Bacteriophage disease-resistant alkali-resistant marssonia sp. And application thereof

Abstract

The invention relates to an alkali-resistant massinia sp., in particular to a separated alkali-resistant massinia sp. P2-6 with broad-spectrum phagocytosis activity on pathogenic bacteria, and the alkali-resistant massinia sp. P2-6 is used for phagocytosis in a bacterium splitting manner, and can be used for preparing the alkali-resistant massinia sp. P2-6 with broad-spectrum phagocytosis activity on pathogenic bacteria. The compound has phagocytosis activity on pathogenic bacteria such as aeromonas veronii, aeromonas hydrophila, aeromonas cainea, pseudomonas aeruginosa, shewanella alginate or photobacterium damsela and the like; the alkali-resistant Marsinia sp. P2-6 disclosed by the invention can be applied to inhibition of pathogenic bacteria in an aquaculture water body; alkali-resisting marssonia sp. P2-6 or a microbial agent thereof is added into an aquaculture feed, so that the organism immunity and / or oxidation resistance and / or disease infection resistance of aquaculture animals can be enhanced; the alkali-resistant Marsinia sp. P2-6 provided 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 an alkali-resistant Marseillais, specifically to an alkali-resistant Marseillais P2-6 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 reproduction of potential pathogens in water bodies seriously threatens the health of aquatic animals. For example, Aeromonas vermiformis can cause septicemia in Chinese mitten crabs (Jiang Guangming, Qian Caiyuan, Gu Xuelin, et al. Study on the etiology of bacterial septicemia in Chinese mitten crabs and Procambarus clarkii [J]. Aquaculture, 2016, 37(4): 46-51. Zhou Huihua, Huang Xiaodong, An Jian, et al. Isolation and identification of pathogenic Aeromonas vermiformis in Chinese mitten crabs, drug sensitivity characteristics and histopathological observation [J]. Journal of Southern Agriculture, 2019, 50(8): 1851-1859.); Aeromonas hydrophila can cause tremor disease and edema in Chinese mitten crabs (Cheng Chao, Xiao Min, Li Jing, et al. Aeromonas hydrophila stimulates the growth of Chinese mitten crabs and Procambarus clarkii. The influence of the immune function of crabs on the immune system of the crabs [J]. Fisheries Science, 2020, 39(4): 465-475.); Aeromonas caviae can cause swelling of shrimp gill filaments, enlargement and rot of the hepatopancreas and mass death (Yuan Zhonghua, Song Haichao, Liu Juntong, et al. Research progress of Aeromonas caviae [J]. Advances in Veterinary Medicine, 2024, 45(6): 106-110.); Pseudomonas aeruginosa can cause skin ulcer disease in largemouth sea bass (Yao Qing, et al. Isolation and identification of Pseudomonas aeruginosa in largemouth sea bass and screening of antimicrobial drugs. Jiangsu Agricultural Sciences, 2024.); Shewanella salina can cause ulcer disease in American eels (HuicongWang, Ying Gu, Jun Chen, Haipeng Cao*. Shewanella algae: an emerging causative agent for ulcer disease in freshwater-farmed American eel Anguilla rostrata[J]. Israel Journal of Aquaculture-Bamidgeh, 2020, IJA_72.2020.964361.).

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

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

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

[0006] In view of the above problems and / or other problems in the related art, the present invention isolates and identifies an alkali-resistant Marseilles bacteria P2-6 with broad-spectrum phagocytic activity from aquaculture sludge.

[0007] The first aspect of the present invention provides an alkali-resistant Massilia P2-6, which is deposited in Guangdong Provincial Microbiological Culture Collection Center with a deposit number of GDMCC No: 66024 and a deposit date of March 17, 2025.

[0008] A second aspect of the present invention provides a microbial agent, wherein the microbial agent comprises the aforementioned alkali-resistant Massimo Bacillus P2-6. 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.

[0009] Preferably, the microbial agent comprises the fermentation broth of the alkali-resistant Marseille bacteria P2-6, or a bacterial liquid preparation prepared from the fermentation broth, or a bacterial powder preparation prepared by drying the fermentation broth.

[0010] The third aspect of the present invention provides the use of the above-mentioned alkali-resistant Marseille bacteria P2-6 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.

[0011] Preferably, the pathogenic bacteria are selected from Aeromonas vermiformis, Aeromonas hydrophila, Aeromonas caviae, Pseudomonas aeruginosa, Shewanella algae or Photobacterium mermanii.

[0012] A fourth aspect of the present invention provides a product for inhibiting pathogenic bacteria in aquaculture water, wherein the product comprises the alkali-resistant Marseille bacteria P2-6 or the microbial agent described above.

[0013] The fifth aspect of the present invention provides the use of the above-mentioned alkali-resistant Marseillani P2-6 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 alkali-resistant Marseillani P2-6 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 and / or hepatopancreas of river crabs.

[0014] The sixth aspect of the present invention provides an aquaculture feed, wherein the aquaculture feed comprises a feed base component and the alkali-resistant Marseillais P2-6 as described above; preferably, the aquaculture feed is a river crab farming feed; preferably, the alkali-resistant Marseillais P2-6 is added to the aquaculture feed at a final dosage of 6.0×10 5 CFU / g feed and above.

[0015] The seventh aspect of the present invention provides the use of the above-mentioned alkali-resistant Marseille bacteria P2-6 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.

[0016] An eighth aspect of the present invention provides a biocontrol product for aquaculture, wherein the biocontrol product comprises the alkali-resistant Marseille bacteria P2-6 as described above or the microbial agent as described above.

[0017] The alkali-resistant Marseille bacteria P2-6 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 Aeromonas vernix, Aeromonas hydrophila, Aeromonas caviae, Pseudomonas aeruginosa, Shewanella algae or Photobacterium mermanii. The alkali-resistant Marseille bacteria P2-6 of the present invention can be used to inhibit pathogenic bacteria in aquaculture water bodies.

[0018] Adding alkali-resistant Marseille bacteria P2-6 or its bacterial agent to aquaculture feed can enhance the innate immunity and / or antioxidant capacity and / or disease resistance of aquaculture animals, especially in improving the activity of acid phosphatase, alkaline phosphatase, superoxide dismutase, lysozyme, catalase in the serum and / or hepatopancreas of river crabs; the alkali-resistant Marseille bacteria P2-6 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 aquaculture animal diseases, especially hepatopancreatic necrosis syndrome of river crabs. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 is the colony morphology of strain P2-6;

[0020] Figure 2 This is a photo of the bacterial morphology of strain P2-6 under an optical microscope (oil objective);

[0021] Figure 3 This is a phylogenetic tree constructed based on the 16S rRNA gene sequence of strain P2-6 (Note: the serial numbers in brackets represent the GenBank accession numbers of the strains; the numbers on the branch points represent the step size values of the neighbor-joining method for calculating 1000 replicates of the data set; the scale of 0.01 represents sequence divergence);

[0022] Figure 4 Heat map analysis of genetic similarity between strain P2-6 and different Marseilles type strains;

[0023] Figure 5 The phagocytic activity of strain P2-6 against Aeromonas vernii, Aeromonas caviae, Aeromonas hydrophila, Pseudomonas aeruginosa, Shewanella algae, and Photorhabdus mermanii;

[0024] Figure 6 The morphology of the plaques formed by strain P2-6 on a double-layer agar plate;

[0025] Figure 7 is the inhibition rate of strain P2-6 against Aeromonas verticillata in artificial simulated aquaculture water. (Note: CON group did not add P2-6 bacterial solution, T1 group added 5.0×10 4 CFU / mL of P2-6 bacterial solution, and the T2 group was supplemented with a concentration of 5.0×10 5 CFU / mL of P2-6 bacterial solution, T3 was added with a concentration of 5.0×10 6 CFU / mL of P2-6 bacterial solution) DETAILED DESCRIPTION

[0026] The present invention is further described below through specific embodiments, but the present invention is not limited to these specific embodiments.

[0027] 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.

[0028] The following describes the process by which the inventors of the present application isolated and obtained the alkali-resistant Marseille bacteria P2-6 of the present invention from the natural environment.

[0029] 1. Experimental Materials

[0030] Aquaculture sludge was collected from the shrimp ponds of Jinhaiwan Seedling Farming Co., Ltd. in Laoshan District, Qingdao City, Shandong Province; Aeromonas vileri HXH1, Aeromonas vileri ST-10, Aeromonas vileri HXY1, Aeromonas hydrophila PX2-6, Aeromonas hydrophila PX2-8, Aeromonas caviae PX2-5, Aeromonas caviae PX3-1, Pseudomonas aeruginosa FJ1-2, Pseudomonas aeruginosa FJ1-5, Pseudomonas aeruginosa FJ1-9, Pseudomonas aeruginosa FJ2-7, Pseudomonas aeruginosa FJ3-2, Pseudomonas aeruginosa FJ3-3, Shewanella salina RZ2-1, Shewanella salina RZ2- 5. Shewanella algae RZ3-4 and Photobacterium mermanii YJ-1 were provided by the National Aquatic Animal Pathogen Bank; healthy river crabs with an average mass of 20.45 ± 1.78 g, housed in the laboratory for 14 days, were provided by Jiangsu Nantong Duoruixian E-Commerce Co., Ltd.; river crab compound feed, free of any microorganisms, was purchased from Jiangsu Hongxiang Feed Technology Co., Ltd. and sterilized at 121°C for 20 minutes, then dried at 45°C for 24 hours, and naturally cooled for use as a basic feed; the glass aquarium was 190 cm × 130 cm × 80 cm and provided by this laboratory; API produced by BioMérieux, France 20NE bacterial biochemical identification reagent strips were purchased from Shanghai Aozhishan Industrial Development Co., Ltd.; Ezup column-type genomic DNA extraction kit (bacteria) was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.; acid phosphatase (ACP), alkaline phosphatase (AKP), superoxide dismutase (SOD), catalase (CAT), lysozyme (LZM) and other detection kits were purchased from Nanjing Jiancheng Bioengineering Institute.

[0031] 2. Preparation of pathogenic bacteria suspension (host bacteria suspension)

[0032] Aeromonas vickers HXH1, Aeromonas vickers ST-10, Aeromonas vickers HXY1, Aeromonas hydrophila PX2-6, Aeromonas hydrophila PX2-8, Aeromonas caviae PX2-5, Aeromonas caviae PX3-1, Pseudomonas aeruginosa FJ1-2, Pseudomonas aeruginosa FJ1-5, Pseudomonas aeruginosa FJ1-9, Pseudomonas aeruginosa FJ2-7, Pseudomonas aeruginosa FJ3-2, Pseudomonas aeruginosa FJ3-3, Pathogenic bacteria strains including Shewanella algae RZ2-1, Shewanella algae RZ2-5, Shewanella algae RZ3-4, and Photobacterium mermanii YJ-1 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 20 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.

[0033] 3. Isolation of bacteriophage microorganisms

[0034] Weigh 1.0 g of aquaculture sludge and dissolve it in 9 mL of sterile water. Oscillate the mixture on an oscillator for 30 minutes to prepare a 1:10 dilution. Use a 1 mL sterile pipette to draw 1 mL of the 1:10 dilution and slowly pipette it along the tube wall into a centrifuge tube containing 9 mL of sterile water. Shake the tube for 30 seconds to mix thoroughly to prepare a 1:100 dilution. Repeat this process to prepare 1:1000, 1:10000, 1:100000, and 1:1000000 dilutions. Aeromonas virens HXH1 was used as the screening host bacteria. 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 placed at 30°C for 24 hours. After the growth of bacterial single colonies, a sterile inoculation loop was used to select single colonies with different colors, morphological characteristics, and sizes. After purification on nutrient agar plates, each isolated strain was placed in sterile nutrient broth and cultured at 30°C and 180 r / min for 24 hours. The culture was then centrifuged at 4°C and 8000 r / min for 20 minutes. After washing the precipitate three times with sterile distilled water, the concentration of each bacterial suspension was adjusted to 1.0×10 9 CFU / mL. Then, Aeromonas wieldii HXH1 was used as the host bacteria, and the tap water double-layer agar plate method was used to detect the isolated strains (1.0×10 9 CFU / mL) of phagocytic ability.

[0035] A strain P2-6 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.

[0036] 4. Phage spectrum analysis of isolated superior strain P2-6

[0037] 4.1 Experimental process of phage spectrum analysis

[0038] Using Aeromonas vickers HXH1, Aeromonas vickers ST-10, Aeromonas vickers HXY1, Aeromonas hydrophila PX2-6, Aeromonas hydrophila PX2-8, Aeromonas caviae PX2-5, Aeromonas caviae PX3-1, Pseudomonas aeruginosa FJ1-2, Pseudomonas aeruginosa FJ1-5, Pseudomonas aeruginosa FJ1-9, Pseudomonas aeruginosa FJ2-7, Pseudomonas aeruginosa FJ3-2, Pseudomonas aeruginosa FJ3-3, Shewanella marine algae RZ2-1, Shewanella marine algae RZ2-5, Shewanella marine algae RZ3-4, and Photobacterium mermanii YJ-1 as host bacteria, the double-layer agar plate method was used to determine the phagocytic activity of the strain P2-6 with excellent phagocytic activity isolated above against the above host bacteria.

[0039] According to the above method, the concentration of 1.0×10 10 CFU / mL of host bacterial suspension and 1.0×10 9 Take 200 μL of the bacterial suspension of strain P2-6 and 200 μL of the host bacterial suspension and add them to semi-solid soft agar. After mixing, pour them onto the agar powder plate. After solidification, incubate at 30°C and observe the formation of plaques.

[0040] The phagocytic activity of strain P2-6 against the above host bacteria was analyzed by the paper disc method: 100 μL of 1.0×10 10 CFU / mL of each host bacterial suspension was spread on the upper layer of the nutrient agar plate, and immediately affixed with 1.0×10 9 The paper discs were soaked with the bacterial suspension of strain P2-6 at 100 CFU / mL and cultured at 30°C for 24 h, and then the inhibition zones were observed.

[0041] By observing the formation of plaques and inhibition zones, the mode of action of the isolated strain P2-6 with excellent phagocytic activity was determined.

[0042] 4.2 Experimental results of phage spectrum analysis

[0043] See also Figure 5 , the phagocytic activity diagram of the strain P2-6 with excellent phagocytic activity isolated above against various aquatic pathogens.

[0044] from Figure 5 It can be seen that strain P2-6 has good phagocytic activity against various pathogenic bacteria (Aeromonas vickers HXH1, Aeromonas vickers ST-10, Aeromonas vickers HXY1, Aeromonas hydrophila PX2-6, Aeromonas hydrophila PX2-8, Aeromonas caviae PX2-5, Aeromonas caviae PX3-1, Pseudomonas aeruginosa FJ1-2, Pseudomonas aeruginosa FJ1-5, Pseudomonas aeruginosa FJ1-9, Pseudomonas aeruginosa FJ2-7, Pseudomonas aeruginosa FJ3-2, Pseudomonas aeruginosa FJ3-3, Shewanella marine algae RZ2-1, Shewanella marine algae RZ2-5, Shewanella marine algae RZ3-4, Photobacterium mermanii YJ-1).

[0045] See also Figure 6 , strain P2-6 (plated together with the host bacteria) can form round, clear, smooth, and neatly edged plaques on double-layer agar plates within 36 h.

[0046] During the above-mentioned experimental process, the inventors of the present application unexpectedly discovered that strain P2-6 could not produce inhibition zones (only plaques) against these pathogenic bacteria (Aeromonas vernix HXH1, Aeromonas vernix ST-10, Aeromonas vernix HXY1, Aeromonas hydrophila PX2-6, Aeromonas hydrophila PX2-8, Aeromonas caviae PX2-5, Aeromonas caviae PX3-1, Pseudomonas aeruginosa FJ1-2, Pseudomonas aeruginosa FJ1-5, Pseudomonas aeruginosa FJ1-9, Pseudomonas aeruginosa FJ2-7, Pseudomonas aeruginosa FJ3-2, Pseudomonas aeruginosa FJ3-3, Shewanella algae RZ2-1, Shewanella algae RZ2-5, Shewanella algae RZ3-4, and Photobacterium mermanii YJ-1).

[0047] These results indicate that strain P2-6 does not inhibit bacteria by producing antimicrobial metabolites, but rather by lysing bacteria.

[0048] 5. Identification of strain P2-6

[0049] 5.1 Molecular Biological Identification

[0050] 1) Homology comparison analysis of 16S rRNA sequences

[0051] Strain P2-6 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 8000 rpm for 1 min, and the supernatant was discarded. The genomic DNA of the isolated strain with excellent phagocytic activity was then extracted using the Ezup column-type genomic DNA extraction kit (bacteria). The 16S rRNA gene was then amplified by PCR using the genomic DNA as a template. The forward primer was 27F: 5′-AGAGTTTGATCCTGGCT CAG-3′, and the reverse primer was 1492R: 5′-GGTTACCTTGTTACGACTT-3′. The total PCR reaction volume was 20 μL, and the reaction solution consisted of 2 μL of 10× Ex Taq buffer, 0.2 μL of 5 U / μL Ex Taq, 1.6 μL of 2.5 mM dNTP mix, 1.0 μL each of the upstream and downstream primers, 0.5 μL of template DNA, and 13.7 μL of ddH2O. PCR amplification conditions were as follows: initial denaturation at 95°C for 5 min; denaturation at 95°C for 30 s; annealing at 56°C for 30 s; extension at 72°C for 1.5 min for 25 cycles; and a final extension at 72°C for 10 min. The PCR products were verified by 1.0% agarose gel electrophoresis and sent to Shanghai Maipu Biotechnology Co., Ltd. for sequencing.

[0052] The sequence of 16S rRNA of strain P2-6 was determined as follows (SEQ ID NO. 1):

[0053]

[0054] 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. It was found that the 16S rRNA gene sequences of strain P2-6 and the alkali-resistant Marseille strain were naturally clustered.

[0055] 2) Phylogenetic analysis

[0056] 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 P2-6 is most closely related to Massilia alkalitolerans strain 262XG3' (GenBank accession number: KF954551).

[0057] 3) ANI and dDDH analysis based on whole genome sequence

[0058] Aseptically inoculate isolated strains with excellent phagocytic activity into sterile nutrient broth. Cultures were shaken at 30°C and 180 rpm for 24 hours, followed by centrifugation at 8000 rpm for 20 minutes to collect the cells. The cells were then 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.

[0059] Whole-genome sequencing revealed that strain P2-6 has a total genome length of 5,953,321 bp with an average GC content of 65.26%. It consists of one chromosome and one plasmid. The genome contains 75 tRNAs, 7 5S rRNAs, 7 16S rRNAs, 7 23S rRNAs, 34 sRNAs, 5,219 coding genes, 20 total genomic islands, no prophages, 24 insertions, and 102 repeats. The overall genome relatedness indices (OGRIs) of isolates with excellent phagocytic activity were analyzed relative to those of closely related species using BLAST+ and MUMmner tools in JSpecesWS (https: / / ggdc.dsmz.de / ggdc.php) and GGDC 3.0 (https: / / ggdc.dsmz.de / ggdc.php#). The taxonomic status of isolates with excellent phagocytic activity was determined based on the ANI and dDDH.

[0060] See also Figure 4 , heat map analysis of genetic similarity between strain P2-6 and different Marseilles type strains, from Figure 4 The results show that strain P2-6 and Massilia alkalitolerans DSM17462 T The similarity is the highest, with an ANI value of 95.59% and a dDDH value of 77.10%.

[0061] 5.2 Morphological identification

[0062] See also Figure 1 The colony characteristics of strain P2-6 on nutrient agar plates are yellow, darker in the middle, lighter at the edges, round, with neat, smooth edges, and moist and sticky texture.

[0063] See also Figure 2 The strain P2-6 bacteria appeared rod-shaped under the oil objective lens of an ordinary optical microscope (100×).

[0064] 5.3 Physiological and biochemical identification

[0065] API 20NE bacterial biochemical identification reagent strips were used to perform physiological and biochemical identification of strain P2-6.

[0066] The results of physiological and biochemical identification showed that strain P2-6 could utilize nitrate, assimilate glucose, ferment maltose and arabinose, but could not ferment mannose, mannitol and glucose, possess cytochrome oxidase and β-galactosidase, tested positive for esculin and malic acid, and tested negative for urea, gelatin, L-tryptophan, arginine, phenylacetic acid, N-acetylglucosamine, potassium gluconate, capric acid, adipic acid and sodium citrate. Except for nitrate reduction, glucose fermentation, urea and other physiological and biochemical characteristics, the references are as follows (the "a" in Table 1 below is from these references: (CHAUDHARY, DK, KIM J "Massiliaagri sp.nov., isolated from reclaimed grassland soil." International journal of systematic and evolutionary microbiology 67.8 (2017): 2696-2703. WEON, HANG-YEON, et al. "Massiliajejuensis sp.nov. and Naxibacter suwonensis sp.nov., isolated from air samples." International journal of systematic and evolutionarymicrobiology 60.8 (2010): 1938-1943. Du Y, Yu X, Wang G ."Massilia tieshanensis sp.nov.,isolated from mining soil."Internationaljournal ofsystematic andevolutionary microbiology 62.Pt_10(2012):2356-2362.ORTHOVA,IVANA,et al."Massilia norwichensis sp.nov.,isolated from an air sample."InternationalJournal ofSystematic and Evolutionary Microbiology 65.Pt_1(2015):56-64.LEONARDO JIBLR,Olivares MCFL,HartmannM S A.35The FamilyOxalobacteraceae[J].2014.), except that the physiological and biochemical characteristics of the strain are different from those of the alkali-resistant Massilia tieshanensis reported by the National Natural Science Foundation of China.

[0067] The physiological and biochemical identification results of strain P2-6 are shown in Table 1.

[0068] Table 1

[0069]

[0070] In Table 1, “+” indicates positive; “-” indicates negative; “±” indicates positive or negative; and “ND” indicates no record in the literature.

[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 P2-6 isolated and obtained in the present invention is Massilia alkalitolerans.

[0072] Performance data

[0073] 1. In vitro antibacterial effect of strain P2-6 on pathogenic bacteria in aquaculture water

[0074] Using Aeromonas vernix as the indicator pathogenic bacteria, the in vitro antibacterial effect of strain P2-6 on pathogenic bacteria in aquaculture water was detected and analyzed.

[0075] The in vitro antibacterial effect analysis of strain P2-6 on Aeromonas vickers HXH1 in aquaculture water was carried out in a glass conical flask.

[0076] According to the above method, the concentration of 1.2×10 9 CFU / mL Aeromonas vickers bacterial suspension and 1.0×10 9 , 1.0×10 8 , 1.0×10 7 CFU / mL bacterial suspension of strain P2-6.

[0077] Take 1mL 1.2×10 9 CFU / mL Aeromonas welchii suspension was added into a sterile conical flask, and 1 mL of 1.0×10 9 , 1.0×10 8 , 1.0×10 7 CFU / mL bacterial suspension of strain P2-6 was finally dilute to 200 mL with sterilized filtered aquaculture water to make the final concentration of Aeromonas wilkeri 6.0×10 6 CFU / mL, and the final concentrations of strains P2-6 were 5.0×10 6 , 5.0×10 5 , 5.0×10 4 CFU / mL. Filtered aquaculture water supplemented with only Aeromonas vickers HXH1 under the same conditions served as a control. Each treatment was replicated three times. Each group was shaken at 30°C and 180 rpm, and the concentration of Aeromonas vickers HXH1 in the aquaculture water (CFU / mL) was measured after four days. The concentration of Aeromonas vickers HXH1 was determined using the dilution-coating TCBS plate method. The inhibition rate was calculated according to formula (1).

[0078] The concentration of Aeromonas victoriae in the control group-the concentration of Aeromonas victoriae in the treatment group

[0079]

[0080] Results see Figure 7 , strain P2-6 (the initial concentrations of the three groups were 5.0×10 4 , 5.0×10 5 , 5.0×10 6 CFU / mL) after treating Aeromonas vernix for 4 days, the concentration of Aeromonas vernix was 8.0×10 5 CFU / mL, 6.0×10 5 CFU / mL, 5.0×10 4 CFU / mL was significantly lower than that of the control group (7.0×10 6 CFU / mL)(P<0.05).

[0081] The above inhibition rate formula was used to calculate the three groups of strains P2-6 (the initial concentrations of the three groups were 5.0×104 , 5.0×10 5 , 5.0×10 6 CFU / mL) against Aeromonas welchii reached 86.57%, 91.43% and 99.29% respectively.

[0082] Figure 7 The results indicate that strain P2-6 has a significant inhibitory effect on the proliferation of Aeromonas vernix in aquaculture water. Strain P2-6 can be used for ecological control of Aeromonas vernix in aquaculture water and for inhibiting pathogenic bacteria in aquaculture water. Strain P2-6 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.

[0083] 2. Effects of strain P2-6 on immunity and antioxidant capacity of aquatic animals

[0084] Using river crab as a representative farmed animal, the effects of strain P2-6 on the immunity and antioxidant capacity of aquatic animals were detected and analyzed.

[0085] 1) Breeding experiment

[0086] Before the experiment, the final concentration of 6.0×10 9 CFU / mL bacterial suspension of strain P2-6 was prepared, and then diluted with sterile distilled water. The dilutions of different concentrations were added to the basic feed to prepare the final concentration (the final concentration of strain P2-6) of 6.0×10 5 CFU / g feed, 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 four experimental groups, each containing three parallel aquariums. Tiles were placed in each aquarium to prevent crab aggression. 120L of aerated tap water and 50 crabs were also added. The experimental groups were fed the four experimental diets daily, while the control group was fed a basal diet for 20 consecutive days, twice daily (8:00 and 18:00). During the experiment, each aquarium was continuously aerated, the water temperature was set at 28°C, and the light-dark cycle was 12h:12h. The feed intake was 3% of the crab's total body weight. Following the aquaculture experiment, the nonspecific immunity and antioxidant capacity of each group of crabs were immediately measured.

[0087] 2) Determination of nonspecific immunity and antioxidant capacity

[0088] Immediately after the above-mentioned breeding experiment, 5 crabs were randomly selected from each aquarium, and their hemolymph and hepatopancreas samples were collected to analyze the relevant indicators of antioxidant capacity and nonspecific immune capacity.

[0089] Specific processing procedures: (I) Blood was drawn from the root of the third abdominal foot of the test crab and collected in a 1 mL sterile centrifuge tube containing an equal amount of sterile anticoagulant (acid citrate dextrose ACD). The blood was centrifuged at 4000 r / min at 4°C for 20 min. The supernatant was collected and stored at -80°C for later use. The activities of nonspecific immune enzymes such as AKP, ACP, and LZM and antioxidant immune enzymes such as SOD and CAT were determined according to the instructions of each detection kit. (II) The hepatopancreas was isolated from each test crab and washed in sterile saline. The hepatopancreas and pre-cooled sterile saline were mixed at a mass to volume ratio of 1:9 and homogenized for 30 s using a homogenizer to obtain a homogenate. The homogenate was centrifuged at 2500 r / min at 4°C for 10 min, and the supernatant was collected. The activities of nonspecific immune enzymes such as AKP, ACP, and LZM and antioxidant immune enzymes such as SOD and CAT were determined according to the instructions of each detection kit.

[0090] 3) Effect data of strain P2-6 on the nonspecific immunity and antioxidant capacity of river crabs

[0091] The effects of adding strain P2-6 to the feed on the activities of AKP, ACP, SOD, LZM, and CAT in the serum of river crabs are shown in Table 2.

[0092] Table 2

[0093]

[0094] Note: CON group was fed with basic feed, T1 group was fed with 6.0×10 5 CFU / g feed, T2 group was fed 6.0×10 6 CFU / g feed, T3 feeding 6.0×10 7 CFU / g feed, T4 feeding 6.0×10 8 CFU / g feed. Different letters in the same row indicate significant differences (P<0.05), and the same letters indicate no significant differences (P>0.05).

[0095] The effects of strain P2-6 on the activities of AKP, ACP, SOD, LZM, and CAT in the hepatopancreas of Eriocheir sinensis are shown in Table 3.

[0096] Table 3

[0097]

[0098] Note: CON group was fed with basic feed, T1 group was fed with 6.0×10 5CFU / g feed, T2 group was fed 6.0×10 6 CFU / g feed, T3 feeding 6.0×10 7 CFU / g feed, T4 feeding 6.0×10 8 CFU / g feed. Different letters in the same row indicate significant differences (P<0.05), and the same letters indicate no significant differences (P>0.05).

[0099] Table 2 shows that compared with the control crabs, the 5 ~6.0×10 8 The activities of AKP, ACP, SOD, LZM and CAT in the serum of river crabs fed with strain P2-6 at CFU / g feed increased by 28.57% (P<0.05)~85.71% (P<0.05), 33.33% (P>0.05)~100.00% (P<0.05), 11.14% (P<0.05)~15.04% (P<0.05), 12.14% (P>0.05)~22.47% (P>0.05) and 22.28% (P>0.05)~37.13% (P>0.05), respectively.

[0100] Table 3 shows that compared with the control crabs, the 5 ~6.0×10 8 The activities of AKP, ACP, SOD, LZM and CAT in the hepatopancreas of river crabs fed with strain P2-6 at CFU / g feed increased by 13.33% (P<0.05) to 33.33% (P<0.05), 9.09% (P<0.05) to 36.36% (P<0.05), 4.11% (P>0.05) to 13.70% (P>0.05), 46.81% (P<0.05) to 248.94% (P<0.05) and 18.18% (P>0.05) to 54.55% (P>0.05), respectively.

[0101] The above results show that strain P2-6 can significantly improve the nonspecific immunity and antioxidant capacity of river crabs.

[0102] 3. Effects of strain P2-6 on the resistance of aquatic animals to disease infection

[0103] 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 P2-6 to resist disease infection in aquatic animals was tested and analyzed.

[0104] 1) Breeding experiment

[0105] Same as the breeding experiment in the second part above.

[0106] 2) Determination of anti-disease infection ability

[0107] The ability of river crabs to resist hepatopancreatic necrosis syndrome caused by Aeromonas vermiformis HXH1 was used as an analysis of their resistance to disease infection.

[0108] Before the experiment, a final concentration of 5.0×10 9 CFU / mL Aeromonas vickers HXH1 bacterial suspension. The obtained Aeromonas vickers HXH1 bacterial suspension was diluted with sterile distilled water and added to the basal feed to prepare a final concentration of 5.0×10 8 CFU / g feed containing poison bait containing Aeromonas virensii HXH1. Immediately after the aquaculture experiment, uniformly sized, healthy, and disease-free river crabs were randomly selected from each aquarium for artificial infection. A total of 1 control group and 5 experimental groups were set up, with 3 parallel aquariums in each group. Each aquarium contained 120L of aerated tap water and 10 uniformly sized river crabs from the control group and the experimental group after the aquaculture experiment. Each group of river crabs was fed with poison bait containing Aeromonas virensii HXH1 for artificial infection. Feeding was done twice a day (8:00 and 18:00). The morbidity and mortality of the river crabs were observed for 10 consecutive days, 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℃, the light-dark cycle was 12h:12h, and the feeding amount was 3% of the total crab weight.

[0109]

[0110] 3) Results of the protection rate of strain P2-6 against disease infection of river crabs

[0111] The protection rate of strain P2-6 against hepatopancreatic necrosis syndrome in river crabs by adding it to feed is shown in Table 4 below.

[0112] Table 4

[0113]

[0114] Different letters in the same column indicate significant differences (P<0.05), and the same letters indicate no significant differences (P>0.05).

[0115] Table 4 shows that after oral infection with Aeromonas vermiformis, crabs fed with basal feed suffered a large number of deaths, with a mortality rate of 83.33%. 5 , 6.0×10 6 , 6.0×10 7 , 6.0×10 8The mortality of crabs fed with the experimental feed containing 6.0×10-1 strain P2-6 was significantly lower, namely 40.00%, 23.33%, 13.33% and 0.00%, respectively, which were 43.33% (P<0.05), 60.00% (P<0.05), 70.00% (P<0.05) and 83.33% (P<0.05) lower than those of crabs fed with the basic feed. 5 , 6.0×10 6 , 6.0×10 7 , 6.0×10 8 The protection rates of strain P2-6 in preventing hepatopancreatic necrosis syndrome in river crabs were 52.00%, 72.00%, 84.00% and 100% respectively.

[0116] Therefore, strain P2-6 can be used for ecological prevention and control of hepatopancreatic necrosis syndrome in river crabs.

[0117] Experimental data and processing

[0118] 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.

[0119] 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.

[0120] 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. An alkali-resistant Marseille bacteria P2-6, wherein The alkali-resistant Massilia alkalitolerans P2-6 is deposited in the Guangdong Provincial Microbial Culture Collection Center with the deposit number GDMCC No: 66024 and the deposit date of March 17, 2025.

2. A microbial agent, characterized in that: The microbial agent comprises the alkali-resistant Marseille bacteria P2-6 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 alkali-resistant Marseille bacteria P2-6 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 alkali-resistant Marseille bacteria P2-6 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 Aeromonas vermiformis, Aeromonas hydrophila, Aeromonas caviae, Pseudomonas aeruginosa, Shewanella algae or Photobacterium mermanii.

6. A product for inhibiting pathogenic bacteria in aquaculture water, characterized by: The product comprises the alkali-resistant Marseille bacteria P2-6 as claimed in claim 1 or the microbial agent as claimed in claim 2 or 3.

7. Use of the alkali-resistant Marseillais P2-6 according to claim 1 or the microbial agent according to 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 an Eriocheir sinensis; preferably, use of the alkali-resistant Marseillais P2-6 according to claim 1 or the microbial agent according to any one of claims 2-3 in increasing the activity of acid phosphatase, alkaline phosphatase, superoxide dismutase, lysozyme and catalase in the serum and / or hepatopancreas of Eriocheir sinensis.

8. An aquaculture feed, characterized by: The aquaculture feed comprises a feed base component and the alkali-resistant Marseillais P2-6 according to claim 1; preferably, the aquaculture feed is a crab farming feed; preferably, the alkali-resistant Marseillais P2-6 is added to the aquaculture feed at a final dosage of 6.0×10 5 CFU / g feed and above.

9. Use of the alkali-resistant Marseille bacteria P2-6 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 alkali-resistant Marseille bacteria P2-6 according to claim 1 or the microbial agent according to claim 2 or 3.

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