Brevibacillus brevis for preventing and controlling tobacco black shank and application thereof

A technology of Bacillus brevis, Tobacco Blackleg, applied in the fields of application, bacteria, fungicides, etc., can solve the problem that the biological control research of Tobacco Blackleg has not yet been systematically reported, and achieves great application potential, obvious inhibitory effect, Efficiently produced effects

Inactive Publication Date: 2015-12-09
TOBACCO RES INST CHIN AGRI SCI ACAD +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Despite the importance of the broad-spectrum bactericidal ability of chitinase, the research on the biological control of tobacco black shank by Bacillus brevis and its chitinase has not been systematically reported

Method used

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  • Brevibacillus brevis for preventing and controlling tobacco black shank and application thereof
  • Brevibacillus brevis for preventing and controlling tobacco black shank and application thereof
  • Brevibacillus brevis for preventing and controlling tobacco black shank and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Embodiment 1: the separation of Bacillus brevis bacterial strain of the present invention

[0028] Isolation Medium: Induced Enzyme Production Medium, 0.1% Polypeptone, 0.05% MgSO 4 7H 2 O, 0.1% KH 2 PO 4 , 0.2% NaCl, 0.2% 1% colloidal chitin, pH adjusted to 6.8. Production of 1% colloidal chitin: weigh 10g of chitin (Sigma, St.Louis, MO, USA) in 100mL of 85% H 3 PO 4 , fully dissolve, overnight at 4°C, add 500mL of ultrapure water, stir with electric power to fully disperse, wash with ultrapure water to about pH 5.0, and store at 4°C for later use.

[0029] Proliferation medium: MRS liquid medium, 10g casein peptone; 10g beef extract; 5g yeast extract; 20g glucose, constant volume 1000mL, for antibacterial determination.

[0030] Separation method: Weigh 1g of diseased soil into a 15mL test tube with a stopper, add 9mL of water, shake on a shaker at room temperature at 200r / min for 1h. Take 1 mL of the supernatant and dilute to 10 -5 times, in the induced enzyme...

Embodiment 2

[0031] Embodiment 2: the culture character of Bacillus brevis bacterial strain of the present invention

[0032] The purified strains were transferred to chitin and nutrient (NA) medium, and cultivated in a 37°C incubator. On the 3rd day, observe the color of the colony and whether there is a transparent circle, and measure the size of the colony and the diameter of the transparent circle. Gram staining. Bacteria are identified, thin sections are made through four steps of primary staining, mordant staining, decolorization, and counterstaining, dried, and microscopically examined.

[0033] Bacillus brevis GB6-1 was cultured on NA medium to produce soluble reddish-brown colonies ( figure 1 ), a transparent circle appeared around the colony in the medium containing 0.2% chitin ( figure 2 ). Bacillus brevis is a Gram-positive bacterium, oval 1.63-2.60 (±1.99)*0.78-1.47 (±1.03) ( image 3 ).

Embodiment 3

[0034] Embodiment 3: Molecular biology identification, 16SrRNA gene fragment sequence alignment and phylogenetic analysis of the Bacillus brevis bacterial strain of the present invention

[0035] Synthesis of amplification primers: synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd.

[0036] Universal primers:

[0037] 27F: 5''

[0038] 1429R: 5''

[0039] Genomic DNA extraction: Bacteria were cultured on PDA medium by streaking method, and cultured in a constant temperature incubator at 37°C for 3 days. Afterwards, the bacteria were transferred to the MRS culture medium, placed in a shaker, and cultured at 37°C and 230r / min for 36h to multiply. After multiplication, take 1ml of the culture solution and add it to the enzyme-inducing medium, place it in a shaker, and cultivate it under the same conditions for 2 days. Use a suction filter to remove the culture medium, collect the bacteria into a mortar, add liquid nitrogen and grind them into powder fo...

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Abstract

The invention provides brevibacillus brevis for preventing and controlling tobacco black shank and application thereof and belongs to the fields of microorganisms and microorganism application. According to the classification and naming, 16S rRNA sequence (1031bp) of B. brevis GB6-1 of the brevibacillus brevis is already uploaded to a GenBank database (serial number: KP 715564) of the NCBI and is preserved in the China General Microbiological Culture Collection Center (CGMCC) of China Committee for Culture Collection of Microorganisms, and the preservation number of the brevibacillus brevis is CGMCC No.11135. A lot of biological experiments prove that the brevibacillus brevis has the characteristics of efficiently secreting chitinase and Beta-1,3-glucosanase and has the obvious inhabiting effect on the tobacco black shank.

Description

technical field [0001] The invention relates to biological control of crop diseases, in particular to a bacillus brevis that efficiently produces chitinase and has obvious inhibitory effect on tobacco black shank, and belongs to the field of microorganisms and their application. Background technique [0002] tobacco black shank Phytophthora parasitica f.sp. nicotianae It is the main disease that threatens the tobacco production in my country, mainly harming the rhizome of the plant, seriously reducing the yield and quality of tobacco. Traditional control measures mainly adopt chemical control, cultivation control and selection of disease-resistant varieties. Tobacco is an economic crop for leaves. The long-term and large-scale use of chemical fungicides to prevent and control tobacco black shank can lead to pesticide residues, drug resistance and environmental pollution, which will reduce the quality of tobacco leaves and limit the domestic and foreign trade of tobacco le...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20A01N63/00A01P3/00C12R1/01
Inventor 陈雪徐同伟陈泽鹏刘勇陈德鑫武侠喻会平游兴琳
Owner TOBACCO RES INST CHIN AGRI SCI ACAD
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