Antagonistic fungus of alginic acid degrading bacteria and application of antagonistic fungus

Abstract

The invention belongs to the technical field of disease prevention and control of cultured seaweed, and particularly relates to antagonistic fungi of alginic acid degrading bacteria and application of the antagonistic fungi. The antagonistic fungus is Aspergillus niger F-DL4, and is preserved in the China General Microbiological Culture Collection Center on August 30, 2024, the preservation number is CGMCC No.41493, the classification name is Aspergillus niger, and the preservation address is Beijing, China.

Description

Technical Field

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

[0002] There are many microorganisms attached or symbiotic on the surface of kelp. During its growth, kelp continuously releases a large amount of organic matter, which is beneficial to the growth of microorganisms and attracts them to attach to the kelp and continue to grow. At the same time, kelp produces chemical defense agents such as antibiotics or peroxides to fight against microbial attachment and protect its own growth. On the one hand, microorganisms use the nutrients released by or on the surface of kelp to grow and produce enzymes, toxins and other substances to affect the host during the growth process. On the other hand, they adapt to the environment to fight against the chemical defense produced by kelp and develop new metabolic pathways. The formation of this kelp algae microbial environment is the result of the co-evolution and mutual selection of kelp and microorganisms.

[0003] Studies have shown that alginate-degrading bacteria are the main epiphytic bacteria on kelp algae. The large-scale reproduction of alginate-degrading bacteria often causes kelp to rot during the cultivation process. Since alginate-degrading bacteria can produce alginate, alginate can enzymatically hydrolyze the cell wall of kelp and the alginate in the kelp algae, thus causing kelp to rot and cause great harm to kelp cultivation. At present, the prevention and control of kelp diseases in China still remains in the screening of kelp disease-resistant varieties, optimizing kelp culture conditions, and using chemical substances for prevention and control. For example, the antibacterial effect of kelp brown algae polyphenols is used to prevent and control kelp diseases, but pathogens proliferate rapidly and the antibacterial spectrum of chemical defense substances is limited. The prevention and control methods for kelp diseases still need further research.

[0004] Biological control technology refers to the use of the antagonism between microbial biocontrol bacteria and pathogens to change the microbial ecological environment on the surface of the host, promote the proliferation of biocontrol bacteria and inhibit the growth of pathogens, thereby achieving the purpose of reducing the occurrence of diseases. The antagonism between kelp biocontrol bacteria and alginate-degrading bacteria can reduce the proliferation of alginate-degrading bacteria during kelp seedling cultivation and reduce the harm it brings. At the same time, the use of kelp biocontrol bacteria is an environmentally friendly disease prevention and control strategy that can promote the growth and development of kelp or improve the disease resistance of kelp. It can not only reduce the occurrence of kelp diseases and promote the expansion of kelp aquaculture, but also avoid the use of chemical pesticides and other environmental hazards. Summary of the invention

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

[0006] To achieve the above object, the technical solution adopted by the present invention is: An antagonistic fungus to alginic acid-degrading bacteria, 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.

[0007] The Aspergillus niger F-DL4 can be used to alleviate the kelp disease and rot caused by alginate-degrading bacteria.

[0008] The culture temperature of the Aspergillus niger F-DL4 is preferably 30°C.

[0009] An application of the alginate-degrading bacteria as antagonists to fungi, and an application of the Aspergillus niger F-DL4 in alleviating kelp disease and rot.

[0010] The Aspergillus niger F-DL4 is used for alleviating kelp disease and rot caused by alginate-degrading bacteria.

[0011] The alginic acid degrading bacteria is Vibrio freundii Vibrio fumissii .

[0012] A bacterial agent for preventing or alleviating kelp seedling disease and rot, the bacterial agent contains the antagonistic fungus.

[0013] The bacterial agent is the fermentation liquid, fermentation culture liquid or fermentation bacterial suspension of the bacteria.

[0014] An application of the bacterial agent, which is application of the bacterial agent in preventing or alleviating disease and rot of kelp seedlings.

[0015] The bacterial agent is used in preventing or alleviating kelp seedling disease and rot caused by alginate-degrading bacteria.

[0016] The alginic acid degrading bacteria is Vibrio freundii Vibrio fumissii .

[0017] The Vibrio flexneri was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on April 23, 2021, with a deposit number of CGMCC No. 22230 and a classification name of Vibrio flexneri. Vibrio fumissii , the storage address is Beijing, China.

[0018] 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.

[0019] The advantages of the present invention are: The strain antagonism experiment confirmed that the Aspergillus niger F-DL4 strain screened by the present invention can antagonize Vibrio freundii 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

[0020] Figure 1 The F-DL4 provided in the embodiment of the present invention is effective against the alginic acid degrading bacteria Vibrio freundii Vibrio fumissii The antagonistic effect of F-DL4 on the alginic acid-degrading bacteria Vibrio freundii is shown in Figure 2. Vibrio fumissii The inhibition effect is good, and the radius of the inhibition zone is 5mm.

[0021] Figure 2 This is a colony morphology diagram of F-DL4 provided in an embodiment of the present invention.

[0022] 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

[0023] The present invention is described in detail below with reference to specific embodiments and accompanying drawings.

[0024] Example 1: Isolation and purification of strains In September 2023, fresh seaweed was collected from the coastline of Yantai, Shandong, stored in a sampling box with ice packs, and transported back to the laboratory to separate and purify surface epiphytes. The kelp seedlings were rinsed with sterile seawater to remove surface impurities. An appropriate amount of seaweed (kelp and its associated algae, such as red algae, brown algae, short dragon beard algae, etc.) was ground with a mortar and quartz sand was added. When the grinding was homogenized, the seaweed tissue fragmentation liquid was washed with sterile water and transferred to a 1 mL eppendorf tube. The speed was selected to be 3000 rpm / min and centrifuged at room temperature for 5 minutes. Finally, the supernatant and lower tissue fluid obtained by centrifugation were quantitatively coated on the surface of the PDA solid culture medium, with the upper supernatant liquid taking 100 μL and the lower tissue liquid taking 75 μL. The PDA solid culture medium was placed in a 30°C constant temperature incubator for culture, and observation was made every day to see whether colonies grew. After colonies grew, single colonies were picked out, purified twice, and inoculated into the PDA solid culture medium. The colonies were preserved using the test tube slant method and placed in a 4°C refrigerator.

[0025] PDA solid culture medium is potato (peeled) 200.0 g / L, glucose: 20.0 g / L, agar: 14.0-20.0 g / L.

[0026] Example 2: Screening of antagonistic bacteria against pathogenic bacteria (alginic acid degrading bacteria) of kelp albinism / green rot (alginic acid degrading bacteria strains were provided by our laboratory) The plate confrontation method was used to test whether the fungi that produced filamentous colonies were antagonists of alginate-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. Vibrio fumissii The bacterial suspension was shaken and cultured until OD 600 The value is about 0.5-1 (the culture conditions are 180rpm / min, 30℃). When the fungal mycelium grows more luxuriantly, use a scalpel to take a bacterial disc with a diameter of 10mm (including the culture medium), and take 100μL of the alginate-degrading bacterial suspension and apply it to the sodium alginate solid culture medium, and then place the fungal 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 it for 48h. Observe whether there is an antibacterial ring and measure the radius of the antibacterial ring. Among them, F-DL4 has the largest inhibition circle, and the radius of the inhibition circle 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 freundii. Vibrio fumissii The radius of the inhibition zone on the Figure 1 ), which has significant antibacterial activity.

[0027] 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 dipotassium hydrogen phosphate, and 0.01 g / L ferrous sulfate per liter of distilled water.

[0028] Vibrio flexneri Vibrio fumissii Vibrio freundii Vibrio fumissii Inoculate into sodium alginate liquid medium at 2% volume concentration, culture in a constant temperature oscillator at 30°C and 180 r / min for 24 h, collect the bacteria by centrifugation at 8000 r / min 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).

[0029] The Vibrio flexneri was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on April 23, 2021, with a deposit number of CGMCC No. 22230 and a classification name of Vibrio flexneri. Vibrio fumissii , the storage address is Beijing, China.

[0030] Example 3: Molecular identification of antagonistic strains of Laminaria pathogenic bacteria The F-DL4 strain was cultured in PDA medium. The mycelium grew densely, the colony morphology was circular, and black spores were produced. The strain F-DL4 was identified to the genus based on the ITS sequence sequencing results combined with the colony morphology observation. After NCBI comparison, the selected strain F-DL4 strain 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 Microorganism Culture Collection Administration, with the deposit number CGMCC No.41493, the deposit date is August 30, 2024, and the deposit address is Beijing, China.

[0031] Example 4: Antagonistic experiment of kelp tissue The above-obtained high antibacterial activity biocontrol antagonistic bacteria strain F-DL4 and Vibrio freundii were inoculated on the kelp tissue block and the antagonistic effect was observed to complete the re-test. The specific experimental method is as follows: A control group was set up, which consisted of kelp tissue blocks with surface disinfection (soaked in 75% alcohol for 2 minutes and then rinsed with sterile water for 5 times) (the inoculation operation was as follows: use a sterilized scalpel to make a 2-4 mm incision on the kelp tissue block, place filter paper with an appropriate amount of sterile seawater under the culture dish for moisturizing, place the kelp tissue block in the culture dish and place it in a 20°C constant temperature incubator without direct light for static cultivation).

[0032] The infection group consisted of kelp tissue blocks inoculated with Vibrio flexneri after surface disinfection (the inoculation procedure was as follows: a 2-4 mm incision was made on the kelp tissue block using a sterilized scalpel, and then OD 600 200 μL of the above-mentioned Vibrio freundii bacterial suspension with a concentration of 1 was inoculated into the incision, 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).

[0033] The experimental group was a kelp tissue block inoculated with antagonistic bacteria F-DL4 and Vibrio freundii after surface disinfection (the inoculation operation was: a 2-4 mm incision was made on the kelp tissue block with a sterilized scalpel, and then 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 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).

[0034] 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 then dilute with distilled water to make OD 600 A bacterial suspension of 1.

[0035] The antagonistic effect was tested by observing tissue sections: tissue sections were taken from the kelp tissue blocks on the 0th and 8th days, respectively, and the tissue sections of about 3 mm × 3 mm were cut with a sterilized scalpel, and fixed with Carnoy's solution for 3 hours. The fixed tissue sections were stained with lactic acid fuchsin stain overnight, and the tissue sections were observed under a microscope at 100 times the oil lens, and then microphotographed to observe whether the kelp disease process was slowed down histologically.

[0036] Experimental Results 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 is observed. In contrast, the internal distribution of kelp tissue cells in the control group on day 8 became uneven, and plasmolysis occurred. The shapes of organelles such as chromatosomes, nuclei, and mitochondria were irregular, and the chromatosome lamellar structure was blurred. The internal contents of the lighter-colored gametocytes gathered in the middle of the cells, the colors at both ends of the cells became lighter, and the cytochrome distribution was uneven. The whitened gametocytes were vacuolated internally, the organelles were completely dissolved, or only a small amount of chromatosomes remained, and the edges of the cell walls became rough.

[0037] Further comparison of the kelp tissue cells of the infection group and the control group on the 8th day showed that the kelp cells in the infection 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 endodermis of the infected kelp, which were oval or round in shape. The intracellular structure was destroyed, and even cell death was caused. The dead cells fell off to form holes on the thallus.

[0038] Compared with the infected group, although the kelp tissue cells in the experimental group were still arranged in disorder and the cells were uneven in size on the 8th day, the storage substances flowing out of the cell gaps were reduced and the number of ruptured cells decreased, indicating 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 and rot.

3. The use of the antagonistic fungi of alginic acid degrading bacteria according to claim 2, characterized in that: The Aspergillus niger F-DL4 is used for alleviating kelp disease and rot caused by alginate-degrading bacteria.

4. The use of the antagonistic fungus of alginate-degrading bacteria according to claim 3, characterized in that: The alginic acid degrading bacteria is Vibrio freundii Vibrio fumissii .

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

6. The bacterial agent according to claim 5, characterized in that: The bacterial agent is the fermentation liquid, fermentation culture liquid or fermentation bacterial suspension of the bacteria according to claim 1.

7. An application of the bacterial agent according to claim 5, characterized in that: The bacterial agent is used in preventing or alleviating kelp seedling disease and rot.

8. The use according to claim 7, characterized in that: The bacterial agent is used in preventing or alleviating kelp seedling disease and rot caused by alginate-degrading bacteria.

9. The use according to claim 8, characterized in that: The alginic acid degrading bacteria is Vibrio freundii Vibrio fumissii strains.

10. The use according to claim 8, 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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