Application of Pseudomonas ias03

A technology for Pseudomonas and mold, applied in the field of microorganisms, can solve problems such as residual risk, malachite green is not easy to degrade, and mutagenic

Active Publication Date: 2019-11-19
JINAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Malachite green was once widely used in the prevention and treatment of saprolegniasis in aquaculture, but malachite green is not easy to degrade, has high residues in aquatic animals, and has teratogenic, carcinogenic and mutagenic risks to mammals and humans. Therefore, since the 1990s, it has been banned by various countries in fishery production.
Although chemical drugs such as formaldehyde, potassium permanganate, chlorine dioxide and hydrogen peroxide also have certain control effects on saprolegniasis, the use of these chemical drugs also has problems such as residual risks and environmental pollution.

Method used

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  • Application of Pseudomonas ias03
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  • Application of Pseudomonas ias03

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1 Isolation of Pseudomonas strain IAS03.

[0026] Test material: pond bottom mud.

[0027] Preparation of medium:

[0028] Beef extract peptone solid medium: peptone 10.0g, beef extract 3.0g, sodium chloride 5.0g, agar powder 15.0-20.0g, distilled water 1000mL, pH 7.2, sterilized at 121°C for 20min (beef extract peptone liquid medium is not Add agar powder).

[0029] KB solid medium: peptone 20.0g, glycerol 10.0mL, K 2 HPO 4 1.5g, MgSO 4 .7H 2 O1.5g, agar powder 15.0g-20.0g, deionized water 1000mL, pH 7.2, high temperature sterilization at 121°C for 20min.

[0030] experiment method:

[0031] (1) Weigh 10g of pond sediment into an Erlenmeyer flask, add 90mL of sterilized water, culture at 28°C, 160r / min constant temperature shaking for 30min, let stand for 5min, take the supernatant, and use the gradient dilution method to dilute the supernatant to 10 3 times, take 0.15mL of the diluted solution and spread it evenly on the beef extract peptone solid me...

Embodiment 2

[0035] The 16S rDNA species identification of embodiment 2 Pseudomonas bacterial strain IAS03

[0036] Test materials: bacterial universal primers (27F: 5'-AGAGTTTGATCCTGGCTCAG-3'; 1492R: 5'-TACGGTTACCTTGTTACGACTT-3'), synthesized by Guangzhou Huada Gene Company.

[0037] experiment method:

[0038] (1) Amplification of 16S rDNA. The preparation of the seed solution of Pseudomonas IAS03 strain is the same as that in Example 1. The seed liquid of the strain was taken for colony PCR, and the primers were universal primers (concentration 10 μM): 27F and 1492R; the reaction system was 25 μL, including ddH 2 O 15.00 μL, KOD-Plus buffer 2.5 μL, dNTP mix (200 mmol / L) 2.5 μL, primer 27F 1.0 μL, primer 1492R 1.0 μL, bacterial solution 1.0 μL, KOD-Plus enzyme 0.5 μL, MgSO 4 (25mmol / L) 1.5μL. The reaction process is as follows: pre-denaturation: 95°C, 5min; denaturation: 94°C, 1min; annealing: 54°C, 1min; extension: 72°C, 90s; re-extension: 72°C, 10min. The PCR product was subjected...

Embodiment 3

[0042] The toxicity experiment of embodiment 3 bacterial strains

[0043] Experimental materials: 105 grass carp (body weight 30.0±5.5g, body length 12±2.5cm), 0.9% sterile saline.

[0044] Preparation of medium: the preparation of beef extract peptone medium is the same as in Example 1.

[0045] Preparation of injection bacterial solution:

[0046] Take the Pseudomonas IAS03 strain, and use an inoculation loop to streak and activate it on the beef extract peptone solid medium. After cultivating at a constant temperature at 28°C for 16 hours, use an inoculation loop to pick a single colony and inoculate it in the beef extract peptone liquid medium, at 28°C , 160r / min constant temperature shaking culture for 12h, then centrifuged at 4°C at 7500r / min for 15min, washed three times with 0.9% sterile normal saline, resuspended with 0.9% sterile normal saline, and finally passed the ultraviolet spectrum Photometer to measure OD 600 value, the concentration of the bacterial soluti...

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Abstract

The invention discloses application of Pseudomonas protegens IAS03 and belongs to the field of microorganism. The sterile cell disruption supernatant of the Pseudomonas protegens IAS03 can remarkably inhibit the growth of water mold hyphae on a flat plate, the minimum effective hyphae inhibition concentration of the supernatant is 6.25 mg/mL, and the minimum effective spore germination inhibition concentration of the supernatant is 3.125 mg/mL. It is found through a strain toxicity experiment that the Pseudomonas protegens IAS03 is safe to grass carps. The Pseudomonas protegens IAS03 is screened from pond bottom mud, does not cause harm to the surrounding environment and ecological balance and conforms to biological safety regulations. The Pseudomonas protegens IAS03 can be applied to the prevention and control of water mold only by needing a single strain and has the advantages of safety, high efficiency, environmental protection compared with traditional measures for prevention and control of antibiotics and disinfectants.

Description

technical field [0001] The invention belongs to the field of microorganisms, and in particular relates to an application of Pseudomonas protegens IAS03. Background technique [0002] Saprolegniasis is a common fungal parasitic disease in brackish water aquaculture production. It has the characteristics of wide epidemic range, long time span, no host selectivity, and difficult to cure. Therefore, the outbreak of Saprolegniasis often leads to a large number of fish deaths. , thus causing huge economic losses to the aquaculture industry. Saprolegnia spp. fungi are important pathogens that cause saprolegnia. Therefore, only by killing the saprolegnia in the water from the source can the prevention and control be achieved. [0003] Malachite green was once widely used in the prevention and treatment of saprolegniasis in aquaculture, but malachite green is not easy to degrade, has high residues in aquatic animals, and has teratogenic, carcinogenic and mutagenic risks to mammals a...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20A61K35/74A61P31/10
CPCA61K35/74
Inventor 张其中方伟王飞飞
Owner JINAN UNIVERSITY
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