CN111662843B - A strain of Bacillus amyloliquefaciens and its isolation and screening method and application - Google Patents

Abstract

The present invention discloses a strain of Bacillus amyloliquefaciens, which was preserved in the General Microbiology Center of the China Microorganism Culture Collection Administration Committee on April 13, 2020, and the preservation number is CGMCC No: 19565; Bacillus amyloliquefaciens, the inhibition rate of coconut stem rot and peanut fruit rot reaches 79%. The inhibition rate of pathogenic bacteria was more than 70%, and the antibacterial activity of the fermentation broth of the strain did not change significantly after being treated at 80 °C for 120 min, and the thermal stability was strong. The strain has great application potential in biological control of crop diseases in hot regions.

Description

A strain of Bacillus amyloliquefaciens and its isolation and screening method and application

technical field

The invention belongs to the technical field of microbial resources, in particular to a strain of Bacillus amyloliquefaciens and a separation and screening method and application thereof.

Background technique

Bacillus has the characteristics of heat resistance, drought resistance, ultraviolet resistance, electromagnetic radiation resistance and tolerance to adverse conditions caused by certain types of chemicals. The use of Bacillus to control plant diseases is currently a commonly used biological control technology in agricultural production. one. Tea dry is the residue left after oil extraction from tea seeds. The annual output in my country exceeds 1 million tons, and it is a commonly used organic fertilizer raw material. Tea kudzu is rich in tea saponin, which is a hemolytic toxin that is often used to prevent snail damage. Combining tea bacillus with biocontrol Bacillus, and exerting the antibacterial effect of tea saponin and Bacillus at the same time, is beneficial to improve the control effect of plant diseases and insect pests. Therefore, it is of great significance to screen Bacillus with broad-spectrum antibacterial activity and resistance to tea saponin.

SUMMARY OF THE INVENTION

At present, no Bacillus resource with broad-spectrum antibacterial activity that is resistant to tea saponin at the same time has been reported. According to the invention, a strain of Bacillus amyloliquefaciens is isolated and screened from Camellia sinensis, which has broad-spectrum antibacterial activity and is heat-resistant and resistant to tea saponin.

In view of this, the present invention provides a strain of Bacillus amyloliquefaciens, which is classified and named Bacillus amyloliquefaciens, which was preserved in the General Microbiology Center of the China Committee for the Collection of Microorganisms on April 13, 2020, address: Beijing No. 3, No. 1 Courtyard, Beichen West Road, Chaoyang District, preservation number: CGMCC No: 19565.

The invention also provides a strain of Bacillus amyloliquefaciens in the prevention and treatment of banana fusarium wilt, coconut stem rot, peanut fruit rot, betel nut anthracnose, coconut gray spot, betel nut heart rot, oil palm heart rot, chestnut blight, Application of one or more of Camellia oleifera leaf spot, Camellia oleifera anthracnose and Chinese cabbage anthracnose.

The present invention also provides a method for separation and screening of Bacillus amyloliquefaciens, characterized in that it comprises the following steps:

1) Camellia oleifera was fermented and cultured at 45°C for 24 hours to prepare Camellia oleifera cake fermentation broth, and the concentration of tea saponin was 20g/L.

2) Weigh 1g of Camellia oleifera fermentation broth, add it to 10ml sterile water, shake and cultivate for 30min, then dilute to ^10-1 , ^10-2 , ^10-3 times with sterile water successively, take 0.1ml respectively and apply it to LB On the medium, culture at 45°C for about 2 days, and select a single colony for purification and culture.

Beneficial effects:

In the mycelial growth rate method test, the inhibition rate of ck-05 strain fermentation broth to banana fusarium wilt reached 82%, the inhibition rate of coconut stem rot and peanut fruit rot reached 79%, and the inhibition rate of betel nut anthracnose, The inhibition rate of pathogenic bacteria such as coconut gray spot, betel nut heart rot, oil palm heart rot, chestnut blight, peanut fruit rot, and oil tea leaf spot is over 70%. The antibacterial activity of the fermentation broth of the ck-05 strain did not change significantly after being treated at 80 °C for 120 min, and the thermal stability was strong.

Description of drawings

Fig. 1 is the colony morphology diagram of Bacillus amyloliquefaciens ck-05 of the present invention on LB plate;

Fig. 2 is the microscopic morphological diagram of Bacillus amyloliquefaciens ck-05 of the present invention;

Fig. 3 is a phylogenetic tree diagram of Bacillus amyloliquefaciens ck-05 of the present invention;

Fig. 4 is the thermostability data diagram of Bacillus amyloliquefaciens ck-05 fermentation broth of the present invention.

Detailed ways

Below in conjunction with example, the present invention is further described:

Example 1:

Isolation and Identification of Bacillus amyloliquefaciens ck-05

1) Camellia oleifera was fermented and cultured at 45°C for 24 hours to prepare Camellia oleifera cake fermentation broth, and the concentration of tea saponin was 20g/L.

2) Weigh 1g of Camellia oleifera fermentation broth, add it to 10ml sterile water, shake and cultivate for 30min, then dilute to 10-1, 10-2, 10-3 times with sterile water successively, take 0.1ml and apply it to LB On the medium, culture at 45°C for about 2 days, and select a single colony for purification and culture.

3) Morphology: After culturing on LB medium for 2 days, as shown in Figures 1 and 2, the colonies are white, round, dry, rough and wrinkled on the surface, small and protruding, with neat edges, which are observed under an optical microscope as Short rods, Gram-positive, oval spores.

4) Main physiological and biochemical characteristics: strain ck-05 contact enzyme test, catalase test, V-P test, indole test, hydrolyzed starch and casein test, nitrate reduction test, citrate utilization test, glucose, sucrose, Maltose fermentation test was positive; methyl red test was negative. The physiological and biochemical characteristics of strain ck-05 are basically the same as those of Bacillus amyloliquefaciens.

5) Molecular biological identification: The DNA of the target strain was extracted using a bacterial genomic DNA extraction kit as a template, and the universal primers 27F and 1492R were used for PCR amplification. 50 ml of PCR products were taken and detected by 1% agarose gel electrophoresis, and the PCR products were sent to Beijing Liuhe Huada Gene Technology Co., Ltd. for sequencing. The sequences were compared by Blast on the NCBI website, and the strains were phylogenetically analyzed by software such as BioEdit and Mega5.0, and a phylogenetic tree was constructed by using Neighbour-joining (NJ method), as shown in Figure 3. The results showed that the strains ck-05 is Bacillus amyloliquefaciens (Accession No.: MK592621).

Example 2: Antimicrobial spectrum determination of Bacillus amyloliquefaciens ck-05 strain fermentation broth

1) Using the mycelial growth rate method. Take 10ml of sterile fermentation filtrate of strain ck-05 and mix it with 90ml of PDA medium cooled to 40°C to prepare a medium plate containing sterile fermentation broth of strain ck-05. Into the center of the plate were tested pathogenic bacteria blocks (5mm), each pathogen was one treatment, and each treatment was repeated 3 times. Taking sterile water mixed with PDA medium as a control, it was placed in a constant temperature incubator at 28 °C for cultivation. When the colonies of the control group were covered with the petri dish, the colony diameter of the treatment group was measured by the cross method, and the bacteriostatic effect of the sterile filtrate was calculated. Rate.

Bacteriostatic rate=(control colony growth diameter-treatment colony growth diameter)/control colony growth diameter×100%

As can be seen from Table 1, the ck-05 bacterial strain fermentation broth has a strong inhibitory effect on 11 kinds of tested pathogens, except that the antibacterial effect on cabbage anthracnose is 43.47%, and the other 10 kinds of tested pathogenic bacteria are inhibited. Among them, the inhibitory effect on banana fusarium wilt was the strongest, with a bacteriostatic rate of 82.22%, followed by peanut fruit rot and coconut stem rot, with bacteriostatic rates of 79.44% and 78.89%, respectively. It can be seen that the ck-05 strain fermentation broth has a wide antibacterial range.

Table 1 Inhibitory effect of ck-05 strain fermentation broth on 11 pathogenic bacteria

Example 3: Determination of thermostability of antibacterial effect of Bacillus amyloliquefaciens ck-05

1) Under the conditions of 40, 60, 80, and 100 °C, the aseptic fermentation broth of the ck-05 strain was treated for 30, 60, 120 min and 121 °C, respectively, for 30 min, and the banana wilt Fusarium oxysporum was used as an indicator. The bacteria were used to determine the bacteriostatic rate of the sterile fermentation broth after treatment at different temperatures. Each treatment was repeated 3 times, and the untreated fermentation broth was used as a control. When the mycelium of the control group covered the petri dish, the colony diameter of the treatment group was measured, and then the bacteriostatic rate was calculated.

It can be seen from Figure 4 that the fermentation broth of ck-05 strain still has a strong antibacterial effect after being treated at different temperatures. Its bacteriostatic activity decreased significantly, and the bacteriostatic rate still reached 41.48% when treated at 100°C for 30 minutes, while the bacteriostatic rate was 31.48% when treated at 100°C for 120 minutes. In addition, the bacteriostatic activity of the fermentation broth of ck-05 strain decreased rapidly after being sterilized by high pressure steam at 121℃ for 20min, and the bacteriostatic rate was only 19.26%. Figure 4 clearly shows that the bacteriostatic rate of the fermentation broth of the ck-05 strain decreases significantly when the temperature rises to 100°C, which may be due to the bacteriostatic activity in the fermentation broth of the ck-05 strain when the temperature is higher than 100°C The material structure changed, which eventually led to the decline of antibacterial activity, but when the temperature was lower than 80 °C, it had better thermal stability, indicating that the antibacterial material of ck-05 strain had strong thermal stability.

sequence listing

<110> Coconut Research Institute, Chinese Academy of Tropical Agricultural Sciences

<120> A strain of Bacillus amyloliquefaciens and its isolation and screening method and application

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1435

<212> DNA

<213> Bacillus amyloliquefaciens

<400> 1

cggcctatac tgcaagtcga gcggacagat gggagcttgc tccctgatgt tagcggcgga 60

cgggtgagta acacgtgggt aacctgcctg taagactggg ataactccgg gaaaccgggg 120

ctaataccgg atggttgtct gaaccgcatg gttcagacat aaaaggtggc ttcggctacc 180

acttacagat ggacccgcgg cgcattagct agttggtgag gtaacggctc accaaggcga 240

cgatgcgtag ccgacctgag agggtgatcg gccacactgg gactgagaca cggcccagac 300

tcctacggga ggcagcagta gggaatcttc cgcaatggac gaaagtctga cggagcaacg 360

ccgcgtgagt gatgaaggtt ttcggatcgt aaagctctgt tgttagggaa gaacaagtgc 420

cgttcaaata gggcggcacc ttgacggtac ctaaccagaa agccacggct aactacgtgc 480

cagcagccgc ggtaatacgt aggtggcaag cgttgtccgg aattattggg cgtaaagggc 540

tcgcaggcgg tttcttaagt ctgatgtgaa agcccccggc tcaaccgggg agggtcattg 600

gaaactgggg aacttgagtg cagaagagga gagtggaatt ccacgtgtag cggtgaaatg 660

cgtagagatg tggaggaaca ccagtggcga aggcgactct ctggtctgta actgacgctg 720

aggagcgaaa gcgtggggag cgaacaggat tagataccct ggtagtccac gccgtaaacg 780

atgagtgcta agtgttaggg ggtttccgcc ccttagtgct gcagctaacg cattaagcac 840

tccgcctggg gagtacggtc gcaagactga aactcaaagg aattgacggg ggcccgcaca 900

agcggtggag catgtggttt aattcgaagc aacgcgaaga accttaccag gtcttgacat 960

cctctgacaa tcctagagat aggacgtccc cttcgggggc agagtgacag gtggtgcatg 1020

gttgtcgtca gctcgtgtcg tgagatgttg ggttaagtcc cgcaacgagc gcaacccttg 1080

atcttagttg ccagcattca gttgggcact ctaaggtgac tgccggtgac aaaccggagg 1140

aaggtgggga tgacgtcaaa tcatcatgcc ccttatgacc tgggctacac acgtgctaca 1200

atggacagaa caaagggcag cgaaaccgcg aggttaagcc aatcccacaa atctgttctc 1260

agttcggatc gcagtctgca actcgactgc gtgaagctgg aatcgctagt aatcgcggat 1320

cagcatgccg cggtgaatac gttcccgggc cttgtacaca ccgcccgtca caccgacgag 1380

agtttgtaac acccgaagtc ggtgaggtaa ccttttatgg agccagcccg cccct 1435

Claims (2)

1. A strain of Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens ) ck-05, characterized in that, the strain was preserved in the General Microbiology Center of the China Microorganism Culture Collection Administration Committee on April 20, 2020, and the deposit number: CGMCCNo: 19565. 2. Bacillus amyloliquefaciens as claimed in claim 1 is in the control of banana fusarium wilt, coconut stem rot, peanut fruit rot, betel nut anthracnose, coconut gray spot, betel nut heart rot, oil palm heart rot, Chinese chestnut The application of one or more bacterial agents in blight, Camellia oleifera, Camellia anthracnose and Chinese cabbage anthracnose.

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