Heat-resistant bacillus orientalis with phagocytosis activity and application of heat-resistant bacillus orientalis

Abstract

The invention belongs to the technical field of bioengineering, and relates to bacillus orientalis and application thereof in bacteriostasis and disease prevention, in particular to bacillus orientalis P2-1 and a microbial agent thereof, and application of the bacillus orientalis P2-1 and the microbial agent in inhibition of pathogenic bacteria in water and prevention of river crab diseases. The bacillus subtilis has a good phagocytosis effect on pathogenic bacteria such as pseudomonas aeruginosa, shewanella alga, citrobacter freundii, klebsiella pneumoniae and enterobacter glycosides, and can be used for remarkably removing the pathogenic bacteria such as the pseudomonas aeruginosa, the shewanella alga, the citrobacter freundii, the klebsiella pneumoniae and the enterobacter glycosides in a water body; the pseudomonas disease of the river crabs is effectively prevented and controlled, and a good effect is achieved on simultaneously preventing black gill disease, hepatopancreas necrosis disease, water shriveled disease, enteritis disease, edema disease and trembling disease in the culture and production of the river crabs.

Description

Technical Field

[0001] The invention relates to a Bacillus orientalis and application thereof in inhibiting bacteria and preventing diseases, in particular to Bacillus orientalis P2-1 and a microbial agent thereof, and application thereof in inhibiting pathogenic bacteria in water bodies and preventing crab diseases, belonging to the technical field of bioengineering. Background Art

[0002] The presence of potential pathogens in water bodies poses a serious threat to the health of aquatic animals and even humans. For example, Pseudomonas aeruginosa can cause skin ulcer disease in largemouth bass (Yao Qing, et al. Isolation and identification of Pseudomonas aeruginosa in largemouth bass and screening of antimicrobial drugs Jiangsu Agricultural Sciences, 2024), Klebsiella pneumoniae can cause ascites in largemouth bass (Cao Xinyue, et al. Isolation and identification of a largemouth bass-derived ST-873 type Klebsiella pneumoniae and study of its biological characteristics [J]. 2023, 54(3): 836-847), and Shewanella algae can cause ulcer disease in American eel (Wang H, et al. Shewanella algae: an emerging causative agent for ulcer disease in freshwater-farmed American eel Anguilla rostrata [J]. Israeli Journal ofAquaculture-Bamidgeh, 2020, IJA_72.2020.964361.), Citrobacter freundii can cause crayfish tail rot (Liu Zhanghuai, et al. Isolation and identification of the pathogen of crayfish tail rot and analysis of its related characteristics [J]. Fisheries Science, 2022, 41(1): 102-109), and Enterobacter bugandum can cause seahorse enteritis. Therefore, the control of these pathogens in water bodies is extremely important.

[0003] Bacteriophagic probiotics are a type of microorganisms with bacteriophagic properties, and are known as natural biological control factors for pathogenic bacteria. For example, bacteriophagic probiotics such as Bdellovibrio and Bacteroides have been proven to have good effects in aquaculture antibacterial and disease prevention applications (Chen Kangyong, et al. Research progress on the application of Bdellovibrio in aquaculture [J]. Fisheries Science, 2018, 37(2): 283-288. Cao Haipeng, et al. Bacteroides, a type of bacteriophagic probiotic that is seriously underestimated [J]. Contemporary Aquatic Products, 2024, 49(7): 68-69.), but these bacteriophagic probiotics are Gram-negative bacteria with low heat resistance and difficulty in solid fermentation, which increases the production cost of their powders and their survival rate during the high temperature (70-90°C) pelleting process of feed as feed raw materials. Therefore, exploring Gram-positive bacteria with bacteriophagic activity has broader application prospects. Summary of the invention

[0004] In view of the above problems and / or other problems in the related art, the present invention separates and identifies a heat-resistant non-antagonistic Bacillus sp. P2-1 with broad-spectrum phagocytic activity from aquaculture sludge.

[0005] In a first aspect, the present invention provides a thermotolerant non-antagonistic Bacillus toyota P2-1 having bacteriophagic activity, wherein the 16S rRNA sequence thereof is shown in SEQ ID NO:1.

[0006] Among them, Bacillus toyonensis P2-1 is deposited in Guangdong Microbiological Culture Collection Center with the deposit number GDMCC 65407.

[0007] A second aspect of the present invention provides a microbial agent, wherein the microbial agent comprises the above-mentioned Bacillus subtilis P2-1.

[0008] Preferably, the microbial agent comprises the fermentation broth of the aforementioned Bacillus subtilis P2-1, or a bacterial liquid preparation made from the fermentation broth, or a bacterial powder preparation made by drying the fermentation broth.

[0009] The third aspect of the present invention provides the use of the above-mentioned Bacillus subtilis P2-1 or the above-mentioned microbial agent in inhibiting pathogenic bacteria in water and preventing crab diseases.

[0010] Among them, the function of inhibiting pathogenic bacteria in water bodies relies on the phagocytic effect of Bacillus sp. P2-1 to lyse bacteria.

[0011] The pathogenic bacteria include Pseudomonas aeruginosa, Shewanella algae, Citrobacter freundii, Klebsiella pneumoniae, and Enterobacter burgdorferi.

[0012] A fourth aspect of the present invention provides a product having a bacteriophagic effect on pathogenic bacteria, wherein the product comprises the aforementioned Bacillus subtilis P2-1 or the aforementioned microbial agent.

[0013] A fifth aspect of the present invention provides the use of the aforementioned Bacillus subtilis P2-1 or the aforementioned microbial agent in the preparation of a biological control product for preventing and / or treating river crab diseases.

[0014] The river crab diseases include Pseudomonas disease of river crabs, black gill disease, hepatopancreatic necrosis disease, water shriveling disease, enteritis disease, edema disease, and tremor disease.

[0015] The invention relates to a Japanese bacillus, in particular to a screened heat-resistant Japanese bacillus P2-1 with phagocytic activity against pathogenic bacteria; the Japanese bacillus has good phagocytic effects on pathogenic bacteria such as Pseudomonas aeruginosa, Shewanella algae, Citrobacter freundii, Klebsiella pneumoniae, Enterobacter burgandii and the like, can significantly remove pathogenic bacteria such as Pseudomonas aeruginosa, Shewanella algae, Citrobacter freundii, Klebsiella pneumoniae, Enterobacter burgandii and the like in water bodies, effectively prevents and controls Pseudomonas disease in river crabs, and also has good effects on preventing black gill disease, hepatopancreatic necrosis disease, water shriveling disease, enteritis disease, edema disease and tremor disease in river crab breeding and production.

[0016] Preservation certificate: strain Bacillus toyonensis P2-1, received and registered by Guangdong Microbiological Culture Collection Center (GDMCC) on November 4, 2024, and the registration number of the collection center is GDMCC 65407. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 is the colony morphology of strain P2-1;

[0018] Figure 2 The phagocytic activity of strain P2-1 against Pseudomonas aeruginosa FJ2-7, Shewanella algae RZ3-4, Citrobacter freundii HY-6, Klebsiella pneumoniae HY-2, and Enterobacter bugandum H4;

[0019] Figure 3 This is a photo of the plaques formed by strain P2-1 on a double-layer agar plate containing tap water;

[0020] Figure 4 This is a photo of the bacterial morphology of strain P2-1 under an optical microscope after Gram staining;

[0021] Figure 5 This is the phylogenetic tree constructed based on the 16S rRNA gene sequence of strain P2-1;

[0022] Figure 6 This is the inhibition rate of strain P2-1 against Pseudomonas aeruginosa FJ2-7, Shewanella algae RZ3-4, Citrobacter freundii HY-6, Klebsiella pneumoniae HY-2, and Enterobacter bugandii H4 in artificial simulated aquaculture water.

[0023] Figure 7 Schematic diagram of the mortality rate of river crabs treated with different doses of strain P2-1 due to Pseudomonas disease. DETAILED DESCRIPTION

[0024] The present invention is further described below through specific implementation modes, but the present invention is not limited to these specific implementation modes.

[0025] Unless otherwise specified, the materials, reagents, etc. used in the following specific embodiments can be obtained from commercial sources. If no specific technology or conditions are specified, the technology or conditions described in the literature in the field or the product instructions are used.

[0026] Example 1

[0027] 1 Experimental Materials

[0028] Aquaculture sludge was collected from a farm in Shandong; Pseudomonas aeruginosa FJ2-7, Shewanella algae RZ3-4, Citrobacter freundii HY-6, Klebsiella pneumoniae HY-2, and Enterobacter bugandii H4 were provided by the National Aquatic Animal Pathogen Bank of Shanghai Ocean University; River crabs were healthy and free of disease and injury, with an average mass of 13.26±2.34g, and were temporarily raised in the laboratory for 14 days, and were provided by a nursery and breeding base in Sihong, Jiangsu.

[0029] 2 Preparation of pathogenic bacterial suspension

[0030] Each pathogenic strain was inoculated into sterile nutrient broth and cultured at 30°C and 200 r / min on a shaker for 24 h. The culture was then centrifuged at 4°C and 8000 r / min for 20 min. After washing twice with sterile saline, the concentration of each pathogenic bacteria suspension was adjusted to 1.0 × 10 10 CFU / mL, stored in a 4℃ refrigerator for later use.

[0031] 3. Isolation and screening of bacteriophage microorganisms

[0032] Weigh 1.0 g of aquaculture sludge and suspend it in 9 mL of sterile water. After oscillating and mixing, place it in a 100°C water bath for 5 min. Then, the thermotolerant strains were isolated and purified on nutrient agar plates using the dilution plate method. The isolated thermotolerant strain colonies were resuspended in sterile saline to a concentration of 1.0 × 10 6 CFU / mL of bacterial solution, and then the tap water double-layer agar plate method was used to determine the phagocytic activity of the heat-resistant strains against Pseudomonas aeruginosa FJ2-7, Shewanella algae RZ3-4, Citrobacter freundii HY-6, Klebsiella pneumoniae HY-2, and Enterobacter burgandii H4, and the paper disk method was used to analyze the antagonistic activity of the heat-resistant strains with excellent phagocytic activity against Pseudomonas aeruginosa FJ2-7, Shewanella algae RZ3-4, Citrobacter freundii HY-6, Klebsiella pneumoniae HY-2, and Enterobacter burgandii H4. The action mode of the heat-resistant strains with excellent phagocytic activity was determined by observing the production of plaques and antagonistic inhibition zones.

[0033] 4 Identification of bacteriophage microorganisms

[0034] 4.1 Molecular identification

[0035] The heat-resistant strain with excellent phagocytic activity was inoculated into 100 mL sterile nutrient broth, and after shaking culture at 30°C and 200 r / min for 24 hours, it was centrifuged at 4°C and 12000 r / min for 1 minute, the supernatant was discarded, and then the genomic DNA was extracted using the Ezup column genomic DNA extraction kit (bacteria), and then the 16S rRNA was amplified by PCR using the genomic DNA as a template. Among them, the forward primer is 27F: 5′-AGAGTTTGATCCTGGCTCAG-3′ (SEQ ID NO: 2), and the reverse primer is 1492R: 5′-GGTTACCTTGTTACGACTT-3′ (SEQ ID NO: 3); the PCR amplification conditions are: 94°C 3min; 94°C 1min, 60°C 1min, 72°C 1min, for a total of 35 cycles. After sequencing the 16S rRNA gene of the thermotolerant strains with excellent phagocytic activity by Shanghai Maipu Biotechnology Co., Ltd., a phylogenetic tree was constructed using the neighbor-joining method using Mega 5.0 software.

[0036] 4.2 Physiological and biochemical identification

[0037] The Gram-stained heat-resistant strains with excellent phagocytic activity were observed for bacterial morphology using an optical microscope, and then the heat-resistant strains with excellent phagocytic activity were physiologically and biochemically identified with reference to the Manual of Identification of Common Bacterial Systems.

[0038] 5. Analysis of heat resistance of bacteriophage microorganisms

[0039] The concentration was 1.0×10 6 1 mL of bacterial suspension of the heat-resistant strain with excellent phagocytic activity at 100 CFU / mL was inoculated into 49 mL of sterile nutrient broth, treated in a water bath at 80°C for 20 min, 90°C for 10 min, and 100°C for 5 min, respectively, and then cultured in a shaker at 30°C and 200 r / min for 24 h to observe the growth of the heat-resistant strain with excellent phagocytic activity.

[0040] Analysis of the in vitro antibacterial effect of 6 phage microorganisms on pathogenic bacteria in artificial simulated aquaculture water

[0041] Before the experiment, the heat-resistant strain with excellent phagocytic activity was inoculated into a solid fermentation medium (bran: corn flour: starch: peptone: water = 350:150:1:1:300) at a 30% inoculum. After solid fermentation at 30°C for 5 days, it was dried and ultrafinely ground to produce a culture medium with a bacterial content of 6×10 10CFU / g of bacterial powder. The in vitro antibacterial effect analysis experiment of bacteriophagic microorganisms on pathogenic bacteria in artificial simulated aquaculture water was carried out in a glass conical flask. Each pathogen was added to 200mL of filtered aquaculture water treated with high pressure sterilization (121℃, 20min) to a concentration of 5×10 6 CFU / mL, and then immediately add the powder of heat-resistant strains with excellent phagocytic activity at a final concentration of 0.2 mg / L. At the same time, the filtered aquaculture water treated with high pressure sterilization (121°C, 20 min) with only the pathogens added under the same conditions was used as a control. Each treatment was repeated 3 times. The concentration (CFU / mL) of each pathogen in the aquaculture water was measured after the treatment group and the control group were shaken at 30°C and 180 r / min for 5 days. Among them, the concentration of pathogens was determined by the dilution coating TCBS plate method. The inhibition rate was calculated according to formula (1).

[0042]

[0043] Analysis on the control effect of phage microorganisms on Pseudomonas infection in river crabs

[0044] The experiment included 1 control group and 2 experimental groups, with three parallel aquariums in each group. Tiles were placed in each aquarium to prevent crabs from fighting, and 8L of breeding water and 10 crabs were added. Before the experiment, the powder of heat-resistant strains with excellent phagocytic activity was added to the compound feed (purchased from Nantong Zhengda Feed Co., Ltd.) to prepare the test feed containing the powder of heat-resistant strains with excellent phagocytic activity at a final dose of 5g / kg feed and 10g / kg feed. The crabs in the experimental group were fed with the test feed containing 5g / kg feed and 10g / kg feed of heat-resistant strains with excellent phagocytic activity every day, and the control group was fed with blank compound feed for 10 consecutive days, twice a day (8:00 and 15:00). Then, each group of crabs was fed with 2.0×10 8 CFU / mL Pseudomonas aeruginosa feed was used for pseudomonas infection, and the crabs were fed twice a day (8:00 and 15:00). The morbidity and mortality of the crabs were observed for 7 consecutive days, and the mortality rate was calculated. The protection rate was calculated according to formula (2). During the experiment, each aquarium was continuously oxygenated, the water temperature was set at 28°C, the light-dark cycle was 12h:12h, and the feeding amount was 3% of the total crab weight.

[0045]

[0046] Analysis on the application effect of phage microorganisms in disease prevention and control in river crab breeding production

[0047] The experiment set up a control pond and a test pond, and each breeding pond (10 mu / pond) was stocked with 12,000 crab seedlings. The river crabs in the control pond were fed with compound feed (purchased from Nantong Zhengda Feed Co., Ltd.) normally, twice a day (6:00 and 17:00), with a feeding amount of 3% of the total body weight of the crabs, and disinfected with bleaching powder once every half a month; the river crabs in the test pond were fed with test feed containing 10g / kg feed of heat-resistant bacterial strain powder with excellent phagocytic activity, twice a day (6:00 and 17:00), with a feeding amount of 3% of the total body weight of the crabs, and 0.2mg / L of heat-resistant bacterial strain powder with excellent phagocytic activity was sprinkled every 10 days. The experimental period was 120 days. During the experiment, the morbidity and mortality of river crabs in each pond were observed.

[0048] 9 Experimental data and processing

[0049] Data are presented as mean ± standard deviation.

[0050] Example 2 Experimental results

[0051] 1. Isolation and identification of bacteriophage microorganisms

[0052] A thermotolerant bacterium P2-1 with excellent phagocytic activity was isolated and screened, forming round, concave, and neatly edged white colonies on nutrient agar plates ( Figure 1 ). Strain P2-1 has good phagocytic activity against Pseudomonas aeruginosa FJ2-7, Shewanella algae RZ3-4, Citrobacter freundii HY-6, Klebsiella pneumoniae HY-2, and Enterobacter burgdorferi H4 ( Figure 2 ), and can form round, clear, smooth, and neatly edged plaques on a double-layer agar plate containing tap water within 24 hours ( Figure 3 ), but unexpectedly found that strain P2-1, unlike traditional antibacterial Bacillus, could not produce antagonistic inhibition zones against Pseudomonas aeruginosa FJ2-7, Shewanella algae RZ3-4, Citrobacter freundii HY-6, Klebsiella pneumoniae HY-2, and Enterobacter bugandii H4, indicating that strain P2-1 did not produce antagonistic inhibition by producing antibacterial active metabolites, but rather phagocytized by lysing bacteria.

[0053] Strain P2-1 is long rod-shaped and Gram-positive ( Figure 4 ), the 16S rRNA gene sequence is as follows:

[0054]

[0055] The 16S rRNA gene sequence of strain P2-1 was compared with the 16S rRNA gene sequences of known strains in the GenBank database. It was found that the 16S rRNA gene sequences of strain P2-1 and Bacillus toyonensis strains were naturally clustered. Then, the phylogenetic tree of strain P2-1 was constructed by the neighbor-joining method ( Figure 5 ) showed that strain P2-1 was most closely related to Bacillus toyonensis strain Bto853 (GenBank accession number: OQ410456). In addition, strain P2-1 VP reaction was positive, could liquefy gelatin, hydrolyze starch, and grow under anaerobic conditions, but could not utilize citrate, propionate, and nitrate, and could not ferment D-xylose, L-arabinose, and D-mannitol, which basically met the physiological and biochemical characteristics of Bacillus toyonensis. Therefore, strain P2-1 was preliminarily determined to be Bacillus toyonensis.

[0056] 2 Analysis of heat resistance of bacteriophage microorganisms

[0057] Strain P2-1 was treated in a water bath at 80°C for 20 min, 90°C for 10 min, and 100°C for 5 min, and then cultured in a shaker at 30°C and 200 r / min for 24 h. The culture solution was turbid and the strain grew well (as shown in Table 1), indicating that strain P2-1 has good heat resistance.

[0058] Table 1 Growth of strain P2-1 under different heat treatment conditions

[0059] Heat treatment conditions Growth 80℃ water bath treatment for 20min + 90℃ water bath treatment for 10min + 100℃ water bath treatment for 5min +

[0060] Note: + indicates that the culture medium is turbid and the bacteria grow well

[0061] 3 Analysis of the in vitro antibacterial effect of phage microorganisms on pathogenic bacteria in artificial simulated aquaculture water

[0062] The strain P2-1 powder had a good inhibitory effect on the proliferation of Pseudomonas aeruginosa FJ2-7, Shewanella algae RZ3-4, Citrobacter freundii HY-6, Klebsiella pneumoniae HY-2, and Enterobacter burgdorferi H4 in artificial simulated aquaculture water, with the inhibition rates reaching 99.95%, 99.54%, 98.72%, 97.85%, and 95.97%, respectively (e.g. Figure 6 As shown), strain P2-1 can be used for ecological control of pathogenic bacteria such as Pseudomonas aeruginosa, Shewanella algae, Citrobacter freundii, Klebsiella pneumoniae, and Enterobacter burgdorferi in water bodies.

[0063] 4. The effect of phages on the prevention and control of Pseudomonas infection in river crabs

[0064] The crabs fed with blank formula feed all died in large numbers after oral infection with Pseudomonas aeruginosa, with a mortality rate of 100%, while the mortality rate of the crabs fed with strain P2-1 powder containing 5g / kg feed and 10g / kg feed was significantly reduced, which was 30.0% and 13.3%, respectively. It was concluded that the protection rate of strain P2-1 powder containing 5g / kg feed and 10g / kg feed against Pseudomonas disease in crabs reached 70.0% and 86.7%, respectively. Therefore, strain P2-1 can be used for ecological prevention and control of Pseudomonas disease in crabs.

[0065] 5. The application effect of bacteriophage microorganisms in disease prevention and control in river crab farming production

[0066] The river crabs in the control pool without the bacterial powder of strain P2-1 developed black gill disease, hepatopancreatic necrosis, water shrunk disease, enteritis, edema, and tremor disease during the entire breeding cycle, and the total number of crab deaths reached 8306; the river crabs in the test pool using the bacterial powder of strain P2-1 did not develop black gill disease, hepatopancreatic necrosis, water shrunk disease, enteritis, edema, and tremor disease during the entire breeding cycle, and only 9 died due to failed molting during the entire experimental cycle (as shown in Table 2). This shows that strain P2-1 can simultaneously prevent the occurrence of black gill disease, hepatopancreatic necrosis, water shrunk disease, enteritis, edema, and tremor disease in river crab breeding and production.

[0067] Table 2 Effect of strain P2-1 on disease prevention and control in river crab farming

[0068]

[0069] Note: + indicates disease occurrence, - indicates no disease occurrence

[0070] 6 Conclusion

[0071] 6.1 The present invention isolates and identifies a thermotolerant Bacillus japonensis P2-1 with broad-spectrum phagocytic properties, which can simultaneously exert phagocytic activity against pathogenic bacteria such as Pseudomonas aeruginosa, Shewanella algae, Citrobacter freundii, Klebsiella pneumoniae, and Enterobacter burgdorferi.

[0072] 6.2 The antibacterial mode of Bacillus subtilis P2-1 is different from the antagonistic mode of traditional antibacterial Bacillus. It cannot secrete metabolites with antibacterial activity, but instead phagocytose bacteria by lysing them.

[0073] 6.3 Bacillus subtilis P2-1 can be used for the ecological control of pathogenic bacteria such as Pseudomonas aeruginosa, Shewanella algae, Citrobacter freundii, Klebsiella pneumoniae, and Enterobacter burgdorferi in water bodies, and can also be used for the ecological prevention and control of Pseudomonas disease in river crabs.

[0074] 6.4 Bacillus subtilis P2-1 can simultaneously prevent the occurrence of black gill disease, hepatopancreatic necrosis, water shriveling disease, enteritis, edema and tremor disease in river crab farming.

[0075] Those skilled in the art can obtain the fermentation broth of Bacillus subtilis P2-1 or the bacterial broth preparation prepared from the fermentation broth or the bacterial powder preparation prepared by drying the fermentation broth according to conventional microbial industrial production methods.

[0076] It should be understood that although this specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description 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 mode may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

[0077] 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 broad-spectrum bacteriophagic Bacillus sp. P2-1, whose 16S rRNA sequence is shown in SEQ ID NO:

1.

2. The Toyo Bacillus P2-1 according to claim 1, characterized in that The Bacillus toyota P2-1 is deposited in Guangdong Microbiological Culture Collection Center with the deposit number of GDMCC 65407.

3. A microbial agent, characterized in that: The microbial agent comprises the Bacillus sp. P2-1 according to claim 1 or 2.

4. The microbial agent according to claim 3, characterized in that: The microbial agent comprises the fermentation liquid of Bacillus toyota P2-1 as claimed in claim 1 or 2, or a liquid preparation made from the fermentation liquid, or a powder preparation made by drying the fermentation liquid.

5. Use of the Bacillus subtilis P2-1 as claimed in claim 1 or the microbial agent as claimed in claim 3 in inhibiting pathogenic bacteria in water and preventing crab diseases.

6. The use according to claim 5, characterized in that The method of inhibiting pathogenic bacteria in water is the phagocytic effect of lysing bacteria.

7. The use according to claim 5, characterized in that: The pathogenic bacteria include Pseudomonas aeruginosa, Shewanella algae, Citrobacter freundii, Klebsiella pneumoniae, and Enterobacter burgdorferi.

8. A product having a bacteriophagic effect on pathogenic bacteria, characterized in that: The product comprises the Bacillus subtilis P2-1 as claimed in claim 1 or the microbial agent as claimed in claim 3.

9. Use of the Bacillus subtilis P2-1 according to claim 1 or the microbial agent according to claim 3 in the preparation of a biological control product for preventing and / or treating crab diseases.

10. The use according to claim 9, characterized in that The river crab diseases include Pseudomonas disease of river crabs, black gill disease, hepatopancreatic necrosis disease, water shriveling disease, enteritis disease, edema disease, and tremor disease.

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