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Detection method of biocontrol bacterium volatile metabolites

A detection method and metabolite technology, applied in the field of active metabolite detection, can solve the problems of insufficient control of experimental conditions, inability to accurately reflect the composition of target metabolites, poor stability of active substances, etc., long time to achieve, many steps, guaranteed The effect of accuracy

Active Publication Date: 2017-04-26
TOBACCO RES INST CHIN AGRI SCI ACAD
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, in the existing research methods for microbial volatile products, most of the control of experimental conditions is not strict enough, and it is not suitable for the analysis of active substances with poor stability. The ratio of the peak area of ​​the product to the total peak area indicates the relative content of the metabolite, which cannot accurately reflect the true composition of the target metabolite in the sample

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  • Detection method of biocontrol bacterium volatile metabolites
  • Detection method of biocontrol bacterium volatile metabolites
  • Detection method of biocontrol bacterium volatile metabolites

Examples

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Embodiment 1

[0037] The detection of embodiment 1 biocontrol bacteria AR03 volatile metabolites

[0038] 1. Instruments and reagents:

[0039] American Agilent 7890B-5975C gas chromatography mass spectrometer; biocontrol bacteria AR03 from the Plant Protection Research Center, Tobacco Research Institute, Chinese Academy of Agricultural Sciences; manual extraction handle, 50 / 30μm DVB / CAR / PDMS extraction head, 20mL brown headspace bottle, purchased from U.S. Supelco Company; 1-pentadecene, normal alkanes (C10-C20) were purchased from Beijing Bailingwei Company; dichloromethane and methanol were both chromatographically pure.

[0040] 2. Bacterial culture and volatile matter collection

[0041] (1) Medium production: Beef extract peptone agar (NA) medium is prepared as follows: 15g glucose, 1g yeast powder, 5g tryptone, 3g beef extract, add water to 1000mL, adjust pH to 7.0, and extinguish at 121°C Bacteria 20min.

[0042] (2) Blank sample preparation: Take 3 mL of uncooled NA medium (abou...

Embodiment 2

[0067] Example 2 Antagonistic effect of volatile metabolite β-sesquiphellandrene on tobacco fungi and bacterial pathogens

[0068] The pure β-sesquiphellandrene used in this example was purchased from International laboratory.

[0069] The antibacterial activity of β-sesquiphellandrene was determined by filter paper strip method. The sample is made into a methanol solution with a mass concentration of 10 mg / ml, and its antagonistic activity to tobacco black shank bacteria, tobacco bacterial wilt, red spot bacteria and anthracnose bacteria is measured according to the filter paper strip method. The specific methods are as follows: Evenly drop 50 μl of paper strips (7mm×3mm), carefully place the filter paper strips in the petri dish according to the direction of the cross, and then inoculate the above-mentioned bacteria in the space of the filter paper strips at equal distances, and incubate at 30°C for 5 days; the control group Add methanol; observe the antagonistic activity o...

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Abstract

The invention discloses a detection method of biocontrol bacterium volatile metabolites. The detection method comprises the following steps: (1) smearing a bacterium fermenting solution on a culture medium, and placing the culture medium into a head space bottle; (2) collecting bacterium volatile matters by using a solid-phase micro extraction technology; (3) identifying the bacterium volatile matters by using a gas chromatograph-mass spectrometer with a multi-mode sample inlet in combination with commercial spectrum library searching and an n-alkanes retention index under a full-scanning mode, and quantitatively analyzing the content of the bacterium volatile matters according to a compound molecular identification result. By adopting the detection method, the problems for collecting, identifying and quantitatively detecting the unstable and easy-to-degrade active volatile matters can be effectively solved, the space-time dynamic information of the extracellular volatile metabolite molecules can be grasped, support is provided for researching the antibacterial mechanism, an acting way and application of the biocontrol bacterium in a biological control field, and a theoretical foundation is provided for enriching the type of the antibacterial substances and digging novel antibacterial genes.

Description

technical field [0001] The invention belongs to the technical field of active metabolite detection, and relates to a detection method for volatile metabolites of biocontrol bacteria. Background technique [0002] The biocontrol bacterium AR03 involved in the present invention is a bacterial strain with good properties screened out from soil rhizosphere bacteria, which is identified as Bacillus pumilus. This bacterial strain and its application in plant disease biocontrol have been Obtained the national invention patent (ZL.201110146683.8). The strain is effective against fruit and vegetable crops such as Ralostonia Solanacearum, Alternaria alternata, Phytophoranicotianae, Colletotrichum nicotianae Av.-Sacca and Erysiphe cichoracearum DC. Plant pathogenic fungi and bacteria have obvious inhibitory effect; in addition, the AR03 strain also has a growth-promoting effect on the tobacco strain. Under sterilized soil conditions, the dry weight of the whole plant and root dry weig...

Claims

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Application Information

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IPC IPC(8): G01N30/02
CPCG01N30/02
Inventor 曹建敏王静邱军于卫松林璎楠孙娜庞雪莉
Owner TOBACCO RES INST CHIN AGRI SCI ACAD
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