CN105349463A - Pseudomonas strain and application thereof - Google Patents
Pseudomonas strain and application thereof Download PDFInfo
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- CN105349463A CN105349463A CN201510856000.6A CN201510856000A CN105349463A CN 105349463 A CN105349463 A CN 105349463A CN 201510856000 A CN201510856000 A CN 201510856000A CN 105349463 A CN105349463 A CN 105349463A
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Abstract
The invention discloses a pseudomonas strain and application thereof. The strain is pseudomonas protegens IAS03, and is preserved in the China center for type culture collection on November 27th, 2015, and the preservation number is CCTCC NO:M 2015711. The strain is screened from pond bottom sludge and then a backwater body is used, so that ecological safety risks do not exist, and biological safety regulations are conformed. The strain has the specific antimicrobial function on aeromonas hydrophila and aeromonas sobria, has no inhibition effect on other bacteria and has no pathogenicity on fishes. Only one strain can be used for inhibiting aeromonas hydrophila and/or aeromonas sobria. Compared with traditional antibiotic and sterilizing agent prevention measures, the strain has the advantages of being safe, environmentally friendly and efficient. The strain can be developed into a microecological preparation to control aeromonas sobria and aeromonas hydrophila in the breeding environment and prevent hemorrhagic septicemia caused when the fishes are infected with aeromonas sobria and aeromonas hydrophila.
Description
Technical field
The invention belongs to microorganism field, relate to a kind of pseudomonad strain, particularly a kind of pseudomonad strain and application thereof.
Background technology
Aeromonas hydrophila (Aeromonashydrophila) and Aeromonas sobria (Aeromonassobria) are present in cultivation water environment, nearly all cultured freshwater fish is often caused to suffer from hueppe's disease, sickness rate is high, mortality ratio is also very high, causes heavy economic losses to culture fishery.Prevent and treat this disease in the past and mainly adopt microbiotic of throwing something and feeding, disinfectant for external use kills the pathogenic bacteria in water body, so not only can produce drug residue for a long time, inducible resistance pathogenic bacteria produces and polluted water environment, and sterilizing agent non-selectivity kills probiotics in water, destroy water body microecological balance, for follow-up outburst bacterial septicemia has buried hidden danger.
The pathogenic bacteria (Aeromonas hydrophila and Aeromonas sobria) of hueppe's disease is suffered from from aquaculture water owing to causing fish, so, need single-mindedly when not affecting probiotics to kill Aeromonas hydrophila in water surrounding and Aeromonas sobria, eliminate the cause of disease source of cultured fishes, prevention hueppe's disease occurs.This is the effective way solved the problem.
Summary of the invention
In order to overcome the shortcoming of prior art with not enough, primary and foremost purpose of the present invention is to provide a kind of pseudomonad strain.Active substance in the fermented liquid of this bacterial strain has the effect that specificity suppresses Aeromonas hydrophila (Aeromonashydrophila) and Aeromonas sobria (Aeromonassobria), other bacterium be separated in water surrounding is had no adverse effects, just in time solves the problems of the prior art.This pseudomonad strain can be developed into probiotics and controls cause of disease Aeromonas sobria in breeding environment and Aeromonas hydrophila, prevents it to infect causing bleeding property of fish septicemia.
Another object of the present invention is to the application that above-mentioned pseudomonad strain is provided.
Object of the present invention is achieved through the following technical solutions:
For achieving the above object, the invention provides a kind of pseudomonas, bacterial classification called after PseudomonasprotegensIAS03, obtains from sediment of pond separation and purification.
The preservation information of described pseudomonas (Pseudomonasprotegens) IAS03: depositary institution: China typical culture collection center (CCTCC), preservation date: on November 27th, 2015, preservation address: China. Wuhan. Wuhan University, deposit number: CCTCCNO:M2015711.
The liquid nutrient medium of described pseudomonas (Pseudomonasprotegens) IAS03 bacterial strain is beef extract-peptone liquid nutrient medium, beef extract-peptone solid medium then liquid medium within is filled a prescription on basis, adds agar 15 ~ 20g heating and melting and make in every 1000ml liquid nutrient medium.
Described beef extract-peptone liquid nutrient medium: peptone 10g/L, extractum carnis 3g/L, sodium-chlor 5g/L, agar 15 ~ 20g/L, pH7.0 ~ 7.2,121 DEG C, the process of 20min high-temperature sterilization; Beef extract-peptone liquid nutrient medium does not add agar.
The fermention medium of described pseudomonas (Pseudomonasprotegens) IAS03 bacterial strain is LB substratum, its component is: peptone 10g/L, yeast extract powder 5g/L, sodium-chlor 10g/L, pH value 7.0 ~ 7.2,121 DEG C, the process of 20min high-temperature sterilization.
Pseudomonas provided by the invention (Pseudomonasprotegens) IAS03 bacterial strain is applied to and suppresses Aeromonas hydrophila and/or Aeromonas sobria.
The application of described pseudomonas (Pseudomonasprotegens) IAS03 in prevention Aeromonas hydrophila and/or Aeromonas sobria infect.
Described pseudomonas (Pseudomonasprotegens) IAS03 is preparing the application in the product suppressing Aeromonas hydrophila and/or Aeromonas sobria.
Described pseudomonas (Pseudomonasprotegens) IAS03 bacterial strain suppresses the principle of Aeromonas hydrophila and Aeromonas sobria to be, and Rhodopseudomonas had both suppressed the growth of pathogenic bacteria by nutrient competition, secondary metabolite and the antimicrobial polypeptide addicted to iron element, prussic acid, oomycetes element, phenazine-1-carboxylic acid and luteolin etc. with bacteriostatic action can also be produced, but single-mindedly kill pathogenic bacteria, and other bacterium or algae are not damaged, thus ensure that under the prerequisite not destroying water ecosystem balance, kill pathogenic bacteria from source.
The present invention, relative to prior art, has following advantage and effect:
(1) the invention provides pseudomonas (Pseudomonasprotegens) IAS03 bacterial strain.
(2) pseudomonas of the present invention (Pseudomonasprotegens) IAS03 bacterial strain obtains from sediment of pond screening, more there is not ecological safety risk with backwater body, meets Biosafety regulation.
(3) pseudomonas IAS03 bacterial strain of the present invention effectively can suppress aquatic pathogenic bacterium: Aeromonas hydrophila and/or Aeromonas sobria.
(4) pseudomonas IAS03 bacterial strain of the present invention produces fungistatic effect to Aeromonas hydrophila and Aeromonas sobria, and to other bacterium unrestraint effects, has narrow spectrum bacteriostatic action, and this bacterium is to fish no pathogenicity.
(5) namely pseudomonas IAS03 bacterial strain of the present invention only must can be applicable to suppress Aeromonas hydrophila and/or Aeromonas sobria by single bacterial strain, relative to traditional microbiotic and sterilizing agent prophylactico-therapeutic measures, has safety, green, efficient advantage.
Accompanying drawing explanation
Fig. 1 is the inhibition of bacterium liquid to Aeromonas hydrophila and Aeromonas sobria of pseudomonas IAS03 bacterial strain; Wherein, Figure 1A is that the bacterium liquid of pseudomonas IAS03 bacterial strain is to the inhibition of Aeromonas hydrophila; Figure 1B is that the bacterium liquid of pseudomonas IAS03 bacterial strain is to the inhibition of Aeromonas sobria.
Fig. 2 be pseudomonas IAS03 bacterial strain without the inhibition of fermented liquid 10 times of concentrated solutions to Aeromonas hydrophila and Aeromonas sobria; Wherein, Fig. 2 A be pseudomonas IAS03 bacterial strain without fermented liquid 10 times of concentrated solutions to the inhibition of Aeromonas sobria; Fig. 2 B be pseudomonas IAS03 bacterial strain without fermented liquid 10 times of concentrated solutions to the inhibition of Aeromonas hydrophila.
Fig. 3 is the colony growth situation of pseudomonas IAS03 bacterial strain on PDA substratum and the fluorescence phenomenon on KB solid medium; Wherein, Fig. 3 A is the cultivation results of pseudomonas IAS03 bacterial strain on PDA solid medium; Fig. 3 B is the cultivation results of pseudomonas IAS03 bacterial strain on KB solid medium, under the ultraviolet lamp of 302nm, observe blue-fluorescence.
Fig. 4 is pseudomonas IAS03 bacterial strain 16SrDNAPCR amplification gel electrophoresis figure; Wherein, swimming lane M is DNAmarkerDL2000; Swimming lane 1 ~ 6 is the pseudomonas IAS03 bacterial strain 16SrDNA band of amplification.
Fig. 5 is 10ppm pseudomonas IAS03 fermented liquid to the inhibition of Aeromonas hydrophila and Aeromonas sobria; Wherein, Fig. 5 A is Aeromonas hydrophila; Fig. 5 B is Aeromonas sobria.
Fig. 6 is 15ppm pseudomonas IAS03 fermented liquid to the inhibition of Aeromonas hydrophila and Aeromonas sobria; Wherein, Fig. 6 A is Aeromonas hydrophila; Fig. 6 B is Aeromonas sobria.
Fig. 7 is 20ppm pseudomonas IAS03 fermented liquid to the inhibition of Aeromonas hydrophila and Aeromonas sobria; Wherein, Fig. 7 A is Aeromonas hydrophila; Fig. 7 B is Aeromonas sobria.
Fig. 8 is 25ppm pseudomonas IAS03 fermented liquid to the inhibition of Aeromonas hydrophila and Aeromonas sobria; Wherein, Fig. 8 A is Aeromonas hydrophila; Fig. 8 B is Aeromonas sobria.
Fig. 9 is 30ppm pseudomonas IAS03 fermented liquid to the inhibition of Aeromonas hydrophila and Aeromonas sobria; Wherein, Fig. 9 A is Aeromonas hydrophila; Fig. 9 B is Aeromonas sobria.
Note: * represents that the amount of the Aeromonas hydrophila of the group (test group) adding pseudomonas IAS03 fermented liquid or Aeromonas sobria is significantly lower than control group (p<0.05), there is significant difference (p<0.05) between the amount that different lowercase represents test group different time points Aeromonas hydrophila or Aeromonas sobria, and same letter indicates without significant difference.
Embodiment
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 thereto.
Aeromonas sobria (Aeromonassobria) described in embodiment document " change [J] of the Spinibarbus sinensis peripheral blood immune indexes of inactivated vaccine immunity. aquatic product journal, 2010,04:626-634. " in open.
Aeromonas hydrophila (Aeromonashydrophila) document " 42 kinds of herbal medicine are to the extracorporeal bacteria inhibitor test [J] of Aeromonas hydrophila. fresh water fishery, 2011,03:61-65. " in open.
The preservation information of described pseudomonas (Pseudomonasprotegens) IAS03: depositary institution: China typical culture collection center (CCTCC), preservation date: on November 27th, 2015, preservation address: China. Wuhan. Wuhan University, deposit number: CCTCCNO:M2015711.
Embodiment 1
The selection systems method of described pseudomonas (Pseudomonasprotegens) IAS03, step is as follows:
(1) take bed mud 10g from pond, Guangzhou in Erlenmeyer flask, add 90mL aqua sterilisa, 28 DEG C, 160r/min, constant-temperature table vibration 30min, gets supernatant after leaving standstill 5min, adopts gradient dilution method by supernatant dilution 10
3doubly, 0.15mL diluent is got in the dull and stereotyped even spread of beef extract-peptone solid medium, 28 DEG C of constant temperature culture 16h.Beef extract-peptone solid medium: peptone 10g, extractum carnis 3g, sodium-chlor 5g, agar 15 ~ 20g, add water to 1000mL, pH7.0,121 DEG C, the process of 20min high-temperature sterilization.The liquid nutrient medium of same recipe does not add agar.
(2) the single bacterium colony on picking step (1) middle plateform is in beef extract-peptone liquid nutrient medium, and 28 DEG C, 160r/min, constant-temperature table vibration 8h, treats OD
600when reaching 0.8, draw the bacterium liquid of 10 μ L, by gradient dilution 10
4doubly, get 0.1mL in the dull and stereotyped even spread of beef extract-peptone solid medium, whether single 28 DEG C of constant temperature culture 16h, observe bacterium colony, repeat 3 times, and carry out mark.
(3) by single colony inoculation in beef extract-peptone liquid nutrient medium, 28 DEG C, 160r/min, constant-temperature table vibration 8h, as seed liquor, the Erlenmeyer flask of the 250mL that 50mL beef extract-peptone liquid nutrient medium is housed is inoculated in, 160r/min constant-temperature shaking culture 24h at 28 DEG C, as bacterium liquid to be measured according to the ratio of 2% (v/v).
(4) using Aeromonas sobria (Aeromonassobria) and Aeromonas hydrophila (Aeromonashydrophila) as indicator, its conservation from this laboratory (-80 DEG C, 20% glycerine is preserved).The bacterial classification deposited go bail for respectively by transfering loop at the flat lining out of beef extract-peptone solid medium, and 28 DEG C of constant temperature culture 12h, picking list bacterium colony activates in beef extract-peptone liquid nutrient medium respectively, until OD
600=0.8, lay at 4 ~ 8 DEG C of refrigerators for subsequent use.
(5) Antagonistic Fungi of Aeromonas sobria and Aeromonas hydrophila is screened by agar plate diffusion process.Be that the punch tool of 0.8mm punches in the central authorities of beef extract-peptone solid medium flat board with diameter, the bacterium liquid 200 μ L got in step (3) adds in this hole, 28 DEG C of constant temperature culture 12h, then the indicator liquid 5 μ L in step (4) is got, add sterilized water until 100 μ L, and with glass stick even spread onboard, 28 DEG C of constant temperature culture 12h.Each bacterium three repetition.
(6) use vernier caliper measurement antibacterial circle diameter, and make a record, filter out Antagonistic Fungi.
(7) amplification of 16SrDNA, the seed liquor of getting the Antagonistic Fungi in step (3) carries out bacterium colony PCR, its primer is universal primer: 27F:5 '-AGAGTTTGATCCTGGCTCAG-3 ', 1492R:5 '-TACGGTTACCTTGTTACGACTT-3 '; Reaction system is 25 μ L, comprises ddH2O15.00 μ L, KOD-Plusbuffer2.5 μ L, dNTPmix (200mmol/L) 2.5 μ L, primer 2 7F1.0 μ L, primer 1492R1.0 μ L, bacterium liquid 1.0 μ L, KOD-Plus enzyme 0.5 μ L, MgSO4 (25mmol/L) 1.5 μ L.Reaction process is as follows: denaturation: 95 DEG C, 5min, sex change: 94 DEG C, 1min, annealing: 54 DEG C, and 1min extends: 72 DEG C, 90s, then extends: 72 DEG C, 10min.Agarose nucleic acid electrophoresis (the 100V of 1.2% is carried out after PCR completes, 30min), fragment is approximately 1500bp, cut glue and reclaim PCR primer, be cloned in carrier T, transformation of E. coli DH5 α (EscherichiacoliDH5 α) (commercially available), PCR detects positive colony, and send Shenzhen Hua Da gene sequencing by bacterium liquid.As shown in Figure 4, wherein, swimming lane M is DNAmarkerDL2000 to result; Swimming lane 1 ~ 6 is the pseudomonas IAS03 bacterial strain 16SrDNA band of amplification.
(8) foundation of evolutionary tree.The sequence of 16SrDNA is imported in EzXtaon specialized database and carries out online compare of analysis, the arrangement of multisequencing coupling is carried out with Cluxtalx1.83 software, analytical results adopts ortho position phase connection (Neighbor-joiningmethod) in Mega5 software to make systematic evolution tree, and carry out bootstrap analyses (Bootstrap) and degree of confidence detection, bootstrapping data set 1000 times.Determine the genus of antagonistic strain and possible kind.
(9) morphologic qualification.The seed liquor of getting Antagonistic Fungi in step (3) with transfering loop is rule on KB solid plate, and parallel edition is PDA solid medium.After 28 DEG C of constant temperature culture 24h, be observe under the ultraviolet lamp of 302nm whether to have fluorescence to occur in wavelength.KB solid medium: peptone 20g, glycerine 10mL, K
2hPO
41.5g, MgS0
4.7H
2o1.5g, agar 15 ~ 20g, water 1000mL, pH7.2,121 DEG C, the process of 20min high-temperature sterilization.PDA solid medium: peeled potatoes 200g, glucose 20g, agar 15 ~ 20g, water 1000mL, pH value 7.0 ~ 7.2.Concrete grammar is clean potato, and stripping and slicing (1 × 1 × 1cm) poach, after 20 minutes, by 4 layers of filtered through gauze, adds glucose 20g and agar 15 ~ 20g, 121 DEG C of sterilising treatment 20min in 1000 milliliters of filtrates.
The 16SrDNA sequence of pseudomonas (Pseudomonasprotegens) IAS03 bacterial strain of separation and purification and the similarity of the 16SrDNA sequence of Pseudomonasprotegens are 99%, showing that this bacterial strain IAS03 and Pseudomonasprotegens gathers based on 16SrDNA cluster analysis is one, and sends blue-fluorescence on KB flat board.As shown in Figure 3, wherein, Fig. 3 A is the cultivation results of pseudomonas IAS03 bacterial strain on PDA solid medium to result; Fig. 3 B is the cultivation results of pseudomonas IAS03 bacterial strain on KB solid medium, under the ultraviolet lamp of 302nm, observe blue-fluorescence.
The antibacterial circle diameter of bacterium liquid to Aeromonas sobria and Aeromonas hydrophila life adopting the agar plate diffusion process of (5) to record pseudomonas IAS03 is respectively 40.29 ± 0.85mm and 38.40 ± 0.69mm.As shown in Figure 1, wherein, Figure 1A is that the bacterium liquid of pseudomonas IAS03 bacterial strain is to the inhibition of Aeromonas hydrophila to result; Figure 1B is that the bacterium liquid of pseudomonas IAS03 bacterial strain is to the inhibition of Aeromonas sobria.
In laboratory conditions, the activated seed liquid getting pseudomonas IAS03 bacterial strain is inoculated in the Erlenmeyer flask (1000mL) containing 250mLLB liquid nutrient medium according to the inoculum size of 2% (v/v), culture condition is 28 DEG C, 160r/min shaking culture 48h, after fermentation ends, fermentation liquor refrigerated centrifuge carries out the centrifugal 15min of 7500r/min, remove thalline, get supernatant, carry out vacuum lyophilization, get xeraphium sterilized water to dissolve, centrifugal again, get the supernatant metre filter of 0.22 μm, obtain 10 times of aseptic Fermented Condensed liquid, then lysoplate assay is adopted, measure its antibacterial circle diameter to Aeromonas sobria and Aeromonas hydrophila.At 28 DEG C after constant temperature culture 12h, respectively Aeromonas sobria and Aeromonas hydrophila are given birth to the inhibition zone of diameter 16.88 ± 0.65mm and 14.03 ± 0.35mm.Result as shown in Figure 2, wherein, Fig. 2 A be pseudomonas IAS03 bacterial strain without fermented liquid 10 times of concentrated solutions to the inhibition of Aeromonas sobria; Fig. 2 B be pseudomonas IAS03 bacterial strain without fermented liquid 10 times of concentrated solutions to the inhibition of Aeromonas hydrophila.
The liquid nutrient medium of described pseudomonas IAS03 bacterial strain is beef extract-peptone liquid nutrient medium, and beef extract-peptone solid medium then adds agar 15 ~ 20g/L on beef extract-peptone liquid culture based formulas basis.
Described beef extract-peptone liquid nutrient medium: peptone 10g/L, extractum carnis 3g/L, sodium-chlor 5g/L, pH7.0 ~ 7.2, sterilising treatment 20min at 121 DEG C.
The fermention medium of described pseudomonas IAS03 bacterial strain is LB liquid nutrient medium, and its component is: peptone 10g/L, yeast extract powder 5g/L, sodium-chlor 10g/L, pH value 7.2,121 DEG C of sterilising treatment 20min.
The 16SrDNA sequence of described pseudomonas (Pseudomonasprotegens) IAS03 bacterial strain is as shown in SEQIDNO:1: (1408bp)
TGCAGTCGAGCGGCAGCACGGGTACTTGTACCTGGTGGCGAGCGGCGGACGGGTGAGTAATGCCTAGGAATCTGCCTAGTAGTGGGGGATAACGTCCGGAAACGGGCGCTAATACCGCATACGTCCTACGGGAGAAAGTGGGGGATCTTCGGACCTCACGCTATTAGATGAGCCTAGGTCGGATTAGCTAGTTGGTGAGGTAATGGCTCACCAAGGCGACGATCCGTAACTGGTCTGAGAGGATGATCAGTCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGAAAGCCTGATCCAGCCATGCCGCGTGTGTGAAGAAGGTCTTCGGATTGTAAAGCACTTTAAGTTGGGAGGAAGGGCAGTTACCTAATACGTGATTGTTTTGACGTTACCGACAGAATAAGCACCGGCTAACTCTGTGCCAGCAGCCGCGGTAATACAGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCGCGTAGGTGGTTTGTTAAGTTGGATGTGAAAGCCCCGGGCTCAACCTGGGAACTGCATCCAAAACTGGCAAGCTAGAGTATGGTAGAGGGTGGTGGAATTTCCTGTGTAGCGGTGAAATGCGTAGATATAGGAAGGAACACCAGTGGCGAAGGCGACCACCTGGACTGATACTGACACTGAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCAACTAGCCGTTGGGAGCCTTGAGCTCTTAGTGGCGCAGCTAACGCATTAAGTTGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGCCTTGACATCCAATGAACTTTCTAGAGATAGATTGGTGCCTTCGGGAACATTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGTAACGAGCGCAACCCTTGTCCTTAGTTACCAGCACGTTATGGTGGGCACTCTAAGGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTACGGCCTGGGCTACACACGTGCTACAATGGTCGGTACAAAGGGTTGCCAAGCCGCGAGGTGGAGCTAATCCCATAAAACCGATCGTAGTCCGGATCGCAGTCTGCAACTCGACTGCGTGAAGTCGGAATCGCTAGTAATCGCGAATCAGAATGTCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTGCACCAGAAGTAGCTAGTCTAACCTTCGGGAGGACGGTACCA。
Embodiment 2
Pseudomonas (Pseudomonasprotegens) IAS03 bacterial strain is to the toxicity test of fish
(1) experiment material: grass carp seedling 270 tail (body weight is about 20g)
(2) preparation of substratum:
Beef extract-peptone liquid nutrient medium: peptone 10g/L, extractum carnis 3g/L, sodium-chlor 5g/L, pH value 7.0 ~ 7.2,121 DEG C of sterilizing 20min;
Beef extract-peptone solid medium: add 15 ~ 20g/L agar in beef extract-peptone liquid nutrient medium, 121 DEG C of sterilizing 20min.
(3) preparation of bacterium liquid is injected:
With transfering loop go bail for hide pseudomonas IAS03 bacterial strain line on beef extract-peptone solid medium flat board, after cultivating 16h at 28 DEG C, with transfering loop picking list colony inoculation in beef extract-peptone liquid nutrient medium, through 160r/min shaking culture 12h at 28 DEG C, then centrifugal (7500rpm, 5 minutes), and with physiological saline centrifugal (7500rpm repeatedly, 5 minutes) wash three times, last resuspended bacterium liquid measures OD by ultraviolet spectrophotometer again
600value, is adjusted to 10 with physiological saline respectively by bacterium liquid
6, 10
7, 10
8, 10
9, 10
10cfu/mL, obtains the pseudomonas IAS03 strain suspensions of different concns.
(4) test method:
Get grass carp 90 tail that weight is about 20g, be divided into 6 groups at random, often organize 15 tails, every tail fish abdominal injection 0.2mL pseudomonas IAS03 strain suspensions (contrast injection equivalent stroke-physiological saline solution), the bacteria concentration of 6 group injections is respectively 0 (contrast), 10
6, 10
7, 10
8, 10
9, 10
10cfu/mL, the fish often after group injection is raised respectively in 1 60L white plastic drum, and inflates in inner bucket water with air compressor pump, often organizes 3 parallel repetitions.Continuous nursing 7 days, changes water every day and feeds granulated feed, the behavior of observed and recorded test fish and dead fish number.
(5) experimental result: after 7 days, statistics fish mortality ratio, finds 1 × 10
9the pseudomonas IAS03 bacterial strain of below cfu/mL concentration to grass carp without any detrimentally affect, and 1 × 10
10cfu/mL just causes the fish of 60% dead, and so large bacterium amount in fact can not be had to enter fish body.Then, by above-mentioned same method to 1 × 10 of grass carp seedling abdominal injection 0.2mL
8cfu/mL Aeromonas hydrophila or Aeromonas sobria, in 3 days, fish is entirely dead, and contrast is survived entirely.Therefore, pseudomonas IAS03 bacterial strain is to fish safety.
Embodiment 3
10ppm pseudomonas IAS03 bacterial strain fermentation liquor is to the inhibition test of Aeromonas hydrophila and Aeromonas sobria
(1) preparation of substratum:
Beef extract-peptone liquid nutrient medium: peptone 10g/L, extractum carnis 3g/L, sodium-chlor 5g/L, pH value 7.0,121 DEG C of sterilizing 20min;
Beef extract-peptone solid medium: add 15g/L agar in beef extract-peptone liquid nutrient medium, 121 DEG C of sterilizing 20min, the cultivation of falling 50mL is based on making solid medium flat board in culture dish.
LB liquid nutrient medium: peptone 10g/L, yeast extract powder 5g/L, sodium-chlor 10g/L, pH value 7.2,121 DEG C of sterilizing 20min.
Aeromonas hydrophila or the Aeromonas sobria of-80 DEG C of preservations is got respectively with transfering loop, at the flat lining out of beef extract-peptone solid medium, after 28 DEG C of cultivation 10h, use transfering loop picking list colony inoculation again in beef extract-peptone liquid nutrient medium, 160r/min shaking culture 12h at 28 DEG C, then measures bacterium liquid OD by ultraviolet spectrophotometer
600value.
(2) preparation of pseudomonas IAS03 bacterial strain fermentation liquor:
The pseudomonas IAS03 bacterial strain of-80 DEG C of preservations is got in the flat lining out activation of beef extract-peptone solid medium with transfering loop, cultivate 16h at 28 DEG C after, with transfering loop picking list colony inoculation in beef extract-peptone liquid nutrient medium, at 28 DEG C, 160r/min shaking culture 12h is until OD
600value is 0.8, as fermentation seed liquid.Then be inoculated in (1L) in the Erlenmeyer flask that 250mLLB substratum is housed according to the inoculum size of 2% (v/v), 160r/min shaking culture 48h at 28 DEG C, the liquid of gained is band fermented liquid.
(3) test method:
Get pond water to carry out filtering and autoclaving, as the growth water surrounding of Aeromonas sobria or Aeromonas hydrophila, by the water of sterilizing respectively equivalent add in the glass test tube of 15 × 150mm, then add the bacterium liquid of Aeromonas hydrophila or Aeromonas sobria respectively, make ultimate density be 3 × 10
10cfu/mL, the liquid final volume of each glass test tube is 8mL, then adds 80 μ L pseudomonas IAS03 bacterial strain fermentation liquors (1 × 10
9cfu/mL), make final concentration be 10ppm (experimental group), in control group, add equivalent aqua sterilisa, be then placed in 28 DEG C through 160r/min shaking culture, and sample at 12h, 24h, 36h, 48h, 60h, 72h, 84h.The sample gathered passes through gradient dilution, namely drawing 10 μ L stostes with liquid-transfering gun joins in the stroke-physiological saline solution of 990 μ L, after mixing, drawing 10 μ L again joins in the physiological saline of 990 μ L, then 100 μ L are therefrom drawn, be uniformly coated on beef extract-peptone solid medium with glass stick, each time point 4 repetition, cultivate after 8 hours for 28 DEG C, carry out enumeration (pseudomonas IAS03 now does not also grow), then carried out statistics and the significant difference analysis of data by Spss software.The results are shown in Figure 5A and Fig. 5 B.Fig. 5 result shows, in 24h after adding fermented liquid, the Aeromonas sobria of experimental group and control group and do not have significant difference addicted to water-based Zymomonas mobilis, but when 36h, pseudomonas IAS03 fermented liquid creates retarding effect to Aeromonas sobria, and occurs significance restraining effect to aeromonas hydrophila quantity at 48h.
Embodiment 4
15ppm pseudomonas IAS03 bacterial strain fermentation liquor is to the inhibition of Aeromonas hydrophila or Aeromonas sobria
Experiment material and method, with embodiment 3, add pseudomonas IAS03 bacterial strain fermentation liquor (1 × 10
9cfu/mL) 120 μ L, make final concentration be 15ppm.Concrete inhibition is shown in Fig. 6 A and Fig. 6 B.Fig. 6 result shows: on pretreatment in 36h, Aeromonas sobria or the Aeromonas hydrophila quantity of experimental group and control group all do not have significant difference, after 48h, two kinds of pathogenic bacteria quantity of experimental group are all remarkable in control group, illustrate that pseudomonas IAS03 bacterial strain creates retarding effect to two kinds of pathogenic bacterias really.
Embodiment 5
20ppm pseudomonas IAS03 bacterial strain fermentation liquor is to the inhibition of Aeromonas hydrophila and Aeromonas sobria
Experiment material and method, with embodiment 3, add pseudomonas IAS03 bacterial strain fermentation liquor (1 × 10
9cfu/mL) 160 μ L, make final concentration be 20ppm.Concrete inhibition is shown in Fig. 7 A and Fig. 7 B.Fig. 7 result shows: 20ppm pseudomonas IAS03 bacterial strain fermentation liquor does not also show and produces remarkable restraining effect to Aeromonas hydrophila or Aeromonas sobria in 36h, but after 48h, creates remarkable restraining effect to two kinds of pathogenic bacterias.
Embodiment 6
25ppm pseudomonas IAS03 bacterial strain fermentation liquor is to the inhibition of Aeromonas hydrophila or Aeromonas sobria
Experiment material and method, with embodiment 3, add pseudomonas IAS03 bacterial strain fermentation liquor (1 × 10
9cfu/mL) 200 μ L, make final concentration be 25ppm.Concrete inhibition is shown in Fig. 8 A and Fig. 8 B.Fig. 8 result shows: 25ppm pseudomonas IAS03 bacterial strain fermentation liquor just gives birth to remarkable restraining effect to Aeromonas sobria and Aeromonas hydrophila at 12h, the quantity of ensuing 24h two kinds of pathogenic bacterias has bounce-back again, but after 48h, significantly suppress the quantity of two kinds of cause of diseases.
Embodiment 7
30ppm pseudomonas IAS03 bacterial strain fermentation liquor is to Aeromonas hydrophila and inhibition that is warm or Aeromonas
Experiment material and method, with embodiment 3, add pseudomonas IAS03 bacterial strain fermentation liquor (1 × 10
9cfu/mL) 240 μ L, make final concentration be 30ppm.Concrete inhibition is shown in Fig. 9 A and Fig. 9 B.Fig. 9 result shows: 30ppm pseudomonas IAS03 bacterial strain fermentation liquor just gives birth to remarkable restraining effect to Aeromonas sobria and Aeromonas hydrophila at 12h, although Aeromonas sobria quantity has bounce-back in ensuing 24h, but decline to a great extent in 48h latter two pathogenic bacteria quantity, pseudomonas IAS03 bacterial strain fermentation liquor restraining effect is remarkable.As can be seen here along with use fermented liquid dosage strengthens, suppress the effect of two kinds of pathogenic bacterias to strengthen, effective time shortens.
Above-described embodiment is the present invention's preferably embodiment; but embodiments of the present invention are not restricted to the described embodiments; change, the modification done under other any does not deviate from spirit of the present invention and principle, substitute, combine, simplify; all should be the substitute mode of equivalence, be included within protection scope of the present invention.
Claims (3)
1. a pseudomonas, it is characterized in that: name is called pseudomonas (Pseudomonasprotegens) IAS03, the China typical culture collection center of Wuhan University of Wuhan, China city is preserved in, deposit number: CCTCCNO:M2015711 on November 27th, 2015.
2. the application of pseudomonas according to claim 1, is characterized in that: the application of described pseudomonas (Pseudomonasprotegens) IAS03 in prevention Aeromonas hydrophila and/or Aeromonas sobria infect.
3. the application of pseudomonas according to claim 1, is characterized in that: described pseudomonas (Pseudomonasprotegens) IAS03 is preparing the application in the product suppressing Aeromonas hydrophila and/or Aeromonas sobria.
Priority Applications (1)
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105861382A (en) * | 2016-05-20 | 2016-08-17 | 南京林业大学 | Bacterium pseudomonas NLX-4 capable of efficiently corroding silicate rocks and application of bacterium pseudomonas NLX-4 |
| CN106138108A (en) * | 2016-06-16 | 2016-11-23 | 暨南大学 | The application of pseudomonas IAS03 |
| CN111100824A (en) * | 2020-01-21 | 2020-05-05 | 暨南大学 | Bacillus and application thereof in denitrification and desulfurization in aquaculture water |
| CN112410247A (en) * | 2020-11-08 | 2021-02-26 | 领先生物农业股份有限公司 | Pseudomonas defensins and application thereof in down feather detergent |
| CN114615884A (en) * | 2019-08-22 | 2022-06-10 | 内盖夫国家生物技术研究所有限公司 | Composition for inoculating aquatic animals |
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 |
|---|
| WANG FF等: "In Vitro Inhibition of Saprolegnia sp by an Antifungal Peptide from Pseudomonas protegens XL03", 《NORTH AMERICAN JOURNOL OF AQUACULTURE》 * |
| 徐丽娟等: "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", 《中国水产科学》 * |
| 蒋启欢: "嗜水气单胞菌的分离鉴定及控制方法研究", 《中国优秀硕士学位论文全文数据库》 * |
| 马朝彬: "鱼类暴发性流行病的预防与治疗", 《现代农业科技》 * |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105861382A (en) * | 2016-05-20 | 2016-08-17 | 南京林业大学 | Bacterium pseudomonas NLX-4 capable of efficiently corroding silicate rocks and application of bacterium pseudomonas NLX-4 |
| CN105861382B (en) * | 2016-05-20 | 2019-07-12 | 南京林业大学 | A kind of efficient corrosion bacterium pseudomonad NLX-4 of silicate rock and its application |
| CN106138108A (en) * | 2016-06-16 | 2016-11-23 | 暨南大学 | The application of pseudomonas IAS03 |
| CN106138108B (en) * | 2016-06-16 | 2019-11-19 | 暨南大学 | The application of pseudomonad IAS03 |
| CN114615884A (en) * | 2019-08-22 | 2022-06-10 | 内盖夫国家生物技术研究所有限公司 | Composition for inoculating aquatic animals |
| 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 |
| CN111100824A (en) * | 2020-01-21 | 2020-05-05 | 暨南大学 | Bacillus and application thereof in denitrification and desulfurization in aquaculture water |
| CN111100824B (en) * | 2020-01-21 | 2021-11-05 | 暨南大学 | Bacillus and application thereof in denitrification and desulfurization in aquaculture water |
| CN112410247A (en) * | 2020-11-08 | 2021-02-26 | 领先生物农业股份有限公司 | Pseudomonas defensins and application thereof in down feather detergent |
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