CN102017912A - Water-saving Haliotis diversicolor aquatilis larvae cultivation method - Google Patents
Water-saving Haliotis diversicolor aquatilis larvae cultivation method Download PDFInfo
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Abstract
The invention discloses a water-saving Haliotis diversicolor aquatilis larvae cultivation method, which comprises the following steps: (1) seaweed cultivation; (2) nutritive salt adding; (3) larvae selection and oosperm distribution: after the water in a seaweed cultivation pool is changed, adding the bacteria liquid of Bdellovibro nectophore into the seaweed cultivation pool, so that the initial concentration of the Bdellovibro nectophore in the water in the pool reaches 10 to 107pfu/mL; then putting the larvae into the seaweed cultivation pool, wherein 40 to 50 thousand oosperms of Haliotis diversicolor aquatilis are distributed in each pool; (4) water changing: changing the water in the pool every 5 to 30 days, and adding the bacteria liquid of Bdellovibro nectophore into the seaweed cultivation pool after changing the water each time, so that the initial concentration of the Bdellovibro nectophore in the water in the pool reaches 10 to 107pfu/mL; and (5) stripping and harvest. The method disclosed by the invention has the advantages of playing an obvious role in promoting the growth of the Haliotis diversicolor aquatilis larvae, greatly improving the survival rate of the Haliotis diversicolor aquatilis larvae, and improving the water quality of the cultivation environment. Because the Bdellovibro nectophore has no toxicity and side effects on the Haliotis diversicolor aquatilis, the method is suitable to be used in the large-scale factory cultivation of abalone, and provides a new cultivation technique for promoting the cultivation of the Haliotis diversicolor aquatilis larvae.
Description
Technical field
The invention belongs to aquaculture shellfish hatchery method field, relate to a kind of aquaculture shellfish hatchery method, the seedling-cultivating method of particularly a kind of water saving, energy-conservation, the haliotis diversicolor Reeve (Haliotis diversicolor aquatilis) that reduces discharging.
Background technology
Haliotis diversicolor Reeve is one of important breed variety in marine site, China south, and annual production once reached 4000 tons.But since 2000, the coastal haliotis diversicolor Reeve artificial culture seedling mortality phenomenon that taken place from Fujian to Hainan, haliotis diversicolor Reeve seedling and poor-performed almost can't be cultivated in the Bao Miao field more than 90%, and this forces many raisers to abandon culturing haliotis diversicolor Reeve.Therefore, how research improves the haliotis diversicolor Reeve seedling is cultivated ecotope, reduces the generation of board falling phenomenon, improves its survival rate and growth rate, all has crucial meaning in the production technology of haliotis diversicolor Reeve seed and even abalone culture industry.
Discover that pathogenic infection is the main cause that causes board falling, propagating artificially under the condition mainly is bacteriosis and viral disease.Common bacterial pathogen has vibrio parahaemolytious, vibrio fluvialis, secondary vibrio alginolyticus etc.Vibrio parahaemolytious mainly causes the abalone muscular dystrophy, and lethality can reach about 50%, and the infection that vibrio fluvialis and secondary vibrio alginolyticus cause can cause the lethality of 60-70%.The method of conventional control abalone board falling mainly is regular administration of antibiotics at present, and antibiotic advantage is the direct entry of medicine, concentration height, fast, the good effect of absorption; Shortcoming is may produce excitant to abalone, cause new illness.If long-term a large amount of antibiotic that uses not only can make the pathogenic bacteria pesticide resistance strengthen, some Bao cases are become be difficult to cure.Antibiotic residue can influence the abalone quality in the Bao body in addition, is unfavorable for selling, and then causes culturist's economic loss.Utilizing the board falling of herbal control abalone also is a kind of common way, though have drug residue free, advantage that economic benefit is high, the slow shortcoming that takes effect is arranged also.Therefore seeking a kind of more efficient, economic seedling-cultivating method seems very urgent.
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; The Bdellovibrio telotroch is meant the growth forms before Bdellovibrio is invaded host bacteria.It is very fast that the Bdellovibrio telotroch is invaded the process of host bacteria, can finish in general several seconds.For Bdellovibrio leech plastid, though exist the weakness that preparation is loaded down with trivial details, be difficult for preservation, the Bdellovibrio telotroch has rapid-action, energetic advantage aspect the potentially pathogenic organism of control breeding water body and aquatic livestock enteron aisle.In addition, traditional haliotis diversicolor Reeve artificial breeding mainly adopts the outer seawater of extraction to enter through husky filter post precipitation and cultures the pond, waste water directly enters the sea area through pipeline, thisly directly take out in line mode and have the energy consumption height, to the heavy-polluted shortcoming in sea area, not only increase the weight of aquaculture cost, also become the big hidden danger that disease takes place Bao Rihou.And the present invention combines the use of Bdellovibrio telotroch, and the employing cycle is changed the mode of water, so not only reduces breeding water and discharge of wastewater, reduces energy consumption especially, has reduced aquaculture cost, is that a kind of facility is feasible, the green seedling raising manners of energy-saving and emission-reduction.
No matter existing studies show that as a kind of effective microorganism preparation, is in food industry or in fields such as (ocean) aquacultures, the application of Bdellovibrio all is safe.For example: abroad, Lenz and Hespell (1978) discovers, Bdellovibrio and animal and people's cell do not had an infectivity [Lenz R.W., Hespell R.B.Attempts to grow bedellovibrios micurgically-injected into animal cells.Archives of Microbiology, 1978,119 (3): 245-248].At home, Lin Mao etc. (2006) have studied the effect of Bdellovibrio to fish cell, find that it does not have dissemination [Lin Mao, Yang Xianle, Xue Hui, Cao Haipeng, Qiu Junqiang to the fish bacterium.The effect of 02 pair of fish cell of Bdellovibrio BDH21 and pathogen.The microbiology circular, 2006,33 (1): 7-11].
Summary of the invention
For overcoming the deficiency that exists in the above-mentioned existing seedling growing process, the object of the present invention is to provide a kind of water-saving haliotis diversicolor Reeve seedling-cultivating method.
Purpose of the present invention realizes by following technical proposals: a kind of haliotis diversicolor Reeve seedling-cultivating method of energy-saving and emission-reduction comprises following operating procedure:
(1) training algae: the beginning in preceding 30～45 days of collecting seedling is trained algae in training algae pond; The control intensity of illumination is at 2000～5000lux in the training algae process, and the control water temperature is at 15～28 ℃;
(2) Ensure Liquid salt: during the training algae, according to 3～5g/m
3Breeding water body, the mode of adding once in 2～4 days is added nutritive salt;
(3) the cloth ovum of collecting seedling: will train the algae pond and change Chi Shui, and add Bdellovibrio telotroch bacterium liquid, and make the initial concentration of the Bdellovibrio telotroch of Chi Shuizhong reach 10～10
7Pfu/mL; Every pond cloth haliotis diversicolor Reeve fertilized egg is 4～50,000 during following seedling, and according to aeration rate 10～60L/h/m
3Carry out aeration, illumination is controlled at 200～3000lux, and water temperature is controlled at 15～28 ℃;
(4) change water: changed water once every 5～30 days, change at every turn and add Bdellovibrio telotroch bacterium liquid behind the water, make the concentration of the Bdellovibrio telotroch of Chi Shuizhong reach 10～10
7Pfu/mL;
(5) peel off and gather: following seedling 30～50 days, young Bao shell reach to peel off behind 3.0～6.5mm gathers.
The described training of step (1) algae pond washes the back and adopts liquor potassic permanganate to carry out disinfection, and injects the seawater through sand filtration and precipitation process then; A filter bag was wrapped up at the pond water inlet pipe mouth in training algae pond before injecting seawater, prevent that fine sand from going into the pond.
The described algae of step (1) is a benthic diatom.Described benthic diatom is a month shape algae, rhombus algae, boat-shaped algae and avette algae.
The described training of step (1) algae is an adherance with the water white transparency polyethylene film.
It is 5: 1: 1 that the described nutritive salt of step (2) contains mass ratio: 1 N element, P element, Si element and Fe element.
Step (3) and (4) described Bdellovibrio are Bdellovibrio (Bdellovibrio sp.) BDFM05, described Bdellovibrio is preserved in Chinese typical culture collection center in the Wuhan University of Luojia Mountain, Wuhan, Hubei Province on August 7th, 2009, and deposit number is CCTCC NO:M 209172.
Described Bdellovibrio BDFM05 carries out carrying out morphologic observation: BDFM05 after the negative staining under electron microscope and is unicellular, ellipse, and size is 1.43 * 0.53um, and end is given birth to flagellum, and flagellum length is 2um at least; Described Bdellovibrio BDFM05 cultivates the transparent circular plaque that can form diameter 2～3mm in three days with the double-layer plate method in 28 ℃;
Described Bdellovibrio telotroch bacterium liquid prepares in accordance with the following methods: the host is in the nutrient broth liquid nutrient medium in inoculation, at 28 ℃ and 200rpm shaking table cultivation 18h, 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 under 4 ℃ of temperature with 6000～8000rpm behind centrifugal 15～20min, with the gained supernatant behind the centrifugal 15～20min of 16000～18000rpm, precipitation DNB liquid nutrient medium, water, physiological saline or 0.2mol/L pH value are 7.2～7.6 phosphate buffer suspension, and obtaining concentration is 10
6～10
9The Bdellovibrio telotroch bacterium liquid of pfu/mL.
Described host is Aeromonas hydrophila (Aeromonas hydrophila); Described DNB liquid nutrient medium is that nutrient broth 0.8g, caseinic acid hydrolysate 0.5g and yeast extract 0.1g are dissolved in the 1000mL distilled water, regulates pH value to 7.2～7.6.
The present invention has following advantage and effect with respect to prior art:
(1) Bdellovibrio telotroch of the present invention causes a disease to aquatic products or potentially pathogenic organism has strong cracking strength, and Bdellovibrio telotroch itself can improve the aquaculture organism intestinal environment, promotes growth, improves Bao Miao immunity.Therefore, compare with other microorganism formulation, the Bdellovibrio telotroch among the present invention has the immunity that improves Bao Miao self, improves its survival rate, accelerates the characteristics such as growth rate of Bao Miao.
What (2) existing haliotis diversicolor Reeve seedling growing process adopted is the mode of flowing water, thus, has not only expended huge human and material resources, financial resources, also can cause certain pollution to environment.And the employing cycle change water and carry out haliotis diversicolor Reeve and grow seedlings, can reduce the demand of output, saved production cost, and can not cause harmful effect environment, the sustainable development of Bao Ye and other aquacultures is had great significance.
Embodiment
Below in conjunction with embodiment the present invention is done further detailed description, but embodiments of the present invention are not limited thereto.Technological parameter for not indicating especially can carry out with reference to routine techniques.
It is to the invention process abalone culture field, Fujian.Culture the pond and build in the breed booth, each pond specification is 2 * 2 * 0.75m, and the depth of water is 0.45m.Disinfecting solution of potassium permanganate is all used in each pond, rinses well repeatedly.It is 70% gobo that pond top covers obscurity, avoids illumination strong excessively.Intensity of illumination generally is controlled at (200-5000) lux.
Embodiment 1
(1) preparation of Bdellovibrio telotroch bacterium liquid
Used Bdellovibrio telotroch ferments by the fermentation process of high density Bdellovibrio telotroch: inoculation 0.5mL 10 in the conical flask that 100mL nutrient broth liquid nutrient medium (sodium chloride 5g, pH 7.4 ± 0.2 for peptone 10g, beef extract powder 3g) are housed
7Cfu/mL Aeromonas hydrophila (Aeromonas hydrophila, bacterium numbering: GIM 1.172 derives from microorganism fungus kind preservation center, Guangdong Province), 200rpm, 28 ℃ of shaking tables were cultivated 18 hours, culture fluid is abandoned supernatant respectively at 4 ℃, the centrifugal 15min of 5000rpm.With 5mL physiological saline suspension precipitation thalline, join one afterwards 100mL DNB (dilute nutrientbroth) liquid nutrient medium (nutrient broth 0.8g is housed, caseinic acid hydrolysate 0.5g, yeast extract 0.1g, sea crystal 28g, be dissolved in 1000mL distilled water, the pH value is 7.2～7.6) conical flask in, insert 1mL again and contain 10
3Pfu/mL Bdellovibrio BDFM05 (CCTCC M209172).Constant temperature shaking table 250rpm, 28 ℃ of cultivation 36h.Culture fluid is got supernatant in 4 ℃ of centrifugal 20min of 6000rpm, again with supernatant in 4 ℃ of centrifugal 20min of 16000rpm, keep precipitation, add 10mL DNB liquid nutrient medium suspended sediment again, obtaining concentration is 10
9The Bdellovibrio telotroch bacterium liquid of pfu/mL.
(2) Bdellovibrio telotroch bacterium liquid is in haliotis diversicolor Reeve (Haliotis diversicolor aquatilis) the middle application of growing seedlings.
The present invention can be divided into two stages altogether.Phase I is the training algae, and the training algae time is 40 days, and this stage is not added Bdellovibrio telotroch bacterium liquid; Second stage is the stage of growing seedlings, and content of the present invention is mainly launched in this stage.Seedling raise period is 40 days, and this stage experimental group is added Bdellovibrio telotroch bacterium liquid immediately behind the water of at every turn changing full pond.With different exchange water cycles, throw the Bdellovibrio telotroch bacterium liquid of variable concentrations respectively to breeding water body.Exchange water cycle is respectively: 5,10,15,20,25,30 days, each quantity of exchanged water was a range.All experiment pool environmental condition of living in comprises that temperature, illumination, pH value, salinity etc. are identical.All test tanks are divided into 25 groups, are A, B-1, B-2, B-3, B-4, C-1, C-2, C-3, C-4, and D-1, D-2, D-3, D-4, E-1, E-2, E-3, E-4, F-1, F-2, F-3, F-4, G-1, G-2, G-3, G-4,2 every group are parallel.The exchange water cycle of each group and to add bacteria concentration as follows:
Following mask body is described this implementation step:
A group: normal flowing water, day quantity of exchanged water is 3 times of the pond water yield, every morning, 7:00 used the high pressure water washing pond, during do not add Bdellovibrio telotroch bacterium liquid, be the plant produced mode;
B organizes (B-1, B-2, B-3, B-4): water once to change (full pond) in 5 days;
C organizes (C-1, C-2, C-3, C-4): water once to change (full pond) in 10 days;
D organizes (D-1, D-2, D-3, D-4): water once to change (full pond) in 15 days;
E organizes (E-1, E-2, E-3, E-4): water once to change (full pond) in 20 days;
F organizes (F-1, F-2, F-3, F-4): water once to change (full pond) in 25 days;
G organizes (G-1, G-2, G-3, G-4): water once to change (full pond) in 30 days;
B-1, C-1, D-1, E-1, F-1, G-1 group be for change behind the water immediately the Bdellovibrio telotroch bacterium liquid of splashing in the pond at every turn, makes that Bdellovibrio telotroch cell concentration reaches 10pfu/mL in the water body.
B-2, C-2, D-2, E-2, F-2, G-2 group be for change behind the water immediately the Bdellovibrio telotroch bacterium liquid of splashing in the pond at every turn, makes that Bdellovibrio telotroch cell concentration reaches 10 in the water body
3Pfu/mL.
B-3, C-3, D-3, E-3, F-3, G-3 group be for change behind the water immediately the Bdellovibrio telotroch bacterium liquid of splashing in the pond at every turn, makes that Bdellovibrio telotroch cell concentration reaches 10 in the water body
5Pfu/mL.
B-4, C-4, D-4, E-4, F-4, G-4 group is for change behind the water immediately the Bdellovibrio telotroch bacterium liquid of splashing in the pond at every turn, makes that Bdellovibrio telotroch cell concentration reaches 10 in the water body
7Pfu/mL.
In phase I training algae process, every pond cloth adheres to 20 of basement membranes, connects algae kind (giving birth to naturally in the local seawater), and the control intensity of illumination is at 2000～5000lux, and water temperature is at 15～25 ℃; According to the algae upgrowth situation, according to 5g/m
3Water body, the mode of adding once in 4 days is added nutritive salt, the ratio that applies of nutritive salt is N: P: Si: Fe=5: 1: 1: 1 (ppm), after 10 days, can observe and be pale green and golden yellow edematus on whole the film, it takes a morsel at the microscopically microscopy for adhering to the skim algae, the finding algae mainly contains large-scale boat-shaped algae, small-sized boat-shaped algae, rhombus algae and avette algae etc., and ratio differs.
In the second stage seedling raising process, also need rationally to regulate illumination, illumination generally is controlled between the 200-2000lux, for algae provides more modest condition, crosses slow and is consumed by Bao Miao to prevent grow too fast accelerated ageing or growth rate of algae.
After algae is trained, the fertilized egg of haliotis diversicolor Reeve is splashed in the pond.Every pond (also being the cloth ovum) 50,000 of splashing.Cloth ovum water temperature is controlled at about 17-22 ℃, in the cloth ovum 3 days, can observe a lot of trochophores that swim in the water body, changes water this period and need filter with 200 mesh filter screens, runs off to prevent larva.After seven days, larva begins metamorphosis, can see being attached with the small young that crawls on adherance, carefully scrapes and gets the microscopically microscopy, observes its vigor.
From training algae to receiving seedling, time is 80 days, young Bao body grows to 3.4～6.4mm, unicellular alga on the adherance is partly aging, algae on the minority adherance is eaten up, and all ponds do not find blank or take off the plate phenomenon, and the spatfall amount on every film is 500～800 and does not wait, can arrive about 1300 on the maximum plates, the index of specifically growing seedlings and measuring method are as follows:
1) haliotis diversicolor Reeve Bao Miao growth and survival condition
A Bao Miao primary quantity, every pond 50,000 seedlings.
It is long that b receives the average shell of seedling, randomly draws 100 Bao Miao, and it is long to measure shell, averages.
The long average daily growth amount of c shell, it is long to receive the average shell of seedling, gets 40 days mean value.
The average individual weight of d Bao Miao is got 100 Bao Miao at random, claims to average after its gross weight.
E receives the seedling amount, takes by weighing 10g Bao Miao, calculates its number, the Bao Miao that collects is claimed the grain number of estimation Bao Miao after its gross weight.
The f survival rate, Bao Miaoliang accounted for the percentage of this stage throwing seedling amount when certain stage finished.
2) the Bao Miao immune indexes is measured
100 of every group of off-test samplings, 20/pipe are put in 5 Eppendoff pipes that fill 1mL Hank ' s buffer solution (pH=7.8) respectively, store in-80 ℃ of mensuration in order to anti-immune indexes.Take out sample during mensuration, it is being melted on ice.Carry out homogenate, 6000r/min then, 4 ℃ of centrifugal 5min get the mensuration that supernatant is used for immune indexes.
With reference to the Bradford method (document Bradford M.A rapid and sensitive method for thequantification of microgram quantities of protein[J] .Analytical Biochemistry, 1976,72:248-254) working sample protein content, with calf serum albumen is standard protein, set up calibration curve, with the Coomassie brilliant blue is developer, reacts 10min in 96 hole ELISA Plate, measures OD in each reaction system with microplate reader
595nmValue.
The mensuration of a, superoxide dismutase (SOD) vigor
The mensuration of 1,2,3,-thrihydroxy-benzene autoxidation speed, under 25 ℃, in 4.5mL 50mmol/L, the K of Ph=8.30
2HPO
4-KH
2PO
4Add 10 μ L 50mmol/L 1,2,3,-thrihydroxy-benzenes in the buffer solution, shake up rapidly, pour in the cuvette of optical path 1cm, under the 325nm wavelength, survey the A value once, require autoxidation speed about 0.070OD/min every 30s.Enzyme assay before adding 1,2,3,-thrihydroxy-benzene, adds SOD sample to be measured (supernatant), records data, is calculated as follows enzymic activity:
Enzymic activity=(0.070-A
325nm)/min * 100% * reactant liquor cumulative volume * sample liquid extension rate/(0.070 * 50% * sample liquid is long-pending)
The enzyme unit definition of living: in every milliliter of reactant liquor, per minute suppresses 1,2,3,-thrihydroxy-benzene autoxidation speed and reaches 50% enzyme amount and be defined as the enzyme unit (U/mL) that lives.
B, antalzyme activity are measured
With the micrococcus lysodeikticus freeze-dried powder is substrate.Use 0.1mol/L, the phosphate buffer of pH=6.4 is made into substrate suspension (OD
570nm≈ 0.3), get this suspension of 3.0mL in the built-in ice bath of test tube, add 50 μ L again and treat test sample, mix, survey A
0Value.Then test solution is moved into 37 ℃ of temperature and bathe mid-30min, place ice bath 10min after the taking-up again,, survey its A value with cessation reaction.Bacteriolyze vigor U
LBy (A
0-A)/calculating of A formula.
Experimental result is by shown in table 1 and the table 2.Table 1 refers to the influence of different seedling raising mannerses to haliotis diversicolor Reeve seedling growing state.As seen, compared with control group (A group), all experimental group improve the survival rate of Bao Miao more significantly, and promote the growth of Bao Miao.Under identical exchange water cycle, along with the rising of the Bdellovibrio telotroch bacterial concentration that adds, the survival rate of Bao Miao and shell are long also in rising trend gradually.
Table 2 refers to the influence of different seedling raising mannerses to haliotis diversicolor Reeve seedling immunity.Data show that compared with control group (A group), the SOD vigor and the antalzyme activity of all experimental group all are significantly improved; Under identical exchange water cycle, along with the rising of the Bdellovibrio telotroch bacterial concentration that adds, Bao Miao immunity also has certain enhancing.
The water quality aspect, ammoniacal nitrogen is received oxidizing process with hypobromous acid and is measured (GB12763.4-91); Nitrite nitrogen diazonium-azo spectrphotometric method for measuring (GB12763.4-91).Through measuring, the every water-quality determination value in the A group pond all is higher than experimental group (B-G group).Wherein, the testing result of A group ammoniacal nitrogen and nitrite nitrogen is respectively 0.083mg/L and 0.011mg/L, and in the experimental group (B-G group), the testing result of ammoniacal nitrogen and nitrite nitrogen is respectively 0.077-0.081mg/L and 0.007-0.009mg/L.In addition, total number of bacteria and vibrios sum context of detection adopt coating nutrient broth flat band method and TCBS flat band method to detect respectively.The result shows that total number of bacteria and vibrios sum in the A group water body reach 10 respectively
5With 10
3The order of magnitude (cfu/mL), the total number of bacteria and the vibrios sum that add the experimental group of Bdellovibrio telotroch bacterium liquid are respectively 10
2～10
3With 10
1～10
2The order of magnitude (cfu/mL), lower than control group, illustrate that Bdellovibrio swimming physical efficiency effectively controls the bacterial number of water body, improve water quality.
Comprehensive each side result adds Bdellovibrio telotroch bacterium liquid, can significantly improve the growth rate of abalone, improves survival rate, enhancing immunity, and can improve water quality, reach the good result of saving water and energy.
The different seedling raising mannerses of table 1 are to the influence of haliotis diversicolor Reeve seedling growing state
The different seedling raising mannerses of table 2 are to the influence of haliotis diversicolor Reeve seedling immunity
The foregoing description is a preferred implementation of the present invention; but embodiments of the present invention are not restricted to the described embodiments; other any do not deviate from change, the modification done under spiritual essence of the present invention and the principle, substitutes, combination, simplify; all should be the substitute mode of equivalence, be included within protection scope of the present invention.
Claims (9)
1. water-saving haliotis diversicolor Reeve seedling-cultivating method is characterized in that comprising following operating procedure:
(1) training algae: the beginning in preceding 30～45 days of collecting seedling is trained algae in training algae pond; The control intensity of illumination is at 2000～5000lux in the training algae process, and the control water temperature is at 15～28 ℃;
(2) Ensure Liquid salt: during the training algae, according to 3～5g/m
3Breeding water body, the mode of adding once in 2～4 days is added nutritive salt;
(3) the cloth ovum of collecting seedling: will train the algae pond and change Chi Shui, and add Bdellovibrio telotroch bacterium liquid, and make the initial concentration of the Bdellovibrio telotroch of Chi Shuizhong reach 10～10
74～50,000 in every pond cloth haliotis diversicolor Reeve fertilized egg during seedling under the pfu/mL, and according to aeration rate 10～60L/h/m
3Carry out aeration, illumination is controlled at 200～3000lux, and water temperature is controlled at 15～28 ℃;
(4) change water: changed water once every 5～30 days, change at every turn and add Bdellovibrio telotroch bacterium liquid behind the water, make the concentration of the Bdellovibrio telotroch of Chi Shuizhong reach 10～10
7Pfu/mL;
(5) peel off and gather: following seedling 30～50 days, young Bao shell reach to peel off behind 3.0～6.5mm gathers.
2. a kind of water-saving haliotis diversicolor Reeve seedling-cultivating method according to claim 1 is characterized in that: the described training of step (1) algae pond washes the back and adopts liquor potassic permanganate to carry out disinfection, and injects the seawater through sand filtration and precipitation process then; A filter bag was wrapped up at the pond water inlet pipe mouth in training algae pond before injecting seawater, prevent that fine sand from going into the pond.
3. a kind of water-saving haliotis diversicolor Reeve seedling-cultivating method according to claim 1 is characterized in that: the described algae of step (1) is a benthic diatom.
4. a kind of water-saving haliotis diversicolor Reeve seedling-cultivating method according to claim 3 is characterized in that: described benthic diatom is a month shape algae, rhombus algae, boat-shaped algae and avette algae.
5. a kind of water-saving haliotis diversicolor Reeve seedling-cultivating method according to claim 1 is characterized in that: the described training of step (1) algae is an adherance with the water white transparency polyethylene film.
6. a kind of water-saving haliotis diversicolor Reeve seedling-cultivating method according to claim 1 is characterized in that: it is 5: 1: 1 that the described nutritive salt of step (2) contains mass ratio: 1 N element, P element, Si element and Fe element.
7. a kind of water-saving haliotis diversicolor Reeve seedling-cultivating method according to claim 1, it is characterized in that: step (3) and (4) described Bdellovibrio are Bdellovibrio (Bdellovibrio sp.) BDFM05, described Bdellovibrio is preserved in Chinese typical culture collection center on August 7th, 2009, and deposit number is CCTCC NO:M 209172.
8. a kind of water-saving haliotis diversicolor Reeve seedling-cultivating method according to claim 1, it is characterized in that: described Bdellovibrio telotroch bacterium liquid prepares in accordance with the following methods: the host is in the nutrient broth liquid nutrient medium in inoculation, at 28 ℃ and 200rpm shaking table cultivation 18h, 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 under 4 ℃ of temperature with 6000～8000rpm behind centrifugal 15～20min, with the gained supernatant behind the centrifugal 15～20min of 16000～18000rpm, precipitation DNB liquid nutrient medium, water, physiological saline or 0.2mol/L pH value are 7.2～7.6 phosphate buffer suspension, and obtaining concentration is 10
6～10
9The Bdellovibrio telotroch bacterium liquid of pfu/mL.
9. a kind of water-saving haliotis diversicolor Reeve cultural method according to claim 8 is characterized in that: described host is an Aeromonas hydrophila (Aeromonas hydrophila); Described DNB liquid nutrient medium is that nutrient broth 0.8g, caseinic acid hydrolysate 0.5g and yeast extract 0.1g are dissolved in the 1000mL distilled water, regulates pH value to 7.2～7.6.
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| CN101638629A (en) * | 2009-08-28 | 2010-02-03 | 华南理工大学 | Bdellovibrio bacteriovorus for preventing and curing rice bacterial diseases and application thereof |
| CN101647406A (en) * | 2009-09-04 | 2010-02-17 | 福建蓝鲸水产有限公司 | Benthic diatom culture method for growing seedlings and abalone fry culture method |
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