Endophytic bacillius subtilis antibacterial volatile substance and identification method thereof

A technology of Bacillus subtilis and volatile substances, applied in the direction of microorganism-based methods, biochemical equipment and methods, botany equipment and methods, etc., can solve the problems of no relevant reports and no in-depth research on volatile gases, and achieve High research and application value, the effect of inhibiting the micro-sclerotia germination of Verticillium dahliae and inhibiting the growth of mycelium

Inactive Publication Date: 2016-03-16
JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the volatile gas produced by the strain has no

Method used

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  • Endophytic bacillius subtilis antibacterial volatile substance and identification method thereof
  • Endophytic bacillius subtilis antibacterial volatile substance and identification method thereof

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Experimental program
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Effect test

Embodiment 1

[0016] Embodiment 1 Generation and collection of volatile substances

[0017] The Bacillus subtilis (Bacillius subtilis) Jaased1 bacterial strain that produces this volatile substance is inoculated into in the LB culture fluid that adds sterilized magnetic stirrer (the described LB culture fluid is yeast powder 5%, NaCl10%, peptone 10%, pH= 7) Shaking culture at 37°C and 120r / min for 24h, after culture, place the culture bottle on a magnetic stirrer and stir at 50°C for 30min. Aging the DVB / CAR / PDMS extraction head at 270°C for 30 minutes, inserting it vertically into the culture bottle, extending the extraction head so that the extraction head is 2cm above the liquid surface of the culture medium, and performing solid phase analysis on the volatile gas produced by Jaased1. Micro-extraction, after 30 minutes of extraction, volatile gases can be extracted from the extraction head.

Embodiment 2

[0018] The mensuration of embodiment 2 volatile gases

[0019] Volatile gases were measured with a thermoelectric TraceDsq-mass spectrometer. Analyze the extraction head at the inlet at 210°C for 5 minutes, adopt the splitless mode, the carrier gas is He, the flow rate is 1.0ml / min, the heating program is 33°C, 3min, the temperature is raised to 180°C at a speed of 10°C / min, and then The temperature was raised to 220°C at a rate of 40°C / min, and held for 5 minutes; the mass spectrometry conditions were electron ionization of 70eV, source temperature of 200°C, fourth-stage temperature of 150°C, and continuous scanning from 40 to 500 m / z. Compared with the NIST database, it was found that the endophyte Jaased1 produced at least 15 volatile substances (see figure 1 ), respectively 2-undecanone (RT=11.06), 2-tridecanone (RT=11.93), 2-phenylethanol (RT=8.35), 2-heptanone (RT=4.36), 2-nonyl Ketone (RT=8.01), 2-nonanol (RT=8.16), 2-decanol (RT=9.17), 2-undecanol (RT=11.16), 2-dodec...

Embodiment 3

[0020] The analysis of embodiment 3 volatile substance active ingredients

[0021] In the 90mm petri dish that divider is arranged in the middle, one side is poured into PDA culture medium (described PDA culture medium is potato 20%, glucose 2%, agar 1.5-2.0%, natural pH value), inoculate watermelon wilt bacteria bacterium Cake (7 mm in diameter), put sterilized filter paper on the other side, and add 100 μL or 0.2 g of volatile chemical substances dropwise. Add 100 μL sterile water to the control. For each treatment of 3 dishes, repeat 3 times. Seal the petri dish with parafilm to block the circulation of air inside and outside the petri dish, culture the petri dish at 25°C, measure the inhibitory effect of volatile chemical substances on mycelium after the control mycelium grows over a compartment, and calculate the antibacterial effect Rate. The result shows that after the compartments add volatile substances respectively, 10 kinds of volatile substances have different d...

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Abstract

The invention relates to a volatile substance which has antibacterial activity and is produced by a biocontrol endophytic bacillius subtilis strain Jaas ed1 and an identification method thereof. The strain at least can produce 15 volatile substances, and 10 of the 15 volatile substances are bacteriostat effective substances and comprise 2-heptanone, 2-nonanone, 2-nonanol, 2-decanone, 2-decanol, 2-phenethyl alcohol, 2-undecanone, 2-cyclododecanoe, 2-undecanol and 2-tridecanone. The collecting and purifying method includes the steps that endophytic bacillius subtilis Jaas ed1 fermentation liquor is heated and stirred on a magnetic stirrer, solid phase microextraction is conducted on volatile gas through a DVB/CAR/PDMS extraction head, and finally extracted volatile substance ingredients are determined through the GC-MS. The volatile gas can inhibit mycelial growth of pathogens of watermelon fusarium wilt and germination of cotton verticillium dahliae microsclerotia, extremely-high study and application value is achieved on development of broad-spectrum antibacterial chemicals, and wide application prospects are achieved for protecting the natural quality of crops and fruits of the crops and reducing pesticide residues.

Description

Technical field: [0001] The invention relates to a group of antibacterial volatile substances produced by endophytic bacillus subtilis (Bacillius subtilis) Jaased1 and an identification method thereof. Background technique: [0002] The research on the biological control of plant diseases is currently mainly focused on the use of plant secondary substances, inducing host resistance and antagonizing microorganisms. Among them, the development and application of antagonistic bacteria and their non-volatile metabolites are increasingly extensive and in-depth. However, there are few studies on the volatile metabolites produced by bacteria. Many reports have indicated that the volatile gases produced by bacteria are of importance in biological control. A series of volatile substances (VCs) produced by bacteria, including aldehydes, esters, alcohols, acids and ketones, etc., these volatile compounds have a broad-spectrum antibacterial effect, and have a certain inhibitory effect ...

Claims

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Application Information

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IPC IPC(8): C07C49/04C07C31/125C07C33/22C12P7/26C12P7/04C12P7/22A01N31/02A01N35/02A01P3/00C12R1/125
CPCC07C49/04A01N31/02A01N35/02C07C31/125C07C33/22C12P7/04C12P7/22C12P7/26
Inventor 林玲王卿邓晟张昕
Owner JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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