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Bacillus subtilis CF-3 and culture method thereof

A Bacillus subtilis, CF-3 technology, applied to the newly screened Bacillus subtilis CF-3 and its culture field, can solve the problems affecting the commercial application of biocontrol bacteria, less research on control effects, and narrow antibacterial spectrum. , to achieve the effect of high potency, broad antibacterial spectrum and easy preparation

Active Publication Date: 2017-02-22
SHANGHAI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The infection of pathogenic fungi has always been the main reason affecting the postharvest quality of fruits and vegetables. At present, there are many domestic and international literature reports on the use of biocontrol bacteria to control plant diseases. On the whole, narrow antibacterial spectrum and low efficacy are the main problems affecting the commercial application of biocontrol bacteria. Therefore, screening biocontrol bacteria with broad antibacterial spectrum and high efficacy for postharvest biological control of fruits and vegetables has become a new challenge. Research Hotspots

Method used

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  • Bacillus subtilis CF-3 and culture method thereof
  • Bacillus subtilis CF-3 and culture method thereof
  • Bacillus subtilis CF-3 and culture method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Embodiment 1: Separation and purification of Bacillus subtilis CF-3

[0019] The bacillus subtilis strain is isolated and screened from purchased fermented bean curd by means of a dilution plate method. Put every piece of fermented bean curd (10 grams) into the Erlenmeyer flask that 100 milliliters of sterilized waters are housed and shake for 10 minutes, then serially dilute to 10 -6 times, to prepare dilutions; mix 1 ml of suspensions of different concentrations with NA solid medium cooled to 55-60°C and sterilize, pour it into a petri dish, and incubate it in a constant temperature incubator at 30°C for 24 hours; after the bacteria grow on the plate, pick a single colony and inoculate them on the NA solid medium for plate culture; then use the streak method to carry out mycelia inhibition experiments on pathogenic fungi, and select biocontrol bacterial strains with good inhibitory effect, and then In 20mL sterilized NA liquid medium, 37°C, 150rpm, shake flask cultur...

Embodiment 2

[0024] Embodiment 2: Identification of biological traits and physiological and biochemical characteristics of Bacillus subtilis CF-3

[0025] 1. Morphological characteristics:

[0026] The morphology of Bacillus subtilis CF-3 was found in electron microscope figure 1 , the colony of Bacillus subtilis cultured on a plate at 37°C for 24 hours was light yellow, round, smooth and translucent to opaque, with neat edges and thicker than the center; Gram staining was positive, and the cells were rod-shaped, with Flagella throughout, uncapsulated. Microscope photos are attached figure 1 .

[0027] 2. Physiological and biochemical characteristics:

[0028]The optimum temperature for the growth of Bacillus subtilis CF-3 is 37°C, and the optimum pH is 7.0-7.2. The rest are shown in Table 1.

[0029] Table 1 Physiological and biochemical characteristics of Bacillus subtilis CF-3

[0030]

[0031]

[0032] Note: "+" means positive or growth, "-" means negative or no growth

Embodiment 3

[0033] Example 3: Sequence determination and analysis of 16S rDNA of Bacillus subtilis CF-3

[0034] 1. Extraction of PCR template DNA:

[0035] Extracted by the DNA extraction kit produced by TAKARA Biotechnology Company

[0036] 2.16S rDNA gene PCR amplification:

[0037] PCR primers were produced by TAKARA Biotechnology Company

[0038] Upstream primer Primer A: 5'-GAGCGGATAACAATTTCACACAGG-3' (SEQ ID NO:2)

[0039] Downstream primer Primer B: 5'-CGCCAGGGTTTTTCCCAGTCACGAC-3' (SEQ ID NO:3)

[0040] The PCR reaction system is shown in Table 2:

[0041] Table 2 PCR reaction system

[0042]

[0043] PCR amplification cycle conditions: first cycle at 94°C for 5 minutes; then cycle 30 times in the order of 94°C for 1 minute, 50-55°C for 1 minute, and 72°C for 1.5 minutes; finally cycle at 72°C for 5 minutes.

[0044] 3. Sequence determination:

[0045] After the PCR product was purified, it was sent to Sangon Bioengineering (Shanghai) Co., Ltd. for sequencing. The sequen...

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Abstract

The invention discloses bacillus subtilis CF-3 with the preservation number being CCTCC No:M 2016125 and a preparation method thereof. Strains are separated from fermented bean curd. The bacillus subtilis is mainly used for controlling postharvest fruit diseases. The bacillus subtilis CF-3 has good inhibition effect on various postharvest fruit disease pathogenic fungi such as tomato botrytis cinerea, strawberry botrytis cinerea, litchi colletotrichum gloeosporiodies, apple valsa mali miyabe et yamada, apple penicillium expansum, peach Monilinia fracticola, jujube phoma pomirum Thum and jujube Alternaria alternata, is broad in antimicrobial spectrum and high in efficacy and has the advantages of being non-toxic to human and livestock, eco-friendly and the like, and besides, the preparation method of the bacillus subtilis CF-3 adopts a simple process, is short in fermentation period and can be applied to postharvest preservation of fruits and vegetables.

Description

technical field [0001] The invention relates to a newly screened bacillus subtilis CF-3 and a culture method thereof. Background technique [0002] Fruit and vegetable rot caused by pathogenic bacteria after harvest has caused serious economic losses. For a long time, in order to control the occurrence of diseases of harvested fruits and vegetables, the method of chemical fungicides is mainly used, but the use of chemical fungicides often easily causes the generation of drug resistance of pathogenic bacteria, affects the safety of food, and causes environmental pollution and other problems. considered to be one of the most promising approaches. Therefore, postharvest fresh-keeping methods for fruits and vegetables with high efficiency, low toxicity, low residue and good environmental compatibility have become a research hotspot. [0003] Bacillus subtilis is a non-pathogenic microorganism that has antagonistic effects on many plant pathogenic organisms. Its metabolic syste...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12R1/125
CPCC12N1/20C12N1/205C12R2001/125
Inventor 高海燕李培中曾晴徐新星戴悦雯尹京苑
Owner SHANGHAI UNIV
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