Mesoporous silica Bt pesticide capable of producing active oxygen and application of mesoporous silica Bt pesticide to plutella xylostella

Abstract

The invention relates to a nano pesticide prepared by compounding mesoporous silica with Bt raw powder and a preparation method of the nano pesticide, and belongs to the technical field of nano biopesticides. The preparation method comprises the steps of preparing the MSN and preparing the mesoporous silica-containing Bt nano-pesticide, and the obtained mesoporous silica-containing Bt nano-pesticide has better insecticidal activity compared with Bt raw powder. Mesoporous silica is mainly used as a carrier and is combined with Bt raw powder in an intermolecular acting force manner. According to the pesticide, the coverage area of Bt on a carrier is increased, so that the insecticidal effect is enhanced.

Description

Technical Field

[0001] The present invention relates to a Bt nano-biological pesticide, specifically to a nano-pesticide prepared by mesoporous silica compounded with Bt original powder and its preparation method, belonging to the technical field of biological pesticides. Background Art

[0002] Bacillus thuringiensis ( Bacillus thuringiensis , abbreviated as Bt) is one of the most widely used insecticides in the world. Its insecticidal mechanism mainly utilizes the parasporal crystals it produces. When insects ingest the parasporal crystals produced by Bt, the midgut of the insects is damaged into δ-endotoxin under alkaline conditions. These toxins can damage the intestines of insects, causing intestinal damage and necrosis, and then making the insects starve to death because they cannot eat. Although the Bt original powder can kill insects, its insecticidal speed is slow, and it also causes certain damage to plant leaves. Therefore, improving the insecticidal speed of Bt plays an important role in expanding its application and protecting biological safety.

[0003] Mesoporous materials are a type of porous materials with pore sizes ranging from 1 to 50 nm, which can play an important role in adsorption, separation, and catalytic reactions. Currently, their preparation methods mainly include sol-gel method, hydrothermal method, template method, and aluminum sulfate method, etc. As a good carrier, mesoporous materials can improve their effects by compounding corresponding bacteria or pesticides. It has been widely used in the preparation of nano-pesticides to improve their insecticidal, bactericidal, etc. effects, and it has less pollution to the environment, so it is considered an effective way to improve the pesticide effects. Summary of the Invention

[0004] In view of the problems existing in the Bt original powder, the purpose of the present invention is to propose a nano-pesticide prepared by mesoporous silica compounded with Bt original powder and its preparation method to improve the insecticidal effect of the Bt original powder.

[0005] A preparation method of striped mesoporous silica is as follows Take CTAB and NaOH in pure water, mix them evenly, and place them in a round-bottom flask. Heat up to 80 °C and magnetically stir for two hours at a rotation speed of 600 revolutions per minute. At this time, the solution becomes clear and transparent. Then, pour 5 mL of TEOS into a dropping funnel and drop it into the above reaction solution drop by drop. Keep the temperature at 80 °C and continue to stir for 20 h. Subsequently, pour the sample into a 50 mL centrifuge tube in batches, use a refrigerated centrifuge at 4 °C, 10000 rpm for 5 min, and wash it 3 times each with deionized water and absolute ethanol, and dry it in vacuum at 60 °C to obtain a white solid, and grind it to obtain a white solid; calcine it in a muffle furnace at 550 °C for 4 h to obtain MSN; the final concentrations of CTAB and NaOH are 2.08 mg / mL and 0.58 mg / mL Analysis of the compounding conditions of mesoporous silica and Bt powder, and the method is as follows: 1) Put 0.01 g of mesoporous silica into a 50 mL conical flask, add a magnetic stir bar, stir on a magnetic stirrer, control the temperature at 30 °C, and take 0.01 g, 0.1 g, 0.2 g, 0.3 g, 0.4 g, and 0.5 g respectively, stir for two hours, and then centrifuge. The centrifugation conditions are: 10000 rpm, 4 °C, 5 min. Place the precipitate in a -80 °C refrigerator for 12 h, and then take it out and place it in a freeze dryer for 6 h. In the prepared LB medium, configure the above-mentioned nano-pesticides with different ratios into 100 ug / mL and coat the plates, and observe after 12 h; 2) Put 0.01 g of mesoporous silica and 0.3 g into a 50 mL conical flask, add a magnetic stir bar, stir on a magnetic stirrer, control the temperature at 30 °C, and the stirring times are 1 h, 2 h, 4 h, 16 h, and 20 h respectively. Then centrifuge. The centrifugation conditions are: 10000 rpm, 4 °C, 5 min. Take the supernatant and measure the absorbance value of the protein at OD595, and compare it with the absorbance values obtained from other ratios to calculate the adsorption amount (Q); 3) Put 0.01 g of mesoporous silica and 0.3 g into a 50 mL conical flask, add a magnetic stir bar, stir on a magnetic stirrer, control the temperatures at 23 °C, 25 °C, 27 °C, and 30 °C respectively, stir for two hours, and then centrifuge. The centrifugation conditions are: 10000 rpm, 4 °C, 5 min. Take the supernatant and measure the absorbance value of the protein at OD595, and compare it with the absorbance values obtained from other ratios to calculate the adsorption amount (Q); Preparation of the compounded nano-pesticide of mesoporous silica and Bt powder, and the method is as follows: Take 0.01 g of MSN and 0.3 g of Bt powder and put them into a 50 mL conical flask. Then place the conical flask in a magnetic stirrer, set the temperature to 25 °C, and stir magnetically for 20 h. Subsequently, centrifuge at 4 °C, 10000 rpm, and 5 min using a refrigerated centrifuge to obtain a precipitate. Place it in a -80 °C refrigerator for 12 h, and then dry it with a freeze dryer for 6 h to obtain MSN-Bt.

[0006] The Bt powder is obtained by purchasing from the market.

[0007] The technical principle of the present invention: Mesoporous silica and Bt powder are mainly polymerized through intermolecular forces. Using mesoporous silica as a carrier, with its large specific surface area, the Bt powder is more evenly distributed on its surface to enhance the contact area with the insect intestine, so as to achieve the purpose of improving the insecticidal effect. The present invention has the following advantages: This nano-pesticide does not require strict conditions, has a low preparation threshold, and is inexpensive. After the Bt technical powder acts on mesoporous silica, due to the large specific surface area of mesoporous silica, the Bt technical powder can better contact with insect intestinal cells, increasing the insecticidal effect. Mesoporous silica has less environmental pollution, which is beneficial to the green environment. Description of the Drawings

[0008] Figure 1 A) SEM image of MSN (200 nm); B) Enlarged SEM image of MSN (50 nm); C) D) SEM images of MSN-Bt; E) Zeta potential diagrams of MSN, Bt, and MSN-Bt; F) Infrared spectra of MSN, Bt, and MSN-Bt Figure 2 A) Standard curve of Bt technical powder; B) Adsorption analysis diagram of mesoporous silica compounded with Bt technical powder at different times; C) Adsorption analysis diagram of mesoporous silica compounded with Bt technical powder at different temperatures; D) Proportion analysis diagram of mesoporous silica compounded with Bt technical powder at different times Figure 3 A) Bioassay analysis diagram of mesoporous silica compounded with Bt technical powder at different temperatures; B) Bioassay analysis diagram of mesoporous silica compounded with Bt technical powder at different times; C) Bioassay analysis of different proportions of mesoporous silica compounded with Bt technical powder; D) UV absorption comparison diagram of MSN, Bt, and MSN-Bt Figure 4 A) Cell survival rate diagrams of Bt and MSN-Bt irradiated by UV lamp for different times; B) Cell survival rates of Bt and MSN-Bt under light and storage; C) Bioassay activity mortality diagrams of Bt and MSN-Bt Figure 5 A) Analysis diagram of reactive oxygen species production of Bt and MSN-Bt; B) Comparison diagram of midgut damage of Plutella xylostella in H2O, MSN, Bt, and MSN-Bt Figure 6 A) Smurf staining diagrams of MSN-Bt, Bt technical powder, and water; B) Leaf damage diagrams of MSN-Bt, Bt technical powder, water, and MSN; C) Pot bioassay diagrams of MSN-Bt, Bt technical powder, water, and MSN The specific implementation manner of the present invention is as follows: A nano-pesticide of mesoporous silica compounded with Bt technical powder and its preparation method S1: Take CTAB and NaOH in pure water, mix them evenly, place them in a round-bottom flask, heat up to 80 °C, and magnetically stir for two hours at a rotation speed of 600 revolutions per minute. At this time, the solution becomes clear and transparent. Then, pour 5 mL of TEOS into a dropping funnel and gradually drop it into the above reaction solution. Keep the temperature at 80 °C and continue stirring for 20 h. Subsequently, pour the sample into 50 mL centrifuge tubes in batches, and use a refrigerated centrifuge at 4 °C, 10,000 rpm for 5 min. Wash it 3 times each with deionized water and absolute ethanol, and dry it in a vacuum at 60 °C to obtain a white solid. Grind it to obtain a white solid; calcine it in a muffle furnace at 550 °C for 4 h to obtain MSN, where the final concentrations of CTAB and NaOH are 2.08 mg / mL and 0.58 mg / mL; S2: Take 0.01 g of MSN and 0.3 g of Bt original powder and place them in a 50 mL conical flask. Then, place the conical flask on a magnetic stirrer, set the temperature to 25 °C, and magnetically stir for 20 h. Subsequently, centrifuge to obtain a precipitate under the conditions of 4 °C, 10,000 rpm for 5 min using a refrigerated centrifuge. Place it in a -80 °C refrigerator for 12 h, and then dry it with a freeze dryer for 6 h to obtain MSN-Bt; S3: Scanning electron microscopy (SEM): Prepare samples by performing conventional SEM treatment (sampling / washing, fixing, dehydration, drying and replacement, sticking and fixing, and coating with gold) on the samples, and use SEM to observe the surface structure and particle size of MSN and MSN-Bt; S4: Zeta potential analysis: Take appropriate solutions of MSN, Bt, and MSN-Bt and measure the potential in a Zeta potential analyzer; S5: Fourier transform infrared spectroscopy (FT-IR) analysis: Qualitatively analyze the functional group compositions of MSN, MSN-Bt, and Bt using a Fourier transform infrared spectrometer; S6: Mix the Bt original powder with pure water to prepare a solution, and dilute it to 100 μg / ml, 60 μg / ml, 40 μg / ml, 20 μg / ml, and 10 μg / ml respectively. Detect the protein concentration using an enzyme-linked immunosorbent assay (ELISA) at a wavelength of OD595 nm. Draw a standard curve of the Bt original powder according to the data shown by the ELISA; S7: Place 0.01 g of mesoporous silica in a 50 mL conical flask, add a magnetic stir bar, and stir on a magnetic stirrer. Control the temperature at 30 °C. Respectively take 0.01 g, 0.1 g, 0.2 g, 0.3 g, 0.4 g, and 0.5 g and stir for two hours, then centrifuge. The centrifugation conditions are: 10,000 rpm, 4 °C, 5 min. Place the precipitate in a -80 °C refrigerator for 12 h, and then take it out and place it in a freeze dryer for 6 h. In the prepared LB medium, configure the above different ratios of nano-pesticides into 100 μg / mL and coat the plates, and observe after 12 h; S8: Place 0.01 g of mesoporous silica and 0.3 g in a 50 mL conical flask, add a magnetic stir bar, stir on a magnetic stirrer, control the temperature at 30 °C, and stir for 1 h, 2 h, 4 h, 16 h, and 20 h respectively. Then centrifuge under the conditions of 10000 rpm at 4 °C for 5 min. Take the supernatant and measure the absorbance of the protein at OD595, and compare it with the absorbance values obtained from other ratios to calculate the adsorption capacity (Q); S9: Place 0.01 g of mesoporous silica and 0.3 g in a 50 mL conical flask, add a magnetic stir bar, stir on a magnetic stirrer, control the temperature at 23 °C, 25 °C, 27 °C, and 30 °C respectively, and stir for two hours. Then centrifuge under the conditions of 10000 rpm at 4 °C for 5 min. Take the supernatant and measure the absorbance of the protein at OD595, and compare it with the absorbance values obtained from other ratios to calculate the adsorption capacity (Q); S10: Place the pesticides with different ratios obtained in S7 above in a -80 °C refrigerator for refrigeration. Take them out after 12 h, place them in a freeze dryer for drying for 6 h and then take them out. Configure them into the same concentration respectively, soak the prepared Plutella xylostella feed in the solution for 20 min, wait for it to dry and then put it into a bioassay cup. Make 3 replicate groups respectively, put 10 Plutella xylostella in each group, observe the number of dead ones, and calculate the mortality rate; S11: Place the pesticides with different temperatures obtained in S8 above in a -80 °C refrigerator for refrigeration. Take them out after 12 h, place them in a freeze dryer for drying for 6 h and then take them out. Configure them into the same concentration respectively, soak the prepared Plutella xylostella feed in the solution for 20 min, wait for it to dry and then put it into a bioassay cup. Make 3 replicate groups respectively, put 10 Plutella xylostella in each group, observe the number of dead ones, and calculate the mortality rate; S12: Place the pesticides with different times obtained in S9 above in a -80 °C refrigerator for refrigeration. Take them out after 12 h, place them in a freeze dryer for drying for 6 h and then take them out. Configure them into the same concentration respectively, soak the prepared Plutella xylostella feed in the solution for 20 min, wait for it to dry and then put it into a bioassay cup. Make 3 replicate groups respectively, put 10 Plutella xylostella in each group, observe the number of dead ones, and calculate the mortality rate; S13: Configure Bt, MSN-Bt, and MSN into solutions with the same concentration, place them in an ultraviolet box, and irradiate for 2 h, 12 h, 48 h, and 72 h respectively. Take 100 uL of each solution into a well plate, place the treated well plate in an enzyme-linked immunosorbent assay instrument, and detect its absorbance at OD595; S14: Prepare Bt and MSN-Bt solutions with the same concentration. Expose a part of the solutions to sunlight for three days and place another part indoors for three days. After three days, conduct plate coating and bioassay analysis experiments respectively. For plate coating, use LB medium and incubate it in a bacterial incubator for 12 h, and compare with the treatment group of the original solution to observe the survival quantity. For the bioassay analysis experiment, soak the above treatment groups and the original solution in feed, select 10 diamondback moths each, and observe the death quantity; S15: Prepare Bt and MSN-Bt solutions with the same concentration, place them at room temperature for 3 days, coat plates on LB solid medium, incubate in a bacterial incubator for 12 h, compare with the original treatment group to observe the survival quantity; at the same time, soak the prepared feed in the solution for bioactivity determination, with 3 replicates for each, 10 diamondback moths in each group, compare with the original treatment group, and calculate the mortality rate of diamondback moths; S16: Cut the prepared feed into small pieces of the same volume, soak them in the medicinal liquid for 15 - 20 min, air-dry and then put them into bioassay cups, with 10 insects in each group. After feeding for 12 h, dissect them under a stereomicroscope with forceps, hold one end of the head and tail respectively and dissect out the complete intestine forcefully (remove redundant tissues such as Malpighian tubules), put it into a prepared sterile aqueous solution with DCFH-DA staining solution and incubate and stain in a 37 °C cell incubator for 20 min (add Rouseup to the positive control to stimulate the intestine to produce reactive oxygen species and continuously treat for 20 min), then suck away the staining solution, replace it with clear water to suspend the intestine, repeat washing 2 - 3 times, and observe under a laser confocal microscope; S17: Soak feed with water, MSN, MSN-Bt, and Bt of the same concentration, feed the 4th - 5th instar larvae of diamondback moths, with 15 in each cup. After feeding for 12 h, dissect the tea geometrid larvae treated differently, place them on ice for 20 min to stun them by low temperature, put them on ice and dissect the diamondback moths with dissecting forceps to take out the complete midgut and quickly transfer the midgut to a 1.5 mL EP tube (sterilized). After sample preparation, observe the damage of the midgut tissue of diamondback moth larvae fed with different treatment samples by TEM, and whether there is any digestion phenomenon in the midgut intima cells; S18: Use larger 3rd instar larvae as experimental subjects. After feeding the 3rd instar larvae with feed soaked in Bt and MSN-Bt of the same concentration for 12 h, replace the feed with feed containing edible bright blue dye (Smurf) and feed for 2 h to ensure that all larvae have eaten the feed containing the dye. Then observe the body color of the insects 12 h later, with clean feed treated with sterile water as the control, and each replicate sample contains the midguts of 15 diamondback moth larvae; S19: Prepare sterile water, MSN, Bt powder, and MSN-Bt (converted according to the adsorption capacity) solutions with the same concentration respectively, spray them on the leaves of pakchoi planted in pots, and after drying, select 15 diamondback moths on each leaf. Observe the leaf conditions after 48 h. At the same time, select leaves with roughly the same size and shape, place them in glass petri dishes, spray the solutions on the leaves, and after drying, place 10 diamondback moths in each petri dish. Observe the leaf conditions after 24 h and calculate the leaf damage rate.

Claims

1. A mesoporous silica composite capable of killing insects, characterized in that, The mesoporous silica composite includes a carrier and a compound; the carrier is striated mesoporous silica; the compound is Bt technical powder; the mesoporous silica is MCM-41; the preparation method for preparing the composite of the MCM-41 type mesoporous silica and Bt technical powder is as follows: S1: Take CTAB and NaOH in pure water, mix them evenly, place them in a round-bottom flask, heat up to 80 °C, and magnetically stir for two hours at a rotation speed of 600 revolutions per minute. At this time, the solution becomes clear and transparent. Then, pour 5 mL of TEOS into a dropping funnel and drop it into the above reaction solution drop by drop. Keep the temperature at 80 °C and continue stirring for 20 h. Subsequently, pour the sample into a 50 mL centrifuge tube in batches, use a refrigerated centrifuge at 4 °C, 10,000 rpm for 5 min, and wash it 3 times each with deionized water and absolute ethanol, and vacuum dry at 60 °C to obtain a white solid, which is ground to obtain a white solid; calcine it in a muffle furnace at 550 °C for 4 h to obtain MSM-41; S2: Place 0.01 g of mesoporous silica in a 50 mL conical flask, add a magnetic stir bar, stir with a magnetic stirrer, control the temperature at 30 °C, and take 0.01 g, 0.1 g, 0.2 g, 0.3 g, 0.4 g, and 0.5 g respectively, stir for two hours, and then centrifuge. The centrifugation conditions are: 10,000 rpm, 4 °C, 5 min. Place the precipitate in a -80 °C refrigerator for 12 h, and then take it out and place it in a freeze dryer for 6 h. In the prepared LB medium, configure the above nano-pesticides with different ratios into 100 μg / mL and coat the plates, and observe after 12 h; S3: Place 0.01 g of mesoporous silica and 0.3 g in a 50 mL conical flask, add a magnetic stir bar, stir on a magnetic stirrer, control the temperature at 30 °C, and stir for 1 h, 2 h, 4 h, 16 h, and 20 h respectively. Then centrifuge under the following conditions: 10,000 rpm, 4 °C, 5 min. Take the supernatant to measure the absorbance of the protein at OD 595 and compare the absorbance values obtained with other ratios to calculate the adsorption capacity (Q); S4: Place 0.01 g of mesoporous silica and 0.3 g in a 50 mL conical flask, add a magnetic stir bar, stir on a magnetic stirrer, control the temperature at 23 °C, 25 °C, 27 °C, and 30 °C respectively, stir for two hours, then centrifuge, centrifuge conditions: 10,000 rpm, 4 °C, 5 min, take the supernatant and measure the absorbance of the protein at OD 595 value, and compare it with the absorbance values obtained from other ratios to calculate the adsorption capacity (Q).

2. The method according to claim 1, characterized in that, The final concentrations of CTAB and NaOH in the mesoporous silica are 2.08 mg / mL and 0.58 mg / mL.

3. The method according to claim 1, characterized in that, The washing with deionized water and absolute ethanol is to remove the template CTAB.

4. The method according to claim 1, wherein Freeze drying includes freezing the precipitate at -80 °C until there is no flowing component and then freeze drying for 12 h.

5. The method according to claim 1, characterized in that, Compared with other ratios, 0.01 g of MSN and 0.3 g of Bt technical powder obtained the largest number of bacteria, which was 610.

6. The method according to claim 1, characterized in that, 0.01 g of MSN and 0.3 g of Bt technical powder obtained the largest adsorption amount of 19 mg / mL at 30 °C for 2 h.

7. The method according to claim 1, characterized in that 0.01 g of MSN and 0.3 g of Bt technical powder obtained the largest adsorption amount of 24 mg / mL at 30 °C for 20 h.

8. According to the nano-pesticide with mesoporous silica compounded with Bt technical powder and its preparation method described in claim 1, apply the nano-Bt biological pesticide to Plutella xylostella. This nano-Bt pesticide uses mesoporous silica as a carrier and is compounded with Bt technical powder through intermolecular forces to enhance the damage of Bt to the intestine of Plutella xylostella, thereby achieving the effect of enhancing insecticidal activity. Therefore, this mesoporous Bt biological pesticide provides a new reference for the application of enhancing Bt insecticides.

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