CN102037917A - Application of bdellovibrio sp. leech plasmid bacterium solution in cultivating penaeus monodon - Google Patents
Application of bdellovibrio sp. leech plasmid bacterium solution in cultivating penaeus monodon Download PDFInfo
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Abstract
The invention in particular relates to an application of a bdellovibrio sp. leech plasmid in cultivating marine products such as penaeus monodon, and the like. The invention is realized by adding a bdellovibrio sp. leech plasmid to the water body and the feeds. The invention is realized specifically based on cultivation pond conditions, preparation before stocking, shrimp postlarvae selecting and stocking, feeding management and water environment management in the cultivation period. The invention is characterized by adding a bdellovibrio sp. leech plasmid bacterium solution to the water body and ensuring the concentration of the bdellovibrio sp. leech plasmid in the water body to be 101-107pfu/mL, simultaneously soaking the feeds in the bdellovibrio sp. leech plasmid bacterium solution with the concentration of 101-107pfu/mL for 30 minutes and changing water once every 30 days in the cultivation period. The invention provides a method for cultivating organisms. The method has the advantages of environment friendliness, safety, no pollution, obvious effect and capabilities of improving the water quality, promoting growth of penaeus monodon, improving the immunity and improving the survival rate, thus having wonderful development prospect. The invention has very important significance in improving the economic benefits and eating safety and guaranteeing human health.
Description
Technical field
The invention belongs to biological technical field, relate to the application of a kind of bacterium, the particularly application of Bdellovibrio leech plastid bacterium liquid in marine products such as Penaeus monodon are cultured.
Background technology
Penaeus monodon (Penaeus monodon) is commonly called as terrible shrimp, grass shrimp, flower shrimp, ring shrimp, spot joint shrimp, ox shape prawn, and big Tiger Prawns is generally called in FAO (Food and Agriculture Organization of the United Nation).Be a kind of of individual maximum in the prawn, the ripe individual general long 22.5cm～32cm of body, body weight 137g～211g is one of general kind of shrimp culture in the world today.
Present without exhaust waste breeding way, cause debirs such as the excreta of prawn, remaining feed, planktonic residual body all in the pond, to accumulate, arrived the middle and later periods of culturing, substrate pollution appears inevitably, the water body quality descends, conditioned pathogen and pathogenic bacteria are increased, the deterioration of the ecological environment, thereby the generation of initiation prawn disease.Therefore, seeking a kind of new method in breeding production, both can reduce and change water, can improve its survival rate and growth rate again, is that numerous culturists' institute expects.
Chinese herbal medicine has drug residue free, advantage that economic benefit is high to the Penaeus monodon growth performance, but the slow shortcoming that takes effect is arranged.Antibiotic is to suppressing the breeding of pathogenic microorganism, and the aspects such as toxic action that reduce its metabolism toxin prawn have played positive effect, but the long-term antibiotic, particularly abuse of antibiotics of using, and its all drawback also reveals gradually.As destroying balance of nature in the shrimp pool, the immunity of organisms of prawn produces harmful effect, produces side effects such as drug resistance and medicament residue, gives aquaculture and has all brought potential harm as consumer's human beings'health.Therefore, the Penaeus monodon of developing the pollution-free healthy of a kind of simple and easy to do, safety, high-quality, nutrition is cultured the common recognition that new technology has become current society.
Bdellovibrio is to parasitize other bacteriums, and can cause a bacterioid of host bacteria cracking; Littler than general bacterium, can pass through bacterial filter, the effect of similar phage is arranged; Gram is negative, and can cracking comprise that vibrio parahaemolytious, vibrio alginolyticus etc. cause the potentially pathogenic organism of shrimp disease.
Can be divided into the leech plastid state that breaks away from host bacteria, has the telotroch state of flagellum, free swimming and in host bacteria, grow the history of life of Bdellovibrio.When the Bdellovibrio telotroch is invaded the host, on the host cell wall, produce a hole, invade host's periplasmic space when Bdellovibrio after hole seal again.Telotroch loses flagellum and host cell is called as " leech plastid " together.Compare with telotroch, the leech plastid not only has preparation, preservation is more convenient, and the environment tolerance is strong, though omit characteristics more of a specified duration slowly action time, also has potential inactivated vaccine function simultaneously.Behind Bdellovibrio telotroch invasion host cell, the respiration of host cell (respiration) is stopped very soon.Bdellovibrio also enters leech plastid state, begins to secrete plurality of enzymes, and the big molecule of clearing up host cell is a small-molecule substance, and then is integrated into the nutriment of self, makes self growth, and is elongated.Finally, the leech plastid of strip is divided into the sections of a lot of weak points, grows flagellum, becomes telotroch again.Afterwards, the essential enzyme of they secretions, broken wall and going out becomes the telotroch that breaks away from host bacteria, has flagellum, free swimming once more.Between this leech plastid puberty, Bdellovibrio does not change the surface texture of host cell, makes former host still have immunogenicity, but the stimulating organism body produces immune response.This function makes uses Bdellovibrio leech plastid in Penaeus monodon is cultured, and can improve the prawn resistance against diseases, effectively prevents vibriosis penaeus.
These characteristics make Bdellovibrio leech plastid be suitable as a kind of microorganism formulation, in the breeding process that is applied to prawn.The author has proved that Bdellovibrio leech plastid can improve survival rate, the immunity of shrimp significantly and promote the shrimp growth in becoming the shrimp aquaculture process by experiment.
Summary of the invention
The objective of the invention is to overcome the drawback of prior art, the application method of Bdellovibrio leech plastid bacterium liquid in Penaeus monodon is cultured is provided.Its feature is learned principle according to breeding ecological, halobiontic growing environment requirement, correlation between the biology and nuisanceless breed standard, the artificial breeding environment of creating reduces input cost, improves Penaeus monodon immunity, promote Penaeus monodon to grow fast, increase economic efficiency.
Purpose of the present invention realizes by following proposal:
The application of Bdellovibrio leech plastid bacterium liquid in Penaeus monodon is cultured.
Described Bdellovibrio Bdellovibrio was (Bdellovibrio sp.) BDFM05, was preserved in Chinese typical culture collection center on August 7th, 2009, and the preservation address is Chinese Wuhan Wuhan University, and deposit number is CCTCC NO:M 209172.
The application of described Bdellovibrio leech plastid bacterium liquid in Penaeus monodon is cultured is that feed is soaked 15-45min in Bdellovibrio leech plastid bacterium liquid.
The application of described Bdellovibrio leech plastid bacterium liquid in Penaeus monodon is cultured is to add Bdellovibrio leech plastid bacterium liquid in water body, makes that Bdellovibrio leech plastid concentration is 10 in the water body
1～10
7Pfu/mL.
The preparation method of described Bdellovibrio leech plastid bacterium liquid is:
The host is in the nutrient broth liquid nutrient medium in inoculation, at 35 ℃ and 200rpm shaking table cultivation 12h, culture fluid is through 4 ℃, behind the centrifugal 10～20min of 5000～8000rpm, precipitation is added in the DNB liquid nutrient medium, insert Bdellovibrio BDFM05, under 25～35 ℃ of temperature, cultivate 36～48h with 150～300rpm shaking table, obtain containing the culture fluid of Bdellovibrio leech plastid and Bdellovibrio telotroch, culture fluid behind centrifugal 15～20min, is removed the supernatant that contains the Bdellovibrio telotroch with 6000～8000rpm under 4 ℃ of temperature, the precipitation that obtains is Bdellovibrio leech plastid; Is that 7.2～7.6 phosphate buffer suspends with Bdellovibrio leech plastid with DNB liquid nutrient medium, water, physiological saline or 0.2mol/ L pH value, and obtaining concentration is 10
11～10
12The Bdellovibrio leech plastid bacterium liquid of pfu/mL.
The present invention has following advantage and effect with respect to prior art:
1, Bdellovibrio leech plastid has stronger cracking strength to pathogenic or potentially pathogenic organism, and can improve the aquaculture organism intestinal environment, promotes growth.Characteristics such as therefore, compare with existing Penaeus monodon cultural method, it is strong that the present invention has the shrimp of making resistance against diseases, and survival rate improves, and it is fast to grow.
2, the present invention adopts the scheme that the plastid prevention of Bdellovibrio leech and adjusting, environment conditioning combine, reached the immunity that improves Penaeus monodon, promoted Penaeus monodon to ingest and growth fast, the longlyest reached 30 days and do not change water, reduce production costs the effect of increasing economic efficiency.
3, Bdellovibrio leech plastid bacterium liquid of the present invention safety in the application of Penaeus monodon is good, existingly studies show that Bdellovibrio to the shrimps free of toxic effects, to human no pathogenicity.
4, the potential inactivated vaccine function of Bdellovibrio leech plastid of the present invention can stimulate prawn to produce immune response, can improve the prawn resistance against diseases, effectively prevents vibriosis penaeus.
Therefore, the method also can be generalized to the breed of other shrimp or aquatic livestock, increases economic benefit, is that the robust techniques of new period fishery synergy increasing peasant income supports.
Embodiment
The present invention is described in further detail below in conjunction with embodiment and form, but embodiments of the present invention are not limited thereto.
The preparation of embodiment 1 Bdellovibrio leech plastid bacterium liquid
Described leech plastid obtains by the following method: (inoculum density is 10 containing vibrio parahaemolytious
7The bacterium 1mL of cfu/mL) (Vibrio parahaemolyticus, purchase in U.S. typical case species preservation center American Type Culture Collection, ATCC 17802) 1L DNB (dilute nutrient broth) liquid nutrient medium (nutrient broth 0.8g, caseinic acid hydrolysate 0.5g, yeast extract 0.1g, be dissolved in the 1000mL distilled water, the pH value is 7.6) in to insert 1mL concentration be 10
3The pfu/mL Bdellovibrio is cultivated 48h in 28 ℃, 250rpm, and culture fluid is removed supernatant behind 4 ℃ of centrifugal 15min of 6000rpm, and precipitation suspends with 100mL DNB, promptly gets the leech plastid; The concentration of Bdellovibrio BDFM05 leech plastid thalline reaches 10
9Pfu/mL.
The application experiment of embodiment 2 Bdellovibrio leech plastid bacterium liquid in the common breed of Penaeus monodon
Concrete enforcement of the present invention place is a South China plant, and the enforcement time is 3 months.
(1) cultivating pool condition, culturing the pond floor space is 10 mu, the dark 2m in pond is an argillo arenaceous at the bottom of the pond, and is smooth at the bottom of the pond, cultures the pond and is equipped with water pump, aerator, and be equipped with pumping plant gate and more complete culvert system;
(2) put preceding preparation in a suitable place to breed, experimental field the source quality in marine site meets State Standard of the People's Republic of China's water quality standard for fishery (GB11607-89) requirement, enter after the sand filtration of seawater process, the precipitation process and culture the pond, meet (NY5052-2001) requirement of Ministry of Agriculture's " pollution-free food mariculture water water quality ".Put seedling preceding 10 days, and, executed nitrogen, phosphorus, K composite fertilizer cultivation food organisms simultaneously, make water transparence reach 40cm, culturing pool salinity 10 ‰ with intaking behind the 80 purposes sleeve shape net filtration;
(3) the shrimp seedling is selected and is put in a suitable place to breed, the shrimp seedling selects individual size neat, the black shell shrimp seedling of the long 1.1-1.3cm of body, and astogeny is neat, muscle is full transparent, pigment is normal in the appendage, and stomach and intestine are full of food, and the shrimp seedling is movable active, to current irritant reaction sensitivity, no ectoparasitism thing and adhere to dirt, water temperature is 20 ℃ when putting seedling, puts seedling on March 20.The growing pond water colour is yellow green, transparency 30cm, and pH is 8.5, and throwing in density is 50,000/mu.The shrimp seedling is transported to the breed pool side, with filling oxygen head oxygenation half an hour in the seedling bag, progressively adds Chi Shui in the pool simultaneously, makes and progressively dwindles the temperature difference and awake seedling;
(4) throw the feeding tube reason, manufactured feed (is bought in Guangdong Hengxing Group Co., Ltd. for No. 1, be conventional prawn culturing feed down together), meet People's Republic of China's agricultural industry criteria " pollution-free food fishing manufactured feed safety limit " regulation (NY5072-2002).Before each bait throwing in, No. 1, manufactured feed is soaked 30min with Bdellovibrio leech plastid bacterium liquid, the concentration of the Bdellovibrio leech plastid in the bacterium liquid reaches 10
1, 10
3, 10
5, 10
7Pfu/mL cultures early stage and mid-term, when the long 6cm of shrimp average body is following, day throwing 2 times, day injected volume be controlled at shrimp heavy 15%; Culture the later stage, when average body reaches 6cm, day throw 5 times, day injected volume be controlled at shrimp heavy 8%;
(5) water environment of culturing the phase is managed, and normal aeration makes oxygen in water reach 5mg/L during the breed.Take different exchange water cycles, changed water once in 5,10,15,20,25,30 days, change water management in the dark water yield of 25cm at every turn, transparency remains on 30cm, adds Bdellovibrio leech plastid bacterium liquid after changing water at every turn, makes the cell concentration in the water body reach 10
1, 10
3, 10
5, 10
7Pfu/mL adds the bacterium method and is: behind corresponding Chi Shui dilution bacterium liquid, the full pond of evenly splashing mixes bacterium liquid.
The experiment grouping
The design of employing randomized block experiment is equally divided into A-D4 experimental group, and B, C, each experimental group of D have 6 groups, are respectively B1, C1, D1; B2, C2, D2; B3, C3, D3; B4, C4, D4; B5, C5, D5; B6, C6, D6; The every group of B1-D6 is further divided into 4 groups, i.e. B1-1-B1-4...D6-1-D6-4, every group of 2 repetitions.5 groups of initial body weight of test prawn are carried out the check of variance regularity, and group difference is remarkable (P＞0.05) not.The processing of five groups of experimental group is as follows:
The A group: the breeding way of plant produced, when being lower than 20cm, the pond water transparency changes water, and change water at every turn and change the dark water of 50cm (amount), keep water transparence at 35cm, throw in manufactured feed No. 1
B group: the Bdellovibrio leech plastid bacterium liquid of splashing in water body makes that leech plastid cell concentration reaches 10 respectively in the water body
1, 10
3, 10
5, 10
7Pfu/mL throws in manufactured feed No. 1
The C group: throwing something and feeding through concentration is 10
1, 10
3, 10
5, 10
7No. 1, the manufactured feed after the Bdellovibrio leech plastid bacterium liquid of pfu/mL soaks
D group: in water body, add Bdellovibrio leech plastid bacterium liquid, make cell concentration reach 10 respectively
1, 10
3, 10
5, 10
7Pfu/mL, throwing something and feeding through concentration simultaneously respectively is 10
1, 10
3, 10
5, 10
7No. 1, the manufactured feed after the Bdellovibrio leech plastid bacterium liquid of pfu/mL soaks
B1, C1, D1 organize per 5 days and change water once, change the dark water of 25cm (amount)
B2, C2, D2 organize per 10 days and change water once, change the dark water of 25cm (amount)
B3, C3, D3 organize per 15 days and change water once, change the dark water of 25cm (amount)
B4, C4, D4 organize per 20 days and change water once, change the dark water of 25cm (amount)
B5, C5, D5 organize per 25 days and change water once, change the dark water of 25cm (amount)
B6, C6, D6 organize per 30 days and change water once, change the dark water of 25cm (amount)
B1-1, C1-1, D1-1...D6-1 change and add Bdellovibrio leech plastid bacterium liquid behind the water and make that cell concentration is 10 in the water body
1Pfu/mL
B1-1, C1-2, D1-2...D6-2 change and add Bdellovibrio leech plastid bacterium liquid behind the water and make that cell concentration is 10 in the water body
3Pfu/mL
B1-1, C1-3, D1-3...D6-3 change and add Bdellovibrio leech plastid bacterium liquid behind the water and make that cell concentration is 10 in the water body
5Pfu/mL
B1-1, C1-4, D1-4...D6-4 change and add Bdellovibrio leech plastid bacterium liquid behind the water and make that cell concentration is 10 in the water body
7Pfu/mL
The Penaeus monodon experiment effect detects
(1) mensuration of growth indexes
Randomly draw 50 tail prawns for every group and detect growing state, it is the slide measure tolerance of 0.1mm that the prawn length measurment adopts precision, and body weight adopts electronic balance weighing.The test growth indexes is calculated as follows:
Survival rate (%)=N
t/ N
o* 100%;
Rate of body weight gain (%)=(W
t-W
o)/W
o* 100%;
The long growth rate (%) of body=(L
t-L
o)/L
o* 100%;
N in the formula
oThe total mantissa of Penaeus monodon during for on-test; N
tThe total mantissa of Penaeus monodon during for off-test; W
oPenaeus monodon body weight during for on-test; W
tPenaeus monodon body weight during for off-test; The Penaeus monodon body was long when Lo was on-test; The Penaeus monodon body was long when Lt was off-test;
The every growth indexes of Penaeus monodon that test records sees Table one～table four: compare with the A group, the survival rate of B, C, D group, rate of body weight gain, the long growth rate of body all are significantly improved, and B, C, D form the value added difference 〉=25.08%, 25.78%, 28.55% of motility rate; The value added difference 〉=40.54%, 65.62%, 86.35% of rate of body weight gain; The long growth rate value added difference 〉=0.09%, 0.17%, 3.19% of body.
From table one-table four as can be seen, in the prawn culturing process, under identical exchange water cycle, concentration rising along with Bdellovibrio leech plastid, survival rate, rate of body weight gain, the long growth rate of body all obviously raise, and all apparently higher than plant produced contrast A group, the method that adopts novel interpolation Bdellovibrio leech plastid is described, can obviously improve the long growth rate of survival rate, rate of body weight gain, body of Penaeus monodon, this has not only saved water consumption greatly, and has saved great amount of manpower in actual production process, material resources has been saved aquaculture cost; From different exchange water cycles, organize respectively that the long growth rate of prawn survival rate, rate of body weight gain, body is not significant to be changed under the same concentrations.Illustrate under the novel cultural method, do not change leech vibrios leech plastid and can both urge the into growth of prawn in 5-30 days.With regard to B, C, three groups of different disposal modes of D, all improve the long growth rate of breed survival rate, rate of body weight gain and body of prawn, but in water body and feed, add Bdellovibrio leech plastid bacterium liquid best results simultaneously.
Therefore, no matter add Bdellovibrio leech plastid bacterium liquid in water, making water body Bdellovibrio leech plastid bacterial concentration is 10
1-10
7Pfu/mL, or interpolation concentration is 10 in feed
1-10
7Pfu/mL Bdellovibrio swimming plastid bacterium liquid, or both dual-purposes do not change water though reach 30 days, all can promote ingesting and growth fast of Penaeus monodon, and simultaneously in water with to add the effect of bacterium liquid best in the feed.
The detection of table one spot joint prawn growth indexes
The detection of table two Penaeus monodon growth indexes
The detection of table three Penaeus monodon growth indexes
The detection of table four Penaeus monodon growth indexes
(2), the detection of immune indexes
Extract blood with the asepsis injector of 1mL from the prawn heart, every group extract 50 tail prawn blood merge place 4 ℃ of standing over night of aseptic centrifuge tube after, the centrifugal 10min of 3000r/min, getting upper serum, to place-80 ℃ of ultra low temperature freezers to preserve to be measured.
1, antalzyme activity measure serum lysozyme active by the turbidity colorimetric method for determining (Ellis A E.Lysozyme assays.[M] //Stolen J S, Fletcher T C, Anderson D P, et al.Techniques in Fish Immunology I.USA:SOS Publications, 1990:101-103.)
Under the experiment condition, it is 1 unit of enzyme activity (U) that the every min light absorption value of every mL serum reduces 0.001.
2, the mensuration of phenol oxidase (PO) vigor
Phenoloxidase activity is measured (Hern á ndez-L ó pezJ such as adopting Hern á ndez-L ó pez, Gollas-Galvan T.Vargas-Albores F.Activation of the prophenoloxidasesystem of the brown Penaeus californiensis Holmes[J] .Comp BiochemPhysiol, method 1996.113C:61-66.).Under experiment condition, the every min extinction of every mL serum increases by 0.001, is defined as 1 unit of enzyme activity (U).
3, superoxide dismutase (SOD) vigor
Superoxide dismutase (SOD) vitality test adopts the method for YI Sun etc. (A Simple Methodfor Clinical Assay of Superoxide Dismutase[J] .Clin.Chem.1988:497-500).An enzyme activity unit is defined as: under experimental condition, nitro tetrazole solution (NBT) is suppressed 50% required zymoprotein amount by SOD.
The measurement result of the every immune indexes of Penaeus monodon sees Table five～table eight:
As can be seen from the table, compare with the A group, antalzyme activity, superoxide dismutase activity, the phenol oxidase vigor of B, C, D group all are significantly improved.B, C, D group antalzyme activity value added difference 〉=8.19U/mL, 9.28U/mL, 9.9U/mL; Superoxide dismutase activity value added difference 〉=53.58U/mL, 79.98U/mL, 97.48U/mL; Phenol oxidase vigor value added difference 〉=10.1U/mL, 10.8U/mL, 11U/mL.
From table five～table eight as can be seen, in the Penaeus monodon breeding process, under identical exchange water cycle, concentration rising along with Bdellovibrio leech plastid, survival rate all obviously raises, and all apparently higher than plant produced contrast A group, the method that adopts novel interpolation Bdellovibrio leech plastid is described, can obviously improve the right immunity of spot joint, strengthen the vigor of enzyme, this has not only saved aquaculture cost in actual production process, and strengthen the immunocompetence of prawn, promote growth; From different exchange water cycles, the prawn enzyme activity of respectively organizing under the same concentrations does not have significant difference, illustrates under the novel prawn culturing method, does not change leech vibrios leech plastid and can both improve prawn immunity in 5-30 days.With regard to B, C, three groups of different disposal modes of D, all improve immunity, but in water body and feed, add Bdellovibrio leech plastid bacterium liquid best results simultaneously.
Therefore, no matter add Bdellovibrio leech plastid bacterium liquid in water, making water body Bdellovibrio leech plastid cell concentration is 10
1-10
7Pfu/mL, or interpolation concentration is 10 in feed
1-10
7Pfu/mL Bdellovibrio leech plastid bacterium liquid, or both dual-purposes, do not change water though reach 30 days, can both significantly improve the immune indexes such as antalzyme activity, superoxide dismutase activity, phenol oxidase vigor of Penaeus monodon, add simultaneously in water and in the feed, effect is better.Change water between 5-30 days, antalzyme activity, superoxide dismutase activity, phenol oxidase vigor are not all had obvious influence.
The measurement result of table five immune indexes
The measurement result of table six immune indexes
The measurement result of table seven immune indexes
The measurement result of table eight immune indexes
(3), water quality detection
1, ammoniacal nitrogen is received oxidizing process with hypobromous acid and is measured (GB12763.4-91);
2, nitrite nitrogen diazonium-azo spectrphotometric method for measuring (GB12763.4-91)
3, H
2S uses dimethylamino-aniline spectrophotometry (GB16489-1996)
Process mensuration, the every water-quality determination value in the A group pond all are higher than experiment group B, C, D's.Wherein, the testing result of A group ammoniacal nitrogen is 0.506mg/L, is higher than the 0.431-0.456mg/L of B group, the 0.467-0.492mg/L of C group, the 0.403-0.441mg/L of D group; A group nitrite nitrogen testing result is 0.015mg/L, is higher than the 0.008-0.009mg/L of B group, the 0.011-0.013mg/L of C group, the 0.007-0.008mg/L of D group; A organizes H
2The S testing result is 0.2mg/L, is higher than the 0.056-0.137mg/L of B group, the 0.114-0.179mg/L of C group, the 0.024-0.106mg/L of D group; By above interpretation of result as can be known, using concentration is 10
1-10
7Pfu/mL Bdellovibrio leech plastid can effectively improve the breeding water body environment, regulates ammoniacal nitrogen, nitrite nitrogen, H in the water
2The content of S, thus the water body environment that is beneficial to Penaeus monodon existence provided, and the best results of in water body and feed, adding Bdellovibrio leech plastid simultaneously.
(4), bacterial population is measured:
1, total plate count detects (GB4789.2-2010) with nutrient agar
2, the vibrios sum detects (SN/T 0173-2010) with the TCBS medium
3, pH measures with the portable pH meter of PHB-3
Total bacterial population of plant produced contrast A group and total vibrios number are respectively up to 4.63 * 10
3Cfu/mL and 6.09 * 10
2Cfu/mL.Total bacterial population of B, C, D group is respectively 4.15 * 10
2Cfu/mL, 4.34 * 10
2Cfu/mL, 4.21 * 10
2Cfu/mL; Total several 8.2 * 10cfu/mL of vibrios, 7.8 * 10cfu/mL, 6.6 * 10cfu/mL have obviously reduced by 1 order of magnitude with A group ratio.The pH of each group between 7.5-9, changes little.
Illustrate that Bdellovibrio leech plastid can effectively control the bacterial population of water body, and reach the purpose of improving water quality.In general, add Bdellovibrio leech plastid, can significantly improve Penaeus monodon, strengthen the immunity of prawn, and can improve water quality, reach the good result of saving water and energy, improve survival rate.
Simultaneously, no matter add Bdellovibrio leech plastid bacterium liquid as can be known by experimental data in water, making water body Bdellovibrio leech plastid bacterial concentration is 10
1-10
7Pfu/mL, or interpolation concentration is 10 in feed
1-10
7Pfu/mL Bdellovibrio leech plastid bacterium liquid, or both dual-purposes do not change water though reach 30 days, can promote that prawn survives yet, and enhancing immunity improves breed phase resistance against diseases, and it is best to add Bdellovibrio leech plastid bacterium liquid effect in water body and feed simultaneously.
(5) infection experiment
Infection experiment is carried out in the pond that fills 5L sand filtration seawater (salinity 10 ‰) of sterilization in advance.From experiment A group, B1-1, B1-2...D6-3, D6-4 totally 73 groups each group get 50 of prawns at random, the long 20～25cm of specification body.Add vibrio parahaemolytious (Vibrio parahaemolyticus purchases in U.S. typical case species preservation center AmericanType Culture Collection, and ATCC 17802) in water body to each group and make that vibrio parahaemolytious concentration reaches 10 in the water body
8Cfu/mL; The A group is the control group of this experiment.The normal bait throwing in of experimental session, continuous charge is cultivated.Behind the artificial infection, observed continuously 7 days, finally be calculated to be motility rate, the employing relative survival rate (Relative Percentage Survival, RPS) calculating formula:
RPS (%)=(1-immune group lethality/control group lethality) * 100%
Table nine is attacked malicious infection experiment relative survival rate
The result of infection experiment shows, (Relative PercentSurvival is 0 RPS) to the relative survival rate of A group, compares with the A group, the relative survival rate of B, C, D group all is significantly improved, the value added difference 〉=93.2%, 86.6%, 100% of B, C, D group relative survival rate.
From table as can be seen, in prawn infection experiment process, under identical exchange water cycle, concentration rising along with Bdellovibrio leech plastid, relative survival rate raises gradually, and all apparently higher than plant produced contrast A group, the method that adopts novel interpolation Bdellovibrio leech plastid is described, can obviously improve the relative survival rate of Penaeus monodon, promptly improve prawn immunity resistance; From different exchange water cycles, the prawn relative survival rate of respectively organizing under the same concentrations does not change significantly.Illustrate under the novel cultural method, do not change leech vibrios leech plastid and can both strengthen the living immunocompetence of prawn in 5-30 days.With regard to B, C, three groups of different disposal modes of D, all improve the relative survival rate of prawn, but in water body and feed, add Bdellovibrio leech plastid bacterium liquid best results simultaneously.
Therefore, no matter add Bdellovibrio leech plastid bacterium liquid in water, making water body Bdellovibrio leech plastid bacterial concentration is 10
1-10
7Pfu/mL, or interpolation concentration is 10 in feed
1-10
7Pfu/mL Bdellovibrio leech plastid bacterium liquid, or both dual-purposes do not change water though reach 30 days, all can improve Penaeus monodon immunity resistance, and simultaneously in water with to add the effect of bacterium liquid best in the feed.Thereby show that further Bdellovibrio leech plastid has potential vaccine effect, can stimulate prawn to produce immune response, resist the infringement of vibrio parahaemolytious, help to improve the survival rate of prawn.
The application experiment of embodiment 3 Bdellovibrio leech plastids in the Penaeus monodon high-elevation breeding pond
(1) the shrimp pool structure is 88m * 83m * 2.2m, and four jiaos is fillet, and each shrimp pool area is 10 mu, is the horizontal yi word pattern of east-west direction and arranges.Distance is 5m between the shrimp pond, and height above sea level is 10m, adopts the cement concrete bank protection around the shrimp pond, lays plastic film at the bottom of the pond, covers husky 35cm then on film again.
Sea area lowest low water line digs a well, and tubing is to pump, and the room distance is 72m, adopts the 100cm pipe of concrete; It is that 8BA18, power are the suction pump of 22kW/ platform that pump house adopts 3 models, draws water to the shrimp pond, and distance is 110m, adopts 60cm * 70cm open channel, enters each shrimp pond by 40cm * 60cm open channel diversion again.Adopt waterwheel aerator, power is the 11kW/ platform, and every mu of water surface is placed 1.
(2) put seedling preceding 1 month,, and clear up with mud at the bottom of the giant flushing pool, after doing pool solarization pond 15d then, the every mu of quicklime 150kg that splashes, water inlet 30cm soaking disinfection continues to add water 90cm, and apply fertilizer simultaneously, the main urea 15kg/ mu of throwing in, phosphate fertilizer 10kg/ mu carries out rich water, about 3d, water transparency reach 35cm just can put seedling.
(3) the shrimp seedling is selected and is put in a suitable place to breed, and the shrimp seedling selects individual size neat, the black shell shrimp seedling of the long 1.0-1.2cm of body, astogeny is neat, muscle is full transparent, and pigment is normal in the appendage, and stomach and intestine are full of food, the shrimp seedling is movable active, to current irritant reaction sensitivity, no ectoparasitism thing and adhere to dirt, water temperature is 20 ℃ when putting seedling, culturing pool salinity 10 ‰ is put the seedling time and common breed is carried out synchronously.The growing pond water colour is yellow green, transparency 30cm, and pH is 8.5, and throwing in density is 5.5 ten thousand/mu.The shrimp seedling is transported to the breed pool side, with filling oxygen head oxygenation half an hour in the seedling bag, progressively adds culturing pool simultaneously in bag, makes and progressively dwindles the temperature difference and awake seedling;
(4) throw the feeding tube reason, feed (No. 1, manufactured feed) is conventional prawn culturing feed, meets People's Republic of China's agricultural industry criteria " pollution-free food fishing manufactured feed safety limit " regulation (NY5072-2002).Before each bait throwing in, feed is soaked 30min with Bdellovibrio leech plastid bacterium liquid, the concentration of the Bdellovibrio leech plastid in the bacterium liquid reaches 10
1, 10
3, 10
5, 10
7Pfu/mL cultures early stage and mid-term, when the long 6cm of shrimp average body is following, day throwing 2 times, day injected volume be controlled at shrimp heavy 15%; Culture the later stage, when body on average reaches 6cm, day throw 5 times, day injected volume be controlled at shrimp heavy 8%;
(5) water environment of culturing the phase is managed, and normal aeration makes oxygen in water reach 5mg/L during the breed.Take different exchange water cycles, changed water once in 5,10,15,20,25,30 days, change the dark water of 25cm (amount) at every turn, transparency remains on 30cm, adds Bdellovibrio leech plastid bacterium liquid after changing water at every turn, makes the cell concentration in the water body reach 10
1, 10
3, 10
5, 10
7Pfu/mL adds the bacterium method and is: behind corresponding Chi Shui dilution bacterium liquid, the full pond of evenly splashing mixes bacterium liquid.
The experiment grouping
The design of employing randomized block experiment is equally divided into 4 experimental group of A-D, and B, C, each experimental group of D have 6 groups, are respectively B1, C1, D1; B2, C2, D2; B3, C3, D3; B4, C4, D4; B5, C5, D5; B6, C6, D6; The every group of B1-D6 is further divided into 4 groups, i.e. B1-1-B1-4...D6-1-D6-4, every group of 2 repetitions.4 groups of initial body weight of test prawn are carried out variance test, and group difference is remarkable (P＞0.05) not.The processing of four groups of experimental group is as follows:
The A group: the breeding way of plant produced, raise early stage, every 7d blowdown is once raised every 2d blowdown in mid-term once, and the raising later stage increases the blowdown number of times, just blowdown when water colour is deepened to become dark brown.For supplying the water yield, add fresh water to test tank because of evaporation and blowdown loss.Throw in pellet (No. 1, manufactured feed)
B group: the Bdellovibrio leech plastid bacterium liquid of splashing in water makes that leech plastid cell concentration reaches 10 respectively in the water body
1, 10
3, 10
5, 10
7Pfu/mL throws in pellet (No. 1, manufactured feed)
The C group: the concentration of throwing something and feeding is 10
1, 10
3, 10
5, 10
7Pellet (No. 1, manufactured feed) after the Bdellovibrio leech plastid thalline of pfu/mL soaks
D group: in water, add Bdellovibrio leech plastid bacterium liquid, make cell concentration reach 10 respectively
1, 10
3, 10
5, 10
7Pfu/mL, the concentration of throwing something and feeding respectively simultaneously is 10
1, 10
3, 10
5, 10
7Pellet (No. 1, manufactured feed) after the Bdellovibrio leech plastid bacterium liquid of pfu/mL soaks;
B1, C1, D1 organize per 5 days and change water once, change the dark water of 25cm (amount)
B2, C2, D2 organize per 10 days and change water once, change the dark water of 25cm (amount)
B3, C3, D3 organize per 15 days and change water once, change the dark water of 25cm (amount)
B4, C4, D4 organize per 20 days and change water once, change the dark water of 25cm (amount)
B5, C5, D5 organize per 25 days and change water once, change the dark water of 25cm (amount)
B6, C6, D6 organize per 30 days and change water once, change the dark water of 25cm (amount)
B1-1, C1-1, D1-1...D6-1 change and add Bdellovibrio leech plastid behind the water and make that cell concentration is 10 in the water body
1Pfu/mL
B1-2, C1-2, D1-2...D6-2 change and add Bdellovibrio leech plastid behind the water and make that cell concentration is 10 in the water body
3Pfu/mL
B1-3, C1-3, D1-3...D6-3 change and add Bdellovibrio leech plastid behind the water and make that cell concentration is 10 in the water body
5Pfu/mL
B1-4, C1-4, D1-4...D6-4 change and add Bdellovibrio leech plastid behind the water and make that cell concentration is 10 in the water body
7Pfu/mL
The Penaeus monodon experiment effect detects
(1) mensuration of growth indexes
Randomly draw 50 tail prawns for every group and detect growing state, it is the slide measure tolerance of 0.1mm that the prawn length measurment adopts precision, and body weight adopts electronic balance weighing.The test growth indexes is calculated as follows:
Survival rate (%)=N
t/ N
o* 100%;
Rate of body weight gain (%)=(W
t-W
o)/W
o* 100%;
The long growth rate (%) of body=(L
t-L
o)/L
o* 100%;
N in the formula
oThe total mantissa of Penaeus monodon during for on-test; N
tThe total mantissa of Penaeus monodon during for off-test; W
oPenaeus monodon body weight during for on-test; W
tPenaeus monodon body weight during for off-test; L
oThe Penaeus monodon body is long during for on-test; L
tThe Penaeus monodon body is long during for off-test;
The every growth indexes of Penaeus monodon that test records sees Table ten-table ten three: as can be seen from the table, compare with the A group, the survival rate of B, C, D group, rate of body weight gain, the long growth rate of body all are significantly improved, and B, C, D form the value added difference 〉=43.58%, 44.58%, 48.55% of motility rate; The value added difference 〉=55.54%, 80.62%, 101.3% of rate of body weight gain; The long growth rate value added difference 〉=5.04%, 4.99%, 4.99% of body.
From table ten-table ten three as can be seen, in prawn high-elevation breeding pond process, under identical exchange water cycle, concentration rising along with Bdellovibrio leech plastid, survival rate, rate of body weight gain, the long growth rate of body all obviously raise, and all apparently higher than plant produced contrast A group, the method that adopts novel interpolation Bdellovibrio leech plastid is described, can obviously improve the long growth rate of survival rate, rate of body weight gain, body of Penaeus monodon, this has not only saved water consumption greatly, and has saved great amount of manpower in actual production process, material resources has been saved aquaculture cost; From different exchange water cycles, organize respectively that the long growth rate of prawn survival rate, rate of body weight gain, body is not significant to be changed under the same concentrations.Illustrate under the novel cultural method, do not change leech vibrios leech plastid and can both urge the into growth of prawn in 5-30 days.With regard to B, C, three groups of different disposal modes of D, all improve the long growth rate of breed survival rate, rate of body weight gain and body of prawn, but in water body and feed, add Bdellovibrio leech plastid bacterium liquid best results simultaneously.
Therefore, no matter add Bdellovibrio leech plastid bacterium liquid in water, making water body Bdellovibrio leech plastid cell concentration is 10
1-10
7Pfu/mL, or interpolation concentration is 10 in feed
1-10
7Pfu/mL Bdellovibrio swimming plastid bacterium liquid, or both dual-purposes do not change water though reach 30 days, all can promote ingesting and growth fast of Penaeus monodon, and simultaneously in water with to add the effect of bacterium liquid best in the feed.
The detection of table ten Penaeus monodon growth indexes
The detection of table ten one spot joint prawn growth indexes
The detection of table ten two Penaeus monodon growth indexes
The detection of table ten three Penaeus monodon growth indexes
(2), the detection of immune indexes
Extract blood with the asepsis injector of 1mL from the prawn heart, every group extract 50 tail prawn blood merge place 4 ℃ of standing over night of aseptic centrifuge tube after, the centrifugal 10min of 3000r/min, getting upper serum, to place-80 ℃ of ultra low temperature freezers to preserve to be measured.
1, antalzyme activity measure serum lysozyme active by the turbidity colorimetric method for determining (Ellis A E.Lysozyme assays.[M] //Stolen J S, Fletcher T C, Anderson D P, et al.Techniques in Fish Immunology I.USA:SOS Publications, 1990:101-103.)
Under the experiment condition, it is 1 unit of enzyme activity (U) that the every min light absorption value of every mL serum reduces 0.001.
2, the mensuration of phenol oxidase (PO) vigor
Phenoloxidase activity is measured (Hern á ndez-L ó pezJ such as adopting Hern á ndez-L ó pez, Gollas-Galvan T.Vargas-Albores F.Activation of the prophenoloxidasesystem of the brown Penaeus californiensis Holmes[J] .Comp BiochemPhysiol, method 1996.113C:61-66.).Under experiment condition, the every min extinction of every mL serum increases by 0.001, is defined as 1 unit of enzyme activity (U).
3, superoxide dismutase (SOD) vigor
Superoxide dismutase (SOD) vitality test adopts the method for YI Sun etc. (A Simple Methodfor Clinical Assay of Superoxide Dismutase[J] .Clin.Chem.1988:497-500).An enzyme activity unit is defined as: under experimental condition, nitro tetrazole solution (NBT) is suppressed 50% required zymoprotein amount by SOD.
The measurement result of the every immune indexes of Penaeus monodon sees Table 14～table ten seven:
As can be seen from the table, compare with the A group, antalzyme activity, superoxide dismutase activity, the phenol oxidase vigor of B, C, D group all are significantly improved.B, C, D group antalzyme activity value added difference 〉=11.09U/mL, 12.18U/mL, 12.8U/mL; Superoxide dismutase activity value added difference 〉=53.58U/mL, 79.98U/mL, 97.48U/mL; Phenol oxidase vigor value added difference 〉=12.1U/mL, 11.8U/mL, 12U/mL.
In Penaeus monodon high-elevation breeding pond process, under identical exchange water cycle, along with the concentration rising of Bdellovibrio leech plastid, survival rate all obviously raises, and all apparently higher than plant produced contrast A group, the method that adopts novel interpolation Bdellovibrio leech plastid is described, can obviously improves the right immunity of spot joint, strengthen the vigor of enzyme, this is in actual production process, not only save aquaculture cost, and strengthened the immunocompetence of prawn, promoted growth; From different exchange water cycles, the prawn enzyme activity of respectively organizing under the same concentrations does not have significant difference, illustrates under the novel prawn culturing method, does not change leech vibrios leech plastid and can both improve prawn immunity in 5-30 days.With regard to B, C, three groups of different disposal modes of D, all improve immunity, but in water body and feed, add Bdellovibrio leech plastid bacterium liquid best results simultaneously.
Therefore, no matter add Bdellovibrio leech plastid bacterium liquid in water, making water body Bdellovibrio leech plastid cell concentration is 10
1-10
7Pfu/mL, or interpolation concentration is 10 in feed
1-10
7Pfu/mL Bdellovibrio leech plastid bacterium liquid, or both dual-purposes, do not change water though reach 30 days, can both significantly improve the immune indexes such as antalzyme activity, superoxide dismutase activity, phenol oxidase vigor of Penaeus monodon, add simultaneously in water and in the feed, effect is better.Change water between 5-30 days, antalzyme activity, superoxide dismutase activity, phenol oxidase vigor are not all had obvious influence.
The measurement result of table ten four immune indexes
The measurement result of table ten five immune indexes
The measurement result of table ten six immune indexes
The measurement result of table ten seven immune indexes
(3), water quality detection
1, ammoniacal nitrogen is received oxidizing process with hypobromous acid and is measured (GB12763.4-91);
2, nitrite nitrogen diazonium-azo spectrphotometric method for measuring (GB12763.4-91)
3, H2S uses dimethylamino-aniline spectrophotometry (GB16489-1996)
Process mensuration, the every water-quality determination value in the A group pond all are higher than experiment group B, C, D's.Wherein, the testing result of A group ammoniacal nitrogen is 0.516mg/L, is higher than the 0.441-0.466mg/L of B group, the 0.47-0.502mg/L of C group, the 0.413-0.451mg/L of D group; A group nitrite nitrogen testing result is 0.016mg/L, is higher than the 0.009-0.01mg/L of B group, the 0.012-0.014mg/L of C group, the 0.008-0.009mg/L of D group; A group H2S testing result is 0.21mg/L, is higher than the 0.066-0.147mg/L of B group, the 0.124-0.189mg/L of C group, the 0.034-0.116mg/L of D group; By above interpretation of result as can be known, using concentration is 10
1-10
7Pfu/mL Bdellovibrio leech plastid can effectively improve the breeding water body environment, regulates ammoniacal nitrogen, nitrite nitrogen, H in the water
2The content of S, thus the water body environment that is beneficial to Penaeus monodon existence provided, and the best results of in water body and feed, adding the Bdellovibrio telotroch simultaneously.
(4), bacterial population detects:
1, total plate count detects (GB4789.2-2010) with nutrient agar
2, the vibrios sum detects (SN/T 0173-2010) with the TCBS medium
3, pH measures with the portable pH meter of PHB-3
Total bacterial population of plant produced A group and total vibrios number are respectively up to 5.33 * 10
3Cfu/mL and 7.89 * 10
2Cfu/mL.Total bacterial population of B, C, D group is respectively 4.14 * 10
2Cfu/mL, 3.79 * 10
2Cfu/mL, 4.11 * 10
2Cfu/mL; Total several 6.5 * 10cfu/mL of vibrios, 6.4 * 10cfu/mL, 6.1 * 10cfu/mL have obviously reduced by 1 order of magnitude with A group ratio.Illustrate that Bdellovibrio leech plastid can effectively control the bacterial population of water body, and reach the purpose of improving water quality.In general, add Bdellovibrio leech plastid, can significantly improve the physique of prawn, strengthen the immunity of prawn, and can improve water quality, reach the good result of saving water and energy, improve survival rate.
Simultaneously, no matter add Bdellovibrio leech plastid bacterium liquid as can be known by experimental data in water, making water body Bdellovibrio leech plastid cell concentration is 10
1-10
7Pfu/mL, or interpolation concentration is 10 in feed
1-10
7Pfu/mL Bdellovibrio leech plastid bacterium liquid, or both dual-purposes do not change water though reach 30 days, can promote that Penaeus monodon survives yet, and enhancing immunity improves breed phase resistance against diseases, and it is best to add Bdellovibrio leech plastid bacterium liquid effect in water body and feed simultaneously.
(5) infection experiment
Infection experiment is carried out in the pond that fills 5L sand filtration seawater (salinity 10 ‰) of sterilization in advance.From experiment A group, B1-1, B1-2...D6-3, D6-4 totally 73 groups each group get 50 of prawns at random, the long 20～25cm of specification body.Add vibrio parahaemolytious (Vibrio parahaemolyticus purchases in U.S. typical case species preservation center AmericanType Culture Collection, and ATCC 17802) in water body to each group and make that vibrio parahaemolytious concentration reaches 10 in the water body
8Cfu/mL; The A group is the control group of this experiment.The normal bait throwing in of experimental session, continuous charge is cultivated.Behind the artificial infection, observed continuously 7 days, finally be calculated to be motility rate, the employing relative survival rate (Relative Percentage Survival, RPS) calculating formula:
RPS (%)=(1-immune group lethality/control group lethality) * 100%
Table ten eight is attacked malicious infection experiment relative survival rate
The result of infection experiment shows, (Relative PercentSurvival is 0 RPS) to the relative survival rate of A group, compares with the A group, the relative survival rate of B, C, D group all is significantly improved, the value added difference 〉=93.2%, 86.6%, 100% of B, C, D group relative survival rate.
From table as can be seen, in prawn infection experiment process, under identical exchange water cycle, concentration rising along with Bdellovibrio leech plastid, relative survival rate raises gradually, and all apparently higher than plant produced contrast A group, the method that adopts novel interpolation Bdellovibrio leech plastid is described, can obviously improve the relative survival rate of Penaeus monodon, promptly improve prawn immunity resistance; From different exchange water cycles, the prawn relative survival rate of respectively organizing under the same concentrations does not change significantly.Illustrate under the novel cultural method, do not change leech vibrios leech plastid and can both strengthen the living immunocompetence of prawn in 5-30 days.With regard to B, C, three groups of different disposal modes of D, all improve the relative survival rate of prawn, but in water body and feed, add Bdellovibrio leech plastid bacterium liquid best results simultaneously.
Therefore, no matter add Bdellovibrio leech plastid bacterium liquid in water, making water body Bdellovibrio leech plastid bacterial concentration is 10
1-10
7Pfu/mL, or interpolation concentration is 10 in feed
1-10
7Pfu/mL Bdellovibrio leech plastid bacterium liquid, or both dual-purposes do not change water though reach 30 days, all can improve Penaeus monodon immunity resistance, and simultaneously in water with to add the effect of bacterium liquid best in the feed.Thereby show that further Bdellovibrio leech plastid has potential vaccine effect, can stimulate prawn to produce immune response, resist the infringement of vibrio parahaemolytious, help to improve the survival rate of prawn.
No matter this shows, be common breed or high-elevation breeding pond, adds Bdellovibrio leech plastid bacterium liquid in water, and making water body Bdellovibrio leech plastid cell concentration is 10
1-10
7Pfu/mL, or with feed with 10
1-10
7Pfu/mL Bdellovibrio leech plastid bacterium liquid soaks; can both reach the immunity that improves shrimp, the purpose that promotion is ingested and grown fast; and can degradation of ammonia nitrogen, increase the bacterium of curing the disease in oxyty, the splitting water; have the immune resistance potent simultaneously, thereby reach the effect of improving water quality, save groundwater resources vibrio parahaemolytious; the protection environment; save cost, increase economic efficiency, promote the fast development of aquaculture.Use simultaneously with feed in water, effect is better.
Claims (5)
1. the application of Bdellovibrio leech plastid bacterium liquid in Penaeus monodon is cultured.
2. according to the application of the described Bdellovibrio leech of claim 1 plastid bacterium liquid in Penaeus monodon is cultured, it is characterized in that: described Bdellovibrio Bdellovibrio is (Bdellovibrio sp.) BDFM05, be preserved in Chinese typical culture collection center on August 7th, 2009, deposit number is CCTCC NO:M 209172.
3. according to the application of the described Bdellovibrio leech of claim 1 plastid bacterium liquid in Penaeus monodon is cultured, it is characterized in that: the application of described Bdellovibrio leech plastid bacterium liquid in Penaeus monodon is cultured is that feed is soaked 15-45min in Bdellovibrio leech plastid bacterium liquid.
4. according to the application of the described Bdellovibrio leech of claim 1 plastid bacterium liquid in Penaeus monodon is cultured, it is characterized in that: the application of described Bdellovibrio leech plastid bacterium liquid in Penaeus monodon is cultured is to add Bdellovibrio leech plastid bacterium liquid in water body, makes that Bdellovibrio leech plastid concentration is 10 in the water body
1～10
7Pfu/mL.
5. according to the application of the described Bdellovibrio leech of claim 1 plastid bacterium liquid in Penaeus monodon is cultured, it is characterized in that: the preparation method of described Bdellovibrio leech plastid bacterium liquid is:
The host is in the nutrient broth liquid nutrient medium in inoculation, at 35 ℃ and 200rpm shaking table cultivation 12h, culture fluid is through 4 ℃, behind the centrifugal 10～20min of 5000～8000rpm, precipitation is added in the DNB liquid nutrient medium, insert Bdellovibrio BDFM05, under 25～35 ℃ of temperature, cultivate 36～48h with 150～300rpm shaking table, obtain containing the culture fluid of Bdellovibrio leech plastid and Bdellovibrio telotroch, culture fluid behind centrifugal 15～20min, is removed the supernatant that contains the Bdellovibrio telotroch with 6000～8000rpm under 4 ℃ of temperature, the precipitation that obtains is Bdellovibrio leech plastid; Is that 7.2～7.6 phosphate buffer suspends with Bdellovibrio leech plastid with DNB liquid nutrient medium, water, physiological saline or 0.2mol/L pH value, and obtaining concentration is the Bdellovibrio leech plastid bacterium liquid of 1011～1012pfu/mL.
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| CN113475440A (en) * | 2021-07-21 | 2021-10-08 | 新荣腾种业有限公司 | Method for breeding penaeus vannamei boone seedlings with glass seedling disease bacterium resistance |
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