A conditional pathogen of Porphyra

Abstract

The present invention provides a conditional pathogenic bacterium of laver thallus and filaments, which is Vibrio ( Vibrio sp. ) NH-13 strain, whose deposit number is CGMCC No.25727. The Vibrio spp. conditionally pathogenic bacteria of laver screened and obtained by the present invention can cause the disintegration of laver pigment bodies and cell vacuolization. The isolation and discovery of the strain is conducive to the advancement of laver disease early warning work and can be used to screen laver strains with disease resistance.

Description

Technical Field

[0001] The invention belongs to the technical field of disease prevention and control of cultured seaweed, and in particular relates to conditional pathogenic bacteria of laver thallus and filaments. Background Art

[0002] Laver ( Porphyra Sensu lato is an important cultivated seaweed in my country, with an annual output value of over 10 billion yuan. Its production process includes two stages: indoor cultivation of filaments and cultivation of thallus in the sea. As the scale of production continues to expand, the laver industry is also facing many difficulties. First, problems such as germplasm degradation and improper management are becoming more prominent. Second, diseases frequently occur during laver seedling cultivation and sea cultivation, resulting in a decline in yield and quality. According to incomplete statistics, the economic losses caused by laver diseases exceed 100 million yuan each year.

[0003] The causes of laver diseases are complex and are usually caused by a variety of pathogens. The early symptoms of the disease are slow and difficult to detect. However, when lesions or rots are visible, the disease has often entered a stage of rapid deterioration. At this time, the disease develops very rapidly and is difficult to control. In addition, the symptoms of laver diseases caused by different pathogens may be the loss of color of laver cells, the appearance of yellow or white spots on shell filaments, and leaf rot. The symptoms of different types of diseases may be difficult to distinguish. During the occurrence of the disease, the algal microbial flora will undergo drastic changes. Environmental changes and imbalances in microbial communities may cause some microorganisms that were initially harmless to algae growth to become pathogens, that is, conditional pathogens. This makes the pathogenic factors of laver diseases more complex and changeable, bringing great difficulties to the prevention and control of laver diseases. Therefore, the discovery of conditional pathogens is of great significance to the prevention and control of laver diseases. Summary of the invention

[0004] The purpose of the present invention is to provide a conditional pathogen of laver thallus and filaments. Research on the pathogenic bacteria can be used to explore the causes of complex diseases of laver, which is beneficial to the optimization of prevention and control technologies for potential diseases of laver.

[0005] The present invention first provides a conditional pathogenic bacterium Vibrio ( Vibrio sp. ) NH-13 strain, deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number CGMCC No.25727 and the deposit date September 13, 2022; the deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing.

[0006] The 16S sequence of the NH-13 strain is SEQ ID NO: 1;

[0007] The strain provided by the present invention can be used in the screening and evaluation of the culture method for detecting the thallus and filaments of Porphyra, and can be used to determine the culture conditions for avoiding the disease of Porphyra.

[0008] The strain provided by the present invention can also be used for screening of algae disease-resistant strains.

[0009] The strain provided by the invention can be applied to the preparation of algae disease diagnosis chips.

[0010] The conditional pathogenic vibrio of laver thallus and filaments screened by the present invention can cause the disintegration of laver chromatosomes and cell vacuolization. The isolation and discovery of the strain is conducive to the advancement of laver disease early warning work and can be used to screen laver strains with disease resistance. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 : Photographs of the course of disease observed when strain NH-13 infected Porphyra thallus;

[0012] Figure 2 :Photographs of the course of disease observed in the case of infection of Porphyra conchocelis with strain NH-13;

[0013] Figure 3 : Maximum likelihood phylogenetic tree of 16S rRNA gene sequences of strain NH-13 and its closely related species, where Vibrio gallicus For the outer group.

[0014] Figure 4 : UPGMA tree and heat map generated based on OrthoANI values ​​of NH-13 strain genome;

[0015] Figure 5 : Transmission electron microscopy image of Vibrio NH-13;

[0016] Figure 6 : Transmission electron microscopy of Vibrio NH-13, where C: chloroplast; M: mitochondria; P: pyrenoid; V: Vibrio Vibrio mytili NH-13; W: cell wall; N: nucleus; Fs: starch core; Plg: plastid globules; Pit: pore connection. DETAILED DESCRIPTION

[0017] The present invention is described in detail below in conjunction with specific embodiments and drawings.

[0018] Example 1: Isolation and purification of pathogenic bacteria

[0019] In November 2021, bacteria were isolated from the healthy laver "Zhedong No. 1" in a laver nursery in Zhanqi, Zhejiang. The laver surface was rinsed 3 to 5 times with sterile seawater (filtered with a glass fiber filter, heated to boiling, boiled for 5 min, and then cooled for use), and then wiped dry with sterile cotton balls (sterilized by wet heat). 10 mg of laver was placed in a homogenate grinding tube filled with 1 mL of sterile seawater for grinding. The homogenate was then diluted to 10 with sterile seawater. -2times.

[0020] Prepare R2A oligonutrient solid and liquid culture media with a salinity of 28‰ respectively. Sterilize 1 / 10 of R2A liquid culture medium (powder weighed in 1 L volume; R2A components: tryptone, acid hydrolyzed casein, yeast extract powder, soluble starch, dipotassium hydrogen phosphate, magnesium sulfate, sodium pyruvate, peptone, glucose; pH 7.2 ± 0.2), sea salt solution (to make the final salinity of the culture medium 28‰) and agar liquid respectively, and the total volume of the above three liquids is 1 L. After sterilization, mix all the liquids and pour them on the plate, which is the R2A oligonutrient solid culture medium with a salinity of 28‰. The purpose of separate sterilization is to prevent the agar and phosphate in the culture medium from producing hydrogen peroxide (H2O2) in a high temperature environment, inhibiting bacterial growth. Mix 1 / 10 of the R2A liquid culture medium with the sea salt solution with a total volume of 1 L, which is the R2A oligonutrient liquid culture medium with a salinity of 28‰.

[0021] Use the oligotrophic enrichment method to culture the epiphytic bacteria of Porphyra. Take 500 μL of the diluted algae homogenate solution and inoculate it into 50 mL of sterilized 1 / 10 R2A liquid medium, and enrich and culture it at 25°C for 1d to 15d. At 5d, 100 μL of the enriched culture solution was diluted and spread on R2A oligotrophic solid medium, and cultured in a constant temperature incubator at 25°C for 24h to 48h. Then, use the streak inoculation method to pick a single colony to purify the isolated strain, and number the obtained strain. After the purified strain is numbered, store it in 20% glycerol at -80°C.

[0022] The infection experiments of laver thallus and shell filaments showed that the strain NH-13 obtained by culturing for 5 days and purifying and isolating by the above oligotrophic enrichment method was pathogenic.

[0023] Example 2: Study on pathogenicity of bacterial strains

[0024] Remove the NH-13 strain from -80°C, inoculate it into 1 / 10 R2A liquid culture medium, and culture it overnight at 25°C and 150 r / min. Inoculate the strain into a new liquid culture medium at a ratio of 1% (1:100) and culture it for 16 hours. Collect the bacterial solution in a sterile centrifuge tube and centrifuge it at 5000 g for 5 minutes. After centrifugation, discard the supernatant and take the precipitate. Resuspend and wash with an appropriate amount of sterile nutrient seawater, and repeat 3 times. After resuspending and washing, add an appropriate amount of sterile nutrient seawater to resuspend and make a bacterial suspension. Adjust the bacterial concentration to 10 7 CFU / ml or so for future use.

[0025] The thallus of Porphyra haitanensis used for infection was provided by the algae seed bank of the Zhejiang Provincial Key Laboratory of Marine Bioengineering. The thallus was cultured in sterile seawater and Ningda No. 3 mother solution was added. The nutrient concentration in the final culture medium was 100 mg / L KNO3, 10 mg / L KH2PO4, 2.5 mg / L FeSO4, 0.25 mg / L MnSO4 and 20 mg / L EDTA-Na2, culture conditions were 20℃, light intensity was 30 μmol·photons·m -2 ·s -1 and photoperiod of 12 h:12 h (L:D). Antibiotics were used to sterilize the Porphyra thallus. The final concentration of antibiotics was 300 μg / mL ampicillin, 100 μg / mL kanamycin and 100μg / mL gentamicin, and the culture was carried out in the dark for 18 h. Then, the culture medium was washed three times with sterile seawater to remove the antibiotic residues, transferred to fresh culture medium and continued to be cultured under the normal photoperiod of 12 h:12 h (L:D). Fresh culture medium was replaced again after 12 h. After standing for 2 days, the Porphyra thallus was taken and a hole was punched in the middle of the leaf using a 6 mm hole puncher. The Porphyra thallus discs were cultured in a six-well plate, and 3 thallus discs were added to each well. The above bacterial suspension was added to the six-well plate, and the infection conditions were: temperature 25℃, light intensity 30 µmol photons·m -2 ·s -1 , photoperiod 12 h: 12 h (L:D). Figure 1 As shown in the figure, 3 days after the NH-13 strain was used to infect the thallus of Porphyra, cells in some parts of the center of the Porphyra leaf began to die, and cells at the edge of the leaf became vacuolated. After 7 days of infection, almost all Porphyra cells died, and the edges of the leaves became ulcerated and white.

[0026] The shell filaments of Porphyra haitanensis used for infection were provided by the algae seed bank of the Zhejiang Provincial Key Laboratory of Marine Biotechnology. Take three or more shell filaments of Porphyra haitanensis with the same growth, add the above-mentioned concentration of bacterial solution until the shells are submerged, and replenish the bacterial suspension in time during the infection process. The infection conditions are the same as above. Observe the morphology of the shell filaments every day after infection and take photos to record. Figure 2 As shown in the figure, in the early stage of infection (0 d ~ 10 d), bleached stripes or patches appeared in the center or edge of the shell of the Porphyra filaments. The course of the disease progressed slowly. In the late stage of the disease (20 d ~ 30 d), the shells were completely bleached, with a rough surface and severe damage to the nacre. The shells of the control group at 30 d did not show bleaching.

[0027] Example 3: 16S rRNA coding sequence analysis, whole genome sequencing and strain characteristics of NH-13

[0028] Single colonies on NH-13 plates were picked for colony PCR, and the following primers (sequence information: 27F: 5'-GAGTTTGATCCTGGCTCAG-3'; 1429R: 5'-GGTTACCTTGTTACGACTT-3') were used for PCR amplification of 16S rRNA and sent for testing. The sequencing results were compared with NCBI and phylogenetic trees ( Figure 3 ), the bacteria were identified as Vibrio The 16S sequence of the bacterium is SEQ ID NO: 1:

[0029]

[0030] 50 mL of NH-13 bacterial solution was sent to Wuhan Fraser Gene Information Co., Ltd. for whole genome third-generation sequencing. The sequencing results were analyzed by ANI Calculator and the strain was named Vibrio Vibrio mytili NH-13 strain ( Figure 4 The strain is deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, with the deposit number CGMCC No.25727 and the deposit date September 13, 2022; the deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing.

[0031] Vibrio Vibrio mytili ) The colonies of NH-13 strain are opaque, milky white, with convex surfaces and regular edges. Transmission electron microscopy shows that Vibrio NH-13 is oval in shape and has a single, thick flagellum ( Figure 5 The biochemical test was performed using the Vibrio family bacterial biochemical identification tube (Hangzhou Microbiological Reagent Co., Ltd., China) according to the instructions to evaluate the growth and metabolic characteristics of Vibrio NH-13, and the results of glucose, sucrose, arabinose, and arginine dihydrolase were all positive.

[0032] Example 4: Observation of pathological characteristics of NH-13 based on transmission electron microscopy

[0033] The free filaments of Porphyra haitanensis used for infection were provided by the algae seed bank of Zhejiang Provincial Key Laboratory of Marine Bioengineering. Before the infection experiment, the filaments were broken into a length of 200 µm ~ 300 µm, and after one week of recovery, they were sterilized with antibiotics. The culture conditions and antibiotic sterilization process were the same as in Example 1. Take an appropriate amount of pretreated free filaments of Porphyra haitanensis in a conical flask, add 50 mL of bacterial solution and co-culture with it. Add 50 mL of sterile nutrient seawater to the blank experimental group for co-culture. The culture conditions are: temperature 25°C, light intensity 30 μmol·photons·m -2 ·s -1 The photoperiod was 12 h: 12 h (L:D). Free filaments of Porphyra were collected for transmission electron microscopy at 24 h and 48 h of culture, and it was found that Vibrio NH-13 secreted extracellular substances on the surface of the filaments to form a biofilm-like structure ( Figure 6 The contents of the filamentous cells attached to the fungus flowed out, the chromatin bodies were severely disintegrated, and the cells eventually became vacuolated.

[0034] The filaments of Porphyra infected by Vibrio NH-13 were selected, and the isolation and purification steps of the pathogenic bacteria in Example 1 were repeated. The same bacteria were identified by 16SrRNA molecule. Koch's postulates verified that the strain was the pathogenic bacteria of Porphyra.

[0035] The strain Vibrio NH-13 of the present invention is separated from healthy laver thallus, and infection experiments have confirmed that it can cause the death of laver thallus and filaments, and is a conditional pathogen. The pathogen can be used to explore the cause of complex laver diseases, and the strain provided by the present invention can be used for algae disease prevention and control and screening of disease-resistant strain breeding.

Claims

1. A conditionally pathogenic strain of Porphyra thallus and filaments, characterized in that: The conditionally pathogenic strain is Vibrio ( Vibrio sp. ), and its deposit number is CGMCC No.25727.

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