KR101957208B1 - Pseudoruegeria sp. M32A2M strain having algicidal activity against Alexandrium tamarense and uses thereof - Google Patents

Abstract

The present invention relates to a Pseudoruegeria sp. M32A2M strain having algicidal activity against Alexandrium tamarense and uses thereof. More specifically, since the Pseudoruegeria sp. M32A2M strain of the present invention selectively exhibits algicidal activity only against Alexandrium tamarense which generates paralytic shellfish poison, it is possible to minimize disturbance of aquatic ecology if red tide is controlled. Also, it is possible to maintain algicidal activity relatively stably even if cultural filtrate or heat-treated cultural filtrate is treated without directly treating a strain, thereby being more useful for killing Alexandrium tamarense, which is toxic plankton.

Description

To Shodoru having a killing activity against Alexandrium tamarens, the genus Leica M32A2M and its use {Pseudoreregeria sp. M32A2M strain having algicidal activity against Alexandrium tamarense and uses thereof}

The present invention relates to a strain Shirodoru having a killing activity against Alexandrium tamarense and a use thereof.

In addition, not only the frequency and duration of pollution caused by pollution of the marine environment but also the social and economic problems that cause huge damage are widened. Red tide is a phenomenon that occurs when a marine environment such as an excess supply of growth promoting substances in seawater and an ocean environment such as solar radiation and water temperature is suitable for the growth conditions of red tide creatures, and red tide creatures consume dissolved oxygen in the water during breathing and carcass decomposition It inhibits the survival of other organisms in the water and releases harmful substances and toxins, resulting in the death of fish and shellfish. In addition, some of the red tide creatures are poisoned by fish and shellfish, and ingestion of these poached shellfish by humans causes paralysis and diarrhea poisoning, threatening human health, which is a serious social and economic problem.

More than 34 species of red tide creatures appearing on the Korean coast have been reported. Until the 1980s, Caterpillar Chitose Ross (Chaetoceros), skeletal Loreto nematic (Skeletonema), Tala Please Syrah (Thalassiosira) and Chemnitz sheet Ah (Nitzschia) yieoteuna Diatoms are a major cause of creatures, such as speed, since 1981 Alexandria Solarium (Alexandrium), kokeulrodinium ( Cochlodinium ), Gymnodinium ( Gymnodinium ) and Heterosigma genus are the major red tide organisms. Particularly, about 30 species of toxin-dominant Alexandrium genus are reported worldwide, and some species produce about 20 chemically similar neurotoxins with different toxicities.

remind Alexandria Solarium (Alexandrium) and toxic species of the genus is Alexandria Solarium Tama Lawrence (A.tamarense), Alexandria Solarium categories Nella (A. catenella) and Alexandria Solarium minu Tomb (A. minutum), such as is known, is in the country and Jinhae Alexandria Solarium Tama lances Yeosu only (A. tamarense), Alexandria Solarium Nella category (A. catenella) have been reported that such the appearance.

Recently, research on biological control using marine microorganisms or viruses has been actively conducted as part of a method for eliminating or eliminating red tide organisms to minimize the damage of red tide. However, the development of methods to effectively control red tide has not been developed yet. In Korea, the 'Horticulture Spraying Act' has been implemented since the late 90s. Although green loam produced by nature is more environmentally friendly than other methods, secondary pollution caused by loam is also a new problem. In order to reduce the red tide damage, it is a fundamental solution to expand the sewage treatment facilities and to make the facilities of the cage farm more environmentally friendly and reduce the cause of eutrophication, but this is also a real cost and time. Recently, a method using marine microorganisms existing in a red tide area has been recognized as a safer and environmentally friendly red tide control method and many studies have been conducted. In addition, studies on microorganisms that control A. tamarense in the study of tidal microorganisms against red tide organisms reported so far are insufficient.

On the other hand, Korean Patent No. 1661543 discloses a Pseudomonas sp. Strain having a fungicidal action and a tidal microorganism preparation using the same. Korean Patent No. 1746667 discloses a chimeric peptide having an anti-algae activity and a red tide control ≪ / RTI > However, there has not yet been disclosed a strain of Lie genus M32A2M and its use for Shodoru having algae activity against Alexandrium tamarense of the present invention.

The present inventors have found that Pseudoruegeria sp. M32A2M strains are effective against Shudoru strain of Roseobacter strain isolated purely from seawater near Geoje in Korea, Alexandrium tamarens, which produces podok ( Alexandrium tamarense ), exhibiting selective killing activity only against Shudoru , and the culturing filtrate of the strain M32A2M of Luax genus and the heat treatment thereof, as well as maintaining a relatively stable killing activity, and a strain showing a killing activity against alexandrite tamarense , The present invention was completed.

In order to solve the above-mentioned problems, the present invention provides Pseudorugeia sp. M32A2M strains to Schodorus having a killing activity.

In addition, the present invention provides a composition for controlling red tide, which comprises the strain Pseudoruegeria sp. M32A2M, a culture thereof, a culture filtrate thereof, or a culture filtrate obtained by heat treatment thereof, as an active ingredient.

A shoe bases of the present invention in RIA (Pseudoruegeria sp.) Strains M32A2M Alexandria Solarium Tama lances (Alexandrium producing paralytic Paddock tamarense ), it is possible to minimize the disturbance of the aquatic ecosystem during red tide control, and even when the culture filtrate and the heat-treated culture filtrate are treated without the direct treatment of the strain, the tidal activity is maintained relatively stably, It can be used more effectively to alleviate the toxic plankton, Alexandrite tamarense.

FIG. 1 is a graph showing the results obtained when Shdooru was treated with a strain of Pseudoriegeria sp. M32A2M to produce Alexandrium tamarense , which produces paralytic podocyte , in a cell of Alexandrite tamarens Chlorophyll-a (Chl-a) and carotenoid concentrations were measured to confirm the killing activity through the reduction of chlorophyll-a and carotenoid concentration compared with the control (control). Control is a control group in which the concentration of chlorophyll-a and carotenoid in the cells of Alexandrium tamarencis was measured without treating the strain Shiaodoru of the present invention with genus M32A2M.
FIG. 2 is a graph showing the results of immunohistochemical staining of a Schizorrhoeae strain of A. pseudomonas sp. M32A2M strain (A) before treatment with Alexandrium tamarense producing paralytic podocyte , , Alexandrite tamarense cells (B) and killed Alexandrium tamarense cells (C) in the post-treatment killing process.
Figure 3 is when each treatment the culture filtrate and the culture filtrate is heat treated them in a shoe bases according to one embodiment of the present invention Ria in (Pseudoruegeria sp.) M32A2M strain in Alexandria Solarium Tama lances (Alexandrium tamarense), Alexandria Solarium Tama The result of confirming the killing activity through the reduction rate of chlorophyll-a concentration in the cells of lance.
FIG. 4 is a graph showing the distribution of the Pseudoruegeria sp. M32A2M strain according to an embodiment of the present invention to the strain Shiaoru, sp.) of other strains of Alexandrium tamarens ( Alexandrium tamarense ) were compared. M32A2M is the genus Leia M32A2M to Shudoru of the present invention; The KCTC42111 is used for Shudoru by Pseudoruegeria sabulilitoris ) KCTC42111 strain; KCTC12737 has been used to treat Shudoru as a Pseudoruegeria aquinaris ) KCTC12737 strain; The 32N101M was the first of its kind in Ruegeria mobilis ) and a strain with a high degree of similarity of 99%; And M22A1M are strains showing a high degree of similarity of 99% to L. atlantica ( Ruegeria atlantica ).
FIG. 5 is a graph showing the results of a comparison of the results of a comparison between the two species of Ketoseolus belonging to the diatom alga, a total of five microalgae isolated from Korean waters, in addition to Alexandrium tamarens, according to one embodiment of the present invention. ( Chaetoceros simplex and Chaetoceros sp.) And Skeletonema costatum and alexandria belonging to the epiphyte algae Alexandrium insuetum ) and Heterocapsa triquetra ( Heterocapsa triquetra ).

In order to achieve the object of the present invention, the present invention provides Pseudorugeeria sp. M32A2M strain to Schodorus having a killing activity.

The Pseudoruegeria sp. M32A2M strain was isolated from seawater near Geoje in the southern coast of Korea and was selected as a strain with excellent killing activity against Alexandrium tamarense . The Pseudoruegeria sp. M32A2M strain was deposited at the Korean Collection for Type Culture (KCTC) on Sep. 19, 2017 (deposit number: KCTC18616P).

In a strain according to one embodiment of the present invention, the killing activity may be, but is not limited to, killing activity against Alexandrium tamarense .

In addition, the present invention provides a composition for controlling red tide, which comprises the strain Pseudoruegeria sp. M32A2M, a culture thereof, a culture filtrate thereof, or a culture filtrate obtained by heat treatment thereof, as an active ingredient.

The term " red tide control " of the present invention is used in various terms such as anti red tide, anti red tide, tidal red tide, and anti tide, and is used for suppressing the growth of algae causing red tide, This means eliminating the phenomenon of causing red tides by preventing the growth of algae. It also includes broader means of killing algae causing red tides or inhibiting growth.

The composition for preventing red tide according to the present invention can be used as an effective ingredient to stably express the effect of Shodoru in a range that does not inhibit the effect of Pseudorugeria sp. M32A2M strain, to improve adhesion to the target organism, For example, a surfactant such as a pharmaceutically acceptable solid carrier, liquid carrier, liquid diluent, liquefied gas diluent, solid diluent, or other suitable adjuvant such as an emulsifying agent, a dispersing agent or a foaming agent.

The control composition of the present invention is preferably formulated into emulsions, wettable powders, granules, powders, capsules and gels, and is preferably provided as a contact agent through a formulation such as a donut type for buoyancy of the formulation .

In addition, the present invention provides a method for controlling red tides, which comprises treating the Shudoru with an effective amount of Pseudoruegeria sp. M32A2M strain, a culture solution thereof, a culture filtrate thereof, or a culture filtrate obtained by heat treatment thereof do. At this time, it is preferable to block the mass proliferation in advance by treating the Shudoru with an effective amount of Pseudoruegeria sp. M32A2M strain, the culture solution thereof, the culture filtrate thereof, or the culture filtrate subjected to the heat treatment thereof in the early stage when harmful algae are generated , The harmful bird is Alexandrium tamarens ( Alexandrium tamarense ) is most preferable.

In addition, the present invention provides a method for preparing a red tide controlling composition comprising the step of culturing a strain of Pseudorugeeria sp. M32A2M in Shudoru .

Any method known in the art can be used for culturing the strain, and the method is not particularly limited.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are merely illustrative of the present invention and that the scope of the present invention is not limited thereto.

Example  One. Shudoru Ria  genus( Pseudoruegeria sp .) M32A2M  Isolation and Identification of Strain

In order to isolate various strains from seawater near Geoje in Korea, seawater was cultured on marine broth 2216 medium for 24 ~ 48 hours at 25 ℃. After isolating various microorganisms, the microorganisms having excellent lyophilicity were finally selected from the microorganisms which were separated purely three times through the passage. As a result of the identification by the analysis of the 16S rDNA gene (SEQ ID NO: 1) of the selected strains, ( Pseudoruegeria sp.), And it was named Shuduoru Leia genus M32A2M. The Pseudoruegeria sp. M32A2M strain was deposited at the Korean Collection for Type Culture (KCTC) on Sep. 19, 2017 (deposit number: KCTC18616P).

Example  2. Shudoru Ria  genus M32A2M  Strain Alexandrium Tamarens  Analysis of Killing Force

A shoe bases according to the invention in Liao (Pseudoruegeria sp.) Alexander Solarium Tama lances (Alexandrium causing toxic blooms of M32A2M strain to confirm that the live joneung for tamarense), Alexandria Solarium Tama lances the F / 2 medium and inoculated to ℃ 20, L: D = 12: 12 (12 hours light conditions / 12 hr dark condition period) and 250μEm ^-2 s ^-1 light condition for 15 days or more. Shadoru was inoculated on F / 2 medium so as to have OD ₆₀₀ value of 0.3 or more and then preincubated at 25 ° C for 24 hours. Subsequently, the strain Shia Dori, which had been pre-cultured, was inoculated into the culture medium of Alexandrium tamarencense at a concentration of 10% (v / v), and the mixture was cultured. After 24 hours, the concentration of dye in the cells of Alexandrium tamarenc was measured Compared with the control (control), the ability of killing was confirmed by the decrease of the pigment concentration. As a result, as shown in Fig. 1, when compared with the control (Control), when the strain of Leia genus M32A2M and the strain of Alexandrium tamarencense producing paralytic podox were mixed with Shudoru of the present invention, the cell of Alexandrite tamarens It was confirmed that the concentration of chlorochlorine-a was decreased by 80% and the concentration of carotenoid was decreased by 75%.

Further, in the same manner as the above-mentioned method, the shadoru of the present invention was treated with the Alexandrite tamarense cells (FIG. 2A) before the treatment with the Alexandrite tamarense of the genus Leah M32A2M, the Alexandrite tamarense cells 2B) and killed Alexandrium tamarens cells (Fig. 2C) were confirmed by microscopy. Microscopic observation showed that the cell membrane of Alexandrite tamarense was destroyed and the brown cytoplasm was leaked out. After complete killing, there was no cytoplasm and only cell membrane was observed.

Example  3. The Shudoru Ria  genus M32A2M  Strain Culture filtrate  And the change of the viability of the culture filtrate after heat treatment

In order to confirm the killing activity of the culture filtrate of the genus Leah M32A2M strain of the present invention against the Shudoru of the present invention, the killing activity of the culture filtrate passed through a filter of 0.4 mu m and the culture filtrate after heat treatment at 105 DEG C for 5 minutes was evaluated, , The chlorophyll-a concentration in the cells of Alexandrium tamarenc was decreased by 61% in the culture filtrate and decreased by 63% in the heat-treated culture filtrate. Thus, the shudoru of Example 1 was compared with the culture medium of Leia M32A2M strain And it was confirmed that the lyophilization activity remained relatively stable.

Example  4. The Shudoru Ria  genus M32A2M  Of strains and other strains Alexandrium Tamarens  About A life force  compare

The shaking activity of Shadoru of the present invention against A. india M32A2M strain, C. shudoru, C. india and C. lu other strains was compared. The comparative strains used were Shodoru , Pseudoruegeria sabulilitoris (KCTC 42111) and Shudoru to Pseudorue geria aquinaris , KCTC 12737) and rubies that have already been reported to have the ability to kill mobilis ) and 32N101M and M22A1M strains, which showed high similarity of 99% to L. atlantica ( Ruegeria atlantica ). As a result, as shown in Fig. 4, Shodoru had a higher killing effect than the other comparative strains in the case of Pseudoruegeria sabulilitoris (KCTC 42111), whereas the strain Shialoru of the present invention was resistant to the genus Lea M32A2M It was confirmed that the lyophilicity was remarkably excellent.

Example  5. The Shudoru Ria  genus M32A2M  For other microalgae of the strain A life force  Confirm

In addition to Alexandrite Tamarens, the strain M32A2M of the present invention belongs to five species of microalgae ( Chaetoceros simplex and Chaetoceros sp.) Belonging to the diatom, in Alexandria Solarium argument that belongs to tatum (Skeletonema costatum) and dinoflagellates Tomb (Alexandrium insuetum ) and Heterocapsa triquetra were tested for their ability to kill . As a result of measuring the concentration of intracellular chlorophyll-a in the microalgae, as shown in FIG. 5, the Shia doru of the present invention showed a strong survival rate against A. alemanum only, And very low lyophilization ability of less than 20% for algae. Therefore, the strain Liao M32A2M of the present invention selectively kills only alexandrite tamarens, so that the red tide can be efficiently controlled while minimizing the influence on the primary producer ecosystem.

Korea Biotechnology Research Institute KCTC18616P 20170919

<110> Korea Research Institute of Bioscience and Biotechnology <120> Pseudoruegeria sp. M32A2M strain having algicidal activity          against Alexandrium tamarense and uses thereof <130> PN17440 <160> 1 <170> KoPatentin 3.0 <210> 1 <211> 1331 <212> DNA <213> Pseudorgegeria sp. <400> 1 tgcagtcgag cgcactcttc ggagtgagcg gcggacgggt tagtaacgcg tgggaatatg 60 cccaaaggta gggaatagcc actggaaacg gtgagtaata ccctatgtgc ccttcggggg 120 aaagatttat cgcctttgga ttagcccgcg ttggattagg tagttggtga ggtaatggct 180 caccaagccg acgatccata gctggtttga gaggatgatc agcaacactg ggactgagac 240 acggcccaga ctcctacggg aggcagcagt ggggaatctt agacaatggg ggcaaccctg 300 atctagccat gccgcgtgag tgatgaaggc cctagggtcg taaagctctt tcgcctgtga 360 agataatgac ggtagcaggt aaagaaaccc cggctaactc cgtgccagca gccgcggtaa 420 tacggagggg gttagcgttg ttcggaatta ctgggcgtaa agcgcacgta ggcggattag 480 tcagtcaagg gtgaaatccc agggctcaac cctggaactg cctttgatac tgctagtctt 540 gagttcgaga gaggtgagtg gaattccgag tgtagaggtg aaattcgtag atattcggag 600 gaacaccagt ggcgaaggcg gctcactggc tcgatactga cgctgaggtg cgaaagtgtg 660 gggagcaaac aggattagat accctggtag tccacaccgt aaacgatgaa tgccagtcgt 720 cgggtagcat gctattcggt gacacaccta acggattaag cattccgcct ggggagtacg 780 gtcgcaagat taaaactcaa aggaattgac gggggcccgc acaagcggtg gagcatgtgg 840 tttaattcga agcaacgcgc agaaccttac caacccttga catcctgatc gcggttttcc 900 gagaggaatt ccttcagttc ggctggatca gtgacaggtg ctgcatggct gtcgtcagct 960 cgtgtcgtga gatgttcggt taagtccggc aacgagcgca acccacgtcc ttagttgcca 1020 gcagttcggc tgggcactct aaggaaactg ccggtgataa gccggaggaa ggtgtggatg 1080 acgtcaagtc ctcatggccc ttacgggttg ggctacacac gtgctacaat ggcagtgaca 1140 atgggttaat cccaaaaagc tgtctcagtt cggattgggg tctgcaactc gaccccatga 1200 agtcggaatc gctagtaatc gcgtaacagc atgacgcggt gaatacgttc ccgggccttg 1260 tacacaccgc ccgtcacacc atgggagttg ggtctacccg aagacggtgc gccaaccttt 1320 acagggggca g 1331

Claims (5)

The Pseudoruegeria sp. M32A2M strain, which has the killing activity KCTC18616P against Alexandrium tamarense . delete delete A method for controlling redness caused by Alexandrium tamarense , which comprises the strain of Pseudoruegeria sp. M32A2M, a culture thereof, a culture filtrate thereof, or a culture filtrate obtained by heat treatment thereof, Composition. delete

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