Kelp green rot pathogenic bacteria and application thereof
By screening and identifying *Pseudomonas alterniflora* HKCH18 as the pathogen of kelp green rot, its degradation activity was revealed, solving the unknown problem of the pathogenesis of kelp green rot, realizing the screening of disease-resistant strains and the preparation of vaccines, and reducing losses in kelp farming.
Patent Information
- Application Number
- CN202511541860.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-10-27
- Publication Date
- 2026-02-24
AI Technical Summary
The pathogen causing kelp green rot disease is not fully identified, leading to frequent outbreaks, rapid spread, and severe economic losses during kelp farming. Existing research has not yet explored its pathogenic mechanism in depth.
Pseudoalteromonas sp. was identified as the pathogen causing green rot in kelp. Its nucleic acid sequence was used to identify its degradation activities with sodium alginate, agar, sodium carboxymethyl cellulose, and starch, which can be used to screen disease-resistant strains and prepare vaccines.
This study provides a foundation for understanding the pathogenesis of kelp green rot disease, enables rapid screening of resistant strains, reduces economic losses, and facilitates the development of effective vaccines to prevent kelp green rot disease.
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Figure CN121555347A_ABST
Abstract
Description
Technical Field
[0001] This application relates to the field of microbial disease prevention and control technology in aquaculture kelp, specifically to a pathogen causing kelp green rot and its application. Background Technology
[0002] Kelp is one of the main algae products in my country's fishery production, belonging to the low-temperature macroalgae category. In 2018, my country's total algae production was 2.35 million tons, of which kelp accounted for 1.52 million tons, or 64.68% of the total algae production. The national algae cultivation area was 150,000 hectares, with kelp cultivation accounting for 31.29%, reaching 45,100 hectares. During kelp cultivation, pathogenic microorganisms or external environmental factors such as unsuitable light and temperature, nutrient imbalances, and other plankton can cause invasive or physiological diseases.
[0003] Kelp green rot is a common and serious disease during the cultivation of summer kelp seedlings. It is characterized by frequent outbreaks, short duration of illness, rapid spread, and the ability to cause mass mortality of seedlings in a short period, resulting in significant economic losses for kelp farming enterprises and seriously threatening the completion of kelp farming production targets. The causes of green rot are related to environmental factors such as insufficient sunlight and excessively high water temperatures, as well as the types and quantities of epiphytic fungi on the kelp.
[0004] To investigate the microbial species responsible for kelp green rot, researchers isolated, purified, identified, and re-stained epiphytic microorganisms from diseased kelp, obtaining a certain number of pathogens causing kelp green rot. However, further investigation is needed to identify the pathogens causing kelp green rot and to further explore their pathogenic mechanisms. Summary of the Invention
[0005] To address the issue that the pathogen causing kelp green rot is not yet fully identified, this application provides a pathogen causing kelp green rot and its application.
[0006] The first aspect of this application provides a pathogen causing green rot in kelp, namely *Pseudoalteromonas* sp. HKCH18. This strain has been deposited at the China General Microbiological Culture Collection Center (CGMCC) on September 4, 2023, with accession number CGMCC NO.28348. This strain can cause green rot symptoms in healthy kelp and is the causative agent of kelp green rot. The screening and identification of this strain provides an important theoretical basis and practical evidence for the prevention and control of kelp green rot, contributing to in-depth research on the pathogenesis of kelp green rot and offering new ideas for its prevention and control.
[0007] Furthermore, the nucleic acid sequence of strain HKCH18 is shown in SEQ ID NO: HKCH18. The determination of this nucleic acid sequence provides a molecular biological basis for the accurate identification of the strain and subsequent research, facilitating rapid and accurate identification of the strain and providing technical support for the early diagnosis and prevention of kelp green rot disease.
[0008]
[0009] Furthermore, strain HKCH18 exhibits activities degrading sodium alginate, agar, sodium carboxymethyl cellulose, and starch. These enzymatic activities reveal the pathogenic mechanism of this strain, namely, it causes kelp tissue destruction by degrading the main components of the kelp cell wall, thereby triggering green rot disease. This provides an important basis for further research into the pathogenic mechanism and the development of targeted prevention and control measures.
[0010] The second aspect of this application provides an application of the pathogen causing kelp green rot disease, specifically the application of strain HKCH18 in screening kelp strains resistant to kelp green rot disease. By artificially infecting kelp with this pathogen, kelp strains resistant to green rot disease can be rapidly screened, providing an effective tool for kelp disease-resistant breeding.
[0011] Furthermore, the application of screening kelp strains resistant to green rot disease involves soaking healthy kelp seedlings in HKCH18 bacterial solution to screen for disease-resistant kelp strains. This screening method is simple to operate, effective, and can screen disease-resistant strains on a large scale in a short time, thus improving breeding efficiency.
[0012] Furthermore, the soaking concentration is 10. 5 -10 9 The concentration of CFU / mL and the soaking time are 2-60 min. By optimizing the soaking parameters, it is possible to ensure the pathogenicity effect while avoiding excessive damage to the kelp, thus providing standardized operating conditions for the screening process.
[0013] A third aspect of this application provides the application of a pathogen causing kelp green rot disease, specifically the application of strain HKCH18 in vaccine preparation. Using this strain to prepare a vaccine can effectively prevent the occurrence of kelp green rot disease and reduce economic losses during aquaculture.
[0014] Furthermore, the vaccine preparation method comprises at least one of the following: inactivated bacterial cells, bacterial erosion components, attenuated bacterial strains, protective antigens, antigenic subunits, antigenic determinants, or expression products of antigen gene expression vectors. The choice of multiple preparation methods provides diverse technical pathways for vaccine development, allowing the selection of the most suitable method based on actual needs.
[0015] Furthermore, the vaccine products are of the following three types: (1) a vaccine made from a single component of the antigen prepared using this strain; (2) a combined vaccine produced by mixing the antigen prepared using this strain with antigens from other bacteria; and (3) a vaccine produced by adding an adjuvant to the antigen of a prepared single or combined vaccine. Different forms of vaccine products can meet different breeding environments and needs, improving the applicability and effectiveness of the vaccine.
[0016] Furthermore, the vaccine is administered via immersion immunization. This immunization method is simple to operate, suitable for large-scale farms, reduces the difficulty and cost of immunization operations, and improves the practicality of the vaccine.
[0017] The beneficial effects of this invention are:
[0018] 1. This invention is the first to screen out a strain of *Pseudoalteromonas* HKCH18, which exhibits good activity in degrading sodium alginate, agar, sodium carboxymethyl cellulose, and starch. In experiments infecting healthy kelp, it can induce green rot symptoms in healthy kelp, thus acting as the pathogen causing kelp green rot.
[0019] 2. By using this bacterium, we can screen kelp strains resistant to kelp green rot disease and conduct in-depth research on the pathogenesis of kelp green rot disease.
[0020] 3. This bacterium can be used to prepare a vaccine for kelp green rot disease, reducing losses caused by diseases in aquaculture. Attached Figure Description
[0021] Figure 1 This is a photograph of the colony morphology of strain HKCH18.
[0022] Figure 2 This is a transmission electron microscope image of strain HKCH18.
[0023] Figure 3 OD600 was measured under different pH conditions.
[0024] Figure 4 OD600 was measured for cultures with different concentrations of NaCl.
[0025] Figure 5 The activity of four substrate enzymes in strain HKCH18 was measured.
[0026] Figure 6 Photographs showing the appearance of healthy kelp.
[0027] Figure 7 Cell images of healthy kelp under 100x oil immersion.
[0028] Figure 8 Photographs showing the appearance of seaweed infected by strain HKCH18.
[0029] Figure 9 Cell images of kelp infected by strain HKCH18 under 100x oil immersion. Detailed Implementation
[0030] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0031] The present invention will be further illustrated below with reference to the embodiments:
[0032] Example 1: Isolation and Identification of Pathogens Causing Green Rot Disease in Kelp
[0033] This embodiment provides a method for isolating and identifying the pathogenic bacterium *Pseudomonas alterniflora* HKCH18, which causes green rot in kelp.
[0034] Isolation and purification of pathogens causing kelp green rot disease: Kelp samples with green rot disease were collected from the Rongcheng sea area of Shandong Province and placed in sample bags. The vesicles, rotten parts of the kelp algae, and residual liquid in the sample bags were mixed and placed in 100 mL of sterilized seawater containing glass beads. After incubation at 18℃ and 200 rpm for 1 h, serial dilutions were performed, with 50 μL of each diluted in 10 mL increments. -2 10 -4 10 -6 10 -8 10 -10 The serially diluted solutions were spread on 2216E agar plates and incubated at 18°C for approximately one week. After incubation, the total colony count was recorded, and strains with different morphologies were picked and streaked onto 2216E agar plates for purification. This process was repeated twice until single colonies were obtained. Verification using a healthy kelp infection experiment confirmed that the isolated colony HKCH18 exhibited pathogenicity for green rot disease. The final colony morphology was recorded, and the isolated strain was stored at -80°C.
[0035] The 2216E culture medium formula (g / L) is as follows: peptone 5.0, yeast extract 1.0, ferric citrate 0.1, sodium chloride 19.45, magnesium chloride 5.98, sodium sulfate 3.24, calcium chloride 1.8, potassium chloride 0.55, sodium carbonate 0.16, potassium bromide 0.55, strontium chloride 0.034, boric acid 0.022, sodium silicate 0.004, sodium fluoride 0.0024, ammonium nitrate 0.0016, disodium hydrogen phosphate 0.008, pH 7.6.
[0036] Upon observation, the colony diameter of strain HKCH18 was approximately 3.5 mm, grayish-black in color, with a darker center and lighter periphery. Gram staining and scanning electron microscopy revealed that the strain was a Gram-negative bacterium with a single flagellum, and the bacterial cells were rod-shaped, approximately 2 μm long and 0.5 μm wide.
[0037] Example 2: Molecular identification of strain HKCH18
[0038] The purified strain was inoculated into liquid 2216E medium and cultured at 18°C and 200 rpm for 24 h. The 16S rDNA sequence was amplified using primers 27F and 1492R and sent to a sequencing company for sequencing, yielding the nucleic acid sequence shown in SEQ ID NO: HKCH18. Sequence alignment was performed in the NCBI and EzBioCloud databases, and the strain was identified based on the sequence with the highest similarity. This strain was identified as belonging to the genus *Pseudomonas* and was deposited on September 4, 2023, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.28348.
[0039] Example 3: Physiological and biochemical characteristics of strain HKCH18
[0040] (1) Physiological and biochemical characteristics were determined according to the "Common Bacterial System Identification Manual". The results of oxidase, catalase, lipase, nitrate reduction and H2S were positive, while the results of methyl red, VP, indole and citrate utilization were negative. It can use fructose, maltose, sucrose, starch, glucose, dextrin and sorbitol as the only carbon source for growth. The glucose oxidation fermentation type is oxidative. The litmus in the milk decomposition experiment fades and turns white, indicating the reducing type.
[0041] (2) pH experiment: The frozen strain HKCH18 was revived on 2216E solid plates and cultured at 18℃ for about 2-3 days to grow single colonies. Colonies were picked and transferred to test tubes containing liquid 2216E medium, incubated overnight at 18℃, and then inoculated into 20mL of liquid 2216E medium, and cultured overnight to obtain seed culture. During this period, 20mL to 50mL of 2216E liquid medium with different pH values were prepared in Erlenmeyer flasks, with pH values set sequentially to 6, 7, 8, 9, and 10, for a total of 5 groups, each with 3 replicates. The seed culture (inoculum amount 2%) was inoculated into the above Erlenmeyer flasks and cultured at 18℃ with shaking for 26h. During this period, samples were taken at 0h, 2h, 4h, 6h, 8h, 10h, 24h, and 26h to measure the average OD600 value of each group. The results showed that strain HKCH18 can grow under pH conditions of 6-10.
[0042] (3) Salt tolerance test: Colonies from the plate were inoculated into test tubes containing liquid 2216E medium. After incubation at 18°C overnight, the culture was transferred to 20 mL of liquid 2216E medium and cultured overnight to obtain the seed culture. During this period, 20 mL to 50 mL of 2216E liquid medium with different salt concentrations were prepared in Erlenmeyer flasks. The salt concentration was adjusted by NaCl, and the concentrations were 0%, 0.5%, 1%, 1.5%, 2%, 3%, 4%, 5%, 6%, and 7%, for a total of 10 groups with 3 replicates per group. The seed culture (inoculation amount of 2%) was inoculated into the above Erlenmeyer flasks and cultured at 18°C with shaking for 26 h. During this period, samples were taken at 0 h, 6 h, 8 h, 10 h, 24 h, and 26 h to measure the average OD600 value of each group. The results showed that strain HKCH18 could tolerate 0-6% NaCl and did not grow in 7% NaCl.
[0043] Example 4: Enzyme activity assay of strain HKCH18
[0044] The activity of various substrate enzymes was determined using the clear zone method. Glycerol-preserved bacterial cultures were streaked onto 2216E plates for recovery and incubated at 18°C for approximately 2-3 days to produce single colonies. Solid culture media were prepared using sodium alginate, agar, sodium carboxymethyl cellulose, and starch. Each culture medium plate was divided into six sections, with one colony inoculated into each section. After 7 days of colony incubation, chromogenic reagents were added, and the colony diameter (d) and clear zone (D) size were observed and recorded. Enzyme activity was expressed as D / d.
[0045] Agar medium is 2216E medium with 2% agar powder added. Sodium alginate medium is agar medium with 1% sodium alginate added. Sodium carboxymethyl cellulose medium is agar medium with 0.5% sodium carboxymethyl cellulose added. Starch medium is agar medium with 0.2% starch added.
[0046] The results showed that the enzyme activity was greater than 1, indicating that strain HKCH18 had good degradation activity against sodium alginate, agar, sodium carboxymethyl cellulose, and starch.
[0047] Example 5: Kelp Infection Experiment
[0048] The cryopreserved strain HKCH18 was revived on 2216E agar plates and cultured at 18°C for 2-3 days until single colonies grew. Colonies were picked and transferred to 1 mL of liquid 2216E medium and incubated overnight at 18°C and 200 rpm. The culture was then transferred to 100 mL of liquid 2216E medium and incubated at 18°C and 200 rpm with shaking for 12 hours to obtain a bacterial suspension in the logarithmic growth phase. The suspension was then diluted sequentially with sterile water to a concentration of 9.8 × 10⁻⁶. 6 CFU / mL, 9.8×10 5 CFU / mL, 9.8×10 4CFU / mL, 9.8×10 3 CFU / mL.
[0049] Four groups of healthy kelp seedlings with good growth were selected, with 10 seedlings in each group. Their appearance was recorded by photography, and cell morphology was observed under a 100x oil immersion microscope. The 10 kelp seedlings in each group were then infected with different dilutions of the bacterial solution. The seedlings were cultured under light in sterile seawater supplemented with the bacterial solution. After 14 days of culture, their appearance and cell morphology were recorded again. The results showed that after 14 days of culture, the kelp turned greenish-white, with blisters and decay. Microscopic observation revealed that the cell protoplasm was not abundant, the fucoidan was destroyed, leaving only chlorophyll, and the tissue appeared green. This indicates that strain HKCH18 can induce green rot disease in healthy kelp and is the pathogenic bacterium of kelp green rot disease.
[0050] Example 6: Screening of strains resistant to kelp green rot disease
[0051] The cryopreserved strain HKCH18 was revived on 2216E agar plates and incubated at 18°C for 2-3 days until single colonies grew. Colonies were picked and transferred to test tubes containing 1 mL of liquid 2216E medium, incubated overnight at 18°C and 200 rpm, then transferred to 100 mL of liquid 2216E medium and incubated at 18°C and 200 rpm with shaking for 12 hours to obtain the logarithmic growth phase bacterial culture. The bacterial culture was then diluted to 10⁻¹⁰ with sterile water. 7 CFU / mL.
[0052] Five groups of 20 kelp seedlings of different strains were selected, and their appearance was recorded by photography. The 20 seedlings from each group were then soaked in a bacterial solution for 30 minutes. The seedlings were then cultured under light in sterile seawater supplemented with the bacterial solution. After 14 days of culture, the disease incidence rate was recorded. Based on the disease incidence rate, kelp strains with strong disease resistance were selected: strain A had a disease incidence rate of 5%, strain B 15%, strain C 30%, strain D 50%, and strain E 85%. The results indicate that strain A has the strongest resistance to green rot disease and is suitable as a disease-resistant breeding material.
[0053] Example 7: Preparation of inactivated vaccine
[0054] HKCH18 was first inactivated using formalin inactivation. The final concentration of inactivated bacteria in the vaccine was 10. 8 CFU / mL, the bacterial suspension was serially diluted with sterile water to a concentration of 9.8 × 10⁻⁶ CFU / mL. 6 CFU / mL, 9.8×10 5 CFU / mL, 9.8×10 4 CFU / mL, 9.8×10 3CFU / mL. In the application, five groups of healthy kelp seedlings with good growth were selected, with 10 seedlings in each group. The first four groups were soaked in different concentrations of sterilized bacterial solutions and cultured normally, respectively. The fifth group was cultured directly in sterilized seawater. The incidence rate was counted after 15 days.
[0055] Artificial infection experiments were then conducted using fresh HKCH18 bacterial suspension at 10 times the LD50 dose, with infection performed via immersion. Fifteen days post-infection, the morbidity rate was calculated. A morbidity rate below 30% was considered indicative of good immunization efficacy.
[0056] Example 8: Preparation of Attenuated Vaccine
[0057] HKCH18 was attenuated by ultraviolet irradiation to obtain an attenuated strain. This attenuated strain was inoculated into liquid 2216E medium and cultured for 24 hours. The bacterial cells were collected by centrifugation, washed three times with PBS buffer, resuspended in PBS, and the bacterial concentration was adjusted to 10⁻⁶. 7 CFU / mL. Two groups of healthy kelp seedlings with good growth were selected, with 10 seedlings in each group. The experimental group was soaked in attenuated bacterial solution for 30 min, while the control group was soaked in PBS for 30 min. Afterward, they were cultured normally for 15 days. Then, an artificial infection experiment was conducted using fresh HKCH18 bacterial solution at 10 times the LD50, with infection performed by immersion. Fifteen days after infection, the morbidity rate was calculated. The morbidity rate in the experimental group was 20%, while the morbidity rate in the control group was 100%, indicating that the attenuated vaccine had a good immunizing effect.
[0058] Example 9: Enzyme activity assay and comparison of inactivated vaccines
[0059] This embodiment aims to verify whether the inactivated vaccine against kelp rot prepared according to the present invention retains the original enzymatic degradation activity of strain HKCH18. By comparing it with live HKCH18 bacteria in parallel, the effect of inactivation treatment on the enzyme activity of the strain is evaluated, thereby further demonstrating the safety of the vaccine.
[0060] Experimental Materials and Grouping: Test Sample: HKCH18 inactivated vaccine prepared according to the method in Example 7. Positive Control: HKCH18 live bacterial suspension isolated, purified, and cultured according to the method in Example 1, with the concentration adjusted to the same as the inactivated vaccine (approximately 10) using sterile seawater. 8 CFU / mL). Negative control: sterile 2216E medium. Enzyme activity assay medium: same as in Example 4, 2216E solid medium plates containing 1% sodium alginate, 2% agar, 0.5% sodium carboxymethyl cellulose and 0.2% starch were prepared.
[0061] Enzyme activity was determined using a clear zone method similar to that in Example 4, and the specific steps are as follows:
[0062] (1) On the above four enzyme activity assay culture medium plates, use a sterile punch to uniformly punch three round holes with a diameter (d) of 6 mm.
[0063] (2) In each of the three wells of each plate, add 50 μL of test sample (inactivated vaccine), positive control (live bacterial suspension) and negative control (sterile culture medium).
[0064] (3) Place all plates in a constant temperature incubator at 18°C for 7 days, keeping the same culture conditions as in Example 4.
[0065] (4) After incubation, remove the plates. For starch degradation active plates, add Lugol's iodine solution; for the other three types of plates, observe directly. Measure the diameter (D) of the transparent hydrolysis ring formed around each well.
[0066] (5) Calculate the D / d ratio, which directly reflects the strength of enzyme activity. If no clear zone is formed, then D=d, D / d=1.0, indicating no corresponding enzyme activity.
[0067] After 7 days of incubation, the enzyme activity assay results for each group are shown in the table below.
[0068] Table 2 Comparison of enzyme activities between inactivated vaccines and live bacteria
[0069]
[0070] The experimental results clearly show that the live HKCH18 bacteria in the positive control group exhibited good degradation ability on all four substrates, forming clear and transparent hydrolysis zones, and its D / d ratio was highly consistent with the results of Example 4.
[0071] In stark contrast, the inactivated vaccine prepared by this invention did not produce any visible clear zones on any of the four enzyme activity assay media (D / d ratio was 1.0), and its performance was exactly the same as that of the negative control group.
[0072] The data from this embodiment strongly demonstrate that formalin inactivation treatment effectively kills pathogens while completely inactivating the enzyme systems that degrade sodium alginate, agar, sodium carboxymethyl cellulose, and starch. This indicates that the inactivated vaccine provided by this invention retains the complete structure of the bacteria as antigens to stimulate an immune response while eliminating their biological activity and potential pathogenicity as live bacteria, thereby greatly improving the safety of the vaccine in practical applications.
[0073] Comparative Example 1: Non-pathogenic kelp infection experiment
[0074] A non-pathogenic Pseudomonas sp., a common epiphytic fungus of kelp, was selected as a control. A bacterial suspension was prepared according to the method in Example 5, with a concentration of 9.8 × 10⁻⁶. 6 CFU / mL. A group of 10 healthy kelp seedlings with good growth were selected. Their appearance was recorded by photography, and cell morphology was observed under a 100x oil immersion microscope. The 10 kelp seedlings were then infected with a bacterial solution and cultured under light in sterile seawater supplemented with the bacterial solution. After 14 days of culture, their appearance and cell morphology were recorded again. The results showed that the kelp had a normal appearance after 14 days of culture, with no obvious lesions. Microscopic observation revealed intact cell structure and normal distribution of fucoidan and chlorophyll. This indicates that non-pathogenic bacteria do not cause green rot disease in kelp.
[0075] Comparative Example 2: Kelp Infection Experiment with Different Concentrations of Pathogenic Bacteria
[0076] HKCH18 bacterial suspension was prepared according to the method in Example 5, but the bacterial suspension concentration was reduced to 9.8 × 10⁻⁶. 2 CFU / mL. A group of 10 healthy kelp seedlings with good growth were selected. Their appearance was recorded by photography, and cell morphology was observed under a 100x oil immersion microscope. The 10 kelp seedlings were then infected with bacterial solution and cultured under light in sterile seawater supplemented with the bacterial solution. After 14 days of culture, their appearance and cell morphology were recorded again. The results showed that the kelp morphology was basically normal after 14 days of culture, with only minor lesions. Microscopic observation revealed that the cell structure was basically intact, with slight damage to fucoidan. This indicates that excessively low concentrations of pathogenic bacteria are insufficient to cause obvious symptoms of green rot disease.
[0077] Performance testing:
[0078] Strain growth performance test: The growth adaptability of the strain was evaluated by measuring the OD600 value under different pH and salt concentration conditions.
[0079] Enzyme activity assay: The ability of the strain to degrade sodium alginate, agar, sodium carboxymethyl cellulose and starch was determined by the clear zone method, and the enzyme activity was expressed as D / d value.
[0080] Pathogenicity test: The pathogenicity of the strain is assessed by observing the changes in appearance and cell structure of kelp after it is infected by the strain.
[0081] Vaccine efficacy testing: The immunization effect of the vaccine is evaluated by statistically analyzing the incidence rate of reinfection after kelp immunization.
[0082] Table 1 Test results of the examples and comparative examples
[0083]
[0084] Data Analysis:
[0085] Analysis of the test results from the examples and comparative examples shows that *Pseudomonas alterniflora* HKCH18 has strong environmental adaptability and can grow under conditions of pH 6-10 and 0-6% NaCl, which is consistent with the growth environment of kelp and is conducive to its colonization and reproduction on the kelp surface. This strain exhibits good activity in degrading sodium alginate, agar, sodium carboxymethyl cellulose, and starch. These enzymatic activities may be the main factors leading to kelp tissue destruction, by degrading the main components of the kelp cell wall, thus disrupting cell structure and causing green rot disease.
[0086] In the kelp infection experiment, strain HKCH18 was effective at a concentration of 9.8 × 10⁻⁶. 6 At a concentration of CFU / mL, it can cause 100% disease in kelp, manifesting as an overall greenish-white discoloration, the presence of blisters and rotting. Microscopic observation reveals insufficient protoplasm in the cells, destruction of fucoidan, leaving only chlorophyll, resulting in a greenish tissue appearance. In contrast, the non-pathogenic *Pseudomonas* bacterium in Comparative Example 1 does not cause disease in kelp, and at a low concentration (9.8 × 10⁻⁶) in Comparative Example 2... 2 HKCH18 (CFU / mL) only caused disease in 10% of kelp, and the symptoms were mild, indicating that pathogenicity is related to strain concentration.
[0087] In vaccine trials, both the inactivated and live attenuated vaccines prepared using HKCH18 showed good immunogenicity. Among them, the high-concentration (9.8 × 10⁻⁶) vaccine... 6 CFU / mL and 9.8×10 5 An inactivated vaccine at a concentration of CFU / mL can completely protect kelp from infection, and a low concentration (9.8 × 10⁻⁶ CFU / mL) is even more effective. 4 CFU / mL and 9.8×10 3 The inactivated vaccine (CFU / mL) protected 90% of kelp from infection, while the live attenuated vaccine protected 80%. In contrast, the morbidity rate in the unimmunized control group was as high as 100%, indicating that the vaccine prepared by HKCH18 has a significant immunoprotective effect.
[0088] In summary, *Pseudomonas alterniflora* HKCH18 is the pathogen of kelp green rot disease. It has good enzyme activity and pathogenicity and can be used to screen kelp strains resistant to kelp green rot disease. It can also be used to prepare vaccines to effectively prevent the occurrence of kelp green rot disease.
[0089] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, all variations falling within the meaning and scope of equivalents of the claims are intended to be included within the present invention, and no reference numerals in the claims should be construed as limiting the scope of the claims.
Claims
1. A pathogen causing green rot in kelp, characterized in that, The pathogen causing the green rot of kelp is Pseudoalteromonas sp. HKCH18, which has been deposited at the China General Microbiological Culture Collection Center on September 4, 2023, with accession number CGMCC NO.28348.
2. The pathogen causing kelp green rot according to claim 1, characterized in that, The nucleic acid sequence of strain HKCH18 is shown in SEQ ID NO: HKCH18.
3. The application of a pathogen causing kelp green rot, characterized in that, Application of the HKCH18 strain, the pathogen of kelp green rot disease, in screening kelp strains resistant to kelp green rot disease.
4. The application of the pathogenic bacterium causing kelp green rot according to claim 3, characterized in that, The application of screening kelp strains resistant to green rot disease involves soaking healthy kelp seedlings in HKCH18 bacterial solution to screen for kelp strains resistant to green rot disease.
5. The application of the pathogenic bacterium causing kelp green rot according to claim 4, characterized in that, The soaking concentration is 10. 5 -10 9 CFU / mL, soaking time is 2-60 min.
6. The application of a pathogen causing kelp green rot disease, characterized in that, Application of the HKCH18 strain of kelp green rot pathogen in vaccine preparation.
7. The application of the pathogenic bacterium causing kelp green rot according to claim 6, characterized in that, The vaccine is prepared by at least one of the following methods: inactivated bacterial cells, bacterial molting components, attenuated bacterial strains, protective antigens, antigenic subunits, antigenic determinants, or expression products of antigen gene expression vectors.
8. The application of the pathogenic bacterium causing kelp green rot according to claim 7, characterized in that, The vaccine products are of the following three types: a. A vaccine made from a single component of an antigen prepared using this strain; b. Combined vaccines produced by mixing antigens prepared using this strain with antigens from other bacteria; c. Vaccines produced by adding adjuvants to the antigens of prepared single or combined vaccines.
9. The application of the pathogenic bacterium causing kelp green rot according to claim 7 or 8, characterized in that, The vaccine is administered via immersion immunization.
10. The pathogen causing kelp green rot according to claim 1, characterized in that, The strain HKCH18 exhibits activity in degrading sodium alginate, agar, sodium carboxymethyl cellulose, and starch.