A strain of Bacillus paramycoides capable of inhibiting asexual reproduction of jellyfish polyps and its application

Abstract

The present invention discloses a Bacillus paramycoides having the function of inhibiting the asexual reproduction of the polyps of the jellyfish, and its application. The strain was deposited in the China Center for Type Culture Collection (CCTCC) on May 8, 2023, at Wuhan University, Wuhan, China, with the deposit number: CCTCC NO: M 2023702. The strain is isolated from the rhizosphere of healthy eelgrass, is safe and non-toxic; can inhibit the asexual reproduction of the polyps of the jellyfish, the moon jellyfish; the strain is easy to culture, and can be further applied to the research and development of bacterial agents for preventing and controlling the outbreak of jellyfish. The comprehensive Bacillus paramycoides YTUSO 00005 has the function of inhibiting the asexual reproduction of the polyps of the jellyfish, can be applied to the preparation of marine microbial functional bacterial agents, has certain economic and ecological benefits, and therefore has the potential to be applied to the prevention and control of the outbreak of jellyfish.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a paramycoides bacillus capable of inhibiting jellyfish polyps and an application thereof. Background Art

[0002] Jellyfish outbreaks are a prominent ecological disaster problem facing the coastal waters of my country and even the world. Jellyfish outbreaks can cause changes in the structure and function of marine ecosystems, have a negative impact on coastal tourism, fisheries, and coastal industries, and pose a serious threat to ecological security and the sustainable development of the marine economy (Dong et al., 2010; Dong et al., 2019). Moon jellyfish are typical disaster jellyfish that frequently break out in the northern East my country Sea, the Yellow Sea, and the Bohai Sea. Moon jellyfish invasions have caused power plant unit shutdowns in Qingdao, Weihai, and Dongying, seriously endangering the safety of cold sources in Shandong coastal facilities (Dong et al., 2010; Dong et al., 2019). At present, the prevention and control technologies and response measures for disaster jellyfish at home and abroad are mainly to intercept and salvage adult jellyfish after an outbreak (Foster et al., 2000), which have problems such as high cost and incomplete elimination. There is still a lack of early prevention and control technologies and response strategies for jellyfish disasters.

[0003] The life history of moon jellyfish is complex, among which the asexual reproduction of jellyfish polyps is the key to the explosive growth of jellyfish numbers and the basis for jellyfish population outbreaks. Studies have revealed that jellyfish are closely related to microorganisms. The asexual reproduction rate of moon jellyfish polyps treated with antibiotics has significantly decreased, indicating that microorganisms can affect the asexual reproduction process of jellyfish ( et al., 2020). Microorganisms also play an important ecological role in the early life history of jellyfish by producing metabolites such as vitamins and essential amino acids and nutrients (Guo et al., 2017; Guo et al., 2019; et al., 2020; Jensen et al., 2023; Peng et al., 2023; Peng et al., 2024). Therefore, it is urgent to screen and obtain strains that have the ability to inhibit the asexual reproduction of jellyfish polyps and are non-toxic, so as to provide technical support for the biological control of jellyfish outbreaks in my country's coastal waters. Summary of the invention

[0004] The object of the present invention is to provide a strain of Bacillus paramycoides SG15 isolated from the rhizosphere sediments of eelgrass in Swan Lake, Rongcheng, Shandong Province, China, which has the effect of inhibiting the asexual reproduction of the jellyfish Aurelia jellyfish polyps. The strain was deposited in the China Center for Type Culture Collection (CCTCC) on May 8, 2023, address: Wuhan University, Wuhan, China, and the deposit number is CCTCC NO: M2023702.

[0005] The second object of the present invention is to provide a microbial preparation having the effect of inhibiting asexual reproduction of jellyfish polyps, which contains Bacillus paramycoides SG15 as an active ingredient.

[0006] The third object of the present invention is to provide the use of the above-mentioned Bacillus paramycoides SG15 or a microbial preparation containing Bacillus paramycoides SG15 in inhibiting the asexual reproduction of the polyps of the harmful jellyfish.

[0007] Preferably, the jellyfish polyp is a moon jellyfish (Aurelia aurita) polyp.

[0008] The fourth object of the present invention is to provide the use of the above-mentioned Bacillus paramycoides SG15 or a microbial preparation containing Bacillus paramycoides SG15 in preventing and controlling the explosive reproduction of jellyfish polyps.

[0009] Preferably, the disaster jellyfish is the moon jellyfish (Aurelia aurita).

[0010] A fifth object of the present invention is to provide a method for inhibiting the asexual reproduction of the polyps of the jellyfish, which comprises exposing the above-mentioned Bacillus paramycoides SG15 or a microbial preparation containing Bacillus paramycoides SG15 to the living environment of the polyps of the moon jellyfish to inhibit the asexual reproduction of the polyps of the jellyfish.

[0011] Preferably, the polyp of the disaster jellyfish is a polyp of the moon jellyfish (Aurelia aurtia).

[0012] The present invention separates and purifies Bacillus paramycoides SG15 from rhizosphere sediments of eelgrass in Swan Lake, Rongcheng, Shandong Province, China. The bacterium is safe and non-toxic; it can inhibit the asexual reproduction of the polyps of the jellyfish Aurelia fasciata; the strain is easy to culture, and can be further applied to the research and development of bacterial agents for preventing and controlling the outbreak of jellyfish. In summary, Bacillus paramycoides YTUSO 00005 has the function of inhibiting the asexual reproduction of the polyps of the jellyfish Aurelia fasciata, can be applied to the preparation of marine microbial functional bacterial agents, has certain economic and ecological benefits, and therefore has the potential to be applied to the prevention and control of the outbreak of jellyfish Aurelia fasciata.

[0013] Bacillus paramycoides SG15, the strain was deposited in the China Center for Type Culture Collection (CCTCC) on May 8, 2023, address: Wuhan University, Wuhan, China, Postal Code: 430072, Deposit Number: CCTCC NO: M2023702. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 is an optical micrograph of Bacillus paramycoides SG15 (100×);

[0015] Figure 2 is the phylogenetic tree of Bacillus paramycoides SG15;

[0016] Figure 3 It is the polyp form of the moon jellyfish (Aurelia aurtia).

[0017] Figure 4 The effect of Bacillus paramycoides SG15 on the asexual reproduction rate, body length, body width and tentacle length of the hydra state of the moon jellyfish (Aureliaaurtia). DETAILED DESCRIPTION

[0018] The present invention is specifically described below in conjunction with specific embodiments:

[0019] Example 1: Isolation and purification of Bacillus paramycoides SG15

[0020] Bacillus paramycoides SG15 was isolated and purified from the rhizosphere sediments of Eelgrass in Swan Lake, Rongcheng, Shandong Province, China in July 2021, and stored in glycerol at -80°C.

[0021] 1. Morphological characteristics:

[0022] Under an optical microscope, the cells of Bacillus paramycoides SG15 are rod-shaped and are Gram-positive bacteria. Figure 1 shown.

[0023] 2. Identification of strain 16s rDNA sequence:

[0024] Identification of strain 16s sequence: The genomic DNA of Bacillus paramycoides SG15 strain was extracted using a bacterial DNA extraction kit (Takara, Japan). The purity and concentration of the DNA were determined by Nannodrop1000. The qualified DNA was used to amplify the 16srRNA gene. The amplification primers were: forward primer 27F (5'-AGAGTTTGATCCTGGCTCA G-3'), reverse primer 1492R (5'-CGGTTACCTTGTTACGACTT-3'); the PCR amplification system was: TaqP CR mix 12.5μl, DNA template 1μl, primers 27F / 1492R 0.5μl each, ddH 2 O was added to 25 μl. The PCR amplification conditions were: pre-denaturation at 95°C for 10 min, denaturation at 9°C for 1 min, annealing at 54°C for 1 min, extension at 72°C for 2 min, 30 cycles, and final extension at 72°C for 10 min. Electrophoresis was performed on a 1.2% agarose gel and observed under a 260nm gel imager to confirm the target band (about 1500 bp). The PCR product containing the target band was sent to the company for sequencing (Sangon Biotechnology Co., Ltd., Qingdao). After quality control of the obtained sequence (as shown in SEQ ID NO.1), it was uploaded to the EZbioclound platform to obtain closely related sequences, and multiple sequence alignment was performed using Align in the MEG7.0 software. The phylogenetic tree was constructed using the Neighbor-joining method. The results are shown in the figure. Figure 2 As shown: the closely related sequence of Bacillus paramycoides SG15 is Bacillus paramycoides NH24A2, and the similarity thereof reaches 99.11%. The strain belongs to the phylum Firmicutes, the class Bacilli, the order Bacillales, the family Bacillaceae, and the genus Bacillus.

[0025] In summary, Bacillus paramycoides SG15 was named Bacillus paramycoides SG15, and was deposited in the China Center for Type Culture Collection (CCTCC) on May 8, 2023, address: Wuhan University, Wuhan, China, Postal Code 430072, Deposit Number: CCTCC NO: M2023702.

[0026] Embodiment 2:

[0027] 1. Activation of strains:

[0028] Take out the strain stored at -80℃, inoculate it on 2216E solid medium, place it in a 28℃ incubator for culture, pick a single colony, streak it on a plate, and set it aside. The components of 2216E solid medium are as follows:

[0029] The composition of 2216E solid medium: 5g tryptone, 1g yeast extract, FePO4·2H 2 O 0.1g, NaCl 25g, agar 18g, ddHO 2 O 1000mL; mix the above culture medium components, adjust the pH to 7.2-7.8, and sterilize.

[0030] 2. Preparation of Bacillus paramycoides SG15

[0031] Pick a single colony of Bacillus paramycoides SG15 on the plate and inoculate it into MA liquid culture medium, and culture it in a shaker (32°C, 180rpm). When the bacterial liquid is cultured to a turbid state, centrifuge it at 8000rpm for 5min to remove the supernatant to obtain the experimental bacteria. The components of MA liquid culture medium are as follows:

[0032] Composition of MA liquid medium: Difco Marine Broth 18g, NaCl 9g, agar 18g, ddH 2 O1000mL; mix the above culture medium components, adjust the pH to 7.2-7.8, and sterilize.

[0033] 3. Bacillus paramycoides SG15 inhibits asexual reproduction of jellyfish polyps

[0034] Pick a single colony of Bacillus paramycoides SG15 and inoculate it into MA liquid medium, and place it in a shaker for culture (32°C, 180 rpm). When the bacterial solution is cultured to the stationary phase, use a microplate reader to measure the OD at 600nm to be about 1.0, and add the bacterial solution to a disposable 48-well culture plate to make the final concentration of the strain 1.0×10 7 cell / mL, each well contained one jellyfish polyp, and each 48-well culture plate was used as a biological replicate. An equal volume of sterilized seawater was added to the control group instead of the bacterial solution. The 48-well plate was placed in an incubator and the temperature was controlled at 19°C. Sterile seawater and SG15 bacterial solution were replaced every 4 days, the number of polyps was observed every four days, the asexual reproduction rate of polyps was calculated, and the morphological changes of polyps were observed ( Figure 3 ) and observed for 28 days. Figure 4 As shown in Figure 2, the asexual reproduction rate of the jellyfish polyps in the bacterial solution group was significantly lower than that in the blank control group ( Figure 4 A), 16 days after the strain treatment, the length and tentacles of the polyp were significantly shortened, and the width was significantly narrowed ( Figure 4B-4D). In summary, the concentration is 1.0x10 7 cell / mL of Bacillus paramycoides SG15 bacterial solution has the effect of inhibiting the asexual reproduction of jellyfish polyps.

[0035] The above are only preferred embodiments of the present invention. It should be noted that the above embodiments do not limit the present invention in any form. Any technical solutions obtained by equivalent replacement or equivalent transformation fall within the protection scope of the present invention.

[0036] SEQ ID NO.1

[0037]

Claims

1. Bacillus paramycoides ( Bacillus paramycoides ) SG15 or a microbial preparation containing Bacillus paramycoides SG15 in inhibiting asexual reproduction of jellyfish polyps, wherein the Bacillus paramycoides SG15 has a preservation number of CCTCC NO: M2023702, and the jellyfish polyps are moon jellyfish ( Aurelia aurita ) polyps.

2. A method for inhibiting asexual reproduction of jellyfish polyps, characterized in that: The invention relates to exposing Bacillus paramycoides SG15 or a microbial preparation containing Bacillus paramycoides SG15 to the living environment of the jellyfish polyp of the jellyfish Aurelia fasciata to inhibit the asexual reproduction of the jellyfish polyp. The Bacillus paramycoides SG15 has a preservation number of CCTCC NO: M2023702, and the jellyfish polyp is the Aurelia fasciata ( Aurelia aurtia ) polyps.

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