Pathogenic bacteria of laver macular brown shell disease

Abstract

The pathogenic bacterium of the laver protonema macular brown shell disease provided by the invention is Vibrio stannaloensis NH-7-1 strain, and the preservation number of the pathogenic bacterium is CCTCC (China Center for Type Culture Collection) M 20252514. The strain can cause macular and rough shell symptoms of shell protonema, and also can cause greening, ulceration and holes of thallus tissues. Meanwhile, the bacterium has infection capability on various economic red algae and shows broad-spectrum algae-lysing activity. Separation and identification of the strain are beneficial to screening of excellent nori strains resistant to maculopathy and maculopathy, and the strain can be used for establishing an early warning and rapid detection system for algal diseases.

Description

Technical Field

[0001] This invention belongs to the field of prevention and control technology of diseases in aquaculture, and specifically relates to a pathogen of yellow spot and rough shell disease in laver. Background Technology

[0002] seaweed ( Porphyra Laver (Porphyra yezoensis) is an important economic seaweed widely cultivated in the coastal areas of Southeast Asia. It not only possesses rich nutritional and economic value but also plays a crucial role in marine carbon sequestration, exhibiting significant ecological benefits. The cultivation and production of laver covers almost the entire year, including indoor seedling cultivation of the sporophyte generation (shell-like filaments) (March to mid-September each year) and marine cultivation of the gametophyte generation (thallus) (late September to December each year, and even February to March of the following year). During this period, laver experiences the high temperatures of summer and autumn, as well as extreme weather events such as torrential rains and typhoons. This not only adversely affects the physiology of laver but also alters the composition and function of environmental microorganisms in the water, making them pathogens or opportunistic pathogens, greatly increasing the risk of disease in aquaculture.

[0003] Among the reported diseases of laver, yellow spot disease has received considerable attention. Outbreaks of yellow spot disease can lead to large-scale death of the laver filaments, making it a significant factor affecting the success or failure of laver seedling cultivation. Yellow spot disease is most prevalent during the summer seedling stage. Typical initial symptoms include pinhead-sized yellow spots appearing on the surface of the laver filaments. As the disease progresses, the spots gradually enlarge, and the color of the filaments changes successively from red to yellow, then to green, eventually bleaching completely. In severely affected areas, the laver surface becomes rough and dull, and the filaments cannot grow. Currently, the identified pathogen of laver yellow spot disease is *Vibrio thaliana*. Vibrio mediterranei 117-T6, *Pseudomonas aeruginosa* PZ201809121102, and *Pseudomonas* HYWX-1 and HYWX-2, among others, were identified. These findings indicate that macular degeneration is not caused by a single specific pathogen, and its etiology is complex. Therefore, it is urgent to further expand the scope of potential pathogen identification and improve the pathogen database to provide a scientific basis for the comprehensive prevention and control of this disease. Summary of the Invention

[0004] This invention provides a pathogenic bacterium of laver that can simultaneously cause yellow spot disease and rough shell symptoms in the filamentous body of laver shells, thus providing core experimental material for pathogen research and integrated control technology development of laver yellow spot disease.

[0005] The pathogen of yellow spot and rough shell disease of laver filaments provided in this invention is Vibrio sinaloa ( Vibrio sp. NH-7-1 strain, deposited at the China Center for Type Culture Collection, accession number CCTCC NO: M 20252514, deposited on November 10, 2025; deposit address: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0006] The optimal growth conditions for the NH-7-1 strain are 30℃, 30 salinity, and 7.0; the conditions most conducive to infection of Porphyra filaments are 28℃, 30 salinity, and 7.0.

[0007] The strains provided by this invention can be used as experimental strains for screening drugs or biocontrol agents for the prevention and treatment of yellow spot disease in seaweed.

[0008] The strains provided by this invention can be used in breeding to screen and identify superior laver strains with resistance to yellow spot disease.

[0009] This invention screened and obtained a Vibrio strain Vibrio sp. NH-7-1, verified by Koch's postulates, is the causative agent of yellow spot disease in laver (Porphyra oleracea). It causes yellow spots and rough shell symptoms on the filamentous shells, and leads to greening, rotting, and pores in the thallus tissue. The acquisition of this strain provides a crucial pathogen model for early warning of laver diseases and for disease-resistant breeding. Attached Figure Description

[0010] Figure 1 Photograph of the filamentous structure of a laver shell suffering from yellow spot disease; Figure 2 : Observation of symptoms of laver shell filaments caused by strain NH-7-1 (A) and scanning electron micrograph (B); Figure 3 Symptoms observed in various macroalgae infected by strain NH-7-1; Figure 4 Colony morphology (A), transmission electron microscopy (B), and genome UPGMA and ANI analysis (C) of strain NH-7-1; Figure 5 Figure 1: Optimal growth (A, B) and infection conditions (C) of strain NH-7-1. Detailed Implementation

[0011] The present invention will now be described in detail with reference to specific embodiments and accompanying drawings.

[0012] Example 1: Isolation and purification of pathogenic bacteria The filamentous bodies of *Porphyra yezoensis* shells suffering from yellow spot disease were collected in August 2022 from a *Porphyra yezoensis* seedling farm in Ganyu, Jiangsu Province, China (34°18'N, 119°18'E). During the sampling period, the temperature fluctuated between 26°C and 30°C, the seawater salinity was 28, and the pH was 8. In shells with milder yellow spot disease, yellow spots appeared on the filamentous parts, and in some areas, the color of the filamentous bodies changed from dark brown to orange-red. Figure 1 In severe cases, large areas of the shell are bleached, the surface is rough and dull, and filamentous structures cannot grow.

[0013] Rinse the shell filaments with sterile seawater and collect the seawater. Scrape the lesion site with a sterile scalpel and mix it with the seawater. Dilute the sample 1000 times, and spread 200 μL of the diluted solution onto ZoBell 2216E, TSA, TSB, R2A, and TCBS agar media with a salinity of 28. After bacterial growth, pick single colonies and streak them at least twice to isolate and purify the strain. Preserve the obtained strain using 20% ​​glycerol and store at -80℃ until use.

[0014] The strain NH-7-1 obtained through the above isolation and purification was verified by Koch's postulate to cause yellow spots and rough shell symptoms in the filamentous body of seaweed shells, and thus has pathogenicity.

[0015] Example 2: Koch's postulates verify the pathogenicity of NH-7-1 Frozen NH-7-1 was inoculated into ZoBell 2216E liquid medium and incubated at 25°C and 150 r·min. -1 Activation culture for 12 h. Add to fresh liquid culture medium at a ratio of 1:100 (v / v) and incubate until the logarithmic growth phase. Centrifuge at 2775 ×g for 5 min, discard the supernatant, and wash twice with sterile seawater. Finally, resuspend in NBU-3 medium, adjusting the bacterial suspension concentration to approximately 1.0 × 10⁻⁶. 7 CFU / mL.

[0016] Laver ( Neoporphyra haitanensis cv. ZD-1) free filaments were obtained from the large seaweed germplasm bank of Ningbo University. The free filaments of *Porphyra yezoensis* were broken into fragments of approximately 200 μm to 300 μm using a mixer, and then evenly sprayed onto sterile seashells. After dark treatment for 48 h, they were then exposed to light at 20℃ and 30 μmol·photons·m⁻¹. -2 ·s -1 They were cultured under a photoperiod of 12 h: 12 h (L: D) until the filaments covered the shells and the shell surface turned purplish-red.

[0017] Collect shell filaments and immerse the shells in bacterial suspension. Infection conditions were: 25℃, light intensity 30 μmol·photons·m⁻¹. -2 ·s -1 The photoperiod was 12 h:12 h (L:D). Algae cultured in NBU-3 medium served as a control group. Each treatment was performed in triplicate. Symptoms were observed every 5 days. The results showed that, in addition to causing typical yellow spot symptoms in the shell filaments, strain NH-7-1 also resulted in a rough shell surface.

[0018] like Figure 2As shown in Figure A, yellow spots began to appear on the edges of the shell filaments 10 days after NH-7-1 infection; 30 days after infection, the yellow spots on the edges of the shell filaments gradually enlarged, and a larger yellow patch appeared in the center of the shell, with the overall color of the shell filaments becoming lighter. At 60 days, the entire shell filament turned yellow, indicating that all the filaments had died.

[0019] Meanwhile, shell filaments infected with NH-7-1 for 30 days also showed symptoms of rough shells. Figure 2 B). Under infection conditions at 25℃, the shell surface loses its normal luster, and the shell appears porous and rough under a scanning electron microscope. At 30℃, the shell surface becomes extremely rough and uneven, and the pores under the electron microscope are even larger, with the rough shell symptoms becoming more pronounced.

[0020] From the shell filaments exhibiting typical symptoms, the strain was re-isolated and purified (using the same method as in Example 1). 16S rRNA gene sequence alignment confirmed that the re-isolated strain was the same as the initially inoculated strain NH-7-1, thus verifying Koch's postulate.

[0021] Example 3: Broad-spectrum algicidal function of NH-7-1 ZD-1 *Porphyra yezoensis* thallus was collected from aquaculture areas in the waters off Ningbo, Zhejiang Province (121°54′N, 29°43′E). *Asparagus cochinchinensis* (also known as *Asparagus setaceus*) Gracilariopsis lemaneiformis cv. 981) was from a farm in Xiapu, Fujian (26°65′N, 119°66′E). *Cyclocarya longicornis* ( Kappaphycus alvarezii ) and Heterocarya kappa ( Kappaphycus striatum The algae were provided by the Hainan Provincial Academy of Marine and Fisheries Sciences. The algae were aseptically treated with a compound antibiotic (final concentration of ampicillin 300 μg / mL; kanamycin 100 μg / mL; gentamicin 100 μg / mL) for 16 h, followed by washing off the antibiotic. The infection experiment was performed according to Example 2, with an algal:bacterial suspension ratio of 1 g:1 L (w:v).

[0022] like Figure 3 As shown, NH-7-1 can infect various large algae, including *Porphyra yezoensis*, *Kappa algae*, and *Gracilaria sanguinalis*. The strain causes yellowing of *Porphyra yezoensis* leaves, followed by rotting and dissolution starting from the leaf edges, with holes appearing in the center of the leaves, and the leaf tissue around the edges and holes turning green and rotting. Three days after infection, the tips of *Kappa algae* branches fade from green to yellow, with severely affected areas becoming bleached. The branches of *Kappa algae* become entirely discolored, with large areas of bleached white patches appearing in severely affected areas. Three days after infection, the tips of *Gracilaria sanguinalis* branches fade, with yellow lesions appearing in severely affected areas.

[0023] Example 4: Whole genome sequencing, transmission electron microscopy, and biochemical identification of NH-7-1 NH-7-1 was sent to the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20252514 and deposit date of November 10, 2025. The deposit address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0024] On ZoBell 2216E agar medium, strain NH-7-1 forms milky white, round colonies with regular edges, smooth surfaces, and regular margins. Figure 4 A). Under transmission electron microscopy, strain NH-7-1 is elliptical, ranging from 1.0 μm to 2.0 μm in length and 0.5 μm to 1 μm in width, and possesses a single, robust flagellum (A). Figure 4 B). Biochemical tests showed positive reactions for glucose, sucrose, mannose, arginine dihydrolase, and 3% and 6% NaCl, while negative reactions were observed for arabinose, inositol, glucosamine hydrochloride (VP), peptone hydrochloride, lysine, and 0%, 8%, and 10% NaCl.

[0025] Total DNA was extracted from strain NH-7-1 and sent to Wuhan Fraser Gene Information Co., Ltd. The whole genome of the strain was sequenced and assembled using the PacBio sequencing platform. The genomic data were uploaded to the NCBI GenBank database (Bioproject number SAMN50932771). UPGMA and ANI analyses of the NH-7-1 genome showed that this strain is related to strains in the NR database. Vibrio sinaloensis The OrthoANI value was 97.59%, indicating a high degree of genetic similarity. Figure 4 C).

[0026] Based on the above identification results, NH-7-1 was named Vibrio sp. NH-7-1.

[0027] Example 5: Optimal growth and infection conditions of NH-7-1 Through single-factor experiments, it was clarified that V. sinaloensis The optimal growth temperature range for NH-7-1 is 25℃-35℃, with a salinity of 20-35 and a pH of 6.5-7.5. Figure 5 A). Response surface methodology was then used for testing. V. sinaloensis The optimal growth conditions for NH-7-1 were determined. The quadratic polynomial regression equation established after quadratic polynomial regression analysis is as follows: OD 600 = 0.7680 + 0.0065 × A + 0.0065 × B + 0.0074 × C + 0.1289 × A × B + 0.0239 × A × C - 0.0357 × B × C - 0.4516 × A 2 -0.1371×B2 -0.0710×C 2 ( p <0.01). Experimental results show that 30℃, salinity 30, and pH 7.0 are the optimal growth conditions for NH-7-1. Figure 5 B). The influence of the three environmental factors on the growth of strain NH-7-1 was in the following order: temperature > salinity > pH.

[0028] Based on the optimal growth of the strain and the suitable growth conditions of laver, further design Vibrio sp. Response surface methodology for optimal infection conditions of NH-7-1. The regression model obtained from the response surface methodology for optimal infection conditions is: Disease erosion rate (%) = 22.33 + 22.083 × A + 2.5 × B + 2.9167 × C + 10 × AB - 1.5825 + BC + 15.50 × A 2 +6.33×B²-7.8333×C² ( p <0.01). Experimental results showed that the most susceptible conditions for infection of strain NH-7-1 were: 28℃, salinity 30, and pH 7.0, which were similar to its optimal growth conditions. Figure 5 C).

[0029] The strain of Vibrio sinaloa of this invention Vibrio sp. NH-7-1 is the pathogen of yellow spot and rough shell disease in laver (Porphyra yezoensis) and also has an algicidal effect on various economically important red algae. This strain can be used to establish pathogen detection methods for yellow spot and rough shell disease in laver, assess the disease resistance of laver germplasm resources, and screen antagonistic agents or drugs that can inhibit this pathogen, providing direct targets and effective means for the control of this disease.

Claims

1. A strain of Porphyridium pathogenic bacteria, characterized in that, The pathogenic strain of laver is Vibrio sinaloa, with the preservation number CCTCC NO:M 20252514.

2. The pathogenic strain of laver as described in claim 1, characterized in that, The optimal growth conditions for the pathogenic strain of laver are a temperature of 30℃, a salinity of 30, and a pH of 7.

0.

3. The application of the strain described in claim 1 in infecting porphyria filaments to establish a model of yellow spot disease and rough husk symptom.

4. A method for preparing a model of yellow spot disease and rough shell of filamentous laver shells, characterized in that, The method described in claim 1 involves infecting the filamentous tissue of *Porphyra yezoensis* to prepare a model.

5. The method as described in claim 4, characterized in that, The infection conditions were a temperature of 28°C, a salinity of 30, and a pH of 7.

0.

6. The application of the strain described in claim 1 as an experimental strain for screening drugs or biocontrol agents for the prevention and treatment of yellow spot disease in laver.

7. The application of the strain described in claim 1 in the screening and identification of laver strains with resistance to yellow spot disease in breeding.

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