Antagonistic bacteria to alginic acid-degrading bacteria and its application

Abstract

The present invention belongs to the technical field of disease prevention and control of cultured kelp, and specifically relates to an antagonistic bacterium of alginate-degrading bacteria and its application. The antagonistic bacterium is Klebsiella aerogenes B-S13, 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.31792, and is classified as Klebsiella aerogenes Klebsiella aerogenes The deposit address is Beijing, China. The 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.

Description

Technical Field

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

[0002] Kelp is an important component of marine aquatic resources. Kelp contains alginate, alginic acid and other substances, which can be used to produce iodine, mannitol, alginate food additives, etc. During the microbial detection of kelp seedling system, a large number of alginate-degrading bacteria were found during the peak period of kelp spore disintegration disease and green rot disease. During the onset of kelp spore disintegration disease, the number of alginate-degrading bacteria can reach 109. Because they are conditional pathogens, under certain conditions, they will cause kelp to rot during the breeding process, causing great harm to kelp breeding. At present, the prevention and control of kelp diseases in China still remains at the stage of screening kelp disease-resistant varieties, optimizing kelp culture conditions, and using chemical substances. There is no effective biological control technology to prevent or alleviate the problem of kelp disease and rot.

[0003] In recent years, seaweed beneficial microorganisms (SBM) have been found to promote the growth and development of seaweed or improve the disease resistance of seaweed. The way to improve the commercial cultivation of seaweed through microbial flora manipulation is to identify and apply seaweed beneficial microorganisms in seaweed aquaculture, which is beneficial to the prevention and control of seaweed diseases and the expansion of seaweed aquaculture. 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 its parasitic host, promote the proliferation of biocontrol bacteria and inhibit the growth of pathogens, so as to achieve 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

[0004] The purpose of the present invention is to provide an antagonistic bacterium to alginate-degrading bacteria and application thereof.

[0005] To achieve the above object, the technical solution adopted by the present invention is:

[0006] The antagonistic bacteria is Klebsiella aerogenes B-S13, which was deposited in the General Microbiological Center of China Microbiological Culture Collection Administration on August 30, 2024, with the deposit number CGMCC No.31792, and the classification name is Klebsiella aerogenes Klebsiella aerogenes , the storage address is Beijing, China.

[0007] The culture temperature of the aerogenes Klebsiella B-S13 is preferably 30°C.

[0008] An application of antagonistic bacteria to the alginate-degrading bacteria, and an application of the aerogenes Klebsiella B-S13 in alleviating kelp rot.

[0009] The application of the aerogenes Klebsiella B-S13 in alleviating kelp disease and rot caused by alginate degrading bacteria.

[0010] The alginic acid degrading bacteria is Vibrio freundii Vibrio fumissii strains.

[0011] A bacterial agent for preventing or alleviating kelp seedling disease and rot, the bacterial agent contains the antagonistic bacteria.

[0012] The bacterial agent is the fermentation liquid, fermentation culture liquid or fermentation bacterial suspension of the bacteria.

[0013] An application of the bacterial agent, which is application of the bacterial agent in preventing or alleviating disease and rot of kelp seedlings.

[0014] The bacterial agent is used in preventing or alleviating kelp seedling disease and rot caused by alginate-degrading bacteria.

[0015] The alginic acid degrading bacteria is Vibrio freundii Vibrio fumissii strains.

[0016] 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.

[0017] 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.

[0018] The advantages of the present invention are:

[0019] The experiment of infecting young spores of healthy kelp confirmed that the aerogenes Klebsiella B-S13 strain obtained by the present invention can alleviate the Vibrio fumissii Symptoms of kelp disease and rot caused by the strain. The 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 This is a diagram showing the antagonistic effect of the strain B-S13 provided in an embodiment of the present invention on Vibrio flexneri, wherein the transparent circle represents the inhibitory effect of B-S13 on Vibrio flexneri, Bar: 17 mm.

[0021] Figure 2 The colony morphology of strain B-S13 provided in the embodiment of the present invention.

[0022] Figure 3 A phylogenetic tree constructed based on the 16S rRNA gene sequence of strain B-S13 provided in an embodiment of the present invention.

[0023] Figure 4 A photograph of healthy kelp young sporophytes infected with the embodiments of the present invention.

[0024] Figure 5 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 of antagonistic bacteria

[0027] 1) Isolation and purification of strains

[0028] 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 isolate and purify surface epiphytes. Rinse the kelp seedlings with sterile seawater to remove surface impurities. Cut the seedlings into small pieces and grind them into homogenate. Then dilute the homogenate ten times with distilled water, and take 120 μL of each dilution and spread it on LB solid culture medium, and culture at 30°C for 24-48 hours. According to the morphological characteristics, different single colonies were selected and purified by streaking at least three times on a new LB solid culture medium. In this purification experiment, a total of 27 purified bacterial strains were isolated and stored in glycerol and placed in a -80 degree refrigerator.

[0029] 2) Screening of antagonistic bacteria

[0030] The isolated bacterial strain was inoculated into LB liquid culture medium, and the culture medium was placed in a 30°C constant temperature shaking incubator and cultured at 180 rpm / min for 48 h. Vibrio fumissii The bacterial suspension was shaken and cultured until OD 600The value is about 0.5-1 (the culture conditions are 180rpm / min, 30℃). Use a puncher to punch out a filter paper with a diameter of 6 mm, sterilize the filter paper and dry it, take 10μL of the separated bacterial solution and drop it on the filter paper, take 100μL of the alginate-degrading bacterial suspension and apply it to the sodium alginate solid culture medium, and then place the filter paper in the center of the sodium alginate solid culture medium. The sodium alginate solid culture medium was placed in a 30℃ constant temperature incubator for 48 h, and the antibacterial ring was observed and the radius of the antibacterial ring was measured. The antibacterial ring radius ≥5mm indicates antibacterial activity. It has been verified that the antibacterial ring radius of the B-S13 strain is 17mm, which has obvious antibacterial activity ( Figure 1 ).

[0031] Sodium alginate solid culture medium is as follows: add 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 freundii Vibrio fumissii Inoculate into sodium alginate medium at a concentration of 2% by volume, 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).

[0033] 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.

[0034] Example 2: Molecular identification of antagonistic strains of alginic acid-degrading bacteria

[0035] The B-S13 strain was cultured in LB medium, and the colony color was white, opaque, convex, and with regular edges. The strain B-S13 was identified to the genus based on the sequencing results of the 16S rDNA sequence combined with the observation of colony morphology. After comparison with NCBI, the selected strain B-S13 strain was found to be similar to Klebsiella aerogenes- Klebsiella aerogenes The strain is Klebsiella aerogenes Klebsiella aerogenes , deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number CGMCC No.31792, the deposit date is August 30, 2024, and the deposit address is Beijing, China.

[0036] Example 3: Re-testing of biocontrol antagonists with high antibacterial activity

[0037] Antagonistic experiment of kelp tissue

[0038] The above-obtained high antibacterial activity biocontrol antagonistic bacteria strain B-S13 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:

[0039] 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).

[0040] 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 bacterial suspension of Vibrio flexneri obtained above 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).

[0041] The experimental group was a kelp tissue block inoculated with antagonistic bacteria B-S13 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 B-S13 obtained above 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).

[0042] The bacterial suspension of B-S13 is Klebsiella aerogenes Klebsiella aerogenes Inoculate into LB 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).

[0043] Antagonistic effects were tested by observing tissue morphology and tissue sections:

[0044] (1) Tissue morphology observation: Directly observe and record the color, thickness and other conditions of the kelp tissue blocks in the control group and the experimental group, and observe the location and time of the occurrence of green spots, green rot and white rot, so as to confirm whether the disease and rot process of the kelp tissue blocks has been slowed down.

[0045] (2) Tissue section observation: Take the kelp tissue blocks on the 0th day and the 8th day for tissue section, use a sterilized scalpel to cut the tissue sections of about 3mm×3mm, and fix them with Carnoy's solution for 3h. Stain the fixed tissue sections with lactic acid fuchsin stain overnight, observe the tissue sections under a microscope with a 100x oil lens, and then take microphotographs to observe whether the kelp rot process has slowed down histologically.

[0046] Experimental Results

[0047] from Figure 4 It can be seen from the control group that under healthy conditions, the disease and decay process of the kelp tissue blocks is relatively slow. Compared with the kelp tissue blocks on the 0th and 3rd days, the kelp tissue blocks in the control group on the 5th day only showed obvious green spots on the edges, and the overall tissue was relatively firm, with normal color and a certain thickness.

[0048] In the infection group inoculated with Vibrio freundii, the kelp tissue blocks on the third day showed obvious green spots on the incision, indicating that Vibrio freundii decomposed the alginate in the kelp algae by secreting alginate, causing the water-soluble brown algae in the kelp algae to dissolve first, causing the kelp tissue at the site of disease to change from brown to green and form green spots, which gradually evolved into a phenomenon of green rot in the kelp tissue. In the infection group, the kelp tissue blocks on the fifth day showed obvious green rot at the inoculation site of the bacteria, the kelp tissue blocks were obviously rotten, the edges showed white rot, and the thickness of the tissue blocks decreased.

[0049] In comparison, the disease and decay process of the kelp tissue blocks in the experimental group inoculated with antagonistic bacteria B-S13 and Vibrio freundii was slowed down. On the third day, the kelp tissue blocks only showed obvious green spots at the edges, and the decay at the incision was relatively mild, indicating that the antagonistic bacteria B-S13 played a role in antagonizing alginate-degrading bacteria in the disease and decay process of the kelp. Although the kelp tissue blocks on the fifth day also showed obvious green rot at the inoculation site of the incision, and the tissue blocks were obviously rotten, with obvious white rot at the edges, and the thickness of the tissue blocks decreased, the degree of disease and decay of the kelp tissue blocks was reduced compared with the infection group, indicating that the disease and decay process was slowed down.

[0050] Since the changes in the kelp disease and rot process at the tissue cell level are not obvious, the culture time of the kelp tissue blocks was extended, and the kelp tissue blocks on the 0th and 8th days were selected for microscopic observation. From the kelp tissue sections of the three groups on the 0th day, it can be seen that the tissue cells of healthy kelp are arranged more regularly, and the cells are light red after being stained with lactic acid fuchsin dye. The cell size and morphology are basically the same, and no storage substances flow out. In the control group, the kelp tissue cells on the 8th day still have a certain regularity in arrangement, but after staining, the cells are dark red, the cell size is different, and a small amount of storage substances flow out in the gaps between cells, showing black granules.

[0051] Comparing the kelp tissue cells of the infection group with those of the control group on the 8th day, it was found that the sizes of the kelp tissue cells were different, and the disorder of the cells increased significantly. In addition, the gaps between tissue cells increased, cavities appeared, and some tissue cells ruptured, causing more storage materials to flow into the cavities, indicating that the kelp tissue infected by Vibrio freundii was more seriously rotten.

[0052] When the kelp tissue cells of the experimental group on the 8th day were compared with those of the infected group, it was found that the arrangement of the kelp tissue cells was still disordered and the cells were of different sizes, but the amount of storage substances flowing out of the gaps between cells was reduced, and the number of ruptured tissue cells decreased, indicating that the disease and decay of the kelp tissue was significantly alleviated.

Claims

1. An antagonistic bacterium to alginic acid-degrading bacteria, characterized in that: The antagonistic bacteria is Klebsiella aerogenes B-S13, which was deposited in the General Microbiological Center of China Microbiological Culture Collection Administration on August 30, 2024, with the deposit number CGMCC No. 31792, and the classification name is Klebsiella aerogenes Klebsiella aerogenes , the storage address is Beijing, China.

2. An application of antagonistic bacteria to the alginate-degrading bacteria according to claim 1, characterized in that: The application of the aerogenes Klebsiella B-S13 in alleviating kelp disease caused by alginate degrading bacteria; the alginate degrading bacteria is Vibrio freundii Vibrio fumissii strains.

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

4. The bacterial agent according to claim 3, characterized in that: The bacterial agent is the fermentation culture liquid or fermentation bacterial suspension 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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