CN117821292A - Bacillus amyloliquefaciens JK-2 and culture method and application thereof - Google Patents
Bacillus amyloliquefaciens JK-2 and culture method and application thereof Download PDFInfo
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Abstract
The invention discloses bacillus amyloliquefaciens JK-2, a culture method and application thereof, and belongs to the technical field of microorganisms. The invention screens an antagonistic strain JK-2 from honeysuckle leaves, has good antagonistic effect on pathogenic bacteria Fusarium equisetum causing honeysuckle diseases, and is identified as bacillus amyloliquefaciens by morphological, physiological and biochemical and molecular biological methodsBacillus amyloliquefaciens) The effective antagonistic components and the safety of the strain are detected, and the strain has remarkable inhibition effect on pathogenic fungi such as gray mold which causes gray mold of strawberries, tomatoes and the like, pathogenic fungi such as Pycnogen grifola which causes pear ring rot, and harmful fungi such as aspergillus niger and penicillium and the like, and the strain JK-2 is proved to haveHas antibacterial broad spectrum. Therefore, the bacillus amyloliquefaciens JK-2 has great potential as a microbial control microbial agent for improving the yield and quality of crops.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to bacillus amyloliquefaciens JK-2, a culture method and application thereof.
Background
Honeysuckle is a traditional Chinese herbal medicine in China, has the effects of clearing heat and detoxicating, benefiting gallbladder and protecting liver, dissipating cold wind, resisting viruses and the like, and is generally used for treating various diseases such as respiratory diseases, dysentery, sores, headache and fever. Along with the expansion of the cultivation area and the change of the ecological environment, the occurrence of honeysuckle diseases such as brown spot, anthracnose, powdery mildew, root rot and the like is more serious, and the development of the honeysuckle industry is severely restricted.
After the honeysuckle is found to be ill through field investigation, a plurality of leaves are provided with a little disease spots, fade green and turn yellow, and the disease is rapid, spread rapidly and has stronger hazard, thereby seriously affecting the yield and quality of the honeysuckle in farmers and planting parks. The pathogenic bacteria of the pathogenic sample are separated, purified and identified, and the pathogenic bacteria causing the pathogenic bacteria are found to be Fusarium equisetum. At present, honeysuckle disease control mainly adopts chemical control and is supplemented with other control methods (including disease-resistant variety screening, agronomic measure management and the like). Although the chemical control has better control effect on honeysuckle diseases, the pathogenic bacteria resistance is improved, the biological community diversity of the soil of the honeysuckle fields is reduced, and the environmental quality of the soil of the honeysuckle fields is affected. In recent years, the quality of honeysuckle and the quality of soil in fields are increasingly emphasized, and the reduction of foods and use for chemical control is a necessary trend.
Disclosure of Invention
The invention screens the strain antagonizing the fusarium equiseti, researches the antagonism, the safety and the bacteriostasis broad spectrum of the strain, optimizes the fermentation condition of the strain, lays a foundation for the preparation of green and environment-friendly biocontrol agents, and has important significance for the development of the honeysuckle economic industry.
The invention provides the following technical scheme:
bacillus amyloliquefaciens (Bacillus amyloliquefaciens) JK-2 for antagonizing fusarium equiseti (Fusarium equiseti) and with the preservation number of CGMCC No.29211.
Preferably, the 16S rDNA sequence of the bacillus amyloliquefaciens JK-2 is shown in SEQ ID No: 1.
The invention also provides a culture method of the bacillus amyloliquefaciens JK-2, which comprises the following steps: and (3) preparing single bacterial colony of the bacillus amyloliquefaciens JK-2 into seed bacterial suspension, and then inoculating the seed bacterial suspension into an LB liquid culture medium for shake culture.
Preferably, the seed bacterial suspension is inoculated in an amount of 2%.
Preferably, the shaking culture comprises shaking culture at 37 ℃ for 18-48 hours along with 200 r/min.
The invention also provides application of the bacillus amyloliquefaciens JK-2 or the culture solution obtained by the culture method in preparation of an antibacterial agent.
Preferably, the active ingredient of the antibacterial agent comprises thalli of the bacillus amyloliquefaciens JK-2 or bacterial liquid containing the bacillus amyloliquefaciens JK-2.
The invention also provides a broad-spectrum antibacterial agent, which takes the bacterial cells of the bacillus amyloliquefaciens JK-2 or bacterial liquid containing the bacterial cells as an active ingredient.
Preferably, the antimicrobial spectrum comprises: fusarium equisetum, aspergillus niger, botrytis cinerea and Penicillium viridis.
The invention also provides a method for preventing and treating honeysuckle diseases, which comprises the step of applying the broad-spectrum antibacterial agent to the honeysuckle tree body.
The beneficial effects are that: the invention provides an antagonistic strain bacillus amyloliquefaciens JK-2 capable of preventing and controlling honeysuckle pathogenic bacteria Fusarium equisetum (Fusarium equiseti), and the bacillus amyloliquefaciens JK-2 strain is found to have no hemolytic toxicity through preliminary safety experiments. The result shows that the strain meets the safety requirements in biological control and antifungal drug research and development.
The antibacterial effect of the JK-2 is verified through an antibacterial ring experiment, the inhibition rate of the JK-2 is found to be 67% on Fusarium equisetum, and the JK-2 has antibacterial effects on pathogenic bacteria such as aspergillus niger, gray mold, grape trellis bacteria, penicillium and the like, so that the bacillus amyloliquefaciens JK-2 strain has broad-spectrum antibacterial activity, has great potential as a microbial control microbial agent, and is used for improving crop yield and quality.
Preservation certificate
Bacillus amyloliquefaciens (Bacillus amyloliquefaciens), the strain of which is designated JK-2, was deposited at the China general microbiological culture Collection center, accession number: beijing, chaoyang area, north Chen Xi Lu No. 1, 3, china academy of sciences microbiological institute, post code: 100101, accession number 29211;
drawings
FIG. 1 shows the result of the culture of strain JK-2 against Fusarium equisetosum;
FIG. 2 shows colony morphology of endophytic bacterium JK-2;
FIG. 3 shows the microstructure morphology of endophyte JK-2;
FIG. 4 is a phylogenetic tree of strain JK-2;
FIG. 5 shows the growth curve and pH monitoring of strain JK-2;
FIG. 6 is a diagram for identifying antibacterial active substances of the strain JK-2;
FIG. 7 is a hemolysis assay of strain JK-2, left: a front face; right: a back surface;
FIG. 8 is a graph showing the bacteriostatic effect of strain JK-2 on other fungi, a: aspergillus niger; b: ash mold; c: the Grignard grape vine fruit bacteria; d: penicillium sp.
Detailed Description
The following examples are provided to illustrate the invention in more detail, but are not to be construed as limiting the scope of the invention.
Example 1 separation and purification of endophytic bacteria from honeysuckle
To screen antagonistic strains of fusarium equiseti, a number of healthy plants were collected from the field of honeysuckle with leaf spot plants in the giant deer county, river north, 9 months, and brought back into the laboratory. Firstly, carrying out leaf surface disinfection by adopting a tissue separation method, soaking an acquired honeysuckle leaf sample in 75% alcohol for 6min, washing residual alcohol for 7 times by using sterile water, carrying out surface disinfection for 30s by using 5% sodium hypochlorite solution, sucking surface liquid by using filter paper, shearing the disinfected honeysuckle leaf into blocks with the size of 2-3cm by using sterile scissors, inoculating the blocks onto a flat plate of an LB solid culture medium, placing the flat plate in a constant temperature incubator at 37 ℃, carrying out stationary culture for 3-5d, taking out thalli after the thalli grow out around the honeysuckle tissue, transferring the thalli into the LB solid culture medium, carrying out separation and purification for a plurality of times, obtaining an endophytic bacterium which is named as JK-2, carrying out slant culture, and preserving the endophytic bacterium at the temperature of 4 ℃ for standby. Sterile water for washing honeysuckle tissues for the last time is coated on the surface of LB solid medium by a sterile coating rod, and is subjected to dark culture at a constant temperature of 37 ℃ to serve as a control group.
LB liquid medium:
10g/L of Tryptone (Tryptone) and 5g/L of Yeast extract (Yeast extract) as well as 10g/L of sodium chloride (NaCl) were weighed, the volume was set to 1L by distilled water, and the pH of the medium was adjusted to 7.2, and the medium was autoclaved (121 ℃ C., 20 min).
LB solid medium:
10g/L of Tryptone (Tryptone) and 5g/L of Yeast extract (Yeast extract) as well as 10g/L of sodium chloride (NaCl) were weighed, the volume was adjusted to 980mL with distilled water, the pH of the medium was adjusted to 7.2, and 15g of agar was added. Autoclaving (121 ℃ C., 20 min).
The endophytes are separated from healthy fresh honeysuckle leaves, repeated experiments are carried out, no bacteria grow out in the negative control plate and the liquid culture medium, and the separated bacteria are plant endophytes and are not surface epiphytes.
EXAMPLE 2 antagonism of Strain JK-2 against the pathogenic bacteria Fusarium equisetum
The JK-2 strain screened and separated from the honeysuckle leaves is subjected to counter culture with the fusarium equiseti, and whether a bacteriostasis ring appears or not is observed.
Activating the JK-2 strain and preparing bacterial liquid. Streaking the frozen and preserved strain into LB plate culture medium, and culturing at 37 ℃ for 24 hours; picking single colony of JK-2 strain in LB plate, inoculating into 100mL LB liquid medium, shake culturing at 37deg.C under 200r/min overnight to obtain 1×10 strain 6 CFU/mL of bacterial suspension.
Making Fusarium equisetum cake. Adding 4mL of sterile water into the fusarium oblique strains of the horsetail respectively, fully oscillating, preparing bacterial suspension, taking 1mL of the bacterial suspension, coating the bacterial suspension in a PDA solid culture medium flat plate, culturing for 3-4d at 28 ℃, and taking out bacterial cakes at the edge of the flat plate bacterial colony by using a sterile puncher with the diameter of 6mm for later use.
The plates were cultivated in opposition. Inoculating Fusarium equisetum cakes (6 mm) at the center point of a PDA flat plate with 90mm, and inoculating 10 mu L of JK-2 bacterial suspension into agar (after gently pricking the agar with a gun head and then pouring the bacterial suspension) at four positions up, down, left and right about 15cm away from the center point, wherein 3 repetitions are set as a treatment group; pumping equal amount of sterile water into four directions of the central point of the PDA flat plate, and setting 3 repetitions as a control group; and (3) culturing the control group and the treatment group at a constant temperature of 28 ℃ and observing whether a transparent inhibition zone appears.
Potato dextrose agar medium (PDA): peeling 200g of potato and cutting (1 cm) 3 ) Putting the potato residue in a cold water pot (about 1300mL of distilled water for evaporation), boiling with strong fire, boiling with mild fire for 30min, filtering potato residue with 8 layers of gauze, adding 20g of glucose and 18g of agar powder into 1000mL of distilled water to fix the volume of the supernatant, packaging, and sterilizing with high-pressure steam (121 ℃ for 20 min).
Results: after being cultivated for 3-4 days at 28 ℃, four positions inoculated with JK-2 are provided with obvious transparent circles, which are antibacterial circles, and the growth of Fusarium equisetum can be inhibited, the diameter of each antibacterial circle is 32mm, and the inhibition rate is 67%. As shown in fig. 1.
Example 3 morphological observations and physiological Biochemical identification of Strain JK-2
The colony morphology of the strain JK-2 in the LB plate is shown in FIG. 2, and the single colony is small, round, opaque, milky white and thick and regular in edge. The microscopic morphology of the strain JK-2 is shown in FIG. 3, and the strain is rod-shaped, motile and sporulated. The physiological and biochemical identification of strain JK-2 was performed using the HBI bacillus biochemical identification strip (Qingdao high-tech industrial park Haibo biotechnology Co., ltd.) and the results are shown in Table 1.
TABLE 1 physiological and biochemical identification results of strain JK-2
EXAMPLE 4 molecular biological characterization of Strain JK-2
Single colony of strain JK-2 is inoculated into LB culture solution at 37 ℃ for 12 hours after shaking culture at 200r/min to obtain JK-2 bacterial suspension, PCR amplification is carried out to obtain an amplified fragment near 1400bp, the amplified fragment is sent to Shanghai industrial bioengineering company for sequencing, and bacterial universal primer 16S rDNA sequence 16S-r RNA-F (SEQ ID NO: 2): 5'-AGAGTTTGATCCTGGCTCAG-3',16s-R RNA-R (SEQ ID NO: 3): 5'-TACGGCTACCTTGTTACGACTT-3', the length of the obtained sequence is 1452bp, and the sequencing result is shown as SEQ ID NO:1 is shown as follows: GCCGTGGCCGGGGTGCTATA CATGCAAGTCGAGCGGACAGATGGGAGCTTGCTCCCTGATGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATGGTTGTTTGAACCGCATGGTTCAGACATAAAAGGTGGCTTCGGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTGCCGTTCAAATAGGGCGGCACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCTCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAATCCTAGAGATAGGACGTCCCCTTCGGGGGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACAGAACAAAGGGCAGCGAAACCGCGAGGTTAAGCCAATCCCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTATGGAGCCAGCCGCCGAAGGGGACCC.
Sequencing results were analyzed by BLAST and aligned with sequences in GenBank, and phylogenetic tree of strain JK-2 was constructed by MEGA-X software, and the construction results are shown in FIG. 4. The results showed that the 16S rDNA sequence of strain JK-2 has 99% similarity with Bacillus amyloliquefaciens (OK 178286.1) and is aggregated in the same branch with more than 80% support rate, and that endophytic bacterium JK-2 was identified as Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) in combination with morphological observation and physiological biochemical experiments.
EXAMPLE 5 monitoring of Strain JK-2 growth Curve and pH
After single bacterial colony of bacillus amyloliquefaciens (Bacillus amyloliquefaciens) JK-2 obtained by streaking in an LB plate is prepared into seed bacterial suspension, the seed bacterial suspension is added into 30mL of LB liquid medium according to 2% of inoculation amount, shake culture is carried out for 12 hours at 37 ℃ in a shaking table at 200r/min, and 3 repeats are set. The absorbance of the bacterial liquid was measured every 2 hours at a wavelength of 600nm using a spectrophotometer, and the corresponding change in pH was also measured.
The specific monitoring results are shown in FIG. 5, and the growth curve of the bacillus amyloliquefaciens (Bacillus amyloliquefaciens) JK-2 strain of the invention is characterized in that: 0-2h is incubation period, 2-12h is exponential growth phase, 18h reaches maximum, and 20-48h is stationary growth phase (FIG. 5); the initial pH of the LB culture solution is 6.98, the pH value starts to rise when the pH value is 4h, the pH value reaches 8.56 in 18h, and the pH value is in a stationary phase in 18h-48h, so that the pH value does not rise any more.
EXAMPLE 6 identification of bacteriostatic Activity of Strain JK-2
The whole fermentation liquid thalli of the strain JK-2, thalli after fermentation liquid centrifugation and supernatant fluid after centrifugation are respectively cultivated in the same flat plate in a counter manner, and the position of bacteriostatic substances is determined to belong to the thalli or to be secreted outside the thalli through the bacteriostatic effects of three different parts of the strain JK-2.
As shown in FIG. 6, the fermentation broth and the bacteria after centrifugation of the fermentation broth show a zone of inhibition around, which indicates that the bacteria has remarkable antibacterial activity, while the supernatant after centrifugation is completely covered by fusarium mycelia of horsetail, which indicates that the antibacterial substance produced by JK-2 is the bacteria itself and does not have the characteristic of extracellular secretion.
Example 7 safety assay of Strain JK-2
According to the invention, a hemolysis test is carried out on bacillus amyloliquefaciens JK-2 strain, single colony of the JK-2 strain is streaked and inoculated in a blood agar plate, and the bacillus amyloliquefaciens is cultivated for 24 hours at a constant temperature of 37 ℃ to observe whether a transparent ring appears around the thallus.
As can be seen from the results of FIG. 7, no transparent ring appeared around the cells, indicating that the cells were safe to human animals.
EXAMPLE 8 antibacterial broad-spectrum assay of Strain JK-2
To examine the antibacterial broad spectrum of Bacillus amyloliquefaciens JK-2 strain, it was cultivated in opposition to Aspergillus niger, which is a pathogenic fungus previously selected by all the Proc. Of the Ministry of microbiology, hebei province, and Aspergillus fumigatus (B.cirear) B16, botrytis cinerea (B.dothidea) D330, and Penicillium distension (P.expansum) HH1, respectively, which were previously selected by the Proc. Of the Ministry of the agriculture and forestry, hebei province, and 3 fungi selected by the Proc. Of the food science, respectively, were cultivated in the articles "Volatile organic compounds produced by Bacillus aryabhattai AYG1023 against Penicillium expansum causing blue mold on the Huangguan pear", and Bacillus amyloliquefaciens JK-2 strain and the above 4 fungi were co-cultivated with the above 4 fungi, respectively, with sterile water as a control, and 3 parallel experiments were performed per group.
And (3) adopting a punching method, punching holes in four directions of a position which is 15cm away from a central point in a 90mm PDA flat plate, respectively, 6mm, taking out the agar after punching, inoculating 20 mu L of JK-2 bacterial suspension into the holes, inoculating a Fusarium equisetum bacterial cake (6 mm) on the other side, culturing the two strains of bacteria on the same line at the constant temperature of 28 ℃ for 3-4d, and observing whether a transparent bacteriostasis ring appears.
As shown in FIG. 8, the bacteria inhibition zones were observed in the plates of the 4 fungi which were cultivated in opposition to the Bacillus amyloliquefaciens JK-2 strain, respectively, while the control was grown normally without inhibition zones. Therefore, the bacillus amyloliquefaciens JK-2 strain has bacteriostasis broad spectrum.
The bacillus amyloliquefaciens JK-2 strain (Bacillus amyloliquefaciens) separated from honeysuckle tissues has broad-spectrum antibacterial activity and great potential as a microbial control microbial agent, so that diseases caused by pathogenic bacteria such as fusarium equisetum, aspergillus niger, gray mold, grape vine cavity bacteria, penicillium and the like are inhibited, and the crop yield and quality are improved.
Although the foregoing embodiments have been described in some, but not all, embodiments of the invention, it should be understood that other embodiments may be devised in accordance with the present embodiments without departing from the spirit and scope of the invention.
Claims (10)
1. Antagonizing Fusarium equisetumFusarium equiseti）Bacillus amyloliquefaciens @Bacillus amyloliquefaciens) JK-2, characterized in that the preservation number is CGMCC No.29211.
2. The bacillus amyloliquefaciens JK-2 of claim 1, wherein the 16S rDNA sequence of the bacillus amyloliquefaciens JK-2 is set forth in SEQ ID No: 1.
3. The method for culturing bacillus amyloliquefaciens JK-2 according to claim 1 or 2, comprising the steps of: and (3) preparing single bacterial colony of the bacillus amyloliquefaciens JK-2 into seed bacterial suspension, and then inoculating the seed bacterial suspension into an LB liquid culture medium for shake culture.
4. A method of culturing according to claim 3, wherein the seed bacterial suspension is inoculated at 2%.
5. The method according to claim 3, wherein the shaking culture comprises shaking culture at 37℃for 18 to 48 hours with 200 r/min.
6. Use of bacillus amyloliquefaciens JK-2 according to claim 1 or 2 or a culture solution obtained by the culture method according to any one of claims 3 to 5 for preparing an antibacterial agent.
7. The use according to claim 6, wherein the active ingredient of the antibacterial agent comprises a bacterial cell of the bacillus amyloliquefaciens JK-2 or a bacterial liquid containing the bacillus amyloliquefaciens JK-2.
8. A broad-spectrum antibacterial agent comprising the bacterial cells of Bacillus amyloliquefaciens JK-2 or a bacterial liquid containing the bacterial cells as claimed in claim 1 or 2 as an active ingredient.
9. The broad spectrum antimicrobial agent of claim 7, wherein the antimicrobial spectrum comprises: fusarium equisetum, aspergillus niger, botrytis cinerea and Penicillium viridis.
10. A method for controlling honeysuckle diseases, which comprises applying the broad-spectrum antibacterial agent of claim 8 or 9 to the tree body of honeysuckle.
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