CN108728380B - Strain with antagonistic action on Chinese cabbage sclerotinia sclerotiorum and culture method and application thereof - Google Patents
Strain with antagonistic action on Chinese cabbage sclerotinia sclerotiorum and culture method and application thereof Download PDFInfo
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- CN108728380B CN108728380B CN201810571631.7A CN201810571631A CN108728380B CN 108728380 B CN108728380 B CN 108728380B CN 201810571631 A CN201810571631 A CN 201810571631A CN 108728380 B CN108728380 B CN 108728380B
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- chinese cabbage
- sclerotinia sclerotiorum
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/10—Animals; Substances produced thereby or obtained therefrom
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
Abstract
The invention discloses a bacterial strain with antagonistic action on Chinese cabbage sclerotinia sclerotiorum, which is a Paenibucillus kribbensis (Paenibucillus kribbensis), has the diameter of a single bacterial body of 0.5-0.7 × 2-3 microns, is free of capsule, is provided with periphytic flagella, is gram-positive, has the diameter of 0.7-0.9 × 1.1.1-1.4 microns, is oval, has rough and opaque surface, is white or yellowish, is provided with yellowish color after being cultured for 72 hours by shaking in a potato glucose liquid culture medium, is stored in a Chinese typical culture preservation center in 26 months of 2018, and has the preservation number of CCTCC NO: M2018236.
Description
Technical Field
The invention relates to the field of strains, in particular to a strain with antagonistic action on Chinese cabbage sclerotinia sclerotiorum and a culture method and application thereof.
Background
Paenibacillus (Paenibacillus) bacteria have been isolated from a variety of environments, many of which are related to humans, animals, plants and the environment. Most of the bacillus bacteria are found in soil, usually plant root growth-promoting bacteria, and have the function of promoting plant growth and can be used in agricultural production. Some Paenibacillus bacteria produce antibacterial compounds that are useful in medicine or pesticides, and some Paenibacillus bacteria are used to produce enzymes for bioremediation or to produce valuable chemicals. The Paenibacillus polymyxa can generate a plurality of bioactive substances such as polypeptide antibiotics, antagonistic proteins, enzymes, plant hormones, flocculating agents and the like, has the functions of biological pesticides and biological bacterial manure, and is widely applied to the fields of agriculture, industrial and mining industry, wastewater treatment and the like. The United states Environmental Protection Agency (EPA) has listed it as one of the commercially applicable microbial species, and the Ministry of agriculture in China also listed it as a primary strain without safety certification. The excessive use of chemical pesticides in traditional agriculture not only increases the pesticide residue, but also causes the drug resistance of many crop pests and causes pollution to soil, water and ecological environment. Along with the progress of society, the living standard of people is improved, the consciousness on food safety is continuously strengthened, and under the condition, the prevention and the treatment of plant epidemic diseases by applying beneficial microorganisms with high efficiency, broad spectrum, safety and no toxicity are increasingly paid more attention by people. The polypeptide antibacterial substance generated by the paenibacillus bacteria has good antagonistic activity on plant pathogenic fungi, has stable property, and can be used for biological control of various plant diseases. Therefore, the paenibacillus bacteria are biocontrol bacteria with great development prospect and application value.
Disclosure of Invention
The invention aims to provide a strain with antagonistic action on sclerotinia sclerotiorum of Chinese cabbage, a culture method and application thereof.
In order to achieve the purpose, the inventor separates a strain with antagonistic action on sclerotinia sclerotiorum of the Chinese cabbage from the Chinese cabbage, and verifies that the strain is bacillus Paenibacillus kribbensis (Paenibacillus kribbensis), and the strain is preserved in a China typical culture collection at 26 months and 4 months in 2018, with the preservation number of CCTCC NO: M2018236, and the address: eight paths of Wuhan university No. 299 in Wuchang district, Wuhan City, Hubei province, the strain DG1 is aerobic bacteria, the diameter of each bacteria is 0.5-0.7 × 2-3 microns, the strain is non-capsule, periphytic flagella, gram-positive, spores 0.7-0.9 × 1.1-1.4 microns, oval, the surface of the colony is rough, opaque, white or yellowish, and the strain is yellowish after being cultured in potato dextrose liquid culture medium for 72 hours.
The 16SrDNA sequence GenBank sequence number of the strain is SEQ MH 351014.
The invention also provides a culture method of the strain with antagonistic effect on the sclerotinia sclerotiorum of the Chinese cabbage, which comprises the following steps: the strain is inoculated into a 180mm gamma 18mm test tube containing 5ml PS (potato sucrose) culture gene, shaking table constant temperature culture is carried out at 28 ℃ and 180rpm for 2d, then the fermentation liquor is centrifuged for 10min at 4 ℃ and 10000 gamma g, supernatant is collected, and filtration sterilization is carried out by adopting a 0.22 mu m microporous filter membrane, thus obtaining the fermentation liquor of the strain.
The application of the strain with antagonism on the sclerotinia sclerotiorum of the Chinese cabbage is characterized in that the strain can be used for preventing and treating plant diseases. The fermentation liquor of the strain can inhibit the growth of hypha of sclerotinia sclerotiorum of Chinese cabbage.
The strain disclosed by the invention is simple to prepare, low in cost, non-toxic, harmless and free of environmental pollution, is beneficial to safely and effectively inhibiting the growth of sclerotinia sclerotiorum hyphae of the Chinese cabbage, is beneficial to green cultivation of the Chinese cabbage and protects a farmland ecosystem.
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FIG. 1 shows a Neighbor-Joining phylogenetic tree constructed by strain DG1 based on the 16S rRNA gene sequence alignment using Thermobacillus xylanicyticus (AJ005795.1) as an outgrowth.
Detailed Description
In order that the objects and advantages of the invention will be more clearly understood, the invention is further described in detail below with reference to examples. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Effect of antagonistic bacteria fermentation broth on hypha growth of sclerotinia sclerotiorum of Chinese cabbage
Three treatments were set up in total: treatment 1 was control (ck) Potato Sucrose (PS) liquid medium 300 ul; treatment 2 was 150ul broth +150ul Potato Sucrose (PS) broth; treatment 3 was 300ul of fermentation broth. Repeating the treatment for 3 times, respectively coating on PSA culture medium, then punching bacterial cake on the colony edge of Sclerotinia sclerotiorum with 5mml puncher, inoculating in the center of the coated PSA culture medium plate, culturing at 28 deg.C for 72 hr, respectively measuring colony diameter, and calculating inhibition rate. The formula for calculating the inhibition rate is as follows: inhibition = (control-treatment)/control 100; the calculation results are shown in table 1.
TABLE 1 inhibition calculation results for three groups of treatments
| Treatment of | Diameter cm of bacterial colony | Inhibition ratio% |
| Control (ck) | 3.1 | |
| 150ul fermentation liquor/dish | 2.5 | 19.4 |
| 300ul fermentation liquor/dish | 1.77 | 42.9 |
Comparison of the width of the antibacterial bands of the antagonistic bacteria on different plant pathogenic bacteria
The plate confronting method is adopted to measure the bacteriostatic zones of antagonistic bacteria to different phytopathogens, Chinese cabbage sclerotinia sclerotiorum, tomato fusarium, tomato black spot and cauliflower black spot are respectively inoculated to the center of a PSA culture medium plate, the joint of the antagonistic bacteria is connected to the periphery 2cm away from the pathogenic bacteria, after culture is carried out for 72 hours at 28 ℃, the widths of the bacteriostatic zones are respectively measured, and the results are shown in Table 2.
TABLE 2 width of the zone of inhibition of antagonistic bacteria against different phytopathogens
| Different pathogenic bacteria | Sclerotium rot of Chinese cabbage | Fusarium solani | Alternaria leaf spot of tomato | Cauliflower scab fungus |
| Width cm of bacteriostatic zone | 0.9 | 0.3 | 0.7 | 0.83 |
The foregoing is only a preferred embodiment of the present invention, and it should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be construed as the protection scope of the present invention.
Claims (5)
1. A strain with antagonism on Chinese cabbage sclerotinia sclerotiorum is characterized in that the strain is identified as Paenibacillus kribbensis (Paenibacillus kribbensis), is preserved in China center for type culture collection (CCTCC NO: M2018236) in 2018, 4 and 26 months.
2. The strain of claim 1 having antagonistic effect against sclerotinia sclerotiorum of chinese cabbage, wherein the 16SrDNA sequence GenBank of said strain has sequence number SEQ MH 351014.
3. A culture method of a strain fermentation liquor with antagonism to Chinese cabbage sclerotinia sclerotiorum is characterized by comprising the following steps: inoculating the strain having antagonistic action against sclerotinia sclerotiorum of chinese cabbage as described in claim 1 into a 180mm gamma 18mm test tube containing 5ml of PS (potato sucrose) medium, shake culturing at 28 deg.C and 180rpm for 2d, centrifuging the fermentation broth at 4 deg.C and 10000 gamma g for 10min, collecting the supernatant, and filtering and sterilizing with a 0.22 μm microporous filter membrane to obtain a strain fermentation broth having antagonistic action against sclerotinia sclerotiorum of chinese cabbage.
4. The use of a strain of Chinese cabbage sclerotinia sclerotiorum as claimed in claim 1, for the control of plant diseases.
5. The use of a strain of Chinese cabbage sclerotinia sclerotiorum as claimed in claim 4, wherein the fermentation broth of said strain inhibits the hyphal growth of Chinese cabbage sclerotinia sclerotiorum.
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| CN201810571631.7A CN108728380B (en) | 2018-06-06 | 2018-06-06 | Strain with antagonistic action on Chinese cabbage sclerotinia sclerotiorum and culture method and application thereof |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102352333B (en) * | 2011-10-21 | 2013-01-16 | 中国农业大学 | Nitrogen-fixing Paenibacillus sp. 1-49 and application thereof |
| CN102250809B (en) * | 2011-07-15 | 2013-03-20 | 中国农业科学院农业资源与农业区划研究所 | Wheat rhizosphere azotobacter for antagonism of various pathogenic fungi and application of wheat rhizosphere azotobacter |
| CN103756931B (en) * | 2013-12-05 | 2016-09-07 | 江西天人生态股份有限公司 | One strain Ke Liben series bacillus and application thereof |
| CN106929457A (en) * | 2017-05-10 | 2017-07-07 | 吉安职业技术学院 | A kind of Ke Liben series bacillus granule and preparation method thereof |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102250809B (en) * | 2011-07-15 | 2013-03-20 | 中国农业科学院农业资源与农业区划研究所 | Wheat rhizosphere azotobacter for antagonism of various pathogenic fungi and application of wheat rhizosphere azotobacter |
| CN102352333B (en) * | 2011-10-21 | 2013-01-16 | 中国农业大学 | Nitrogen-fixing Paenibacillus sp. 1-49 and application thereof |
| CN103756931B (en) * | 2013-12-05 | 2016-09-07 | 江西天人生态股份有限公司 | One strain Ke Liben series bacillus and application thereof |
| CN106929457A (en) * | 2017-05-10 | 2017-07-07 | 吉安职业技术学院 | A kind of Ke Liben series bacillus granule and preparation method thereof |
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