Bacillus amyloliquefaciens JK-2 and its culture method and application

Abstract

The invention discloses a strain of Bacillus amyloliquefaciens JK-2, a cultivation method and an application thereof, and belongs to the field of microbial technology. The invention screens an antagonistic strain JK-2 from honeysuckle leaves, which has a good antagonistic effect on the pathogen Fusarium equisetifolia that causes honeysuckle diseases. The strain is identified as Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens ), and tested its effective antagonistic components and safety. It has a significant inhibitory effect on the pathogen Botrytis cinerea that causes gray mold in strawberries and tomatoes, the pathogen Botrytis cinerea that causes pear ring rot, as well as Aspergillus niger and Penicillium, proving that strain JK‑2 has a broad spectrum of antibacterial activity. Therefore, Bacillus amyloliquefaciens JK‑2 has great potential as a microbial control agent to improve crop yield and quality.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and specifically relates to a strain of Bacillus amyloliquefaciens JK-2 and a cultivation method and application thereof. Background Art

[0002] Honeysuckle is a traditional Chinese herbal medicine with the effects of clearing away heat and detoxifying, promoting bile secretion and protecting the liver, dissipating heat and cooling the wind, and resisting viruses. It is usually used to treat respiratory diseases, dysentery, sores, headaches and fever, etc. With the expansion of honeysuckle cultivation area and changes in the ecological environment, the occurrence of honeysuckle diseases is becoming more and more serious, such as brown spot disease, anthracnose, powdery mildew, root rot, etc., which seriously restrict the development of the honeysuckle industry.

[0003] Field investigations found that after the onset of honeysuckle disease, many leaves had some spots, faded green and turned yellow, and the disease was rapid, spread quickly, and was highly harmful, seriously affecting the yield and quality of honeysuckle in farmers and plantations. By isolating, purifying and identifying the pathogens of the diseased samples, it was found that the pathogen causing the disease was Fusarium equisetifolia. At present, chemical control is the main method for the prevention and control of honeysuckle diseases, supplemented by other prevention and control methods (including disease-resistant variety screening and agronomic measures management). Although chemical control has a good control effect on honeysuckle disease, it will increase the resistance of pathogenic bacteria, reduce the diversity of soil biological communities in honeysuckle fields, and affect the environmental quality of honeysuckle field soil. In recent years, the quality of honeysuckle and its field soil quality have received more and more attention, and reducing the food and use of chemical control is an inevitable trend. Summary of the invention

[0004] The invention screens strains antagonistic to Fusarium equiseti, studies the antagonism, safety and broad-spectrum antibacterial activity of the strains, and optimizes the fermentation conditions, thus laying a foundation for the preparation of green and environmentally friendly biocontrol agents and having important significance for the development of the honeysuckle economic industry.

[0005] The present invention provides the following technical solutions:

[0006] The invention discloses a Bacillus amyloliquefaciens JK-2 strain antagonistic to Fusarium equiseti, with the deposit number being CGMCC No.29211.

[0007] Preferably, the 16S rDNA sequence of the Bacillus amyloliquefaciens JK-2 is shown as SEQ ID No: 1.

[0008] The present invention also provides a method for culturing the Bacillus amyloliquefaciens JK-2, comprising the following steps: preparing a single colony of the Bacillus amyloliquefaciens JK-2 into a seed bacterial suspension, inoculating the suspension into an LB liquid culture medium, and culturing the suspension on a shaking table.

[0009] Preferably, the inoculation amount of the seed bacteria suspension is 2%.

[0010] Preferably, the shaking culture comprises shaking culture at 37° C. with a speed of 200 r / min for 18 to 48 h.

[0011] The present invention also provides the use of the Bacillus amyloliquefaciens JK-2 or the culture solution obtained by the culture method in preparing an antibacterial agent.

[0012] Preferably, the active ingredient of the antibacterial agent includes the bacterial cells of the Bacillus amyloliquefaciens JK-2 or a bacterial solution containing the Bacillus amyloliquefaciens JK-2.

[0013] The present invention also provides a broad-spectrum antibacterial agent, which uses the bacterial cells of the Bacillus amyloliquefaciens JK-2 or a bacterial liquid containing the bacterial cells as an effective ingredient.

[0014] Preferably, the antimicrobial spectrum includes: Fusarium equisetum, Aspergillus niger, Botrytis cinerea, Botrytis cinerea and Penicillium.

[0015] The invention also provides a method for preventing and treating honeysuckle diseases, comprising applying the broad-spectrum antibacterial agent to the honeysuckle tree.

[0016] Beneficial effects: The present invention provides an antagonistic strain Bacillus amyloliquefaciens JK-2 that can prevent and treat Fusarium equiseti, a pathogen of honeysuckle. The Bacillus amyloliquefaciens JK-2 strain has undergone preliminary safety experiments and was found to have no hemolytic toxicity. The results indicate that the strain meets the safety requirements in the development of biological control and antifungal drugs.

[0017] The present invention also verifies the antibacterial effect of JK-2 through an inhibition zone experiment, and finds that the inhibition rate of Fusarium equisetifolia is 67%, and it has an antibacterial effect on pathogenic bacteria such as Aspergillus niger, Botrytis cinerea, Botrytis cinerea, Penicillium, etc. Therefore, the Bacillus amyloliquefaciens JK-2 strain has a broad-spectrum antibacterial activity and has great potential as a microbial control agent for improving crop yield and quality.

[0018] Deposit certificate

[0019] Bacillus amyloliquefaciens, whose strain is named JK-2, has been deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on December 1, 2023, with the deposit address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code: 100101, and the deposit number is 29211; BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1This is the result of confrontation culture between strain JK-2 and Fusarium equisetum;

[0021] Figure 2 This is the colony morphology of the endophytic bacterium JK-2;

[0022] Figure 3 This is the microstructural morphology of endophyte JK-2;

[0023] Figure 4 is the phylogenetic tree of strain JK-2;

[0024] Figure 5 Growth curve and pH value monitoring of strain JK-2;

[0025] Figure 6 This is the identification diagram of antibacterial active substances of strain JK-2;

[0026] Figure 7 This is the hemolytic assay of strain JK-2, left: front; right: back;

[0027] Figure 8 The antibacterial effect diagram of strain JK-2 on other fungi: a: Aspergillus niger; b: Botrytis cinerea; c: Botrytis cinerea; d: Penicillium. DETAILED DESCRIPTION

[0028] In order to further illustrate the present invention, the scheme provided by the present invention is described in detail below in conjunction with embodiments, but they should not be understood as limiting the protection scope of the present invention.

[0029] Example 1 Isolation and purification of endophytic bacteria from honeysuckle

[0030] In order to screen for antagonistic strains of Fusarium equisetifolia, several healthy plants were collected from the honeysuckle field with leaf spot disease in Julu County, Hebei Province in September 2022 and brought back to the laboratory. The tissue separation method was used. First, the leaf surface was disinfected. The collected honeysuckle leaf samples were soaked in 75% alcohol for 6 minutes, the residual alcohol was rinsed with sterile water 7 times, and then the surface was disinfected in 5% sodium hypochlorite solution for 30 seconds. The surface liquid was blotted with filter paper. The disinfected honeysuckle leaves were cut into 2-3 cm squares with sterile scissors, inoculated on a plate of LB solid culture medium, placed in a 37°C constant temperature incubator, and cultured for 3-5 days. When the bacteria grew around the honeysuckle tissue, the bacteria were picked and transferred to LB solid culture medium. After multiple separation and purification, an endophytic bacterium was obtained and named JK-2. It was cultured on a slant and stored at 4°C for later use. The sterile water used for the last flushing of the honeysuckle tissue was spread on the surface of the LB solid culture medium with a sterile spreading rod and cultured in the dark at 37°C as a control group.

[0031] LB liquid medium:

[0032] Weigh 10 g / L of tryptone, 5 g / L of yeast extract, and 10 g / L of sodium chloride (NaCl), and make up to 1 L with distilled water. Adjust the pH of the culture medium to 7.2, and sterilize with high-pressure steam (121°C, 20 min).

[0033] LB solid medium:

[0034] Weigh 10 g / L of tryptone, 5 g / L of yeast extract, and 10 g / L of sodium chloride (NaCl), dilute to 980 mL with distilled water, adjust the pH of the medium to 7.2, and add 15 g of agar. Sterilize with high-pressure steam (121°C, 20 min).

[0035] Endophytes were isolated from healthy and fresh leaves of Honeysuckle and the experiment was repeated many times. No bacteria grew in the negative control plates and liquid culture medium, proving that the isolated bacteria were endophytes rather than epiphytes on the surface.

[0036] Example 2 Antagonistic effect of strain JK-2 on pathogenic bacteria Fusarium equiseti

[0037] The JK-2 strain isolated from the leaves of honeysuckle was cultured against Fusarium equisetum to observe whether an inhibition zone appeared.

[0038] Activation of JK-2 strains and preparation of bacterial solution. The frozen strains were streaked onto LB plate culture medium and cultured at 37°C for 24 hours; a single colony of JK-2 strain on the LB plate was picked and inoculated into 100 mL of LB liquid culture medium and cultured overnight at 37°C and 200 r / min to prepare a concentration of 1×10 6 CFU / mL of bacterial suspension.

[0039] Preparation of Fusarium equisetifolia cakes. Add 4 mL of sterile water to the Fusarium equisetifolia slant strains, shake thoroughly to make a bacterial suspension, then apply 1 mL to a PDA solid culture medium plate, culture at 28°C for 3-4 days, and use a sterile puncher with a diameter of 6 mm to punch a bacterial cake at the edge of the colony on the plate for later use.

[0040] Plate confrontation culture. Inoculate Fusarium equisetifolia cake (6mm) at the center of a 90mm PDA plate, and inoculate 10μL of JK-2 bacterial suspension in the agar at four locations about 15cm from the center (use a gun tip to gently pierce the agar and inject the bacterial suspension), with 3 replicates as the treatment group; inject an equal amount of sterile water at four locations at the center of the PDA plate, with 3 replicates as the control group; both the control group and the treatment group were cultured at 28℃ to observe whether a transparent antibacterial zone appeared.

[0041] Potato dextrose agar (PDA): 200 g of peeled potatoes were cut into 1 cm 3 ), put it in a pot with cold water (to reserve water for evaporation, about 1300mL of distilled water), boil it over high heat, then simmer it over low heat for 30 minutes, filter out the potato residue with 8 layers of gauze, make up the supernatant to 1000mL with distilled water again, add 20g of glucose and 18g of agar powder, adjust the pH value to natural, package it, and sterilize it with high-pressure steam (121℃, 20min).

[0042] Results: After 3-4 days of confrontation culture at 28℃, the four parts of the inoculated JK-2 showed obvious transparent circles, which were the inhibition zones, and could inhibit the growth of Fusarium equisetifolia. The diameter of the inhibition zone was 32mm, and the inhibition rate was 67%. Figure 1 shown.

[0043] Example 3 Morphological observation and physiological and biochemical identification of strain JK-2

[0044] The colony morphology of strain JK-2 on LB plate is as follows Figure 2 As shown in the figure, the single colony is small and round, opaque, milky white, and has regular edges. Figure 3 As shown, the strain is rod-shaped, motile, and can produce spores. The strain JK-2 was physiologically and biochemically identified using HBI Bacillus biochemical identification strips (Qingdao High-Tech Industrial Park Haibo Biotechnology Co., Ltd.), and the results are shown in Table 1.

[0045] Table 1 Physiological and biochemical identification results of strain JK-2

[0046]

[0047]

[0048] Example 4 Molecular Biological Identification of Strain JK-2

[0049]

[0050] The sequencing results were analyzed by BLAST and compared with the sequences in GenBank. The phylogenetic tree of strain JK-2 was constructed using MEGA-X software. The construction results are shown in Figure 4 The results showed that the 16S rDNA sequence of strain JK-2 had a 99% similarity with Bacillus amyloliquefaciens (OK178286.1), and they were clustered in the same branch with more than 80% support rate. Combined with morphological observation and physiological and biochemical experiments, the endophytic bacterium JK-2 was identified as Bacillus amyloliquefaciens.

[0051] Example 5 Monitoring of growth curve and pH value of strain JK-2

[0052] After the single colony of Bacillus amyloliquefaciens JK-2 obtained by streaking on the LB plate was prepared into a seed suspension, it was added to 30 mL of LB liquid culture medium at a 2% inoculum, and cultured at 37°C, 200 r / min shaking for 12 hours, with 3 replicates. The absorbance of the bacterial solution was measured at a wavelength of 600 nm using a spectrophotometer every 2 hours, and the corresponding pH value change was also measured.

[0053] Specific monitoring results include Figure 5 As shown, the growth curve characteristics of the Bacillus amyloliquefaciens JK-2 strain of the present invention are: 0-2h is the incubation period, 2-12h is the exponential growth period, 18h reaches the maximum, and 20-48h is the stable growth period ( Figure 5 ); The initial pH of the LB culture medium was 6.98, the pH value began to increase at 4h, reached 8.56 at 18h, and was in a stable period from 18h to 48h and no longer increased.

[0054] Example 6 Identification of antibacterial active substances of strain JK-2

[0055] The whole fermentation broth of strain JK-2, the bacteria after centrifugation of the fermentation broth, and the supernatant after centrifugation were cultured opposite to Fusarium equiseti in the same plate. The antibacterial effect of three different parts of JK-2 was used to determine whether the antibacterial substance was located inside the bacteria or secreted outside the bacteria.

[0056] The results are as follows Figure 6 As shown, antibacterial zones appeared around the fermentation broth and the bacteria after centrifugation of the fermentation broth, indicating significant antibacterial activity, while the supernatant after centrifugation was completely covered by Fusarium equiseti hyphae, indicating that the antibacterial substance produced by JK-2 was the bacteria itself and did not have the characteristics of extracellular secretion.

[0057] Example 7 Safety determination of strain JK-2

[0058] The present invention conducts a hemolysis test on the Bacillus amyloliquefaciens JK-2 strain, streaks and inoculates a single colony of the JK-2 strain on a blood agar plate, cultures the plate at 37° C. for 24 hours, and observes whether a transparent circle appears around the bacterial body.

[0059] from Figure 7 The results showed that no transparent circle appeared around the bacteria, indicating that the bacteria is safe for humans and animals.

[0060] Example 8 Determination of broad-spectrum antibacterial activity of strain JK-2

[0061] In order to detect the broad-spectrum antibacterial activity of Bacillus amyloliquefaciens JK-2 strain, it was respectively co-cultured with the pathogen Aspergillus niger screened in the early stage by Hebei Institute of Microbiology Co., Ltd. and gray mold (B. cirerea) B16, Botrytis cinerea (B. dothidea) D330 and Penicillium expansum (P. expansum) HH1 screened in the early stage by the Institute of Biotechnology and Food Science of Hebei Academy of Agricultural and Forestry Sciences. Among them, the three fungi screened by the Institute of Biotechnology and Food Science of Hebei Academy of Agricultural and Forestry Sciences were published in the article "Volatile organic compounds produced by Bacillus aryabhattai AYG1023 against Penicillium expansum causing blue mold on the Huangguan pear". Bacillus amyloliquefaciens JK-2 strain and the above four fungi were cultured in confrontation, and sterile water was used instead of Bacillus amyloliquefaciens JK-2 strain to co-culture with the above four fungi as a control, and three parallel experiments were conducted in each group.

[0062] Using the punching method, 6 mm holes were punched in four locations 15 cm away from the center point of a 90 mm PDA plate. The agar was taken out after the holes were inoculated into the holes, and 20 μL of the JK-2 bacterial suspension was inoculated into the holes. On the other side, a 6 mm cake of Fusarium equisetifolia was inoculated. The two strains were placed on the same line and cultured at a constant temperature of 28°C for 3-4 days to observe whether a transparent inhibition zone appeared.

[0063] Depend on Figure 8 It can be seen that the four fungi and Bacillus amyloliquefaciens JK-2 strains were cultured on the plates with inhibition zones, while the controls grew normally without inhibition zones. Therefore, Bacillus amyloliquefaciens JK-2 strains have a broad spectrum of antibacterial properties.

[0064] The invention separates a Bacillus amyloliquefaciens JK-2 strain from honeysuckle tissue, which has a broad-spectrum antibacterial activity and great potential as a microbial control agent, thereby inhibiting the occurrence of diseases caused by pathogenic bacteria such as Fusarium equisetifolia, Aspergillus niger, Botrytis cinerea, Botrytis cinerea, Penicillium and the like, and improving crop yield and quality.

[0065] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A strain of Bacillus amyloliquefaciens JK-2 antagonistic to Fusarium equiseti, characterized in that: The deposit number is CGMCC No.29211.

2. The method for culturing Bacillus amyloliquefaciens JK-2 according to claim 1, characterized in that: The following steps are involved: A single colony of the Bacillus amyloliquefaciens JK-2 was prepared into a seed bacterial suspension, which was then inoculated into an LB liquid culture medium and cultured on a shaking platform.

3. The culture method according to claim 2, characterized in that: The inoculation amount of the seed bacteria suspension is 2%.

4. The culture method according to claim 2, characterized in that: The shaking culture includes shaking culture at 37° C. with a speed of 200 r / min for 18 to 48 hours.

5. Use of the Bacillus amyloliquefaciens JK-2 according to claim 1 or the culture solution obtained by the culture method according to any one of claims 2 to 4 in the preparation of an antibacterial agent, characterized in that: The antimicrobial agent has an antimicrobial spectrum of Fusarium equiseti, B. cirerea B16, B. dothidea D330 and P. expansum HH1.

6. The use according to claim 5, characterized in that: The active ingredient of the antibacterial agent includes the bacterial body of the Bacillus amyloliquefaciens JK-2 or a bacterial solution containing the Bacillus amyloliquefaciens JK-2.

7. A broad-spectrum antibacterial agent, characterized in that: The active ingredient is the bacterial body of Bacillus amyloliquefaciens JK-2 or a bacterial solution containing the bacterial body as described in claim 1.

8. The broad-spectrum antibacterial agent according to claim 7, characterized in that: The antibacterial spectrum is: Fusarium equiseti, B. cirerea B16, B. dothidea D330 and P. expansum HH1.

9. A method for preventing and treating honeysuckle diseases, characterized in that: The method comprises applying the broad-spectrum antibacterial agent according to claim 7 or 8 to the tree of honeysuckle.

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