KR20100014998A - Marine bacteria having algicidal activity against cochlodinium sp - Google Patents

Abstract

PURPOSE: A marine bacteria Micrococcus luteus SY-13 is provided to ensure algicidal activity, suppress tide, and reduce damage to fishing industry. CONSTITUTION: A marine bacteria Micrococcus luteus SY-13(deposit number: KCTC 11361BP) has algicidal activity to Cochlodinium sp. The 10% of culture medium of the marine bacteria Micrococcus luteus SY-13 has algicidal activity to Cochlodinium sp. including Cochlodinium polykrikoides. The marine bacteria Micrococcus luteus SY-13 also has algicidal activity to Chattonella sp., Akashiwo sanguinea, Gymnodinium impudicum, Heterosigma akashiwo, Prorocentrum micans, Prorocentrum minimum, or Scrippsiella trochoidea. An algicidal composition contains the marine bacteria Micrococcus luteus SY-13 and nutrition medium.

Description

Marine bacteria having algicidal activity against Cochlodinium sp.

The present invention relates to marine bacteria having algae activity against algae of Coclodinium. More particularly micro Caucus Lou Proteus (Micrococcus luteus) SY-13 (accession number: KCTC 11361BP) The kokeulrodinium in having saljo activity against avian Lou marine bacteria and the micro Caucus Proteus (Micrococcus luteus) SY-13 named ( Accession No .: KCTC 11361BP) relates to an algae composition containing as an active ingredient.

Harmful algae (HABs) are a natural phenomenon occurring all over the world. Hazardous algae occur frequently and have a large impact on the marine aquaculture industry. The incidence of harmful algae over the last three decades has increased due to a number of factors including environmental pollution and global warming. In Korea, severe red-tailed algae occur each year, which causes enormous damage to fisheries. In particular, Cochlodinium polykrikoides red tide causes enormous damage to aquaculture in Korea, Japan and other countries. The economic losses incurred by Coclodinium Polycricoides in the Republic of Korea are estimated at US $ 95.5 million and US $ 7.2 million in 1995 and 2003, respectively. Coclodinium polycricoides is an indwelling marine plankton and flagella species and photosynthetic algae with an unusual spiral shape. The feeder cells of Coclodinium polycricoides generally form long cell chains. Since its inception in 1982, hazardous colodinium red tide has occurred annually in the coast of Korea. Coclodinium polycricoides usually kills fish and shellfish through the production of large amounts of mucus and oxygen consumption.

Many scientists have implemented physiological and ecological inventions to reduce fishery damage caused by harmful algae. Some chemicals mitigate harmful algae, but the side effects of chemical reagents as environmental pollutants necessitate the invention of safer algae reagents for biological control. Recently, many inventions have been carried out on the interaction between various bacteria during the extinction of harmful birds. Many algae bacteria are isolated from various sources. These saljo bacteria, for example, includes a variety in the species Alteromonas (Alteromonas), Bacillus (Bacillus), Saito wave (Cytophaga), Flavobacterium (Flavobacterium), micro Rhodococcus (Micrococcus), Pseudomonas Monastir ( Pseudomonas), Pseudomonas al interrogating a Pseudomonas (Pseudoalteromonas), Vibrio (Vibrio). These findings have improved the likelihood that harmful algae can be controlled by bactericidal bacteria.

The screening process of algae bacteria isolated seven marine bacteria against coclodinium polycricoides. Seawater samples were collected during the extinction of the Coclodinium Polycricoides red tide at Gamak Bay, South Korea. The population of algae bacteria against coclodinium polycricoides was high during the period of red tide extinction (3.7 × 10 ³ mL ⁻¹ ). Algal bacteria were classified based on biochemical and chemical classification characteristics and 16S rDNA sequence analysis.

Therefore, the present inventors confirmed that the strongest bacterium strain among the isolated marine bacteria was Micrococcus luteus SY-13, and that the secondary metabolite produced by the strain was a substance which inhibits growth against Coclodinium polycricoides. And to complete the present invention. The inventors also describe the classification of the bactericidal bacteria and have determined the range of algalicides against algal activity and harmful algal species.

The problem to be solved by the present invention is to isolate the strongest microbial strain strain of the isolated marine bacteria. It is also intended to confirm that the secondary metabolite produced by the isolated marine microbial strain is a substance that inhibits the growth of coclodinium polycricoides. In addition, another problem to be solved by the present invention is to describe the classification of the bactericidal bacteria and to measure the algae activity and the range of the algae against the harmful algal species.

It is an object of the present invention to provide a marine bacterium Micrococcus luteus SY-13 (Accession No .: KCTC 11361BP) which has an algicidal activity against algae of Coclodinium.

In addition, the 10% culture filtrate of the micrococcus luteus SY-13 is characterized by having algae activity in the genus Cochlodinium containing Cochlodinium polykrikoides ( Cochlodinium polykrikoides ).

In addition, the Micrococcus luteus SY-13 (Accession Number: KCTC 11361BP) is Chattonella sp. , Akashiwo sanguinea , Gymnodinium impudicum , Hetero Characterized by algae activity in algae selected from Heterosigma akashiwo , Prorocentrum micans , Prorocentrum minimum and Scrippsiella trochoidea . .

Still another object of the present invention is to provide an algae composition containing the marine bacteria Micrococcus luteus SY-13 (Accession No .: KCTC 11361BP) and a nutrient medium.

The effect of the present invention is to provide micrococcus luteus SY-13, the strongest bacteriostatic strain among the isolated marine bacteria. In addition, it was confirmed that the secondary metabolite produced by the isolated bacterial strain is a substance that inhibits the growth of Coclodinium polycricoides, and describes the classification of the bactericidal bacterium, and measured the range of the algae against the algal activity and harmful algal species will be.

It is an object of the present invention to isolate and identify algae bacteria against dinoflagellate coclodinium polycricoides and to measure algal activity and algae range. Seven bacteriobacteria were isolated during the reduction period of Coclodinium polycricoides red tide. Algal bacteria against Coclodinium polycricoides were calculated using the optimal spread method (MPN).

At the time of red tide extinction, the number of bacteria in the field was high (3.7 × 10 ³ mL ⁻¹ ). Algal bacteria were classified based on biochemical and chemical classification characteristics and 16S rDNA sequence analysis. Seven bactericidal bacteria isolated from the present invention belong to the genus Bacillus, Dietzia , Janibacter and Micrococcus .

It was confirmed that the algae bacteria designated as Micrococcus luteus SY-13 (Accession No .: KCTC 11361BP) produced secondary metabolites. When a 5% culture filtrate of this strain is applied to the coclodinium polycricoides culture, more than 90% of the coclodinium polycricoides cells are destroyed within 6 hours. Micrococcus Luteus SY-13 exhibits significant algal activity against Coclodinium polycricoides and a broad range of algae against various harmful algae (HAB) species.

Experimental results of the present invention confirmed that Microcaucus Luteus SY-13 is a candidate substance for controlling harmful algae.

Accordingly, the present inventors deposited the micrococcus luteus SY-13 with the accession number KCTC 11361BP to the Microbial Deposit Center, Korea Research Institute of Bioscience and Technology, located at 111, Gwahakro, Yuseong-gu, Daejeon, July 7, 2008.

Hereinafter, the present invention will be described in more detail.

Seawater samples were collected in the Coclodinium Polycricoides red tide quencher at Gamak Bay for the population calculation and separation of algae bacteria. The number of bactericidal bacteria against Coclodinium polycricoides was calculated by the MPN method. The predominant noxious species at the sample collection site was coclodinium polycricoides (1,320 cells mL ⁻¹ ). The number of killing bacteria (size larger than 0.2 μm and smaller than 0.8 μm) against Coclodinium polycricoides was high (3.7 × 10 ³ cells mL ⁻¹ ). At this time, the viable cell density was 4.4 × 10 ⁴ cells mL ⁻¹ .

To isolate bacterial strains with algae activity, we used positive wells in which Coclodinium polycricoides cells were completely dead: 53 colonies with different colony colors and morphological forms were isolated. Was screened for. Populations of each strain were calculated and again inoculated with coclodinium polycricoides to confirm algal activity against coclodinium polycricoides. As a result, seven algae strains were isolated in the present invention. Sarcobacteria strains were not isolated in wells inoculated with seawater samples smaller than 0.2 μm in size.

Algal Activity of Seven Algal Strains Against Coclodinium Polycricoides

Algae activity of seven algae strains against Coclodinium polycricoides was invented. As shown in FIG. 1, all strains significantly inhibit growth and motility of coccaloid coclodinium polycricoides. In particular, strain SY-13 exhibited the strongest killing activity and was selected for later experiments. After 6 hours of treatment, all strains inhibited at least 79% coclodinium polycricoides at 10% filtrate concentration. Based on the motility of the cells, at least 50% of Coclodinium polycricoides cells lost motility within 1 hour of treatment with 10% culture filtrate. Interestingly, the algicidal activity of all strains used in the present invention was found in the culture filtrate and not in the cell culture.

Identification of Algae Bacteria

Morphological and biochemical characteristics of the seven bactericidal bacteria are summarized in Table 1. All strains were Gram-positive and Catalase-positive. Scanning electron microscopy revealed that strains SY-11, SY-13, SY-31 and SY-47 are cocci and strains SY-19, SY-20 and SY-21 are rods (Figure 2). ). Strain SY-47 is the smallest with a cell size of 0.6-0.7 μm. All strains except for SY-21 hydrolyzed gelatin. Strains SY-19, SY-20 and SY-47 also hydrolyzed starch, casein and esculin. The strain does not include arginine dehydrolase or urease. Strains SY-19 and SY-20 absorb D-glucose, D-maltose, potassium gluconate, N-acetyl-D-glucosamine, malate and trisodium citrate, D-glucose, D-fractose, glycerol, Acids are generated from D-Maltose, D-Trehalose, and D-Sucrose.

Table 2 shows the cell fatty acid analysis of the bactericidal bacteria. Strains SY-11, SY-13 and SY-31 are characterized in that they contain a large amount of iso- and anteiso-methyl-branched-chains, including iso-C _{15: 0} and anteiso-C _{15: 0} It is similar in that it contains acid. Strains SY-19 and SY-20 include anteiso-C _{15: 0} and anteiso-C _{17: 0} as major constituents of cellular fatty acids. Strain SY-21 contains C _{17: 0} as its main component. The main components of strain SY-47 were iso-C _{16: 0} and antheiso-C _{17: 1} ω8c. Based on the fatty acid analysis table, strains SY-11, SY-13 and SY-31 are similar to the genus Micrococcus. Strains SY-19 and SY-20 showed a fatty acid analysis table similar to the genus Bacillus. However, strains SY-21 and SY-47 could not identify genus levels using a MIDI system.

16S rDNA sequencing was performed to identify seven bactericidal bacteria at the species level. The sequences of strains SY-11, SY-13 and SY-31 shared the highest similarities (98.1%, 98.6% and 98.7%) with Micrococcus luteus ATCC 381 ^T , respectively. The sequences of strains SY-19 and SY-20 showed at least 99% homology with Bacillus atrophaeus ATCC 51189. Strains SY-21 and SY-47 showed at least 98% 16S rDNA sequence similarity with Dietzia maris DSM 43102 and Janibacter brevis DSM 13953 ^T , respectively.

Table 1 is a table showing the morphological and biochemical analysis of the bactericidal bacteria against Coclodinium polycricoides.

Table 2 shows the fatty acid composition of the bactericidal bacteria. Fatty acid methyl esters were prepared from biomass obtained from trypticase soy agar (TSA) supplemented with aged seawater. After incubation for 3 days at 25 ℃ was analyzed using gas chromatography. It is measured by Sherlock Microbial Identification System (MIDI) and represents% values for total fatty acids.

Physiological Characteristics and Phylogenetic Analysis of Strain SY-13

Strain SY-13, the strongest bacterium, was Gram-positive, non-motor cocci. Growth of strain SY-13 was observed at 15-45 ° C., pH 5-10. This strain was resistant to 10% NaCl concentration but did not require NaCl for growth. This strain, however, showed weak growth at 15 ° C and 45 ° C, pH 10 and 6% NaCl concentration. Optimal growth conditions for strain SY-13 were 25 ° C., pH 8.0, 3.0% NaCl. Strains SY-13 and SY-31 showed very similar characteristics in many respects. However, strains SY-13 and SY-31 showed obvious macrostructural and biochemical differences (Table 1). The colony of strain SY-13 was convex and smooth, while the strain SY-31 was protruding. These strains differed from each other in the assimilation of N-acetyl-D-glucosamine and acid products from phenylacetic acid and D-maltose. Moreover, strain SY-13 and strain SY-31 showed 99.3% 16S rDNA sequence similarity. Among them, SY-13 and strain SY-31 were found to show a significant difference in the algae activity (Fig. 1).

Based on morphology, biochemistry and chemical classification, these data enabled the identification of strain SY-13 as a member of the genus Micrococcus. Moreover, the nearly-complete 16S rDNA sequence (1,409 bp) of these strains indicates that the sequence is very closely related to Micrococcus luteus ATCC 381 ^T (98.6% homology, M38242) (FIG. 3). We therefore selected and selected strains that are Micrococcus luteus SY-13. The 16S rDNA sequence of strain SY-13 was deposited in the GenBank database under accession number EU021048.

Algal Effect of Culture Filtrate of Micrococcus Luteus SY-13 Against Coclodinium Polycricoides

Agrochemical effects were determined according to the concentration of bacterial culture filtrate of Micrococcus luteus SY-13 against Coclodinium polycricoides (FIG. 4). Coclodinium polycricoides cells and Micrococcus Luteus SY-13 were cultured under the conditions described above. Culture filtrate of Micrococcus Luteus SY-13 was added to Coclodinium polycricoides cultures in medium-exponential growth (ca. 1.0 × 10 ⁴ cells mL ⁻¹ ) at concentrations of 1.25, 2.5, 5 and 10%, respectively. It became. Some algae activity was observed in control wells to which 10% filtrate of Zobell 2216E medium was added, but this activity was not significant. There was a significant difference between control and filtrate after 3 hours of incubation at concentrations higher than 1.25%. After 6 hours, at least 90% of coclodinium polycricoides cells were destroyed at a concentration of 5%. At 10% concentration, at least 98% coclodinium polycricoides cells were destroyed within 6 hours. These results suggest that the filtrate of Micrococcus luteus SY-13 inhibits the growth of Coclodinium polycricoides in a concentration and time-dependent manner.

In wells containing 10% culture filtrate of Micrococcus Luteus SY-13, coclodinium polycricoides cells lose motility; All non-kinetic Coclodinium polycricoides chains were separated and the resulting single cells and abnormally expanded Coclodinium polycricoides cells finally degraded within 6 hours (FIG. 5). After 6 hours, some coclodinium polycricoides cells remained in the culture medium without rupturing, and these cells eventually also rounded up and expanded so that most of the coclodinium polycricoides cells degraded within 9 hours after treatment.

Agrochemical range of Micrococcus luteus SY-13 against other pest species

The effect of additional agrochemicals of the culture filtrate of micrococcus luteus SY-13 against harmful algal species was measured (FIG. 6). The present inventors used three shells and three shells in the present invention, Dinophyceae, two needle-like steels (Raphidophyceae), and one Bacillariophyceae. The 10% culture filtrate of Micrococcus luteus SY-13 exhibited a wide range of algicidal activity against all tested cotyledon and cotyledons. Algal activity against harmful algae species is as follows: Chattonella sp. (6 hours after treatment: 90.3% killed), Akashiwo sanguinea (71.8%), Gnodium impudi Gymnodinium impudicum (44.3%), Heterosigma akashiwo (25.6%), Prorocentrum micans (47.8%), P. minimum (19.7%) ) And Scrippsiella trochoidea (48.2%). However, the filtrate of Micrococcus luteus SY-13 did not show a clear inhibitory effect on the growth of Skeletonema costatum . Micrococcus Luteus SY-13 is believed to have a broad range of algae against harmful algal species except diatoms.

Many inventions have been carried out regarding the control of harmful algae. In fact, however, little progress has been made in the invention of the control of harmful birds. In Korea, ocher was the only method used to control and mitigate harmful algae. However, this method was limited and not very effective. Moreover, it is controversial because of the potential for adverse effects on marine ecosystems. Therefore, alternative measures need to be invented for controlling harmful birds. Recently, many scientists have focused on bactericidal bacteria for the control of harmful algae.

In the present invention, we isolated algae bacteria and measured field populations in fractions of size smaller than 0.8 μm (mainly including bacteria and bacteria) and 0.2 μm (mainly including viruses and virus-like particles). Furthermore, the present inventors have isolated and identified algal bacteria that effectively inhibit the growth of coclodinium polycrycoides. In the extinction of Coclodinium polycricoides red tide, the number of algae bacteria against Coclodinium polycricoides was high (3.7 × 10 ³ cells mL ⁻¹ ). On the other hand, the effect of the virus and virus-like particles may be negligible because almost no algal activity was detected in fractions smaller than 0.2 μm.

Seven bactericidal bacteria isolated in the present invention belong to the genus Bacillus, dietezia, Zanibacter and micrococcus. All known bacteriophages are phylogenetically one of the γ-proteobacteria or cytopaga-flavobacterium-bacteriodes (CFB) group. γ-proteobacteria include Altereromonas, Pseudomonas, Pseudo-Altermonas and Vibrio species. Moreover, the genus Pseudoaltermonas includes many species that produce biologically active extracellular products against several types of red tide species. However, the algae bacteria proposed by the present invention, Dieggia, Zanibacter and micrococcus, belong to Actinbacteria, and Bacillus species belong to Firmicutes. To date, no radiobacterium and pyrumik have been reported as bactericidal bacteria.

All the algal bacteria of the present invention show significant algal activity against coclodinium polycricoides. Furthermore, algal activity against Coclodinium polycricoides was detected in the filtrate of all strains, not cells. These results indicate that all seven types of algae strains secrete some algae compound into the culture. It is therefore believed that all agicide strains of the present invention act as indirect attackers. Some bacteria directly attack algae cells. For example, Imai et al. (1993) report that cytoplasmic species directly attack algae cells. Indirect attacks generally occur through chemical mediators and are species-specific.

The strongest strain, Micrococcus luteus SY-13, indirectly inhibits the growth of Coclodinium polycricoides but this strain does not exhibit species-specificity. In particular Micrococcus Luteus SY-13 exhibits strong activity against Chattonella spp. And Coclodinium polycricoides. This strain represents a broad host range against all pest species tested. However, this strain shows weak activity against relatively small algal cells (<20 μm cell size), such as heterosigma acacio and prorocentrum minimum. Moreover, these algal cells regenerate within two days. Interestingly, Microcaucus Luteus SY-13 shows no inhibitory effect on the growth of diatom skeletonoma costumum. Micrococcus luteus SY-13 is believed to have only harmful algal species-specific algae activity. Thus, these results indicate that Micrococcus Luteus SY-13 plays an important role in controlling harmful algae associated with Coclodinium polycricoides.

Although bactericidal bacteria are not the best way to control large numbers of harmful algae, they are effective in small red tides, such as those occurring in Maryland in 1997. However, before the killing bacteria can be applied to the control of harmful birds, further invention is needed. Although knowledge of these compounds is biologically important to marine ecosystems, little screening of algae compounds to control harmful algae has yet been made.

It is important to characterize the properties and functions of the algae compounds before they are applied to biological environmental purification in the natural environment. Unfortunately, most conventional inventions related to the control of noxious algae through the application of agrochemical effects have been limited to freshwater ecosystems, with few inventions having little environmental side effects on marine algae. Recently, characteristics of secondary metabolites purified from algae bacteria have been reported. Furthermore, there have been reports of algae that have little effect on marine life, such as bacillamide and phlorotannins. In conclusion, the inventors have invented seven kinds of bacteriobacteria including micrococcus luteus SY-13. The results suggest that algal bacteria are potential candidates for the control of harmful algae. Moreover, the invention requires the identification of the algal compound structure and the precise mechanism identification.

The present invention will be described in more detail with reference to the following examples. However, these examples do not limit the scope of the present invention.

Example 1 Isolation of Algal Bacteria Against Coclodinium Polycricoides

Seawater samples from Gamak Bay were collected from a depth of 1 m using Van Dorn sampler. Sampling was carried out during the extinction of the Coclodinium polycricoides red tide in mid-September 2002 to isolate and count populations of algae bacteria against Coclodinium polycricoides. The number of coclodinium polycricoides cells was calculated using an inverted microscope.

The harmful coarse algae used in the present invention was collected from Gamak Bay in mid-September 2002. Sterile clones were obtained through repeated passages with capillary pipettes and repeated passages using enriched seawater medium containing antibiotic complexes. The concentration of the antibiotic complex in the medium was: 100 μg mL ^-1 ampicillin, 10 μg mL ^-1 streptomycin, 10 μg mL ^-1 chloramphenicol, 10 μg mL ^-1 penicillin G, 50 μg mL ^-1 neomycin, 50 μg mL ^-1 gentamicin, 10 μg mL ^-1 kanamycin and 1.5 μg mL ^-1 nystatin. All antibiotics and f / 2-Si medium were purchased from Sigma (St. Louis, MO). Coclodinium polycricoides was incubated and maintained in f / 2-Si medium made of GF / F-filtration seawater. f / 2-Si components of the medium _{is: 75 mg NaNO 3, 5 mg} NaH 2 PO 4 · H 2 O, 4.36 mg Na 2 EDTA, 3.15 mg FeCl 3 · 6H 2 O, 0.01 mg CuSO 4 · 5H 2 O , 0.022 mg ZnSO ₄ · 7H ₂ O, 0.01 mg CoCl ₂ · 6H ₂ O, 0.18 mg MnCl ₂ · 4H ₂ O, 0.006 mg Na ₂ MoO ₄ · 2H ₂ O, 0.1 mg thiamineHCl, 0.5 μg Biotin and 0.5 Μg 1 liter of GF / F-filtered seawater each containing vitamin B12. Cultures at 20 ° C. were grown in disposable tissue culture flasks (Iwaki, Chiba, Japan) sterilized with light intensity 120 μmol photon m ⁻² s ⁻¹ under 12 hour light: 12 hour dark cycle.

The first screening for the bactericidal bacteria was carried out with only Coclodinium polycricoides cells. The population of algae bacteria against Coclodinium polycricoides was calculated using the optimal spread method (MPN). Red algae cultured in medium-exponential growth were ca. Diluted 1.0 mL 10 ⁴ cells mL ⁻¹ and some 0.5 mL of Coclodinium PolyCrycoides culture was aseptically transferred to a 48-well microplate (Costar, Corning, NY). Seawater samples were filtered through 0.8 or 0.2 μm membrane pore-sized membrane filters (Advantec, Tokyo, Japan) and then diluted with autoclaved seawater. The 0.8 μm filter removes larger particles than bacteria. A portion of each diluted seawater sample (0.5 mL) was inoculated into each well of a 48-well microplate containing Coclodinium Polycricoides culture. Cultures in microplates were cultured under the conditions as described above and the viability of Coclodinium polycricoides cells in each well was analyzed daily using an inverted microscope. Wells in which more than 99% of Coclodinium polycricoides cells were destroyed were considered “positive” and the MPN method was calculated using a microcomputer program to determine the population of the bactericidal bacteria against Coclodinium polycricoides. At this time the autoclave and filtered (0.1 μm membrane pore-size membrane filter) seawater was inoculated as a negative control.

Potential algae strains against Coclodinium polycricoides were isolated from positive wells in which Coclodinium polycricoides cells were completely destroyed. Wells with the highest dilution factor possible were selected to isolate dominant species and subsamples were plated on Zobell 2216E agar plates. The number of bacteria visible to the naked eye was measured by colony forming unit (CFU) after 1 week of growth on a Zobell 2216E agar plate at 25 ° C. All 53 colonies with different colony colors and morphological forms were selected for isolation. Each strain was incubated and inoculated again with coclodinium polycricoides to confirm algal activity against coclodinium polycricoides. As a result, seven algae strains were isolated in the present invention.

Example 2 Determination of Activity of Isolated Strain

These strains were cultured in Zobell 2216E medium to measure the algae activity of seven strains against Coclodinium polycricoides. The bacteria were incubated at 25 ° C., 200 rev min ⁻¹ for 3 days. The bacterial cells were then removed by centrifugation (5,000 g at 4 ° C. for 20 minutes) and filtration (0.2 μm membrane pore-size filters) and the culture filtrate was used to measure algal activity. Coclodinium polycricoides cells were cultured in 300 mL tissue culture flasks containing 100 mL f / 2-Si medium. Coclodinium polycricoides (180 μl) was then inoculated into each well of a 24-well tissue culture plate (NUNCLON, Copenhagen, Denmark) in the mid-exponential growth phase (12 days post culture). 20 μl of each bacterial culture filtrate was added to a 24-well plate containing coclodinium polycricoides. 20 μl of fresh Zobell 2216E medium was added as a control to exclude the activity of bacterial culture medium. The biological assay plate was then incubated under the conditions as described above. After 6 hours, the number of viable cells was calculated directly on the Sedgewick-Rafter grater at × 200 magnification using an inverted microscope. Algal activity was calculated as follows: Algal activity (%) = {1-(initial entity of coclodinium polycrycoides subject / coclodinium polycricoides visible visually after treatment) x 100}. All experiments were repeated three times and the results are expressed as mean and standard deviation of the raw data.

Example 3 Identification of Algal Bacteria

Seven algae strains were grown on Zobell 2216E agar medium at 25 ° C. for 3 days. Morphological features were observed with a scanning electron microscope (Hitachi S3500N, Japan). Standard physiology and biochemical characteristics were tested according to the method of MacFaddin. Additional biochemical tests were performed using API kits (API 20E, API 50CHB / E, API 50 CHL, and API Staph; BioMerieux, Marcy L'Etoile, France). Furthermore, cellular fatty acid methyl esters are described in Huys et al. (1997) and analyzed by Sherlock Microbial Identification System (MIDI; Hewlett Packard 6890, Los Angeles, CA).

PCR amplification of the gene of 16S rRNA was performed with Takara PCR heat cycler. Genomic DNA was prepared using AccuPrep Genomic DNA Extraction Kit Bioneer, Daejeon, Republic of Korea) and PCR was prepared using template and universal primer 27F (5'-AGAGTTTGATCMTGGCTCAG-3 ') (SEQ ID NO: 1) and 1492R (5'-GATTACCTTGTTACGACTT -3 ') (SEQ ID NO: 2) using genomic DNA. These primers were commonly synthesized (Bioneer, Republic of Korea). The gene was amplified by 30 cycles at 94 ° C. for 20 seconds, 55 ° C. for 1 minute, 72 ° C. for 1 minute and then elongated at 72 ° C. for 10 minutes. The 1.5 kb PCR product was cloned from the pGEM-T easy vector (Promega, Madison, WI) and sequenced using Termination Sequencing Ready Reaction Kit (Perkin Elmer, Foster City, Calif.) And using an ABI 377 Genetic Analyzer (Perkin Elmer). Was analyzed.

Almost fully revealed 16S rDNA sequences were obtained from CLUSTAL W software Ver. Sort using 1.7. The 16S rDNA sequence used for phylogenetic analysis was derived and compared with the 16S rDNA sequence of other bacteria available in the DDBJ / EMBL / GenBank database. Kimura's two-parameter model was applied to the calculation of evolutionary distance. The phylogenetic tree was performed by a neighbor-joining method. Bootstrap analysis of 1000 copies was performed using MEGA version 2.0.

Example 4 Agrochemical Effects of SY-13 Culture Filtrate of Strain Against Coclodinium Polycricoides

The agrochemical effect of strain SY-13 filtrate against Coclodinium polycricoides was invented at various filtration concentrations. Bacterial and coclodinium polycricoides cultures were prepared as described above. Culture filtrate of strain SY-13 was added to coclodinium polycricoides culture (ca. 1.0 × 10 ⁴ cells mL ⁻¹ ) at concentrations of 1.25, 2.5, 5 and 10%, respectively. Zobell 2216E medium was added to the control and the biological assay plate was also incubated as described above. After 3, 6 and 9 hours of incubation, live stream cells in each well were counted on a Sedgewick-Rafter chamber using an inverted microscope.

Example 5 Agrochemical Range of Strain SY-13 Against Other Harmful Bird Species

The following red tide species were used to investigate the algae range of strain SY-13 against other harmful algae species: Chatonella spp. And Heterosigma Akassio provided by National Fisheries Research and Development Institute Footycomb, Pro-Rocentrum Mikans, Pro-Rocentrum Minimum, Scripsiella Trocoidea and Scretonema Costatum were provided by the South Sea Institute of Korea Ocean Invention. The cultures of five cotyledon, two needles, and one diatom were monocultured but showed some bacterial contamination.

All but the skeletal retrograde nematic course tatum algae culture solution is in the 20 12 hours ℃ name: 12 hours female cycle, the roughness 120 μmol photons m ^-2 s ^{- 1} were incubated at f / 2-Si medium under. To invent the acaricide range of strain SY-13, cultures of harmful algae except bacteria and Skeletonema costumum were prepared in the same manner as the coclodinium polycricoides assay. 10% concentration of the filtrate was added to each algal culture during the medium-exponential growth phase. After 6 hours of culture, live stream cells were calculated as described above.

An agar overlay assay was used to assess the killing effect of strain SY-13 on the growth of S. costatum . An 8 mm diameter filter paper disc (Advantec), aseptically buried with 10% culture filtrate, was placed in the center of the S. costatum lawn. The algal ron plates were incubated in f / 2 agar plates for 2 weeks at 20 ° C., roughness 30 μmol photons m ⁻² s ⁻¹ . Growth inhibition was measured by the size of the clear inhibition area around the filter paper disc.

Figure 1 shows the algae activity of 10% culture filtrate of seven algae strains against Coclodinium polycricoides. All strains were incubated at 25 ° C. for 3 days in Zobell 2216E medium. A 10% culture filtrate of each strain was added to Coclodinium polycricoides cell cultures (ca. 1.0 × 10 ⁴ cells mL ⁻¹ ) incubated with medium-exposure metabolites. Filtrate of Zobell 2216E medium was used as a control. Data is shown as mean ㅁ standard deviation from three analyzes.

Figure 2 is a scanning micrograph of the algae strain isolated in the present invention. All strains were grown at 25 ° C. for 3 days in Zobell 2216E agar. a. Strain SY-11; b. Strain SY-13; c. Strain SY-19; d. Strain SY-20; e. Strain SY-21; f. Strain SY-31; g. Strain SY-47. Scale bar = 5 μm.

3 shows a phylogenetic tree based on comparison with 16S rRNA gene sequence to indicate taxonomic location of strain SY-13. The phylogenetic tree was prepared using the neighbor-joining method. Bootstrap values expressed as percentages of 1000 copies were given at branches.

Figure 4 shows the algae activity of the culture filtrate of Micrococcus luteus SY-13 against Coclodinium polycricoides at various concentrations (●, control; ○, 1.25%; ▼, 2.5%; Δ, 5%, ■, 10%). Micrococcus Luteus SY-13 was incubated at 25 ° C. for 3 days in Zobell 2216E medium. Each culture filtrate was added to coclodinium polycricoides cell cultures (ca. 1.0 × 10 ⁴ cells mL ⁻¹ ) incubated with a medium-exponent. Filtrate of Zobell 2216E medium was used as a control. Data is shown as mean ± standard deviation from three analyzes.

FIG. 5 is a micrograph of the coclodinium polycricoides digestion process treated with 10% culture filtrate of Micrococcus luteus SY-13. a. Living cells of coclodinium polycricoides forming eight chains. b. Rounded and expanded cells 30 minutes after exposure to 10% culture filtrate. c, d. Severely damaged cells after 3 and 6 hours, respectively. Ruptured and expanded cells were observed (arrows). It should be noted that the nucleus and protoplasts of Coclodinium polycricoides cells are degenerated. Once Coclodinium Polycricoides cells rupture, Coclodinium Polycricoides cells do not recover to live cells before two days. Scale bar = 30 μm.

Figure 6 shows the algae range of Micrococcus luteus SY-13 against other pest species. A 10% culture filtrate of Micrococcus luteus SY-13 was added to each red tide culture cultured with a medium-large exponent. After incubation for 6 hours, algal activity was measured. Data is shown as mean ± standard deviation from three analyzes.

<110> Pusan National University Industry-University Cooperation Foundation <120> Marine bacteria having algicidal activity against Cochlodinium          sp. <160> 2 <170> KopatentIn 1.71 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 1 agagtttgat cmtggctcag 20 <210> 2 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 2 gattaccttg ttacgactt 19  

Claims (4)

Marine bacterium Micrococcus luteus SY-13 with algae activity against algae in Coclodinium (Accession No .: KCTC 11361BP) The method of claim 1, wherein the 10% culture filtrate of the micrococcus Luteus SY-13 is marine bacteria Micrococcus Luteus SY-13 having algae activity in the algae of Cochlodinium containing Cochlodinium polykrikoides The method according to claim 1 or 2, wherein the Micrococcus luteus SY-13 (Accession Number: KCTC 11361BP) is Chattonella sp., Akashiwo sanguinea , Kimnodium Algae selected from Gymnodinium impudicum , Heterosigma akashiwo , Prorocentrum micans , Prorocentrum minimum and Scrippsiella trochoidea Marine bacterium Micrococcus luteus SY-13 An algae composition comprising the marine bacterium Micrococcus luteus SY-13 (Accession No .: KCTC 11361BP) and a nutrient medium

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