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Bacillus thuringiensis for killing mosquito larvae and application of bacillus thuringiensis

A technology of Bacillus aureus and spore thuringiensis, which can be used in applications, pesticides, bacteria, etc., can solve the problems of natural environment biological chain and soil damage, and achieve the effect of reducing risks

Inactive Publication Date: 2017-10-17
浙江翠溪农业开发有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the residues of these chemical pesticides have had extremely negative impacts on organisms other than mosquitoes, including humans and domestic animals, and have also caused great damage to the biological chain and soil in the natural environment

Method used

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  • Bacillus thuringiensis for killing mosquito larvae and application of bacillus thuringiensis
  • Bacillus thuringiensis for killing mosquito larvae and application of bacillus thuringiensis
  • Bacillus thuringiensis for killing mosquito larvae and application of bacillus thuringiensis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Collection of Soil Samples and Isolation and Culture of Bt Strains

[0024] The soil sampling time is selected in summer and autumn (May to October), and the sunny slope of Dawangling Nature Reserve in Guangxi is selected. In order to increase the probability of obtaining new strains, the sampling points should be selected with complete vegetation protection, no human intervention in the ecological environment, and rich soil humus. best place. Bt strain screening adopts sodium acetate + antibiotic + temperature screening method, weighs 1g of soil sample into 10mL of BPA culture solution, then adds penicillin sodium salt and gentamicin sulfate to 400μg / mL, and then cultures on a shaking table at 30°C and 220rpm 4h. Let it stand for 30 minutes, take 10mL of soil suspension, put it in a 75°C water bath for 20 minutes, and shake it every few minutes. Take 1mL of the supernatant and perform a 10-fold serial dilution operation, setting up 4 dilution gradients (10 -1 ,10 -...

Embodiment 2

[0026] Scanning Electron Microscopy Observation of Crystal Protein of Bt S2160-1 Strain

[0027] The Bt strain was cultured in G-Tris medium at 30°C until the spores were completely formed, and 500 μL of bacteria were collected by centrifugation at 12,000 g for 10 minutes at 4°C, and then washed with 1M ice-cold NaCl for 3 times, and finally the bacteria were suspended in 500 μL of ice-cold ultrapure in the water. Take 10 μL of the mixture of spores and paraspore crystals and carefully spread it on a clean cover glass, then dry it in vacuum at 4 °C, and then wash it with 1% osmium tetroxide (OsO 4 ) to fix the sample, the sample is placed on a metal sample stage, and put into a vacuum evaporator to spray a metal film with a thickness of about 20nm. The prepared sample was placed in a S3-400N scanning electron microscope (HITACHI Ltd, Japan), and the image was observed and recorded under the voltage condition of 20kV.

Embodiment 3

[0029] SDS-PAGE electrophoresis analysis of crystal protein of Bt S2160-1 strain

[0030] The Bt strain was cultured in 200 mL of G-Tris medium for more than 3 days, and the culture conditions were 30° C. and 220 rpm shaking culture. Optical microscope observation confirmed that the spores were fully formed, and the paraspore crystals and spore mixture were collected by centrifugation at 12,000g for 10 minutes at 4°C. The precipitate was washed three times with an equal volume of ice-cold 1M NaCl, and then the cells were sonicated for 20 min (Model VC-130, Sonics and Materials Inc, USA). Centrifuge at 12,000g for 10min at 4°C, collect the precipitate, dissolve in 5mL of 50mM Na 2 CO 3 solution (PH=10.0). Generally, 7.5% or 12% SDS-PAGE can be used to analyze the crystal protein composition of Bt bacteria, 5uL prepared parasporal crystal protein and 5uL 2×SDS-PAGE sample Buffer [0.075M Tris Cl, (pH6.8); 0.020M EDTA; 2% SDS; 0.02% bromophenol blue; 0.25M dithiothreitol (DTT)...

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Abstract

The invention belongs to the technical field of biological prevention and control of sanitary insect pests, and mainly discloses a material and a method for preventing and controlling larvae of mosquitoes which are the leader of 'four pests'. The invention discloses a novel strain Bt S2160-1 of Bacillus thuringiensis, Bt for killing mosquito larvae, wherein the preservation number of the novel strain is CGMCC No.13274. The strain Bt S2160-1 has very high insecticidal activity upon larvae of culex fatigans and aedes albopictus, and compared with a Bt mosquito strain Bti recommended by WHO (World Health Organization), the Bt S2160-1 has relatively high activity upon larvae of aedes albopictus. The Bacillus thuringiensis Bt S2160-1 disclosed by the invention can be made into an insecticide for large-scale prevention and control on larva populations of culex fatigans and aedes albopictus. Therefore, the strain can be powerful supplement as a mosquito control microbial agent in addition to Bti, or even can be used as a substitution of Bti for controlling populations of aedes albopictus, and the risk that mosquitoes have resistance to a Bti crystal protein which is used for a long time to control mosquito populations can be reduced.

Description

technical field [0001] The invention belongs to the technical field of biological control of sanitary pests. The present invention relates to a bacillus thuringiensis strain with high insecticidal activity against mosquito larvae, a method for analyzing the genomics of the strain, a method for preparing bacillus thuringiensis for killing mosquito larvae and its application in the field of mosquito biological control . Background technique [0002] Bacillus thuringiensis (Bt) was discovered in Japan in 1901, and was isolated by Berliner from the diseased larvae of the Mediterranean mealylosia moth in 1911, and was named after the place where it was found in Thuringiensis, Germany. It is a Gram-positive bacterium that is widely distributed in a variety of habitats and is by far the safest and most widely used insecticidal bacterium. The parasporal crystal protein (parasporal crystal protein) formed in the stable growth stage, also known as insecticidal crystal protein (ICP),...

Claims

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Application Information

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IPC IPC(8): C12N1/20A01N63/00A01P7/04C12R1/07
CPCA01N63/00C12N1/205C12R2001/07
Inventor 方宣钧张文飞
Owner 浙江翠溪农业开发有限公司
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