KR101971250B1 - Olleya sp. M5A2M strain having algicidal activity against Alexandrium tamarense and Cochlodinium polykrikoides and uses thereof - Google Patents

Abstract

The present invention relates to an Olleya sp. M5A2M strain having algicidal activity against Alexandrium tamarense and Cochlodinium polykrikoides, and to uses of the same. More specifically, the Olleya sp. M5A2M strain of the present invention exhibits selective algicidal activity against Alexandrium tamarense and Cochlodinium polykrikoides, which are harmful red tide organisms, and thus, when used to control red tide, may minimize disturbance of the ecosystem. Also, even residual culture broth of the strain, without direct application of the strain, maintains a relatively reliable algicidal activity, and thus may be beneficially used in controlling Alexandrium tamarense producing paralytic shellfish poison, and Cochlodinium polykrikoides causing significant damage in domestic fish farms.

Description

Ollea genus M5A2M strains having killing activity against Aleurandium tamarences and Cochlonidium polycricoides and their uses {Olleya sp. M5A2M strain having algicidal activity against Alexandrium tamarense and Cochlodinium polykrikoides and uses thereof}

The present invention relates to an oleander genus M5A2M strains having a killing activity against alexandrite tamarenes and cochlearidium polycricoides and their uses.

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.

The kokeulrodinium (Cochlodinium) is a genus of phytoplankton that are dormant spores, the dormant state of the water temperature start activity increased more than 23 ℃ Celsius haejimyeon nutrient-rich and rapidly growing twice in 24 hours in seawater. It has photosynthetic action, but it also has animal characteristics because it can move by causing vortex flow. One or several cells are connected and the size of the cells is 20 ~ 40㎛ (1m is one millionth of a meter), but it does not have its own toxicity. However, if there are more than 3,000 per 1ml of mucous component, Can be killed by dyspnea or the like. In the coastal area of Korea, livestock and shellfish have been killed since 1989. In particular, fish and shellfish have been consumed from Chungmu coast, Yantai-Gimimdo area, Samyang area and Namhae Mizogi line between August and October, I was killed. Currently, the main provinces are Yeosu Bay, Namhae, Tongyoung, Busan coast and South Sea of the East Sea in Gohung Nara Road in Jeonnam.

In order to cope with the enormous economic damage of the aquaculture industry due to such harmful red tide creatures, researches on numerous red tide phenomena have been actively carried out. There are physical, biological and chemical control methods for the remediation and removal of red tide organisms for harmful tidal control. Typical examples thereof include an air injection device or a filtration device, a method using an ultrasonic treatment and an ozone-153 treatment method, a method in which red tide creatures are filtered or centrifuged and recovered from the sea surface, a method in which red tide creatures are adsorbed and coagulated by sedimentation using clay or polymer flocculant A method using a bacterium of blooming-causing organisms, a method using a bacterium or the like, a method of treating a chemical substance (eg, hydrogen peroxide, copper sulfate, chlorine, herbicide, etc.) . Among the control methods, the relatively effective method is the method using the clay adsorption method using the clay so that the clay is sprayed in seawater to adsorb and precipitate the red tide organisms, and the aluminum ions in the clay are eluted and destroy the cells of the red tide organism. This method has been mainly studied in Japan and has been carried out in Korea since 1985 by the National Fisheries Research and Development Agency. However, this method is non - selective, and when the flow of the algae is strong, the effect of controlling is low, and when the amount of sunshine and water temperature is high, red tide occurs again. Conventional red tanning agents have difficulties in control because they have disadvantages such as non-selective red tide, sunlight and water temperature, safety for non-target organisms, environmental pollution, human toxicity and difficulty in mass production. Therefore, it is required to develop eco - friendly technology that can overcome these shortcomings.

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, the oleander genus M5A2M strains which have aldosteric activity against Alexandrite tamarenes and Cochlondinium policlciodes of the present invention and their uses have not yet been disclosed.

The present inventors have found that the Flavobacter strain Olleya sp. Strain M5A2M isolated purely from seawater near Geoje in the southern coast of Korea is a harmful red tide organism It exhibits selectively killing activity only against Alexandrium tamarense and Cochlodinium polykrikoides , and even when the cultured filtrate of strain Oleyae M5A2M is treated, the killing activity remains relatively stable , Olleya sp.). The present inventors have completed the present invention by confirming that they exhibit significantly higher tearing activity against Alexandrite tamarens and Cochlodinium policycloides , as compared with other strains of Olleya sp.

In order to solve the above problems, the present invention provides an Olleya sp. M5A2M strain (KCTC18617P) having a killing activity.

The present invention also provides a composition for controlling red tide comprising the above-mentioned Olleya sp. M5A2M strain, a culture thereof or a culture filtrate thereof as an active ingredient.

Since the Olleya sp. M5A2M strain of the present invention exhibits selective killing activity only against the harmful red tide creatures Alexandrium tamarense and Cochlodinium polykrikoides , It is possible to minimize the disturbance of the ecosystem and maintain relatively stable lyocell activity even when the culturing filtrate is treated without directly treating the strain. Therefore, it is possible to produce cochlearnidium polycryocides and paralyzed podox, It can be used more useful for chewing lance.

1 is oleic I in accordance with one embodiment of the present invention (Olleya sp.) A toxic red tide organisms M5A2M strain of Alexandria Solarium Tama lances (Alexandrium (Chl-a: Chlorophyll-a) of alexandrite tamarens and cochlearidium polychloroides, respectively, when treated with tamarense (A) and cochlodinium polykrikoides (B) The carotenoid concentration was measured and compared with the control (control), the result of confirming the killing activity through the degree of reduction of chlorophyll-a and carotenoid concentration. Control is a control group in which the concentrations of chlorophyll-a and carotenoid in cells of Alexandrium tamarense (A) and Cochlondinium polycrycoides (B) were measured without treating the strain of Oleaceae M5A2M of the present invention.
FIG. 2 is a graph showing the results of immunization of alexandrite tamarense cells (A) and killed Alexandrium tamarense cells (B) before treatment of an Olleya sp. M5A2M strain according to an embodiment of the present invention with alexandrite tamarens It is the result of confirmation through a microscope.
FIG. 3 is a graph showing the results of the measurement of the activity of the Cochrlidium polycrycoides cell (A) and the killed Cochlonidium Polychrycides cell (A) before the treatment of the Olleya sp. M5A2M strain according to an embodiment of the present invention, B) was confirmed through a microscope.
FIG. 4 is a graph showing the effect of the culture filtrate of the strain Olleya sp. M5A2M according to an embodiment of the present invention on Cochlonidium policycloides as compared to that of Chlorophyll-a ) Concentration. The results are as follows.
FIG. 5 is a graph comparing the killing activity of a strain of M5A2M strain of the genus Olleya according to an embodiment of the present invention and a strain of Alexandria tamarens (A) and Cochlonidium polycrycoides (B) This is a result. M5A2M is an oleander genus M5A2M strain of the present invention; KCTC23673 is a registered trademark of Olleya namhaensis ) KCTC23673 strain; And 32N3Y are strains showing a high degree of similarity of 99% with Olleya marilimosa .
FIG. 6 is a graph showing the results of a comparison of two strains belonging to the diatom alga, a total of six microalgae isolated from Korean waters, in addition to alexandrite tamarens and cochlearidium polychloroides , according to one embodiment of the present invention. It is known that Chaetoceros simplex and Chaetoceros sp., Thalassiosira sp. And Skeletonema costatum and alexandria belonging to the epiphyte algae Alexandrium insuetum ) and Heterocapsa triquetra ) in the case of the present invention.

In order to achieve the object of the present invention, the present invention provides an Olleya sp. Strain M5A2M having killing activity.

The above-mentioned Olleya sp. M5A2M strain was isolated from the seawater near Geoje in the southern coast of Korea and was selected as a strain having excellent killing activity against Alexandrium tamarense and Cochlodinium polykrikoides . . The above-mentioned strain Olleya sp. M5A2M was deposited on September 19, 2017 by the Korean Collection for Type Culture (KCTC) (Accession No .: KCTC18617P).

In a strain according to an embodiment of the present invention, the killing activity may be, but is not limited to, tear activating activity against Alexandrium tamarense or Cochlodinium polykrikoides .

The present invention also provides a composition for controlling red tide comprising the above-mentioned Olleya sp. M5A2M strain, a culture thereof or a culture filtrate 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 of the present invention is used as an effective ingredient to stably express the effect within a range that does not inhibit the effect of the strain Olleya sp. M5A2M, to improve adhesion to the target organism, It may further comprise a surfactant such as a pharmaceutically acceptable solid carrier, a liquid carrier, a liquid diluent, a liquefied gas diluent, a 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 .

The present invention also provides a method for controlling red tides, comprising treating an effective amount of an Olleya sp. Strain M5A2M, a culture thereof or a culture filtrate thereof in a red tide occurrence area. At this time, the effective amount of the above-mentioned Olleya sp. M5A2M strain, the culture solution thereof or the culture filtrate thereof is preferably treated early in the generation of the harmful algae to prevent mass proliferation in advance, Lens most preferably (Alexandrium tamarense) or poly kokeulrodinium Cri Koh des (Cochlodinium polykrikoides).

The present invention also provides a method for preparing a red tide controlling composition comprising the step of culturing an Olleya sp. M5A2M strain.

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 1 [ Olleya  sp.) Isolation and identification of M5A2M 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 separation of the various microorganisms, were final screening excellent microbial ability saljo in a purified microorganism through three times subculture, was identified by the 16S rDNA gene (SEQ ID NO: 1) analysis of the selected strains results in oleic I (Olleya sp.), which was named Oleayaceae M5A2M. The above-mentioned strain Olleya sp. M5A2M was deposited on September 19, 2017 by the Korean Collection for Type Culture (KCTC) (Accession No .: KCTC18617P).

Example 2 Analysis of Salmonella of Alexandrite tamarense and Cochlonidium Polycrycoides in the Strain M5A2M of Oleaceae

In order to confirm the survival ability against the harmful red tide creatures Alexandrite tamarense and Cochlodinium polykrikoides of the Olleya sp. M5A2M strain according to the present invention, Were inoculated into F / 2 medium and cultured for 15 days or more at 20 ° C, L: D = 12: 12 (12 hrs condition / 12 hrs dark condition cycle) and 250 μEm ^-2 s ^-1 light condition. Oleacean M5A2M The strain was inoculated on F / 2 medium with an OD ₆₀₀ value of 0.3 or more and then pre-cultured at 25 ° C for 24 hours. Thereafter, the pre-cultured oleoresin M5A2M strain was inoculated into the above culture medium of Alexandrium tamarencense and the culture solution of Corynebacterium polycrycoides at a concentration of 10% (v / v), followed by mixing and culturing. After 24 hours, a mixture of alexandrite tamarens and cochlodinium By measuring the concentration of the pigment in the cells of the Polycrycoides, the ability of lyophilization was confirmed by the decrease of the pigment concentration as compared with the control (control). As a result, as shown in Fig. 1, when the oleander genus M5A2M strain of the present invention, alexandrite tamarense, and cochlearnidium polychloroiodes were mixed and cultured, respectively, as compared with the control (control), in the case of alexandrite tamarense, It was confirmed that the concentration of chlorophyll-a was reduced by 79%, the concentration of carotenoid was decreased by 76%, and the concentration of chlorocholin-a in the case of Cochlonidium polychloroides was decreased by 84% and the concentration of carotenoid was decreased by 86%.

In addition, the Alexandrite tamarense cells (FIG. 2A) and the killed Alexandrium tamarense cells (FIG. 2B) before the treatment of the Aleuria genus M5A2M strain of the present invention with Alexandrium tamarens were confirmed by the same microscope (FIG. 3A) and killed Coclodinium Polycycloidos cells (FIG. 3B) before the treatment of Oleacea M5A2M strain with Cochlonidium policycloides. In the microscopic observation, it was confirmed that brown cell cytoplasm was leaked as the cell membrane of alexandrite tamarens and cochlearidium polychloroides was destroyed.

Example 3: Changes in the viability of the culture filtrate of the strain Oleya M5A2M of the present invention

In order to confirm the killing activity of the cultured filtrate of the strain Oleya sp. M5A2M of the present invention, the killing activity of the culture filtrate passed through a filter of 0.4 mu m was evaluated. As a result, as shown in Fig. 4, the cell of Cochlodinium polychloroides Was reduced by 77%, indicating that the killing activity was relatively stable as compared with the culture medium of strain Oleya M5A2M.

Example  4. The Oleya  genus M5A2M  Of strains and other strains Alexandrium  Comparison of live performance against tamarens and cochlearidium polycrycoides

In the present invention, the strain M5A2M of the genus Oleya and the strain Alexandrium It was compared tamarense) and poly kokeulrodinium Cri Koh des saljo active for (Cochlodinium polykrikoides). The comparative strains used were Olleya namhaensis KCTC23673 strain and Olenya marilimosa were selected as the 32N3Y strain which showed a high degree of similarity of 99%. As a result, it was confirmed that the oleander genus M5A2M strain of the present invention was significantly superior in lyophilicity to alexandrite tamarens (FIG. 5A) and cochlonidine polycrycoides (FIG. 5B) compared to the comparative strains.

Example 5. Identification of the viability of the other microalgae of the strain Oleya M5A2M of the present invention

In addition to Alexandrium tamarense and Cochlodinium polykrikoides , the strain of M5A2M strain of the present invention belongs to six species of Ketocephalus belonging to the diatom, a total of six microalgae isolated from the Korean waters Chaetoceros simplex and Chaetoceros sp.), Tala Please Syrah in (Thalassiosira sp.) and skeletal Margarito nematic course tatum (Skeletonema costatum) and Khartoum to Alexandria Solarium argument belongs to the dinoflagellates (Alexandrium insuetum) and heterocyclic kaepsa tree Quetta Trapani (Heterocapsa triquetra ) were tested and compared for the presence of killing activity. As a result of measuring the concentration of intracellular chlorophyll-a in the microalgae, it was found that the strain of Oleay genus M5A2M of the present invention showed a strong survival against only alexandrite tamarens and cochlearidium polychloroides, , And very low lyophilization ability of less than 20% for other marine microalgae. Therefore, the strain of Oleaceae M5A2M of the present invention selectively alleviates the action of only Alexandrite tamarense and Cochlonidium polycrycoides, so that the red tide can be efficiently controlled while minimizing the influence on the primary producer ecosystem.

Korea Biotechnology Research Institute KCTC18617P 20170919

<110> Korea Research Institute of Bioscience and Biotechnology <120> Olleya sp. M5A2M strain having algicidal activity against          Alexandrium tamarense and Cochlodinium polykrikoides and uses          the <130> PN17441 <160> 1 <170> KoPatentin 3.0 <210> 1 <211> 1046 <212> DNA <213> Olleya sp. <400> 1 gcaggaagac tgccctatgg gttgtaaact gcttttatac aggaagaaac actgctacgt 60 ggtagcagct tgacggtact gtaagaataa ggatcggcta actccgtgcc agcagccgcg 120 gtaatacgga ggatccaagc gttatccggg aatcattggg tttaaagggt ccgtaggtgg 180 ataattaagt cagaggtgaa atcctgcagc tcaactgtag aattgccttt gatactggtt 240 atcttgagtt attatgaagt agttagaata tgtagtgtag cggtgaaatg catagatatt 300 acatagaata ccaattgcga aggcagatta ctaataatat actgacactg atggacgaaa 360 gcgtggggag cgaacaggat tagataccct ggtagtccac gccgtaaacg atggtcacta 420 gctgttcgaa cttcggtttg agtggctaag cgaaagtgat aagtgaccca cctggggagt 480 acgttcgcaa gaatgaaact caaaggaatt gacgggggcc cgcacaagcg gtggagcatg 540 tggtttaatt cgatgatacg cgaggaacct taccagggct taaatgtaga ttgacaggac 600 tagagatggt tttttcttcg gacaatttac aaggtgctgc atggttgtcg tcagctcgtg 660 ccgtgaggtg tcaggttaag tcctataacg agcgcaaccc ctgttgttag ttgccagcga 720 gtcatgtcgg gaactctaac aagactgcca gtgcaaactg tgaggaaggt ggggatgacg 780 tcaaatcatc acggccctta cgtcctgggc tacacacgtg ctacaatggt agggacagag 840 agcagccact gggcgaccag gagcgaatct ataaacccta tcacagttcg gatcggagtc 900 tgcaactcga ctccgtgaag ctggaatcgc tagtaatcgc atatcagcca tgatgcggtg 960 aatacgttcc cgggccttgt acacaccgcc cgtcaagcca tggaagctgg gagtgcctga 1020 agtccgtcgc cgtaaggagc ggccta 1046

Claims (5)

Olleya sp. M5A2M strain with accession number KCTC18617P which has tear activating activity against Alexandrium tamarense or Cochlodinium polykrikoides . delete delete The present invention relates to a method for preventing red blood cell infection caused by Alexandrium tamarense or Cochlodinium polykrikoides containing Olleya sp. Strain M5A2M of the present invention, a culture solution thereof or a culture filtrate thereof as an active ingredient, / RTI &gt; delete

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