Bacillus thuringiensis MB-15 strain and preparation method of its wettable powder

A technology of Bacillus aureus and wettable powder, which is applied in the field of biological control of agricultural pests, can solve the problems of unstable performance, short duration, and slow onset of effect, and achieve good control effect, long duration, and insecticidal broad spectrum effect

Inactive Publication Date: 2013-06-19
HEBEI AGRICULTURAL UNIV.
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The second object of the present invention is to provide a Bacillus thuringiensis MB-15 wettable powder and its preparation method in view of the problems of short duration of action, unstable performance and slow onset of action in previous Bacillus thuringiensis preparations

Method used

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  • Bacillus thuringiensis MB-15 strain and preparation method of its wettable powder
  • Bacillus thuringiensis MB-15 strain and preparation method of its wettable powder
  • Bacillus thuringiensis MB-15 strain and preparation method of its wettable powder

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Embodiment 1, the isolation and identification of Bacillus thuringiensis MB-15 bacterial strain

[0054] The strain of the present invention is isolated from the soil Bt MB-15 strain by the present inventors themselves, and the soil comes from Hebei Province.

[0055] Weigh 10g soil sample and put it into 50mL sodium acetate liquid medium (0.5g peptone, 0.25g yeast powder, 0.5g NaCl, 1.8g sodium acetate, 50ml water, adjust pH value to 7.2-7.4, 121℃, autoclave 30min), penicillin sodium salt was added to a final concentration of 400ug / mL, and cultured on a shaking table (200r / min, 30°C) for 4h. After the cultivation, take 10mL of the soil suspension, put it into a sterile centrifuge tube and centrifuge at 3000r / min for 15min, take 1.5mL of the upper layer of turbid solution in a 65°C water bath for 15min, take 0.2mL of the heat-treated turbid solution, and apply LB solid medium (2g of peptone, 1g of yeast powder, 2g of NaCl, 3.2g of agar powder, 200ml of water, adjust th...

Embodiment 2

[0057] Embodiment 2, bacillus thuringiensis MB-15 is to the determination of insecticidal activity of Lepidoptera vegetable pest larvae

[0058] A single colony of Bt MB-15 was inoculated in LB liquid medium, and fermented in a shaking flask at 30°C and 200 r / min until most of the paraspora crystals fell off, and the culture was stopped to obtain the fermentation broth. Put the fermentation broth into a centrifuge tube, centrifuge (4°C, 12 000 r / min, 5 min), discard the supernatant and keep the precipitate, fully suspend and wash the precipitate with sterile water, repeat twice, and weigh the obtained precipitate. Wet weight, and finally suspended and diluted with sterile water into different mass concentrations for biological assays.

[0059] For the bioassay of the second instar larvae of Spodoptera litura, diamondback moth and Pieris rapae, the soaking method was adopted. Fresh leaves of Eucommia litura or cabbage leaves of the same size were immersed in the test solution o...

Embodiment 3

[0060] The SDS-PAGE analysis of embodiment 3, crystal protein

[0061]Observe the colony with separated cell crystals under an oil microscope, scrape a small amount of bacterial lawn and dissolve it in 100 μL sterilized water, shake and resuspend, centrifuge (4°C, 12 000 r / min, 5 min), discard the supernatant, and use 200 mL After resuspension in 1M NaCl, centrifuge (4°C, 12 000 r / min, 5 min), discard the supernatant, resuspend with sterilized water, centrifuge (4°C, 12 000 r / min, 5 min), discard For the supernatant, resuspend the pellet in 20 μL sterilized water, add the same volume of 2× loading buffer, mix well, boil at 100°C for 5 min, centrifuge (4°C, 12 000 r / min, 1 min), and take 20mL supernatant was used for SDS-PAGE detection (see figure 2 ).

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Abstract

The invention discloses a Bacillus thuringiensis MB-15 strain which has a broad spectrum and high virulence on vegetable lepidoptera pests, and the preservation number of the strain is CGMCC No. 5255. The strain can form a diamond insecticidal crystal protein, contains cry1I, cry1Ac, cry2Ac and vip3Aa genes, and has high-efficiency insecticidal activity on plutella xylostella, Pieris brassicae, beet armyworm, Spodoptera litura, Helicoverpa armigera and other lepidoptera pests. The invention also relates to a preparation method of the wettable powder of strain. The wettable powder comprises the following content of main components: 40.0-50.0% of Bacillus thuringiensis MB-15 strain powder, 5.0-10.0% of a dispersant, 5.0-10.0% of a wetting agent, 0.5-1.0% of a stabilizer, 0.5-1.0% of an ultraviolet light protection agent, and 28.0-49.0% of a carrier. The wettable powder can be used to control a plurality of lepidoptera pests on vegetables, and has the characteristics of broad spectrum, high efficiency, and safety, etc.

Description

technical field [0001] The invention belongs to the technical field of biological control of agricultural pests. The invention relates to a bacillus thuringiensis strain with broad-spectrum and high toxicity to lepidopteran pests on vegetables, and also relates to a preparation method of a wettable powder of the strain. technical background [0002] Bacillus thuringiensis ( Bacillus thuringiensis , referred to as Bt), is a widely distributed Gram-positive bacterium, and its preparation is currently the world's largest microbial insecticide (Yu Ziniu. Production and application of Bacillus thuringiensis preparations [M]. Beijing: Agricultural Press, 1993.). It is widely used to control various insects of 9 orders including Lepidoptera, Diptera, Coleoptera and Orthoptera (Schnepf E., Crickmore N., Van Rie, et al . Bacillus thuringiensis and its pesticide crystal proteins[J]. Microbiol Mol Biol Rev , 1998, 62:775-806.). The main active ingredient of Bt preparation is i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20A01N63/00A01N25/14A01P7/04C12R1/07
Inventor 王勤英葛东华宋萍南宫自艳
Owner HEBEI AGRICULTURAL UNIV.
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