CN106138108A - The application of pseudomonas IAS03 - Google Patents

The application of pseudomonas IAS03 Download PDF

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CN106138108A
CN106138108A CN201610438651.8A CN201610438651A CN106138108A CN 106138108 A CN106138108 A CN 106138108A CN 201610438651 A CN201610438651 A CN 201610438651A CN 106138108 A CN106138108 A CN 106138108A
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pseudomonas
ias03
bacterial strain
water
supernatant
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CN106138108B (en
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张其中
方伟
王飞飞
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Jinan University
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Jinan University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria

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Abstract

The present invention discloses the application of a kind of pseudomonas IAS03, belongs to microorganism field.In the present invention, the steril cell of pseudomonas (Pseudomonas protegens) IAS03 crushes supernatant and can significantly inhibit the growth of saprolegnia hypha on flat board, it is 6.25mg/mL to the minimum effectively inhibition concentration of mycelia, and the minimum effectively inhibition concentration to spore germination is 3.125mg/mL.By the toxicity test of bacterial strain, find that pseudomonas IAS03 is safe to Ctenopharyngodon idellus.Pseudomonas IAS03 is to obtain from sediment of pond screening, will not work the mischief surrounding with ecological balance, meet bio-safety regulation.Pseudomonas IAS03 only must i.e. can be applicable to prevent and treat fish molds by single bacterial strain, relative to traditional antibiotic and disinfectant prophylactico-therapeutic measures, has safe efficient, the advantage of environmental protection.

Description

The application of pseudomonas IAS03
Technical field
The invention belongs to microorganism field, be specifically related to a kind of pseudomonas (Pseudomonas protegens) IAS03 Application.
Background technology
Fish molds is a kind of common fungi autoeciousness disease during brackish culture produces, it have Epidemic Scope wide, time Between span is big, heal etc. feature without host selection, refractory, therefore the outburst of fish molds frequently results in Fish mortality, thus Tremendous economic is brought to lose to culture fishery.Saprolegnia (Saprolegnia spp.) fungus is the important cause causing fish molds Pathogenic bacteria, therefore, only presses down the water mo(u)ld killing in water body from source, can be only achieved and prevent and treat purpose.
Malachite green oxalate was once widely used in the preventing and treating of fish molds in aquaculture, but malachite green oxalate is the most degradable, There is high residue phenomenon in aquatic animal body, and mammal and the mankind are existed teratogenesis, risk carcinogenic, mutagenic, therefore Disabled in fish production by various countries the most successively from the nineties in last century.Although formaldehyde, potassium permanganate, chlorine dioxide and peroxide Change the chemicalses such as hydrogen and fish molds is also had certain prevention effect, but the use of these chemicalses there is also residual risk With pollute the problem such as environment.Therefore, find a kind of selectivity safe efficient, environmental protection to press down and kill the technology of water mo(u)ld and become more Important.
Microbial control, is to utilize the interaction relationship between microorganism to press down to kill pathogen, thus protects host to exempt from Infected by pathogen, be there is safe efficient and environmental protection advantage, Japan than the good EM technology according to husband professor be exactly one good Example.
Summary of the invention
In order to overcome the shortcoming of prior art with not enough, it is an object of the invention to provide a kind of pseudomonas IAS03's Application.There is the active substance of water resistant mycete in its intracellular, lays the foundation for being developed further into microbial ecological agent.
The purpose of the present invention is achieved through the following technical solutions:
The present invention provides a kind of pseudomonas (Pseudomonas protegens) IAS03 in prevention of water fungal infection Application.
Described pseudomonas (Pseudomonas protegens) IAS03 is in the product of preparation suppression water mo(u)ld Application.
Described pseudomonas (Pseudomonas protegens) IAS03 is by entering the bacterial strain in sediment of pond Obtain after row is isolated and purified.Its preservation information: depositary institution: China typical culture collection center (CCTCC), preservation day Phase: on November 27th, 2015, preservation address: China. Wuhan. Wuhan University, deposit number: CCTCC NO:M 2015711.? Through disclosed in patent " application number: 201510856000.6, title: a kind of pseudomonad strain and application thereof ".
Described pseudomonas (Pseudomonas protegens) IAS03, by the following method isolated:
After being dissolved by the sediment of pond sterilized water gathered, take supernatant dilution on beef extract-peptone solid medium Carrying out line to separate, repeat 2～3 times, until being separated to form, uniform bacterium colony, then this bacterium colony being trained at KB solid Support in base and cultivate, and expose under the uviol lamp that wavelength is 366nm, the form of perusal bacterium colony.
Through Hua Da genome company to the order-checking of this bacterium colony 16S rDNA gene and the comparison of EzTaxon data base, this The classification position of the pseudomonas IAS03 of bright institute isolated belongs to: Rhodopseudomonas protegens bacterium (Pseudomonas protegens)。
Described beef extract-peptone solid medium: peptone 10.0g/L, Carnis Bovis seu Bubali cream 3.0g/L, sodium chloride 5.0g/L, Agar powder 15.0～20.0g/L, pH 7.0～7.2,121 DEG C, the process of 20min high temperature sterilize；Beef extract-peptone liquid culture Base is not added with agar powder.
This bacterial strain is on the flat board of beef extract-peptone solid medium, and bacterium colony is circular, upwards swells, neat in edge, table Face is smooth moistening, and in faint yellow, size is homogeneous, and on KB solid medium, under the uviol lamp that wavelength is 366nm, sends out Go out the fluorescence of blueness.
Described pseudomonas (Pseudomonas protegens) IAS03 can effectively suppress the growth of water mo(u)ld, Thus protect Fish infecting from water mo(u)ld.
Pseudomonad strain IAS03, is a strain antibacterial isolated and purified from sediment of pond, and water mo(u)ld is had efficiently by it Inhibitory or killing effect, and it is from water body environment, there will be no problems of ecological security for water body environment, therefore, selects false single It will be a kind of green, efficient and feasible mode that born of the same parents bacterium IAS03 prevents and treats by the fish molds caused by Saprolegnia fungus.
The present invention, relative to prior art, has such advantages as and effect:
(1) in the present invention, the steril cell of pseudomonas (Pseudomonas protegens) IAS03 crushes supernatant flat Can significantly inhibit the growth of saprolegnia hypha on plate, it is 6.25mg/mL to the minimum effectively inhibition concentration of mycelia, and sprouts spore The minimum effectively inhibition concentration sent out is 3.125mg/L.By the toxicity test of bacterial strain, find that Ctenopharyngodon idellus is by pseudomonas IAS03 Safety.
(2) pseudomonas IAS03 obtains from sediment of pond screening, will not work the mischief surrounding with ecological balance, Meet bio-safety regulation.
(3) pseudomonas IAS03 can press down and kills aquatic pathogenic bacterium: water mo(u)ld.
(4) pseudomonas IAS03 only must i.e. can be applicable to prevent and treat fish molds by single bacterial strain, relative to traditional antibiotic and Disinfectant prophylactico-therapeutic measures, has safe efficient, the advantage of environmental protection.
Accompanying drawing explanation
Fig. 1 is the fluorescence phenomenon of pseudomonas IAS03；Wherein, figure A is beef extract-peptone solid medium；Figure B is KB Solid medium.
Fig. 2 is the molecular biology identification of pseudomonas IAS03；Wherein, figure A is the electrophoretogram of 16S rDNA；Figure B is The systematic evolution tree of 16S rDNA gene.
Detailed description of the invention
Below in conjunction with embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention are not limited to This.
The separation of embodiment 1 pseudomonad strain IAS03.
Test material: sediment of pond.
The preparation of culture medium:
Beef extract-peptone solid medium: peptone 10.0g, Carnis Bovis seu Bubali cream 3.0g, sodium chloride 5.0g, agar powder 15.0～ 20.0g, 7.2,121 DEG C of high temperature sterilizes of distilled water 1000mL, pH process 20min, and (beef extract-peptone fluid medium is added without Agar powder).
KB solid medium: peptone 20.0g, glycerol 10.0mL, K2HPO41.5g, MgSO4.7H2O1.5g, agar powder 15.0g～20.0g, 7.2,121 DEG C of high temperature sterilize 20min of deionized water 1000mL, pH.
Test method:
(1) weighing the bed mud 10g in pond in conical flask, add 90mL aquesterilisa, 28 DEG C, 160r/min constant temperature oscillation is trained After supporting 30min, after standing 5min, take supernatant, use gradient dilution method that supernatant is diluted to 103Times, take 0.15mL diluent in cattle Meat extract peptone solid medium flat board even spread, 28 DEG C of constant temperature culture 16h.
(2) the single bacterium colony on picking step (1) middle plateform is in beef extract-peptone fluid medium, 28 DEG C, 160r/ Min constant-temperature shaking culture 8h, until OD600When value reaches 0.8, draw the bacterium solution of 10 μ L with liquid-transfering gun, by gradient dilution extremely 104Times, taking 0.1mL in beef extract-peptone solid medium flat board even spread, 28 DEG C of constant temperature culture 16h, observation bacterium colony is No single, repeat 2～3 times.
(3) by single colony inoculation in beef extract-peptone fluid medium, 28 DEG C, 160r/min constant temperature oscillation is trained After supporting 8h, bacterium solution is as activated seed liquid.
(4) seed liquor taking step (3) with inoculating loop is inoculated on KB solid medium by the way of line, 28 DEG C, After constant temperature culture 24h, perusal colonial morphology, under wavelength is 366nm uviol lamp, observe fluorescence phenomenon simultaneously, result is shown in figure 1.From Figure 1A, this bacterial strain is on the flat board of beef extract-peptone solid medium, and bacterium colony is circular, upwards swells, and edge is whole Together, smooth surface moistens, and in faint yellow, size is homogeneous, but on KB solid medium, under the uviol lamp that wavelength is 366nm, Send the fluorescence (Figure 1B) of blueness.
The 16S rDNA Species estimation of embodiment 2 pseudomonad strain IAS03
Test material: bacterial universal primers (27F:5 '-AGAGTTTGATCCTGGCTCAG-3 '；1492R:5 '- TACGGTTACCTTGTTACGACTT-3 '), Guangzhou Hua Da genome company synthesize.
Test method:
(1) amplification of 16S rDNA.The preparation of the seed liquor of pseudomonas IAS03 bacterial strain is with embodiment 1.Take this bacterial strain Seed liquor carries out bacterium colony PCR, and its primer is universal primer (concentration 10 μMs): 27F and 1492R；Reaction system is 25 μ L, including ddH2O 15.00 μ L, KOD-Plus buffer 2.5 μ L, dNTP mix (200mmol/L) 2.5 μ L, primer 2 7F are 1.0 μ L, Primer 1492R is 1.0 μ L, bacterium solution 1.0 μ L, KOD-Plus enzyme 0.5 μ L, MgSO4(25mmol/L)1.5μL.Course of reaction is as follows: Denaturation: 95 DEG C, 5min；Degeneration: 94 DEG C, 1min；Annealing: 54 DEG C, 1min；Extend: 72 DEG C, 90s；Re-extend: 72 DEG C, 10min.PCR primer carries out the agarose nucleic acid electrophoresis (100v, 30min) of 1.2%, and 16S rDNA fragment is about 1500bp, Seeing Fig. 2 A, the product selecting band single and bright send Hua Da gene sequencing.
(2) foundation of systematic evolution tree.The gene order of 16S rDNA is imported in EzTaxon data base and carries out in line sequence Row comparison, and carry out multisequencing coupling arrangement with Cluxtalx 1.83 software, analysis result uses the neighbour in Mega 5.0 software Systematic evolution tree is made in position phase connection (Neighbor-joining method), and analysis (Bootstrap) of booting carries out confidence Degree detection, bootstrapping data set 1000 times.Primarily determine that the possible genus of antagonistic strain and kind.Result is shown in Fig. 2 B.
From the 16S rDNA full length gene about 1500bp of Fig. 2 A, pseudomonas ISA03, and sequencing result obtains length For the sequence of 1408bp, in EzTaxon specialized database, comparison result shows, with pseudomonas Pseudomonas The similarity of protegens 16S rDNA gene order is 99%, and aligned sequences length accounts for 16S rDNA gene order total length 96.51%, thus Preliminary Identification bacterial strain ISA03 is Pseudomonas protegens.Named pseudomonas (Pseudomonas protegens)IAS03。
The 16S rDNA sequence such as SEQ ID of described pseudomonas (Pseudomonas protegens) IAS03 bacterial strain Shown in NO:1 (1408bp).
The toxicity test of embodiment 3 bacterial strain
Experiment material: Ctenopharyngodon idellus 105 tail (body weight 30.0 ± 5.5g, body length 12 ± 2.5cm), the physiological saline solution of 0.9%.
The preparation of culture medium: the preparation of beef-protein medium is with embodiment 1.
The preparation of injection bacterium solution:
Take pseudomonas IAS03 bacterial strain, on beef extract-peptone solid medium, carry out line with inoculating loop and activate, 28 After DEG C constant temperature culture 16h, with inoculating loop picking list colony inoculation in beef extract-peptone fluid medium, 28 DEG C, 160r/ After min constant-temperature shaking culture 12h, then carry out 4 DEG C of 7500r/min, 15min and be centrifuged, and with the physiological saline solution of 0.9% Centrifuge washing three times, then the physiological saline solution with 0.9% is resuspended, measures OD finally by ultraviolet spectrophotometer600Value, uses The concentration of bacterium solution is adjusted to 10 by normal saline respectively6, 107, 108, 109, 1010, 1011cfu/mL。
Test method:
Take body weight suitable and healthy Ctenopharyngodon idellus 35 tail, be randomly divided into 7 groups, often organize 5 tails, be respectively labeled as 106Group, 107Group, 108Group, 109Group, 1010Group, 1011Group and matched group, each group three parallel, raises in the drum of 10L (warp before Shi Yonging respectively Potassium permanganate processes), 106Group, 107Group, 108Group, 109Group, 1010Group, 1011Group is respectively by lumbar injection 200 μ L bacterium solution, dense Degree is respectively 106Cfu/mL, 107Cfu/mL, 108Cfu/mL, 109Cfu/mL, 1010Cfu/mL and 1011Cfu/mL bacterium solution, comparison The physiological saline solution of group injection equivalent 0.9%, feeds 7 days continuously, entirely changes water and pellet of throwing something and feeding every day.Every day record and The OAS of fish in statistical experiment group and matched group, record seven days, finally add up the death toll of fish, result such as table 1 continuously.
The toxicity test of table 1 pseudomonas IAS03 bacterial strain
By table 1, by the data analysis of SPSS software, the half lethal concentration of Ctenopharyngodon idellus is by pseudomonas IAS03 bacterial strain (LC50) it is 7.34 × 109Cfu/mL, it can thus be assumed that this bacterial strain is safe to Ctenopharyngodon idellus.
Embodiment 4 antagonistic strain inhibition to saprolegnia hypha
Test material: water mo(u)ld (Saprolegnia sp.) is from the conservation of this laboratory.Described water mo(u)ld (Saprolegnia sp.) document " screening of Saprolegnia antagonist and antagonistic activity material stability preliminary study [J] thereof. micro- Biology is circulated a notice of, and 2015,42 (6): 1067-1074. " disclosed in.
The preparation of culture medium:
Rhizoma Solani tuber osi dextrose solid medium (PDA): peeled potatoes 200.0g, glucose 20.0g, agar 15.0～20.0g, Water 1000mL, pH are natural.Boiling water boils 20min, then by four layers of filtered through gauze, remove residue, filtrate add glucose and Agar powder, 121 DEG C of high temperature sterilizes process 20min.The preparation of beef extract-peptone fluid medium is with embodiment 1.
Test method:
(1) steril cell crushes the preparation of supernatant.The seed liquor taking this bacterial strain is inoculated in equipped with 50mL according to the ratio of 2% The conical flask of the 250mL of beef extract-peptone fluid medium, 28 DEG C, after 160r/min constant-temperature shaking culture 48h, will fermentation Liquid is in 4 DEG C, and 7500rpm is centrifuged 15min, collects thalline, with sterilized water cyclic washing twice, centrifugal, then suspends with PBS, carries out Ultrasonic disruption (super 3s, stop 5s), the endochylema liquid of gained is in 4 DEG C, and 12000rpm is centrifuged 30min, collects supernatant, and with 0.45 The sterilised membrane filter in μm aperture filters and i.e. obtains steril cell and crush supernatant (CFDS).
(2) steril cell crushes the supernatant inhibitory action to saprolegnia hypha.By the aseptic bacterial cell disruption that concentration is 50mg/mL Supernatant is diluted to the Concentraton gradient of 1.5625～50mg/mL, then uses agar hole diffusion method, measures its anti-saprolegnia mycelial growth Activity, i.e. utilizes aseptic card punch to punch (diameter 8mm) in PDA plate central authorities, inoculation four pieces respectively at the about 3cm of the surrounding in hole Fish molds block (diameter 8mm), after 25 DEG C of constant temperature culture 24h, the steril cell being separately added into 200 μ L variable concentrations in hole crushes Clearly, then 48h is cultivated in 25 DEG C of continuation.Matched group adds sterile PBS buffer, treats that matched group fish molds covers with flat board, measure real Test the diameter of inhibition zone in group.Each group three parallel.Result shows that the steril cell of concentration >=6.25mg/mL crushes Supernatant just can suppress the growth of saprolegnia hypha.
Embodiment 5 antagonistic strain inhibition to fish molds spore germination
Test material:
The preparation of fish molds spore suspension: take fresh fish molds plate and punch with aseptic card punch, then with tweezers gripping 10～ 12 pieces of fish molds blocks are equipped with in the conical flask of 150mL sterilized water, after 25 DEG C of constant temperature culture 2d, filter with 8 layers of sterile gauze, Remove mycelia, finally by method of dilution butteron on plate, fish molds spore is counted, be adjusted to 5 × 10 with sterilized water3Cfu/mL, 4 DEG C of ice Case preserves.
The preparation of culture medium:
The compound method of PDA culture medium and beef extract-peptone fluid medium is with embodiment 4.
Test method:
(1) steril cell crushes the preparation of supernatant with embodiment 4.
(2) steril cell crushes the supernatant inhibitory action to fish molds spore germination.It is that 50mg/mL steril cell breaks by concentration Broken supernatant is diluted to the Concentraton gradient of 1.5625～50mg/mL.200 μ L concentration are respectively 1.5625～50mg/mL aseptic Cell breakage supernatant joins in EP pipe, and the EP pipe of each concentration is separately added into spore suspension (about 50 spores of 10 μ L Son), after 25 DEG C of constant temperature culture 16h, use dilution plating procedure, the sprouting quantity of statistics spore, and calculate spore germination Rate.Every hexad is parallel.Result shows that the steril cell of concentration >=3.125mg/mL crushes supernatant and just can suppress fish molds spore The sprouting of son.
Above-described embodiment is the present invention preferably embodiment, but embodiments of the present invention are not by above-described embodiment Limit, change that other is any is made under spirit and the principle without departing from the present invention, modify, substitute, combine, simplify, All should be the substitute mode of equivalence, within being included in protection scope of the present invention.

Claims (2)

1. pseudomonas (Pseudomonas protegens) IAS03 application in prevention of water fungal infection.
Application the most according to claim 1, it is characterised in that: described pseudomonas (Pseudomonas Protegens) IAS03 application in the product of preparation suppression water mo(u)ld.
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Cited By (1)

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CN110205264A (en) * 2019-05-22 2019-09-06 暨南大学 The application of Saprolegnia antagonist JD03 and its fermentation liquid and reactive compound in the product of preparation prevention and treatment saprolegniasis

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110205264A (en) * 2019-05-22 2019-09-06 暨南大学 The application of Saprolegnia antagonist JD03 and its fermentation liquid and reactive compound in the product of preparation prevention and treatment saprolegniasis
CN110205264B (en) * 2019-05-22 2020-09-04 暨南大学 Saprolegnia antagonist JD03 and application of fermentation liquor and active compound thereof in preparation of products for preventing and treating saprolegniasis

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