a specific embodiment, the extracellular polysaccharide is obtained by removing residual protein from the extract, desalting, anion exchange chromatography and gel filtration chromatography purification; the weight average molecular weight of the extracellular polysaccharide is 71.484 kDa, the number average molecular weight of the extracellular polysaccharide is 66.625 kDa, the average molecular weight of the extracellular polysaccharide is 76.316 kDa, and the polydispersity coefficient of the extracellular polysaccharide is 1.073. Taking the total sugar content of the extracellular polysaccharide as 100%, mannose is 78.52%, glucose is 12.19%, arabinose is 4.41%, glucuronic acid is 3.53%, and galactose is 1.34%.

[0019] The second aspect of the present invention provides a composition, which comprises the exopolysaccharide or the extract in the application as described in any one of the first aspect of the present invention, and an insecticidal active component, wherein the insecticidal active component is a Bacillus thuringiensis fermentation product or an insecticidal protein derived from Bacillus thuringiensis. The Bacillus thuringiensis fermentation product comprises an insecticidal protein derived from the Bacillus thuringiensis.

[0020] In a specific embodiment, the insecticidal active component is a fermentation product of strain G033A.

[0021] In one specific embodiment, the insecticidal active ingredient is contained in G033A wettable powder.

[0022] In a specific embodiment, the mass ratio of the extract to the G033A wettable powder is 1:1 to 3:1.

[0023] The third aspect of the present invention provides the use of the composition as described in the second aspect of the present invention in controlling fall armyworm.

[0024] Beneficial effects of the present invention: The present invention finds that the extract containing extracellular polysaccharides (i.e., crude extracellular polysaccharides) extracted from Bacillus thuringiensis G033A can significantly reduce the degree of degradation of Bacillus thuringiensis insecticidal protein after being irradiated with ultraviolet light or sunlight, and the higher the content of crude extracellular polysaccharides, the stronger the protection ability for insecticidal protein, which is conducive to the G033A wettable powder still being able to better exert its efficacy after being irradiated with sunlight, and prolonging the effective period of the G033A wettable powder. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 A bottom-up schematic diagram of the experimental setup for simulating outdoor UV exposure is shown.

[0026] Figure 2 The SDS-PAGE electrophoresis results of the insecticidal protein of the wettable powder G033A in Example 1 after irradiation at four ultraviolet wavelengths for 12 hours are shown, wherein the amount of powder loaded in each lane is the same.

[0027] Figure 3 The degradation rate curve of the insecticidal protein of the wettable powder G033A in Example 1 after irradiation at four ultraviolet wavelengths for different time periods is shown.

[0028] Figure 4 The SDS-PAGE of the bacterial agent and crude sugar mixture of Example 2 after UV irradiation for 12 hours is shown, wherein the amount of powder loaded in each lane is the same.

[0029] Figure 5 The degradation rate of insecticidal protein in Example 2 of the mixed solution of bacterial agent and raw sugar after UV irradiation for different time periods is shown. Among them, the data were analyzed by one-way ANOVA, **** ( p <0.0001),***( p <0.001), marked ** ( p <0.01) indicates a significant difference at the corresponding level, and ns (p>0.05) indicates no significant difference.

[0030] Figure 6 The degradation rate of the insecticidal protein at different times in each treatment of Example 3 is shown. Among them, the difference was analyzed by one-way ANOVA, **** ( p <0.0001),***( p <0.001), **( p <0.01) indicates a significant difference at the corresponding level, and ns (p>0.05) indicates no significant difference.

[0031] Figure 7 The results of the determination of the biological activity of each treatment in Example 3 against fall armyworm are shown. Among them, the difference was analyzed by one-way ANOVA, **** ( p <0.0001) and **( p <0.01) indicated that there were significant differences at the corresponding levels.

[0032] Figure 8 It shows the changes in Yunnan's natural environment from October 18, 2024 to November 01, 2024.

[0033] Fig. 9 Davis scores are shown.

[0034] Fig.10 The field control effects of each treatment in Example 4 are shown. Among them, the difference was analyzed by one-way ANOVA, **** ( p <0.0001),***( p <0.001), **( p<0.01) indicates a significant difference at the corresponding level, and ns (p>0.05) indicates no significant difference.

[0035] Fig.11 The damage levels of corn leaves treated in Example 4 are shown. DETAILED DESCRIPTION

[0036] The above contents of the present invention are further described in detail below in the form of preferred implementation cases, but they do not constitute a limitation to the present invention.

[0037] Unless otherwise specified, the strains, plasmids and reagents in the embodiments of the present invention can be purchased through commercial channels.

[0038] G033A and 32000IU / mg G033A Bacillus thuringiensis engineered bacteria wettable powder (pesticide registration number: PD20171726) are produced by Wuhan Kono Biotechnology Co., Ltd.

[0039] The fall armyworm (Spodoptera frugiperda) was provided by the Institute of Plant Protection, Chinese Academy of Agricultural Sciences.

[0040] Artificial feed formula for fall armyworm larvae: 200 g corn flour, 100 g soybean flour, 90 g yeast powder, 50 g sucrose, 15 g agar, 1.8 g sorbic acid, 1.8 g methyl parahydroxybenzoate (paraben), and 1000 ml water.

[0041] The rearing conditions of Spodoptera frugiperda larvae were as follows: 27 ± 1 degrees Celsius, (65 ± 5)% relative humidity, and 14L:10D light intensity. Example 1: Analysis of the Degradation of 130 kD Protein in G033A WP after UV Irradiation

[0042] The wavelength of ultraviolet light that can penetrate the atmosphere ranges from 290 nm to 400 nm. Based on this, a set of ultraviolet experimental equipment simulating outdoor ultraviolet radiation was customized. Each module contains ultraviolet light sources of 345 nm, 365 nm, 380 nm and 395 nm, and each wavelength can be individually adjusted and switched on and off. At a distance of 30 cm, the illumination intensity of the 345 nm ultraviolet light source is 772 microwatts / square centimeter; the illumination intensity of the 365 nm ultraviolet light source is 1425 microwatts / square centimeter; the illumination intensity of the 380 nm ultraviolet light source is 105 microwatts / square centimeter; and the illumination intensity of the 395 nm ultraviolet light source is 52 microwatts / square centimeter. See the top view of the experimental device. Figure 1 .

[0043] The influence of different ultraviolet wavelengths: Weigh multiple portions of G033A wettable powder at 10 mg / portion, place each portion in a culture dish with a diameter of 6 cm, add 2 mL of ultrapure water and mix well to obtain a bacterial suspension with a final concentration of 5 mg / mL (field application concentration) of G033A wettable powder. Each bacterial suspension was placed 30 cm below four light sources of 345 nm, 365 nm, 380 nm and 395 nm in the ultraviolet experimental device for irradiation for 12 hours. The bacterial suspension that was protected from light for 12 hours was used as a positive control. After irradiation, the water in each bacterial suspension was evaporated, so each treated sample was resuspended with 1 mL of ultrapure water, shaken evenly, and then subjected to SDS-PAGE, see Figure 2 ImageJ software (version 1.42I) was used to analyze the gray value of the 130 kDa insecticidal protein band in the SDS-PAGE electrophoresis results, and the gray value was converted into a peak area value. The protein degradation rate was calculated according to the following formula: degradation rate (%) = (peak area value of positive control treatment-peak area value of UV irradiation treatment) / peak area value of positive control treatment × 100%. The results showed that the 130 kDa insecticidal protein in G033A wettable powder would be degraded to varying degrees under the four different wavelengths of UV irradiation. When irradiated for 12 h, the degradation rate of 130 kDa insecticidal protein was 77.41% at 345 nm, 90.51% at 365 nm, 89.18% at 380 nm, and 68.80% at 395 nm. It can be seen that the degradation degree of insecticidal protein in G033A WP was the highest at 365 nm and 380 nm.

[0044] Effect of different irradiation durations: Weigh 10 mg of G033A WP and place each portion in a 6 cm diameter culture dish. Add 2 mL of ultrapure water and mix well to obtain a bacterial suspension with a final concentration of 5 mg / mL of G033A WP. Each bacterial suspension was placed 30 cm below four different wavelengths of 345 nm, 365 nm, 380 nm and 395 nm in a UV experimental device and irradiated for 4 h, 7 h, 10 h, 13 h and 16 h. After irradiation, the water in each bacterial suspension was evaporated, so each treated sample was resuspended with 1 mL of ultrapure water, shaken evenly, and then subjected to SDS-PAGE and analyzed using ImageJ software (version 1.42I) for the gray value of the 130 kDa insecticidal protein band in the SDS-PAGE electrophoresis results. The gray value was converted into a peak area value, and the degradation rate of the 130 kDa insecticidal protein was calculated using the same formula as above. The degradation rate curves of the 130 kDa insecticidal protein at each wavelength are shown in Figure 2. Figure 3 The results showed that after irradiation for 16 h, the 130 kDa insecticidal protein was completely degraded at wavelengths of 365 nm and 380 nm, the degradation rate of the 130 kDa insecticidal protein was 91.82% at a wavelength of 345 nm, and the degradation rate of the 130 kDa insecticidal protein was 84.67% at a wavelength of 395 nm.

[0045] In summary, the degradation degree of insecticidal protein in G033A WP at 365 nm and 380 nm was higher than that at 345 nm and 395 nm. Example 2: Analysis of the effect of G033A extracellular polysaccharide on the resistance of G033A wettable powder to UV degradation

[0046] Industrial culture medium formula: 5wt% starch, 0.5wt% corn steep liquor, 1wt% peptone, 0.1wt% calcium chloride, 0.3wt% potassium dihydrogen phosphate, 0.1wt% magnesium sulfate, and the balance is water.

[0047] G033A was cultured in LB medium, crude exopolysaccharides of G033A were extracted based on the procedure of Example 1 of CN2024110563571, and crude exopolysaccharides (crude sugar) of G033A were quantified based on the phenol-sulfuric acid method.

[0048] G033A was cultured in industrial culture medium, crude exopolysaccharides of G033A were extracted based on the operation of Example 1 of CN2024110563571, and crude exopolysaccharides of G033A were quantified based on the phenol-sulfuric acid method.

[0049] Weigh several portions of G033A wettable powder at 10 mg / portion, place each portion in a culture dish with a diameter of 6 cm, add G033A crude extracellular polysaccharide extracted from LB culture product, add 2 mL of ultrapure water, mix well, make the final concentration of G033A wettable powder to 5 mg / mL, the final concentration of G033A crude extracellular polysaccharide to 5 mg / mL, prepare bacterial agent and crude sugar mixed solution 1#; make the final concentration of G033A wettable powder to 5 mg / mL, the final concentration of G033A crude extracellular polysaccharide to 10 mg / mL, prepare bacterial agent and crude sugar mixed solution 2#; make the final concentration of G033A wettable powder to 5 mg / mL, the final concentration of G033A crude extracellular polysaccharide to 20 mg / mL, prepare bacterial agent and crude sugar mixed solution 3#.

[0050] Weigh several portions of G033A wettable powder at 10 mg / portion, place each portion in a culture dish with a diameter of 6 cm, add G033A crude extracellular polysaccharide extracted from the culture product of industrial culture medium, add 2 mL of ultrapure water, mix well, make the final concentration of G033A wettable powder to be 5 mg / mL, the final concentration of G033A crude extracellular polysaccharide to be 5 mg / mL, and prepare bacterial agent crude sugar mixed solution 4#; make the final concentration of G033A wettable powder to be 5 mg / mL, the final concentration of G033A crude extracellular polysaccharide to be 10 mg / mL, and prepare bacterial agent crude sugar mixed solution 5#; make the final concentration of G033A wettable powder to be 5 mg / mL, the final concentration of G033A crude extracellular polysaccharide to be 20 mg / mL, and prepare bacterial agent crude sugar mixed solution 6#.

[0051] The mixture of inoculum and crude sugar 1# to 6# was placed in a UV experimental device at 365 nm wavelength and irradiated at 30 cm for 12 hours. The bacterial suspension treated in dark for 12 hours was used as a positive control, and the bacterial suspension without crude sugar and with a final concentration of G033A wettable powder of 5 mg / mL was irradiated at 365 nm wavelength and 30 cm for 12 hours as a negative control. After treatment, each treated sample was suspended in 1 mL of ultrapure water, shaken evenly, and then subjected to SDS-PAGE analysis. Figure 4 , and the degradation rate of 130 kDa insecticidal protein was calculated based on the software and formula in Example 1. The results showed that the degradation rate of 130 kDa protein in the negative control was 99.25%, the degradation rates of bacterial agent crude sugar mixture 1# to 3# were 90.59%, 81.48% and 64.96% respectively; the degradation rates of bacterial agent crude sugar mixture 4# to 6# were 71.02%, 67.28% and 56.06% respectively. The above results show that crude exopolysaccharides have a protective effect on insecticidal proteins. However, the crude exopolysaccharides extracted from industrial culture medium fermentation broth have better UV resistance than the crude exopolysaccharides extracted from LB culture medium fermentation broth.

[0052] The bacterial agent and crude sugar mixture 4# to 6# were placed in an ultraviolet experimental device and irradiated at 30 cm at a wavelength of 365 nm for 4h, 7h, 10h, 13h and 16h. The bacterial suspension treated in the dark for the same time (4h, 7h, 10h, 13h and 16h) was used as a positive control, and the bacterial suspension without adding crude sugar and with a final concentration of 5 mg / mL of G033A wettable powder was irradiated at 30 cm at a wavelength of 365 nm for 4h, 7h, 10h, 13h and 16h as a negative control. After treatment, each treated sample was suspended in 1 mL of ultrapure water, shaken evenly, and then subjected to SDS-PAGE analysis and the degradation of 130 kDa insecticidal protein was calculated based on the software and formula in Example 1. The results are shown in Table 1. Figure 5. The results showed that after 4 h of ultraviolet treatment at 365 nm, the degradation rate of 130 kD insecticidal protein in the negative control was 67.95%; the degradation rate of 130 kDa insecticidal protein in the bacterial agent and crude sugar mixture 4# was 54.46%; the degradation rate of 130 kDa insecticidal protein in the bacterial agent and crude sugar mixture 5# was 47.25%; the degradation rate of 130 kDa insecticidal protein in the bacterial agent and crude sugar mixture 6# was 17.88%, and the content of undegraded 130 kD insecticidal protein was 2.6 times that of the negative control. In addition, for the negative control group, the 130 kD insecticidal protein was completely degraded after 16 h of ultraviolet treatment; while the degradation rate of 130 kDa protein in the bacterial agent and crude sugar mixture 4# was 91.72%; the degradation rate of 130 kDa protein in the bacterial agent and crude sugar mixture 5# was 72.69%; and the protein degradation rate of the bacterial agent and crude sugar mixture 6# was reduced to 56.37%. This indicates that crude extracellular polysaccharide of G033A can significantly reduce the degradation of 130 kDa insecticidal protein in wettable powder by ultraviolet light under ultraviolet irradiation, and the higher the concentration of crude extracellular polysaccharide, the stronger its protective ability against insecticidal protein. Example 3: Analysis of the effect of G033A extracellular polysaccharide on the UV degradation resistance of G033A wettable powder in Yunnan

[0053] From October 18 to 24, 2024, the average UV intensity of outdoor sunlight in Yunnan at 12:00 noon was 4542 microwatts / square centimeter, and the maximum UV intensity was 5140 microwatts / square centimeter. Among them, the highest UV intensity in Yunnan is more than three times that in Beijing.

[0054] Weigh 8 portions of G033A wettable powder at 200 mg / portion, and place each portion in a 9 cm diameter culture dish. Add 4 mL of 100 mg / mL crude exopolysaccharide (extracted from industrial culture medium fermentation broth) solution to each of 4 portions, mix well, and the final concentration of the powder is 50 mg / mL, as the mixed irradiation group; add 4 mL of ultrapure water to each of the other 4 portions, mix well, and make the final concentration of the powder 50 mg / mL, as the powder control group-1. From October 18 to 24, 2024, the samples were placed in outdoor sunlight in the high ultraviolet area of ​​Yunnan. Both the treatment group and the control group were irradiated for 1 day, 2 days, 5 days and 7 days. The treated samples were suspended again with 4 mL of ultrapure water to make the final concentration of the powder 50 mg / mL, and then divided into two portions. One portion was used for the degradation rate determination of 130 kDa insecticidal protein; the other portion was used for the biological activity determination of fall armyworm.

[0055] Take 4 4 ​​mL of 50 mg / mL bacterial suspension and treat it in the dark for 1 day, 2 days, 5 days and 7 days as the powder control group-2 for the bioactivity test of fall armyworm. Take 4 4 ​​mL of 100 mg / mL crude exopolysaccharide solution and place it in a 9 cm diameter culture dish. From October 18 to 24, 2024, place it in the outdoor sunlight in the high ultraviolet area of ​​Yunnan for 1 day, 2 days, 5 days and 7 days as the crude exopolysaccharide control group for the bioactivity test of fall armyworm.

[0056] Degradation rate of 130 kDa insecticidal protein: A sample from the mixed irradiation group and the powder control group-1 was diluted with ultrapure water to a final powder concentration of 10 mg / mL, and then SDS-PAGE analysis was performed and the degradation rate of 130 kDa insecticidal protein was calculated based on the software and formula in Example 1. The results are shown in Figure 6 The results showed that after one day of outdoor sunlight treatment, the degradation rate of 130 kD insecticidal protein in the powder control group-1 was 68.33%, and the protein degradation rate in the mixed irradiation group was 50.86%; after 7 days of outdoor sunlight treatment, the protein degradation rate of 130 kD protein in the powder control group-1 was 92.67%, and the protein degradation rate in the mixed irradiation group was 81.99%. The above results once again show that crude extracellular polysaccharides can significantly reduce the degradation of 130 kDa insecticidal protein by ultraviolet rays.

[0057] Determination of biological activity against fall armyworm: The artificial diet of fall armyworm larvae was dispensed into culture dishes at a dosage of 15 g / dish. The samples in the mixed irradiation group were added to the artificial feed and mixed evenly to make the concentration of G033A wettable powder 200 μg / g and the concentration of crude extracellular polysaccharide 400 μg / g. This feed group was called the mixed irradiation group; the mixed samples of the powder control group-1 and the crude extracellular polysaccharide control group were added to the artificial feed and mixed evenly to make the concentration of G033A wettable powder 200 μg / g and the concentration of crude extracellular polysaccharide 400 μg / g. This feed group was called the single irradiation group; the samples in the powder control group-1 were added to the artificial feed and mixed evenly to make the concentration of G033A wettable powder 200 μg / g. This feed group was called the powder control group-1; the samples in the powder control group-2 were added to the artificial feed and mixed evenly to make the concentration of G033A wettable powder 200 μg / g. This feed group was called the powder control group-2. After evaporating appropriate amounts of water from each test feed group, the corresponding test samples were obtained and evenly distributed in a 24-well plate. Then, one newly hatched larva of Spodoptera frugiperda was inoculated into each well and covered with a paper towel. Each treatment was repeated 3 times, with 24 newly hatched larvae in each repeat. The sample in which only 3 ml of ultrapure water was added to the artificial feed was used as a negative control. The larvae were observed every day, and the number of dead insects was counted after 7 days. The corrected mortality rate was calculated based on the negative control. The results are shown in Figure 7. The results showed that in the samples of 1 day, the corrected mortality of fall armyworm in the powder control group-2 was 85.33%, the corrected mortality of fall armyworm in the mixed irradiation group was 63.49%, the corrected mortality of fall armyworm in the single irradiation group was 43.93%, and the corrected mortality of fall armyworm in the powder control group-1 was 42.86%. It can be seen that the corrected mortality of fall armyworm in the single irradiation group was equivalent to that of the powder control group-1, and was significantly lower than that of the mixed irradiation group. The trends of samples on other days were consistent with this. In summary, G033A crude extracellular polysaccharide can effectively reduce the degradation of G033A wettable powder protein by sunlight, which is conducive to the G033A wettable powder to still exert its efficacy better after strong sunlight irradiation. Example 4: Field control effect in Yunnan

[0058] Experimental location: Experimental base of the Institute of Plant Protection, Academy of Agricultural Sciences, Jiangcheng District, Pu'er City, Yunnan Province (N: 22°55′, E: 101°49′). The experimental site is flat, and the soil type is loam with medium fertility.

[0059] The corn variety planted was XianDa. When the corn growth period was the five-leaf stage, that is, on October 18, 2024, 300g / mu of G033A Bacillus thuringiensis engineered bacteria wettable powder and 300g / mu of G033A crude extracellular polysaccharide (extracted from industrial culture medium fermentation broth) were applied to the treatment group; only 300g / mu of G033A Bacillus thuringiensis engineered bacteria wettable powder was applied as control-1; and neither G033A Bacillus thuringiensis engineered bacteria wettable powder nor G033A crude extracellular polysaccharide was applied as control-2. During the entire period, the water and fertilizer management of all treatments was the same, and no other pesticides were applied. Each treatment was replicated three times, and each replicate was 30 m 2 The width of the different treatments was set at about 1.5 m, that is, the area of ​​the plot was 30 m 2 , protect the row spacing of 1.5m. Record the weather conditions every day during the treatment period, see Figure 8 From October 18 to November 1, 2024, the weather was mostly sunny, with temperatures between 25 and 30°C and no large-scale rainfall. The number of live fall armyworms in each treatment was investigated using a five-point sampling method at 1 d, 3 d, 5 d, 7 d, 10 d, and 14 d after application. The damage to corn leaves in each treatment was investigated using a five-point sampling method at 0 d, 1 d, 3 d, 5 d, 7 d, 10 d, and 14 d after application, with a focus on the trumpet and sheath areas. Ten plants were investigated at each point. The insect population reduction rate (%) and control effect (%) of each treatment were calculated according to formulas (1) and (2); the damage to corn leaves was calculated according to Fig. 9 The Davis score was used to analyze the leaf damage classification. The results of the control effect are shown in Fig.10; The damage level of corn leaves is shown in Fig.11 . The formula is as follows: Insect population reduction rate = (number of live insects before application - number of live insects after application) × 100% / number of live insects before application Formula (1) Control effect = (insect population reduction rate of treatment group - insect population reduction rate of control group) × 100% / (1 - insect population reduction rate of control group) Formula (2)

[0060] according to Fig.10 The results showed that the control-1 (only wettable powder was applied) achieved the highest control effect of 77.84% on the 5th day, and began to decline on the 7th day. The control effect was only 45.88% after 14 days. After adding crude sugar, the control effect was higher than that of the treatment with only wettable powder at 6 time points, and reached the highest control effect of 89.46% on the 7th day. Compared with the treatment with only wettable powder, the control effect was increased by 11.62%, and began to decline on the 7th day. The control effect was 65.33% at 14 days, which was not significantly different from the control effect of only using wettable powder for 7 days, and the effective period was extended by 7 days.

[0061] according to Fig.11 The results show that the leaf damage of the control group-2 (neither wettable powder nor crude sugar was applied) continued to worsen, and by the 14th day, the leaf damage level was close to 9 (the highest damage level). The leaf damage level of the control group-1 (only wettable powder was applied) was reduced to 6.55 on the 14th day; the leaf damage level of the treatment group was further reduced to 5.17 on the 14th day.

Claims

1. Use of an exopolysaccharide or an extract containing the exopolysaccharide in improving the resistance of insecticidal proteins to sunlight or ultraviolet degradation, wherein the exopolysaccharide or the extract is extracted from Bacillus thuringiensis G033A; and / or The insecticidal protein is expressed by Bacillus thuringiensis G033A.

2. The use according to claim 1, characterized in that: The extract is obtained by extracting through the following steps: 1) adjusting the pH value of the fermentation broth of Bacillus thuringiensis G033A to 8 to 9, then performing a first centrifugation, and collecting a first supernatant after the first centrifugation; 2) adding trichloroacetic acid or an aqueous solution of trichloroacetic acid to the first supernatant, thereby treating the first supernatant with the trichloroacetic acid to obtain a first treated liquid, and then performing a second centrifugation to collect a second supernatant after the second centrifugation; 3) After adjusting the pH value of the second supernatant to 6 to 7, ethanol is added to the second supernatant to obtain a second treatment liquid, the second treatment liquid is precipitated, and then a third centrifugation is performed, and the precipitate after the third centrifugation is collected, and the precipitate is the wet extract containing extracellular polysaccharides.

3. The use according to claim 1, characterized in that: The culture medium for culturing the Bacillus thuringiensis G033A is one of an industrial culture medium, an LB culture medium, a sugar-producing culture medium with glucose as a substrate, and a sugar-producing culture medium with sucrose as a substrate.

4. The use according to claim 2, characterized in that: In step 2), the mass / volume content of the trichloroacetic acid in the first supernatant is 0.4% to 0.5%; and / or In step 3), the volume of ethanol added to the second supernatant is 3 to 3.5 times the volume of the second supernatant.

5. The use according to claim 2, characterized in that: In step 2), the first treatment solution is placed at 25 to 35° C. for 1.5 to 3 hours; and / or In step 3), the second treatment solution is placed at 2 to 8° C. for 12 to 20 hours.

6. A composition, characterized in that The invention comprises the extracellular polysaccharide or the extract in the application as claimed in any one of claims 1 to 5, and an insecticidal active component, wherein the insecticidal active component is a fermentation product of Bacillus thuringiensis or an insecticidal protein derived from Bacillus thuringiensis.

7. The composition according to claim 6, characterized in that The insecticidal active component is a fermentation product of the G033A strain.

8. The composition according to claim 7, characterized in that The insecticidal active ingredient is contained in the G033A wettable powder.

9. The composition according to claim 8, characterized in that The mass ratio of the extract to the G033A wettable powder is 1:1 to 3:

1.

10. Use of the composition according to any one of claims 6 to 9 in controlling fall armyworm.

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