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Bacillus subtilis and antibacterial protein thereof

A Bacillus subtilis, protein technology, applied in the directions of bacteria, fungicides, biocides, etc., can solve the problems of agricultural pesticide residues, environmental pollution, and high production costs, and achieve stable properties and significant bacteriostatic effects.

Inactive Publication Date: 2013-06-05
SICHUAN NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the use of chemical organic pesticides not only has high production costs, but also brings problems such as environmental pollution, pesticide residues in agricultural products, and various diseases

Method used

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  • Bacillus subtilis and antibacterial protein thereof
  • Bacillus subtilis and antibacterial protein thereof
  • Bacillus subtilis and antibacterial protein thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 Isolation and identification of Bacillus subtilis

[0034] 1. Strain isolation

[0035] Samples were collected from farmland soil in Longquanyi District, Chengdu City, Sichuan Province, and beef extract peptone medium (NA) was prepared. The dilution coating method and single colony plate streaking method were used to purify and preserve the strains. Prepare potato medium (PDA), and use the confrontation method to screen for antagonistic bacteria, that is, inoculate the mycelial block of indicator bacteria with a diameter of 6 mm in the center of the plate, and inoculate the isolated strain at 3 cm in front of the indicator bacteria colony at equidistant points, and culture at a constant temperature of 28 °C On day 3, measure and calculate the diameter of the inhibition zone. A strain with strong antibacterial activity against Botrytis cinerea, rice blast fungus, Curvularia maize, wheat head blight, Fusarium graminearum, and Rhizoctonia solani was obtained, na...

Embodiment 2

[0070] Example two Antibacterial experiments of Bacillus subtilis Loq18

[0071] 1. Live bacteria antibacterial experiment

[0072] Take the botrytis cinerea mycelium block with a diameter of 6mm and the preservation number ACCC36415 in a 50mL Erlenmeyer flask filled with 20mL potato liquid medium, and at the same time insert the two-ring Loq18 strain, and culture it at a constant temperature of 28°C for 72 hours.

[0073] After testing, the botrytis cinerea mycelium block does not grow, and its spores do not germinate, and the inhibition rate of live bacteria is 100%.

[0074] 2. The relationship between culture time and antibacterial activity of Bacillus subtilis Loq18

[0075] Use an inoculation loop to pick a Loq18 single colony and inoculate it into a 15mL test tube with a liquid volume of 30%, place at 37°C, and shake at 180r / min for 18h. Inoculate in a 250mL fermentation bottle according to 5% inoculum amount, and inoculate an equal volume of sterile water as a blank ...

Embodiment 3

[0086] Example 3 Preparation and Properties of Antibacterial Proteins

[0087] 1. Preparation of antibacterial crude extract of Bacillus subtilis Loq18

[0088] Streak culture of Bacillus subtilis Loq18 on NA medium for 18 hours, take a single colony to NA liquid medium for 24 hours at 37°C and shake at 200r / min, then inoculate 1000mL Erlenmeyer flask at 30°C with 10% inoculum amount , 160r / min fermentation 72h to obtain fermentation broth.

[0089] The fermentation broth was centrifuged at 4°C and 6000r / min for 30min to remove bacteria, and the supernatant was collected. Using the salting-out method, add 30% solid ammonium sulfate to the fermented liquid, and let stand overnight at 4°C. Centrifuge at 12000r / min at 4°C for 10min, collect the supernatant and the precipitate respectively, and use Botrytis cinerea with preservation number ACCC36415 as the indicator bacteria to detect the antibacterial activity of the supernatant and the precipitate, respectively. The results sh...

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Abstract

The invention discloses bacillus subtilis and an antibacterial protein thereof, and relates to the field of biological pesticides, in particular to the bacillus subtilis and the antibacterial protein thereof for resisting plant pathogenic fungi. The bacillus subtilis provided by the invention is a strain which is preserved in the China Center for Type Culture Collection and has a preserving number of CCTCC NO:M2011017. The invention also provides a plant pathogenic fungus resistant protein generated by the bacillus subtilis. The bacillus subtilis has a significant bacteriostatic effect; and the antibacterial protein generated by the bacillus subtilis is very stable in property, has the advantages of very wide temperature adaptation range, acid and alkali resistance, ultraviolet stability,tolerance of organic solvents and protease, and the like, can adapt to a variety of natural conditions, can be used for preparing pesticides for controlling the plant pathogenic fungi, and has a verygood application prospect.

Description

technical field [0001] The invention relates to the field of biological pesticides, in particular to a bacillus subtilis resistant to plant pathogenic fungi and an antibacterial protein thereof. Background technique [0002] Plant diseases have always been one of the important restrictive factors for high-quality and high-yield crops. It is estimated that the average loss of major crops in the world accounts for about 10-15% of the total output, and the annual direct economic loss is as high as hundreds of billions of dollars. Among plant diseases, 70-80% of the diseases are caused by the infection of pathogenic fungi. Plant pathogenic bacteria not only directly cause crop yield decline, some pathogenic fungi will also secrete a variety of toxins and metabolites harmful to humans and animals during the process of infecting crops, posing a great threat to the safety of crops. [0003] The control of crop fungal diseases often relies on chemical control. However, the use of ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20C07K14/32C12P21/02A01N63/02A01P3/00C12R1/125
Inventor 张晓喻黄春萍刘刚张宏
Owner SICHUAN NORMAL UNIV
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