CN106222104A - One strain fish molds Antagonistic Actinomycetes QHV2, isolation and identification method and application - Google Patents
One strain fish molds Antagonistic Actinomycetes QHV2, isolation and identification method and application Download PDFInfo
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Abstract
The invention discloses a strain fish molds Antagonistic Actinomycetes QHV2, isolation and identification method and application.This strain fish molds Antagonistic Actinomycetes QHV2 belongs to streptomyces, and Classification And Nomenclature is massif streptomyceteStreptomyces collinus, deposit number is CCTCC NO:M2016140, is deposited in China typical culture collection center on March 23rd, 2016, and preservation address is Wuhan University of Wuhan City of Hubei China province.Antibacterial activity test shows, face-off growth 35 days, and fish molds growth of pathogenic bacteria is substantially suppressed, and ferments 57 days, has preferable anti-saprolegnia activity.Detection of Stability shows, after continuous passage 10 generation, the QHV2 in 35 days can substantially suppress saprolegnia hypha to grow.The actinomycetes QHV2 of the present invention has obvious antagonism to fish molds pathogen, provides certain reference frame for fish molds prevention and control.
Description
Technical field
The present invention relates to strain fish molds Antagonistic Actinomycetes QHV2 and an isolation and identification method thereof, and application.
Background technology
Fish molds (Saprolegnia) be aquaculture causes management of parasitic diseases fish molds (Saprolegniasis) master
Want pathogen.Fish molds without strict selectivity to host, is the worldwide disease of serious threat cultured freshwater fish, especially exists
In the viscid egg hatching later stage, fish molds even can infect fertilization the fish roe grown, and mycelia is wound around and makes germ cell death by suffocation.At fish molds
In the production practices of sick preventing and treating, once used multiple medical treatment, such as salicylic acid, " Galla Chinensis ", malachite green oxalate etc., its
In, maximally efficient medicine is malachite green oxalate, but because of its high residue and " three cause " (carcinogenic, teratogenesis, cause are dashed forward) effect, the most by
It is classified as disabling fisheries drug both at home and abroad.
For finding new fish molds prevention and control active drug, recent domestic scholar is at screening anti-saprolegnia chemicals and biology
The report copper sulfate such as active substance aspect has carried out positive exploration and research, Yang Xianle can suppress the growth of saprolegnia hypha,
Reducing the fish molds infection rate of Grass carp, Ali etc. reports and shows that boric acid can be as the drug candidate of fish molds prevention and control, and Singh etc. reports
Road black substance Semen Brassicae Campestris (Brassica nigra) etc. Chinese herbal medicine all have the higher external fish molds activity that presses down, the report such as Firouzbakhsh
Use probiotic bacteriaEnterococcus faeciumWith probiotics Fructooligosaccharide (FOS)) combine to produce and assist
Survival rate of fish fry and anti-Saprolegnia parasitica infection ability etc. can be significantly improved with effect.But about actinomycetes in fish molds prevention and control
Apply rare report, the fish molds antagonism unwrapping wire that this research separation screening antibacterial activity from Margarita healthy aquaculture Sediments is stable
Bacterium, and it is carried out form and molecular biology identification, to providing some skills for the research and development of fish molds biological control of diseases medicine
Art is supported.
Summary of the invention
In order to overcome at present relevant actinomycetes blank problem present in the fish molds prevention and control, it is an object of the invention to provide a strain
Fish molds Antagonistic Actinomycetes QHV2 and isolation and identification method thereof, and application.
For achieving the above object, the technical solution used in the present invention is as follows:
One of scheme is to provide a strain fish molds Antagonistic Actinomycetes QHV2, this actinomyces streptomyces, and Classification And Nomenclature is massif chain
MyceteStreptomyces collinus, deposit number is CCTCC NO:M2016140, is deposited on March 23rd, 2016
China typical culture collection center, preservation address is Wuhan University of Wuhan City of Hubei China province.
Described fish molds Antagonistic Actinomycetes QHV2 biological property is as follows: bacterium colony circular white in Gause I culture medium,
Flat, diameter 5-9mm, produce purple pigment, outer rim has a ring purple annulus, and edge is more neat but rough；And at glucose ferment
Female cream agar culture medium upper surface is white, and back side purple grows luxuriant, and aerial hyphae is flourishing, examines under a microscope its spore
Sub-silk crimps in the shape of a spiral, and spore is rounded.
The two of scheme are to provide the authentication method of fish molds Antagonistic Actinomycetes QHV2, the method with AF1/AR1 as primer, PCR
Amplification QHV2 bacterial strain 16 S rDNA gene, amplified production carries out 1% agarose gel electrophoresis, and deposition condition is 90v voltage stabilizing,
30min, obtains the amplified production of 1435bp, and PCR primer purified Hou Song Sangon Biotech (Shanghai) Co., Ltd. surveys
Sequence, sequencing result is compared by GenBank blast, and downloads similarity sequence more than 98%, uses
MEGA5.2 software, phylogenetic tree construction；Wherein the sequence of AF1 is that the sequence of agagtttgatcctggctcag, AR1 is
acggctaccttgttacgactt。
The amplified matter of 1435bp is 16S rDNA sequence PCR result.
The three of scheme are to provide the separation method of fish molds Antagonistic Actinomycetes QHV2, comprise the steps:
(1) mud sample is gathered: mud sample picks up from the lakebed bed mud in Margarita freshwater mussel healthy aquaculture base, by room temperature natural for the mud sample gathered
Air-dry, then with mortar, air-dried mud sample is ground, standby；
(2) preparation culture medium: a, Gause I culture medium: medium component is soluble starch 2g, KNO3 0.1g 、K2HPO4
0.05g 、MgSO4· 7H2O 0.05g、 NaCl 0.05g 、FeSO4· 7H2O 0.001g, agar 1.8 g, first use 10-
20 mL cold water, by starch furnishing pasty state, are poured in 60-70 mL boiling water and are heated, and stirring, to transparence, adds culture medium after cooling
Other in addition to starch composition in formula, is settled to 100 mL after dissolving, adjust pH to 7.4～7.6,121 DEG C of sterilizing 20min；b、
The dual anti-culture medium of Gause I: adding nalidixic acid in Gause I culture medium, the final concentration of nalidixic acid reaches 80-120
Mg/L, is simultaneously introduced cycloheximide, and cycloheximide final concentration reaches 30-60 mg/L, for actinomycetic separation and screening；c、
Potato dextrose agar (PDA): fresh peeled potatoes 100.0 g, glucose 10.0 g, agar powder 9.0 g, steaming
Distilled water 500 ml, Rhizoma Solani tuber osi elder generation heated and boiled, then remove slag with filtered through gauze, the benefit that adds water to commercial weight and adds agar powder, 121 DEG C
Sterilizing 20min, for cultivation and the preservation of fish molds；
(3) actinomycetic separation, purification and preservation: take in the triangular flask that 1g mud sample adds 10ml sterilized water, shake 30 min, adopt
With gradient dilution spread plate, sample is applied on the flat board of the dual anti-culture medium of Gause I, 28 DEG C of constant temperature culture 7-10
My god, the actinomycetes choosing bacterium colony dry tack free are forwarded on the flat board of Gause I culture medium, repeatedly after purification 2-3 time, and picking
Single colony inoculation is to Gause I test tube slant, and 28 DEG C of constant temperature culture 6-8 days, 4 DEG C of Storage in refrigerator are standby.
The four of scheme are to provide fish molds Antagonistic Actinomycetes QHV2 application in fish molds prevention and control.
Beneficial effects of the present invention:
(1) the fish molds Antagonistic Actinomycetes QHV2 that the present invention relates to is that can having fish molds growth of pathogenic bacteria of finding first is substantially pressed down
That make and that antibacterial activity is stable fish molds Antagonistic Actinomycetes, has filled up the blank of current research；
(2) being separated into of fish molds Antagonistic Actinomycetes QHV2 builds the phylogenetic tree of QHV2 bacterial strain and provides the foundation guarantee.
Accompanying drawing explanation
Fig. 1 is the actinomycetes QHV2 fungistatic effect to fish molds；Wherein A: Pelteobagrus fulvidraco ovum fish molds pathogen compares；B: actinomycetes
QHV2 and Pelteobagrus fulvidraco fish molds pathogen face-off growth 3 days；C, actinomycetes QHV2 and Pelteobagrus fulvidraco fish molds pathogen face-off growth 5 days.
Fig. 2 is that actinomycetes QHV2 compares with fish molds face-off 3 days, the bacteriostasis rate of 5 days.
Fig. 3 is to use after test tube slant continuous passage 10 generation the fungistatic effect figure to fish molds, wherein A: Pelteobagrus fulvidraco ovum fish molds
Pathogen compares；B: actinomycetes QHV2 grows 3 days with the face-off of Pelteobagrus fulvidraco fish molds pathogen；C: actinomycetes QHV2 with Pelteobagrus fulvidraco water
Mould pathogen face-off growth 5 days.
Fig. 4 is the different fermentations time actinomycetes QHV2 to be produced the impact of fish molds antagonistic activity material；Wherein A:PDA puts down
The Pelteobagrus fulvidraco ovum fish molds pathogen comparison of 3 days is grown on plate；B: ferment 3 days actinomycetes QHV2 and fish molds face-off growth 3 days；C: send out
5 days actinomycetes QHV2 of ferment and fish molds face-off growth 3 days；D: ferment 7 days actinomycetes QHV2 and fish molds face-off growth 3 days.
Fig. 5 is that the bacteriostasis rate that fish molds stands facing each other 3 days with the actinomycetes QHV2 of different fermentations time compares.
Fig. 6 is actinomycetes QHV2 anti-saprolegnia active substance heat stability test results, wherein A: cultivate the Pelteobagrus fulvidraco ovum of 3 days
Fish molds pathogen compares；B: actinomycetes QHV2 grows 3 days with fish molds face-off；A: sterile supernatant after heat treatment；B: after heat treatment
Bacterium solution；C: bacterium solution before heat treatment.
Fig. 7 is the colonial morphology microscopy results of actinomycetes QHV2.
Fig. 8 is mycelia, fibrillae of spores and the spore shape that actinomycetes QHV2 grows on glucose yeast cream agar culture medium.
Fig. 9 is the 16S rDNA gene PCR amplification product gel electrophoresis detection result of actinomycetes QHV2, M:1 kb
DNA Marker；1-3: annealing temperature be respectively 58 DEG C, 60 DEG C, 62 DEG C time QHV2 16S rDNA gene PCR amplified production.
Figure 10 is the phylogenetic tree of actinomycetes QHV2 16S rDNA gene order.
Detailed description of the invention
Below in conjunction with embodiment, the present invention will be further described, the experiment side of unreceipted actual conditions in the following example
Method, is typically considered to the known approaches of this area.And following example are only one of preferred embodiment of the invention, and unrestricted
The protection domain of invention.
Embodiment one
(1) collection of mud sample and process: mud sample picks up from Hunan the Miluo River and the lakebed bed mud in numerous mountains red Margarita freshwater mussel healthy aquaculture base,
The mud sample at room temperature natural air drying that will gather, then grinds air-dried mud sample with mortar, standby.
(2) culture medium preparation
Gause I culture medium: composition has soluble starch 2g, KNO3 0.1g 、K2HPO4 0.05g 、MgSO4· 7H2O
0.05g、 NaCl 0.05g 、FeSO4· 7H2O 0.001g, agar 1.8g, first stick with paste starch furnishing with 10-20 cold water mL
Shape, pours in 60-70 mL boiling water, heated and stirred to transparence, and other added in culture medium prescription in addition to starch after cooling becomes
Point, it being settled to 100 mL after dissolving, adjust pH to 7.4～7.6,121 DEG C of sterilizing 20min, for actinomycetic purification and cultivation.
The dual anti-culture medium of Gause I: add nalidixic acid (final concentration 80-120 mg/L) in Gause I culture medium
With cycloheximide (final concentration 30-60mg/L), for actinomycetic separation and screening.
Sucrose Cha Shi agar culture medium: observe for actinomycetes morphological characteristic, sucrose 3 g, FeSO4· 7H2O 0.001
G, K2HPO4 0.1g, MgSO4·7H2O 0.05g, KCl 0.05g, NaNO3 0.2 g, distilled water 100 ml, accurately weigh
Above-mentioned formula materials, after dissolving constant volume, adjusts PH to 7.2, in subpackage to triangular flask, adds agar powder 2g, 121 DEG C of sterilizings
20min。
Glucose yeast cream agar culture medium: observe for actinomycetes morphological characteristic, glucose 1g, yeast extract 1 g,
K2HPO40.1 g, distilled water 100 ml, accurately weigh above-mentioned formula materials, after dissolving constant volume, adjusts PH to 7.2, subpackage to three
In the bottle of angle, add agar powder 2 g, 121 DEG C of sterilizing 20min.
Number agar culture medium of kirschner: observe for actinomycetes morphological characteristic, glucose 2 g, KNO3 0.1 g,Nacl
0.05 g,K2HPO4 0.1 g, FeSO4· 7H2O0.001 g,MgCO3·7H2O 0.03 g,CaCO30.05 g, distilled water
100 ml, accurately weigh above-mentioned formula materials, after dissolving constant volume, adjust PH to 7.2, in subpackage to triangular flask, add agar powder 2
G, 121 DEG C of sterilizing 20min.
Potato dextrose agar (PDA): fresh peeled potatoes 100 g, glucose 10 g, agar powder 9 g,
Distilled water 500 ml, Rhizoma Solani tuber osi elder generation heated and boiled, then remove slag with filtered through gauze, the benefit that adds water to commercial weight and adds 10 g agar
Powder, for cultivation and the preservation of fish molds.
(3) tested water mo(u)ld: Pelteobagrus fulvidraco ovum fish molds pathogen is separated by Shanghai Ocean University Yang Xianle professor's laboratory and protects
Hide.
(4) actinomycetic separation, purification and preservation: take in the triangular flask that 1g mud sample adds 10 mL sterilized water, shakes 30
Min, uses gradient dilution spread plate that sample is applied to the flat board of the dual anti-culture medium of Gause I, 28 DEG C of constant temperature culture 7-
10 days, choosing the actinomycetes of bacterium colony dry tack free and be forwarded to Gause I flat board, repeatedly after purification 2-3 time, picking list bacterium colony connects
Kind to Gause I test tube slant, 28 DEG C of constant temperature culture about 7 days, 4 DEG C of Storage in refrigerator are standby.
(5) antibacterial activity test and Detection of Stability
It is tested bacterium with Pelteobagrus fulvidraco ovum fish molds pathogen, uses agar to move block method and institute's separating payingoff bacteria does flat board face-off and grows real
Test, the fish molds antagonistic activity of detection institute separating payingoff bacteria, with bacteriostasis rate for the examination actinomycetic antagonistic activity of Indexs measure, antibacterial
Rate=(pathogen diameter after comparison pathogen diameter-process)/comparison pathogen diameter), result is shown in Fig. 1, Fig. 2.Actinomycetes antagonism
The stability of activity is forwarded to Gause I culture medium and ferments 5 days after using flat board continuous passage 10 generation, pipette agar block and target
Mark water mo(u)ld carries out face-off growth 3 days, and the result of suppression fish molds growth is shown in Fig. 3.QHV2 bacterial strain carries out 3 on Gause I flat board
My god, 5 days, 7 days cultivation and fermentation, with Pelteobagrus fulvidraco ovum fish molds pathogen face-off growth 3 days, explore fermentation time to produce antagonistic activity thing
The impact of matter, result is shown in Fig. 4, Fig. 5.The heat stability test of active substance, actinomycetes the most to be measured are trained at Gause I liquid
After supporting base fermentation 7 days, 10000 r/min high speed centrifugations, take aseptic 80 DEG C of water-bath 1h of cleer and peaceful bacterium solution respectively standby, then use nothing
Bacterium card punch makes a call to 6 holes around PDA plate uniformly, takes one piece of fish molds block afterwards and is connected on PDA plate central authorities, then right to three
Hole is separately added into the bacterium solution of the supernatant of heat treatment, heat treatment, with actinomycete fermentation stock solution for comparison, flat board is placed in 28 DEG C of perseverances
Temperature cultivate 3 days, 5 days, after 7 days observe saprolegnia hypha growth inhibited situation, result such as Fig. 6.
As shown in Figure 1, make, for examination target bacterium, to grow 3-5 days with actinomycetes QHV2 face-off with Pelteobagrus fulvidraco ovum fish molds pathogen,
Fish molds growth of pathogenic bacteria is the most substantially suppressed, and creates certain anti-saprolegnia active matter after this explanation actinomycetes QHV2 fermentation
Matter.
As shown in Figure 2, the relatively different face-off growth time actinomycetes QHV2 bacteriostasis rate to fish molds, face-off growth 3 days, presses down
Bacterium rate reaches 68%, and face-off growth 5d bacteriostasis rate is 64%.
From the figure 3, it may be seen that after continuous passage 10 generation, in 3-5 days, actinomycetes QHV2 can substantially suppress saprolegnia hypha to grow, this
Illustrate that actinomycetes QHV2 produces the hereditary stability of fish molds antagonistic activity material preferably, and the bacteriostasis rate of actinomycetes QHV2 is still up to
67%。
From Fig. 4, Fig. 5, actinomycetes QHV2 fermentation 5d suppression fish molds effect is best, saprolegnia hypha and actinomycetes truffle it
Between transparent distance maximum, along with its fish molds antagonistic activity of prolongation of fermentation time significantly reduces, but the fermentation actinomycetes of 7 days are also
Being demonstrated by preferable anti-saprolegnia activity, this explanation fermentation time has difference to the production of actinomycetes QHV2 fish molds antagonistic activity material
Impact, its active substance stability there is also bigger difference；Fermentation culture 3 days, 5 days, the actinomycetes QHV2 of 7 days and fish molds pair
The bacteriostasis rate cultivated 3 days that stands erect is respectively 67%, 71% and 56%.Go back on agar block during in view of actinomycetes with fish molds face-off growth
May proceed to produce active substance, it is proposed that the fermentation time producing anti-saprolegnia active substance is 5-7 days.
As seen from Figure 6, compared with the control, either QHV2 bacterial strain fermentation liquor stock solution, or the aseptic supernatant after heat treatment
Liquid or bacterium solution, the most fairly obvious to the fungistatic effect of Pelteobagrus fulvidraco ovum water mo(u)ld, bacteriostasis rate is up to more than 62%.This illustrates actinomycetes
The fish molds antagonistic activity material that QHV2 fermentation produces is heat stable compound.
(6) actinomycetic form and molecular biology identification
Actinomycetic morphologic observation uses Leica M205 Stereo microscope to carry out observing and microscopic photography, and actinomycetes are at Gao Shi mono-
The colonial morphology after 7d is cultivated as shown in Figure 7 on number flat board；Actinomycetes QHV2 base on glucose yeast cream agar culture medium
Interior mycelia, aerial hyphae and fibrillae of spores spore shape are as shown in Figure 8；Actinomyces strain QHV2 cultural characteristic on different culture media
As shown in Table 1.Molecular Identification uses PCR amplification technique, and bacterial genomes DNA prepared with pyrolysis method is as template, and use is drawn
Thing AF 1/ AR1 expands actinomycetes 16S rDNA sequence, and PCR reaction system (50 μ L) is: 2X GC BufferI 25 μ l,
DNTP 3 μ l, primer AF1 1.5 μ l, AR1 1.5 μ l, DNA profiling 1 μ l, La Taq enzyme: 0.5 μ l, dd H2O 17.5μl。
PCR reaction condition: after 94 DEG C of denaturation 5 min, 90 DEG C of degeneration 30 s, 58 DEG C of annealing 30 s, 72 DEG C are prolonged
Stretching 1.5min, 30 circulations, last 72 DEG C extend 10 min, and amplified production carries out 1% agarose gel electrophoresis detection
(90v, 30min), result shows, it is thus achieved that the clear band of about 1.5kb size, concrete electrophoresis result is as shown in Figure 9.PCR
The order-checking of product purified Hou Song Sangon Biotech (Shanghai) Co., Ltd., sequencing result is entered by GenBank blast
Row comparison is analyzed, and downloads similarity sequence more than 98%, uses MEGA5.2 software, and phylogenetic tree construction, result is shown in
Figure 10.
Table one: actinomycetes QHV2 cultural characteristic on different culture media:
Known to Fig. 7, QHV2 bacterium colony circular white, flat, diameter 5-9mm, produce purple pigment, outer rim has a ring purple annulus.
White at glucose yeast cream agar culture medium upper surface from Fig. 8 and Biao mono-, actinomycetes QHV2, the back side is purple
Color, grows luxuriant, and aerial hyphae is flourishing.Examining under a microscope its fibrillae of spores to crimp in the shape of a spiral, spore is rounded.According to it
In the morphological characteristic (table one) identified in culture medium, tentatively actinomycetes QHV2 is belonged to streptomyces.
As shown in Figure 9, annealing temperature be respectively 58 DEG C, 60 DEG C, 62 DEG C time, PCR has amplified the special of about 1.5kb
Property band, and during annealing temperature 58 DEG C, amplified production band is the brightest, effect is best.Above-mentioned PCR primer is sent to after purification raw
Work biological engineering (Shanghai) limited company checks order, and through GenBank blast comparison on NCBI, chooses in GenBank
With the bacterial strain of QHV2 bacterial strain 16S rDNA sequence similarity more than 98%, utilize MEGA5.2 software, build the system of QHV2 bacterial strain
Grow tree.
As seen from Figure 10: based on 16S rDNA build phylogenetic tree, QHV2 with Streptomyces collinus
NR-041063 is polymerized to cluster, and sibship is nearest, thus identifies that actinomycetes QHV2 isStreptomyces collinus。
[0001]
＜ 110 ＞ Hunan University of Arts and Science
＜ 120 ＞ mono-strain fish molds Antagonistic Actinomycetes QHV2, isolation and identification method and application
〈160〉1
〈210〉1
〈211〉1435
〈212〉DNA
＜ 213 ＞ massif streptomycete (Streptomyces collinus)
〈220〉
〈223〉16S rDNA
〈400〉1
GGCCGGTGGC GGGCTGCTTA CCATGCAAGT CGAACGATGA ACCACTTCGG TGGGGATTAG 60
TGGCGAACGG GTGAGTAACA CGTGGGCAAT CTGCCCTGCA CTCTGGGACA AGCCCTGGAA 120
ACGGGGTCTA ATACCGGATA CTGATCCGTC TGGGCATCCA GACGGTTCGA AAGCTCCGGC 180
GGTGCAGGAT GAGCCCGCGG CCTATCAGCT TGTTGGTGAG GTAGTGGCTC ACCAAGGCGA 240
CGACGGGTAG CCGGCCTGAG AGGGCGACCG GCCACACTGG GACTGAGACA CGGCCCAGAC 300
TCCTACGGGA GGCAGCAGTG GGGAATATTG CACAATGGGC GAAAGCCTGA TGCAGCGACG 360
CCGCGTGAGG GATGACGGCC TTCGGGTTGT AAACCTCTTT CAGCAGGGAA GAAGCGAAAG 420
TGACGGTACC TGCAGAAGAA GCGCCGGCTA ACTACGTGCC AGCAGCCGCG GTAATACGTA 480
GGGCGCGAGC GTTGTCCGGA ATTATTGGGC GTAAAGAGCT CGTAGGCGGC TTGTCACGTC 540
GGTTGTGAAA GCCCGGGGCT TAACCCCGGG TCTGCAGTCG ATACGGGCAG GCTAGAGTTC 600
GGTAGGGGAG ATCGGAATTC CTGGTGTAGC GGTGAAATGC GCAGATATCA GGAGGAACAC 660
CGGTGGCGAA GGCGGATCTC TGGGCCGATA CTGACGCTGA GGAGCGAAAG CGTGGGGAGC 720
GAACAGGATT AGATACCCTG GTAGTCCACG CCGTAAACGG TGGGCACTAG GTGTGGGCAA 780
CATTCCACGT TGTCCGTGCC GCAGCTAACG CATTAAGTGC CCCGCCTGGG GAGTACGGCC 840
GCAAGGCTAA AACTCAAAGG AATTGACGGG GGCCCGCACA AGCGGCGGAG CATGTGGCTT 900
AATTCGACGC AACGCGAAGA ACCTTACCAA GGCTTGACAT ACACCGGAAA GCATTAGAGA 960
TAGTGCCCCC CTTGTGGTCG GTGTACAGGT GGTGCATGGC TGTCGTCAGC TCGTGTCGTG 1020
AGATGTTGGG TTAAGTCCCG CAACGAGCGC AACCCTTGTC CCGTGTTGCC AGCAGGCCCT 1080
TGTGGTGCTG GGGACTCACG GGAGACCGCC GGGGTCAACT CGGAGGAAGG TGGGGACGAC 1140
GTCAAGTCAT CATGCCCCTT ATGTCTTGGG CTGCACACGT GCTACAATGG CCGGTACAAT 1200
GAGCTGCGAT ACCGCGAGGT GGAGCGAATC TCAAAAAGCC GGTCTCAGTT CGGATTGGGG 1260
TCTGCAACTC GACCCCATGA AGTCGGAGTC GCTAGTAATC GCAGATCAGC ATTGCTGCGG 1320
TGAATACGTT CCCGGGCCTT GTACACACCG CCCGTCACGT CACGAAAGTC GGTAACACCC 1380
GAAGCCGGTG GCCCAACCCC TTGTGGGAGG AGCTTCGAAA GGTGGAACCT GGGGG 1435
Claims (4)
1. a strain fish molds Antagonistic Actinomycetes QHV2, this actinomyces streptomyces, Classification And Nomenclature is massif streptomyceteStreptomyces collinus, deposit number is CCTCC NO:M2016140, is deposited in China on March 23rd, 2016
Type Tissue Collection, preservation address is Wuhan University of Wuhan City of Hubei China province.
The authentication method of fish molds Antagonistic Actinomycetes QHV2 the most according to claim 1, it is characterised in that be with AF1/AR1
Primer, PCR expands QHV2 bacterial strain 16 S rDNA gene, and amplified production carries out 1% agarose gel electrophoresis, and deposition condition is 90v
Voltage stabilizing, 30min, obtain the amplified production of 1435bp, wherein the sequence of AF1 is the sequence of agagtttgatcctggctcag, AR1
For acggctaccttgttacgactt.
The separation method of fish molds Antagonistic Actinomycetes QHV2 the most according to claim 1, it is characterised in that include walking as follows
Rapid:
(1) mud sample is gathered: mud sample picks up from the lakebed bed mud in Margarita freshwater mussel healthy aquaculture base, by room temperature natural for the mud sample gathered
Air-dry, then with mortar, air-dried mud sample is ground, standby；
(2) preparation culture medium: a, Gause I culture medium: medium component is soluble starch 2g, KNO3 0.1g 、K2HPO4
0.05g 、MgSO4· 7H2O 0.05g、 NaCl 0.05g 、FeSO4· 7H2O 0.001g, agar 1.8 g, first use 10-
20 mL cold water, by starch furnishing pasty state, are poured in 60-70 mL boiling water and are heated, and stirring, to transparence, adds culture medium after cooling
Other in addition to starch composition in formula, is settled to 100 mL after dissolving, adjust pH to 7.4～7.6,121 DEG C of sterilizing 20min；b、
The dual anti-culture medium of Gause I: adding nalidixic acid in Gause I culture medium, the final concentration of nalidixic acid reaches 80-120
Mg/L, is simultaneously introduced cycloheximide, and cycloheximide final concentration reaches 30-60 mg/L, for actinomycetic separation and screening；
(3) actinomycetic separation, purification and preservation: take in the triangular flask that 1g mud sample adds 10ml sterilized water, shake 30 min, adopt
With gradient dilution spread plate, sample is applied on the flat board of the dual anti-culture medium of Gause I, 28 DEG C of constant temperature culture 7-10
My god, the actinomycetes choosing bacterium colony dry tack free are forwarded on the flat board of Gause I culture medium, repeatedly after purification 2-3 time, and picking
Single colony inoculation is to Gause I test tube slant, and 28 DEG C of constant temperature culture 6-8 days, 4 DEG C of Storage in refrigerator are standby.
Fish molds Antagonistic Actinomycetes QHV2 the most according to claim 1, it is characterised in that QHV2 answering in fish molds prevention and control
With.
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| CN107412322A (en) * | 2017-08-22 | 2017-12-01 | 中国水产科学研究院珠江水产研究所 | It is a kind of to be used to prevent and treat composition of aquaculture saprolegniasis and preparation method thereof |
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| CN101985605A (en) * | 2010-08-24 | 2011-03-16 | 上海海洋大学 | Saprolegnia antagonist and application thereof |
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| CN101985605A (en) * | 2010-08-24 | 2011-03-16 | 上海海洋大学 | Saprolegnia antagonist and application thereof |
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| 贺凤: "水霉拮抗菌的筛选及其拮抗活性物质稳定性初步研究", 《微生物学通报》 * |
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| CN107412322A (en) * | 2017-08-22 | 2017-12-01 | 中国水产科学研究院珠江水产研究所 | It is a kind of to be used to prevent and treat composition of aquaculture saprolegniasis and preparation method thereof |
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