CN110396482B - Antagonistic bacterium bacillus amyloliquefaciens X8 strain and application thereof - Google Patents

Abstract

The invention discloses an antagonistic bacterium Bacillus amyloliquefaciens X8 strain and application thereof, wherein the strain is named as (Bacillus amyloliquefaciens strain X8), and the strain is preserved in the China center for type culture collection (CCTCC No): m2018830. The antagonistic bacteria separated and screened by the method lays a foundation for preventing and treating loach rotten skin disease and developing related biological agents.

Description

Antagonistic bacterium bacillus amyloliquefaciens X8 strain and application thereof

Technical Field

The invention belongs to the technical field of microorganisms, and particularly relates to an antagonistic bacterium bacillus amyloliquefaciens X8 strain and application thereof.

Background

Misgurnus anguillicaudatus (Misgurnus anguillicaudatus) with the reputation of 'ginseng in water' is a common breed in aquaculture, and farmers are located all over the country except special areas such as Qinghai-Tibet plateau areas. The loach meat is delicious and rich in nutrition, so that the loach meat is extremely good in sales. At present, China is a large country for loach export, and a large amount of loaches need to be exported abroad every year. The Liyun harbor is the biggest loach breeding and exporting base in China and is the only national level export loach quality safety demonstration area in China. The loach is exported about 1 ten thousand tons every year, and the output value reaches more than 4000 ten thousand dollars. The total export of the Onagawa in 1-2 months of 2017 from the Hongyun harbor exceeds 8.112 multiplied by 10⁵kg, 60.58% of the total aquatic organism in the total outlet. The Chinese hong Kong and Macau frequently send demand information to the continent, however, the supply and demand gaps always exist, and the reason is that once the loaches are attacked, the loaches are not treated timely and correctly, the loach yield and plasmids are seriously affected, and the export demand cannot be met. Paramisgurnus dabryanus (Paramisgurus dabryanus) becomes a new breeding variety in recent years due to the advantages of large size, fast growth, strong stress resistance, high yield and the like. However, as the cultivation area is enlarged and the cultivation density is increased, the occurrence of diseases is more frequent. The most common disease with great harm is loach rotten diseaseSkin disease.

Every spring and summer, loaches are easy to attack diseases in the processes of feeding, transporting and the like. Loach rotten skin disease is a common disease with extremely high infectivity, long incubation period, great harm and high incidence, and after loach rotten skin disease occurs, the loach rotten skin disease can cause the conditions of body rotting, fin rotting and inappetence, ulcer of the whole body, large-area blood spots distributed on the surfaces of diseased fishes, and the like. What is worse, after the diseased loaches die, the internal organ systems of the diseased loaches are damaged, most of the dead loaches sink to the water bottom and even are buried in silt, and can float out of the water surface only after being swelled by water, and a large amount of germs exist in abdominal cavities of the dead loaches and spread to the whole culture pond, so that greater pollution and pathogen transmission are caused. As for the research on the pathogenic factor of loach rotten skin disease, only Aeromonas sobria reported by Xiaokeyu et al.

The prevention, control and treatment of the skin rot disease mainly adopt chemical drugs (including antibiotics and the like) at present, and the result is that pathogenic bacteria can generate drug resistance, drug residues can interfere the growth and reproduction of aquatic animal intestinal probiotics, and disinfectant and antibiotics remained in water destroy aquaculture water environment, influence the quality of aquatic products, and even bring potential danger to eaters. The method for preventing and treating the diseases by utilizing antagonistic bacteria to inhibit the generation of pathogenic bacteria is an environment-friendly method for preventing and treating the diseases, and some reports are provided in the prevention and treatment of other aquatic diseases at present. For example, Kozasa uses Bacillus megaterium (Bacillus toyoi) to treat Japanese eels, so that the death rate of eels caused by Edwardsiella is reduced; marine actinomycetes screened by Zhaoshujiang and the like have strong inhibiting effect on large yellow croaker pathogenic vibrio, vibrio parahaemolyticus (V.parahaemolyticus) and vibrio harveyi (V.harveyi). The method works in the early stage, a batch of pathogenic bacteria of the loach rotten skin disease are separated from a diseased loach pond, and then antagonistic bacteria are separated and screened by taking the pathogenic bacteria as indicator bacteria, so that a foundation is laid for preventing and treating the loach rotten skin disease and developing related biological preparations.

Disclosure of Invention

The invention aims to provide an antagonistic bacterium bacillus amyloliquefaciens X8 strain and application thereof.

The specific technical scheme is as follows:

an antagonistic bacterium, namely a Bacillus amyloliquefaciens strain X8, which is named as (Bacillus amyloliquefaciens strain X8) and is preserved in China Center for Type Culture Collection (CCTCC) in 11-month and 26-month in 2018, wherein the preservation number is CCTCC No: m2018830. And (4) storage address: wuhan university school (the first small facing of Wuhan university), Wuhan university collection, eight Wuhan district 299, Wuhan City, Hubei province.

Furthermore, the nucleotide sequence of the strain is shown as SEQ ID NO 1.

The antagonistic bacterium bacillus amyloliquefaciens X8 strain is applied to the preparation process of a biological agent for preventing and treating loach rotten skin disease.

Compared with the prior art, the invention has the beneficial effects that:

the antagonistic bacteria X8 separated and screened by the invention has stronger bacteriostatic action on Aeromonas salmonicida D5-7 causing loach rotten skin disease, the diameter of a bacteriostatic zone reaches 18.6 +/-0.25 mm, and the antagonistic bacteria X8 also has stronger bacteriostatic action on other pathogens causing aquatic diseases such as Aeromonas veronii (A.veronii), Aeromonas sobria (A.sobria), Aeromonas hydrophila (A.hydrophilum), Edwardsiella tarda (Edwards) and Vibrio harveyi (Vibrio veyi) Vibrio anguillarum (V.anguillarum) and lays a foundation for the prevention and treatment of the loach rotten skin disease and the development of related biological preparations.

Drawings

FIG. 1 shows primary screening (A) and secondary screening (B) for antagonistic bacteria;

FIG. 2 colony morphology (A) and gram stain results (B) of X8;

gelatin (a) and starch (B) hydrolysis experiments of fig. 3X 8;

FIG. 4X 8(MH128163) phylogenetic tree constructed based on 16S rDNA;

FIG. 5X 8 shows the inhibition of different pathogenic bacteria, CK is aseptic LB, A group is fresh water pathogenic bacteria, B group is sea water pathogenic bacteria.

Detailed Description

The technical solution of the present invention will be described in further detail with reference to the accompanying drawings and specific embodiments.

1 isolation of antagonistic bacteria

Collecting LianyunWater sample and bottom mud of loach culture pond of certain aquaculture company in Gansheng elm region of Hongkong city are mixed with sterile physiological saline according to the ratio of 10^-1～10^-7Gradient dilution, coating on LB culture medium, culturing at 28 deg.C for 24H, picking single colony with inoculating loop, streaking to obtain pure culture, and separating out 11 strains with numbers of L11, L12, L13, L14, L15, L17, L18, L19, L20, H25 and X8. A single colony was picked for liquid culture, added with 15% final concentration of glycerol, and stored at-20 ℃ for later use.

2 screening of antagonistic bacteria

The experiment adopts a filter paper sheet bacteriostasis ring method. Inoculating pathogenic bacteria D5-7 in LB liquid culture medium, and culturing at 28 deg.C for 16 h. 10 μ L of the culture solution was poured into a petri dish, and LB solid medium cooled to room temperature was poured in, and mixed by gentle shaking. Placing an aseptic filter paper sheet with the diameter of 6mm in a culture dish, dripping 20 mu L of bacteria to be detected on the filter paper sheet, culturing at 28 ℃ for 24h, and observing and determining the size of a bacteriostatic circle. And re-screening the strains with the larger inhibition zone again, and screening out the strains with the larger inhibition zone and stably appearing for subsequent experiments.

The primary selection result shows that bacterial strains L11, X8, L18 and H25 have inhibition zones, and the diameters are 11.2 +/-0.11 mm, 14.2 +/-0.15 mm, 11.9 +/-0.14 mm and 9.9 +/-0.25 mm respectively. And (3) re-screening the strains with the inhibition zones, and screening out the strain X8 (shown in figure 1) with larger inhibition zones and stability for subsequent experiments.

3. Identification of antagonistic bacterium X8

3.1 morphological feature Observation

The three regions of antagonist bacteria X8 were streaked on LB plate, and after 24 hours of incubation at 28 ℃ they were observed. The X8 bacterial colony is yellow white, polygonal, irregular in edge, dry and uneven in surface and opaque; gram-positive bacteria with a size of (2.99 + -0.28) μm × (1.17 + -0.17) μm when the gram stain is purple (as shown in FIG. 2); has spore, no capsule and no motility.

3.2 physiological and Biochemical experiments

(1) Hydrolysis test of macromolecules

The results of observation with Bacillus pumilus E14 reported by this experimental group as a control show that antagonistic bacteria X8 has the ability to hydrolyze starch and gelatin, and cannot hydrolyze casein and oil, as shown in FIG. 3.

(2) Bacterial micro biochemical reaction tube experiment

According to the operation and observation records of Hangzhou microbial reagent limited company's ' bacterial micro biochemical reaction tube instruction '. The results are shown in Table 1.

TABLE 1 antagonistic bacteria X8 micro biochemical tube reaction results

3.3 molecular characterization

And (3) analyzing and constructing a phylogenetic tree by adopting a 16S rDNA sequence. The 16S rRNA gene of the antagonistic strain X8 is amplified, and agarose gel electrophoresis shows that the size of the 16S rDNA fragment is about 1500 bp. The PCR product was sent to Biotechnology engineering (Shanghai) GmbH for sequencing, the obtained sequence was used to search for model strain sequence and downloaded by EZ.BioCloud website, MEGA 6.06 software was used to construct X8 strain phylogenetic clade by the temporary method (repetition number is 1000, step value is percentage), and the result is shown in FIG. 4.

As can be seen from FIG. 4, the antagonistic bacterium X8 has the same branch as strain Bacillus amyloliquefaciens strain D15, and the genetic distance is the closest, and the homology is 98%. By integrating colony and cell morphology observation, physiological and biochemical experiments and 16S rRNA gene sequence analysis, antagonistic bacteria X8 is identified as Bacillus amyloliquefaciens (Bacillus amyloliquefaciens), so the antagonistic bacteria is tentatively named as B. amyloliquefaciens X8.

4. Study on antagonistic spectrum of antagonistic bacterium X8

The results are shown in FIG. 5, using the bacteriostatic circle method with filter paper (CK is sterile LB) and the same method as described for antagonism D5-7.

As can be seen from the results in FIG. 5, X8 has antagonistic activity against Aeromonas veronii, Aeromonas sobria, Aeromonas hydrophila, Aeromonas salmonicida, Edwardsiella tarda, Vibrio harveyi and Vibrio anguillarum with the diameters of the zones of inhibition being 14.7 + -0.15 mm, 16.8 + -0.15 mm, 11.2 + -0.1 mm, 15.9 + -0.15 mm, 8.3 + -0.2 mm, 11.8 + -0.13 mm and 8.2 + -0.15 mm, respectively.

The above description is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited thereto, and any person skilled in the art can obviously obtain simple changes or equivalent substitutions of the technical solutions within the technical scope of the present invention.

Sequence listing

<110> Liu love people

<120> antagonistic bacterium bacillus amyloliquefaciens X8 strain and application thereof

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1448

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

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cggggcggca gctatacatg caagtcgagc ggacagatgg gagcttgctc cctgatgtta 60

gcggcggacg ggtgagtaac acgtgggtaa cctgcctgta agactgggat aactccggga 120

aaccggggct aataccggat ggttgtctga accgcatggt tcagacataa aaggtggctt 180

cggctaccac ttacagatgg acccgcggcg cattagctag ttggtgaggt aacggctcac 240

caaggcgacg atgcgtagcc gacctgagag ggtgatcggc cacactggga ctgagacacg 300

gcccagactc ctacgggagg cagcagtagg gaatcttccg caatggacga agtctgacgg 360

agcaacgccg cgtgagtgat gaaggttttc ggatcgtaaa gctctgttgt tagggaagaa 420

caagtgccgt tcaaataggg cggcaccttg acggtaccta accagaaagc cacggctaac 480

tacgtgccag cagccgcggt aatacgtagg tggcaagcgt tgtccggaat tattgggcgt 540

aaagggctcg caggcggttt cttaagtctg atgtgaaagc ccccggctca accggggagg 600

gtcattggaa actggggaac ttgagtgcag aagaggagag tggaattcca cgtgtagcgg 660

tgaaatgcgt agagatgtgg aggaacacca gtggcgaagg cgactctctg gtctgtaact 720

gacgctgagg agcgaaagcg tggggagcga acaggattag ataccctggt agtccacgcc 780

gtaaacgatg agtgctaagt gttagggggt ttccgcccct tagtgctgca gctaacgcat 840

taagcactcc gcctggggag tacggtcgca agactgaaac tcaaaggaat tgacgggggc 900

ccgcacaagc ggtggagcat gtggtttaat tcgaagcaac gcgaagaacc ttaccaggtc 960

ttgacatcct ctgacaatcc tagagatagg acgtcccctt cgggggcaga gtgacaggtg 1020

gtgcatggtt gtcgtcagct cgtgtcgtga gatgttgggt taagtcccgc aacgagcgca 1080

acccttgatc ttagttgcca gcattcagtt gggcactcta aggtgactgc cggtgacaaa 1140

ccggaggaag gtggggatga cgtcaaatca tcatgcccct tatgacctgg gctacacacg 1200

tgctacaatg gacagaacaa agggcagcga aaccgcgagg ttaagccaat cccacaaatc 1260

tgttctcagt tcggatcgca gtctgcaact cgactgcgtg aagctggaat cgctagtaat 1320

cgcggatcag catgccgcgg tgaatacgtt cccgggcctt gtacacaccg cccgtcacac 1380

cacgagagtt tgtaacaccc gaagtcggtg aggtaacctt taggagccag ccgccgaagg 1440

tgataaag 1448

Claims (2)

1. An antagonistic bacterium, Bacillus amyloliquefaciens X8 strain, wherein the strain is named asBacillus amyloliquefaciens strain X8, which has been preserved in China center for type culture Collection in 2018, 12 and 11 months, with the preservation number of CCTCC No: m2018830.

2. Use of the antagonistic bacterium, bacillus amyloliquefaciens strain X8, according to claim 1, in the preparation of a biological agent for controlling loach pythium disease caused by aeromonas salmonicida.

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