Bacillus thuringiensis strain and parasporal crystal thereof, extraction method of parasporal crystal, and application of the two in controlling plant nematode disease

A technology of Bacillus thuringiensis and Bacillus aureus, applied in the field of microorganisms and plant protection, can solve the problems of outstanding drug resistance of nematodes, excessive pesticide residues, and limited varieties, so as to reduce pesticide residues, reduce environmental pollution, and inhibit hatching Effect

Active Publication Date: 2012-01-11
HUNAN PLANT PROTECTION INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, chemical nematicides are often used in agricultural production to prevent and control plant parasitic nematode diseases, but the varieties of nematicides on the market today are limited, and most of them are highly toxic and high resi...

Method used

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  • Bacillus thuringiensis strain and parasporal crystal thereof, extraction method of parasporal crystal, and application of the two in controlling plant nematode disease
  • Bacillus thuringiensis strain and parasporal crystal thereof, extraction method of parasporal crystal, and application of the two in controlling plant nematode disease
  • Bacillus thuringiensis strain and parasporal crystal thereof, extraction method of parasporal crystal, and application of the two in controlling plant nematode disease

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Experimental program
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Effect test

Embodiment 1

[0025] A Bacillus thuringiensis bacterial strain of the present invention, the depository unit of the bacterial strain is the Chinese Type Culture Collection Center, the address of the depository unit is located at Wuhan University, China, the preservation date is January 21, 2010, and the preservation name is Bacillus thuringiensis YC-10, The deposit number is CCTCC NO: M 2010023.

[0026] The bacterial strain of this embodiment mainly adopts the plate dilution separation method, utilizes nutrient agar medium to cultivate, and obtains after separating from tobacco, and the specific separation and screening steps are as follows:

[0027] 1. Prepare nutrient agar medium, including the following components in mass fraction: beef extract 0.5%, peptone 1.0%, NaCl 1.0%, adjust the pH value to about 7.2, and sterilize at 121° C. for 30 minutes.

[0028] 2. Isolation of bacterial strains: Collect 36 parts of healthy tobacco plants from root-knot nematode-infested fields, rinse them und...

Embodiment 2

[0035] Example 2: Application of fermentation filtrate made from Bacillus thuringiensis YC-10 in nematode control

[0036] Utilize the Bacillus thuringiensis YC-10 obtained by screening in Example 1 to prepare the fermentation filtrate, the specific preparation method is: inoculate the Bacillus thuringiensis YC-10 obtained in Example 1 into the fermentation medium, and the fermentation medium comprises the following Mass fraction components: beef extract 0.3% to 0.5%, peptone 0.5% to 2.0%, glucose 0.1% to 0.3%, NaCl 0.2% to 1.0%, MgSO 4 ·7H 2 O 0.01%~0.03%, K 2 HPO 4 0.005%~0.03% and MnSO 4 0.003% to 0.005%, the pH value of the fermentation medium is 7.0, 25°C to 37°C, 125r / min to 220r / min shaker culture for 72h to 120h, to obtain bacterial fermentation broth, and then centrifuged at a speed of 10000r / min After 10 minutes, the supernatant was taken as the fermentation filtrate.

[0037] The obtained fermentation filtrate was applied according to the following steps, and t...

Embodiment 3

[0052] Example 3: Application of parasporal crystal solution made of Bacillus thuringiensis YC-10 in nematode control

[0053] Utilize the Bacillus thuringiensis YC-10 obtained by screening in Example 1 to prepare the parasporal crystal solution (i.e. crystal toxin protein solution), the specific preparation method is: the activated bacterial strain Bacillus thuringiensis YC-10 is inserted into the fermentation medium ( Refer to the recipe in Example 2) to carry out fermentation culture, 30 ° C, 220r / min shaker culture for 72h, until the bacteria are basically broken, centrifuge to collect the cell crystal mixture, wash 3 times with 1mol / L NaCl and sterilized water, With 50mmol / L Na containing 2% β-mercaptoethanol 2 CO 3 Suspend in buffer solution (pH 9.5), act at 37°C for 1 hour; centrifuge to take the supernatant, add 1 / 20 volume of 4mol / L sodium acetate solution (pH 4.0), and precipitate on ice; wash the precipitated protein with sterilized water After two times, use 50mm...

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Abstract

The invention discloses a bacillus thuringiensis strain which has a preservation title of bacillus thuringiensis YC-10, is preserved in China Center for Type Culture Collection and has a preservation number of CCTCC NO:M2010023. The bacillus thuringiensis strain can generate a parasporal crystal protein while forming into a gemma. A protein extraction method comprises steps of: inoculating a bacteria liquid with activated bacterial strains to a fermentation medium, culturing with oscillating, and obtaining a mixture of gammae and crystals; washing, suspending with a buffer, centrifuging to obtain a supernatant, precipitating, dissolving, removing impurities by centrifugation and dialysing overnight, wherein the dialysate contains parasporal crystals. The bacillus thuringiensis strain or the parasporal crystals produced therefrom can be effectively applied to plant nematode disease control. The strain and application thereof in the invention have advantages of high efficiency, low toxicity, ecological environment friendliness, reduction of pesticide residuals, etc.

Description

technical field [0001] The invention relates to microorganisms and their application in the technical field of plant protection, in particular to a bacillus thuringiensis and its companion crystals, as well as the corresponding extraction method and application. Background technique [0002] Plant parasitic nematodes are one of the important diseases of crops. The annual crop losses caused by plant parasitic nematodes in the world exceed hundreds of billions of dollars, and nematode diseases have become a prominent problem in agricultural production. At present, chemical nematicides are often used in agricultural production to prevent and control plant parasitic nematode diseases, but the varieties of nematicides on the market today are limited, and most of them are highly toxic and high residual pesticides. Nematicides have not only caused serious pollution to the environment, but also caused a series of environmental and social problems such as excessive pesticide residue...

Claims

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Application Information

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IPC IPC(8): C12N1/20C07K14/325C12P21/02C07K1/36C07K1/34C07K1/30A01N63/00A01N63/02A01P5/00C12R1/07
Inventor 成飞雪刘勇张德咏王忠勇程菊娥罗源华张松柏何明远谭新球朱春晖罗香文戴建平杨春晓
Owner HUNAN PLANT PROTECTION INST
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