An antagonistic bacterium of shewanella putrefaciens and application thereof

Abstract

The application discloses an antagonistic bacterium of pathogenic shewanella of Apostichopus japonicus and application thereof, and belongs to the technical field of aquatic microorganisms. Gilvus japonicus The antagonistic bacterium is separated from the intestinal tract of healthy Apostichopus japonicus and is identified as Japanese Yellow Sea Oceanic Bacterium (Shewanella japonica), has no beta-hemolysis characteristic, is safe and non-toxic to Apostichopus japonicus, and can significantly antagonize the pathogenic bacterium Shewanella of Apostichopus japonicus skin rot syndrome. The application also discloses application of the antagonistic bacterium, which can be prepared into an aquatic microecological preparation for preventing and treating the skin rot syndrome of Apostichopus japonicus, and is applied through intraperitoneal injection or immersion, so that the survival rate of Apostichopus japonicus under exposure of the pathogenic bacterium is significantly improved. The strain has strong salt and alkali resistance, tolerances to various antibiotics and chemical drugs, and strong environmental adaptability, can be applied to the cultivation of Apostichopus japonicus to replace traditional antibiotics, effectively solves the problems of food safety and environmental pollution caused by the abuse of antibiotics, and has good industrial application prospect.

Description

Technical Field

[0001] This invention relates to the field of aquatic microbiology technology, and in particular to a bio-antagonistic bacterium of Shewanella, a pathogen of sea cucumber, and its application. Background Technology

[0002] sea ​​cucumber (imitation sea cucumber) Apostichopus japonicus It belongs to the phylum Echinodermata, class Holothuroidea, order Aspidochirotida, family Stichopodidae, and genus Apostichopus. Apostichopus aquaculture plays an important role in China's aquaculture industry.

[0003] The most common disease among sea cucumbers is skin rot syndrome, which can cause significant economic losses and affect the sustainable development of sea cucumber farming. The causes of sea cucumber diseases include both abiotic and biotic factors. Common abiotic factors include excessively high temperatures and dissolved oxygen stratification in the water, while common biotic factors include parasites, viruses, and bacteria.

[0004] When diseases occur in sea cucumbers, antibiotics, disinfectants, and microecological agents are commonly used for control. However, the overuse of antibiotics and disinfectants not only affects the safety of aquatic organisms but also leads to water quality deterioration and ecosystem imbalance. Therefore, microecological agents that follow the ecological principle of "inclusion and coexistence" are playing an increasingly prominent role in the prevention and control of diseases in sea cucumbers.

[0005] Aquatic animal pathogen antagonists are a class of functional microorganisms isolated from the aquaculture environment or aquatic animals themselves. They have good environmental adaptability and functional specificity, and can directly or indirectly inhibit or kill pathogens that cause diseases in aquatic animals. At the same time, they do not pose a threat to farmed animals, humans and the environment, and maintain the microecological balance and health of the aquaculture system.

[0006] Based on their origin, aquatic animal pathogen antagonistic bacteria can be divided into four categories: The first category originates from the aquatic animals themselves. The body surface, gills, intestines, and other parts of aquatic animals are "natural habitats" for microorganisms. Some of these microorganisms will antagonize pathogens in order to compete for living space and nutrients. The second category originates from the aquaculture environment. Water bodies and bottom sediments are the main breeding grounds for aquatic pathogens. At the same time, there are also a large number of antagonistic bacteria that "compete and coexist" with pathogens. These antagonistic bacteria are highly tolerant to the aquaculture environment and are suitable for the prevention and control of diseases in the entire water body. The third category originates from natural aquatic ecosystems. Unpolluted natural waters have rich microbial diversity and contain a large number of pathogen antagonistic bacteria with unique functions. The fourth category originates from artificial breeding. With the development of microbial technology, pathogen antagonistic bacteria can be obtained in the laboratory through targeted screening, genetic modification, and other means.

[0007] Studies have shown that antagonistic bacteria of aquatic animal pathogens isolated from the environment or healthy aquatic animals are well adapted to the environment and safe for the host, and have great potential for application in the prevention and control of aquatic diseases.

[0008] A strain of Bacillus subtilis isolated from the intestine of sea cucumber ( Bacillus subtili It can inhibit Vibrio splenti, the pathogen of skin rot syndrome, through two mechanisms: competition for nutrients and living space, and secretion of extracellular antagonistic substances. Vibrio splendidus ) growth.

[0009] From grass carp ( Ctenopharyngodon idella A strain of Bacillus belye was isolated from a sample of sediment from an aquaculture pond. Bacillus velezensis ( ), which can antagonize the pathogen of fish-derived Aeromonas hydrophila, thereby preventing and controlling the disease.

[0010] From rainbow trout ( Oncorhynchus mykiss Boswellia (a type of bacteria) isolated from the skin microbiome Bosea ) and Flavobacterium ( Flavobacterium ), can inhibit the pathogenic bacterium Flavovirens psychrophilus (Flavobacterium psychrophilum) of rainbow trout fry syndrome (RTFS). Flavobacterium psychrophilum ) growth.

[0011] Plate antagonism test from Nile tilapia ( Oreochromis niloticus A strain of Bacillus isolated from intestinal mucus ( Bacillus It can inhibit Streptococcus dolphinus ( Streptococcus iniae ) and Edwardsiella tarda ( Edwardsiella piscicida The growth of these two pathogens.

[0012] Therefore, we can try to start with healthy sea cucumbers themselves and attempt to isolate a bacterium that can antagonize Shewanella, the pathogen of skin rot syndrome. Shewanella sp. strain strain B9). Summary of the Invention

[0013] In view of the problems of existing technologies that rely on antibiotics for the prevention and treatment of sea cucumber skin rot syndrome and lack safe and efficient microecological agents, the purpose of this invention is to provide a strain of antagonistic bacteria against the pathogen of sea cucumber skin rot syndrome. This strain is safe and non-toxic to sea cucumber and can significantly antagonize the pathogen Shewanella B9.

[0014] To achieve the above objectives, the present invention provides an antagonistic bacterium against Shewanella, a pathogen of sea cucumbers, wherein the antagonistic bacterium is *Hypertia japonica* (Japanese sea cucumber pathogen). Gilvimarinus japonicus It was deposited on March 19, 2026, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 37977.

[0015] On the other hand, the present invention provides the application of the above-mentioned antagonistic bacteria of *Shewanella spp.*, a pathogen of *Stichopus japonicus*, in the preparation of aquatic formulations for the prevention and treatment of *Stichopus japonicus* skin rot syndrome.

[0016] Preferably, the aquatic preparation is a microecological preparation, and the effective component of the microecological preparation is a bacterial suspension of a herbicidal antagonistic bacterium of the sea cucumber pathogen Shewanella.

[0017] Preferably, the microecological preparation is administered via intraperitoneal injection or immersion.

[0018] Preferably, the active ingredient is the above-mentioned antagonistic bacteria of Shewanella, a pathogen of sea cucumber, and it also includes aquaculture-acceptable auxiliary materials.

[0019] Preferably, the above-mentioned aquatic microecological preparation is applied to the sea cucumber or its aquaculture water by intraperitoneal injection or immersion. The microecological preparation can significantly antagonize Shewanella, the pathogen causing skin rot syndrome in sea cucumbers. Shewanella sp.strain B9 improves the survival rate of sea cucumbers exposed to pathogens.

[0020] Therefore, the present invention provides an antagonistic bacterium against Shewanella, a pathogen of sea cucumber, and its application, which has the following beneficial effects: (1) This invention is the first to isolate and screen a strain from the intestines of healthy sea cucumbers that can produce an inhibition zone against the pathogen of sea cucumber skin rot syndrome, and does not produce a β-hemolytic zone. The strain was identified as *Hypericum japonicum*. Gilvimarinus japonicus ).

[0021] (2) The antagonistic bacteria were used to conduct intraperitoneal injection and immersion challenge tests on sea cucumbers to verify that it was non-toxic to sea cucumbers. Under the condition of using the antagonistic bacteria, the mortality rate of the pathogen challenge was much lower than that of the pathogen challenge alone. The results showed that the strain could effectively inhibit the pathogenicity of the sea cucumber skin rot syndrome pathogen to sea cucumbers. It is expected to be applied to sea cucumber farming and provide an effective measure for the prevention and control of sea cucumber skin rot syndrome.

[0022] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0023] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments of the present invention will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1The results of the plate antagonism test are shown. Among them, A is the antagonistic strain L1-17-1 and B is the antagonistic strain Z3-25. Figure 2 The results are from the hemolysis test of the antagonistic strains; where A is the antagonistic strain L1-17-1 and B is the antagonistic strain Z3-25. Figure 3 Gram staining micrographs (100×) of the antagonistic strains; where A is the antagonistic strain L1-17-1 and B is the antagonistic strain Z3-25; Figure 4 Phylogenetic analysis of the strains based on the full-length 16S rDNA sequence; where A is the antagonistic strain L1-17-1 and B is the antagonistic strain Z3-25; Figure 5 Standard curves for bacterial concentration-cell concentration are shown for strains L1-17-1 and Z3-25; where A is the standard curve for bacterial concentration-cell concentration of strain L1-17-1; and B is the standard curve for bacterial concentration-cell concentration of strain Z3-25. Figure 6 The growth curves are for strains L1-17-1 and Z3-25; where A is the growth curve for strain L1-17-1 and B is the growth curve for strain Z3-25. Figure 7 Survival rate of two antagonistic bacteria injected with *Stichopus japonicus* for 120 hours after infection; Figure 8 To determine the survival rate of sea cucumber subjected to immersion in a poison-treated bath for 120 hours; Figure 9 The results of the in vivo antagonistic test between strain Z3-25 and strain B9 are shown; where A is the intraperitoneal injection group and B is the immersion group. Detailed Implementation

[0025] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0026] To make the objectives, technical solutions, and advantages of this application clearer, more thorough, and more complete, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings and embodiments. The following detailed descriptions are all illustrations of embodiments, intended to provide further detailed explanation of the present invention. Unless otherwise specified, all technical terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0027] The instruments, equipment, reagents, and materials used in the examples were all obtained commercially.

[0028] Example 1 In April 2024, five healthy sea cucumbers were collected from the Shicheng Island sea cucumber farm in Zhuanghe City, Liaoning Province. The weight of each sea cucumber was (1.25±0.35) g. They were transported in ice boxes to Dalian Ocean University (Dalian City, Liaoning Province) for subsequent experiments. The transportation time was 4 hours.

[0029] The Shewanella bacteria used in this embodiment ( Shewanella sp. strain B9) has been verified by experiments to conform to Koch's postulates and is the pathogen of the sea cucumber skin rot syndrome.

[0030] Screening process for antagonistic bacteria of Shewanella, the pathogen of sea cucumber: (1) Isolation of cultured bacteria from the intestine of sea cucumber: The intestines of a healthy sea cucumber were removed using a sterile scalpel and placed in a sterile mortar. An appropriate amount of sterile seawater was added, and the mixture was ground into a homogenate using a sterile pestle. The homogenate was then serially diluted with sterile seawater, with 100 μL diluted to 10 μL each time. -4 10 -5 10 -6 The homogenate was evenly spread onto 2216E agar medium and incubated upside down at 25°C for 48 hours, with checks every 12 hours. Once colonies were visible on the plates, their characteristics were observed, and single colonies were picked and inoculated onto TCBS and 2216E agar medium, respectively. After multiple isolation and purification processes, pure bacterial cultures were obtained.

[0031] (2) Screening of antagonistic bacteria against the pathogen of sea cucumber skin rot syndrome: Using Shewanella B9 as an indicator bacterium, 100 μL of bacterial culture was evenly spread onto 2216E agar medium. After complete absorption, 2.5 μL of isolated culturable bacteria from the gut of *Stichopus japonicus* was spotted onto the medium. After complete absorption, the medium was incubated at 25°C for 48 hours, and the presence of inhibition zones was observed. Strains exhibiting inhibition zones were identified as antagonistic strains to strain B9.

[0032] Eighty culturable bacteria were isolated from the intestines of healthy sea cucumbers. Using a spot inoculation method, bacteria capable of antagonizing the growth of Shewanella B9, the pathogen causing sea cucumber skin rot syndrome, were screened. Two bacterial strains were found to produce inhibition zones against strain B9. Figure 1 As shown, strain L1-17-1 is shown in part A and strain Z3-25 is shown in part B, respectively.

[0033] (3) Hemolysis test: Hemolysis tests were performed on two antagonistic strains of strain B9. Single colonies were picked and inoculated into 2216E liquid medium and cultured at 25°C and 120 r / min for 24 h. 2.5 μL of the bacterial solution was then inoculated onto Columbia blood agar medium. After the bacterial solution was completely absorbed, it was placed in a constant temperature incubator at 25°C and inverted for one week to observe whether a β-hemolytic ring was formed.

[0034] The results are as follows Figure 2 As shown, neither strains L1-17-1 nor Z3-25 produced a clear β-hemolytic ring.

[0035] (4) Morphological observation and molecular identification of strains L1-17-1 and Z3-25.

[0036] On 2216E agar plates, colonies of strain L1-17-1 were pale yellow, with a moist and smooth surface, neat edges, and a rounded shape; colonies of strain Z3-25 were also pale yellow, with a moist and smooth surface, neat edges, and a rounded shape.

[0037] Take OD 600nm 30 μL of liquid culture with a pH of 0.6–0.8 was Gram-stained using a Gram staining kit (purchased from Kingclone (Beijing) Biotechnology Co., Ltd.). Microscopic observation revealed that cells of strains L1-17-1 and Z3-25 were both red, rod-shaped, and short rod-shaped, identifying them as Gram-negative bacilli. Figure 3 As shown.

[0038] (5) Molecular identification of the strain: Total DNA was extracted from strains L1-17-1 and Z3-25 by boiling in water. The full-length 16S rDNA was amplified using universal primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′).

[0039] Reaction system (total 30µL): template DNA 2µL, forward and reverse primers (10µmol / L) 3µL each, 2×EasyTaqPCR superMix 15µL, ddH2O 7µL.

[0040] PCR reaction procedure: pre-denaturation at 95℃ for 2 min; denaturation at 95℃ for 30 s, annealing at 55℃ for 30 s, extension at 72℃ for 90 s, for a total of 35 cycles; further extension at 72℃ for 5 min, and storage at 20℃.

[0041] PCR amplification products were detected by 1.2% agarose gel electrophoresis. Successfully amplified PCR products were sent to BGI Genomics for sequencing. The sequencing results were compared with BLASTn in the NCBI gene bank. Sequences with high homology were selected, and phylogenetic analysis of these homologous 16S rDNA sequences was performed using MEGA11.0 software. The genetic distance was calculated based on the Poisson model using the neighbor-joining method to construct a germline phylogenetic tree and identify the strains.

[0042] like Figure 4 As shown, strain L1-17-1 and Vibrio cyclitrophicus (CP170034.1) showed the highest homology, with a sequence identity of 98.92% at 99% coverage, and it clustered with it in the same branch in the phylogenetic tree. Figure 4 (A). Strain Z3-25 and Gilvimarinus japonicus The strain (AP031500.1) showed the highest homology, with a sequence identity of 99.93% at 100% coverage, and it clustered with the strain in the phylogenetic tree. Figure 4 Based on morphological characteristics and molecular identification results, strain L1-17-1 is presumed to be (B). Vibrio It belongs to the bacteria, strain Z3-25 is Gilvimarinus It belongs to the bacteria.

[0043] On March 19, 2026, strain Z3-25 was deposited at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The identification result was *Hypericum japonicum* (Japanese seaweed). Gilvimarinus japonicus The phylogenetic tree, with accession number CGMCC No. 37977, demonstrates the accuracy of the above results.

[0044] (6) Construction of the bacterial absorbance-cell concentration standard curve: To rapidly and accurately quantify bacterial suspensions of strains that did not produce a β-hemolytic zone, a plate count method was used to construct an absorbance-cell concentration standard curve. Logarithmic growth phase bacterial suspensions were serially diluted, and the absorbance at 600 nm was measured using a 721 spectrophotometer. The diluted bacterial suspensions were plated on 2216E agar plates, with three replicates per group. After incubation at 25°C for 24 h, plates with colony counts between 30 and 300 were selected for counting, and colony forming units (CFU / mL) were calculated. The absorbance was then measured using the OD500 of the same bacterial suspension. 600nm Plot the standard curve using the x-axis and the CFU / mL value as the y-axis to obtain the regression equation.

[0045] The standard curves of bacterial absorbance-cell concentration for the two antagonistic strains are as follows: Figure 5As shown. Using a high goodness-of-fit regression equation, the growth equation for strain L1-17-1 is obtained as follows: y =4×10 11 x -9×10 8 ( R 2 =0.9988); the growth equation for strain Z3-25 is: y =3×10 11 x -8×10 8 (R) 2 =0.999) x For OD 600nm ; y (Bacterial concentration).

[0046] (7) Based on the bacterial absorbance-cell concentration standard curve, plot the growth curves of strain L1-17-1 and strain Z3-25 at 25℃.

[0047] Single colonies of strains L1-17-1 and Z3-25 were inoculated into 2216E liquid medium and cultured at 25°C and 120 rpm for 24 h to prepare a bacterial suspension. 20 mL of the bacterial suspension (10% inoculum) was inoculated into 180 mL of sterile 2216E liquid medium. Samples (5 mL each time) were taken every 6 h, using a blank medium as a control to calibrate the zero point. The absorbance at 600 nm was measured using a 721 spectrophotometer. If the absorbance value exceeded 0.8, the sample was diluted to a value between 0.2 and 0.8. The OD was calculated using a bacterial absorbance-cell concentration standard curve. 600nm The corresponding CFU / mL values ​​were used to plot the bacterial growth curve with sampling time on the x-axis and CFU / mL values ​​on the y-axis.

[0048] The results are as follows Figure 6 As shown. The lag phase of strain L1-17-1 was 0h-12h, the logarithmic growth phase was 12h-27h, and it entered the plateau phase after 36h, with a cell concentration of (3.63×10⁻⁶)⁻¹. 11 ±0.36×10 11 ) CFU / mL - (3.74×10 11 ±0.16×10 11 The lag phase of strain Z3-25 was 0-6 hours, the logarithmic growth phase was 6-36 hours, and it entered the plateau phase after 36 hours. The cell concentration during the plateau phase was 2.78 × 10⁻⁶ CFU / mL. 11 ±0.08×10 11 ) CFU / mL - (2.80×10 11 ±0.12×10 11 CFU / mL.

[0049] (8) Challenge test: In early September 2024, healthy sea cucumbers (average body weight 4.88g ± 0.67g) were collected from the sea cucumber farm on Shicheng Island, Zhuanghe City, Liaoning Province. They were temporarily raised in the laboratory for one week for challenge and in vivo antagonism experiments. During the rearing and challenge processes, the water temperature was kept constant at 25℃, aeration was provided 24 hours a day, and the fish were fed their usual feed once a day, with a complete water change every 7 days.

[0050] Single colonies were picked and inoculated into 2216E liquid medium and cultured at 25℃ and 120 rpm for 24 h. Based on the standard curve of absorbance-cell concentration, the bacterial suspension was quantitatively transferred to sterile centrifuge tubes and centrifuged at 6000 rpm for 5 min. The supernatant was collected and resuspended in an appropriate amount of sterile seawater to prepare a bacterial suspension. Intraperitoneal injection and immersion challenges were then performed on *Stichopus japonicus*. Both the intraperitoneal injection and immersion challenges were divided into 6 groups, including 3 blank control groups and 3 challenge groups. Ten *Stichopus japonicus* were randomly selected from each group and placed in 1L containers containing 500 mL of sterile seawater for the experiment.

[0051] In the intraperitoneal injection challenge test group, each *Stellaria media* was administered 8 × 10 7 The bacterial suspension was injected at a dose of 1 × 10⁻⁶ cells / g. In the blank control group, each sea cucumber was injected with the same volume of sterile seawater as the bacterial suspension in the challenge group. The sea cucumbers were cultured for 120 hours, and observations were recorded. The immersion challenge group received 1 × 10⁻⁶ cells / g of bacterial suspension per group. 8 The bacterial suspension was added to the culture water at a dose of cell / mL, while the blank control group was added to the culture water with the same volume of sterile seawater as the bacterial suspension in the challenge test group. The culture was carried out for 120 hours and the results were observed and recorded.

[0052] Survival rate of sea cucumber (similar to sea cucumber) = (Number of surviving sea cucumbers in the challenge test / Total number of sea cucumbers in the challenge test) × 100%.

[0053] Healthy sea cucumbers were subjected to injection challenge experiments using strains L1-17-1 and Z3-25, respectively, at a dose of 8 × 10⁻⁶. 7 cell / g, results as follows Figure 7 As shown, the survival rate of *Stichopus japonicus* in the blank control group and the injected strain Z3-25 group was 100.0% ± 0.0% at 120 h, while the survival rate of *Stichopus japonicus* in the injected strain L1-17-1 group was only 50.0% ± 10.0% at 120 h, which was significantly lower than the first two groups. P The value <0.05 indicates that strain Z3-25 is non-toxic to sea cucumber, while strain L1-17-1 is toxic to sea cucumber. Therefore, strain Z3-25 was selected for subsequent tests.

[0054] A sea cucumber-like immersion challenge experiment was conducted on strain Z3-25, with an immersion concentration of 1×10⁻⁶.8 cells / mL, results as follows Figure 8 As shown, the survival rate of the sea cucumber strain Z3-25 at 120h was 100.0% ± 0.0% in both the blank control group and the experimental group, further verifying that the strain Z3-25 is safe and non-toxic to the sea cucumber strain Z3-25.

[0055] (9) In vivo antagonistic test for the pathogen of sea cucumber skin rot syndrome: In vivo antagonistic tests against *S. spinosaurus* skin rot syndrome were conducted using temporarily cultured *S. spinosaurus*. Single colonies of antagonistic strains Z3-25 and B9 were inoculated into 2216E liquid medium and cultured at 25°C and 120 rpm for 24 h. Based on the standard curve of absorbance-cell concentration, an appropriate amount of bacterial suspension was transferred to a sterile centrifuge tube and centrifuged at 6000 rpm for 5 min. The supernatant was collected and resuspended in an appropriate amount of sterile seawater to prepare a bacterial suspension, which was used for intraperitoneal injection and immersion antagonistic tests of *S. spinosaurus*. Both the intraperitoneal injection and immersion antagonistic tests were divided into 12 groups, including 3 blank control groups, 3 control groups with only strain B9, 3 control groups with only antagonistic strains, and 3 antagonistic test groups. Ten *S. spinosaurus* were randomly selected from each group and placed in a 1L container containing 500 mL of sterile seawater for the antagonistic test.

[0056] Intraperitoneal injection antagonist test group and single antagonist strain control group: each sea cucumber was first injected with 8×10 7 The antagonistic strains were injected at a dose of 2.5 × 10⁻⁶ cells / g, while the control group (single strain B9) and the blank control group were injected with the same volume of sterile seawater as the antagonistic experimental group. After 24 hours of culture, the entire water volume was changed. Each sea cucumber in the antagonistic experimental group and the control group (single strain B9) was then injected with 2.5 × 10⁻⁶ cells / g of sterile seawater. 6 Each sea cucumber was injected with a dose of cell / g of strain B9. In the control group with antagonistic strain alone and the blank control group, each sea cucumber was injected with an equal volume of sterile seawater as the bacterial suspension of the antagonistic test group. The sea cucumbers were cultured for 120 hours and the results were recorded.

[0057] The survival rate of *Stichopus japonicus* 120 hours after intraperitoneal injection is as follows: Figure 9 As shown in Figure A, the blank control group and the group injected with 8×10 alone... 7 Cell / g strain Z3-25 control group, injected alone with 2.5×10 6 Cell / g strain B9 control group, first injected 8×10 7 Inject 2.5 × 10⁻⁵ cells / g of strain Z3-25. 6 The 120-hour survival rates of the cell / g strain B9 antagonistic test groups were 100.0%±0.0%, 80.0%±10.0%, 23.0%±17.0%, and 80.0%±10.0%, respectively. Among them, the 120-hour survival rate of the antagonistic test groups of the sea cucumber was significantly higher than that of the control group injected with strain B9 alone. P<0.05), indicating that strain Z3-25 significantly reduced the virulence of strain B9 in sea cucumbers after intraperitoneal injection.

[0058] The immersion antagonism test group and the single antagonist strain control group were each initially administered 1×10 8 Antagonistic bacterial suspension was added to the aquaculture water at a dose of 1 × 10⁻⁶ cells / mL. For the control group (single strain B9) and the blank control group, sterile seawater of the same volume as the experimental group's bacterial suspension was added to the aquaculture water. After 24 hours of aquaculture, the entire water volume was replaced. The antagonistic experimental group and the single strain B9 control group were then treated with 1 × 10⁻⁶ cells / mL of sterile seawater. 8 The bacterial suspension of strain B9 was added to the culture water at a dose of cell / mL. The control group and the blank control group were each added with an equal volume of sterile seawater to the culture water. The culture was carried out for 120 hours and the results were recorded.

[0059] The survival rate of sea cucumbers treated with immersion bacteria for 120 hours was as follows: Figure 9 As shown in Figure B, the blank control group and the single addition of 1×10 8 cell / mL strain Z3-25 control group, with 1×10⁻⁶ cells / mL added. 8 cell / mL strain B9 control group, first add strain 1×10 8 Add 1×10⁻⁵ cells / mL Z3-25 after Z3-25. 8 The 120-hour survival rates of the antagonistic groups of strain B9 (cell / mL) were 100.0%±0.0%, 100.0%±0.0%, 38.0%±0.5%, and 75.0%±12.5%, respectively. Similar to the intraperitoneal injection of *Stichopus japonicus*, the 120-hour survival rate of the antagonistic groups was significantly higher than that of the control group receiving strain B9 alone. P <0.05), indicating that the sea cucumber *Stichopus japonicus* immersed in strain Z3-25 is more resistant to the invasion of strain B9.

[0060] It is worth noting that the survival rate of the sea cucumber group injected with high dose of strain Z3-25 alone was lower than that of the blank control group, although the difference was not significant; while the survival rate of the sea cucumber group immersed in high concentration of strain Z3-25 alone was the same as that of the blank control group.

[0061] (10) Physiological and biochemical characteristics analysis of antagonistic strains: Metabolic analysis of antagonistic strains was performed using Biolog GenIII microplates, and the results were compared with Biolog's species database. After 16–24 h of incubation, if the similarity value (SIM) ≥ 0.50, the system automatically provided strain-level identification results.

[0062] The metabolic profile of strain Z3-25 was detected using Biolog GenⅢ microplates, and the results are shown in Table 1.

[0063] Table 1. Biolog Gen III metabolometry profile of strain Z3-25

[0064] Note: + indicates positive; - indicates negative; w indicates weak positive. A1-H12 is a Biolog assay for microplate location.

[0065] Strain Z3-25 does not grow at pH 5, grows weakly at pH 6, and grows normally under NaCl concentrations ranging from 1% to 8%, indicating that this strain has weak acid resistance and strong salt and alkali resistance. The carbon sources that this strain can fully utilize for normal growth include D-maltose, D-cellobiose, β-Methyl-D-glucosinolate, D-salicylic acid, and α-D-glucose. The carbon sources that can be utilized for weak growth include dextran, D-trehalose, D-minobiose, N-Acetyl-D-glucosamine, D-mannose, D-fructose-6-PO4, and L-hydroxysuccinic acid. This strain also exhibits some resistance to antibiotics such as clostridial acid, dimethylaminetetracycline, and aztreonam, and is also resistant to tetrazolium violet, lithium chloride, potassium tellurite, and sodium bromate.

[0066] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A bio-antagonistic bacterium against Shewanella, a pathogen of sea cucumbers, characterized in that: The antagonistic bacteria is *Yellow Ocean Bacterium japonicum* (Japanese Yellow Ocean Bacterium). Gilvimarinus japonicus It was deposited on March 19, 2026, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCCNo. 37977.

2. The application of the antagonistic bacteria of *Shewanella spp.*, the pathogen of *Stichopus japonicus*, as described in claim 1, in the preparation of aquatic formulations for the prevention and treatment of *Stichopus japonicus* skin rot syndrome.

3. The application according to claim 2, characterized in that: The aquatic preparation is a microecological preparation, and the effective component of the microecological preparation is a bacterial suspension of a herb that mimics the antagonistic bacteria of the sea cucumber pathogen Shewanella.

4. The application according to claim 3, characterized in that: The microecological preparation is administered via intraperitoneal injection or immersion.

5. An aquatic microecological preparation for preventing and treating sea cucumber skin rot syndrome, characterized in that: The active ingredient is the antagonistic bacterium against Shewanella pathogen, as described in claim 1, and it also includes aquaculture-acceptable auxiliary materials.

6. A method for preventing and treating sea cucumber skin rot syndrome, characterized in that: The aquatic microecological preparation described in claim 5 is applied to *Stichopus japonicus* or its aquaculture water via intraperitoneal injection or immersion. The microecological preparation significantly antagonizes *Shewanella*, the pathogen causing skin rot syndrome in *Stichopus japonicus*. Shewanella sp. strain B9), to improve the survival rate of sea cucumbers exposed to pathogens.

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