CN105349463B - A kind of pseudomonad strain and its application - Google Patents
A kind of pseudomonad strain and its application Download PDFInfo
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- CN105349463B CN105349463B CN201510856000.6A CN201510856000A CN105349463B CN 105349463 B CN105349463 B CN 105349463B CN 201510856000 A CN201510856000 A CN 201510856000A CN 105349463 B CN105349463 B CN 105349463B
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Abstract
The present invention discloses a kind of pseudomonad strain and its application.The bacterial strain is pseudomonad (Pseudomonas protegens) IAS03, and China typical culture collection center, deposit number are preserved on November 27th, 2015：CCTCC NO:M 2015711.The bacterial strain screens to obtain from sediment of pond, then ecological safety risk is not present with return water body, meets bio-safety regulation；There is the bacteriostasis of specificity to Aeromonas hydrophila and Aeromonas sobria, and to other bacterium unrestraint effects, and the bacterium is to fish no pathogenicity；Only must single bacterial strain can be used to inhibit Aeromonas hydrophila and/or Aeromonas sobria, relative to traditional antibiotic and disinfectant control measure, have the advantages that safety, green, efficient.The Aeromonas sobria in probiotics control breeding environment and Aeromonas hydrophila are can be developed into, prevents it and infects fish causing bleeding property septicemia.
Description
Technical field
The invention belongs to microorganism fields, are related to a kind of pseudomonad strain, more particularly to a kind of pseudomonad strain and
It is applied.
Background technology
Aeromonas hydrophila (Aeromonas hydrophila) and Aeromonas sobria (Aeromonas sobria) are deposited
It is in cultivation water environment, often cause nearly all cultured freshwater fish to suffer from hueppe's disease, incidence is high, and the death rate is also very
Height causes heavy economic losses to culture fishery.The disease is prevented in the past mainly using antibiotic is fed, and disinfectant for external use is killed
Pathogen in water body, it is long-term so not only to will produce medicament residue, inducible resistance pathogen generates and pollute water environment, but also
Disinfectant non-selectivity kills beneficial bacterium in water, destroys water body microecological balance, is buried for follow-up outburst bacterial septicemia
Hidden danger.
Since the pathogen (Aeromonas hydrophila and Aeromonas sobria) for causing fish to suffer from hueppe's disease carrys out autotrophy
Water body is grown, it is therefore desirable to the single-minded Aeromonas hydrophila killed in water environment and mild gas in the case where not influencing beneficial bacterium
Monad eliminates the cause of disease source of cultured fishes, prevents hueppe's disease.This is the effective way to solve the above problems
Diameter.
Invention content
In order to overcome the disadvantages and deficiencies of the prior art, the primary purpose of the present invention is that providing a kind of pseudomonad bacterium
Strain.There is active material in the zymotic fluid of the bacterial strain specificity to inhibit Aeromonas hydrophila (Aeromonas hydrophila)
With the effect of Aeromonas sobria (Aeromonas sobria), have no adverse effects to the other bacterium detached in water environment, just
Solve the problems of the prior art.The pseudomonad strain can be developed into the cause of disease temperature in probiotics control breeding environment
With Aeromonas and Aeromonas hydrophila, prevents it and infect fish causing bleeding property septicemia.
Another object of the present invention is to provide the applications of above-mentioned pseudomonad strain.
The purpose of the invention is achieved by the following technical solution：
To achieve the above object, the present invention provides a kind of pseudomonad, and strain is named as Pseudomonas protegens
IAS03 is isolated and purified from sediment of pond and is obtained.
The preservation information of described pseudomonad (Pseudomonas protegens) IAS03：Depositary institution：Chinese allusion quotation
Type culture collection (CCTCC), preservation date：On November 27th, 2015, preservation address：The Chinese Wuhan Wuhan Universitys,
Deposit number：CCTCC NO:M 2015711.
The fluid nutrient medium of described pseudomonad (Pseudomonas protegens) the IAS03 bacterial strains is beef extract egg
White peptone fluid nutrient medium, beef extract-peptone solid medium then on the basis of liquid medium within formula, are trained per 1000ml liquid
Addition agar 15~20g heating and meltings in base are supported to be made.
The beef extract-peptone fluid nutrient medium：Peptone 10g/L, beef extract 3g/L, sodium chloride 5g/L, agar 15
~20g/L, pH7.0~7.2,121 DEG C, the processing of 20min high-temperature sterilizations；Beef extract-peptone fluid nutrient medium is not added with agar i.e.
It can.
The fermentation medium of described pseudomonad (Pseudomonas protegens) the IAS03 bacterial strains is LB culture mediums,
Its group is divided into：Peptone 10g/L, yeast extract powder 5g/L, sodium chloride 10g/L, 7.0~7.2,121 DEG C of pH value, 20min high temperature
Sterilization treatment.
Pseudomonad (Pseudomonas protegens) IAS03 bacterial strains provided by the invention are applied to inhibit thermophilic aqueous vapor
Monad and/or Aeromonas sobria.
Pseudomonad (Pseudomonas protegens) IAS03 is in prevention Aeromonas hydrophila and/or mildly
Application in Aeromonas infection.
Pseudomonad (Pseudomonas protegens) IAS03 prepare inhibit Aeromonas hydrophila and/or
Application in the product of Aeromonas sobria.
Described pseudomonad (Pseudomonas protegens) IAS03 bacterial strains inhibit Aeromonas hydrophila and mild
The principle of Aeromonas is that pseudomonas can inhibit the growth of pathogen by nutrient competition, moreover it is possible to generate thermophilic iron element,
The secondary metabolites and antibacterial polypeptide with bacteriostasis such as hydrogen cyanide, oomycetes element, phenazine-1-carboxylic acid and luteolin, specially
Pathogen is killed in one suppression, and is not damaged to other bacterium or algae, and water ecosystem balance is not being destroyed to ensure that
Under the premise of, pathogen is killed from source.
The present invention compared with the existing technology, has the following advantages and effect：
(1) the present invention provides pseudomonad (Pseudomonas protegens) IAS03 bacterial strains.
(2) pseudomonad (Pseudomonas protegens) IAS03 bacterial strains of the invention are screened from sediment of pond
It arrives, then ecological safety risk is not present with return water body, meet bio-safety regulation.
(3) pseudomonad IAS03 bacterial strains of the invention can effectively inhibit aquatic pathogenic bacterium：Aeromonas hydrophila and/or
Aeromonas sobria.
(4) pseudomonad IAS03 bacterial strains of the invention generate fungistatic effect to Aeromonas hydrophila and Aeromonas sobria,
And to other bacterium unrestraint effects, the bacteriostasis with specificity, and the bacterium is to fish no pathogenicity.
(5) pseudomonad IAS03 bacterial strains of the invention only must single bacterial strain can be applied to inhibit Aeromonas hydrophila and/
Or Aeromonas sobria has the advantages that safe, green, efficient relative to traditional antibiotic and disinfectant control measure.
Description of the drawings
Fig. 1 is inhibition of the bacterium solution to Aeromonas hydrophila and Aeromonas sobria of pseudomonad IAS03 bacterial strains；Its
In, Figure 1A is inhibition of the bacterium solution to Aeromonas hydrophila of pseudomonad IAS03 bacterial strains；Figure 1B is pseudomonad IAS03
Inhibition of the bacterium solution of bacterial strain to Aeromonas sobria.
Fig. 2 is 10 times of concentrates of without fermented liquid of pseudomonad IAS03 bacterial strains to Aeromonas hydrophila and mild gas list
The inhibition of born of the same parents bacterium；Wherein, Fig. 2A is 10 times of concentrates of without fermented liquid of pseudomonad IAS03 bacterial strains to mild gas unit cell
The inhibition of bacterium；Fig. 2 B are suppression of the 10 times of concentrates of without fermented liquid of pseudomonad IAS03 bacterial strains to Aeromonas hydrophila
Effect processed.
Fig. 3 is bacterium colony growing state of the pseudomonad IAS03 bacterial strains in PDA culture medium and on KB solid mediums
Fluorescence phenomenon；Wherein, Fig. 3 A are cultivation results of the pseudomonad IAS03 bacterial strains on PDA solid mediums；Fig. 3 B are false unit cell
Cultivation results of the bacterium IAS03 bacterial strains on KB solid mediums, blue-fluorescence is observed under the ultraviolet lamp of 302nm.
Fig. 4 is pseudomonad IAS03 bacterial strain 16S rDNA PCR amplification gel electrophoresis figures；Wherein, swimming lane M is DNA
marker DL2000；Swimming lane 1~6 is the pseudomonad IAS03 bacterial strain 16S rDNA bands of amplification.
Fig. 5 is inhibition of the 10ppm pseudomonad IAS03 zymotic fluids to Aeromonas hydrophila and Aeromonas sobria；
Wherein, Fig. 5 A are Aeromonas hydrophila；Fig. 5 B are Aeromonas sobria.
Fig. 6 is inhibition of the 15ppm pseudomonad IAS03 zymotic fluids to Aeromonas hydrophila and Aeromonas sobria；
Wherein, Fig. 6 A are Aeromonas hydrophila；Fig. 6 B are Aeromonas sobria.
Fig. 7 is inhibition of the 20ppm pseudomonad IAS03 zymotic fluids to Aeromonas hydrophila and Aeromonas sobria；
Wherein, Fig. 7 A are Aeromonas hydrophila；Fig. 7 B are Aeromonas sobria.
Fig. 8 is inhibition of the 25ppm pseudomonad IAS03 zymotic fluids to Aeromonas hydrophila and Aeromonas sobria；
Wherein, Fig. 8 A are Aeromonas hydrophila；Fig. 8 B are Aeromonas sobria.
Fig. 9 is inhibition of the 30ppm pseudomonad IAS03 zymotic fluids to Aeromonas hydrophila and Aeromonas sobria；
Wherein, Fig. 9 A are Aeromonas hydrophila；Fig. 9 B are Aeromonas sobria.
Note：* it indicates that the Aeromonas hydrophila of the group (test group) of pseudomonad IAS03 zymotic fluids or mild gas unit cell is added
The amount of bacterium is substantially less than control group (p<0.05), different lowercase letter test group different time points Aeromonas hydrophilas or temperature
There are significant difference (p between the amount of Aeromonas<0.05), and same letter indicates no significant difference.
Specific implementation mode
With reference to embodiment and attached drawing, present invention is further described in detail, but embodiments of the present invention are not limited to
This.
Aeromonas sobria (Aeromonas sobria) described in embodiment is in document " the immune China of inactivated vaccine
Variation [J] aquatic product journals of hangnail Barb peripheral blood immune indexes, 2010,04:626-634. " in disclose.
Aeromonas hydrophila (Aeromonas hydrophila) is in document " body of 42 kinds of Chinese herbal medicines to Aeromonas hydrophila
Outer bacteriostatic test [J] fresh water fisherys, 2011,03:61-65. " in disclose.
The preservation information of described pseudomonad (Pseudomonas protegens) IAS03：Depositary institution：Chinese allusion quotation
Type culture collection (CCTCC), preservation date：On November 27th, 2015, preservation address：The Chinese Wuhan Wuhan Universitys,
Deposit number：CCTCC NO:M 2015711.
Embodiment 1
The screening of described pseudomonad (Pseudomonas protegens) IAS03 and identification method, steps are as follows：
(1) the bed mud 10g from Guangzhou pond is weighed in conical flask, adds 90mL aqua sterilisas, 28 DEG C, 160r/
Min, constant-temperature table vibrate 30min, and supernatant is taken after standing 5min, and supernatant is diluted 10 using gradient dilution method3Times, take 0.15mL
Dilution is in beef extract-peptone solid medium tablets even spread, 28 DEG C of constant temperature incubation 16h.Beef extract-peptone solid is trained
Support base：Peptone 10g, beef extract 3g, sodium chloride 5g, 15~20g of agar, 1000mL, pH7.0,121 DEG C, 20min high are added water to
Warm sterilization treatment.The fluid nutrient medium of same recipe is not added with agar.
(2) single bacterium on picking step (1) middle plateform is fallen in beef extract-peptone fluid nutrient medium, 28 DEG C, 160r/
Min, constant-temperature table vibrate 8h, wait for OD600When reaching 0.8, the bacterium solution of 10 μ L is drawn, gradient dilution 10 is passed through4Times, take 0.1mL in
Beef extract-peptone solid medium tablets even spread, 28 DEG C of constant temperature incubation 16h, whether observation bacterium colony is single, is repeated 3 times,
And it marks.
(3) by single colony inoculation in beef extract-peptone fluid nutrient medium, 28 DEG C, 160r/min, constant-temperature table
Oscillation 8h is inoculated according to the ratio of 2% (v/v) equipped with 50mL beef extract-peptone fluid nutrient mediums as seed liquor
The conical flask of 250mL, at 28 DEG C 160r/min constant-temperature shaking cultures for 24 hours, as bacterium solution to be measured.
(4) with Aeromonas sobria (Aeromonas sobria) and Aeromonas hydrophila (Aeromonas
Hydrophila it) is used as indicator bacteria, the conservation (- 80 DEG C, 20% glycerine preserves) from this laboratory.It goes bail for respectively the bacterium deposited
Kind is crossed by oese in beef extract-peptone solid medium tablets, 28 DEG C of constant temperature incubation 12h, respectively picking single bacterium colony
It is activated in beef extract-peptone fluid nutrient medium, until OD600=0.8, deposit is spare in 4~8 DEG C of refrigerators.
(5) Antagonistic Fungi of Aeromonas sobria and Aeromonas hydrophila is screened by agar plate diffusion method.With a diameter of
The card punch of 0.8mm takes the 200 μ L of bacterium solution in step (3) to add in the center punching of beef extract-peptone solid medium tablets
Enter in the hole, 28 DEG C of constant temperature incubation 12h, then take the 5 μ L of instruction bacterium solution in step (4), sterile water is added up to 100 μ L, and
Onboard with glass bar even spread, 28 DEG C of constant temperature incubation 12h.Three repetitions of each bacterium.
(6) vernier caliper measurement antibacterial circle diameter is used, and is made a record, Antagonistic Fungi is filtered out.
(7) amplification of 16S rDNA, takes the seed liquor of the Antagonistic Fungi in step (3) to carry out bacterium colony PCR, and primer is general
Primer：27F：5 '-AGAGTTTGATCCTGGCTCAG-3 ', 1492R：5′-TACGGTTACCTTGTTACGACTT-3′；Reactant
System is 25 μ L, including 15.00 μ L, KOD-Plus buffer of ddH2O 2.5 μ L, dNTP mix (200mmol/L) 2.5 μ L, is drawn
Object 27F 1.0 μ L of 1.0 μ L, primer 1492R, 1.0 μ L, KOD-Plus enzyme of bacterium solution 0.5 μ L, MgSO4 (25mmol/L) 1.5 μ L.Instead
Answer process as follows：Pre-degeneration：95 DEG C, 5min, denaturation：94 DEG C, 1min, annealing：54 DEG C, 1min, extend：72 DEG C, 90s, then prolong
It stretches：72 DEG C, 10min.1.2% agarose nucleic acid electrophoresis (100V, 30min) is carried out after the completion of PCR, segment is about
1500bp, gel extraction PCR product, is cloned into carrier T, conversion bacillus coli DH 5 alpha (Escherichia coli DH5 α)
(commercially available), PCR detects positive colony, and send bacterium solution to Shenzhen Hua Da gene sequencing.The results are shown in Figure 4, wherein swimming lane M is
DNA marker DL2000；Swimming lane 1~6 is the pseudomonad IAS03 bacterial strain 16S rDNA bands of amplification.
(8) foundation of chadogram.The sequence of 16S rDNA is imported in EzXtaon specialized databases and carries out online comparison point
Analysis carries out multisequencing matching arrangement with 1.83 softwares of Cluxtalx, and analysis result uses the ortho position phase connection in Mega5 softwares
(Neighbor-joining method) makes systematic evolution tree, and carries out bootstrapping analysis (Bootstrap) and confidence level inspection
It surveys, bootstrapping data set 1000 times.Determine antagonistic strain category and possible kind.
(9) morphologic identification.The seed liquor of Antagonistic Fungi in step (3) is taken to cross on KB solid plates with oese,
Parallel edition is PDA solid mediums.28 DEG C of constant temperature incubations for 24 hours after, in wavelength be 302nm ultraviolet lamp under seen whether fluorescence
Occur.KB solid mediums：Peptone 20g, glycerine 10mL, K2HPO4 1.5g、MgS04.7H2O 1.5g, 15~20g of agar,
1000mL, pH7.2,121 DEG C of water, the processing of 20min high-temperature sterilizations.PDA solid mediums：Peeled potatoes 200g, glucose 20g,
15~20g of agar, water 1000mL, pH value 7.0~7.2.Specific method is to clean potato, and stripping and slicing (1 × 1 × 1cm) boiling 20 divides
Glucose 20g and 15~20g of agar, 121 DEG C of sterilization treatments are added with 4 layers of filtered through gauze in Zhong Hou in 1000 milliliters of filtrates
20min。
The 16S rDNA sequences of pseudomonad (Pseudomonas protegens) the IAS03 bacterial strains isolated and purified with
The similarity of the 16S rDNA sequences of Pseudomonas protegens is 99%, this is shown based on 16S rDNA clusterings
It is one that bacterial strain IAS03 and Pseudomonas protegens, which gather, and sends out blue-fluorescence on KB tablets.As a result such as Fig. 3 institutes
Show, wherein Fig. 3 A are cultivation results of the pseudomonad IAS03 bacterial strains on PDA solid mediums；Fig. 3 B are pseudomonad
Cultivation results of the IAS03 bacterial strains on KB solid mediums, blue-fluorescence is observed under the ultraviolet lamp of 302nm.
The bacterium solution of pseudomonad IAS03 is measured to Aeromonas sobria and thermophilic aqueous vapor list using the agar plate diffusion method of (5)
The antibacterial circle diameter that born of the same parents bacterium generates is respectively 40.29 ± 0.85mm and 38.40 ± 0.69mm.The results are shown in Figure 1, wherein figure
1A is inhibition of the bacterium solution to Aeromonas hydrophila of pseudomonad IAS03 bacterial strains；Figure 1B is pseudomonad IAS03 bacterial strains
Inhibition of the bacterium solution to Aeromonas sobria.
In laboratory conditions, the activated seed liquid of pseudomonad IAS03 bacterial strains is taken to be connect according to the inoculum concentration of 2% (v/v)
For kind in the conical flask (1000mL) containing 250mL LB liquid mediums, condition of culture is 28 DEG C, 160r/min oscillation trainings
48h is supported, after fermentation, the chilled centrifuge of zymotic fluid carries out 7500r/min and centrifuges 15min, removes thalline, takes supernatant, into
Row vacuum freeze drying takes the sterile water dissolution of xeraphium, then centrifuges, and takes supernatant to be filtered with 0.22 μm of filter, obtains 10 times
Then sterile Fermented Condensed liquid uses lysoplate assay, measures its suppression to Aeromonas sobria and Aeromonas hydrophila
Bacterium loop diameter.At 28 DEG C after constant temperature incubation 12h, diameter 16.88 is given birth to Aeromonas sobria and Aeromonas hydrophila respectively
The inhibition zone of ± 0.65mm and 14.03 ± 0.35mm.The results are shown in Figure 2, wherein Fig. 2A is pseudomonad IAS03 bacterial strains
Inhibition of the 10 times of concentrates of without fermented liquid to Aeromonas sobria；Fig. 2 B are the sterile hair of pseudomonad IAS03 bacterial strains
Inhibition of the 10 times of concentrates of zymotic fluid to Aeromonas hydrophila.
The fluid nutrient medium of the pseudomonad IAS03 bacterial strains is beef extract-peptone fluid nutrient medium, beef extract egg
15~20g/L of agar is then added in white peptone solid medium on the basis of beef extract-peptone Liquid Culture based formulas.
The beef extract-peptone fluid nutrient medium：Peptone 10g/L, beef extract 3g/L, sodium chloride 5g/L, pH7.0
~7.2, the sterilization treatment 20min at 121 DEG C.
The fermentation medium of the pseudomonad IAS03 bacterial strains is LB liquid medium, and group is divided into：Peptone 10g/
L, yeast extract powder 5g/L, sodium chloride 10g/L, 7.2,121 DEG C of sterilization treatment 20min of pH value.
The 16S rDNA sequences such as SEQ ID of described pseudomonad (Pseudomonas protegens) the IAS03 bacterial strains
NO：Shown in 1：(1408bp)
TGCAGTCGAGCGGCAGCACGGGTACTTGTACCTGGTGGCGAGCGGCGGACGGGTGAGTAATGCCTAGGAATCTGCCT
AGTAGTGGGGGATAACGTCCGGAAACGGGCGCTAATACCGCATACGTCCTACGGGAGAAAGTGGGGGATCTTCGGAC
CTCACGCTATTAGATGAGCCTAGGTCGGATTAGCTAGTTGGTGAGGTAATGGCTCACCAAGGCGACGATCCGTAACT
GGTCTGAGAGGATGATCAGTCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATT
GGACAATGGGCGAAAGCCTGATCCAGCCATGCCGCGTGTGTGAAGAAGGTCTTCGGATTGTAAAGCACTTTAAGTTG
GGAGGAAGGGCAGTTACCTAATACGTGATTGTTTTGACGTTACCGACAGAATAAGCACCGGCTAACTCTGTGCCAGC
AGCCGCGGTAATACAGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCGCGTAGGTGGTTTGTTAAGTT
GGATGTGAAAGCCCCGGGCTCAACCTGGGAACTGCATCCAAAACTGGCAAGCTAGAGTATGGTAGAGGGTGGTGGAA
TTTCCTGTGTAGCGGTGAAATGCGTAGATATAGGAAGGAACACCAGTGGCGAAGGCGACCACCTGGACTGATACTGA
CACTGAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCAACTAGC
CGTTGGGAGCCTTGAGCTCTTAGTGGCGCAGCTAACGCATTAAGTTGACCGCCTGGGGAGTACGGCCGCAAGGTTAA
AACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTAC
CAGGCCTTGACATCCAATGAACTTTCTAGAGATAGATTGGTGCCTTCGGGAACATTGAGACAGGTGCTGCATGGCTG
TCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGTAACGAGCGCAACCCTTGTCCTTAGTTACCAGCACGTTA
TGGTGGGCACTCTAAGGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTAC
GGCCTGGGCTACACACGTGCTACAATGGTCGGTACAAAGGGTTGCCAAGCCGCGAGGTGGAGCTAATCCCATAAAAC
CGATCGTAGTCCGGATCGCAGTCTGCAACTCGACTGCGTGAAGTCGGAATCGCTAGTAATCGCGAATCAGAATGTCG
CGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTGCACCAGAAGTAGCTAGTCT
AACCTTCGGGAGGACGGTACCA。
Embodiment 2
Toxicity test of pseudomonad (Pseudomonas protegens) the IAS03 bacterial strains to fish
(1) experiment material：270 tail of grass carp seedling (weight about 20g)
(2) preparation of culture medium：
Beef extract-peptone fluid nutrient medium：Peptone 10g/L, beef extract 3g/L, sodium chloride 5g/L, pH value 7.0~
7.2,121 DEG C of sterilizing 20min；
Beef extract-peptone solid medium：Addition 15~20g/L agar in beef extract-peptone fluid nutrient medium, 121
DEG C sterilizing 20min.
(3) preparation of bacterium solution is injected：
It is lined in beef extract-peptone solid medium tablets with the go bail for pseudomonad IAS03 bacterial strains of Tibetan of oese,
After cultivating 16h at 28 DEG C, it is inoculated in beef extract-peptone fluid nutrient medium with oese picking single bacterium colony, is passed through at 28 DEG C
160r/min shaken cultivation 12h are then centrifuged for (7500rpm, 5 minutes), be used in combination physiological saline be centrifuged repeatedly (7500rpm, 5 points
Clock) it washs three times, last resuspended bacterium solution measures OD by ultraviolet specrophotometer again600Value, is distinguished bacterium solution with physiological saline
It is adjusted to 106, 107, 108, 109, 1010Cfu/mL obtains the pseudomonad IAS03 strain suspensions of various concentration.
(4) test method：
It is 90 tail of grass carp of 20g or so to take weight, is randomly divided into 6 groups, every group of 15 tails are false per tail fish intraperitoneal injection 0.2mL
Monad IAS03 strain suspensions (control injection equivalent sterile saline), the bacteria concentration of 6 groups injection is respectively 0 (control),
106, 107, 108, 109, 1010Cfu/mL, the fish after every group of injection are raised respectively in 1 60L white plastic drum, and sky is used in combination
Gas compression pump is inflated into inner bucket water, every group of 3 parallel repetitions.It is continuous to feed 7 days, water is changed daily and feeds pellet, observation
Record behavior and the dead fish number of test fish.
(5) experimental result：After 7 days, the fish death rate is counted, finds 1 × 109The pseudomonad of cfu/mL or less concentration
IAS03 bacterial strains to grass carp without any harmful effect, and 1 × 1010Cfu/mL just causes 60% fish dead, it becomes virtually impossible to have
So big bacterium amount enters fish body.Then, grass carp seedling is injected intraperitoneally with above-mentioned same method the 1 × 10 of 0.2mL8cfu/mL
Aeromonas hydrophila or Aeromonas sobria, fish is entirely dead in 3 days, compares full survival.Therefore, pseudomonad IAS03 bacterial strains are to fish
Safety.
Embodiment 3
10ppm pseudomonad IAS03 bacterial strain fermentation liquors test the inhibition of Aeromonas hydrophila and Aeromonas sobria
(1) preparation of culture medium：
Beef extract-peptone fluid nutrient medium：Peptone 10g/L, beef extract 3g/L, sodium chloride 5g/L, pH value 7.0,121
DEG C sterilizing 20min；
Beef extract-peptone solid medium：15g/L agar is added in beef extract-peptone fluid nutrient medium, 121 DEG C go out
Bacterium 20min, the culture of falling 50mL are based on that solid medium tablets are made in culture dish.
LB liquid medium：Peptone 10g/L, yeast extract powder 5g/L, sodium chloride 10g/L, 7.2,121 DEG C of sterilizings of pH value
20min。
Aeromonas hydrophila or the Aeromonas sobria of -80 DEG C of preservations are taken with oese respectively, it is solid in beef extract-peptone
It crosses on body culture medium flat plate, after 28 DEG C of culture 10h, then is inoculated in beef extract-peptone liquid with oese picking single bacterium colony and trains
It supports in base, the 160r/min shaken cultivations 12h at 28 DEG C, bacterium solution OD is then measured by ultraviolet specrophotometer600Value.
(2) preparation of pseudomonad IAS03 bacterial strain fermentation liquors：
Take the pseudomonad IAS03 bacterial strains of -80 DEG C of preservations in beef extract-peptone solid medium tablets with oese
Scribing line activation is inoculated in oese picking single bacterium colony in beef extract-peptone fluid nutrient medium after cultivating 16h at 28 DEG C,
The 160r/min shaken cultivations 12h at 28 DEG C is until OD600Value is 0.8, as fermentation seed liquid.Then according to 2% (v/v's)
Inoculum concentration is inoculated in the conical flask equipped with 250mL LB culture mediums (1L), the 160r/min shaken cultivations 48h at 28 DEG C, institute
The liquid obtained is band fermented liquid.
(3) test method：
Pond water is taken to be filtered and high pressure sterilization, as Aeromonas sobria or the growth water ring of Aeromonas hydrophila
The water difference equivalent of sterilizing is added in the teat glass of 15 × 150mm, is then respectively adding Aeromonas hydrophila or mild by border
The bacterium solution of Aeromonas so that ultimate density is 3 × 1010The liquid final volume of cfu/mL, each teat glass are 8mL, so
After 80 μ L pseudomonad IAS03 bacterial strain fermentation liquors (1 × 10 are added9Cfu/mL) so that final concentration of 10ppm (experimental group), it is right
According in group be added equivalent aqua sterilisa, be subsequently placed in 28 DEG C through 160r/min shaken cultivations, and in 12h, for 24 hours, 36h, 48h, 60h,
72h, 84h are sampled.The sample acquired draws 10 μ L stostes with liquid-transfering gun and is added to the sterile of 990 μ L by gradient dilution
In physiological saline, after mixing, then draws 10 μ L and be added in the physiological saline of 990 μ L, then therefrom draw 100 μ L, use glass
Stick is uniformly coated on beef extract-peptone solid medium, 4 repetitions of each time point, after 28 DEG C are cultivated 8 hours, is carried out
Bacterium colony counts (pseudomonad IAS03 at this time is not grown also), and the statistics and conspicuousness of data are then carried out by Spss softwares
Variance analysis.As a result see Fig. 5 A and Fig. 5 B.Fig. 5 the results show that after zymotic fluid is added for 24 hours in, experimental group and control group
Aeromonas sobria and thermophilic aqueous monad are not significantly different, but in 36h, pseudomonad IAS03 zymotic fluids are to mild
Aeromonas produces depression effect, and conspicuousness inhibiting effect occurs in 48h to aeromonas hydrophila quantity.
Embodiment 4
Inhibition of the 15ppm pseudomonad IAS03 bacterial strain fermentation liquors to Aeromonas hydrophila or Aeromonas sobria
Pseudomonad IAS03 bacterial strain fermentation liquors (1 × 10 are added with embodiment 3 in experiment material and method9cfu/mL)120μ
L so that final concentration of 15ppm.Specific inhibition is shown in Fig. 6 A and Fig. 6 B.Fig. 6 results are shown：Before experiment in 36h, experimental group
It is all not significantly different with the Aeromonas sobria or Aeromonas hydrophila quantity of control group, after 48h, two kinds of experimental group
Pathogen quantity is substantially lower than control group, illustrates that pseudomonad IAS03 bacterial strains produce inhibition effect to two kinds of pathogens really
It answers.
Embodiment 5
Inhibition of the 20ppm pseudomonad IAS03 bacterial strain fermentation liquors to Aeromonas hydrophila and Aeromonas sobria
Pseudomonad IAS03 bacterial strain fermentation liquors (1 × 10 are added with embodiment 3 in experiment material and method9cfu/mL)160μ
L so that final concentration of 20ppm.Specific inhibition is shown in Fig. 7 A and Fig. 7 B.Fig. 7 results are shown：20ppm pseudomonad IAS03 bacterium
Strain zymotic fluid does not show also to significantly inhibit effect to Aeromonas hydrophila or Aeromonas sobria generation in 36h, but in 48h
Afterwards, the effect of significantly inhibiting is produced to two kinds of pathogens.
Embodiment 6
Inhibition of the 25ppm pseudomonad IAS03 bacterial strain fermentation liquors to Aeromonas hydrophila or Aeromonas sobria
Pseudomonad IAS03 bacterial strain fermentation liquors (1 × 10 are added with embodiment 3 in experiment material and method9cfu/mL)200μ
L so that final concentration of 25ppm.Specific inhibition is shown in Fig. 8 A and Fig. 8 B.Fig. 8 results are shown：25ppm pseudomonad IAS03 bacterium
Strain zymotic fluid just significantly inhibits effect, next two kinds of diseases for 24 hours in 12h to Aeromonas sobria and Aeromonas hydrophila life
The quantity of opportunistic pathogen has rebound again, but the quantity of two kinds of cause of diseases is significantly suppressed after 48h.
Embodiment 7
30ppm pseudomonad IAS03 bacterial strain fermentation liquors are to Aeromonas hydrophila and temperature or the inhibition of Aeromonas
Pseudomonad IAS03 bacterial strain fermentation liquors (1 × 10 are added with embodiment 3 in experiment material and method9cfu/mL)240μ
L so that final concentration of 30ppm.Specific inhibition is shown in Fig. 9 A and Fig. 9 B.Fig. 9 results are shown：30ppm pseudomonad IAS03 bacterium
Strain zymotic fluid just significantly inhibits effect in 12h to Aeromonas sobria and Aeromonas hydrophila life, although next interior for 24 hours
Aeromonas sobria quantity has rebound, but declines to a great extent in 48h latter two pathogen quantity, pseudomonad IAS03 strain fermentations
Liquid inhibiting effect is notable.It can be seen that with using zymotic fluid dosage to increase, inhibit the effect enhancing of two kinds of pathogens, when effective
Between shorten.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, it is other it is any without departing from the spirit and principles of the present invention made by changes, modifications, substitutions, combinations, simplifications,
Equivalent substitute mode is should be, is included within the scope of the present invention.
Claims (3)
1. a kind of pseudomonad, it is characterised in that：Entitled pseudomonad Pseudomonas protegens IAS03, in
On November 27th, 2015 China typical culture collection center for being preserved in Wuhan University of Wuhan, China city, deposit number：CCTCC
NO:M 2015711。
2. the application of pseudomonad described in claim 1, it is characterised in that：The pseudomonad Pseudomonas
Applications of the protegens IAS03 in preparing the product for preventing Aeromonas hydrophila and/or Aeromonas sobria infection.
3. the application of pseudomonad described in claim 1, it is characterised in that：The pseudomonad Pseudomonas
Applications of the protegens IAS03 in preparing the product for inhibiting Aeromonas hydrophila and/or Aeromonas sobria.
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| CN106138108B (en) * | 2016-06-16 | 2019-11-19 | 暨南大学 | The application of pseudomonad IAS03 |
| CN114615884B (en) * | 2019-08-22 | 2024-05-14 | 内盖夫国家生物技术研究所有限公司 | Composition for inoculating aquatic animals |
| CN111100824B (en) * | 2020-01-21 | 2021-11-05 | 暨南大学 | Bacillus and application thereof in denitrification and desulfurization in aquaculture water |
| CN112410247B (en) * | 2020-11-08 | 2022-05-10 | 领先生物农业股份有限公司 | Pseudomonas defensins and application thereof in down feather detergent |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101181635A (en) * | 2007-12-10 | 2008-05-21 | 河北师范大学 | Method for preparing pathogenicity hydrosphere unit cell bacterium killed vaccine |
| CN103320365A (en) * | 2013-07-09 | 2013-09-25 | 中国水产科学研究院淡水渔业研究中心 | Fish-sourced aeromonas hydrophila disease antagonistic strain and application thereof |
| CN103719167A (en) * | 2012-10-14 | 2014-04-16 | 青岛医防消毒专业技术中心 | Degerming agent specially used for aeromonas hydrophila water body |
| CN104388339A (en) * | 2014-10-27 | 2015-03-04 | 徐州工程学院 | Microecological preparation suitable for marine culture and application thereof |
-
2015
- 2015-11-27 CN CN201510856000.6A patent/CN105349463B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101181635A (en) * | 2007-12-10 | 2008-05-21 | 河北师范大学 | Method for preparing pathogenicity hydrosphere unit cell bacterium killed vaccine |
| CN103719167A (en) * | 2012-10-14 | 2014-04-16 | 青岛医防消毒专业技术中心 | Degerming agent specially used for aeromonas hydrophila water body |
| CN103320365A (en) * | 2013-07-09 | 2013-09-25 | 中国水产科学研究院淡水渔业研究中心 | Fish-sourced aeromonas hydrophila disease antagonistic strain and application thereof |
| CN104388339A (en) * | 2014-10-27 | 2015-03-04 | 徐州工程学院 | Microecological preparation suitable for marine culture and application thereof |
Non-Patent Citations (4)
| Title |
|---|
| In Vitro Inhibition of Saprolegnia sp by an Antifungal Peptide from Pseudomonas protegens XL03;Wang FF等;《North American Journol of Aquaculture》;20170223;第79卷(第2期);168-175 * |
| 嗜水气单胞菌的分离鉴定及控制方法研究;蒋启欢;《中国优秀硕士学位论文全文数据库》;20130615(第06期);D052-70 * |
| 恩诺沙星控制嗜水气单胞菌性鲫败血症的防耐药用药方案;徐丽娟等;《中国水产科学》;20130530;第20卷(第3期);635-643 * |
| 鱼类暴发性流行病的预防与治疗;马朝彬;《现代农业科技》;20060930;176-177 * |
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