JP2002060307A - Red tide-controlling agent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new red tide-controlling agent, and further to obtain a new microorganism as an active ingredient for the agent. SOLUTION: This red tide-controlling agent contains the microorganisms belonging to the genus Alteromonas as an active ingredient. The new microorganism as the active ingredient therefor is Alteromonas NB3131 (FERM P-17926) of the microorganism belonging to the genus Alteromonas.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The invention of this application relates to a red tide controlling agent and a novel microorganism. For more information,
The invention of this application relates to a red tide controlling agent capable of effectively preventing or reducing damage caused by red tide in a sea area where marine products are harvested and cultured, and a novel microorganism as an active ingredient of the agent.

[0002]

2. Description of the Related Art Red tide (red water, red tide) is a general term for a phenomenon in which seawater discolors with the explosive large proliferation of suspended organisms (mainly phytoplankton). In Japan, various names are left among fisheries stakeholders depending on the region, such as ebb water in the Tohoku region, blue tide and white tide in Tokyo Bay, Napa water in Sagami Bay, kashimi tide in the Seto Inland Sea, Okinawa Star Juice (Pusnusir)
Are the same phenomena as the red tide.

[0003] The content and properties of red tide are not necessarily the same, and the constituent plankton, diatom, dinoflagellum, raphid algae,
It covers a wide area such as green algae and ciliates. The mechanism of red tide generation has been qualitatively clarified by many studies and studies. In other words, red tides are likely to occur when nutrients such as nitrogen and phosphorus contained in domestic wastewater and industrial wastewater flow into rivers and waters in the inner bays and inland waters, and nutrients are eluted from marine sediment that is rich in organic matter. It is a water area where nutrition is advanced. In such waters, plankton multiplication starts when the water temperature and salinity become conditions suitable for the growth of plankton, and when there are growth promoting factors (trace metals such as heavy metals such as iron and special organic substances), these are triggered. It is thought to cause plankton overgrowth. Furthermore, when conditions such as a small amount of zooplankton feeding on phytoplankton or plankton accumulating in a specific place due to tides or winds overlap, a so-called red tide is generated. However, quantitative conditions such as when, where and what kind of red tide are generated are not sufficiently clarified.

The red tide has long caused many fisheries damages in various parts of the world, due to mechanical injuries such as plankton obstructing fish gills, and saxitoxin, goniotoxin, and berevetoxin from plankton. And other harmful substances are secreted. For this reason, various measures have been proposed and implemented in order to prevent and reduce the fishery damage caused by the red tide. For example, a red tide information transmission business is being implemented so that countermeasures can be taken in advance for the occurrence of red tide. In addition, as measures to prevent the occurrence of red tide, technologies for improving water quality and sediment, and development measures for feed, stopping feed, and early shipment are being implemented.

[0005] However, although the conventional measures are effective in reducing the damage caused by the red tide, they have not aimed at eliminating the generated red tide and thereby restoring the water environment to a favorable state.

On the other hand, in recent years, attempts have been made to kill red tide by killing red tide plankton using microorganisms. For example, Japanese Patent Application Laid-Open No. 05-112417 discloses a red tide controlling agent containing a microbial cell belonging to Pseudomonas stutzeri as an active ingredient. Also,
JP-A-07-322875 discloses a red tide controlling agent containing a microorganism belonging to the genus Caulobacter as an active ingredient.

On the other hand, microorganisms belonging to the genus Alteromonas are enzymes-producing bacteria such as protease and lipase (for example, see
No. 38749, Japanese Patent Application Laid-Open No. 11-169177, etc., and production of brown algae decomposition products such as kelp and hijiki (Japanese Patent Application Laid-Open No. 05-030988).
JP-A-09-206067) and components of a crustacean virus control agent (JP-A-09-206067). However, it has not been known at all that the microorganism of the genus Alteromonas has killing ability against phytoplankton causing red tide.

[0008]

SUMMARY OF THE INVENTION The inventors of the present application searched the sea and mud on the coast of Nagasaki Prefecture for the purpose of isolating novel useful aquatic microorganisms, and analyzed the function of the microorganisms obtained. As a result, we succeeded in isolating a microorganism of the genus Alteromonas which has a strong killing ability against plankton causing red tide.

An object of the present invention is to provide a red tide controlling agent using the novel microorganism.

[0010]

SUMMARY OF THE INVENTION The present application discloses an invention which solves the above-mentioned problems, such as Alteromonas.
s) A red tide controlling agent comprising a microorganism belonging to the genus as an active ingredient.

[0011] Further, as one of the microorganisms of the genus Alteromonas, which is an active ingredient of the red tide controlling agent of the present invention, a novel microorganism
Also provides Alteromonas NB3131 (FERM P-17926).

Hereinafter, embodiments of the present invention will be described in detail.

[0013]

BEST MODE FOR CARRYING OUT THE INVENTION The red tide controlling agent of the present invention is characterized by using a microorganism of the genus Alteromonas as an active ingredient. Specifically, as a culture solution itself containing microorganisms, a concentrated solution, separated cells, broken cells, dried cells, or a form in which any of these is adsorbed or mixed with clay powder, fish culture sea areas, etc. Can be sprayed.

As the microorganism of the genus Alteromonas, any kind of Alteromonas may be used as long as it has a killing ability against plankton that causes red tide. Alteromonas NB3131 strain isolated from lagoon is preferred. This Altero
The monas NB3131 strain has been deposited as a patented microorganism with the Institute of Biotechnology and Industrial Technology, National Institute of Advanced Industrial Science and Technology on June 28, 2000 (Accession No. FREM P-17926).

The mycological properties of the Alteromonas NB3131 strain are as follows.

(1) Gram-negative (2) Bacillus (3) Aerobic (4) Oxidase-positive (5) Catalase-positive (6) Salt requirement (grown in seawater medium) (7) Glucose utilization (8) Brown (9) Major quinone Q-8 This Alteromonas NB3131 strain has the nucleotide sequence shown in SEQ ID NO: 1 in a part of its genomic DNA.
In addition, the determination of the genome sequence was performed using the well-known PCR method. That is, PCR was performed using DNA extracted from the cells as a template and oligonucleotides consisting of the nucleotide sequences of SEQ ID NOs: 2 and 3 as primers. Reaction conditions are as shown in Table 1. The sequence of the obtained PCR product was determined by a sequencer.

[0017]

[Table 1]

Mycological properties and genome D as described above
The Alteromonas NB3131 strain having an NA partial sequence can be cultured at about 20 ° C. in a known medium, for example, Marine Ager 2216 (pH7.6: manufactured by Difco). Also this
Alteromonas NB3131 strain can be frozen and stored at a temperature of about -80 ° C.

The results of tests on the properties and the like of Alteromonas NB3131 are shown below. Test Example The killing ability (algicidal properties) against plankton caused by red tide was tested.

In a 24-well plate, 1.5 ml of ESM medium, 0.5 m of Chattonella, a kind of phytoplankton, which causes red tide
l, 25 μl of the bacterial solution or supernatant of the Alteromonas NB3131 strain is added, and a light-dark cycle of 26 ° C., 12 hours / 12 hours (illuminance 3000 l)
Culture was performed for 7 days in x). After the cultivation, algicidal activity was determined by observing the motility and morphology of the chatonella with a microscope, and the appearance of the culture solution was evaluated.

++: A state where Chattonella is almost dead +: A state where floating Chattonella is small and the number of sedimentation and death is large ±: The number of floating is slightly small and the number of sedimentation is increasing, but the number of deaths is changed No state-: The number of suspensions, the number of settling and the number of deaths are almost the same as those of the control. The cells or supernatant of Alteromonas NB3131 strain is 2%.
ESM medium containing glucose, Altero grown at 26 ° C
culture solution of monas NB3131 strain, or centrifugation of the culture solution (1
5,000 rpm, 4 ° C, 10 minutes), and the supernatant
℃, 80 ℃, 100 ℃ heat treatment for 5 minutes each,
And supernatant obtained by filtration with a filter having a pore size of 0.2 μm (New Stelladics; manufactured by Kurabo Co.) (filtration)
Was used.

The results are as shown in Table 2. As shown in Table 2, strong algicidal activity was observed in the bacterial solution and the untreated supernatant. However, when the supernatant is subjected to heat treatment or filtered, the algacidal properties are lost.Therefore, cells remain in the supernatant after centrifugation, and the supernatant is removed by these cells. It is thought that it showed sex.

That is, it was suggested that the Alteromonas NB3131 strain is not a type that produces an algicidal substance outside the cells, but is a strain that exhibits algicidal properties when it comes into contact with Chatonella. Fig. 1 shows normal chatonella (top)
And killed by culturing with a bacterial solution of Alteromonas NB3131 (bottom row).

[0024]

[Table 2]

Next, the number of surviving cells of Châtonella was counted using a hemocytometer, and the algicidal activity of Alteromonas NB3131 strain was quantitatively examined.

The results are as shown in FIG. Control and filtered supernatants did not affect viable counts of Châtonella, while Alteromonas NB31
When 31 strains of the bacterial solution were added, the chutnera almost completely died within 24 hours of the culture. Further, as a result of observation at short time intervals, as shown in FIG. 3, Alteromonas NB31
It was confirmed that 31 strains completely killed Chatnella between 8 hours and 24 hours.

Further, Alteromona to be added to Chatonella
s The concentration of the NB3131 strain was diluted 1- to 1000-fold, and the algicidal change was examined.

The results are as shown in FIG.
The monas NB3131 strain almost completely killed Chatonella within 24 hours up to a 1/100 concentration. Furthermore, even at a concentration of 1/1000
After 48 hours, it was confirmed that she had the ability to kill Chatnella completely.

[0029]

As described above in detail, according to the invention of this application, a novel microorganism Alteromonas NB31 having a strong killing ability against phytoplankton causing red tide.
The present invention provides 31 strains and a morning tide controlling agent comprising this microorganism as an active ingredient.

[0030]

[Sequence List] Sequence Listing <110> Japan Science and Technology Corporation <120> An agent for red tide <130> NP00298YS <140> <141> <160> 3 <170> PatentIn Ver. 2.1 <120> <210> 1 <211> 1491 <212> DNA <213> Alteromonas <400> 1 atrtttgatc ctggctcaga ttgaacgctg gcggcaggcc taacacatgc aagtcgaacg 60 gtaacatttc tagcttgcta gaagatracg agtggcggac gggtragtaa tgcttgggaa 120 cttgcctttg cgagggggac aacagttgga aacgactgct aataccgcat aacgtcttks 180 ggaccaaaac ggggctttta gctctggcgc aaagagaggc ccaagtgaga ttagctagtt 240 ggtgaggtaa aggcttacca aggcgrcgat ctctagctgt tctgagagga agatcagcca 300 cactgggact gagacacggc ccagactcta cgggaggcag cagtgggraa tattgcacaa 360 tggggraaac cmtgatgcag ccatgccgcg tgtgtgaaga aggcwtcggg ttgtaaagma 420 ctwtcagttg tgaggaaggg rtgttggtta atacccaaca tcattgacgt tagcaacaga 480 agaagcaccg gctaactccg tgccagcagc cgcggtaata cggagggtgc gagcgttaat 540 cgraattact gggcgtaaag mgcamgcagg cggtwtgtta agctagatgt gaaagcmccg 600 ggcttaacct ggraattgca tttagaactg gcagactaga gtcatttaga ggggggtgga 660 at accaggtg tagctggtra aatccgtaga gatctggagg aacatcggtg gcgaaggcgg 720 ccccctggag ataractgac gctcatgtgc gaaagcgtgg gtagcgaaca ggatwagata 780 ccctggtagt ccacgccgta aacgctgtct actagctgtg tgttgtwwtt aaacgatgcg 840 tggcgaasta acgcgctaag tagaccgcct ggggagtacg gccgcaaggt taaaactcaa 900 atgatttdac gggggcccgc acaagcggtg gagcatgtgg tttaattcga tgcaacgcga 960 agaaccttac catcmcttga catccagaga attttctaga gatagattag tgcttcggga 1020 actctgaaac aggtgctgca tggctgtcgt cagctcgtgt cgtgagatgt tgggttaagt 1080 cccgcaacga gcgcaaccct tatccttagt tgccagcctt aagttgggca ctctaaggar 1140 actgccggtr acaaaccgga ggaaggtggg gacgacgtca agtcatcatg gcccttacgg 1200 gatgggctac acacgtgcta caatggcagg tacagaggga agcgagactg tkaagtggag 1260 cggaaccctt aaagcttgth gtagtccgga ttggagtctg caactcgact ccatgaagtc 1320 ggartcgcta gtaatcgcag gtcagaatac tgcggtgaat acgttcccgg gccttgtaca 1380 caccgsccgt tacaccatgg gagtgggatg caaaagaagt agttagtcta accttcggga 1440 gggcgattac cactttgtgt ttcatgactg gggtgaagtc gtaacaaggt a 1491 <210 > 2 <211> 20 <212 > DNA <213> Artificial Sequence <220> <213> Artificial Sequence: Synthesized oligonucleotide <400> 2 agagtttgat cctggctcag 20 <210> 3 <211> 19 <212> DNA <213> Artificial Sequence <220> <213> Artificial Sequence : Synthesized oligonucleotide <400> 3 ggctaccttg ttacgactt 19

[Brief description of the drawings]

Fig. 1: Normal chatonella (upper) and Alteromonas NB
It is a chatonella (lower row) that has been killed by culturing with the bacterial solution of 3131 strain.

FIG. 2 is a graph showing the change over time in the number of surviving cells of Châtonella.

FIG. 3 is a graph showing the change over time in the number of surviving cells of Chatnella.

FIG. 4. Number of surviving cells of Chatonella and Alteromonas NB3131
It is the graph which showed the relationship with the density | concentration of the strain.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification code FI Theme coat ゛ (Reference) (C12N 1/00 C12R 1:01) C12R 1:01) (C12N 1/20 (C12N 1/20 C12R 1) : 01) C12R 1:01) C12N 15/00 ZNAA F-term (reference) 4B024 AA10 AA11 CA03 HA14 4B065 AA01X AC20 CA54 4D040 DD01 4H011 AD01 BB21 BC08 DA13 DD01

Claims (3)

[Claims] 1. A red tide controlling agent comprising a microorganism belonging to the genus Alteromonas as an active ingredient. 2. The microorganism belonging to the genus Alteromonas is Al
The red tide controlling agent according to claim 1, which is teromonas NB3131 (FERM P-17926). 3. Alteromonas NB3131 (FERM P-17926).