Psychrobacter flexneri and application of psychrobacter flexneri in induction of juvenile mollusk attachment of mytilus coruscus

Abstract

The invention belongs to the technical field of biology, and particularly discloses a psychrobacter flexneri strain which is named as Psychrobacter fozii M209, is classified and named as Psychrobacter fozii, is preserved in the China Center for Type Culture Collection (CCTCC), and has the preservation number of CCTCC NO: M 20241109. The invention further discloses application of the psychrobacter flexneri M209 in induction of juvenile mollusk attachment of mytilus coruscus, a biofilm formed by the strain has activity of inducing juvenile mollusk attachment of mytilus coruscus and can adapt to extreme environments, and a novel scheme with a wider application range is provided for increasing the attachment rate of mytilus coruscus and increasing the yield of mytilus coruscus.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and relates to a strain of Psychrobacter fozii and an application thereof in inducing attachment of thick-shelled mussel juveniles, and specifically relates to the separation, purification, identification and preservation of Psychrobacter fozii M209 and the influence of the microbial capsule formed by the strain under different initial bacterial density conditions on the attachment of thick-shelled mussel juveniles. Background Art

[0002] Thick-shelled mussels (Mytilus coruscus), belonging to the phylum Mollusca, class Bivalvia, order Mytilodia, family Mytilidae, are important marine shellfish aquaculture species in my country, distributed along the coasts of the Yellow Sea, Bohai Sea and East China Sea. In the life history of thick-shelled mussels, after the fertilized eggs develop into eyed larvae, they undergo metamorphosis through attachment and metamorphosis into juveniles, and finally develop into adult shellfish. In the process of growth and development from juveniles to adult shellfish, when the environment changes, the juveniles of thick-shelled mussels can cut off the byssus by themselves, start crawling and re-select a suitable attachment base to attach again. In the aquaculture method in the sea area, seedling removal is an important factor affecting the seedling survival rate of thick-shelled mussels, so improving the secondary attachment rate is the key to solving industrial problems.

[0003] Patent CN108315280B discloses a marine hydrocarbon-removing marine rod bacterium and its application in inducing the attachment of thick-shelled mussel juveniles, revealing that the attachment of thick-shelled mussel juveniles can be regulated by the interaction between offshore bacteria and mussel hosts. Hadal bacteria are more adaptable to the environment than offshore bacteria. The abyss is the least explored and most mysterious marine environment on Earth, with a depth of approximately 6000m-11000m below sea level. Compared with offshore areas, the high pressure, low temperature, darkness, and oligotrophic environment of the abyss create a unique microbial community. Therefore, it is particularly important to explore and study the application effect of biofilms formed by marine bacteria in the abyss area on offshore mussel farming. 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, the present invention screened deep-sea bacteria to explore the induction activity of deep-sea bacteria on mussel attachment.

[0005] The specific technical solutions of the present invention are as follows:

[0006] In a first aspect, the present invention provides a strain of Psychrobacter fozii M209, which is derived from surface sediments at -6000m underwater at the MT2016 / 12-SMT1 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 PP464215. The bacteria were finally named Psychrobacter fozii M209, and its classification name was Psychrobacter fozii. The bacteria were deposited in the China Center for Type Culture Collection (CCTCC) on May 30, 2024, with the deposit number CCTCC NO: M 20241109, and the deposit 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 above-mentioned Psychrobacterium fowleri in inducing attachment of thick-shelled mussel juveniles.

[0008] Furthermore, the biofilm formed by Psychrobacter fowleri has the activity of inducing the attachment of thick-shelled mussel juveniles.

[0009] Furthermore, the specific induction method is: the Psychrophilic Bacillus fowleri strain is inoculated into the seawater 2216E liquid culture medium for expansion culture, the bacterial precipitate is obtained after centrifugation, it is washed with sterile filtered seawater and made into a suspension, the suspension is diluted and cultured at 18°C ​​in the dark until a biofilm is formed, and then used to induce the attachment of thick-shelled mussel spat.

[0010] 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 26.7%, which was relatively better. The initial density was 1.0×10 6 cells / ml, the biofilm formed had an induction activity of only 15% on juvenile spat.

[0011] The present invention has the following beneficial effects:

[0012] The biofilm formed by the Psychrobacter fozii M209 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 scheme with a wider range of applications for improving the attachment rate of thick-shelled mussels and increasing the yield of mussels. The present invention also finds that the biofilm formed under different initial density conditions has different induction activities on juveniles, which is helpful for the application optimization of Psychrobacter fozii M209. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 These are the experimental results of induction of attachment of thick-shelled mussel spat by microbial capsules formed by different initial bacterial densities in a specific embodiment. DETAILED DESCRIPTION

[0014] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments.

[0015] 1. Isolation and preservation of strains

[0016] The Psychrobacter fozii M209 involved in the present invention originates from surface sediments at -6000m underwater at the MT2016 / 12-SMT1 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 (http: / / www.ncbi.nlm.nih.gov / ) through 16S rRNA gene sequencing, and the sequence number was PP464215. The bacterial name was finally determined to be Psychrobacter fozii M209. The bacteria were deposited in the China Center for Type Culture Collection (CCTCC) on May 30, 2024, with the deposit number CCTCC NO: M20241109, and the deposit address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0017] 2. Experiment on the induction of attachment of Psychrobacter fozii M209 to juvenile mussels

[0018] The main steps are to inoculate the pure strain of Psychrobacter fowleri M209 into the seawater 2216E liquid culture medium for expansion, obtain bacterial sediment after centrifugation, wash with sterile filtered seawater and make a suspension, and repeat the experiment many times. Finally, the obtained bacterial solution is diluted and stained with acridine orange, and 30 fields of view are randomly selected for counting under a fluorescence microscope with a magnification of 1000, and the total density of the bacterial solution is determined to be 4×10 9A sterilized glass slide was placed in each culture dish, and sterilized filtered seawater and bacterial solution were added to the culture dish to make the initial bacterial density 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.

[0019] 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 spat attachment was 26.7% and the initial density was 1.0×10 6 cells / ml, the biofilm formed had an induction activity of only 15% on juvenile spat.

[0020] In summary, the biofilm formed by Psychrobacter fozii M209 has the effect of inducing juvenile spat attachment, 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.

[0021] 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 Psychrobacterium flexneri, characterized in that The name is Psychrobacterfozii M209, the classification name is Psychrobacter fozii, and it is preserved in the China Center for Type Culture Collection with the preservation number CCTCCNO: M 20241109.

2. Use of the Psychrobacterium fowleri described in claim 1 in inducing attachment of thick-shelled mussel juveniles.

3. The use of Psychrobacterium fowleri in inducing attachment of thick-shelled mussel spat according to claim 2, characterized in that: The biofilm formed by Psychrobacter fowleri has the activity of inducing the settlement of juvenile thick-shelled mussels.

4. The use of Psychrobacterium fowleri in inducing attachment of thick-shelled mussel spat according to claim 3, characterized in that: The specific induction method is: the Psychrophilic Bacillus fowleri strain is inoculated into the seawater 2216E liquid culture medium for expansion and culture, the bacterial precipitate is obtained after centrifugation, it is washed with sterile filtered seawater and made into a suspension, the suspension is diluted and cultured at 18°C ​​in the dark until a biofilm is formed, and then used to induce the attachment of thick-shelled mussel spat.

5. The use of Psychrobacterium fowleri in inducing attachment of thick-shelled mussel spat according to claim 4, 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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