CN107964517B - Bacillus capable of antagonizing various aquaculture pathogenic bacteria and application thereof - Google Patents
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Abstract
The invention discloses a bacterial strain capable of antagonizing various aquaculture pathogenic bacteria and application thereof. The antagonistic bacteria of the invention is Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) InAD-160, and the preservation number of the microorganism is CGMCC No. 14030. The bacillus amyloliquefaciens InAD-160 is obtained by separating from deep seawater in Indian ocean, can inhibit the growth of various aquaculture pathogenic bacteria such as Edwardsiella tarda (Edwards siella tarda), Aeromonas hydrophila (Aeromonas hydrophylla), Shewanella putrefaciens (Shewanella putrefacesiens), Vibrio vulnificus (Vibrio vulnificus) and the like, and can be used for preventing and treating aquaculture diseases caused by bacteria such as Edwardsiella tarda, Aeromonas hydrophila, Shewanella putrefaciens, Vibrio vulnificus and the like. The bacillus amyloliquefaciens InAD-160 obtained from deep sea is a biological control potential strain with high control efficiency and wide control range, and has good application and development prospects.
Description
Technical Field
The invention belongs to the technical field of beneficial bacterium screening, and particularly relates to bacillus capable of antagonizing various aquaculture pathogenic bacteria and application thereof.
Background
China is a world aquaculture big country and is the only country in the world with the aquaculture yield exceeding the fishing yield. However, aquaculture in coastal areas and inland areas of China is affected by aquaculture diseases to different degrees every year, and huge losses and damages are caused to the breeding industry. Bacterial diseases are relatively more and popular among aquaculture diseases. For example, Edwardsiella tarda of Edwardsiella of Enterobacteriaceae, which is a gram-negative enteric pathogenic bacterium, has caused diseases in more than twenty kinds of fishes, such as eel, paralichthys olivaceus, tilapia, Chinese soft-shelled turtle, carp and the like, and causes great loss to aquaculture; the Edwardsiella tarda is also a pathogenic bacterium which is commonly suffered by people and fishes and directly poses a threat to human health. For example, aeromonas hydrophila of aeromonas of vibriaceae exists widely in fresh water, sewage and soil, can cause infectious diseases of various aquatic animals to cause hemorrhage of aquatic animals, and is a main pathogen of fulminant infectious diseases of freshwater fish in China; in addition, the main target of aeromonas hydrophila infection is aquatic animal food, which can cause not only animal infection but also acute gastroenteritis, septicemia and the like of human, so that the aeromonas hydrophila infection becomes an important pathogenic bacterium which is commonly concerned by aquaculture and food public health.
At present, the prevention and treatment means aiming at bacterial aquatic diseases in China still mainly depend on chemical drugs such as antibiotics, and the like, and the measures such as antibiotic treatment and the like can achieve the aims of partially controlling the disease occurrence and promoting the growth of cultured animals, but the problems of drug resistance of pathogenic microorganisms, drug residue, water environment pollution, aquatic product quality safety and the like caused by the measures are more and more serious; meanwhile, due to the long-term use of the medicines, the normal microbial barriers owned by the skin, the intestinal tracts and the like of the cultured animals are damaged while pathogenic organisms are killed, so that endogenous pathogenic bacteria or exogenous pathogenic bacteria originally inhibited by the normal flora barriers are greatly propagated, the micro-ecological balance of the normal microbial flora of the epidermis and the intestinal tracts of the cultured animals is destroyed, and the disease occurrence condition of the cultured animals is frosted on snow. Antagonistic microorganisms in the natural environment are utilized to inhibit pathogenic bacteria, so that the pathogenic attack of cultured animals is prevented and controlled, and the disease occurrence is controlled, namely the biological prevention and control is always a research hotspot. The biological preparation or the living bacteria preparation containing the living bacteria and used for animals is prepared by separating and identifying beneficial microorganisms from the nature or artificially building the beneficial microorganisms through bioengineering and developing special processes such as culturing, fermenting, drying, processing and the like, and has important theoretical significance and practical application value.
Disclosure of Invention
The invention aims to provide a marine bacillus amyloliquefaciens strain capable of antagonizing various aquatic pathogenic bacteria; through extensive screening, a bacillus amyloliquefaciens is obtained from an Indian ocean seawater sample, and has obvious inhibition effect on the growth of various aquaculture pathogenic bacteria such as Edwardsiella tarda (Edwardsiella tarda), Aeromonas hydrophila (Aeromonas hydrophylla), Vibrio vulnificus (Vibrio vulnificus) and Shewanella putrefaciens (Shewanella putrefensis).
The Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) InAD-160 strain provided by the invention is a gram-positive bacterium separated from deep seawater samples of Indian ocean. The strain is preserved in China general microbiological culture Collection center (CGMCC), the preservation address is No. 3 of West Lu No.1 of North Chen of the south-facing-Yang district in Beijing, the microorganism research institute of the Chinese academy of sciences, the preservation number is CGMCC No.14030, and the preservation date is No.14 of 04.2017.
The application of the bacillus amyloliquefaciens InAD-160 in preparing biological products for antagonizing marine culture pathogenic bacteria;
in a further aspect, the present invention provides a biological product for antagonizing marine culture pathogens;
the marine culture pathogenic bacteria are Edwardsiella tarda, Aeromonas hydrophila, Vibrio vulnificus or Shewanella putrefaciens;
the biological product is zymogen liquid or bacteria powder.
The bacillus amyloliquefaciens InAD-160 obtained by screening is from deep sea, is suitable for growing at proper temperature and salt and has wide suitable PH range, and the strain liquid, the strain fermentation culture solution and the filtrate of the fermentation culture solution have obvious inhibiting effect on various aquaculture pathogenic bacteria in fresh water and seawater culture environments, thereby having good prospect in application.
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FIG. 1: and (3) the inhibition map of the bacillus amyloliquefaciens InAD-160 on the growth of Edwardsiella tarda.
Detailed Description
In order to better understand the present invention, the following examples are further illustrated.
Example 1 isolation, identification and preservation of Bacillus amyloliquefaciens InAD-160
Step 1, strain purification culture: the original sample collection site of the strain InAD-160 obtained by the invention is bottom seawater at a certain station (E88 degrees; S4 degrees) of the Indian ocean. After the sample is collected, 1 ml of seawater sample is taken and added with sterile glycerol to the final concentration of 20 percent, the seawater sample is immediately placed into a refrigerator at minus 80 ℃ for storage, taken out after being taken back to a laboratory, the sample is taken out, thawed and aseptically coated on MA 2216E culture medium (Difco, the product number: 212185), the coated culture dish is placed upside down at 25 ℃ for culturing for 24-96 hours, the grown single colony is picked up, the streak purification is carried out on the MA 2216E culture medium flat plate, the streak-marked culture dish is placed upside down at 25 ℃ for culturing for 24-96 hours, the grown single colony is picked up, the streak purification is carried out on the MA 2216E culture medium flat plate again, the streak-marked culture dish is placed upside down at 25 ℃ for culturing for 24-96 hours, and the single colony grows out and is purified once. Single colonies which are continuously purified for 3 times are picked into 2ml of MB 2216 culture medium (Difco, product number: 279110) containing 20 percent of glycerin, and the medium is put into a refrigerator at the temperature of minus 80 ℃ for storage and standby, and meanwhile InAD-160 plates which are continuously purified for 3 times are reserved for standby.
Step 2. amplification and sequence analysis of 16S rRNA Gene of Strain
Preparation of 16S rRNA Gene template: picking single colony from InAD-160 storage spare plate to MA
And (3) scribing and purifying on a 2216E culture medium plate, inverting the scribed culture dish at 25 ℃ for culturing for 24-96 hours, picking 2-3 single colonies with sterilized toothpicks in 20 mu L of RNA free water to be mixed uniformly when the single colonies grow out, and obtaining the bacteria liquid with slightly turbid concentration. Placing in a PCR instrument at 99 deg.C for 15 min. Taking the supernatant as a PCR amplification template.
And (3) PCR amplification: primers used for PCR amplification were 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACT T-3'). PCR reaction 60. mu.L: 2 × Taq Mix 30 μ L, primers: 27F, 1492R each 3. mu.L, 3. mu.L of template, and sterile water to 60. mu.L. And (3) PCR reaction conditions: 95 ℃ for 5 min; 94 ℃ for 45 s; 57 ℃, 1min30 s; 30 cycles at 72 ℃ for 1min for 30 s; 72 ℃ for 10 min. Sequencing the PCR product, and comparing the 16S rRNA gene sequence to identify InAD-160 to be closest to Bacillus amyloliquefaciens (Bacillus amyloliquefaciens).
The most similar species of the Bacillus amyloliquefaciens InAD-160 is Bacillus amyloliquefaciens of the Bacillus genus through 16S rDNA gene sequence comparison analysis, the homology is 99.8 percent, and therefore the Bacillus amyloliquefaciens is named as the Bacillus amyloliquefaciens InAD-160.
The strain is preserved in China general microbiological culture Collection center (CGMCC), the preservation address is No. 3 of West Lu No.1 of North Chen of the south-facing-Yang district in Beijing, the microorganism research institute of the Chinese academy of sciences, the preservation number is CGMCC No.14030, and the preservation date is No.14 of 04.2017.
In the laboratory, the strain was stored for a long period in such a manner that the strain was added to MB 2216 medium (Difco, cat # 279110) containing 20% glycerol and stored at-80 ℃; the short-term storage was carried out by inoculating the strain on a MA 2216E medium (Difco, cat # 212185) slant and storing at 4 ℃ until use.
The strain related to the invention has the following characteristics:
1) morphological characteristics: after culturing for 24h at 28 ℃ on MA 2216E medium, the diameter of the colony is 1-2mm, and the colony is milky white, round, bulged and irregular in edge.
2) Physiological and biochemical characteristics: the bacteria can grow with salinity range of 0% -35% (w/v) (optimally 1% -5%), growth pH range of 3.5-8.5 (optimally 7.0-8.0), and growth temperature range of 10-55 deg.C (optimally 25-30 deg.C). The bacterium has negative oxidase reaction and positive catalase reaction, has the activities of protease, lysozyme and urease, and can utilize citrate to reduce nitrate. In the study of bacterial sugar metabolism using API 50CH, the strain produced acid in all of the following substances, and appeared positive: mannitol, L-arabinose, D-ribose, D-glucose, D-fructose, D-mannose, inositol, mannitol, sorbitol, methyl-alpha D-mannopyranoside, amygdalin, arbutin, esculin, salicin, D-cellobiose, D-maltose, D-lactose, D-melibiose, D-sucrose, D-trehalose, D-raffinose, starch, glycogen, D-gentiobiose; acid production was not possible in any of the following substances, and was negative: erythritol, D-arabinose, D-xylose, L-xylose, D-adonitol, methyl-beta-xylopyranoside, D-galactose, L-sorbose, L-rhamnose, dulcitol, methyl-alpha D-glucopyranoside, N-acetylglucosamine, inulin, D-melezitose, xylitol, D-tulose, D-lyxose, D-tagatose, D-fucose, L-fucose, D-arabitol, L-arabitol, potassium gluconate, potassium 2-ketogluconate and potassium 5-ketogluconate.
4) The bacteriostatic characteristic is as follows: the antibacterial property of the bacillus amyloliquefaciens InAD-160 is measured by a perforation method (the aperture is 10mm), the result shows that the bacillus amyloliquefaciens has obvious inhibition effect on the growth of a plurality of aquaculture pathogenic bacteria of Edwardsiella tarda, Aeromonas hydrophila, Vibrio vulnificus and Shewanella putrefaciens, and the inhibition effect is shown in the following table 1:
TABLE 1 determination of bacteriostatic properties of Bacillus amyloliquefaciens InAD-160 on aquaculture pathogenic bacteria by perforation method
Example 2 application of Bacillus amyloliquefaciens InAD-160
Step 1, preparing a bacillus amyloliquefaciens InAD-160 bacterial liquid: streaking strain preserved at-80 deg.C in 2216E seawater solid culture medium, culturing at 25-30 deg.C, selecting single colony, inoculating in fresh 2216E seawater liquid culture medium, fermenting at 25-30 deg.C and 120 rpm for 48 hr to obtain fermentation broth with strain density of 1 × 109/ml。
Step 2, bait preparation: the basic bait used in the experiment was a three-way bioengineering (Weifang) Limited company larval and juvenile bait. The experimental group bait is prepared according to the standard that every 10g of basic bait is mixed with 4ml of fermentation liquor, and the control group bait is mixed with the culture seawater.
Step 3, culturing the turbot: the average body length is 8.37 +/-1.13 cm, the average weight is 9.2 +/-1.99 g, the healthy turbot 180 tails are temporarily cultured in a circulating water culture system for 1 week and then randomly divided into an experimental group and a control group, each treatment group is divided into 3 parallel groups, and 30 tails of fish are placed in each parallel group. The experimental group and the control group are respectively fed with baits added with InAD-160 bacterial liquid and basic baits, the baits are fed for 1 time every day, the feeding amount of each initial group is 10g, and the feeding amount is adjusted according to the ingestion condition of the fed fish in the later period. The experiment period was 2 water changes per week with 2/3 water changes. The culture water temperature is 16-19 ℃, and the air is continuously inflated.
Step 4, measuring the growth index of the turbot: and (3) stopping feeding for 24 hours after 5 weeks of culture test in the step 3 before changing water, and weighing the quality of each group of cultured fish. And (3) counting the weight and the weight gain rate of each group of fishes: the weight gain rate was (final mass-initial mass/initial mass) × 100%, and the experimental group was (80.7 ± 11.2)%, and the control group was (28.3 ± 9.6)%. Therefore, the Bacillus amyloliquefaciens InAD-160 can improve the weight gain rate of the turbot.
Step 5, culturing disease resistance of turbot, namely suspending Edwardsiella tarda (Edwardsiella tarda) cultured for 48 hours on 2216E seawater solid culture medium by using sterile seawater to reach the concentration of 5.8 multiplied by 105CFU/mL is the counteracting toxic bacteria liquid, and each group of experimental fish subjected to the culture test in the step 3 for 6 weeks is injected with 100 mul of the counteracting toxic bacteria liquid per belly muscle. After 3 days, the cumulative mortality of each group of fish was counted: the experimental group is (50.3 + -2.5)%, and the control group is 100%. Therefore, the Bacillus amyloliquefaciens InAD-160 can improve the capability of the turbot in resisting the Edwardsiella tarda infection.
Claims (5)
1. A Bacillus amyloliquefaciens characterized in thatBacillus amyloliquefaciens) The preservation number of (2) is CGMCC number 14030.
2. Use of the bacillus amyloliquefaciens of claim 1 for preparing a biological product that antagonizes a marine culture pathogen that is edwardsiella tarda, aeromonas hydrophila, vibrio vulnificus, or shewanella putrefaciens.
3. The use of claim 2, wherein the biological product is a fermented liquid or a powder.
4. A biological product prepared using the Bacillus amyloliquefaciens strain of claim 1.
5. The biological product according to claim 4, wherein the biological product is a fermented liquid or a fermented powder.
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| 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 |
| 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 |
| 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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| CN104974950A (en) * | 2015-05-11 | 2015-10-14 | 山东大学（威海） | Marine Bacillus amyloliquefaciens and uses thereof |
| CN105238722A (en) * | 2015-11-03 | 2016-01-13 | 江苏省苏微微生物研究有限公司 | Bacillus amyloliquefaciens strain as well as preparation method and application of strain powder preparation of bacillus amyloliquefaciens strain |
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| CN104974950A (en) * | 2015-05-11 | 2015-10-14 | 山东大学（威海） | Marine Bacillus amyloliquefaciens and uses thereof |
| CN105238722A (en) * | 2015-11-03 | 2016-01-13 | 江苏省苏微微生物研究有限公司 | Bacillus amyloliquefaciens strain as well as preparation method and application of strain powder preparation of bacillus amyloliquefaciens strain |
| CN106701645A (en) * | 2017-03-26 | 2017-05-24 | 天津市水生动物疫病预防控制中心 | Bacillus amyloliquefaciens B7 with immune growth promoting effect and application method of bacillus amyloliquefaciens B7 |
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| Effects of potential probiotic Bacillus amyloliquifaciens FPTB16 on systemic and cutaneous mucosal immune responses and disease resistance of catla (Catla catla);Anushree Das等;《Fish & Shellfish Immunology》;20130904;第35卷(第5期);第1547-1553页 * |
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