CN111662843B

CN111662843B - Bacillus amyloliquefaciens and separation and screening method and application thereof

Info

Publication number: CN111662843B
Application number: CN202010556485.8A
Authority: CN
Inventors: 刘小玉; 付登强; 黄丽云
Original assignee: Coconut Research Institute of Chinese Academy of Tropical Agricultural Sciences
Current assignee: Coconut Research Institute of Chinese Academy of Tropical Agricultural Sciences
Priority date: 2020-06-17
Filing date: 2020-06-17
Publication date: 2022-05-31
Anticipated expiration: 2040-06-17
Also published as: CN111662843A

Abstract

The invention discloses a bacillus amyloliquefaciens strain, which is preserved in China general microbiological culture Collection center in 2020, 4 months and 13 days, wherein the preservation number is as follows: CGMCC No: 19565, respectively; by adopting the bacillus amyloliquefaciens provided by the invention, the inhibition rate on coconut stem rot and peanut rot reaches 79%, the inhibition rate on pathogenic bacteria such as areca anthracnose, coconut gray leaf spot, areca core rot, oil palm core rot, Chinese chestnut epidemic disease, camellia oleifera leaf spot and the like exceeds 70%, and the antibacterial activity of the strain fermentation liquor is not obviously changed after being treated for 120min at 80 ℃, and the thermal stability is strong. The strain has great application potential in the biological control of hot-zone crop diseases.

Description

Bacillus amyloliquefaciens and separation and screening method and application thereof

Technical Field

The invention belongs to the technical field of microbial resources, and particularly relates to bacillus amyloliquefaciens and a separation and screening method and application thereof.

Background

The bacillus has the characteristics of heat resistance, drought resistance, ultraviolet resistance, electromagnetic radiation resistance, tolerance to adverse conditions caused by certain types of chemical substances and the like, and the bacillus is used for preventing and treating plant diseases and is one of the commonly used biological prevention and treatment technologies in agricultural production at present. The tea seed cake is residue left after oil extraction of tea seeds, the annual output of China exceeds 100 ten thousand tons, and the tea seed cake is a common organic fertilizer raw material. The tea seed cake is rich in tea saponin, which is a hemolytic toxin and is commonly used for preventing and treating snail and the like. The tea seed cake is combined with the biocontrol bacillus, and the antibacterial effects of the tea saponin and the bacillus are simultaneously exerted, so that the prevention and control effects of plant diseases and insect pests are favorably improved. Therefore, the screening of the bacillus with broad-spectrum antibacterial activity and tea saponin resistance is of great significance.

Disclosure of Invention

At present, no bacillus resource with tea saponin resistance and broad-spectrum antibacterial activity is reported. The invention separates and screens a bacillus amyloliquefaciens strain from tea seed cake, and the bacillus amyloliquefaciens strain has broad-spectrum antibacterial activity and heat resistance and tea saponin resistance.

In view of the above, the invention provides a strain of Bacillus amyloliquefaciens, which is classified and named as Bacillus amyloliquefaciens and is preserved in China general microbiological culture Collection center at 13 months 4 and 2020 with the address: xilu No. 1 Hospital No. 3, Beijing, Chaoyang, on Beijing, with a registration number: CGMCC No: 19565.

the invention also provides application of the bacillus amyloliquefaciens strain in prevention and treatment of one or more of banana vascular wilt, coconut stem rot, peanut rot, areca anthracnose, coconut gray leaf spot disease, areca core rot, oil palm heart rot, Chinese chestnut blight, camellia oleifera leaf spot, camellia oleifera anthracnose and cabbage anthracnose.

The invention also provides a separation and screening method of the bacillus amyloliquefaciens, which is characterized by comprising the following steps of:

1) fermenting and culturing oil tea cake at 45 deg.C for 24h to obtain oil tea cake fermentation liquid with tea saponin concentration of 20 g/L.

2) Weighing 1g of oil tea camellia cumquat fermentation liquid, adding into 10ml of sterile water, performing shake culture for 30min, and sequentially diluting to 10 degrees with sterile water^-1、10^-2、10^-3And (3) coating 0.1ml of the suspension on an LB culture medium respectively, culturing at 45 ℃ for about 2 days, and selecting a single colony for purification culture.

Has the advantages that:

in a hypha growth rate method test, the inhibition rate of the ck-05 strain fermentation liquor on the disease of banana vascular wilt reaches 82%, the inhibition rate on coconut stem rot and peanut kernel rot reaches 79%, and the inhibition rate on pathogenic bacteria such as betelnut anthracnose, coconut gray leaf spot, betelnut heart rot, oil palm heart rot, Chinese chestnut blight, peanut kernel rot, camellia oleifera leaf spot and the like exceeds 70%. The antibacterial activity of the ck-05 strain fermentation liquor is not obviously changed after being treated for 120min at the temperature of 80 ℃, and the thermal stability is strong.

Drawings

FIG. 1 is a colony morphology of Bacillus amyloliquefaciens ck-05 on an LB plate;

FIG. 2 is a microscopic morphology 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 a graph of thermal stability data for a Bacillus amyloliquefaciens ck-05 fermentation broth of the present invention.

Detailed Description

The invention is further described below with reference to examples:

example 1:

separation and identification of bacillus amyloliquefaciens ck-05

2) Weighing 1g of oil tea camellia cumquat fermentation solution, adding into 10ml of sterile water, carrying out shake culture for 30min, sequentially diluting to 10-1, 10-2 and 10-3 times with the sterile water, respectively taking 0.1ml of the diluted fermentation solution, coating the diluted fermentation solution on an LB (lysogeny broth) culture medium, carrying out culture at 45 ℃ for about 2 days, and selecting a single colony for purification culture.

3) The form and the form are as follows: after 2 days of culture on LB medium, the colonies were white, round, dry, rough, wrinkled, small, protruding, and relatively clean, and had short rods, gram-positive, and sporulated shapes when observed under an optical microscope, as shown in FIGS. 1 and 2.

4) The main physiological and biochemical characteristics are as follows: the strain ck-05 catalase test, the V-P test, the indole test, the starch hydrolysis and casein test, the nitrate reduction test, the citrate utilization test and the glucose, sucrose and maltose fermentation test are all positive; the methyl red test is negative. The physiological and biochemical characteristics of the strain ck-05 are basically consistent with those of Bacillus cereus amyloliquefaciens.

5) Molecular biological identification: the DNA of the target strain is extracted by adopting a bacterial genome DNA extraction kit and is used as a template, and PCR amplification is carried out by adopting universal primers 27F and 1492R. 50ml of PCR product is detected by electrophoresis with 1% agarose gel, and the PCR product is sent to Beijing Liuhe Hua Dagen science and technology Co. The sequence is subjected to Blast comparison on NCBI website, phylogenetic analysis is carried out on the strain by adopting software such as BioEdit, Mega5.0 and the like, and a phylogenetic tree is constructed by adopting adjacency (Neighbour-join, NJ method), as shown in FIG. 3, the result shows that the strain ck-05 is Bacillus amyloliquefaciens (accession number: MK 592621).

Example 2: antibacterial spectrum determination of bacillus amyloliquefaciens ck-05 strain fermentation liquid

1) A hypha growth rate method is adopted. Taking 10ml of the ck-05 strain sterile fermentation filtrate and 90ml of PDA culture medium cooled to 40 ℃ to be uniformly mixed, and preparing a culture medium plate containing the ck-05 strain sterile fermentation liquid. Test pathogen clumps (5mm) were inoculated into the center of the plate, with 1 treatment for each pathogen, and 3 replicates for each treatment. And (3) taking the mixture of sterile water and the PDA culture medium as a reference, placing the mixture in a constant-temperature incubator at 28 ℃ for culture, measuring the diameter of the colony of the treatment group by adopting a cross method when the colony of the reference group grows over a culture dish, and calculating the bacteriostasis rate of the sterile filtrate.

The inhibition ratio is (growth diameter of control colony-growth diameter of treated colony)/growth diameter of control colony x 100%

As can be seen from Table 1, the ck-05 strain fermentation broth has a strong inhibition effect on 11 tested pathogenic bacteria, and has an inhibition rate of over 50% on other 10 tested pathogenic bacteria except for 43.47% of anthracnose on cabbage heart, wherein the inhibition effect on banana wilt is strongest, the inhibition rate is 82.22%, and the inhibition rates are 79.44% and 78.89% respectively for peanut rot and coconut stem rot. Therefore, the ck-05 strain fermentation liquid has a wide antibacterial range.

TABLE 1 ck-05 Strain fermentation broth inhibitory action on 11 pathogenic bacteria

Example 3: determination of bacteriostatic action thermal stability of bacillus amyloliquefaciens ck-05

1) Treating the ck-05 strain sterile fermentation liquor for 30min, 60 min, 120min and high-pressure steam sterilization treatment for 30min at the temperature of 40 ℃, 60 ℃, 80 and 100 ℃ respectively, taking fusarium oxysporum f.sp.cubense as an indicator bacterium, and measuring the bacteriostasis rate of the sterile fermentation liquor after different temperature treatments. Each treatment was repeated 3 times, with untreated fermentation broth as control. And measuring the diameter of the colony of the treatment group when the control group mycelium grows full of the culture dish, and then calculating the bacteriostasis rate.

As can be seen from FIG. 4, the ck-05 strain fermentation liquor still has strong bacteriostatic action after being treated at different temperatures, the bacteriostatic action is not obviously changed after being treated at the temperature below 80 ℃ for 30, 60 and 120min, but the bacteriostatic activity is obviously reduced after being treated at the high temperature of 100 ℃, the bacteriostatic rate can still reach 41.48 percent after being treated at the temperature of 100 ℃ for 30min, and the bacteriostatic rate is 31.48 percent after being treated at the temperature of 100 ℃ for 120 min. In addition, the antibacterial activity of the ck-05 strain fermentation liquor is rapidly reduced after the fermentation liquor is subjected to high-pressure steam sterilization treatment at 121 ℃ for 20min, and the antibacterial rate is only 19.26%. Fig. 4 clearly shows that the bacteriostatic rate of the ck-05 strain fermentation liquid is obviously reduced after the temperature is increased to 100 ℃, which may be caused by the structural change of the bacteriostatic activity substance in the ck-05 strain fermentation liquid when the temperature is higher than 100 ℃, and finally cause the bacteriostatic activity to be reduced, but the temperature is lower than 80 ℃, which shows that the ck-05 strain antibacterial substance has stronger thermal stability.

Sequence listing

<110> coconut institute of tropical agricultural academy of sciences of China

<120> bacillus amyloliquefaciens strain and separation and screening method and application thereof

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<170> SIPOSequenceListing 1.0

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<212> DNA

<213> Bacillus amyloliquefaciens (Bacillus amyloliquefaciens)

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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

1. Bacillus amyloliquefaciens strainBacillus amyloliquefaciens) ck-05, characterised byThe strain is preserved in China general microbiological culture Collection center (CGMCC) at 20 days 4 months in 2020, and the preservation number is as follows: CGMCC No: 19565.

2. the application of the bacillus amyloliquefaciens as claimed in claim 1 in a fungicide for preventing and treating one or more of banana vascular wilt, coconut stem rot, peanut rot, areca anthracnose, coconut gray leaf spot disease, areca heart rot, oil palm heart rot, Chinese chestnut blight, camellia oleifera leaf spot, camellia oleifera anthracnose and cabbage anthracnose.