Bacillussubtilis used for inhibiting formation of Colletotrichum capsici appressoria and antibacterial peptides thereof

A technology of Bacillus subtilis, anthrax bacteria, applied in the direction of chemicals for biological control, microbial-based methods, peptides, etc., to achieve the effect of preventing diseases

Inactive Publication Date: 2014-02-19
SHENZHEN POLYTECHNIC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, there are no reports at home and abroad on screening active strains that inhibit the formation of capsicum anthracnose appresses from Bacillus subtilis and applying them to the research of biological control of anthracnose

Method used

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  • Bacillussubtilis used for inhibiting formation of Colletotrichum capsici appressoria and antibacterial peptides thereof
  • Bacillussubtilis used for inhibiting formation of Colletotrichum capsici appressoria and antibacterial peptides thereof
  • Bacillussubtilis used for inhibiting formation of Colletotrichum capsici appressoria and antibacterial peptides thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1 Screening of bacterial strains

[0052] The beef extract peptone medium was used as the separation medium, and the bacterial strains were isolated by the conventional dilution plate separation method. The slide culture method was used for strain screening. The specific method is: from the separation plate cultured at 37°C for 12-20h, use a toothpick to pick up single colonies one by one, inoculate them into 96-well cell culture plates containing 280μL YPD culture medium, culture at 37°C for 18h, and obtain strains for screening of bacterial cultures. Capsicum anthracnose strain 501 was cultured on a PDA plate at 25°C for 7-10 days to produce a large amount of milky yellow myxospore clusters. Pick up the myxospore mass and dissolve it in sterile distilled water to prepare a spore suspension, and count the spores with a hemocytometer to adjust the spore concentration to 1 × 10 6 / mL, add penicillin (100U / mL) and streptomycin (100μg / mL) to the spore solution...

Embodiment 2

[0055] Identification of embodiment 2 strains

[0056] According to the experimental observation, the strain C-D6 has the following microbiological characteristics: when cultured on beef extract peptone medium at 37°C for 18 hours, it forms round protruding colonies with milky white, rough surface and wrinkles, and neat edges; the bacteria are rod-shaped, with a size of 0.89 μm × 2.74 μm. Observation after Gram staining: spores are middle-aged, oval, and cysts are not enlarged. Gram stain positive.

[0057] Using conventional PCR method, using

[0058] Universal primers 8F: 5'-GAGAGTTTGATCCTGGCTCAG-3' and 1492R: 5'-GGYTACCTTGTTACGACTT-3' were amplified from the genomic DNA of C-D6 to obtain a 1691bp 16S rDNA sequence. See SEQ ID NO: 1 for the specific sequence.

[0059] Search alignment by Blast, the sequence was compared with two reference strains of Bacillus subtilis in Genbank Bacillus subtilis The sequences of L4 and WD23 (accession numbers: GQ421472, EU780682, res...

Embodiment 3

[0068] Example 3 Determination of Inhibitory Effect of Bacterial Strains

[0069] The activity of bacterial cultures in inhibiting appressoria formation was determined by the slide culture method. The method is: take the bacterial culture solution and the anthrax spore suspension (1×10 6 / mL) each 20 μL, mix and drop on the glass slide, incubate at 25°C for 12 hours, and observe the effect of the bacterial culture solution on the sporulation appressoria under a microscope.

[0070] The above method was used to determine the effect of Bacillus C-D6 culture solution on the appressorium formation of Capsicum anthracis. The inhibitory effect of the culture medium of C-D6 strain on appressorium formation of capsicum anthracnose see image 3 . attached image 3 It shows that the culture solution of this bacterium does not affect the spore germination of Capsicum anthracnose, but it can obviously affect the growth of the germ tube, so that the front end of the mycelia cannot di...

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Abstract

The invention relates to a Bacillussubtilis C-D6 strain used for inhibiting formation of Colletotrichum capsici appressoria and applications. The strain is collected in China General Microbiological Culture Collection Center (CGMCC). The number is CGMCCNo. 5345. The invention also provides a suitable culture method for the strain for generation of active substances for inhibiting formation of appressoria and a separation and purification method for antibacterial proteins. By utilization of the above method, the culture solution of the C-D6 strain can strongly inhibit formation of Colletotrichum capsici 501 appressoria. The separated antibacterial proteins from the culture solution and with a low concentration can completely inhibit formation of the strain appressoria. The C-D6 strain and the produced antibacterial proteins have specific inhibition effects, can effectively interdict invasion of pathogenic bacteria, and can be used for biological prevention and control of crop anthracnose caused by Colletotrichum capsici.

Description

technical field [0001] The invention belongs to the field of biological control of plant diseases, and in particular relates to a bacillus subtilis which inhibits the formation of capsicum anthracnose appresses and its application. Background technique [0002] As an important part of comprehensive management of plant diseases, biological control has attracted much attention from all over the world because of its "compatibility" with ecological environment protection and "unity" with sustainable agricultural development. Biological control is playing an increasingly important role in promoting the development of agricultural and forestry production and protecting the ecological environment. [0003] Capsicum anthracnose has a wide range of hosts and can harm the fruits, stems, leaves and young fruits of various vegetables, flowers, medicinal plants and field crops, causing symptoms such as rotten fruits, dead branches, leaf spots and dead seedlings, and causing serious damag...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20A01P3/00C07K1/30C07K14/32C12R1/125
Inventor 曾大兴吴小丽贾书娟张晓阳
Owner SHENZHEN POLYTECHNIC
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