Byllouin nitrate reduction pelagic bacillus and application thereof in induction of juvenile mollusk attachment of mytilus coruscus

Abstract

The invention belongs to the technical field of biology, and particularly discloses a deep nitrate reducing ocean bacillus, the name of the deep nitrate reducing ocean bacillus is Pelagibacterium nitrareduce M200, the classification name of the deep nitrate reducing ocean bacillus is Pelagibacterium nitrareduce, the deep nitrate reducing ocean bacillus is preserved in the China Center for Type Culture Collection, and the preservation number of the deep nitrate reducing ocean bacillus is CCTCC M 20241110. The invention also discloses an application of the Pelagibacterium nitrareduce M200 in induction of juvenile mollusk adhesion of mytilus coruscus, a biofilm formed by the strain has the activity of inducing juvenile mollusk adhesion of mytilus coruscus and can adapt to an extreme environment, and a new scheme with a wider application range is provided for increasing the adhesion rate of mytilus coruscus and increasing the yield of mytilus coruscus.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and relates to a strain of Pelagibacterium nitratireducens and an application thereof in inducing the attachment of thick-shelled mussel juveniles, and specifically to the separation, purification, identification and preservation of Pelagibacterium nitratireducens M200, and the influence of the biofilm formed by the strain under different initial bacterial density conditions on the attachment of thick-shelled mussel juveniles. Background Art

[0002] The ocean covers more than two-thirds of the Earth's surface, contains a large number of rich microbial resources, and plays an important ecological role. The Mariana Trench is the deepest trench on Earth, located in the western North Pacific Ocean, near the Mariana Islands. The bottom of the trench has ultra-high hydrostatic pressure, low temperature and darkness. Due to the difficulty of sampling, few microorganisms were isolated and cultured from the Mariana Trench in the early days. With the development of deep-sea sampling technology, researchers have gradually carried out research on microorganisms in the Mariana Trench.

[0003] Thick-shelled mussel (Mytilus coruscus), as one of the important economic species of marine aquaculture shellfish in my country, is also a representative attached organism. In the growth history of thick-shelled mussels, the larvae will go through two stages, the planktonic stage and the attached stage. The larvae undergo metamorphosis and develop into juveniles, which change from a planktonic state to an attached life. After the juveniles grow and develop, they become adult shellfish. Thick-shelled mussel juveniles will look for a suitable surface for attachment and growth. When the environment changes, the juveniles will cut off their own byssus and look for a new attachment base for attachment. In recent years, due to the massive destruction and overfishing of thick-shelled mussel natural resources, seedling production has been severely restricted, and wild resources have declined sharply. In the marine aquaculture method, seedling removal is an important factor affecting the seedling survival rate of thick-shelled mussels. Therefore, improving the secondary attachment rate is the key to solving industrial problems. It is particularly necessary to develop deep-sea bacteria and explore the development of technologies to induce thick-shelled mussel attachment using bacteria to form biofilms to solve seedling problems. Summary of the invention

[0004] The phenomenon of seedling shedding is a common problem in mussel farming in both the north and south, and is also an important aspect that affects mussel production. There are many ways to solve the problem of seedling shedding, such as selecting an excellent attachment substrate, appropriate seedling packing density, raft farming, and choosing the right time to pack seedlings. Previous studies have found that attachment substrates with bacterial biofilms can significantly increase the attachment rate of mussel juveniles, and different bacteria have different induction activities. Although some offshore bacteria have induction activity, the bacteria themselves are not very adaptable to extreme environments. Therefore, this study screened deep-sea bacteria to explore the induction activity of deep-sea bacteria on mussel attachment.

[0005] The specific technical solution adopted by the present invention is as follows:

[0006] In a first aspect, the present invention provides a strain of hadal nitrate-reducing pelagic bacterium Pelagibacterium nitratireducens M200, which originates from the surface sediments at -10,900 m underwater at the MT2016 / 12-MTD station (142.36°E, 11.43°N) in the Mariana Trench in the western Pacific Ocean. Pure strains were isolated and purified using ZoBell 2216E solid culture medium, and uploaded to the NCBI database after comparison with the nucleic acid data in GENEBANK through 16S rRNA gene sequencing, obtaining the sequence number PP464210. The bacteria were finally named Pelagibacterium nitratireducens M200, and its classification name was Pelagibacterium nitratireducens. The bacteria were deposited in the China Center for Type Culture Collection (CCTCC) with a preservation date of May 30, 2024 and a preservation number of CCTCC M20241110. The preservation address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0007] In a second aspect, the present invention provides the use of the abyssal nitrate-reducing pelagic bacillus Pelagibacterium nitratireducen M200 in inducing attachment of thick-shelled mussel spat.

[0008] Furthermore, in the application of Pelagibacterium nitratireducen M200, the pure strain of Pelagibacterium nitratireducen M200 was inoculated into the seawater 2216E liquid culture medium for expansion and culture, and the bacterial precipitate was obtained after centrifugation, which was washed with sterilized seawater and made into a suspension. The suspension was diluted and cultured at 18°C ​​in the dark until a biofilm was formed, and then used to induce the attachment of thick-shelled mussel spat.

[0009] Furthermore, the suspension was diluted to an initial bacterial density of 1.0×10 6 ~5×10 8 The biofilms formed under different initial density conditions had different induction activities on juvenile spat. 8 cells / ml, the induction rate of biofilm formed on juvenile spat attachment was 27.78% and the initial density was 1.0×10 6 cells / ml, the induction activity of the biofilm formed on juvenile spat was only 14.4%.

[0010] The present invention has the following beneficial effects:

[0011] The biofilm formed by the abyssal nitrate-reducing pelagic bacterium Pelagibacterium nitratireducen M200 of the present invention has the activity of inducing the attachment of thick-shelled mussel juveniles, and the strain can adapt to extreme environments, providing a new and wider range of solutions for improving the attachment rate of thick-shelled mussels and increasing mussel yields. The present invention also found that the biofilm formed under different initial density conditions has different induction activities on juveniles, which is helpful for optimizing the application of the abyssal nitrate-reducing pelagic bacterium Pelagibacterium nitratireducen M200. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 The results of an experiment on induction of the attachment of thick-shelled mussel spats by biofilms formed by different initial bacterial densities in a specific embodiment. DETAILED DESCRIPTION

[0013] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments.

[0014] 1. Isolation and preservation of strains

[0015] The nitrate-reducing pelagic bacterium Pelagibacterium nitratireducens M200 involved in the present invention is derived from the surface sediments at -10900m underwater at the MT2016 / 12-MTD station (142.36°E, 11.43°N) in the Mariana Trench in the western Pacific Ocean. The pure strain colony is isolated and purified using ZoBell 2216E solid culture medium, and the 16S rRNA gene is sequenced and compared with the nucleic acid data in GENEBANK (http: / / www.ncbi.nlm.nih.gov / ) and uploaded to the NCBI database, and the sequence number is PP464210. The bacterial name is finally determined to be nitrate-reducing pelagic bacterium M200. The bacterium is deposited in the China Center for Type Culture Collection (CCTCC), the deposit date is May 30, 2024, the deposit number is CCTCC M 20241110, and the deposit address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0016] 2. Experiment on the induction of nitrate-reducing Pelagibacterium nitratireducen M200 on the attachment of thick-shelled mussel juveniles

[0017] The main steps are to inoculate the pure strain of nitrate-reducing pelagic bacillus into the seawater 2216E liquid culture medium for expansion, obtain bacterial sediment after centrifugation, wash with sterile seawater and make a suspension, and repeat the experiment many times. Finally, the obtained bacterial solution is diluted and stained with acridine orange, and 10 points are randomly selected for counting under a 1000x fluorescence microscope to determine the total density of the bacterial solution to be 4×10 9 A sterilized glass slide was placed in each culture dish, and sterilized seawater and bacterial solution were added to the culture dish so that the initial bacterial density was 1.0×10 6 cells / ml, 1.0×10 7 cells / ml, 1.0×10 8 cells / ml and 5×10 8 cells / ml. The biofilm was formed by incubation at 18°C ​​in the dark for 48 hours. Finally, the slide with biofilm and 10 thick-shelled mussel spats (shell length 1.31±0.02mm; shell height 0.89±0.02mm) were placed in a culture dish containing sterile filtered seawater, and the attachment rate of spats was recorded at 48 hours, that is, the percentage of spats attached to the slide to the total number of spats in the culture dish.

[0018] The results are as follows Figure 1 The results show that the biofilm formed by the bacteria has the activity of inducing the attachment of thick-shelled mussel juveniles, and the induction activity of the biofilm formed under different initial density conditions on juveniles is also different. 8 cells / ml, the induction rate of biofilm formed on juvenile spat attachment was 27.78% and the initial density was 1.0×10 6 cells / ml, the induction activity of the biofilm formed on juvenile spat was only 14.4%.

[0019] In summary, the biofilm of the abyssal nitrate-reducing pelagic bacterium Pelagibacterium nitratireducens M200 has the effect of inducing the attachment of juvenile spat, and the present invention is of great significance for clarifying the attachment mechanism of juvenile spat of thick-shelled mussel and for the artificial cultivation of thick-shelled mussel.

[0020] This specific implementation is only an explanation of the present invention, not a limitation of the present invention. Any changes made by those skilled in the art after reading the specification of the present invention will be protected by the patent law as long as they are within the scope of the claims of the present invention.

Claims

1. A strain of Abyssal nitrate-reducing Pelagibacterium, characterized in that: The name is Pelagibacterium nitratireducens M200, the classification name is Pelagibacterium nitratireducens, and it is preserved in the China Center for Type Culture Collection with the preservation number CCTCCM20241110.

2. Use of the abyssal nitrate-reducing pelagic bacillus according to claim 1 in inducing attachment of thick-shelled mussel spat.

3. The use of the abyssal nitrate-reducing pelagic bacillus according to claim 2 in inducing attachment of thick-shelled mussel spat, characterized in that: The pure strain of nitrate-reducing pelagic bacillus was inoculated into seawater 2216E liquid culture medium for expansion and culture. The bacterial precipitate was obtained after centrifugation, washed with sterile seawater and made into a suspension. The suspension was diluted and cultured at 18°C ​​in the dark until a biofilm was formed, which was then used to induce the attachment of thick-shelled mussel spat.

4. The use of the abyssal nitrate-reducing pelagic bacillus according to claim 3 in inducing attachment of thick-shelled mussel spat, characterized in that: The suspension was diluted to an initial bacterial density of 1.0×10 6 ~5×10 8 cells / ml.

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