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Endophytic bacillus amyloliquefaciens capable of generating a large number of antagonistic tobacco bacterial wilt active materials

A technology for tobacco bacterial wilt and amylolytic spores, applied in the directions of plant growth regulators, microorganism-based methods, chemicals for biological control, etc. To achieve the effect of expanding production and effective application, good colonization ability, and obvious inhibitory effect

Active Publication Date: 2016-02-03
武汉康绿达生物科技有限公司
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Problems solved by technology

[0004] There are many researches on the biological control of tobacco bacterial wilt, but few report on the specific antibacterial substances of plant endophytes and their control effects on tobacco bacterial wilt. Plant endophytes have greater advantages in plant biological control of exogenous pathogens , the study of its antibacterial substances plays a vital role in the biological control of plant soil-borne diseases

Method used

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  • Endophytic bacillus amyloliquefaciens capable of generating a large number of antagonistic tobacco bacterial wilt active materials
  • Endophytic bacillus amyloliquefaciens capable of generating a large number of antagonistic tobacco bacterial wilt active materials
  • Endophytic bacillus amyloliquefaciens capable of generating a large number of antagonistic tobacco bacterial wilt active materials

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Experimental program
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Effect test

Embodiment 1

[0020] Embodiment 1: screening and identification of antagonistic strains

[0021] The stems of healthy Burley tobacco plants in Daejeon were taken, sterilized on the surface, cut into pieces, enriched in normal saline, placed in a water bath at 80°C, diluted and coated to pick a single colony, and the pathogenic bacteria of tobacco solanacear was used as an indicator bacteria to conduct a plate confrontation experiment. A biocontrol strain BG2 with significant antagonistic effect was screened out. This strain has a rod-shaped body and produces spores. The colonies on the LB plate are round and flat, the surface is rough, white and opaque, the edges are uneven, there is expansion, and no adhesion , easy to pick. By analyzing the 16S rDNA sequence of the BG2 strain, Blast comparison search was carried out in the GenBank nucleotide sequence database, and the sequence with higher homology to BG2 was selected for phylogenetic analysis. The results are as follows: figure 1 shown....

Embodiment 2

[0022] Embodiment 2: Antimicrobial Spectrum Determination, Pot Plant Colonization and Control Effect Detection

[0023] Take 14 strains of pathogenic fungi preserved in the laboratory, use the disc punching method, take a piece of fungus each and place it in the center of the petri dish, and cultivate it at 30°C. Punch holes, add 20 μL filter-sterilized fermentation supernatant of the BG2 strain, and incubate at 30°C until the fungus spreads to the edge of the plate to record the antibacterial situation. The results are shown in Table 1. Pathogenic fungi have a good inhibitory effect.

[0024] Tobacco seedlings of the K326 variety were transplanted into potted plants for testing, and the endophytic tobacco strain BG2 was inserted into the fermentation medium, fermented at 25-40°C, 150-250r / min for 24-60h, and the tobacco bacterial wilt pathogen RS9-2 Insert the strain into NA liquid medium, ferment at 25-35°C, 150-250r / min for 12-24h; dilute the fermentation broth of strain B...

Embodiment 3

[0034] Embodiment 3: identification of antibacterial active substance

[0035] Stability of antibacterial substances to temperature, pH value, and ultraviolet: Treat the antibacterial substances produced by strain BG2 at 30°C, 40°C, 50°C, 60°C, 70°C, 80°C, 100°C, 115°C and 121°C for 30 minutes ; adjust the pH of the antibacterial substance produced by bacterial strain BG2 to 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, 12.0, and treat overnight; place the antibacterial substance produced by bacterial strain BG2 under a 30W ultraviolet lamp, Irradiate at a distance of 15cm for 1h, 2h, 3h, 4h, 5h, and 6h, respectively, and measure the antagonistic activity of the filtrate against the pathogenic bacteria R. solanacearum. The result is as image 3 shown by image 3 a It can be seen that the antibacterial substances produced by the strain BG2 can maintain more than 90% of the antibacterial activity at 30°C-60°C; image 3 It can be seen that the antibacterial substance pro...

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Abstract

The invention relates to the technical field of microbial pesticides and discloses endophytic bacillus amyloliquefaciens BG2 capable of generating a large number of antagonistic tobacco bacterial wilt active materials. The endophytic bacillus amyloliquefaciens BG2 is preserved in the China Center for Type Culture Collection, and the preservation number is CCTCC NO. M 2015173. The invention further discloses the potting application of the bacterial strain, identification of generated antibiotic substances and high-yield fermentation process optimization of the bacterial strain. The bacterial strain has high colonization capacity in tobacco plant tissue and rhizosphere soil of the tobacco plant tissue, can generate antagonistic tobacco bacterial wilt active materials, can restrain various pathogenic fungi, and can be produced more widely at low cost and applied effectively. It is found through isolation purification and identification analysis of antipathogen active materials that the active components of the bacterial strain are mainly lipopeptid surfactin homolog and protein. The invention further provides a high-yield fermentation process of the bacteriostatic active materials. The process is characterized in that 10-100 g / L bean pulp, 5-50 g / L glucose, 1-10 g / L K2HPO4, 0.05-1.00 g / L CaCl2 and 0.05-1.00 g / L MgSO4 are adopted, pH is 7.2-7.4, culture temperature is 25-40 DEG C, inoculum size is 1-5%, liquid volume is 10-100 ml / 250 ml, inoculum age is 6-18 h, and fermentation is conducted for 24-60 h.

Description

technical field [0001] The invention relates to the technical field of using microbial pesticides, in particular to the application of a kind of tobacco endogenous amylolytic spores ( Bacillus amylolique faciens ) Potted application of BG2, optimization of high-yield fermentation process of antibacterial substances, and research and identification of physicochemical properties of antibacterial substances. Background technique [0002] tobacco( Nicotiana tabacum ) has high economic value and can be said to be one of the most important economic crops in my country. Tobacco bacterial wilt (Tobacco Bactrial Wilt) is one of the main diseases of tobacco and is called "smoke cancer". Bacteria ( Ralstonia solanacearum ) caused by a systemic duct destructive disease, is a serious soil-borne disease that commonly occurs in the world, mainly distributed in tropical, subtropical and broad temperate regions. For a long time, the control methods for bacterial wilt of Solanaceae plants...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12P21/00A01N63/02A01P1/00C12R1/07
Inventor 陈守文王晓王昌军余君马昕徐迪红李进平杨欢陈建刚冀志霞杨勇魏雪团
Owner 武汉康绿达生物科技有限公司
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