CN107964517A - It is a kind of can a variety of pathogenic bacterias of antagonism bacillus and its application - Google Patents
It is a kind of can a variety of pathogenic bacterias of antagonism bacillus and its application Download PDFInfo
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- CN107964517A CN107964517A CN201711390818.9A CN201711390818A CN107964517A CN 107964517 A CN107964517 A CN 107964517A CN 201711390818 A CN201711390818 A CN 201711390818A CN 107964517 A CN107964517 A CN 107964517A
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Abstract
The invention discloses one plant can a variety of aquaculture pathogenetic bacterias of antagonism bacterium bacterial strain and its application.The Antagonistic Fungi of the present invention is bacillus amyloliquefaciens (Bacillus amyloliquefaciens) InAD 160, and microbial preservation number is CGMCC No.14030.The bacillus amyloliquefaciens InAD 160 of the present invention is separated from the deep sea water of the Indian Ocean to be obtained, it can suppress the growth of a variety of pathogenic bacterias such as Edwardsiella tarda (Edwardsiella tarda), Aeromonas hydrophila (Aeromonas hydrophila), Shewanella putrefaciens (Shewanella putrefaciens), Vibrio vulnificus (Vibrio vulnificus), available for prevention by the bacterial aquiculture disease such as Edwardsiella tarda, Aeromonas hydrophila, Shewanella putrefaciens, Vibrio vulnificus.The bacillus amyloliquefaciens InAD 160 that the present invention is obtained from deep-sea is one plant of biological control potentiality bacterial strain that prevention is efficient, prevention and treatment range is wide, has good application and development prospect.
Description
Technical field
The invention belongs to beneficial bacterium screening technique field, and in particular to it is a kind of can a variety of pathogenic bacterias of antagonism bud
Spore bacillus and its application.
Background technology
China is world aquaculture big country, is the country that only one cultured output exceedes fishery output in the world.So
It is and either coastal in China or hinterland aquaculture is all influenced be subject to the farming disease harms to some extent every year, provisions
Grow industry and cause huge loss and harm.Bacterial disease is relatively more in aquiculture disease, it is popular also relatively
Extensively.Such as the Edwardsiella tarda of enterobacteriaceae Edwardsiella, it is a kind of Gram-negative enteric pathogen, two
Disease, such as common eel, lefteye flounder, Tilapia mossambica, Shelled Turtle Trionyx Sinensis, carp etc. are caused in more than ten kinds of fish, is caused to aquaculture huge
Loss；Edwardsiella tarda is also a kind of people, fish diseased opportunistic pathogen altogether, and threat is directly caused to human health.Such as arc again
The Aeromonas hydrophila of Cordycepps Aeromonas, is widely present in fresh water, sewage and soil, can trigger a variety of aquatic animals
Infectious disease, causes the haemorrhage of aquatic livestock, is the main pathogen of China's cultured freshwater fish outburst sexually transmitted disease；In addition thermophilic water
The main object of Aeromonas infection is aquatic livestock property food, can not only cause the infection of animal, can also cause that people's is acute
Gastroenteritis, septicemia etc., thus it becomes the important pathogen that aquaculture and food public health are paid close attention to jointly.
The means of prevention of China directed toward bacteria property aquatic products disease still relies primarily on the chemicalses such as antibiotic, antibiosis at present
The measures such as extract for treating can achieve the purpose that part control disease occurs, promotes cultivated animals growth, but the cause of disease thus brought
The problems such as microorganism drug resistance, medicament residue, water environment pollution and fish quality, is increasingly severe；Simultaneously as this
The long-time service of a little medicines, also damaged while causal organism is killed cultivated animals skin, enteron aisle etc. possessed it is normal
Microbial barrier, enables those endogenous pathogens suppressed originally by normal flora barrier or exogenous pathogen a large amount of
Breeding, destroys the microecological balance of cultivated animals epidermis and enteron aisle normal microflora, causing cultivated animals disease, a situation arises
Make the matter worse.Suppress pathogen using the antagonistic microbe in natural environment, prevent the pathogen invasion of cultivated animals, control disease
Occur, i.e. biological control is always a research hotspot.Manually set up from nature separation, identification or by bioengineering beneficial
Microorganism, develop through cultivate, ferment, dry, process etc. special process be made biological agent containing viable bacteria and for animal or
Active bacteria formulation has important theory significance and actual application value.
The content of the invention
It is an object of the invention to provide one plant can a variety of aquatic pathogenic bacteriums of antagonism ocean bacillus amyloliquefaciens；Through wide
General screening, obtains a bacillus amyloliquefaciens from Indian Ocean water sample, and the bacterium is for example slow to a variety of pathogenic bacterias
Slow tarda (Edwardsiella tarda), Aeromonas hydrophila (Aeromonas hydrophila), Vibrio vulnificus
The growth of (Vibrio vulnificus), Shewanella putrefaciens (Shewanella putrefaciens) significantly inhibit work
With.
InAD-160 plants of bacillus amyloliquefaciens (Bacillus amyloliquefaciens) provided by the present invention, are
The one plant of gram-positive bacteria separated from the deep sea water sample of the Indian Ocean.The bacterium has been preserved in Chinese microorganism strain
Preservation administration committee common micro-organisms center (CGMCC), preservation address are Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3s, in
Institute of microbiology of the academy of sciences of state, preserving number are CGMCC No.14030, preservation date for 04 month 2017 No. 14.
The bacillus amyloliquefaciens InAD-160 of the present invention answering in the biological products for preparing antagonism sea-farming pathogen
With；
Another aspect of the present invention provides a kind of biological products, which is used for antagonism sea-farming pathogen；
The sea-farming pathogen, is Edwardsiella tarda, Aeromonas hydrophila, Vibrio vulnificus or corrupt Shiva
Salmonella；
The biological products, are zymocyte liquid or bacterium powder.
The bacillus amyloliquefaciens InAD-160 that present invention screening obtains derives from deep-sea, its growth thermophilic, suitable salt, suitable PH
Scope is wide, its bacterial strain bacterium solution, strain fermentation nutrient solution, fermentation culture filtered fluid to fresh water, sea water culture environment it is a variety of
Pathogenic bacteria significantly inhibits effect, has good prospect in application aspect.
Brief description of the drawings
Fig. 1：The suppression figure that bacillus amyloliquefaciens InAD-160 grows Edwardsiella tarda.
Embodiment
In order to more preferably understand the present invention, it is described further by the following examples.
1 bacillus amyloliquefaciens InAD-160 of embodiment separation identification and preservation
Step 1. bacterial strain divides pure culture：The primary sample collecting location that the present invention obtains bacterial strain InAD-160 is the Indian Ocean
(E88 ° of certain erect-position；S4 °) bottom seawater.After sample collection, 1 milliliter of seawater sample is taken to add sterile glycerol to final concentration of 20%,
- 80 DEG C of refrigerators are immediately placed in preserve, take back take out behind laboratory sample thaw it is sterile be applied to MA 2216E culture mediums (Difco,
Article No.：212185) on, with coated culture dish be inverted in 25 DEG C of culture 24-96 it is small when, single bacterium colony that picking is grown, in MA
It is point pure that line is carried out on 2216E culture medium flat plates, by the culture dish for pulling line be inverted in 25 DEG C of culture 24-96 it is small when, picking length
The single bacterium colony gone out, carrying out ruling again on MA 2216E culture medium flat plates, it is pure to divide, and the culture dish for pulling line is inverted in 25 DEG C
When culture 24-96 is small, treat that single bacterium colony is grown, dividing single time.Picking continuously divides the single bacterium of pure 3 times to fall on 2ml ends containing glycerine
Concentration is 20% MB 2216 culture medium (Difco, article No.：279110) -80 DEG C of refrigerators are put into save backup, and are retained at the same time
Continuously divide the InAD-160 plates of pure 3 times spare.
The amplification and sequence analysis of the 16S rRNA genes of step 2. bacterial strain
The preparation of 16S rRNA gene templates：Picking single bacterium on plate, which is saved backup, from InAD-160 falls on MA
It is pure that line point is carried out on 2216E culture medium flat plates, it is small that the culture dish for pulling line is inverted in 25 DEG C of culture 24-96
When, treat that single bacterium colony is grown, fallen within 20 μ L RNA free water and mixed with 2~3 single bacteriums of toothpick picking of sterilizing, bacterium solution is dense
Degree is slightly muddy.It is placed in PCR instrument, 99 DEG C, 15min.Supernatant is taken as PCR amplification template.
PCR amplification：PCR amplification the primer is 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-
GGTTACCTTGTTACGACT T-3').60 μ L of PCR reaction systems：2 × Taq Mix, 30 μ L, primer：Each 3 μ L of 27F, 1492R,
3 μ L of template, sterile water complement to 60 μ L.PCR reaction conditions：95 DEG C, 5min；94 DEG C, 45s；57 DEG C, 1min30s；72 DEG C,
1min 30s, 30 circulations；72 DEG C, 10min.PCR product is sequenced, by InAD-160 after the comparison of its 16S rRNA gene order
Identification is closest with bacillus amyloliquefaciens (Bacillus amyloliquefaciens).
Bacillus amyloliquefaciens InAD-160 compares analysis by 16S rDNA gene orders
The Bacillus amyloliquefaciens that Bacillus belongs to, homology 99.8%, therefore the bacterium are to be named as Xie Dian
Afnyloliquefaciens InAD-160.
The bacterium has been preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), preservation
Address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica, preserving number CGMCC
No.14030, preservation date for 04 month 2017 No. 14.
In the lab, which is that bacterial strain is added 2216 culture mediums of MB containing 20% glycerine
(Difco, article No.：279110) in culture medium, in -80 DEG C of preservations；Short term storage mode is to train inoculation in MA 2216E
Support base (Difco, article No.：212185) on inclined-plane, saved backup at 4 DEG C.
Bacterial strain of the present invention has following feature：
1) morphological feature：On MA 2216E culture mediums, after 28 DEG C of culture 24h, colony diameter size is 1-2mm, breast
White, it is circular, heave, edge is irregular.
2) physio-biochemical characteristics：It is 0%-35% (w/v) (most suitable 1%-5%) that the bacterium, which can grow salinity range, grows pH
Scope is 3.5-8.5 (most suitable 7.0-8.0), and growth temperature range is 10 DEG C -55 DEG C (25 DEG C -30 DEG C most suitable).The bacterium oxidizing ferment
Reaction negative, catalase reaction is positive, has protease, lysozyme, urease activity, using citrate, can reduce
Nitrate.In the bacterium glycometabolism research carried out using API 50CH, bacterial strain can produce acid in following material, show as
It is positive：Mannitol, L-arabinose, D-ribose, D-Glucose, D-Fructose, D-MANNOSE, inositol, mannitol, sorbierite, first
Base-α D- mannopyranoses glycosides, amarogentin, ursin, aesculin, salicin, D- cellobioses, D-Maltose, D- lactose,
D- melibioses, D- sucrose, D- trehaloses, D- gossyposes, starch, glycogen, D- gentiobioses；It cannot all be produced in following material
Acid, shows as feminine gender：Erythritol, D-arabinose, D- xyloses, L- xyloses, D- adonites, methyl-β-xylopyranose
Glycosides, D- galactolipins, L- sorboses, L- rhamnoses, dulcitol, methyl-α D- glucopyranosides, N-Acetyl-D-glucosamine, chrysanthemum
Powder, D- melezitoses, xylitol, D- Toulons sugar, D- lyxoses, D-Tag, D- fucoses, L-fucose, D-arabitol, L-
Arabite, K-IAO, 2 ketone group K-IAOs and 5 ketone group K-IAOs.
4) antibacterial feature：Antagonistic property survey is carried out to bacillus amyloliquefaciens InAD-160 using punch method (aperture 10mm)
It is fixed, the results showed that the bacterium is to a variety of pathogenic bacteria Edwardsiella tardas, Aeromonas hydrophila, Vibrio vulnificus, corruption
The growth of Shewanella significantly inhibits effect, its is antibacterial as shown in table 1 below：
1 punch method of table measures antagonistic properties of the bacillus amyloliquefaciens InAD-160 to pathogenic bacteria
The application of 2. bacillus amyloliquefaciens InAD-160 of embodiment
The preparation of step 1. bacillus amyloliquefaciens InAD-160 bacterium solutions：The strain of -80 DEG C of preservations is taken to consolidate in 2216E seawater
Body culture medium is rule, and in 25 DEG C of -30 DEG C of cultures, picking single bacterium colony is seeded in fresh 2216E seawater fluid nutrient mediums in 25-30
DEG C, 120-200rpm fermented and cultureds 48h obtain zymotic fluid, zymotic fluid inoculum density is 1 × 109/ml。
It is prepared by step 2. bait：Foundation bait used is threeway bioengineering (Weifang) Co., Ltd larva and juvenile in this experiment
Bait.The standard that 4ml zymotic fluids are mixed by every 10g foundation baits prepares experimental group bait, control group bait breeding seawater spice.
Step 3. turbot cultivates：A length of 8.37 ± the 1.13cm of average body, the health that average weight is 9.2 ± 1.99g are big
180 tail of brill, temporarily supports and is randomly divided into two treatment groups of experimental group and control group after 1 week in circulating water culture system, each processing
Group 3 is parallel, and each parallel group puts 30 tail of fish.Experimental group and control group are fed respectively plus the bait of InAD-160 bacterium solutions and basis
Bait, feeds bait 1 time daily, and initial every group of feeding volume is 10g, and the later stage adjusts bait throwing according to the situation of ingesting of feeding fish
Amount.During the experiment changes weekly water 2 times, quantity of exchanged water 2/3.16-19 DEG C of temperature of cultivation, continuous charge.
Step 4. turbot growth indexes measure：Before water is changed after step 3 culture experiment carries out 5 weeks, stop eating 24h, each group
Cultivation fish claims quality.Count the weight and rate of body weight gain of each group fish：Rate of body weight gain=(whole opisthosoma quality-initial weight/initial constitution
Amount) × 100%, experimental group is (80.7 ± 11.2) %, and control group is (28.3 ± 9.6) %.Therefore, bacillus amyloliquefaciens
InAD-160 can improve turbot rate of body weight gain.
The premunition of step 5. Isolated from Diseased Scophthalmus maximus:Take the slow Edward that 48h is cultivated on 2216E seawater solid mediums
Salmonella (Edwardsiella tarda) is suspended into concentration as 5.8 × 10 with antiseptic sea water5CFU/mL, as attacks toadstool liquid, takes
Each group after step 3 culture experiment carries out 6 weeks tests fish, and per the injection of tail abdominal muscles, 100 μ l are above-mentioned attacks toadstool liquid.After 3 days, system
Count the cumulative mortality of each group fish：Experimental group is (50.3 ± 2.5) %, control group 100%.Therefore, bacillus amyloliquefaciens
InAD-160 can improve the ability that turbot resists Edwardsiella tarda infection.
Claims (6)
1. a kind of bacillus amyloliquefaciens, it is characterised in that the deposit number of the bacillus amyloliquefaciens is CGMCC
No.14030。
2. the answering in the biological products for preparing antagonism sea-farming pathogen of the bacillus amyloliquefaciens described in claim 1
With.
3. application as claimed in claim 2, it is characterised in that the sea-farming pathogen is Edwardsiella tarda,
Aeromonas hydrophila, Vibrio vulnificus or Shewanella putrefaciens.
4. application as claimed in claim 2, it is characterised in that the biological products are zymocyte liquid or bacterium powder.
5. a kind of biological products, it is characterised in that the biological products are the solution starch gemma bars described in usage right requirement 1
Prepared by bacterium.
6. biological products as claimed in claim 5, it is characterised in that the biological products are zymocyte liquid or bacterium powder.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109536418A (en) * | 2018-12-28 | 2019-03-29 | 佛山市艳晖生物科技有限公司 | A kind of microbial bacterial agent and its application in aquaculture |
| CN111040975A (en) * | 2020-01-03 | 2020-04-21 | 广东省微生物研究所（广东省微生物分析检测中心） | Enzyme-producing bacillus halophilus and application thereof in preventing and controlling pathogenic bacteria in saline water aquaculture |
| CN111944715A (en) * | 2020-07-28 | 2020-11-17 | 华东师范大学 | Shewanella putrefaciens and application thereof, aquatic feed and aquaculture method |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109536418A (en) * | 2018-12-28 | 2019-03-29 | 佛山市艳晖生物科技有限公司 | A kind of microbial bacterial agent and its application in aquaculture |
| CN111040975A (en) * | 2020-01-03 | 2020-04-21 | 广东省微生物研究所（广东省微生物分析检测中心） | Enzyme-producing bacillus halophilus and application thereof in preventing and controlling pathogenic bacteria in saline water aquaculture |
| CN111040975B (en) * | 2020-01-03 | 2021-10-15 | 广东省微生物研究所（广东省微生物分析检测中心） | Enzyme-producing bacillus halophilus and application thereof in preventing and controlling pathogenic bacteria in saline water aquaculture |
| CN111944715A (en) * | 2020-07-28 | 2020-11-17 | 华东师范大学 | Shewanella putrefaciens and application thereof, aquatic feed and aquaculture method |
| CN111944715B (en) * | 2020-07-28 | 2022-12-23 | 华东师范大学 | Shewanella putrefaciens and application thereof, aquatic feed and aquaculture method |
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