Bacteriophagous disease-resistant change Marsinia sp. And application thereof

Abstract

The invention relates to a varied massinia, and in particular relates to a separated varied massinia P2-4 with broad-spectrum phagocytosis activity on pathogenic bacteria, and the varied massinia P2-4 is used for phagocytosis in a bacterium cracking manner, so that the activity of the varied massinia P2-4 is improved. The compound has phagocytosis activity on pathogenic bacteria such as pseudomonas aeruginosa, shewanella alginosa, aeromonas cainea, aeromonas hydrophila and photobacterium damsela; the Marsinia variabilis P2-4 provided by the invention can be applied to inhibition of pathogenic bacteria in an aquaculture water body; by adding the varied maleinia sp. P2-4 or the microbial inoculum thereof into the aquaculture feed, the immunity and / or the oxidation resistance and / or the disease infection resistance of the organism of aquaculture animals can be enhanced; the varied marssonia sp. P2-4 disclosed by the invention can be used as a feed additive for aquaculture or a biocontrol product for aquaculture, and can also be used for preventing and / or treating diseases of aquaculture animals.

Description

Technical Field

[0001] The present invention relates to a variant of Marseillaria, specifically to a variant of Marseillaria P2-4 and a microbial agent thereof, as well as their applications in inhibiting pathogenic bacteria in aquaculture water, enhancing the immunity and / or antioxidant capacity and / or disease resistance of aquaculture animals, in aquaculture feed, and in preparing products for preventing and / or treating aquaculture animal diseases, belonging to the technical field of aquatic probiotics. Background Art

[0002] The massive growth of potential pathogens in water bodies seriously threatens the health of aquatic animals. For example, Pseudomonas aeruginosa can cause ulcers on the surface of yellow catfish (Yan Hua, et al. Isolation of Pseudomonas aeruginosa PA-5 from yellow catfish and its effect on host antimicrobial peptide genes [J]. Journal of Southwest Agriculture, 2021, 34(6): 1351-1358.); Shewanella algae can cause ulcer disease in American eel (Huicong Wang, Ying Gu, Jun Chen, Haipeng Cao. Shewanella algae: an emerging causative agent for ulcer disease in freshwater-farmed American eel Anguillarostrata [J]. Israeli Journal of Aquaculture-Bamidgeh, 2020, IJA_72.2020.964361.); Aeromonas caviae can destroy and disintegrate the epithelial cells of the gill tissue of river crabs, causing the rupture and disintegration of the gill septum and mass death (Zhou Liying, et al. Identification and pathogenicity analysis of Aeromonas caviae pathogenic to Chinese mitten crabs [J]. Freshwater Fisheries, 2022, 52(1): 58-65.); Aeromonas hydrophila can cause hepatosplenomegaly, mesenteric and intestinal wall bleeding in mandarin fish (Lai Mingyong. Early immune response of intestinal tissue of mandarin fish to Aeromonas hydrophila infection [J]. Journal of Fujian Agriculture and Forestry University (Natural Science Edition), 2024, 53(6): 763-771.).

[0003] Therefore, the control of potential pathogens in water is crucial to the health of aquatic animals.

[0004] Bacteriophages are a type of probiotic microorganism with bacteriophage properties and are known as natural biocontrol agents for pathogens. For example, bacteriophage probiotics such as Bdellovibrio and Vibrio phagocytophilus have been widely shown to have a positive effect on pathogenic bacteria such as Aeromonas hydrophila and Aeromonas vermifuge in water.

[0005] Therefore, it is desirable in the art to screen out probiotics with bacteriophagic activity. Summary of the Invention

[0006] In view of the above-mentioned problems and / or other problems in the related art, the first aspect of the present invention provides a modified Massilia varians P2-4, wherein the modified Massilia varians P2-4 is deposited in the Guangdong Provincial Microbiological Culture Collection Center with a deposit number of GDMCC No: 66023 and a deposit date of March 17, 2025.

[0007] A second aspect of the present invention provides a microbial agent, wherein the microbial agent comprises the aforementioned variant of Massillaria P2-4. Preferably, the microbial agent is used as a probiotic for aquaculture animals; more preferably, the microbial agent is used as a feed additive for aquaculture animals.

[0008] Preferably, the microbial agent comprises the fermentation broth of the aforementioned variation of Bacillus massiliense P2-4, or a bacterial liquid preparation prepared from the fermentation broth, or a bacterial powder preparation prepared by drying the fermentation broth.

[0009] The third aspect of the present invention provides the use of the above-mentioned variant of Massillaria P2-4 or the above-mentioned microbial agent in inhibiting pathogenic bacteria in aquaculture water bodies; preferably, the use is the use in preparing products for inhibiting pathogenic bacteria in aquaculture water bodies; more preferably, the use is the use in preparing products having phagocytic activity against pathogenic bacteria in aquaculture water bodies.

[0010] Preferably, the pathogenic bacteria are selected from Pseudomonas aeruginosa, Shewanella algae, Aeromonas caviae, Aeromonas hydrophila or Photobacterium mermanii.

[0011] A fourth aspect of the present invention provides a product for inhibiting pathogenic bacteria in aquaculture water, wherein the product comprises the aforementioned modified Massillaria P2-4 or the aforementioned microbial agent.

[0012] The fifth aspect of the present invention provides the use of the above-mentioned modified Marseillaria P2-4 or the above-mentioned microbial agent in enhancing the body immunity and / or antioxidant capacity and / or disease resistance of aquaculture animals; preferably, the aquaculture animal is a river crab; preferably, the above-mentioned modified Marseillaria P2-4 or the above-mentioned microbial agent is used to increase the activity of acid phosphatase, alkaline phosphatase, superoxide dismutase, lysozyme and catalase in the serum of river crab.

[0013] The sixth aspect of the present invention provides an aquaculture feed, wherein the aquaculture feed comprises a feed base component and the modified Massillaria P2-4 as described above; preferably, the aquaculture feed is a river crab farming feed.

[0014] The seventh aspect of the present invention provides the use of the above-mentioned modified Massillaria P2-4 or the above-mentioned microbial agent in the preparation of products for preventing and / or treating diseases of aquatic animals; preferably, the aquatic animals are river crabs; preferably, the diseases include hepatopancreatic necrosis syndrome.

[0015] An eighth aspect of the present invention provides a biocontrol product for aquaculture, wherein the biocontrol product comprises the aforementioned modified Massillaria P2-4 or the aforementioned microbial agent.

[0016] The modified Marseille bacteria P2-4 with broad-spectrum phagocytic activity isolated and obtained by the present invention performs phagocytosis by lysing bacteria, and can have phagocytic activity against pathogenic bacteria such as Pseudomonas aeruginosa, Shewanella algae, Aeromonas caviae, Aeromonas hydrophila, and Photobacterium mermanii. The modified Marseille bacteria P2-4 of the present invention can be used to inhibit pathogenic bacteria in aquaculture water bodies.

[0017] Adding modified Marseillaria P2-4 or its bacterial agent to aquaculture feed can enhance the immunity and / or antioxidant capacity and / or disease resistance of aquaculture animals, especially by improving the activity of acid phosphatase, alkaline phosphatase, superoxide dismutase, lysozyme, catalase, etc. in the serum of river crabs; the modified Marseillaria P2-4 of the present invention can be used as a feed additive for aquaculture, or as a biocontrol product for aquaculture, and can also prevent and / or treat diseases of aquaculture animals, especially hepatopancreatic necrosis syndrome of river crabs. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is the colony morphology of the Marseilles strain P2-4;

[0019] Figure 2 This is a photo of the bacterial morphology of the Marseilles strain P2-4 under an optical microscope (oil objective);

[0020] Figure 3 This is a phylogenetic tree constructed based on the 16S rRNA gene sequence of P. variabilis P2-4 (Note: the numbers in brackets represent the GenBank accession numbers of the strains; the numbers on the branch points represent the step size of the neighbor-joining method for calculating 1000 replicates of the data set; the scale of 0.01 represents the sequence divergence);

[0021] Figure 4 Heat map analysis of genetic similarity between the P2-4 strain of Masseilliae and different Masseilliae model strains;

[0022] Figure 5 The phagocytic activity of the P2-4 strain of Massillaria against Pseudomonas aeruginosa, Shewanella salinarum, Aeromonas caviae, Aeromonas hydrophila, and Photobacterium mermanii is shown;

[0023] Figure 6 This is a morphological diagram of the plaques formed by the Marseilles P2-4 strain on a double-layer agar plate;

[0024] Figure 7 Schematic diagram of the inhibition rate of different doses of Marseille P2-4 bacterial agent on Pseudomonas aeruginosa in aquaculture water (Note: CO group was not added with P2-4 bacterial solution, T1 group was added with a concentration of 1.0×10 3 CFU / mL of P2-4 bacterial solution, and the T2 group was supplemented with a concentration of 1.0×10 4 CFU / mL of P2-4 bacterial solution, T3 was added with a concentration of 1.0×10 5 CFU / mL of P2-4 bacterial solution). DETAILED DESCRIPTION

[0025] The present invention is further described below through specific embodiments, but the present invention is not limited to these specific embodiments.

[0026] Unless otherwise specified, the materials and reagents used in the following embodiments can be obtained from commercial sources. Where specific techniques or conditions are not specified, they were carried out according to the techniques or conditions described in the literature in the field or according to the product instructions.

[0027] The following describes the operation process of the inventor of the present application to separate and obtain the modified Marseille bacteria P2-4 of the present invention from the natural environment.

[0028] 1. Experimental Materials

[0029] Aquaculture sludge was collected from the shrimp ponds of Jinhaiwan Seedling Breeding Co., Ltd. in Laoshan District, Qingdao City, Shandong Province; Pseudomonas aeruginosa HX-1 was isolated from a crab with hepatopancreatic necrosis syndrome by our laboratory; Pseudomonas aeruginosa FJ1-2, Pseudomonas aeruginosa FJ1-5, Pseudomonas aeruginosa FJ1-9, Pseudomonas aeruginosa FJ3-2, Pseudomonas aeruginosa PX3-3, Shewanella algae RZ2-2, Shewanella algae RZ3-4, Shewanella algae RZ2-1, Aeromonas caviae PX2-5, Aeromonas caviae PX3-1, Aeromonas hydrophila PX2-8, and Aeromonas hydrophila Aeromonas PX2-6 and Photobacterium mermanii YJ-1 were provided by the National Aquatic Animal Pathogen Bank. Six hundred river crabs with an average body weight of 17.51 ± 1.28 g were provided by Jiangsu Nantong Duoruixian E-Commerce Co., Ltd. and acclimated for 14 days under experimental conditions. River crab feed was purchased from Jiangsu Hongxiang Feed Technology Co., Ltd., sterilized by autoclaving at 121°C for 20 min, oven-dried at 45°C for 24 h, and then air-dried for use as basal feed. Twenty-four glass aquariums, measuring 190 cm × 130 cm × 80 cm, were provided by our laboratory. API 20NE bacterial biochemical identification reagent strips and the Ezup column-type genomic DNA extraction kit (bacteria) produced by BioMérieux (France) were purchased from Shanghai Aozhishan Industrial Development Co., Ltd. Detection kits for acid phosphatase (ACP), alkaline phosphatase (AKP), superoxide dismutase (SOD), catalase (CAT), and lysozyme (LZM) were purchased from Nanjing Jiancheng Bioengineering Research Institute.

[0030] 2. Preparation of pathogenic bacteria suspension (host bacteria suspension)

[0031] Pseudomonas aeruginosa HX-1, Pseudomonas aeruginosa FJ1-2, Pseudomonas aeruginosa FJ1-5, Pseudomonas aeruginosa FJ1-9, Pseudomonas aeruginosa FJ3-2, Pseudomonas aeruginosa PX3-3, Shewanella algae RZ2-2, Shewanella algae RZ3-4, Shewanella algae RZ2-1, Aeromonas caviae PX2-5, Aeromonas caviae PX3-1, Aeromonas hydrophila PX2-8, Aeromonas hydrophila PX2-6, Photobacterium mermanii YJ-1 and other pathogenic bacteria strains were inoculated into sterile nutrient broth and cultured in a shaker at 30°C and 180 rpm for 24 h. The culture was then centrifuged at 4°C and 8000 rpm for 10 min. The precipitate was washed three times with sterile distilled water, and the concentration of each pathogenic bacteria suspension was adjusted to 1.0 × 10 10 CFU / mL, and stored in a refrigerator at 4°C for future use.

[0032] 3. Isolation of bacteriophage microorganisms

[0033] Weigh 1.0 g of aquaculture sludge (collected from the shrimp ponds of Jinhaiwan Seedling Breeding Co., Ltd., Laoshan District, Qingdao City, Shandong Province) and dissolve it in 9 mL of sterile water. Oscillate the sludge on an oscillator for 30 minutes to prepare a 1:10 uniform dilution. Use a 1 mL sterile pipette to draw up 1 mL of the 1:10 dilution and slowly inject it along the wall of the tube into a centrifuge tube containing 9 mL of sterile water. Shake the tube and mix for 30 seconds to mix evenly to prepare a 1:100 dilution. Repeat this process to prepare 1:1000, 1:10000, 1:100000, and 1:1000000 dilutions. Pseudomonas aeruginosa HX-1 was used as the screening host bacteria, and the tap water double-layer agar plate method was used to detect whether each dilution contained potential phage microorganisms. 100 μL of the dilution that produced phage plaques was taken for plate coating and incubated at 30°C for 24 h. When a single bacterial colony grew, a sterile inoculation loop was used to select single colonies with different colors, morphological characteristics, and sizes. After purification on a nutrient agar plate, Pseudomonas aeruginosa HX-1 was then used as the host bacteria, and the tap water double-layer agar plate method was used to detect the presence of each isolated strain (1.0×10 6 CFU / mL) of phagocytic ability.

[0034] A strain P2-4 with excellent phagocytic activity was isolated by the above method, inoculated onto a nutrient agar slant, cultured at 30°C for 24 hours, and stored at 4°C for later use.

[0035] 4. Phage spectrum analysis of isolated superior strain P2-4

[0036] 4.1 Experimental process of phage spectrum analysis

[0037] Pseudomonas aeruginosa HX-1, Pseudomonas aeruginosa FJ1-2, Pseudomonas aeruginosa FJ1-5, Pseudomonas aeruginosa FJ1-9, Pseudomonas aeruginosa FJ3-2, Pseudomonas aeruginosa PX3-3, Shewanella algae RZ2-2, Shewanella algae RZ3-4, Shewanella algae RZ2-1, Aeromonas caviae PX2-5, Aeromonas caviae PX3-1, Aeromonas hydrophila PX2-8, Aeromonas hydrophila PX2-6, and Photobacterium mermanii YJ-1 were used as host bacteria. The double-layer agar plate method was used to determine the phagocytic activity of the strain P2-4 (1.0×10 6 CFU / mL) against the above host bacteria.

[0038] According to the above method, the concentration of 1.0×10 6 CFU / mL of each pathogenic bacteria suspension (host bacteria suspension) and 1.0×10 6CFU / mL of strain P2-4 suspension; take 200 μL of strain P2-4 suspension and 200 μL of host bacterial suspension, add them to semi-solid soft agar, mix well and pour them onto agar powder plate, incubate at 30°C after solidification, and observe the formation of plaques.

[0039] The phagocytic activity of strain P2-4 against the above host bacteria was analyzed by the paper disc method: 100 μL of 1.0×10 6 CFU / mL of each pathogenic bacteria suspension (host bacteria suspension) were spread on the upper layer of the nutrient agar plate, and immediately affixed with 1.0×10 6 The paper pieces (5 mm in diameter) were soaked in the bacterial suspension of strain P2-4 (CFU / mL) and cultured at 30°C for 24 h, and then the inhibition zones were observed.

[0040] By observing the formation of plaques and inhibition zones, the mode of action of strain P2-4 with excellent phagocytic activity was determined.

[0041] 4.2 Experimental results of phage spectrum analysis

[0042] See also Figure 5 , the phagocytic activity diagram of the bacterial suspension of the strain P2-4 with excellent phagocytic activity isolated above against various aquatic pathogens.

[0043] from Figure 5 It can be seen that strain P2-4 has good phagocytic activity against various pathogenic bacteria (Pseudomonas aeruginosa HX-1, Pseudomonas aeruginosa FJ1-2, Pseudomonas aeruginosa FJ1-5, Pseudomonas aeruginosa FJ1-9, Pseudomonas aeruginosa FJ3-2, Pseudomonas aeruginosa PX3-3, Shewanella algae RZ2-2, Shewanella algae RZ3-4, Shewanella algae RZ2-1, Aeromonas caviae PX2-5, Aeromonas caviae PX3-1, Aeromonas hydrophila PX2-8, Aeromonas hydrophila PX2-6, Photobacterium mermanii YJ-1).

[0044] See also Figure 6 , strain P2-4 (plated together with the host bacteria) can form round, clear, smooth, and neatly edged plaques on double-layer agar plates within 36 h.

[0045] During the above-mentioned experimental process, the inventors of the present application unexpectedly discovered that strain P2-4 could not produce inhibition zones (only plaques) against these pathogenic bacteria (Pseudomonas aeruginosa HX-1, Pseudomonas aeruginosa FJ1-2, Pseudomonas aeruginosa FJ1-5, Pseudomonas aeruginosa FJ1-9, Pseudomonas aeruginosa FJ3-2, Pseudomonas aeruginosa PX3-3, Shewanella algae RZ2-2, Shewanella algae RZ3-4, Shewanella algae RZ2-1, Aeromonas caviae PX2-5, Aeromonas caviae PX3-1, Aeromonas hydrophila PX2-8, Aeromonas hydrophila PX2-6, Photobacterium mermanii YJ-1).

[0046] These results indicate that strain P2-4 does not inhibit bacteria by producing antimicrobial metabolites, but rather by lysing bacteria.

[0047] 5. Identification of strain P2-4

[0048] 5.1 Molecular Biological Identification

[0049] 1) Homology comparison analysis of 16S rRNA gene sequences

[0050] Strain P2-4 was inoculated into 100 mL of sterile nutrient broth and cultured in a shaker at 30°C and 180 rpm for 24 h. The culture was then centrifuged at 4°C and 12,000 rpm for 1 min, and the supernatant was discarded. Genomic DNA was then extracted using the Ezup column-based genomic DNA extraction kit (bacteria), and the 16S rRNA gene was amplified by PCR using the genomic DNA as a template. The forward primer was 27F: 5′-AGAGTTTGATCCTGGCTCAG-3′, and the reverse primer was 1492R: 5′-GGTTACCTTGTTACGACTT-3′. The total PCR reaction volume was 40 μL, consisting of 1 μL template DNA, 1 μL each of the upstream and downstream primers, 20 μL of Premix Taq, and 17 μL of ddH2O. PCR amplification conditions were: initial denaturation at 95°C for 3 min, followed by 35 cycles of denaturation at 95°C for 1 min, annealing at 60°C for 1 min, and extension at 72°C for 1 min, followed by a final extension at 72°C for 10 min. PCR products were analyzed by 1.0% agarose gel electrophoresis and then sent to Shanghai Maipu Biotechnology Co., Ltd. for sequencing.

[0051]

[0052] The 16S rRNA gene sequence measured above was compared with the 16S rRNA gene sequences of known bacteria in the GenBank database through the BLAST search system of NCBI for homology analysis, and it was found that the 16S rRNA gene sequences of strain P2-4 and the variable Marseille strain were naturally clustered.

[0053] 2) Phylogenetic analysis

[0054] The phylogenetic tree was constructed using the neighbor-joining method using MEGA 11.0 software (Replications = 1000, bootstrap values were taken as percentages). Figure 3 , it can be seen that strain P2-4 is most closely related to M. variabilis CCUG 35299 (GenBank accession number: NR_042652).

[0055] 3) ANI and dDDH analysis based on whole genome sequence

[0056] Strain P2-4 was aseptically inoculated into sterile nutrient broth and cultured at 30°C, shaking at 180 rpm for 24 hours. The cells were then harvested by centrifugation at 4000 rpm for 10 minutes and sent to Shanghai Meiji Biopharmaceutical Technology Co., Ltd. for whole-genome sequencing. Raw reads were quality-filtered using Fastp v0.23.0. The filtered reads were assembled using MonoCycle v0.4.8, and the assembly results were then corrected using Illumina sequencing data.

[0057] According to the results of whole genome sequencing, the total length of the whole genome sequence of strain P2-4 is 5,971,490bp, with an average GC content of 65.24%, consisting of 1 chromosome and 1 plasmid; its genome contains 75 tRNAs, 7 5S rRNAs, 7 16S rRNAs, 7 23S rRNAs, 34 sRNAs, 5238 coding genes, 20 total genomic islands, 1 prophage, 23 insertion sequences and 102 repeat sequences.

[0058] The BLAST+ and MUMmner tools in JSpecesWS (https: / / ggdc.dsmz.de / ggdc.php) and the GGDC 3.0 (https: / / ggdc.dsmz.de / ggdc.php#) online tools were then used to analyze the overall genomic relatedness indices (OGRIs) of strain P2-4 and other closely related species, and the taxonomic status of strain P2-4 was determined based on ANIb, ANIm, and dDDH.

[0059] See also Figure 4 , heat map analysis of genetic similarity between strain P2-4 and different Marseilles type strains, from Figure 4 The results showed that strain P2-4 had the highest similarity with the variable Marseilles strain CGMCC 4.7419, with an ANIb value of 94.88%, an ANIm value of 95.93%, and a dDDH value of 79.5%.

[0060] 5.2 Morphological identification

[0061] See also Figure 1 The colonies of strain P2-4 on nutrient agar plates were light yellow, round, slightly convex in the center, with neat edges and a moist surface.

[0062] See also Figure 2 , the strain P2-4 bacteria appeared rod-shaped under the oil immersion lens (100×).

[0063] 5.3 Physiological and biochemical identification

[0064] API 20NE bacterial biochemical identification reagent strips were used to perform physiological and biochemical identification of strain P2-4.

[0065] The results of physiological and biochemical identification showed that strain P2-4 could not reduce nitrate; could not utilize arginine, urea and gelatin; could assimilate glucose, maltose and malic acid; could not assimilate arabinose, mannose, phenylacetic acid, mannitol, N-acetyl-glucosamine, potassium gluconate, caprylic acid, adipic acid and sodium citrate; had cytochrome oxidase; could not ferment glucose; could not utilize L-tryptophan and quinolin; and did not have β-galactosidase. Except for the utilization of gelatin and quinolin, the physiological and biochemical characteristics of arabinose and p-nitro-β-D-methylgalactose fermentation were consistent with the literature (the "a" in Table 1 below is from these literatures: CHAUDHARY DK, KIM J. Massilia agri sp.nov., isolated from reclaimed grassland soil [J]. International journal of systematic and evolutionary microbiology, 2017, 67(8): 2696-703. ORTHOVA I, P,GLAESER SP,et al.Massilianorwichensis sp.nov.,isolated from an air sample[J].International Journal of Systematic and Evolutionary Microbiology,2015,65:56-64.SINGH H,DU J,WON K,etal.Massilia arvi sp.nov.,isolated from fallow-land soil previously cultivatedwith Brassica oleracea,and emended description of the genus Massilia[J].International Journal of Systematic and Evolutionary Microbiology,2015,65:3690-3696.WEON HY,KIM BY,SON JA,et al.Massilia aerilata sp.nov.,isolated from an air sample[J].International journal of systematic and evolutionarymicrobiology,2008,58(6):1422-5.XU P,LI WJ,TANG SK,et al.Naxibacteralkalitolerans gen.nov.,sp.nov.,a novel member of the family'Oxalobacteraceae'isolated from China[J].International journal ofsystematicand evolutionarymicrobiology,2005,55(3):1149-53.),except that the variegata reported in this study is different, the rest of the physiological and biochemical characteristics are the same as those of the variegata.

[0066] The physiological and biochemical identification results of strain P2-4 are shown in Table 1.

[0067] Table 1

[0068]

[0069]

[0070] In Table 1, “+” indicates positive; “-” indicates negative; “±” indicates positive or negative.

[0071] In summary, combined with the above molecular biological identification results, morphological observation and physiological and biochemical identification results, it can be determined that the strain P2-4 isolated and obtained in the present invention is Massilia varians.

[0072] Performance data

[0073] 1. In vitro antibacterial effect of strain P2-4 on pathogenic bacteria in aquaculture water

[0074] Taking Pseudomonas aeruginosa as the representative pathogenic bacteria, the in vitro antibacterial effect of strain P2-4 on pathogenic bacteria in aquaculture water was detected and analyzed.

[0075] The in vitro antibacterial effect of strain P2-4 on Pseudomonas aeruginosa HX-1 in aquaculture water was analyzed in a glass conical flask. Pseudomonas aeruginosa HX-1 suspension was added to filtered aquaculture water that had been sterilized by autoclave (121°C, 20 min) to a final concentration of 1.0×10 5 CFU / mL, and then immediately add the bacterial suspension of strain P2-4 to make the final concentration of 1.0×10 3 CFU / mL, 1.0×10 4 CFU / mL, 1.0×10 5 CFU / mL. Filtered aquaculture water supplemented with only Pseudomonas aeruginosa HX-1 under the same conditions was used as a control. Each treatment was replicated three times. Each treatment was shaken at 30°C and 180 rpm for 5 days, and the concentration of Pseudomonas aeruginosa HX-1 (CFU / mL) was measured daily. The concentration of Pseudomonas aeruginosa HX-1 was determined using the dilution TCBS plate method. The inhibition rate was calculated according to formula (1).

[0076]

[0077] Results see Figure 7 On the 5th day, the concentrations of strains P2-4 (T1-T3 groups were 1.0×10 3 CFU / mL, 1.0×10 4 CFU / mL, 1.0×10 5 CFU / mL) were treated with 6.0×10 3 CFU / mL, 9.0×10 2 CFU / mL, 83.0 CFU / mL, which were significantly lower than the concentration of Pseudomonas aeruginosa in the control group (9.2×10 5 CFU / mL)(P<0.05).

[0078] According to the above antibacterial rate formula, the concentrations were calculated to be 1.0×10 3 CFU / mL, 1.0×10 4 CFU / mL, 1.0×10 5 The inhibition rates of strain P2-4 against Pseudomonas aeruginosa reached 99.35%, 99.90% and 99.99% respectively.

[0079] Figure 7 The results indicate that strain P2-4 has a significant inhibitory effect on the proliferation of Pseudomonas aeruginosa HX-1 in aquaculture water. Strain P2-4 can be used for ecological control of Pseudomonas aeruginosa in aquaculture water and for inhibiting pathogenic bacteria in aquaculture water. Strain P2-4 and its microbial agents can be used to prepare products for inhibiting pathogenic bacteria in aquaculture water, particularly products with phagocytic activity against pathogenic bacteria in aquaculture water.

[0080] 2. Effects of strain P2-4 on immunity and antioxidant capacity of aquatic animals

[0081] Using river crab as a representative farmed animal, the effects of strain P2-4 on the immunity and antioxidant capacity of aquatic animals were detected and analyzed.

[0082] 1) Breeding experiment

[0083] Before the experiment, the final concentration was 6.0×10 11 CFU / mL of the strain P2-4 suspension was then added to the basal feed to prepare a final concentration (the final concentration of the strain P2-4) of 6.0×10 6 CFU / g feed, 6.0×10 7 CFU / g feed, 6.0×10 8 The experiment included one control group and three experimental groups, each with three parallel aquariums. Tiles were placed in each aquarium to prevent crabs from fighting. 120L of aerated tap water at 28°C was added, along with 50 crabs weighing an average of 17.51±1.28g. The experimental groups were fed the three experimental feeds daily, while the control group was fed the basal feed for 40 consecutive days, twice daily (8:00 and 18:00) at a feeding amount of 3% of the crabs' total body weight. The light-dark cycle was 12h:12h. After the aquaculture experiment, the nonspecific immunity and antioxidant capacity of the crabs in each group were immediately measured.

[0084] 2) Determination of nonspecific immunity and antioxidant capacity

[0085] Immediately after the above-mentioned breeding experiment, 5 crabs were randomly selected from each aquarium and their hemolymph was collected to analyze the relevant indicators of antioxidant capacity and nonspecific immune capacity.

[0086] Specific processing process: blood was drawn from the root of the third abdominal foot of river crabs (experimental group and control group), collected in a 1 mL sterile centrifuge tube containing an equal amount of sterile anticoagulant (acid citrate dextrose ACD), centrifuged at 4000 r / min at 4°C for 20 min, and the supernatant was taken and stored at -80°C for later use. Referring to the instructions of each detection kit, the levels of nonspecific immune enzymes such as AKP, ACP and LZM in the serum, as well as the levels of antioxidant enzymes such as SOD and CAT in the serum were determined.

[0087] 3) Effect data of strain P2-4 on the nonspecific immunity of river crabs

[0088] The effects of adding strain P2-4 to feed on the nonspecific immune ability of river crabs are shown in Table 2.

[0089] Table 2

[0090]

[0091] Note: CON: feeding the basic feed without strain P2-4, T1: feeding the basic feed containing 6.0×10 6 CFU / g feed of strain P2-4; T2: feeding the experimental feed containing 6.0×10 7 CFU / g feed of strain P2-4; T3: feeding the experimental feed containing 6.0×10 8 CFU / g of experimental feed of feed strain P2-4; different letters in the same column indicate significant differences (P<0.05), and the same letters indicate no significant differences (P>0.05).

[0092] From the results in Table 2, it can be calculated that compared with the crabs in the control group, the activities of ACP, AKP and LZM in the serum of the crabs fed with the three groups of experimental feeds increased by 117.94% to 290.50% (P<0.05), 34.51% to 56.98% (P<0.05) and 9.09% to 43.94% (P<0.05), respectively.

[0093] The above results show that strain P2-4 can significantly improve the nonspecific immunity of river crabs.

[0094] 4) Effect of strain P2-4 on the antioxidant capacity of river crabs

[0095] The effects of adding strain P2-4 to feed on the antioxidant capacity of river crabs are shown in Table 3.

[0096] Table 3

[0097]

[0098] Note: CON: feeding the basic feed without strain P2-4, T1: feeding the basic feed containing 6.0×10 6 CFU / g feed of strain P2-4; T2: feeding the experimental feed containing 6.0×10 7 CFU / g feed of strain P2-4; T3: feeding the experimental feed containing 6.0×10 8 CFU / g of experimental feed of feed strain P2-4; different letters in the same column indicate significant differences (P<0.05), and the same letters indicate no significant differences (P>0.05).

[0099] From the results in Table 3, it can be calculated that compared with the control group, the serum SOD and CAT activities of the crabs fed with the three experimental feeds increased by 2.32% to 17.16% (P<0.05) and 26.48% to 44.66% (P<0.05), respectively.

[0100] The above results show that strain P2-4 can significantly improve the antioxidant capacity of river crabs.

[0101] 3. Effects of strain P2-4 on the resistance of aquatic animals to disease infection

[0102] Using river crab as a representative farmed animal and the ability to resist hepatopancreatic necrosis syndrome infection as the disease resistance ability, the ability of strain P2-4 to resist disease infection in aquatic animals was detected and analyzed.

[0103] 1) Breeding experiment

[0104] Before the experiment, the final concentration of 6.0×10 8 CFU / mL bacterial suspension of strain P2-4 was diluted with sterile distilled water, and the dilutions of different concentrations were added to the basic feed to prepare a final concentration (the final concentration of strain P2-4) of 6.0×10 3 CFU / g feed, 6.0×10 4 CFU / g feed, 6.0×10 5 CFU / g feed for three groups of experimental feeds.

[0105] The experiment included one control group and three experimental groups, each containing three parallel aquariums. Tiles were placed in each aquarium to prevent crab aggression. Ten crabs, weighing an average of 22.12 ± 0.6 g, were fed 120 L of aerated tap water. The three experimental groups were fed the three experimental diets daily, while the control group was fed a basal diet for 20 consecutive days, twice daily (8:00 AM and 6:00 PM).

[0106] After the breeding experiment, the disease resistance and infection resistance of each group of river crabs were tested.

[0107] 2) Determination of anti-disease infection ability

[0108] Before the experiment, a final concentration of 5.0×10 9 CFU / mL Pseudomonas aeruginosa HX-1 bacterial suspension. The obtained Pseudomonas aeruginosa HX-1 bacterial suspension was diluted with sterile distilled water and added to the basal feed respectively, and finally a final concentration of 5.0×10 6 CFU / g feed of Pseudomonas aeruginosa HX-1 bait.

[0109] Immediately after the experiment, each group of crabs was fed bait containing Pseudomonas aeruginosa HX-1 to artificially infect the crabs with hepatopancreatic necrosis syndrome. The bait was fed twice daily (8:00 and 18:00) and the crabs were observed for 7 consecutive days for morbidity and mortality. The mortality rate was calculated, and the protection rate was calculated according to formula (2). During the experiment, each aquarium was continuously aerated, the water temperature was set at 28°C, the light-dark cycle was 12h:12h, and the feeding amount was 3% of the crabs' total body weight.

[0110]

[0111] 3) Results of the protection rate of strain P2-4 against disease infection of river crabs

[0112] The results of adding strain P2-4 to the feed to resist disease infection of river crabs are shown in Table 4 below.

[0113] Table 4

[0114]

[0115] Different letters in the same column indicate significant differences (P<0.05), and the same letters indicate no significant differences (P>0.05).

[0116] As can be seen from Table 4, after oral infection with Pseudomonas aeruginosa, crabs fed with the basic diet suffered a large number of deaths. The diseased crabs showed typical symptoms of hepatopancreatic necrosis syndrome, with a mortality rate of 86.67%. 3 , 6.0×10 4 , 6.0×10 5 The mortality rate of river crabs fed with the three experimental feeds with the highest CFU / g feed was significantly reduced.

[0117] Calculations show that compared with the control group of crabs fed with basic feed, the mortality rates of crabs fed with the three experimental feeds were reduced by 36.67% (P>0.05), 53.34% (P<0.05) and 63.34% (P<0.05), respectively.

[0118] According to the average mortality results in Table 4, the three groups of experimental feed (containing the final concentration of strain P2-4 6.0×10 3 , 6.0×10 4 , 6.0×10 5 CFU / g feed) and the protection rates of preventing hepatopancreatic necrosis syndrome caused by Pseudomonas aeruginosa in river crabs reached 42.31%, 61.54% and 73.08% respectively.

[0119] Therefore, strain P2-4 can significantly enhance the disease resistance and infection ability of river crabs and can be used to prevent and / or treat hepatopancreatic necrosis syndrome in river crabs.

[0120] Experimental data and processing

[0121] All the above-mentioned effect experimental data were expressed as mean ± deviation and statistically analyzed using SPSS19.0 software. P < 0.05 indicated that the difference was significant.

[0122] It should be understood that although this specification is described in terms of implementation methods, not every implementation method contains only one independent technical solution. This narrative method of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each implementation method can also be appropriately combined to form other implementation methods that can be understood by those skilled in the art.

[0123] The series of detailed descriptions listed above are only specific descriptions of feasible implementation methods of the present invention. They are not intended to limit the scope of protection of the present invention. Any equivalent implementation methods or changes that do not deviate from the technical spirit of the present invention should be included in the scope of protection of the present invention.

Claims

1. A variant of Massilia varians P2-4, wherein The variation Massiliavarians P2-4 was deposited in Guangdong Provincial Microbiological Culture Collection Center with the deposit number GDMCC No: 66023 and the deposit date of March 17, 2025.

2. A microbial agent, characterized in that: The microbial agent comprises the modified Massillaria P2-4 as claimed in claim 1; preferably, the microbial agent is used as a probiotic for aquaculture animals; more preferably, the microbial agent is used as a feed additive for aquaculture animals.

3. The microbial agent according to claim 2, wherein: The microbial agent comprises the fermentation broth of the variation of Massillaria P2-4 as claimed in claim 1, or a bacterial liquid preparation prepared from the fermentation broth, or a bacterial powder preparation prepared by drying the fermentation broth.

4. Use of the modified Massillaria P2-4 according to claim 1 or the microbial agent according to any one of claims 2 to 3 in inhibiting pathogenic bacteria in aquaculture water; preferably, the use is in the preparation of a product for inhibiting pathogenic bacteria in aquaculture water; more preferably, the use is in the preparation of a product having phagocytic activity against pathogenic bacteria in aquaculture water.

5. The use according to claim 4, characterized in that The pathogenic bacteria are selected from Pseudomonas aeruginosa, Shewanella algae, Aeromonas caviae, Aeromonas hydrophila or Photobacterium mermanii.

6. A product for inhibiting pathogenic bacteria in aquaculture water, characterized by: The product comprises the modified Massillaria P2-4 according to claim 1 or the microbial agent according to claim 2 or 3.

7. Use of the modified Marseillaris P2-4 as described in claim 1 or the microbial agent as described in any one of claims 2-3 in enhancing the body immunity and / or antioxidant capacity and / or disease resistance of aquaculture animals; preferably, the aquaculture animal is a river crab; preferably, use of the modified Marseillaris P2-4 as described in claim 1 or the microbial agent as described in any one of claims 2-3 in increasing the activity of acid phosphatase, alkaline phosphatase, superoxide dismutase, lysozyme and catalase in the serum of river crabs.

8. An aquaculture feed, characterized by: The aquaculture feed comprises a basic feed component and the modified Massillaria thaliana P2-4 as claimed in claim 1; preferably, the aquaculture feed is a river crab farming feed.

9. Use of the modified Massillaria P2-4 according to claim 1 or the microbial agent according to any one of claims 2 to 3 in the preparation of a product for preventing and / or treating aquatic animal diseases; preferably, the aquatic animal is a river crab; preferably, the disease includes hepatopancreatic necrosis syndrome.

10. A biocontrol product for aquaculture, characterized by: The biocontrol product comprises the modified Marseilles bacteria P2-4 as claimed in claim 1 or the microbial agent as claimed in claim 2 or 3.

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