Pseudomonas sp. 94-6Y and bacterial agent and application thereof

Abstract

The application discloses a Pseudomonas australis 94-6Y strain, a bacterial agent and application thereof, and belongs to the technical field of microorganisms. Fictibacillus macauensis The Pseudomonas australis 94-6Y strain is classified and named as Pseudomonas australis, the strain number is 94-6Y, the preservation number is CGMCC No.32718, the strain is a gram-positive strain, the colony is milky white, nearly round, opaque, the surface is slightly rough, and has no luster. The Pseudomonas australis 94-6Y strain can significantly inhibit the water production breeding pathogenic bacteria Vibrio vulnificus, Vibrio splendidus and Vibrio alginolyticus; meanwhile, it is proved that the Pseudomonas australis 94-6Y strain has the ability of efficiently reducing ammonia nitrogen, nitrite nitrogen and chemical oxygen demand in breeding water, is helpful to reduce the breeding disease risk coefficient, improves the breeding water environment, and has important significance for strengthening the research and development of microecological preparation products and popularization of the ecological healthy breeding mode.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a strain of *Pseudomonas macranthae* 94-6Y, its inoculum, and its applications. Background Technology

[0002] Vibrio pathogens are a highly dangerous class of pathogens in aquaculture, with common species including Vibrio vulnificus, Vibrio splenti, Vibrio alginolyticus, Vibrio parahaemolyticus, and Vibrio harveyi. They can infect various farmed species such as fish, shrimp, sea cucumbers, and shellfish, causing outbreaks with high mortality rates and severely impacting aquaculture efficiency. Currently, domestic aquaculture practices still heavily rely on antibiotics and other chemical drugs to control vibrio-related aquatic diseases. However, the excessive use of antibiotics easily leads to drug-resistant strains, environmental damage, suppression of the immune systems of farmed species, and even drug residues, seriously threatening food safety and public health.

[0003] Ammonia nitrogen, nitrite, and chemical oxygen demand (COD) are key water quality indicators in aquaculture. In high-density aquaculture, these indicators are prone to being too high, directly causing serious consequences such as reduced dissolved oxygen in the water, poor feed intake of farmed animals, slowed growth, chronic or acute hypoxia, poisoning, and even mass mortality.

[0004] Microecological preparations, composed of beneficial microorganisms and their metabolites, reduce the frequency of disease outbreaks by inhibiting the growth of pathogens and regulating the microbial population structure in aquatic environments, thus promoting the balanced development of aquatic ecosystems. Simultaneously, beneficial microorganisms can effectively remove harmful components such as ammonia nitrogen, nitrite, and phosphate, and decompose excess feed and residue, thereby purifying water quality and improving the aquaculture environment. These advantages have led to the increasingly widespread application of microecological preparations in aquaculture. Therefore, identifying beneficial strains suitable for aquaculture and strengthening the research and development of microecological preparations are of great significance for promoting ecologically healthy aquaculture models and achieving a win-win situation for both economic and ecological benefits. Summary of the Invention

[0005] This invention provides a strain of *Pseudomonas macranthae* 94-6Y, its inoculum, and its application. The *Pseudomonas macranthae* can inhibit aquaculture pathogens such as *Vibrio vulnificus*, *Vibrio brilliantus*, and *Vibrio alginolyticus*, and reduce ammonia nitrogen, nitrite nitrogen, and COD in aquaculture water, thereby helping to reduce aquaculture diseases and improve the aquaculture water environment.

[0006] To achieve the above-mentioned objectives, the present invention is implemented through the following technical solution:

[0007] This invention provides a strain of *Pseudomonas macranthae* 94-6Y, which is classified and named *Pseudomonas macranthae*. Fictibacillus macauensis The accession number is CGMCC No. 32718.

[0008] Furthermore, the *Pseudomonas Macaoensis* 94-6Y is a Gram-positive strain with milky-white, nearly round, opaque colonies that are slightly rough and dull.

[0009] Furthermore, the 16S rDNA sequence of the *Pseudomonas Macaoensis* 94-6Y is shown in SEQ ID No. 1.

[0010] Furthermore, the optimal growth temperature for *Pseudomonas Macaoensis* 94-6Y is 15-37℃, and the salt concentration tolerance is 0%-5%.

[0011] The present invention also provides a microbial agent prepared by fermentation of the aforementioned *Pseudomonas macranthae* 94-6Y.

[0012] Furthermore, the microbial agent includes microbial powder, and the microbial content of the microbial powder is 5 × 10⁻⁶. 10 -10×10 10 CFU / g.

[0013] The present invention also provides the application of *Pseudomonas macranthae* 94-6Y or its agent in the preparation of formulations for inhibiting pathogens in aquaculture.

[0014] Furthermore, the pathogen is one or more of Vibrio vulnificus, Vibrio brilliantus, and Vibrio alginolyticus.

[0015] This invention also provides the application of *Pseudomonas macranthae* 94-6Y or its agent in improving water quality indicators in aquaculture.

[0016] Furthermore, the improvement of water quality indicators in aquaculture includes significantly reducing the content of ammonia nitrogen, nitrite, and COD in the aquaculture system.

[0017] Furthermore, the concentration of *Pseudomonas macranthae* 94-6Y or the inoculant is 0.005%-0.1% of the total volume of the aquaculture system, and the treatment period is 1-5 days.

[0018] Furthermore, the concentration of *Pseudomonas Macaoensis* 94-6Y is 0.01%, the treatment period is 4 days, the removal rate of ammonia nitrogen is 80%-85%, the removal rate of nitrite is 55%-65%, and the removal rate of COD is 70%-80%.

[0019] Furthermore, aquatic animals in aquaculture include bullfrogs and sea cucumber seedlings.

[0020] Furthermore, the sea cucumber seedlings have a body length of 0.5-2 cm, and the microbial agent is an animal feed additive with an effective live bacteria count of 0.5 × 10⁻⁶. 4 CFU / g-3×10 4The animal feed additive is administered at a rate of 1-10 g / day per 100 sea cucumber seedlings (CFU / g).

[0021] Compared with the prior art, the present invention has the following advantages and technical effects:

[0022] 1. This invention provides a strain of *Pseudomonas macranthae* 94-6Y, which has a suitable growth temperature of 37℃ and a salt concentration tolerance of 0-5%. Strain 94-6Y can adapt to both freshwater and common seawater environments and can be used in both freshwater and seawater aquaculture.

[0023] 2. The *Pseudomonas Macaoensis* 94-6Y strain can significantly inhibit aquaculture pathogens such as *Vibrio vulnificus*, *Vibrio splenti*, and *Vibrio alginolyticus*, further broadening the application of the strain in aquaculture.

[0024] 3. The *Pseudomonas macranthae* 94-6Y provided by this invention has the ability to efficiently reduce ammonia nitrogen, nitrite nitrogen, and COD (chemical oxygen demand) in aquaculture water. In aquaculture, when the concentration of the bacterial agent is 0.01%, the ammonia nitrogen removal rate is 84.03%, the nitrite removal rate is 60.38%, and the COD removal rate is 77.34% on the 4th day of treatment. The *Pseudomonas macranthae* 94-6Y has the advantages of short action time and high nitrogen removal rate in aquaculture systems.

[0025] 4. The *Pseudomonas macranthae* 94-6Y provided by this invention, overall, helps reduce the risk of aquatic diseases and improve the aquatic environment for aquaculture. It is of great significance for strengthening the research and development of microecological preparations and promoting ecological and healthy aquaculture models. Attached Figure Description

[0026] Figure 1 Colony characteristics of *Pseudomonas Macaoensis* 94-6Y on LB solid medium.

[0027] Figure 2 Phylogenetic tree of Pseudomonas Macaoense 94-6Y.

[0028] Figure 3 The growth curves of *Pseudomonas macrantha* 94-6Y at different temperatures are shown.

[0029] Figure 4 OD of *Pseudomonas macrantha* 94-6Y after culturing at different salinities for 24 h 600 Numerical value.

[0030] Figure 5 The antibacterial effect of *Pseudomonas macranthae* 94-6Y on *Vibrio vulnificus*.

[0031] Figure 6 The antibacterial effect of *Pseudomonas macrantha* 94-6Y on *Vibrio splenum*.

[0032] Figure 7 The antibacterial effect of *Pseudomonas macrantha* 94-6Y on *Vibrio alginolyticus*.

[0033] Figure 8 The effect of Bacillus pseudosporidis Macao 94-6Y on degrading ammonia nitrogen in aquaculture water samples.

[0034] Figure 9 The effect of Bacillus pseudosporidis Macao 94-6Y on degrading nitrite in aquaculture water samples.

[0035] Figure 10 The effect of Bacillus pseudosporidis Macao 94-6Y on reducing COD in aquaculture water samples.

[0036] Figure 11 The effect of *Pseudomonas macranthae* 94-6Y on ammonia nitrogen content in sea cucumber farming water.

[0037] Figure 12 The effect of *Pseudomonas macranthae* 94-6Y on nitrite content in sea cucumber farming water.

[0038] Figure 13 The effect of *Pseudomonas Macaoensis* 94-6Y on Vibrio counts in sea cucumber aquaculture water. Detailed Implementation

[0039] The present invention will now be described in further detail with reference to specific accompanying drawings and embodiments. These descriptions are intended to explain the invention and not limit it. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments were all purchased from conventional biochemical reagent stores. All quantitative experiments in the following embodiments were performed in at least three replicates, and the results were averaged.

[0040] Example 1: Source, morphology, identification and preservation of the strain

[0041] 1. Strain source and screening: The bottom water sample collected from a mandarin fish farm in Qingdao was used as the isolation sample. The samples were serially diluted and plated on LB medium with appropriate gradients. After incubation at 28°C for 2 days, a single colony was picked and streaked for purification to obtain strain 96-4Y.

[0042] 2. Strain identification

[0043] (1) Morphological identification: After the strain 94-6Y was cultured on LB plate at 28℃ for 2 days, the colonies were milky white, nearly round, opaque, with a slightly rough and dull surface.

[0044] (2) Gene identification: DNA was extracted from strain 94-6Y and its 16S rDNA sequence was amplified. Universal bacterial primers 27F and 1492R were used as primers for PCR amplification of the extracted DNA. The PCR reaction system consisted of 2 μL DNA template, 2 μL each primer, and 44 μL PCR SuperMix. The PCR amplification program was: 94℃ pre-denaturation for 5 min; 94℃ for 1 min, 55℃ for 1 min, and 72℃ for 2 min, for a total of 30 cycles. Sequencing alignment was performed, and a phylogenetic tree was constructed using the Neighbor-Jionig method. The results are shown below. Figure 2 As shown, it was identified as a strain of *Pseudomonas macrantha*, with the taxonomic name [missing information]. Fictibacillus macauensis .

[0045] 3. Strain preservation:

[0046] The selected strain of *Pseudomonas macranthae* 94-6Y was deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, on November 20, 2024. Fictibacillus macauensis The accession number is CGMCC No. 32718.

[0047] Example 2: Investigation of growth conditions of *Pseudomonas macranthae* 94-6Y

[0048] 1. Determination of growth curves of strain 94-6Y at different temperatures

[0049] Strain strain 94-6Y was inoculated into LB liquid medium, and the inoculation amount was controlled to achieve the initial OD. 600 The values ​​were 0.1, and the cells were incubated in shakers at 15℃, 28℃, and 37℃ with a shaker speed of 180 rpm. Three replicates were set up for each temperature, and the OD was measured every 2 h. 600 Numerical values ​​are recorded and growth curves are plotted.

[0050] The results show that ( Figure 3 The strain 94-6Y can grow at three temperatures: 15℃, 28℃ and 37℃, indicating that it can adapt to common aquaculture temperatures in both the north and south. At 37℃, the strain 94-6Y reaches the stable phase first, and its growth rate is significantly faster than at 28℃ and 15℃. Subsequent cultivation and fermentation of this strain should be carried out at 37℃.

[0051] 2. Growth experiment of strain 94-6Y under different salinity conditions

[0052] Prepare LB liquid culture media with salinity of 0%, 1%, 3%, and 5% respectively. Inoculate strain 94-6Y with the culture medium, controlling the inoculum size to achieve the initial OD. 600 The value was 0.1, with 3 replicates for each salinity. The cells were incubated at 37°C with shaking at 180 rpm. The OD was measured after 24 h. 600 Numerical value.

[0053] The results show that ( Figure 4 After 24 h of culture, strain 94-6Y showed OD values ​​at four salinity gradients. 600 The values ​​all showed significant increases, indicating that strain 94-6Y can grow in salinities ranging from 0% to 5%, with the optimal salinity for growth being 1%. At salinities of 0%, 1%, and 3%, its OD values... 600 The numerical differences are not significant; the OD values ​​at 5% salinity are similar. 600 The values ​​are significantly lower, indicating that when the salinity is not higher than 3%, the growth of strain 94-6Y is less affected by salinity changes, and its growth is significantly inhibited when the salinity increases to 5%. Therefore, strain 94-6Y can adapt to freshwater and common seawater environments and can be used in both freshwater and seawater aquaculture.

[0054] Example 3: Safety test of *Pseudomonas macranthae* 94-6Y

[0055] Healthy zebrafish with a body length of 2-4 cm were used as subjects. Three experimental groups and one control group were set up, with three replicates in each group. The experimental period was 7 days. Ten healthy zebrafish were placed in each experimental tank. 94-6Y bacterial solution was added to the experimental groups until a final concentration of 1×10⁻⁶ was achieved. 5 1×10 6 1×10 7 CFU / mL, with no substances added to the control group. Maintain the water temperature at 25℃ and pH 7.0-7.5, and observe the growth of zebrafish.

[0056] The experimental results showed that within 7 days of adding the bacterial solution, all zebrafish in the experimental and control groups grew well, with no abnormalities in respiration or swimming, and no deaths.

[0057] Example 4: Inhibition test of *Pseudomonas macranthae* 94-6Y against common aquatic pathogens

[0058] Common aquatic pathogens such as Vibrio vulnificus, Vibrio splenium, and Vibrio alginolyticus were selected as indicator strains. Vibrio vulnificus, Vibrio splenium, and Vibrio alginolyticus were inoculated into liquid 2216E medium and cultured at 28°C with shaking for 24 h. Then, they were inoculated into liquid LB medium and cultured at 37°C with shaking for 24 h to obtain indicator bacterial suspensions. The indicator bacterial suspensions were serially diluted and spread onto the corresponding 2216E plates. Sterile Oxford cups were placed on the plates, and 100 μL of strain 94-6Y bacterial suspension was added to each cup. A 50 ppm chlortetracycline hydrochloride solution was used as a positive control. The plates were incubated at 28°C for 24 h, and the antibacterial results were observed.

[0059] The results showed that it contained the indicator pathogen Vibrio vulnificus ( Figure 5 Vibrio splendens ( Figure 6 ) and Vibrio alginolyticus ( Figure 7 The presence of inhibition zones on all three Vibrio species indicates that *Pseudomonas macranthae* 94-6Y has a significant inhibitory effect on all three Vibrio species.

[0060] Example 5: Detection of the ability of *Pseudomonas macranthae* 94-6Y to remove ammonia nitrogen, nitrite and COD from aquaculture water samples

[0061] Five L of tailwater samples were collected from a bullfrog breeding pond in Qingdao. The initial ammonia nitrogen content was 7.64 mg / L, nitrite content was 0.53 mg / L, and COD content was 118.7 mg / L. 50 mL of fresh Bacillus maculatus 94-6Y bacterial solution was added, with a bacterial solution concentration of 0.01%. The solution was aerated with sterile air, and samples were taken every 24 h to test the ammonia nitrogen content, nitrite content, and COD content.

[0062] The results showed that after adding 94-6Y bacterial solution, the ammonia nitrogen concentration in the water sample ( Figure 8 ), nitrite concentration ( Figure 9 ) and COD content ( Figure 10 The concentration of ammonia nitrogen in the water sample decreased continuously after 4 days. The concentration of nitrite in the water sample was 1.22 mg / L, with a removal rate of 84.03%; the concentration of nitrite in the water sample was 0.21 mg / L, with a removal rate of 60.38%; and the COD content in the water sample was 26.9 mg / L, with a removal rate of 77.34%.

[0063] Example 6: Application of *Pseudomonas macranthae* 94-6Y in sea cucumber farming

[0064] This embodiment was carried out at a sea cucumber farm in Jimo District, Qingdao City. The volume of a single breeding pond at the farm is 10 cubic meters. The farm raises sea cucumber seedlings with a body length of about 1 cm, with a number of about 12,000. The daily feed amount is 0.5 kg.

[0065] Two breeding ponds from the same batch were used for the experiment. The experimental group was fed with 8×10⁻⁶ microbial powder of strain 94-6Y. 10 Add CFU / g to the feed to achieve an effective live bacteria count of 1×10⁻⁶. 4 The feed was administered at CFU / g, while the control group's feed did not contain any microbial products. All other aquaculture conditions remained unchanged. Water samples from the aquaculture ponds were taken periodically before feeding to test the concentrations of ammonia nitrogen and nitrite, and the samples were spread onto Vibrio chromogenic medium (TCBS agar plates) to detect the number of Vibrio bacteria. The experimental period was 6 months.

[0066] Experimental results showed that during a 6-month breeding cycle, the ammonia nitrogen ( ) in the experimental tanks where strain 94-6Y bacterial powder was added was significantly reduced. Figure 11 ), nitrite ( Figure 12 ) and Vibrio number ( Figure 13 All of these values ​​were lower than those of the control group without added bacterial powder, with ammonia nitrogen concentration decreasing by 5.25-25.19%, nitrite concentration decreasing by 14.29-33.47%, and Vibrio count decreasing by 58.62-74.31%.

[0067] Although the embodiments of the present invention have been described above in conjunction with the examples, it should be noted that the described embodiments are only a part of the specific implementation of the present invention and are not intended to limit the scope of protection of the present invention. Any equivalent substitutions or improvements made within the scope of the technical concept and technical method of the present invention shall fall within the scope of protection and disclosure of the present invention.

Claims

1. A strain of *Pseudomonas Macaoensis* 94-6Y, characterized in that, The strain described is classified as *Pseudomonas macranthum* 94-6Y and named *Pseudomonas macranthum*. Fictibacillus macauensis The accession number is CGMCC No. 32718.

2. The *Pseudomonas Macaoensis* 94-6Y according to claim 1, characterized in that, The *Pseudomonas Macaoensis* 94-6Y strain is a Gram-positive strain with milky-white, nearly round, opaque colonies that are slightly rough and dull.

3. The *Pseudomonas Macaoensis* 94-6Y according to claim 1, characterized in that, The optimal growth temperature for *Pseudomonas Macaoensis* 94-6Y is 15-37℃, and the salt concentration tolerance is 0%-5%.

4. The inoculum prepared by fermentation of *Pseudomonas macranthae* 94-6Y as described in any one of claims 1-3.

5. The use of *Pseudomonas macranthae* 94-6Y as described in any one of claims 1-3 or the bacterial agent as described in claim 4 in the preparation of formulations for inhibiting pathogens in aquaculture, characterized in that... The pathogen is one or more of Vibrio vulnificus, Vibrio brilliantus, and Vibrio alginolyticus.

6. The application of *Pseudomonas macranthae* 94-6Y as described in any one of claims 1-3 or the bacterial agent as described in claim 4 in improving water quality indicators in aquaculture, characterized in that... Improving water quality indicators in aquaculture includes significantly reducing ammonia nitrogen, nitrite, and COD levels in the aquaculture system.

7. The application according to claim 6, characterized in that, The concentration of *Pseudomonas macranthae* 94-6Y or the inoculant is 0.005%-0.1% of the total volume of the aquaculture system, and the treatment period is 1-5 days.

8. The application according to claim 7, characterized in that, The microbial agent includes microbial powder, and the microbial content of the microbial powder is 5 × 10⁻⁶. 10 -10×10 10 CFU / g.

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