Application of exopolysaccharide or extract containing exopolysaccharide in enhancing insecticidal protein against sunlight or ultraviolet degradation

Abstract

The present invention relates to the application of exopolysaccharides or extracts containing exopolysaccharides in enhancing the resistance of insecticidal proteins to sunlight or ultraviolet degradation. The present invention discovers that the extract containing exopolysaccharides extracted from Bacillus thuringiensis G033A can significantly reduce the degradation degree of the insecticidal protein of Bacillus thuringiensis after being irradiated by ultraviolet or sunlight, and the higher the content of the extract, the stronger the protective ability for the insecticidal protein. This is beneficial for the wettable powder of G033A to still exert its efficacy well after being irradiated by sunlight and to extend the effective period of the wettable powder of G033A.

Description

Technical Field

[0001] The present invention relates to the field of biocides, and particularly to the application of extracellular polysaccharides or extracts containing extracellular polysaccharides in enhancing 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 long-term efficacy of the product and the control effect in the field. The ultraviolet rays in natural sunlight are divided into three types: weak-effect wavelength UVA (wavelength 320 to 400 nm), strong-effect wavelength UVB (wavelength 290 to 320 nm), and ultra-strong-effect wavelength UVC (wavelength 190 to 280 nm). UVC is almost absorbed by the atmospheric ozone layer and rarely reaches the ground. Therefore, it is mainly UVA and UVB that cause the degradation of Bt insecticidal crystal proteins.

[0003] In order to protect Bt from the influence of the external environment, extend its long-term efficacy in the field, and enhance the ultraviolet resistance of Bt, it is a very crucial factor. The current methods mainly include the following aspects: 1) adding adjuvants, such as adding biochar can improve the ultraviolet resistance of Cry1Ac protein; 2) changing the dosage form, such as in CN202111025625X, preparing the Bt preparation into a granule.

[0004] Many strains of Bacillus thuringiensis can produce extracellular polysaccharides, and the uses of its extracellular polysaccharides remain to be further developed.

[0005] When preparing a Bacillus thuringiensis preparation, generally, after liquid fermentation, the bacteria produced by liquid fermentation are collected and then further prepared into a preparation form. During this process, the fermentation supernatant after collecting the bacteria is discarded. Since extracellular polysaccharides are secreted into the fermentation supernatant during fermentation, it is also regarded as waste and discarded, which greatly causes waste of extracellular polysaccharides. And 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 aspect of the present invention provides the application of extracellular polysaccharides or extracts containing the extracellular polysaccharides in enhancing the resistance of insecticidal proteins to sunlight or ultraviolet degradation.

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

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

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

[0010] In a specific embodiment, the exopolysaccharide or the extract is obtained by extracting 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:

[0013] 1) Adjust the pH value of the fermentation broth of Bacillus thuringiensis G033A to 8 to 9, then perform the first centrifugation, and collect the first supernatant after the first centrifugation;

[0014] 2) Add 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 treatment solution, then perform the second centrifugation, and collect the second supernatant after the second centrifugation;

[0015] 3) Adjust the pH value of the second supernatant to 6 to 7, then add ethanol to the second supernatant to obtain a second treatment solution, precipitate the second treatment solution, then perform the third centrifugation, and collect the precipitate after the third centrifugation. The precipitate is the wet extract containing exopolysaccharide.

[0016] In a specific embodiment, the medium for culturing Bacillus thuringiensis G033A is one of an industrial medium, an LB medium, a sugar-producing medium with glucose as a substrate, and a sugar-producing medium with sucrose as a substrate.

[0017] 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%.

[0018] 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.

[0019] In a specific embodiment, in step 2), the first treatment solution is placed at 25 to 35 °C for 1.5 to 3 hours.

[0020] In a specific embodiment, in step 3), the second treatment solution is placed at 2 to 8 °C for 12 to 20 hours.

[0021] In a specific embodiment, the exopolysaccharide is obtained by purifying the extract through residual protein removal, desalting, anion exchange chromatography, and gel filtration chromatography; the weight-average molecular weight of the exopolysaccharide is 71.484 kDa, the number-average molecular weight of the exopolysaccharide is 66.625 kDa, the average molecular weight of the exopolysaccharide is 76.316 kDa, the polydispersity coefficient of the exopolysaccharide is 1.073. Taking the total sugar content of the exopolysaccharide as 100%, mannose is 78.52%, glucose is 12.19%, arabinose is 4.41%, glucuronic acid is 3.53%, and galactose is 1.34%.

[0022] The second aspect of the present invention provides a composition, which includes the exopolysaccharide or the extract in any one of the applications described in the first aspect of the present invention, and an insecticidal active component, and the insecticidal active component is a fermentation product of Bacillus thuringiensis or an insecticidal protein derived from Bacillus thuringiensis. Among them, the fermentation product of Bacillus thuringiensis includes an insecticidal protein derived from Bacillus thuringiensis.

[0023] In a specific embodiment, the insecticidal active component is a fermentation product of strain G033A.

[0024] In a specific embodiment, the insecticidal active component is contained in a wettable powder of G033A.

[0025] In a specific embodiment, the mass ratio of the extract to the wettable powder of G033A is 1:1 to 3:1.

[0026] The third aspect of the present invention provides the application of the composition described in the second aspect of the present invention in controlling Spodoptera frugiperda.

[0027] The beneficial effects of the present invention: The present invention discovers that the extract containing exopolysaccharide (i.e., crude exopolysaccharide) extracted from Bacillus thuringiensis G033A can significantly reduce the degradation degree of the insecticidal protein of Bacillus thuringiensis after being irradiated by ultraviolet or sunlight, and the higher the content of the crude exopolysaccharide, the stronger the protection ability for the insecticidal protein. This is beneficial for the wettable powder of G033A to still play its efficacy well after being irradiated by sunlight and extends the effective period of the wettable powder of G033A. Description of the Drawings

[0028] Figure 1 Shows a bottom view schematic diagram of the experimental device for simulating outdoor ultraviolet irradiation.

[0029] Figure 2 Shows the SDS-PAGE electrophoresis results of the insecticidal protein after the wettable powder of G033A in Example 1 is irradiated at four ultraviolet wavelengths for 12 h. Among them, the sample loading amount of the powder in each lane is the same.

[0030] Figure 3 Shows the degradation rate curves of the insecticidal protein in Example 1 Wettable Powder G033A after irradiation at four ultraviolet wavelengths for different times.

[0031] Figure 4 Shows the SDS-PAGE of the crude sugar mixture of the microbial agent in Example 2 after 12 h of ultraviolet irradiation, where the sample loading amount of the wettable powder is the same in each lane.

[0032] Figure 5 Shows the degradation rate of the insecticidal protein in the crude sugar mixture of the microbial agent in Example 2 after ultraviolet irradiation for different times. Among them, one-way ANOVA was used to analyze the differences, **** ( 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.

[0033] Figure 6 Shows the degradation rate of the insecticidal protein in each treatment of Example 3 at different times. Among them, one-way ANOVA was used to analyze the differences, **** ( 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.

[0034] Figure 7 Shows the determination results of the biological activity of each treatment in Example 3 against Spodoptera frugiperda. Among them, one-way ANOVA was used to analyze the differences, **** ( p <0.0001) and ** ( p <0.01) indicate a significant difference at the corresponding level.

[0035] Figure 8 Shows the changes in the natural environment in Yunnan from October 18, 2024 to November 1, 2024.

[0036] Figure 9 Shows the Davis score.

[0037] Figure 10 Shows the field control effects of each treatment in Example 4. Among them, one-way ANOVA was used to analyze the differences, **** ( 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.

[0038] Figure 11 Shows the damage level of corn leaves in each treatment of Example 4. Detailed implementation mode

[0039] The above content of the present invention will be further described in detail below in the form of preferred implementation cases, but it does not constitute a limitation to the present invention.

[0040] Unless otherwise specified, the strains, plasmids and reagents in the embodiments of the present invention can be purchased through commercial channels.

[0041] The wettable powder of Bacillus thuringiensis engineering bacteria G033A and 32000 IU / mg G033A (pesticide registration number: PD20171726) is produced by Wuhan Kenuo Biotechnology Co., Ltd.

[0042] The tested Spodoptera frugiperda is provided by the Institute of Plant Protection, Chinese Academy of Agricultural Sciences.

[0043] Artificial feed formula for Spodoptera frugiperda larvae: 200 g of corn flour, 100 g of soybean flour, 90 g of yeast powder, 50 g of sucrose, 15 g of agar, 1.8 g of sorbic acid, 1.8 g of methyl p-hydroxybenzoate (Nipagin), 1000 ml of water.

[0044] The feeding conditions for Spodoptera frugiperda larvae are as follows: 27 ± 1 °C, (65 ± 5)% relative humidity, 14L:10D light.

[0045] Example 1: Analyze the degradation degree of the 130 kD protein in the G033A wettable powder after being irradiated by ultraviolet light waves

[0046] The wavelength range of ultraviolet light passing through the atmosphere is 290 nm to 400 nm. Based on this, a set of ultraviolet experimental devices for simulating outdoor ultraviolet irradiation is customized. Each module contains ultraviolet light sources of 345 nm, 365 nm, 380 nm and 395 nm, and the switches of each wavelength can be adjusted separately. At a distance of 30 cm, the light intensity of the 345 nm ultraviolet light source is 772 μW / cm²; the light intensity of the 365 nm ultraviolet light source is 1425 μW / cm²; the light intensity of the 380 nm ultraviolet light source is 105 μW / cm²; the light intensity of the 395 nm ultraviolet light source is 52 μW / cm². The top view of the experimental device is shown in Figure 1 .

[0047] Effect of different UV wavelengths: Weigh multiple portions of wettable powder G033A at 10 mg / portion, place each portion in a petri dish with a diameter of 6 cm, and then add 2 mL of ultrapure water to each and mix well to obtain a bacterial suspension with a final concentration of 5 mg / mL of wettable powder G033A (field spraying concentration). Place each bacterial suspension 30 cm below the light sources of four different wavelengths, namely 345 nm, 365 nm, 380 nm, and 395 nm, in the UV experimental device for irradiation treatment for 12 h. Use the bacterial suspension treated in the dark for 12 h as the positive control. After irradiation, the water in each bacterial suspension has evaporated, so each treated sample is resuspended with 1 mL of ultrapure water again, shaken evenly, and then SDS-PAGE is performed. See Figure 2 . Use ImageJ software (version 1.42I) to analyze the gray value of the 130 kDa insecticidal protein band in the SDS-PAGE electrophoresis result, convert the gray value into a peak area value, and calculate the protein degradation rate according to the following formula: Degradation rate (%) = (peak area value of the positive control treatment - peak area value of the UV irradiation treatment) / peak area value of the positive control treatment × 100%. The results show that the 130 kDa insecticidal protein in wettable powder G033A will be degraded to varying degrees under the irradiation of four different wavelengths of UV alone. At 12 h of irradiation, the degradation rate of the 130 kDa insecticidal protein at 345 nm wavelength is 77.41%, at 365 nm wavelength is 90.51%, at 380 nm wavelength is 89.18%, and at 395 nm wavelength is 68.80%. It can be seen that the degradation degree of the insecticidal protein in wettable powder G033A is the highest at 365 nm and 380 nm.

[0048] Effect of different irradiation durations: Weigh multiple portions of wettable powder G033A at 10 mg, place each portion in a petri dish with a diameter of 6 cm, and then add 2 mL of ultrapure water to each and mix well to obtain a bacterial suspension with a final concentration of 5 mg / mL of wettable powder G033A. Place each bacterial suspension 30 cm below the light sources of four different wavelengths, namely 345 nm, 365 nm, 380 nm, and 395 nm, in the UV experimental device for irradiation treatment for 4 h, 7 h, 10 h, 13 h, and 16 h. After irradiation, the water in each bacterial suspension has evaporated, so each treated sample is resuspended with 1 mL of ultrapure water again, shaken evenly, and then SDS-PAGE is performed and ImageJ software (version 1.42I) is used to analyze the gray value of the 130 kDa insecticidal protein band in the SDS-PAGE electrophoresis result, convert the gray value into a peak area value, and calculate the degradation rate of the 130 kDa insecticidal protein using the same formula as above. The degradation rate curves of the 130 kDa insecticidal protein at each wavelength are shown inFigure 3 The results showed that when irradiated 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 345 nm wavelength and 84.67% at 395 nm wavelength.

[0049] In summary, the degradation degree of the insecticidal protein in the G033A wettable powder was higher at 365 nm and 380 nm than at 345 nm and 395 nm.

[0050] Example 2: Analyze the effect of G033A exopolysaccharide on the anti-ultraviolet degradation of G033A wettable powder

[0051] Industrial medium formula: starch 5 wt%, corn steep liquor 0.5 wt%, peptone 1 wt%, calcium chloride 0.1 wt%, potassium dihydrogen phosphate 0.3 wt%, magnesium sulfate 0.1 wt%, and the balance is water.

[0052] G033A was cultured in LB medium, and the crude exopolysaccharide of G033A was extracted based on the operation in Example 1 of CN2024110563571, and the crude exopolysaccharide of G033A (crude sugar) was quantified based on the phenol-sulfuric acid method.

[0053] G033A was cultured in industrial medium, and the crude exopolysaccharide of G033A was extracted based on the operation in Example 1 of CN2024110563571, and the crude exopolysaccharide of G033A was quantified.

[0054] Multiple portions of G033A wettable powder were weighed at 10 mg per portion, and each portion was placed in a 6 cm diameter petri dish. The crude exopolysaccharide of G033A extracted from the LB culture product was added, and then 2 mL of ultrapure water was added and mixed evenly to make the final concentration of G033A wettable powder 5 mg / mL and the final concentration of crude exopolysaccharide of G033A 5 mg / mL, obtaining the crude sugar mixture of the bacterial agent 1#; making the final concentration of G033A wettable powder 5 mg / mL and the final concentration of crude exopolysaccharide of G033A 10 mg / mL, obtaining the crude sugar mixture of the bacterial agent 2#; making the final concentration of G033A wettable powder 5 mg / mL and the final concentration of crude exopolysaccharide of G033A 20 mg / mL, obtaining the crude sugar mixture of the bacterial agent 3#.

[0055] Weigh multiple portions of wettable powder G033A at 10 mg per portion, place each portion in a petri dish with a diameter of 6 cm, add the crude extracellular polysaccharide G033A extracted from the industrial culture medium product, then add 2 mL of ultrapure water, mix well to make the final concentration of wettable powder G033A 5 mg / mL and the final concentration of crude extracellular polysaccharide G033A 5 mg / mL, thus preparing the crude sugar mixture of the bacterial agent 4#; make the final concentration of wettable powder G033A 5 mg / mL and the final concentration of crude extracellular polysaccharide G033A 10 mg / mL, thus preparing the crude sugar mixture of the bacterial agent 5#; make the final concentration of wettable powder G033A 5 mg / mL and the final concentration of crude extracellular polysaccharide G033A 20 mg / mL, thus preparing the crude sugar mixture of the bacterial agent 6#.

[0056] Place the crude sugar mixtures of the bacterial agent 1# to 6# at a distance of 30 cm from the ultraviolet experimental device at a wavelength of 365 nm and irradiate for 12 h. Use the bacterial suspension treated in the dark for 12 h as the positive control, and use the bacterial suspension without adding crude sugar and with a final concentration of wettable powder G033A of 5 mg / mL irradiated at a distance of 30 cm from the ultraviolet experimental device at a wavelength of 365 nm for 12 h as the negative control. After the treatment, each treated sample is suspended in 1 mL of ultrapure water, shaken evenly, and then subjected to SDS-PAGE analysis, as shown in Figure 4 , and calculate the degradation rate of the 130 kDa insecticidal protein based on the software and formula in Example 1. The results show that the degradation rate of the 130 kDa protein in the negative control is 99.25%, and the degradation rates of the crude sugar mixtures of the bacterial agent 1# to 3# are 90.59%, 81.48%, and 64.96% respectively; the degradation rates of the crude sugar mixtures of the bacterial agent 4# to 6# are 71.02%, 67.28%, and 56.06% respectively. The above results indicate that the crude extracellular polysaccharide has a protective effect on the insecticidal protein. However, the crude extracellular polysaccharide extracted from the industrial culture medium fermentation broth has better ultraviolet resistance than the crude extracellular polysaccharide extracted from the LB culture medium fermentation broth.

[0057] Place the crude sugar mixtures of the bacterial agent 4# to 6# at a distance of 30 cm from the ultraviolet experimental device at a wavelength of 365 nm and irradiate for 4 h, 7 h, 10 h, 13 h, and 16 h. Use the bacterial suspension treated in the dark for the same time (4 h, 7 h, 10 h, 13 h, and 16 h) as the positive control, and use the bacterial suspension without adding crude sugar and with a final concentration of wettable powder G033A of 5 mg / mL irradiated at a distance of 30 cm from the ultraviolet experimental device at a wavelength of 365 nm for 4 h, 7 h, 10 h, 13 h, and 16 h as the negative control. After the treatment, each treated sample is suspended in 1 mL of ultrapure water, shaken evenly, and then subjected to SDS-PAGE analysis and calculate the degradation of the 130 kDa insecticidal protein based on the software and formula in Example 1. The results are shown in Figure 5The results showed that after 4 h of ultraviolet treatment at 365 nm, the degradation rate of the 130 kD insecticidal protein in the negative control was 67.95%; the degradation rate of the 130 kDa insecticidal protein in the crude sugar mixture 4# of the bacterial agent was 54.46%; the degradation rate of the 130 kDa insecticidal protein in the crude sugar mixture 5# of the bacterial agent was 47.25%; the degradation rate of the 130 kDa insecticidal protein in the crude sugar mixture 6# of the bacterial agent was 17.88%, and the content of the 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 the 130 kDa protein in the crude sugar mixture 4# of the bacterial agent was 91.72%; the degradation rate of the 130 kDa protein in the crude sugar mixture 5# of the bacterial agent was 72.69%; the degradation rate of the protein in the crude sugar mixture 6# of the bacterial agent decreased to 56.37%. This indicates that the extracellular polysaccharide G033A can significantly reduce the degradation of the 130 kDa insecticidal protein in the wettable powder by ultraviolet light under ultraviolet irradiation, and the higher the concentration of the extracellular polysaccharide, the stronger the protective ability for the insecticidal protein.

[0058] Example 3: Analyze the effect of extracellular polysaccharide of G033A in Yunnan region on the anti-ultraviolet degradation of G033A wettable powder

[0059] From October 18th to 24th, 2024, the average ultraviolet intensity at noon in Yunnan outdoors was 4542 μW / cm², and the highest ultraviolet intensity was 5140 μW / cm². Among them, the highest ultraviolet intensity in Yunnan was more than 3 times that in Beijing.

[0060] Weigh 8 portions of G033A wettable powder at 200 mg / portion, and place each portion in a petri dish with a diameter of 9 cm. Among them, add 4 mL of 100 mg / mL extracellular polysaccharide (extracted from industrial culture medium fermentation broth) solution to 4 portions, mix well, and the final concentration of the powder is 50 mg / mL, serving as the mixed irradiation group; add 4 mL of ultrapure water to the other 4 portions, mix well, and make the final concentration of the powder 50 mg / mL, serving as the powder control group - 1. From October 18th to 24th, 2024, place the samples under outdoor sunlight in the high-ultraviolet region of Yunnan. Irradiate both the treatment group and the control group for 1 day, 2 days, 5 days, and 7 days. Suspend the treated samples with 4 mL of ultrapure water again to make the final concentration of the powder 50 mg / mL, and then divide them into two parts. One part is used for the determination of the degradation rate of the 130 kDa insecticidal protein; the other part is used for the bioactivity determination against Spodoptera frugiperda.

[0061] Take 4 samples of 4 mL of 50 mg / mL bacterial suspension and treat them in the dark for 1 day, 2 days, 5 days, and 7 days as the powder control group - 2 for bioactivity determination of Spodoptera frugiperda. Take 4 samples of 4 mL of extracellular polysaccharide solution with a concentration of 100 mg / mL and place them in a 9 cm diameter Petri dish. Expose them to outdoor sunlight in a high-ultraviolet area in Yunnan from October 18th to 24th, 2024 for 1 day, 2 days, 5 days, and 7 days as the extracellular polysaccharide control group for bioactivity determination of Spodoptera frugiperda.

[0062] Degradation rate of 130 kDa insecticidal protein: Dilute one sample from the mixed irradiation group and the powder control group - 1 with ultrapure water to make the final concentration of the powder 10 mg / mL. Then perform SDS-PAGE analysis and calculate the degradation rate of 130 kDa insecticidal protein based on the software and formula in Example 1. The results are shown in Figure 6 . The results show that when treated with outdoor sunlight for 1 d, the degradation rate of 130 kD insecticidal protein in the powder control group - 1 is 68.33%, and the protein degradation rate in the mixed irradiation group is 50.86%. When treated with outdoor sunlight for 7 d, the degradation rate of 130 kD protein in the powder control group - 1 is 92.67%, and the protein degradation rate in the mixed irradiation group is 81.99%. The above results once again indicate that extracellular polysaccharide can significantly reduce the degradation of 130 kDa insecticidal protein by ultraviolet light.

[0063] Bioactivity determination of Spodoptera frugiperda: Divide the artificial diet for Spodoptera frugiperda larvae into Petri dishes at a dosage of 15 g / dish. Add the samples from the above mixed irradiation group to the artificial diet and mix evenly to make the concentration of G033A wettable powder 200 μg / g and the concentration of extracellular polysaccharide 400 μg / g. This feed group is called the mixed irradiation group. Add the mixed samples from the above powder control group - 1 and the extracellular polysaccharide control group to the artificial diet and mix evenly to make the concentration of G033A wettable powder 200 μg / g and the concentration of extracellular polysaccharide 400 μg / g. This feed group is called the single irradiation group. Add the samples from the above powder control group - 1 to the artificial diet and mix evenly to make the concentration of G033A wettable powder 200 μg / g. This feed group is called the powder control group - 1. Add the samples from the above powder control group - 2 to the artificial diet and mix evenly to make the concentration of G033A wettable powder 200 μg / g. This feed group is called the powder control group - 2. After appropriately evaporating the moisture of each test feed group, obtain the corresponding test samples, and evenly distribute each test sample in a 24-well plate. Then inoculate 1 newly hatched larva of Spodoptera frugiperda into each well and cover it with a paper towel. Each treatment is repeated 3 times, and each repetition has 24 newly hatched larvae. Use the sample with only 3 ml of ultrapure water added to the artificial diet as the negative control. Observe the larvae every day, count the number of dead insects after 7 d, and calculate the corrected mortality rate based on the negative control. The results are shown in Figure 7The results showed that in the samples at 1 d, the corrected mortality rate of Spodoptera frugiperda in the powder control group - 2 was 85.33%, that in the mixed irradiation group was 63.49%, that in the single irradiation group was 43.93%, and that in the powder control group - 1 was 42.86%. It can be seen that the corrected mortality rate of Spodoptera frugiperda in the single irradiation group was comparable to that in the powder control group - 1, and was significantly lower than that in the mixed irradiation group. The trends of the samples on other days were the same as this. In summary, the extracellular polysaccharide of G033A can effectively reduce the degradation of the protein of G033A wettable powder by sunlight, and thus is conducive to the better exertion of the efficacy of G033A wettable powder after strong sunlight irradiation.

[0064] Example 4: Control effect in the fields of Yunnan

[0065] Experimental site: The experimental base of the Plant Protection Institute of the Academy of Agricultural Sciences in Jiangcheng District, Pu'er City, Yunnan Province (latitude N: 22°55′, longitude E: 101°49′). The terrain of the experimental field is flat, and the soil type belongs to loam with medium fertility.

[0066] The corn variety planted was Xianda. At the five - leaf stage of the corn growth period, that is, on October 18, 2024, 300 g / mu of the wettable powder of the engineered Bacillus thuringiensis strain G033A and 300 g / mu of the extracellular polysaccharide of G033A (extracted from the industrial culture medium fermentation broth) were applied to the treatment group; only applying 300 g / mu of the wettable powder of the engineered Bacillus thuringiensis strain G033A was used as control - 1; neither applying the wettable powder of the engineered Bacillus thuringiensis strain G033A nor applying the extracellular polysaccharide of G033A was used as control - 2. During the whole period, the water and fertilizer management of all treatments was the same, and no other pesticides were applied. Each treatment had three replicates, and each replicate was 30 m 2 , with a width of about 1.5 m set between different treatments, that is, the plot area was 30 m 2 , and the protective row spacing was 1.5 m. Record the daily weather conditions during the treatment period, as shown in Figure 8 , from October 18 to November 1, 2024, the weather was mostly sunny throughout the period, the temperature was between 25 and 30 °C, and there was no large - scale rainfall. The number of live Spodoptera frugiperda in each treatment was investigated by the 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 by the five - point sampling method at 0 d, 1 d, 3 d, 5 d, 7 d, 10 d, and 14 d after application, focusing on investigating the trumpet - shaped mouth and leaf sheath parts, and 10 plants were investigated at each point. Calculate the reduction rate of the insect population (%) and the control effect (%) of each treatment according to formulas (1) and (2); the damage to corn leaves was analyzed for leaf damage grading according to Figure 9 the Davis score ofFigure 10 ; The hazard level results of corn leaves are shown in Figure 11 . Among them, the formula is as follows:

[0067] Reduction rate of insect population = (number of live insects before application - number of live insects after application) × 100% / number of live insects before application Formula (1)

[0068] Control effect = (reduction rate of insect population in treatment group - reduction rate of insect population in control group) × 100% / (1 - reduction rate of insect population in control group) Formula (2)

[0069] According to Figure 10 the results, it can be seen that Control - 1 (only applying wettable powder) reached the highest control effect on the 5th day, which was 77.84%, started to decline on the 7th day, and the control effect on the 14th day was only 45.88%; while after adding crude sugar, the control effect was higher than that of the treatment only using wettable powder at 6 time points, reached the highest control effect on the 7th day, which was 89.46%, compared with only using wettable powder, the control effect increased by 11.62%, started to decline on the 7th day, and the control effect on the 14th day was 65.33%, showing no significant difference compared with the control effect of only using wettable powder for 7 days, and the effective period was extended by 7 days.

[0070] According to Figure 11 the results, it was found that the leaf damage of Control - 2 (neither applying wettable powder nor crude sugar) continued to increase, and by the 14th day, the leaf damage degree was close to 9 (the highest damage level). For Control - 1 (only applying wettable powder), the leaf damage degree decreased to 6.55 on the 14th day; for the treatment group, the leaf damage degree further decreased to 5.17 on the 14th day.

Claims

1. Use of an extracellular polysaccharide-containing extract in enhancing the resistance of an insecticidal protein to sunlight or ultraviolet degradation, wherein the extracellular polysaccharide-containing extract is obtained by extracting from the fermentation broth of Bacillus thuringiensis G033A; and the insecticidal protein is expressed by Bacillus thuringiensis G033A; the extracellular polysaccharide-containing extract is obtained by the following steps: 1) The Bacillus thuringiensis G033A is cultured with an industrial culture medium to obtain a fermentation broth of the Bacillus thuringiensis G033A. The pH value of the fermentation broth of the Bacillus thuringiensis G033A is adjusted to 8 to 9, and then subjected to a first centrifugation to collect a first supernatant after the first centrifugation; wherein, The formula of the industrial medium is 5 wt% starch, 0.5 wt% corn steep liquor, 1 wt% peptone, 0.1 wt% calcium chloride, 0.3 wt% potassium dihydrogen phosphate, 0.1 wt% magnesium sulfate, and the balance is water; 2) Add 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 treatment solution, and then perform a second centrifugation to collect the second supernatant after the second centrifugation; 3) Adjust the pH value of the second supernatant to 6 to 7, then add ethanol to the second supernatant to obtain a second treatment solution, precipitate the second treatment solution, and then perform a third centrifugation to collect the precipitate after the third centrifugation. The precipitate is the wet extracellular polysaccharide-containing extract.

2. The application according to claim 1, wherein 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.

3. The application according to claim 1, 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.

4. A composition, characterized in that, It includes the extracellular polysaccharide-containing extract in the application as described in any one of claims 1 to 3, and an insecticidal active component, and the insecticidal active component is a fermentation product of Bacillus thuringiensis G033A or an insecticidal protein derived from Bacillus thuringiensis G033A.

5. The composition according to claim 4, wherein The insecticidal active component is contained in the wettable powder of G033A.

6. The composition according to claim 5, wherein The mass ratio of the extracellular polysaccharide-containing extract to the wettable powder of G033A is 1:1 to 3:

1.

7. Use of the composition as described in any one of claims 4 to 6 in controlling Spodoptera frugiperda.

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