CN113862228B - Broad-spectrum virulent phycophage MinS1 and application thereof - Google Patents
Broad-spectrum virulent phycophage MinS1 and application thereof Download PDFInfo
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Abstract
The invention belongs to the field of phagosome, in particular to a broad-spectrum virulent phagosomeMicrocystis aeruginosa cyanophage MinS1(MinS 1) and application thereof, the phycophage is preserved in the common microorganism center of the microorganism strain preservation management committee at 9 and 7 of 2021, and the preservation number is CGMCC No.23089. After a plurality of blue algae are infected by the lytic phycophage, the blue algae can be proliferated in a host and can be lysed, and the broad spectrum of the blue algae can lead the blue algae to have great application potential; the algae-phagostimulant is separated from natural water, can safely and efficiently crack a plurality of blue algae in fresh water, and does not harm the environment and human beings; meanwhile, the algae phagosome has simple culture conditions and easy expansion, has potential application value in the aspect of preventing and treating the water bloom caused by the blue algae, and can be used as a potential novel algae control product and means for treating the blue algae water bloom.
Description
Technical Field
The invention belongs to the field of phycophage, and particularly relates to a broad-spectrum virulent phycophage MinS1 and application thereof, and relates to biological treatment of cyanobacteria bloom.
Background
The eutrophication of water is a global water pollution problem, and can cause mass propagation of cyanobacteria, the explosive growth of cyanobacteria is easy to form cyanobacteria bloom, the influence range is wide, the hazard is serious, and huge losses are caused to economy and environment. 75% of lakes in China face different degrees of eutrophication pollution, and the method is one of countries with the most serious and most widely distributed blue algae bloom in the world, and has important practical significance for effectively controlling and treating the bloom. The existing technology for controlling algae bloom can be divided into chemical method, mechanical cleaning method, nutrition control method, hydrodynamic control method, biological control method and the like, wherein the biological control method for removing algae by adopting microorganisms has wide application prospect.
Among the several methods available, physical and chemical methods are most commonly used. Although the physical method has quick effect on algae control, a large amount of manpower and material resources are consumed. The chemical method mainly controls algae through chemical agents, and has the defect that the chemical agents can cause pollution in water bodies.
The phagosome is a virus population which specifically infects prokaryotic algae, and the infected blue algae dies through a series of steps of adsorption, invasion, biosynthesis, maturation, release and the like, so that the quantity of the blue algae is effectively reduced, and the generation of water bloom is reduced or eliminated. After blue algae die, the algae phagosome gradually dies due to the loss of hosts, and does not cause secondary pollution and damage to natural water bodies, so the algae phagosome is a harmless biological control factor of blue algae bloom. However, most of the algae phagosome reported at present has strong species and strain specificity, narrow algae killing spectrum and long cracking period, and needs several days or more than ten days. Such as MA-LMM01 isolated from Mikata lake of Japan, can specifically infect only one strain of toxic microcystis aeruginosa and has a long incubation period; liu Xinyao and the like, the discovered short-tail phaeophaga bodies Pf-WMP3 and Pf-WMP4 can infect the same host blue algae but have narrow algae killing spectrum; the S-LBS1 is proved by an infection experiment to be dissolved only in the Synechococcus TCC 793; in addition, newly discovered phaeophagosomes in the last two and three years, such as PA-SR01 phaeophagosome only lyses Pseudoanabaena KCZY-C8, but not the other two Pseudoanabaena strains and 17 other cyanobacteria strains; mic1 has an infectious effect only on its host, microcystis hui FACHB-1339. Blue algae bloom is usually formed by mixing and exploding a plurality of blue algae, and the propagation speed is rapid, so that the application of the existing phaeophagy body for treating the blue algae bloom is limited.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a broad-spectrum cracking phycophage capable of safely, efficiently and rapidly cracking various blue algae and application thereof.
The technical scheme adopted for solving the technical problems is as follows: a broad-spectrum lytic phaeophaeoosome isolated from Microcystis aeruginosa Microcystis aeruginosa FACHB-905 is named Microcystis aeruginosa cyanophage MinS1 (MinS 1 is abbreviated as Siphoviridae, min), and belongs to the family Leptodaceae in classification. The microbial strain is preserved in the general microbiological center of the microbiological strain preservation management committee at 9 and 7 of 2021, the preservation number is CGMCC No.23089, and the preservation organization address is: the institute of microbiology, national institute of sciences, no. 3, north chen west way 1, region of korea, beijing city, postal code: 100101.
the biology characteristics of the phagosome are as follows: the phycophage MinS1 has a head structure with icosahedron and an slender tail, the head diameter is about 54nm (+ -5 nm), and the tail length is 260nm (+ -5 nm); the phycophage MinS1 can form transparent and uniform sized plaques on the algae flat plate of the microcystis aeruginosa Microcystis aeruginosa FACHB-905, and has no halo around and clear and regular edges; the phycophage MinS1 has a wide host range and can cross-mesh lyse a plurality of blue algae strains, including: chlorella, tremella, segment-breeding algae, nostoc, synechococcus;
BLASTN alignment with the whole genome sequence of MinS1 did not find a sequence identical thereto and the highest homology was only 1%, indicating that MinS1 is a new phycophage.
The separation and purification method of the phycophage MinS1 specifically comprises the following steps:
(1) Cultivation of Microcystis aeruginosa Microcystis aeruginosa FACHB-905 as target alga species
5mL of microcystis aeruginosa Microcystis aeruginosa FACHB-905 algae liquid is diluted into a conical flask filled with 500mL of BG11 liquid culture medium according to the volume ratio of 1:100, and is placed in an illumination incubator with the temperature of 25 ℃ and the illumination intensity of 2000lx and the light-dark period of 12h:12h, and the logarithmic phase FACHB-905 algae liquid is obtained after about 10 days, and OD 680 ≈0.6。
(2) Enrichment and screening of phagostimulants
And (3) water sample treatment: collecting water sample of the surface water of Fujian Zhangzhou Ma Yangxi at 11/23/2020, taking the water sample back to the laboratory at low temperature, and centrifuging at 12000g for 20 min at 4deg.C to remove the precipitate. The supernatant was taken for the experiment. The phagosome was harvested in this step.
Taking sterile conical flasks with the number corresponding to the number of samples, and respectively adding 30mL of water sample supernatant with the treated sampling points and 30mL of logarithmic phase FACHB-905 algae liquid into each conical flask. Culturing in an illumination incubator after mixing, and manually shaking the algae for 2-3 times per day. Meanwhile, water taken by the water purifier is used for replacing water sample supernatant as a control. The yellow culture broth was centrifuged at 6000g for 10 minutes, and the supernatant was filtered through a 0.45 μm and 0.22 μm nitrocellulose filter in this order. Adding the filtrate into FACHB-905 algae solution in logarithmic phase of 10 times volume, mixing, and culturing until obvious yellowing phenomenon appears to obtain the primary screening infection solution.
(3) Preliminary purification of phagosome
Preparing concentrated algae liquid (1:20): taking the logarithmic phase FACHB-905 algae liquid prepared in the step (1), centrifuging 10000g for 10 minutes, and retaining supernatant which is equivalent to 1/20 of the original volume for suspension precipitation;
the melted BG11 medium containing 0.7% (W/V) agar is split into 8 mL/tube in an ultra clean bench, and placed in a constant temperature water bath at 45 ℃ for 30min until the temperature is constant.
Taking the initially screened infection solution containing phaga-phaga body obtained in the step (2), and carrying out serial gradient dilution (10) on the infection solution with BG11 liquid culture medium -1 _10 -6 ) The method comprises the steps of carrying out a first treatment on the surface of the Taking the diluted liquid of each dilution according to the volume ratio of 1:9 is added into 0.9mL of concentrated FACHB-905 algae liquid in logarithmic phase, mixed evenly, placed in a shaking table with the rotating speed of about 70rpm, and incubated for 20-30 minutes at 25 ℃ to enable viruses to be fully adsorbed to algae cells;
taking 8 mL/tube of BG11 culture medium containing 0.7% agar from 45 ℃ constant temperature water bath, immediately wiping water outside the tube with paper, pouring the virus-algae mixed solution into the tube, rapidly shaking the tube with a vortex oscillator for 3-5s to fully mix the virus-algae mixed solution, immediately pouring the virus-algae mixed solution onto a BG11 culture substrate layer flat plate (about 110mm in diameter) containing 1.5% (W/V) agar, sealing a sealing film for sealing a sealing hole (at a sealing film at the junction of a dish and a cover) after the virus-algae mixed solution is solidified, and inverting the sealing film at 2000lx and 25 ℃ for light-dark period: culturing in a 12h/12h incubator and observing plaque formation (7 days were required). The independent phycophage formed under the maximum dilution is dug and suspended in 500 mu L of BG11 culture medium, and is placed in a refrigerator at 4 ℃ for overnight or at 25 ℃ for light-proof incubation for 1 hour, so that the phycophage is thoroughly released, 12000g is centrifuged for 10 minutes, and the supernatant is taken to obtain the first generation of phycophage.
(4) Further purification of phagosome
Taking the first-generation phagosome obtained in the step (3), and serially and gradiently diluting the first-generation phagosome into 10 by using BG11 culture medium -1 To 10 -6 Repeating the plaque experiment in the step (3) for at least 3 times to obtain the plaque with uniform shape and size. Single plaques are picked up, suspended in 500 mu L BG11 medium, incubated at 4 ℃ overnight in a refrigerator or at 25 ℃ in the dark for 1 hour, centrifuged at 10000g for 10-20 minutes, and the supernatant is taken as a suspension of purified phaeophagosome MinS1.
(5) Enlarged culture of phaga
Adding 1mL of the purified phagosome MinS1 suspension obtained in the step (4) into 10mL of fresh cultivated logarithmic phase FACHB-905 algae liquid, uniformly mixing, cultivating in an illumination incubator until the algae liquid is yellow to obtain phagosome MinS 1-algae cultivation lysate, centrifuging 6000g of lysate for 10 minutes, and collecting supernatant. The supernatant was added to fresh cultured logarithmic phase FACHB-905 algae liquid at a ratio of 1:10 (V/V), mixed well, and cultured in an illumination incubator for 24 hours to yellow the algae liquid. The expansion culture was repeated in this manner.
(6) Preparation of algae-phagostimulant suspension
Taking the phycophage MinS 1-algae culture lysate prepared in the step (5), centrifuging at 4 ℃ for 10min at 6000g, taking supernatant, and filtering sequentially through nitrocellulose filter membranes with the pore diameters of 0.45 mu m and 0.22 mu m to obtain the phycophage MinS1 suspension.
The invention also provides the application of the broad-spectrum high-efficiency virulent phaeophaeophaeophaeophaeophaeophaeophaeophaea MinS1 in cracking chlorella, tremella, segment-breeding algae, nostoc or synechococcus and in preventing and treating cyanobacteria bloom.
When in use, the phaeophagosome suspension is added into a cyanobacterial bloom sample, and has a cracking effect on the chlorella, the tremella, the segment-cultivated algae, the nostoc and the synechococcus in the cyanobacterial bloom sample.
The phaeophagosome MinS1 has the functions of cracking microcystis aeruginosa FACHB-905, microcystis aeruginosa FACHB-942, microcystis aeruginosa FACHB-469, microcystis aeruginosa FACHB-924, microcystis aeruginosa FACHB-1326, microcystis Hui FACHB-908, microcystis Hui FACHB-1317, microcystis Hui FACHB-1318, microcystis Hui FACHB-929, microcystis viridis FACHB-1342, microcystis viridis FACHB-1337, microcystis FACHB-915, anabaena water bloom FACHB-245, microcystis lupula FACHB-1255, microcystis candidia FACHB-596, fusarium albe FACHB-1166, fusarium garicum FACHB-881, fuscoporia FACHB-708, fuscoparia angusta FACHB-402, fachb-240, fachb and Fachb 805. The broad spectrum virulence is reflected in that MinS1 can infect and lyse the above 22 algae strains, and spans five purposes: synechococcus, botryococcus, nostoc and Chromococcoccoccoccyales are the most broad-spectrum one strain of phaeophaga.
In summary, the invention provides a novel broad-spectrum lytic phaeophaga MinS1, a separation method and application thereof, and the virus can lyse the host algae microcystis aeruginosa, can infect related strains of Synechococcus, phaeophyta, oscillatoria and Nostoc, and has potential application value in preventing and treating water bloom caused by blue algae.
Compared with the prior art, the invention has the advantages that: the invention discloses a novel broad-spectrum lytic phycophage MinS1 capable of lysing a plurality of blue algae across five orders for the first time, and a separation method and application thereof, which make up for the limitation of narrow host range and limited application of the phycophage reported before. After the lytic phycophage is infected with a plurality of blue algae, the blue algae can be proliferated in a host and can be lysed, and the broad spectrum of the blue algae can lead the blue algae to have great application potential; the algae-phagostimulant is separated from natural water, can safely and efficiently crack a plurality of blue algae in fresh water, and does not harm the environment and human beings; meanwhile, the algae phagosome has simple culture conditions and easy expansion, has potential application value in the aspect of preventing and treating the water bloom caused by the blue algae, and can be used as a potential novel algae control product and means for treating the blue algae water bloom.
Drawings
FIG. 1 is a transmission electron microscope image of a negatively stained phagosome MinS 1;
FIG. 2 shows the morphology of the plaques of the phycophage MinS1 on a double-layer plate of Microcystis aeruginosa FACHB-905;
FIG. 3 is a result of Blastn alignment of the genomic sequence of the phagosome MinS1 in Genbank;
FIG. 4 shows the algae liquid under a microscope (10X 40), wherein 905C is control microcystis aeruginosa FACHB-905, and 905T is MinS 1-infected microcystis aeruginosa FACHB-905;469C is control microcystis aeruginosa FACHB-469, 469T is MinS 1-infected microcystis aeruginosa FACHB-469;1337C is control group green microcystis FACHB-1337,1337T is MinS1 infected green microcystis FACHB-1337;596C is control nostoc FACHB-596,596T is MinS 1-infected nostoc FACHB-596;
FIG. 5 shows microcystis aeruginosa FACHB-905 algae solution, control algae solution on the left side, experimental algae solution on the right side (yellowing due to MinS1 addition);
the phycophage MinS1 is preserved in the common microorganism center of the microorganism strain preservation management committee at 2021, 9 and 7 days, the preservation number is CGMCC No.23089, and the preservation organization address is: the institute of microbiology, national institute of sciences, no. 3, north chen west way 1, region of korea, beijing city, postal code: 100101, class name Microcystis aeruginosa cyanophage MinS1.
Detailed Description
The present invention will be described in detail with reference to the following examples and drawings, but the present invention is not limited to the following specific examples.
MinS1 is a high-efficiency broad-spectrum lytic phycophage separated by taking microcystis aeruginosa Microcystis aeruginosa FACHB-905 as a target, and broad-spectrum virulence of the phycophage is shown in that the MinS1 can infect and lyse microcystis aeruginosa FACHB-905, FACHB-942, FACHB-469 and FACHB-924FACHB-1326; microcystis Hui Fachb-908, fachb-1317, FAchb-1318, FAchb-929; green microcystis FACHB-1342, FACHB-1337; microcystis FACHB-915; anabaena fascicularis FACHB-245; alternaria water bloom FACHB-1255; nostoc FACHB-596; fachb-1166; fusarium garicum FACHB-881; planktonic alga FACHB-708; the method comprises the steps of performing FACHB-402 and FACHB-240 on the dinoflagellate; and the chlorella FACHB-805 and FACHB-1061. 22 species of the 32 tested algae strains can be lysed, and five meshes are crossed: synechococcus, botryococcus, nostoc and Chromococcoccoccoccyales are the most broad-spectrum one strain of phaeophaga.
Example 1
Preparation of phycophage MinS1
(1) Activation culture of target algae species
Dipping a proper amount of microcystis aeruginosa FACHB-905 (Microcystis aeruginosa FACHB-905) algae liquid by a sterile inoculating loop, streaking the microcystis aeruginosa FACHB-905 (Microcystis aeruginosa FACHB-905) algae liquid onto a BG11 culture medium plate containing 1.5% (W/V) agar, placing the microcystis aeruginosa on a BG11 culture medium plate at 25 ℃, culturing the microcystis aeruginosa in an illumination incubator with a light-dark period of 12h:12h (namely in one illumination period, 12h is a light-dark period, and circulating the cycle), picking up the monocystis to fall into 5mL fresh sterilized BG11 culture medium for culturing after monocystis grown, manually shaking the microcystis to be 3 times a day until the OD680 is about 0.6 (logarithmic growth period), inoculating the microcystis to the fresh sterilized and cooled BG11 culture medium at the temperature of 25 ℃, and culturing the microcystis 2000lx until the OD680 is about 0.6 to obtain purified target algae liquid;
(2) Enrichment and screening of phagostimulants
And (3) water sample treatment: the surface water sample collected from Fujian Zhangzhou Ma Yangxi is centrifuged at 4 ℃ and 12000g for 20 minutes to remove the precipitate. The supernatant was taken for the experiment.
30mL of the supernatant of the water sample treated by the method and 30mL of the logarithmic phase FACHB-905 algae liquid are evenly mixed and cultured in an illumination incubator, and the algae are manually shaken for 2 to 3 times every day. Meanwhile, water taken by the water purifier is used as a control group instead of water sample supernatant. Compared with a control group, the culture solution of the yellowing experimental group is centrifugated for 10 minutes by 6000g, and the supernatant is sequentially filtered by 0.45 mu m and 0.22 mu m nitrocellulose filter membranes; adding the filtrate into FACHB-905 algae solution in logarithmic phase of 10 times volume, mixing, and culturing until obvious yellowing phenomenon appears to obtain the primary screening infection solution.
(3) Purification of phagosome
Preparing concentrated algae liquid: taking the FACHB-905 algae liquid in logarithmic phase, centrifuging 10000g for 10 minutes, and keeping 1/20 of the supernatant of the original volume for suspension precipitation (for example, taking 20ml of algae liquid for concentration by 20 times, centrifuging to discard the supernatant, and re-suspending the precipitation with 1mL of BG 11);
sterilized BG11 semi-solid medium containing 0.7% (W/V) agar was dispensed into 8 mL/tube in an ultra clean bench and placed in a 45℃constant temperature water bath to prevent clotting.
Taking the preliminary screening infection liquid containing the phaga-bodies obtained in the step (2), and carrying out serial gradient dilution (10) on the preliminary screening infection liquid with BG11 liquid culture medium -1 -10 -6 ) The method comprises the steps of carrying out a first treatment on the surface of the Taking and fully mixing the diluted solutions of all dilutions, and mixing the diluted solutions according to the volume ratio of 1:9 is added into 0.9mL of concentrated FACHB-905 algae liquid, evenly mixed, placed in a shaking table with the rotation speed of 70rpm at the temperature of 25 ℃ and incubated for 25 minutes, so that viruses are fully adsorbed;
adding the above incubation mixed solution into 8 mL/tube BG11 semisolid culture medium, mixing thoroughly by vortex oscillation, immediately pouring onto 1.5% (W/V) agar-containing BG11 solid culture medium plate, sealing with sealing film after solidification, and stamping holes to prevent contamination by bacteria in air, etc., stamping holes to ensure oxygen in culture dish, inverting at 25deg.C, 2000lx, and light-dark period of 12h: culturing in a light incubator for 12h, and observing and recording the formation of the plaque. The independent phycophage formed under the maximum dilution is dug, suspended in 500 mu L of BG11 liquid culture medium, placed in a refrigerator at 4 ℃ overnight to thoroughly release the phycophage, centrifuged for 10 minutes at 10000g, and the supernatant is taken to obtain the first generation of phycophage.
Taking the first generation of phycophage obtained above, and performing 10 steps with BG11 liquid culture medium -1 To 10 -6 Serial gradient dilution, repeating the above steps for at least 3 times to obtain uniform shape and size of plaque. The single plaque was extracted and suspended in 500ul of BG11 liquid medium, placed in a refrigerator at 4℃overnight, centrifuged at 10000g for 10-20 min, and the supernatant was taken to obtain purified suspension of the phycophage MinS1.
(4) Amplification culture of phaga
Adding 1mL of purified phagosome MinS1 suspension obtained in the step (3) into 10mL of freshly cultured logarithmic phase FACHB-905 algae liquid, uniformly mixing, culturing in an illumination incubator until the algae liquid is yellow to obtain phagosome MinS 1-algae culture lysate, centrifuging 6000g of the lysate for 10 minutes, and taking supernatant. Adding the supernatant into fresh cultured FACHB-905 algae liquid in logarithmic phase at a ratio of 1:10 (V/V), mixing, and culturing in an illumination incubator until algae liquid is yellow. As shown in fig. 5. The expansion culture was repeated in this manner.
(5) Preparation of algae-phagostimulant suspension
Taking the phycophage-algae culture lysate prepared in the step (4), centrifuging at 4 ℃ and 6000g for 10min, taking supernatant, and filtering sequentially through nitrocellulose filter membranes with the pore diameters of 0.45 mu m and 0.22 mu m to obtain the phycophage MinS1 suspension.
Wherein the formula of the BG-11 medium is shown in Table 1 below:
table 1 formula of BG-11 Medium
The phycophage is preserved in China general microbiological culture Collection center (China Committee) for culture Collection of microorganisms, and the preservation number is: CGMCC No.23089, the preservation date is 2021, 9 and 7, and the preservation unit address is: beijing, chaoyang area, north Chenxi Lu No. 1, 3, postal code 100101.
Example 2
Morphological observations of phycophage MinS1
Taking 30ml of the phycophage MinS1 suspension separated and purified in the step (5) of the example 1, performing ultracentrifugation (4 ℃,35000g for 1 h), removing supernatant, slowly washing sediment twice by using 1 XPBS, re-suspending and dripping on a copper grid, standing for 10min, and then sucking redundant water from the side by using neutral filter paper; then, a drop of 3% uranyl acetate was dropped on the copper mesh, and after 20s of staining, the stain was sucked off from the side with neutral filter paper. Standing for 10min, air drying, and observing the morphology of the phycophage by using a transmission electron microscope.
As a result, as shown in FIG. 1, the phycophage MinS1 has a head portion having a diameter of 54nm (+ -5 nm) and an elongated tail portion having a length of 260nm (+ -5 nm) in an icosahedral structure.
The phycophage plaque experiment is carried out by taking 2uL purified and concentrated (concentration volume ratio is 20:1), and transparent phycophage plaques with uniform size and shape, no halo around the plaques and clear and regular edges can be obtained as shown in figure 2.
Example 3
Alignment of genomic sequences of phycophage MinS1
Genome extraction: 30ml of the high titer phagosome suspension obtained in step (5) of example 1 was subjected to density gradient centrifugation using freshly prepared 20% (W/V) and 40% (W/V) sucrose solutions for one week, centrifuged at 4℃and 35000g for 1h, the supernatant was discarded, and after resuspension of the pellet with 200ul of 1 XPBS, the phagosome genome was extracted using Roche high purity viral DNA kit.
Genome library construction: a genomic library was constructed using kit NEBNext Ultra II DNA Library Prep Kit for Illumina (#e7645). The DNA content of the library constructed was determined using Qubit.
Sequencing on a machine: diluting the constructed library with HTl buffer solution after thawing at room temperature to a final concentration of 10nM, taking out 2 μL, adding 8 μL buffer solution, and mixing uniformly; adding 8 μl of buffer solution into 2 μl of NaOH, and mixing; the library was denatured for 5min by mixing 10. Mu. L0.2M NaOH and 10. Mu.L of 2nM, at which point the library concentration was 0.1M NaOH and 1 nM. To the 20. Mu.L of the library, 1980. Mu.L of buffer was added, the library was diluted to 10pM, 2000. Mu.L of the diluted library was obtained in total, 600. Mu.L of the library was taken, and the mixture was sequenced on a machine.
Genome assembly: comparing the control group with the experimental group sequencing reads database, and deleting reads which can be matched with the control group from the experimental group sequencing data by using a script; the sequencing data quality was checked using the program FastQC (http:// www.bioinformatics.babraham.ac.uk/subjects/FastQC /), then the experimental group remaining data was low quality value data filtered using trimmatic v0.36, and finally the filtered data was spliced using SPADes v3.13.0 to obtain the MinS1 complete genome sequence.
Genomic sequence homology alignment: the MinS1 genome was aligned to NCBI's nucleic acid database (BLASTN: https:// BLAST. NCBI. Lm. Nih. Gov /), and the BLAST alignment showed that the MinS1 sequence was not present in the existing database (FIG. 3) and that the highest homology was only 1% homologous, which was a novel algae phagosome that was not reported.
Example 4
Algae phagosome MinS1 algae killing range test
The logarithmic phase algae liquid in table 2 and the phycophage MinS1 suspension prepared in the step (5) in example 1 are mixed according to the volume ratio of 2:1 (800 uL of logarithmic phase algae liquid: 400uL of MinS1 suspension) to be used as an experimental group for artificial infection, the control group uses BG11 liquid culture medium to replace the phycophage, and the experimental group is placed in an illumination incubator with the temperature of 25 ℃ and the illumination intensity of 2000lx and the light-dark period of 12h:12h for cultivation. Determination of OD of each group with an ELISA apparatus 680 . Taking the average value of the parallel groups and calculating the ratio of the control group to the experimental group (the ratio is larger than 1.2, the phycophage can be infected by the algae and is a positive result, otherwise, the phycophage can not be infected by the algae and is a negative result); in addition, the infection and the cleavage are observed by naked eyes and a microscope to confirm OD 680 And (5) judging the result of the method. FIG. 4 shows photographs of some microscopic algae infected with and not infected with the phaeophaga MinS1.
TABLE 2 algicidal Range experiment results for phycophage MinS1
“+”suspective“-”unsuspective
MinS1 infects and lyses microcystis aeruginosa FACHB-905, FACHB-942, FACHB-469, FACHB-924, FACHB-1326; microcystis Hui Fachb-908, fachb-1317, FAchb-1318, FAchb-929; green microcystis FACHB-1342, FACHB-1337; microcystis FACHB-915; anabaena fascicularis FACHB-245; alternaria water bloom FACHB-1255; nostoc FACHB-596; fachb-1166; fusarium garicum FACHB-881; planktonic alga FACHB-708; the method comprises the steps of performing FACHB-402 and FACHB-240 on the dinoflagellate; chlorella FACHB-805 and FACHB-1061. 22 species of the 32 tested algal strains were lysed, spanning five orders: synechococcus, botryococcus, nostoc and Chromococcoccoccoccyales are the most broad-spectrum one strain of phaeophaga.
The above description is not intended to limit the invention, nor is the invention limited to the examples described above. Variations, modifications, additions, or substitutions that would be within the spirit and scope of the invention are also within the scope of the invention, which is defined by the following claims.
Claims (6)
1. Broad-spectrum virulent phagosome（Microcystisaeruginosa cyanophage）MinS1, its characterized in that: it is preserved in the common microorganism center of the microorganism strain preservation management committee at the 9 th month of 2021, with the preservation number of CGMCC No.23089.
2. The use of a broad-spectrum virulent phaeophaeoosome MinS1 according to claim 1 for lysing chlorella, tremella, segment-breeding algae, nostoc, synechococcus.
3. A broad-spectrum virulent phaeophaga MinS1 of claim 1 is used in the lysis of microcystis aeruginosa ACHB-905, microcystis aeruginosa FACHB-942, microcystis aeruginosa FACHB-469, microcystis aeruginosa FACHB-924, microcystis aeruginosa FACHB-1326, microcystis Hui FACHB-908, microcystis Huinella FACHB-1317, microcystis Huinella FACHB-1318, microcystis Huinella FACHB-929, microcystis viridis FACHB-1342, microcystis viridis FACHB-1337, microcystis FACHB-915, anabaena water bloom FACHB-245, microcystis water FACHB-1255, candidia FACHB-596, F.alfasciana FACHB-1166, F.praecox FachB-1, F.lanolata FACHB-88708, F.lanugo FACHB-402, F.lanugo FACHB-240, F.lanugo FACHB-1061.
4. The use of a broad-spectrum virulent phaeophaga MinS1 as claimed in claim 1 for the prevention and treatment of cyanobacteria bloom.
5. The use according to claim 4, characterized in that: the algae-phagostimulant suspension is added into the cyanobacteria bloom sample to have a cracking effect on the chlorella, the tremella, the segment-breeding algae, the candida and the synechococcus in the cyanobacteria bloom sample.
6. The use according to claim 5, wherein the phaeophaeoid MinS1 has an effect on microcystis aeruginosa FACHB-905, microcystis aeruginosa FACHB-942, microcystis aeruginosa FACHB-469, microcystis aeruginosa FACHB-924, microcystis aeruginosa FACHB-1326, microcystis Hui FACHB-908, microcystis Hui FACHB-1317, microcystis Hui FACHB-1318, microcystis Hui FACHB-929, microcystis viridis FACHB-1342, microcystis viridis FACHB-1337, microcystis FACHB-915, anabaena water bloom FACHB-245, microcystis water bloom FACHB-1255, candidia FACHB-596, F.alfa FACHB-1166, F.lanuginosus FACHB-881, F-708, F.lanugo FACHB-402, F-240, F-F.lanugo FACHB 1061, F-F.
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| CN111269891A (en) * | 2018-12-05 | 2020-06-12 | 宁波大学 | Broad-spectrum virulent cyanophage Me-ZS1 and application thereof |
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| CN109097339A (en) * | 2017-06-21 | 2018-12-28 | 宁波大学 | A kind of cracking performance cyanophage MACPNOA1 and its application |
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