Broad-spectrum bacteriostatic pseudomonas aeruginosa Jcl-3 and application thereof

Abstract

The invention discloses a broad-spectrum bacteriostatic pseudomonas aeruginosa Jcl-3 and application thereof, the broad-spectrum bacteriostatic pseudomonas aeruginosa Jcl-3 is preserved in the China Center for Type Microbiological Culture Collection, the preservation number is CCTCC NO: M 2024211, and the preservation time is January 25, 2024. A broad-spectrum antibacterial strain Jcl-3 is obtained through separation and screening, and the pseudomonas aeruginosa Jcl-3 provided by the invention has an obvious inhibition effect on aquatic disease bacteria and can be prepared into an aquatic attenuated vaccine, so that diseases of aquatic animals are reduced, and the health state is improved. Meanwhile, the strain has an obvious inhibition effect on hypha growth of plant pathogenic fungi, also has a very good antibacterial effect on staphylococcus aureus and escherichia coli, has a broad-spectrum antibacterial effect, and effectively solves the problem that various pathogenic bacteria are harmful to the environment, crops and human health.

Description

Technical Field

[0001] The present invention relates to the field of microbial antibacterial activity, and specifically relates to a broad-spectrum antibacterial strain of Pseudomonas aeruginosa Jcl-3 and applications thereof. Background Art

[0002] As an important food production industry in the world, aquaculture provides humans with a rich source of protein and plays a key role in ensuring food security and promoting economic development. In recent years, with the continuous growth of people's demand for aquatic products, the scale of aquaculture has continued to expand, and intensive and high-density farming has become the mainstream. However, the deterioration of the farming environment and the increase in farming density have led to the frequent occurrence of various diseases. Various aquatic pathogens, such as Aeromonas vermiformis, can cause diseases such as enteritis, ulcers and bleeding in fish; Vibrio cholerae can be transmitted through water or feed. Infected fish show symptoms such as oligoactivity, loss of appetite, red and swollen mouth, and diarrhea. Severe infection may lead to fish death.

[0003] For a long time, antibiotics have been widely used in the prevention and control of aquaculture diseases. However, the frequent use of antibiotics will not only make these harmful bacteria resistant, but also cause drug residues in aquaculture water and aquatic products. This not only threatens human health, but also destroys the ecological balance of water bodies. Although traditional inactivated vaccines have certain immune effects, they have problems such as weak immune response and short duration. Inactivated vaccines mainly stimulate the body's humoral immunity, have limited activation effects on cellular immunity, and are difficult to provide comprehensive and lasting immune protection. In addition, the preparation process of inactivated vaccines is complicated and costly, which limits their large-scale application. The production of inactivated vaccines requires strict control of inactivation conditions and requires a large amount of antigen preparation, which increases production costs and time costs.

[0004] As a new type of vaccine, attenuated vaccines have unique advantages. They attenuate the pathogens so that they retain their immunogenicity while significantly reducing their pathogenicity. After vaccination, the attenuated strains can reproduce in a limited manner in the host, simulating the natural infection process, and can effectively stimulate the body's humoral and cellular immune responses, producing more lasting and comprehensive immune protection. Compared with inactivated vaccines, attenuated vaccines require fewer doses and times of vaccination to induce a stronger immune response, and the production cost is relatively low.

[0005] Pseudomonas aeruginosa (P.aeruginosa) belongs to the phylum Proteobacteria. It is a Gram-negative, aerobic, long rod-shaped bacterium with only unidirectional motility. It forms smooth, moist, slightly raised, medium-sized colonies with neat wavy edges on ordinary agar plates. It has a certain degree of transparency and is often in a fused state. After a few days, the green color of the culture medium gradually deepens, and the surface of the colony presents a metallic luster. There is a single flagellum at one end of the bacterial body. Under a dark field microscope or phase contrast microscope, it can be seen that the bacteria are active in movement, have no spores, and can form capsules. It is easily stained with common dyes. The optimal growth temperature is 25-30°C.

[0006] Pseudomonas aeruginosa has simple nutritional requirements and grows fast, which makes its production and fermentation cost low, which is conducive to the development of attenuated vaccines. Pseudomonas aeruginosa has good ecological adaptability and can compete with a variety of microorganisms, which helps control pathogens. Its effect is long-lasting and its competitiveness is strong, showing good application prospects.

[0007] The development of attenuated vaccines for aquaculture against Pseudomonas aeruginosa is of great significance to the sustainable development of aquaculture. On the one hand, it can effectively reduce the use of antibiotics, reduce the risk of drug residues and environmental pollution, and ensure the quality and safety of aquatic products. On the other hand, it can provide efficient immune protection for aquatic animals, reduce the incidence and mortality of diseases, increase aquaculture production and economic benefits, and promote the healthy and stable development of aquaculture. Many studies and practices have shown that the rational use of attenuated vaccines can significantly improve the economic and ecological benefits of aquaculture.

[0008] Alternaria alternata is a fungus that causes leaf spots, rots, wilts, and other plant diseases. The texture of the colonies is usually velvety or wooly. The colonies are flocculent, grow rapidly, and are dark white at first, darkening as they age; the underside is brown. It is an opportunistic pathogen that affects more than 380 plant species. It is the most common mold in arid, warm climates. F. commune is a plant root rot pathogen with a wide geographic distribution and host range. It can cause root rot in crops such as white pine, Douglas fir, carnation, corn, carrot, and barley. F. commune is very similar to F. oxsproum in morphological characteristics. The colonies are white, the aerial fungi are velvety, and they can produce conidia. F. oxsproum is a facultative parasitic fungus that can infect plants and survive in the soil. It belongs to the Ascomycetes, the order Psoraleales, the family Nodules, and the genus Fusarium. The colonies are flocculent, and the hyphae are dense white. The colonies are pinkish white, light pink to fleshy. It can cause wilt in many plants such as melons, Solanaceae, bananas, cotton, and legumes. The above three plant pathogenic fungi are extremely harmful to plants, and the commonly used chemical methods will cause drug resistance, pesticide residues and other disadvantages. Biological control is greener and more environmentally friendly. Pseudomonas aeruginosa has an inhibitory effect on plant pathogenic fungi. For example, Pseudomonas aeruginosa CWD-D is made into a biocontrol agent for the prevention and control of plant diseases caused by these pathogens such as Phytophthora taro, Phytophthora litchii, and Phytophthora capsici. In addition, Pseudomonas aeruginosa KK9a has a highly efficient antagonistic effect on Verticillium dahliae, which can be used for the prevention and control of crop wilt caused by Verticillium dahliae, and has a significant control effect on cotton wilt. However, there are currently no studies on Pseudomonas aeruginosa that can simultaneously inhibit Staphylococcus aureus, Escherichia coli, Alternaria alternata, F. commune, and F. oxsproum. Summary of the invention

[0009] Purpose of the invention: In view of the problems existing in the prior art, the present invention provides a broad-spectrum antibacterial Pseudomonas aeruginosa Jcl-3, which has a broad-spectrum antibacterial property and can antagonize the growth of Gram-positive bacteria, Gram-negative bacteria, plant pathogenic fungi and aquatic disease bacteria, such as Staphylococcus aureus, Escherichia coli, Alternaria alternata, F. commune, F. oxsproum, Vibrio cholerae, and Aeromonas veronii, and can prevent and control aquatic disease bacteria and plant pathogenic fungi.

[0010] The present invention also provides application of the broad-spectrum antibacterial Pseudomonas aeruginosa Jcl-3.

[0011] Technical solution: In order to achieve the above-mentioned purpose, the broad-spectrum antibacterial Pseudomonas aeruginosa Jcl-3 described in the present invention has been deposited in the China Center for Type Microorganism Collection with a deposit number of CCTCCNO: M 2024211 and a deposit date of January 25, 2024.

[0012] The screening method of Pseudomonas aeruginosa Jcl-3 of the present invention comprises the following steps:

[0013] Tissue samples were taken from the intestines of dead California sea bass, placed in sterile physiological saline, and shaken thoroughly to prepare a bacterial suspension. The bacterial suspension was spread on LB solid culture medium using the dilution plate method, and inverted overnight culture was used to obtain the primary screening strain. The primary screening strain obtained by separation and screening was used as the test bacteria, Staphylococcus aureus and Escherichia coli were used as indicator bacteria, and Pseudomonas aeruginosa Jcl-3 with antagonistic effects on Staphylococcus aureus and Escherichia coli was screened out using the Oxford cup method.

[0014] The invention discloses an application of the Pseudomonas aeruginosa Jcl-3 in inhibiting or antagonizing Gram-positive bacteria, Gram-negative bacteria, plant pathogenic fungi and aquatic pathogenic bacteria.

[0015] Among them, the Pseudomonas aeruginosa Jcl-3 is used in inhibiting or antagonizing Staphylococcus aureus, Escherichia coli, Alternaria alternata, F. commune, F. oxsproum, Vibrio cholerae, and Aeromonas veronii.

[0016] The fermentation product or bacterial agent containing Pseudomonas aeruginosa Jcl-3 of the present invention.

[0017] The fermentation product or bacterial agent of Pseudomonas aeruginosa Jcl-3 of the present invention is used in inhibiting or antagonizing Gram-positive bacteria, Gram-negative bacteria, plant pathogenic fungi and aquatic disease bacteria.

[0018] The fermentation product is obtained by inoculating Pseudomonas aeruginosa Jcl-3 into a culture medium for fermentation.

[0019] Wherein, the culture medium comprises: 5-10 g / L tryptone, 5-10 g / L yeast extract, 10-15 g / L sodium chloride, and pH 6.8-7.0.

[0020] Furthermore, the culture medium comprises: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, and pH 6.8-7.0.

[0021] The fermentation conditions are as follows: when the seed liquid reaches the logarithmic phase, fresh fermentation medium is inoculated at a volume ratio of 1-2% to start fermentation, the temperature is controlled to be maintained at 32-37°C during the fermentation process, the shaking speed is 150-180r / min, and the fermentation is carried out for 48-60 hours.

[0022] Preferably, fresh fermentation medium is added at a 2% inoculation rate to start fermentation. During the fermentation process, the temperature is controlled to be maintained at 37° C., the shaking speed is 180 r / min, and the fermentation is carried out for 48 hours.

[0023] Among them, the fermentation product or bacterial agent is used to inhibit or antagonize Staphylococcus aureus, Escherichia coli, Alternaria alternata, F. commune, F. oxsproum, Vibrio cholerae, and Aeromonas veronii.

[0024] The invention discloses an application of the Pseudomonas aeruginosa Jcl-3 in the preparation of attenuated aquatic vaccines and biocontrol agents or antimicrobial agents for preventing and controlling plant pathogenic fungi.

[0025] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0026] The present invention obtains a broad-spectrum antibacterial strain Jcl-3 through separation and screening. The Pseudomonas aeruginosa Jcl-3 provided by the present invention exhibits significant inhibitory activity against a variety of aquatic pathogens. Based on this characteristic, the strain has the potential to be prepared into an aquatic attenuated vaccine. This is of great significance to the sustainable development of the aquaculture industry, as it can reduce the risk of drug residues in the breeding environment and aquatic products, effectively guarantee the quality and safety of aquatic products, and reduce pollution to the surrounding ecological environment. In addition, it provides efficient immune protection for aquatic animals, reduces the incidence and mortality of diseases, increases breeding output and economic benefits, and promotes the healthy and stable development of the aquaculture industry.

[0027] In addition, the Pseudomonas aeruginosa Jcl-3 provided by the present invention has a significant inhibitory effect on the mycelial growth of plant pathogenic fungi, can be made into a biocontrol agent for preventing and treating plant pathogenic fungi, reduces plant diseases, or is used for diseases caused by fungi, improves health status, and is more environmentally friendly. At the same time, the strain also has a good antibacterial effect on Staphylococcus aureus and Escherichia coli. The problem of various pathogenic bacteria causing harm to the environment, crops and human health is effectively solved.

[0028] The strain Jcl-3 of the present invention has excellent antibacterial effects on Staphylococcus aureus, Escherichia coli, Alternaria alternata, F. commune, F. oxsproum, Vibrio cholerae and Aeromonas veronii, can prevent and control aquatic disease bacteria and plant pathogenic fungi, and has a very broad-spectrum antibacterial performance. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The morphological and Gram staining images of Pseudomonas aeruginosa Jcl-3;

[0030] Figure 2 To determine the antibacterial activity of Pseudomonas aeruginosa Jcl-3 against Staphylococcus aureus (left) and Escherichia coli (right) using the Oxford cup method;

[0031] Figure 3 This is the predicted map of secondary metabolites of Pseudomonas aeruginosa Jcl-3;

[0032] Figure 4 This is a site analysis map of the secondary metabolite synthesis gene cluster of Pseudomonas aeruginosa Jcl-3;

[0033] Figure 5 To construct a phylogenetic tree based on the gene sequence of Pseudomonas aeruginosa Jcl-316sDNA fragment;

[0034] Figure 6 The plate confrontation method was used to determine the inhibitory effect of Pseudomonas aeruginosa Jcl-3 on the mycelial growth of Alternaria alternata, F. commune, and F. oxsproum (from left to right);

[0035] Figure 7 To determine the antibacterial activity of Pseudomonas aeruginosa Jcl-3 against Vibrio cholerae (left) and Aeromonas vermifuge (right) using the Oxford cup method;

[0036] Figure 8 This is a high performance liquid chromatography detection diagram of Pseudomonas aeruginosa Jcl-3;

[0037] Fig. 9 This is a graph showing the survival rate of California bass. DETAILED DESCRIPTION

[0038] The present invention is further described below in conjunction with the accompanying drawings and embodiments.

[0039] Unless otherwise specified, the materials and reagents used in the examples of the present invention can be obtained from commercial sources. Experimental methods without specific conditions in the examples are usually carried out under conventional conditions or under conditions recommended by the manufacturer.

[0040] The Staphylococcus aureus and Escherichia coli used are wild-type common strains provided by Huaiyin Institute of Technology, and other wild-type strains can be used.

[0041] Vibrio cholerae and Aeromonas vermifuge are both common wild strains provided by Huaiyin Institute of Technology. Other wild strains may be used.

[0042] Alternaria alternata, F. commune, and F. oxsproum are all wild-type known strains donated by Huaiyin Agricultural Science Research Institute (Huai'an Agricultural Science Research Institute) in Xuhuai District, Jiangsu Province, and provided by Huaiyin Institute of Technology. Other wild-type strains may be used.

[0043] Example 1

[0044] Isolation and screening of Pseudomonas aeruginosa Jcl-3 and determination of its antibacterial activity

[0045] The strain Jcl-3 was isolated from the intestines of dead California seabass. The isolation process was as follows: tissue samples were taken from the intestines of dead California seabass, placed in sterile physiological saline, and fully shaken to make a bacterial suspension. 100uL of the diluted test solution was added dropwise to the LB solid culture medium, spread evenly, and inverted to culture overnight at 37°C. The morphology of individual colonies on the culture medium was observed through aseptic operation, including the color, size, edge of the colony, and surface roughness of the colony, to obtain the primary screening strain. The cultured primary screening strain was used as the test bacteria, and Staphylococcus aureus and Escherichia coli were used as indicator bacteria. The Oxford cup method was used to screen out strains with good antagonistic effects on Staphylococcus aureus and Escherichia coli, and the strain was named Jcl-3. The morphology of individual colonies on the culture medium was observed through aseptic operation. The strain Jcl-3 was round, green, smooth, moist, slightly raised, with neat and wavy edges, a certain degree of transparency, and in a fused state on the LB culture medium, such as Figure 1 shown.

[0046] The Oxford cup method was used to determine the antibacterial activity. The specific steps include: taking single colonies of Staphylococcus aureus and Escherichia coli and inoculating them into LB liquid culture medium, culturing them at 37°C and 180r / min until the logarithmic phase, mixing them evenly with the cooled unsolidified LB solid culture medium and pouring them into a plate. After the culture medium solidifies, aseptically clamp the Oxford cup and place it vertically on the plate. Take a single colony of strain Jcl-3 and inoculate it into LB liquid culture medium, culture it at 37°C and 180r / min for 48h. After centrifugation of the fermentation liquid, take 200μl of the supernatant and pour it into the Oxford cup. Culture it at 37°C for 24h and observe the diameter of the inhibition zone around the Oxford cup. Diameter of inhibition zone / mm = total diameter of inhibition zone / mm - diameter of Oxford cup / mm. The results are as follows: Figure 2 As shown, strain Jcl-3 has a good inhibitory effect on both Staphylococcus aureus and Escherichia coli. In summary, the fermentation broth of strain Jcl-3 has a good inhibitory effect on bacterial pathogens.

[0047] Example 2

[0048] Whole genome sequencing and species identification of Pseudomonas aeruginosa Jcl-3.

[0049] The selected strain Jcl-3 was cultured to the logarithmic growth phase, and its genomic DNA was extracted and sequenced using the PacBio RS II and Illumina HiSeq 4000 platforms. The sequencing results showed that the full length of the sequence of Pseudomonas aeruginosa Jcl-3 was 6418249 bp, the GC content was 66.40%, and it contained 5984 genes. Figure 3 As shown in Figure 2, Pseudomonas aeruginosa Jcl-3 contains 16 secondary metabolite encoding gene clusters, among which Region 6 has 100% similarity with the azetidomonamide A / azetidomonamide B synthesis gene cluster from BGC0002037; Region 9 has 100% similarity with the L-2-amino-4-methoxy-trans-3-butenoic acid synthesis gene cluster from BGC0000287; Region 10 has 100% similarity with the hydrogen The similarity between the synthetic gene cluster of Region 1 and BGC0002489 is 100%; the similarity between Region 1 and the synthetic gene cluster of pseudopaline from BGC0002489 is 100%; the similarity between Region 1 and the synthetic gene cluster of pyochelin from BGC0002678 is 92%; the similarity between Region 13 and the synthetic gene cluster of pyocyanine from BGC0000936 is 57%, while the similarity between Region 8 and the synthetic gene cluster of Pf-5pyoverdine from BGC0000413 is 23%; the similarity between Region 11 and the synthetic gene cluster of lankacidin C from BGC0001100 is 13%; the similarity between Region 15 and the synthetic gene cluster of pyoverdine SMX-1 from strain BGC0002693 is 12%, and the similarity between Region 12 and the synthetic gene cluster of oxalomycin from BGC0001106 is 15%. The similarity between Region 3 and the synthetic gene cluster of MA026 from strain BGC0002379 is 6%, and the similarity between Region 3 and the synthetic gene cluster of MA026 from strain BGC0002379 is 5%, indicating that these gene clusters may synthesize new antibacterial substances. Figure 4 As shown, the functions of other unannotated gene clusters are unknown because no closely related sequences have been found.

[0050] The 16srDNA fragment of Jcl-3 (SEQ ID NO.1) was BLASTed online at the NCBI website, and the results were used to construct a phylogenetic tree. Figure 5As shown, it is most closely related to Pseudomonas aeruginosa FDAARGOS 532. Combined with its characteristics, Jcl-3 was identified as Pseudomonas aeruginosa. It has been deposited in the China Center for Type Microorganism Collection in Wuhan, China on January 25, 2024, and the deposit number is CCTCC NO: M 2024211.

[0051] Example 3

[0052] Identification of the inhibitory effect of Pseudomonas aeruginosa Jcl-3 fermentation broth on the mycelial growth of Alternaria alternata, F. commune, and F. oxsproum

[0053] Preparation of culture medium: The basal culture medium of Pseudomonas aeruginosa Jcl-3 is LB medium, including: 10g tryptone, 5g yeast extract, 10g sodium chloride, 1000ml distilled water, pH 6.8, sterilized at 121℃ for 20min; the basal culture medium of Alternariaalternata, F.commune, and F.oxsproum is PDA medium, including 200g potato, 20g glucose, 20g agar, 1000ml distilled water, sterilized at 121℃ for 20min.

[0054] The antibacterial activity was determined by plate confrontation. The specific steps were as follows: a single colony of Pseudomonas aeruginosa Jcl-3 was placed in an LB liquid test tube culture medium to prepare a seed solution. When the seed solution reached the logarithmic phase, it was inoculated with an LB liquid culture medium at a volume ratio of 2% to start fermentation. During the fermentation process, the temperature was controlled to be maintained at 37°C, the shaking speed was 180 r / min, and the fermentation was performed for 48 hours to obtain the fermentation solution; Alternaria alternata, F. commune, and F. oxsproum, which were grown in PDA culture medium, were inoculated in the center of a PDA plate (diameter 9 cm) (logarithmic phase bacteria, about 0.5 cm in size) and the growth medium was used as the culture medium. 3 ), 1 μL of Pseudomonas aeruginosa Jcl-3 fermentation liquid and sterile water were added at 2.5 cm on both sides of the bacterial block, and cultured in a constant temperature incubator at 28°C. Figure 6 As shown, on the side where the fermentation liquid of Pseudomonas aeruginosa Jcl-3 was added, the mycelial growth of Alternaria alternata, F. commune, and F. oxsproum was poor, while on the side where sterile water was added, there was no effect, indicating that Pseudomonas aeruginosa Jcl-3 has a significant antibacterial effect on Alternaria alternata, F. commune, and F. oxsproum.

[0055] Example 4

[0056] Using aquatic pathogens as indicator bacteria, its broad-spectrum antibacterial effect was tested to expand its potential application range.

[0057] Take a single colony of Vibrio cholerae and Aeromonas vermifuge and inoculate it in LB liquid culture medium. Culture it at 37°C and 180r / min until the logarithmic phase. Mix it evenly with the LB solid culture medium that has been cooled to non-solidified and pour it into a plate. After the culture medium solidifies, aseptically clamp the Oxford cup and place it vertically on the plate. Take a single colony of strain Jcl-3 and inoculate it in LB liquid culture medium. Culture it at 37°C and 180r / min for 48h. After centrifugation of the fermentation liquid, take 200μL of the supernatant and pour it into the Oxford cup. Culture it at 37°C for 24h and observe the diameter of the inhibition zone around the Oxford cup. Diameter of inhibition zone / mm = total diameter of inhibition zone / mm - diameter of Oxford cup / mm. The results are as follows: Figure 7 As shown, strain Jc-3 has a good inhibitory effect on the above aquatic pathogens Vibrio cholerae and Aeromonas veronii.

[0058] Example 5

[0059] Identification of Pseudomonas aeruginosa pyochelin in the culture supernatant of Pseudomonas aeruginosa Jcl-3.

[0060] Take a single colony of Pseudomonas aeruginosa Jcl-3 in LB liquid test tube culture medium to make a seed liquid. When the seed liquid reaches the logarithmic phase, it is inoculated with LB liquid culture medium at a volume ratio of 2% to start fermentation. During the fermentation process, the temperature is controlled to be maintained at 37°C, the shaking speed is 180r / min, and the fermentation is carried out for 72 hours to obtain the fermentation liquid. Repeat the same operation in GGP culture medium (each 1L of distilled water contains 30mL glycerol, 10g peptone, 0.5gK2HPO4 and MgSO4). The fermentation liquid obtained from the two culture media is centrifuged at 8000r / min to take the supernatant, and hydrochloric acid is added to acidify it to pH1.2, and 4°C overnight. The liquid is centrifuged at 10000r / min to take the precipitate, put into an ampoule, add 1mL pure methanol, blow and suck, and 4°C overnight. Centrifuge it again, filter the membrane, and take the supernatant to make 60% methanol.

[0061] The detection conditions of pyochelin for HPLC are as follows: the mobile phase is composed of solvent A (10mM NH4H2PO4) and solvent B (95% methanol in 10mM H3PO4), and is used as follows: 0-29 minutes, 20-83% solvent B; the remaining 2 minutes, 83-100% solvent B. Elution is performed at a flow rate of 0.8mL / min at room temperature, and the UV detection wavelength is 254nm. The HPLC detection effect is as follows Figure 8 shown.

[0062] After the standard solution was injected, under the chromatographic conditions, the retention time of the target component pyochelin was 21.8 and 24.3 min. The pretreated sample of the culture medium LB was subjected to HPLC analysis, and a chromatographic peak appeared at the same retention time as the standard product. By comparing the retention time with the standard product, it was preliminarily determined that the peak was the target component peak. Similarly, the sample of the culture medium GGP was subjected to HPLC analysis, and the target component peak was also detected at the corresponding retention time. The pyochelin content in the culture medium LB was 7.26 mg / L, and the content in the culture medium GGP was 10.13 mg / L, which was higher. The GGP culture medium was an optimized culture medium.

[0063] Example 6

[0064] Immunity experiment of attenuated vaccine strain Jcl-3 of Pseudomonas aeruginosa

[0065] High temperature mutagenesis was used, and the strain suspension was placed in a water bath at a certain temperature. The temperature was set to 60°C and the treatment time was 10 minutes. After treatment, it was quickly cooled and diluted for coating culture. The strain treated with physical mutagenesis was inoculated in LB medium for multiple subcultures. Each subculture was transferred at a 1% inoculation amount, and the next subculture was carried out after culturing to the logarithmic growth phase, and the subculture was repeated 5 times. The strains at different treatment stages were prepared into a certain concentration of bacterial suspension, and the bacterial solution was injected into the fish by intraperitoneal injection. The mortality and symptoms of the fish were observed, and compared with the original strain infection group, the 5th generation strain with significantly weakened virulence was screened out.

[0066] The selected weak strain of Pseudomonas aeruginosa (5th generation) was cultured to the logarithmic growth phase, and then the cells were collected by centrifugation. The cells were resuspended in phosphate buffered saline (PBS), and 1% aluminum gel adjuvant and 2% protective skim milk powder were added to prepare a vaccine preparation. The concentration of the vaccine was 10 7 CFU / mL of weak strains.

[0067] Healthy, uniform-sized California bass were selected and temporarily kept in an aquarium or pond with good water quality, suitable temperature and dissolved oxygen for 2 weeks to adapt to the environment. The fish were randomly divided into 2 groups, a vaccination group and a blank control group, with 30 fish in each group.

[0068] The vaccination group was injected intraperitoneally with a dose of 10 7 CFU / mL attenuated vaccine 0.1mL / fish. The blank control group was injected with the same volume of saline. After immunization, the behavior and feeding of the fish were observed for 21 days.

[0069] The virulent strain of Pseudomonas aeruginosa (original Jcl-3 strain) was injected at a concentration of 10 7CFU / mL, 100 μL was injected into each fish. After the challenge, the fish were transferred back to the original breeding environment and the disease and death of the fish were observed continuously for 14 days, and the number of fish deaths and abnormal behaviors were recorded every day.

[0070] Calculate the survival rate: Calculate the survival rate of each group of fish based on the recorded data. The formula for calculating the survival rate is: Survival rate = (number of surviving fish / initial number of fish in the group) × 100%. See the California bass survival rate for more information. Fig. 9 Comparing the survival rates of the vaccination group and the blank control group, the survival rate of the vaccination group was significantly higher than that of the blank control group, indicating that the vaccine made from Pseudomonas aeruginosa has a certain protective effect against infection by its strong strains.

Claims

1. A broad-spectrum antibacterial strain of Pseudomonas aeruginosa (P. aeruginosa) Jcl-3 has been deposited in the China Center for Type Microorganism Collection with the deposit number CCTCC NO: M 2024211 and the deposit date is January 25, 2024.

2. A use of the Pseudomonas aeruginosa Jcl-3 according to claim 1 in inhibiting or antagonizing Gram-positive bacteria, Gram-negative bacteria, plant pathogenic fungi and aquatic pathogens.

3. The use according to claim 2, characterized in that: The Pseudomonas aeruginosa Jcl-3 is used in inhibiting or antagonizing Staphylococcus aureus, Escherichia coli, Alternaria alternata, F. commune, F. oxsproum, Vibrio cholerae, and Aeromonas veronii.

4. A fermentation product or bacterial agent containing the Pseudomonas aeruginosa Jcl-3 according to claim 1.

5. Use of a fermentation product or bacterial agent containing Pseudomonas aeruginosa Jcl-3 according to claim 1 in inhibiting or antagonizing Gram-positive bacteria, Gram-negative bacteria, plant pathogenic fungi and aquatic disease bacteria.

6. The use according to claim 5, characterized in that: The fermentation product is obtained by inoculating Pseudomonas aeruginosa Jcl-3 into a culture medium for fermentation.

7. The use according to claim 6, characterized in that: The culture medium preferably comprises: 5-10 g / L tryptone, 5-10 g / L yeast extract, 10-15 g / L sodium chloride, and pH 6.8-7.

0.

8. The use according to claim 6, characterized in that: The fermentation conditions are as follows: when the seed liquid reaches the logarithmic phase, fresh fermentation medium is added at a volume ratio of 1-2% to start fermentation, the temperature is controlled to be maintained at 32-37° C., the shaking speed is 150-180 r / min, and the fermentation is carried out for 48-60 hours.

9. The use according to claim 5, characterized in that: The fermentation product or bacterial agent is used for inhibiting or antagonizing Staphylococcus aureus, Escherichia coli, Alternaria alternata, F. commune, F. oxsproum, Vibrio cholerae and Aeromonas veronii.

10. Use of the Pseudomonas aeruginosa Jcl-3 according to claim 1 in the preparation of attenuated aquatic vaccines and biocontrol agents or antimicrobial agents for controlling plant pathogenic fungi.

Images