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New bacterial strain of Bacillus thuringiensis for killing grub pest and pest killing protein thereof

A technology of thuringiensis spores and insecticidal crystal protein, which is applied in the directions of insecticides, bacteria, biocides, etc., can solve the problem of small quantity, and achieve the effect of specific insecticidal activity and good control effect.

Active Publication Date: 2012-01-25
QINGDAO AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the number of Bt strains and their products that successfully control grub-like pests is still relatively small. Therefore, Bacillus thuringiensis with a more efficient insecticidal effect is screened and isolated to discover new, efficient and broad-spectrum insecticidal proteins. For grubs The control of underground pests is of great significance

Method used

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  • New bacterial strain of Bacillus thuringiensis for killing grub pest and pest killing protein thereof
  • New bacterial strain of Bacillus thuringiensis for killing grub pest and pest killing protein thereof
  • New bacterial strain of Bacillus thuringiensis for killing grub pest and pest killing protein thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0012] Isolation and biological characteristics of embodiment 1 bacillus thuringiensis bacterial strain B-Y7-1:

[0013] 1.1 Source of soil samples

[0014] Soil samples were collected from various places in Shandong Province. Soil samples from non-cultivated land such as barren grassland and woodland were selected. About 50g of soil 5-10cm below the surface was collected, sealed in bags, numbered, and the collection location was registered.

[0015] 1.2 Medium

[0016] Strains were isolated using NGKG selective medium (NISSUI company, Japan), 3.975g NGKG medium plus 135mL distilled water, sterilized for 20min, cooled to 50°C, added egg yolk liquid 15mL (3mL egg yolk liquid + 12mL 0.85% normal saline), poured flat. Bacterial strain culture adopts LB medium (peptone 1%, yeast powder 0.5%, NaCl 1%, pH 7.0) or beef extract peptone medium (beef extract 0.3%, peptone 0.5%, pH 7.0), solid medium is added 1.3 %~1.5% agar.

[0017] Isolation of 1.3Bt strain

[0018] Suspend 1g of...

Embodiment 2B

[0023] Preparation and bioactivity assay of embodiment 2Bt bacterial strain B-Y7-1 bacterium liquid

[0024] 2.1 Sample preparation

[0025] Pick a single colony of the strain B-Y7-1, inoculate it in 5 mL of LB liquid medium, and incubate with shaking at 30°C 200r / min for 16-17h. Min shake culture for about 72 hours, check under a microscope, after separation of spores and crystals, centrifuge at 10,000r / min for 10min, discard the supernatant, suspend the collected bacteria with sterile water, and make a bacterial liquid preparation for later use.

[0026] 2.2 Biological activity assay

[0027] Using sterile water as a control, mix 5mL of bacterial liquid with 10g of feed (fine potato shreds) and 90g of sterilized soil evenly, put them into plastic boxes with a diameter of 12cm and a height of 9cm, and insert 3-day-old grubs into each box 5 worms, 6 boxes per treatment (30 worms in total), repeated 3 times, reared at room temperature, checked the number of dead worms and liv...

Embodiment 3B

[0028] The biological activity of embodiment 3Bt bacterial strain B-Y7-1 parasporal crystal protein

[0029] 3.1 Preparation of protein samples

[0030] Pick a single colony of strain B-Y7-1, inoculate it in LB liquid medium, and cultivate it at 30°C and 200r / min for about 3-4 days. After the spores and crystals are separated, purify the parasporal crystal protein. The measured sequence is SEQ ID NO :1. After alkaline hydrolysis of the protein, the protein concentration was determined with a Bradford protein concentration assay kit, and the protein samples were stored at -20°C for the determination of biological activity.

[0031] 3.2 Biological activity of parasporal crystal protein

[0032] Dilute the purified parasporal crystal protein to different concentrations, mix 5mL protein solutions of different concentrations with 10g feed (fine potato shreds) and 90g sterilized soil, and put them into a plastic box with a diameter of 12cm and a height of 9cm 5 3-day-old grubs we...

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Abstract

The invention relates to a new bacterial strain of Bacillus thuringiensis for killing grub pest and a pest killing protein thereof. The new bacterial strain has the collection number of CCTCC (China Center For Type Culture Collection) No. M2011267. The bacterial strain and the pest killing protein provided by the invention can be used for preparing the preparation capable of preventing and controlling grub pests. The bacterial strain provided by the invention has specific insecticidal activity on the grub pests. The pest killing crystal protein provided by the invention has good prevention and control effect on main soil insects such as Holotrichia oblita and Holotrichia parallela in China.

Description

technical field [0001] The invention belongs to the technical field of pest biological control, and in particular relates to a new strain of Bacillus thuringiensis and its insecticidal protein for killing grub-like pests, that is, the new strain of Bacillus thuringiensis B-Y7-1 and its insecticidal protein. Background technique [0002] Grubs are the general term for the larvae of the Coleoptera Scaraboideae, which damage various crops, fruit trees, forests, pastures and other plants. control of soil-dwelling pests. According to survey statistics, 86% of the damage to the underground parts of plants is caused by grub hazards. Every year, grubs occur in an area of ​​about 100 million mu in the country, and in severe years, it reached 320 million mu, and the output loss reached 20% to 40%. The Huanghuai River Basin suffered the most serious damage. There are more than 100 species of grubs recorded in my country, of which more than 30 species are common, including Holotrichia...

Claims

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Application Information

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IPC IPC(8): C12N1/20C07K14/325A01N63/02A01P7/04C12R1/07
Inventor 李长友郑桂玲耿以龙周洪旭张媛初立良
Owner QINGDAO AGRI UNIV
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