Antagonistic fungus to alginic acid-degrading bacteria and its application

Abstract

The present invention belongs to the technical field of disease prevention and control of cultivated seaweed, and specifically relates to an antagonistic fungus of alginic acid-degrading bacteria and its application. The antagonistic fungus is Aspergillus niger F-DL4, which was deposited in the General Microbiological Center of China Microbiological Culture Collection Administration on August 30, 2024, with a deposit number of CGMCC No.41493, and is classified as Aspergillus niger Aspergillus niger, The storage address is Beijing, China.

Description

Technical Field

[0001] The invention belongs to the technical field of disease prevention and control of cultured seaweed, and particularly relates to an antagonistic fungus of alginic acid-degrading bacteria and an application thereof. Background Art

[0002] Numerous microorganisms attach to or coexist with the surface of kelp. As kelp grows, it continuously releases large amounts of organic matter, which is beneficial to microbial growth, attracting them to the kelp and allowing them to continue growing. Simultaneously, the kelp produces chemical defenses such as antibiotics and peroxides to counteract microbial attachment and protect its own growth. Microorganisms utilize nutrients released by or on the surface of the kelp to grow, producing enzymes and toxins that affect their hosts. Furthermore, they adapt to the environment, counteracting the kelp's chemical defenses and developing new metabolic pathways. The formation of this microbial environment within the kelp biosphere is the result of coevolution and mutual selection between kelp and microorganisms.

[0003] Studies have shown that alginate-degrading bacteria are the main epiphytic bacteria on kelp. Excessive growth of alginate-degrading bacteria often causes kelp disease and rot during aquaculture. Because alginate-degrading bacteria can produce alginate, they can enzymatically degrade the cell walls and alginate within the kelp, leading to kelp disease and rot, which poses a significant threat to kelp aquaculture. Currently, domestic efforts to prevent and control kelp diseases remain limited to screening disease-resistant kelp varieties, optimizing kelp culture conditions, and using chemical control methods. For example, efforts to control kelp diseases utilize the antibacterial effects of kelp brown algae polyphenols. However, pathogens proliferate rapidly, and the antibacterial spectrum of chemical defense substances is limited. Further research is needed to investigate the prevention and control of kelp diseases.

[0004] Biological control technology utilizes the antagonistic effects between microbial biocontrol bacteria and pathogens to alter the microbial ecosystem on the surface of their host, promoting the proliferation of biocontrol bacteria and inhibiting the growth of pathogens, thereby reducing the incidence of disease. By utilizing the antagonistic effects between kelp biocontrol bacteria and alginate-degrading bacteria, the proliferation of alginate-degrading bacteria can be reduced during kelp seedling cultivation, minimizing the harm they cause. Furthermore, the use of kelp biocontrol bacteria is an environmentally friendly disease control strategy that can promote kelp growth and development or enhance kelp's disease resistance. This not only reduces the occurrence of kelp diseases and promotes the expansion of kelp aquaculture, but also avoids the environmental hazards caused by the use of chemical pesticides. Summary of the Invention

[0005] The purpose of the present invention is to provide an antagonistic bacterium to alginic acid-degrading bacteria and application thereof.

[0006] To achieve the above purpose, the technical solution adopted by the present invention is:

[0007] An antagonistic fungus to alginic acid-degrading bacteria, the antagonistic fungus is Aspergillus niger F-DL4, which was deposited in the General Microbiology Center of the China Microorganism Culture Collection Administration on August 30, 2024, with the deposit number CGMCC No.41493, and is classified as Aspergillus niger Aspergillus niger , the storage address is Beijing, China.

[0008] The Aspergillus niger F-DL4 can be used to alleviate the kelp disease and rot caused by alginate-degrading bacteria.

[0009] The culture temperature of the Aspergillus niger F-DL4 is preferably 30°C.

[0010] An application of the alginic acid-degrading bacteria as an antagonistic fungus, and an application of the Aspergillus niger F-DL4 in alleviating kelp disease and rot.

[0011] The Aspergillus niger F-DL4 is used in alleviating kelp disease and rot caused by alginic acid degrading bacteria.

[0012] The alginic acid degrading bacteria is Vibrio flexneri Vibrio fumissii .

[0013] A bacterial agent for preventing or alleviating kelp seedling disease and rot, the bacterial agent contains the antagonistic fungus.

[0014] The bacterial agent is the fermentation liquid, fermentation culture liquid or fermentation bacterial suspension of the bacteria.

[0015] An application of the bacterial agent, which is an application of the bacterial agent in preventing or alleviating disease and rot of kelp seedlings.

[0016] The bacterial agent is used in preventing or alleviating kelp seedling disease and rot caused by alginic acid-degrading bacteria.

[0017] The alginic acid degrading bacteria is Vibrio flexneri Vibrio fumissii .

[0018] The Vibrio flexneri was deposited in the General Microbiology Center of China Culture Collection Administration on April 23, 2021, with the deposit number CGMCC No. 22230 and the classification name Vibrio flexneri Vibrio fumissii , the storage address is Beijing, China.

[0019] The bacterial agent is used in preparing an antagonistic bacterial agent for pathogenic bacteria of kelp seedling albinism / green rot caused by alginic acid degrading bacteria.

[0020] The advantages of the present invention are:

[0021] The strain antagonism experiment confirmed that the Aspergillus niger F-DL4 strain obtained by the present invention can antagonize Vibrio flexneri Vibrio fumissiiThe strain provided by the present invention can be used to prevent and alleviate the disease and rot process of kelp seedlings, significantly reduce the incidence of kelp albinism or green rot, and provide a new method and new idea for the prevention and treatment of kelp disease and rot. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The F-DL4 provided in the embodiment of the present invention is effective against alginic acid degrading bacteria Vibrio flexneri Vibrio fumissii The antagonistic effect diagram of F-DL4 against alginic acid degrading bacteria Vibrio flexneri is shown in Figure 3, where the transparent circle represents the antagonistic effect of F-DL4 on alginic acid degrading bacteria Vibrio flexneri. Vibrio fumissii The inhibition effect is good, and the radius of the inhibition zone is 5mm.

[0023] Figure 2 This is a colony morphology diagram of F-DL4 provided in an embodiment of the present invention.

[0024] Figure 3 This is a diagram showing the tissue morphology observation of kelp tissue blocks at different stages provided by an embodiment of the present invention. DETAILED DESCRIPTION

[0025] The present invention is described in detail below with reference to specific embodiments and accompanying drawings.

[0026] Example 1: Isolation and purification of strains

[0027] In September 2023, fresh seaweed was collected from the Yantai coastline in Shandong Province, stored in a sampling box with ice packs, and transported back to the laboratory for isolation and purification of surface-attached bacteria. Kelp seedlings were rinsed with sterile seawater to remove surface impurities. Appropriate amounts of seaweed (kelp and its associated algae, such as red algae, brown algae, and short-bearded algae) were ground in a mortar and pestle and then added with quartz sand. Once a homogenate formed, the seaweed tissue fragments were rinsed with sterile water and transferred to a 1 mL eppendorf tube. The tubes were centrifuged at 3000 rpm / min for 5 minutes at room temperature. The supernatant and sub-layer fluid obtained from the centrifugation were quantitatively plated onto the surface of PDA solid culture medium, with a volume of 100 μL of the supernatant and 75 μL of the sub-layer fluid respectively. The PDA solid culture medium was placed in a constant temperature incubator at 30°C and observed every day for the growth of colonies. After the colonies grew, single colonies were picked out and purified twice before being inoculated into the PDA solid culture medium. The colonies were preserved using the test tube slant method and placed in a 4°C refrigerator.

[0028] PDA solid culture medium consists of 200.0 g / L potato (peeled), 20.0 g / L glucose, and 14.0-20.0 g / L agar.

[0029] Example 2: Screening for Antagonistic Bacteria to Kelp Albinism / Green Rot Pathogens (Alginic Acid Degrading Bacteria) (Alginic Acid Degrading Bacteria Strain Provided by Our Laboratory)

[0030] The fungi that produce filamentous colonies were tested using the plate confrontation method to determine whether they were antagonists of alginic acid-degrading bacteria. The fungal strains stored in the test tubes were inoculated on PDA solid culture medium, and the culture medium was placed in a 30°C constant temperature incubator for 48 hours. At the same time, the alginic acid-degrading bacteria Vibrio flexneri were cultured. Vibrio fumissii The bacterial suspension was shaken and cultured until OD 600 The value is about 0.5-1 (culture conditions are 180rpm / min, 30℃). When the fungal hyphae grow more luxuriantly, use a scalpel to take a bacterial disc with a diameter of 10mm (including culture medium), and take 100μL of alginate-degrading bacterial suspension to spread on the sodium alginate solid culture medium, and then place the fungal bacterial disc in the center of the sodium alginate culture medium. Place the sodium alginate solid culture medium in a 30℃ constant temperature incubator and culture for 48h. Observe whether there is an inhibition ring and measure the radius of the inhibition ring. Among them, the inhibition ring of F-DL4 is the largest, and the radius of the inhibition ring is ≥4mm, indicating that it has antibacterial activity. It has been verified that the F-DL4 strain has antibacterial activity against the alginate-degrading bacteria Vibrio flexneri. Vibrio fumissii The radius of the inhibition zone on the Figure 1 ), and has obvious antibacterial activity.

[0031] The sodium alginate solid culture medium is prepared by adding 5 g / L sodium alginate, 5 g / L ammonium sulfate, 1 g / L magnesium sulfate, 2 g / L potassium hydrogen phosphate, and 0.01 g / L ferrous sulfate per liter of distilled water.

[0032] Vibrio flexneri Vibrio fumissii Vibrio flexneri Vibrio fumissii Inoculate into sodium alginate liquid medium at a volume concentration of 2%, culture in a constant temperature shaker at 30°C and 180 rpm for 24 h, collect the bacteria by centrifugation at 8000 rpm for 10 min, wash twice with sterile water, resuspend in sterile water, and adjust the OD of each bacterial suspension. 600 The value is 1.0 (the bacterial concentration is about 8×10 8 CFU / mL).

[0033] The Vibrio flexneri was deposited in the General Microbiology Center of China Culture Collection Administration on April 23, 2021, with the deposit number CGMCC No. 22230 and the classification name Vibrio flexneri Vibrio fumissii , the storage address is Beijing, China.

[0034] Example 3: Molecular identification of antagonistic strains of Laminaria pathogenic bacteria

[0035] The F-DL4 strain was cultured in PDA medium. The mycelium grew densely, the colony morphology was circular, and black spores were produced. Based on the obtained ITS sequence sequencing results and the colony morphology observation, the strain F-DL4 was identified to the genus. After comparison with NCBI, the selected strain F-DL4 was found to be similar to the Aspergillus genus. Aspergillus niger The strain is Aspergillus niger. Aspergillus niger, It is deposited in the General Microbiology Center of China Culture Collection Administration, with the deposit number CGMCC No.41493, the deposit date is August 30, 2024, and the deposit address is Beijing, China.

[0036] Example 4: Antagonistic Experiment of Kelp Tissue

[0037] The highly antibacterial biocontrol antagonistic bacterial strain F-DL4 obtained above was inoculated together with Vibrio flexneri on kelp tissue pieces and the antagonistic effect was observed to complete the retest. The specific experimental method is as follows:

[0038] A control group was set up, which consisted of kelp tissue blocks that were surface-sterilized (soaked in 75% alcohol for 2 minutes and then rinsed with sterile water 5 times) (the inoculation operation was as follows: a 2-4 mm incision was made on the kelp tissue block using a sterilized scalpel, and a filter paper with an appropriate amount of sterile seawater was placed under the culture dish for moisturizing. The kelp tissue block was placed in the culture dish and placed in a 20°C constant temperature incubator without direct light for static culture).

[0039] The infection group was a kelp tissue block inoculated with Vibrio flexneri after surface disinfection (the inoculation operation was: use a sterilized scalpel to cut a 2-4 mm incision on the kelp tissue block, and then take the OD 600 200 μL of the above-mentioned Vibrio flexneri bacterial suspension was inoculated into the incision, and a filter paper with an appropriate amount of sterile seawater was placed under the culture dish to keep it moist. The kelp tissue block was placed in the culture dish and placed in a 20°C constant temperature incubator without direct light for static culture).

[0040] The experimental group was a kelp tissue block inoculated with antagonistic bacteria F-DL4 and Vibrio flexneri after surface disinfection (the inoculation operation was: use a sterilized scalpel to cut a 2-4 mm incision on the kelp tissue block, and then take the OD 600 200 μL of the bacterial suspension of Vibrio flexneri and F-DL4 was inoculated into the incision, and a filter paper with an appropriate amount of sterile seawater was placed under the culture dish to keep it moist. The kelp tissue block was placed in the culture dish and placed in a constant temperature incubator at 20°C without direct light for static culture).

[0041] The bacterial suspension of F-DL4 is Aspergillus niger Aspergillus niger Inoculate onto potato dextrose agar slant and culture until abundant spores are produced. Add distilled water for elution and dilute with distilled water to prepare OD600 A bacterial suspension of 1.

[0042] Antagonistic effects were tested by observing tissue sections: tissue sections were taken from kelp tissue blocks at day 0 and day 8, respectively. Sterilized scalpels were used to cut approximately 3 mm x 3 mm sections, which were then fixed with Carnoy's solution for 3 hours. The fixed sections were then stained overnight with lactofuchin stain and observed under a microscope at 100x oil immersion. Microphotography was then performed to histologically determine whether the kelp rot process was slowed.

[0043] Experimental results

[0044] The kelp tissue blocks on day 0 and day 8 were selected for microscopic observation. Figure 3 It can be seen from the kelp tissue sections on day 0 that healthy kelp cells are neatly arranged. After staining with lactic acid fuchsin dye, the cells appear light red, with consistent size and morphology, and no outflow of stored substances was observed. In contrast, the internal distribution of kelp tissue cells in the control group on day 8 became uneven, and plasmolysis occurred. Organelles such as chromatin, nucleus, and mitochondria were irregular in shape, and the chromatin lamellar structure was blurred. The internal contents of the lighter-colored gametocytes gathered toward the middle of the cell, the color at both ends of the cell became lighter, and the cytochrome distribution was uneven. The interior of the whitened gametocytes became vacuolated, the organelles completely dissolved, or only a small amount of chromatin remained, and the edges of the cell walls became rough.

[0045] Further comparison of the kelp tissue cells of the infected group and the control group on the 8th day showed that the kelp cells in the infected group were of different sizes and the degree of disordered arrangement was significantly increased. The alginate produced by alginate-degrading bacteria caused the kelp algae to soften significantly, resulting in loosening of the intercellular matrix. There were a large number of granular storage substances in the cells of the infected kelp endodermis, which were oval or round in shape. The intracellular structure was destroyed and even led to cell death. The dead cells fell off to form holes on the thallus.

[0046] Compared with the infected group, although the kelp tissue cells in the experimental group were still arranged in disorder and the cell sizes were uneven on the 8th day, the storage substances flowing out of the cell gaps were reduced and the number of ruptured cells was reduced, which indicated that the disease and rot of the kelp tissue had been significantly improved.

Claims

1. An antagonistic fungus to alginic acid-degrading bacteria, characterized in that: The antagonistic fungus is Aspergillus niger F-DL4, which was deposited in the General Microbiological Center of China Microbiological Culture Collection Administration on August 30, 2024, with the deposit number CGMCC No.41493, and the classification name is Aspergillus niger Aspergillus niger , the storage address is Beijing, China.

2. An application of the alginic acid degrading bacteria as claimed in claim 1 as an antagonistic fungus, characterized in that: The application of Aspergillus niger F-DL4 in alleviating kelp disease caused by alginate-degrading bacteria; the alginate-degrading bacteria is Vibrio freundii Vibrio fumissii .

3. A fungal agent for alleviating kelp seedling disease and rot, characterized in that: The bacterial agent contains the antagonistic fungus according to claim 1.

4. The bacterial agent according to claim 3, characterized in that The bacterial agent is the fermentation broth of the bacteria according to claim 1.

5. An application of the bacterial agent according to claim 3, characterized in that: The bacterial agent is used to alleviate the disease and rot of kelp seedlings caused by alginate-degrading bacteria; the alginate-degrading bacteria is Vibrio freundii Vibrio fumissii strains.

6. The use according to claim 5, characterized in that: The bacterial agent is used in preparing an antagonistic bacterial agent for pathogenic bacteria of albinism / green rot of kelp seedlings caused by alginate-degrading bacteria.

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