CN108504594B - Amycolatopsis and application thereof in preparation of pseudo-ginseng root rot resistant agent - Google Patents
Amycolatopsis and application thereof in preparation of pseudo-ginseng root rot resistant agent Download PDFInfo
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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
-
- 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
-
- 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
Abstract
The invention relates to an amycolatopsis YIM PH20520 strain and application thereof in preparation of a pseudo-ginseng root rot resistant agent, belonging to the technical field of microbial pesticides. Amycolatopsis YIM PH20520 (of the present invention)Amycolatopsis speibonae YIM PH20520) which has been deposited in the chinese culture collection at 11/4/2017 with a deposition number of CCTCC NO: and M2017189. The pseudo-ginseng root rot resistant agent prepared by liquid fermentation culture of the bacterial strain has good inhibitory activity on pseudo-ginseng root rot pathogenic fungi, and can be used for preventing and treating pseudo-ginseng root rot. The invention has the advantages of high efficiency, low toxicity, low raw material cost and simple production process, and has potential and practical application value in biological control.
Description
Technical Field
The invention relates to an amycolatopsis and application thereof in preparation of a pseudo-ginseng root rot resistant agent, belonging to the technical field of microbial pesticides.
Background
The rapid development of green biopesticides is a key problem which needs to be solved urgently in national economy and social development, the microbial resources in China are rich, important guarantee is provided for independently researching and developing microbial pesticides, and the microbial pesticides become important and effective ways for preventing and treating plant diseases. Actinomycetes are important producers of agricultural antibiotics, more than 70% of which are produced by actinomycetes, and the agricultural antibiotics derived from actinomycetes mainly include validamycin, cycloheximide, kasugamycin and the like, and play a very important role in biological control of plant diseases.
Because the difficulty and the probability of finding new antibiotics from actinomycetes of common species are getting larger and smaller, people constantly turn their eyes to the research of rare actinomycetes resources such as amycolatopsis, parmonillosis, nocardia, saccharopolyspora and the like, and the role of the rare actinomycetes as a new agricultural antibiotic source becomes more and more important. In earlier researches, an active amycolatopsis is obtained by screening, and fermentation liquor of the active amycolatopsis has good activity of resisting the root rot of panax notoginseng, and has potential and practical application value in biological control of the root rot of panax notoginseng.
Disclosure of Invention
The invention aims to provide a strain of amycolatopsis YIM PH20520(Amycolatopsis speibonae YIM PH20520) and application thereof in preparing pseudo-ginseng root rot resistant agent. YIM PH20520 strain was deposited in the chinese type culture collection on 11/4/2017, address: the preservation number of Wuhan university in Wuhan City of China is CCTCC NO: and M2017189.
The inventionAmycolatopsis speibonae The YIM PH20520 strain has the morphological characteristics that: the colony is grey white, the surface is hairy, and sparse hyphae grow on the edge; the substrate hyphae and the aerial hyphae are filamentous and irregularly branched, and are not broken generally; the aerial hyphae form long spore chains, straight or wave or spiral, and the spores are round, oval or rod-shaped. Amplifying the 16S rRNA gene of the strain by adopting a conventional PCR method, sequencing to obtain a gene sequence, carrying out similarity analysis on the 16S rRNA gene sequence of the strain in an EzBioCloud database, and constructing a strain phylogenetic tree by adopting an adjacent approach (Neighbor-join) to complete phylogenetic analysis of the strain.
Phylogenetic trees constructed from the 16S rRNA gene sequences showed that: strain YIM PH20520 and pseudoamycolatopsis effective seedAmycolatopsis speibonae JS72TClustered in the same evolved branch, their 16S rRNA gene sequence similarity was 99.37%.
FIG. 1 shows the phylogenetic relationship between strain YIM PH20520 and amycolatopsis species constructed by the neighbor joining method, indicating that the strain belongs to one of amycolatopsis, which is named as amycolatopsis YIM PH20520(Amycolatopsis speibonae YIM PH 20520). The strain is preserved in China center for type culture Collection in 2017, 4 month and 11 days, and the address is as follows: the preservation number of Wuhan university in Wuhan City of China is CCTCC NO: and M2017189.
Amycolatopsis YIM PH20520 (of the present invention)Amycolatopsis speibonae YIM PH20520) in preparing anti-Notoginseng radix root rot agent.
The invention is realized by the following steps:
preparing a microbial agent for resisting pseudo-ginseng root rot:
1. culture of test tube species
Inoculating the strain YIM PH20520 on a test tube solid culture medium inclined plane, wherein the culture medium formula is as follows: 4g of yeast powder; 4g of glucose; 5g of malt extract; 3.5mg of compound vitamin; 12g of agar powder; 1000ml of water; the culture was carried out at 28 ℃ for 6 days at pH 7.2 to obtain tube seeds.
2. Liquid seed culture
Adopting a 500ml triangular flask to culture in a shake flask, and the method is to firstly prepare a liquid seed culture medium, wherein the formula of the liquid seed culture medium is as follows: 4g of yeast powder; 4g of glucose; 5g of malt extract; 3.5mg of compound vitamin; 1000ml of water; pH 7.2, 100ml of liquid medium was placed in each flask, sterilized at 120 ℃ for 30 minutes, cooled and inoculated to 1cm2Large test tube species were incubated for 3 days at 28 ℃ on a shaker at 220 rpm.
3. Preparation of pseudo-ginseng root rot resisting agent
Adopting a 1000ml triangular flask for shake flask culture, the method is to firstly prepare a liquid fermentation culture medium, and the formula of the liquid fermentation culture medium is as follows: 10.0g of glucose; 10.0g of starch; peptone 5.0 g; 5.0g of yeast powder; NaCl 4.0 g; k2HPO4 0.5g ；MgSO4·7H2O 0.5g ；CaCO31.0 g; adding water to 1000ml, sterilizing at pH 7.8 and 120 deg.C for 30 min, adding 200ml liquid fermentation medium into each triangular flask, sterilizing at 120 deg.C for 30 min, cooling, inoculating 20ml seed solution obtained by the method 2, and culturing at 28 deg.C on a shaker at rotation speed of 220 rpm for 6 days to obtain strain fermentation liquid. Adding equal volume of ethanol into the fermentation liquid, shaking for 6 hr, filtering to remove mycelium, steaming to remove ethanol in the supernatant to obtain fermented extract, and diluting the fermented extract with water to the volume of the original fermentation liquid to obtain the final product.
The microbial agent for resisting panax notoginseng root rot prepared by the method is tested for preventing and treating panax notoginseng root rot pathogenic bacteria by adopting indoor toxicity measurement (a spore germination inhibiting method and a flat plate bacteriostasis test method).
Indoor toxicity test results show that: in the experiment of the method for inhibiting spore germination, the inhibition rates of the bacterial strain YIM PH20520 anti-panax notoginseng root rot agent on the germination of pathogenic fungi of panax notoginseng root rot, such as fusarium oxysporum, fusarium solani, alternaria ginseng and phoma herbarum, are respectively 98%, 99%, 100% and 99%.
In the plate bacteriostasis test method, the diameters of the bacteriostasis rings of the pathogenic fungi of the root rot of panax notoginseng, such as fusarium oxysporum, fusarium solani, alternaria ginseng and phoma herbarum are respectively 21.6mm, 22.5mm, 27.8mm and 22.5 mm.
Indoor toxicity test shows that the preparation has strong in vitro inhibitory activity on pathogenic bacteria of root rot of panax notoginseng.
The invention provides an amycolatopsis strain YIM PH20520 and application thereof in fermentation preparation of a pseudo-ginseng root rot resistant agent. The invention has the advantages of high efficiency, low toxicity, low raw material cost and simple production process, and has potential and practical application value in biological control.
Drawings
FIG. 1 is a phylogenetic tree constructed based on the 16S rRNA gene sequence of the amycolatopsis YIM PH20520 of the present invention.
Detailed Description
The invention will be further illustrated with reference to specific examples:
the first embodiment is as follows: amycolatopsis YIM PH20520(Amycolatopsis speibonae YIM PH20520) identification
Extracting DNA of the strain by a conventional enzymolysis method, amplifying 16S rRNA gene of the strain by a PCR method, sending the amplified gene to Shanghai' S chemical company for sequencing to obtain a gene sequence, performing 16S rRNA gene sequence similarity analysis (http:// www.ezbiocloud.net/eztaxon) of the strain in an EzBioCloud database, and constructing a strain phylogenetic tree by an adjacent approach method (Neighbor-join) to complete strain phylogenetic analysis. By passingThe phylogenetic tree constructed by the 16S rRNA gene sequence shows that: strain YIM PH20520 and pseudoamycolatopsis effective seedAmycolatopsis speibonae JS72TClustered in the same evolved branch, their 16S rRNA gene sequence similarity was 99.37%. FIG. 1 shows the phylogenetic relationship between strain YIM PH20520 and amycolatopsis species constructed by the neighbor joining method, indicating that the strain belongs to one of amycolatopsis, which is named as amycolatopsis YIM PH20520(Amycolatopsis speibonae YIM PH20520) which has been deposited at the chinese collection of type cultures on day 11, 4 month 2017, address: the preservation number of Wuhan university in Wuhan City of China is CCTCC NO: and M2017189.
Example two: spore germination inhibition assay
1. Preparation of microbial agent for resisting pseudo-ginseng root rot for experiment
The microbial agent for resisting pseudo-ginseng root rot is prepared from amycolatopsis YIM PH20520 (C)Amycolatopsis speibonae YIM PH20520) strain was prepared by a liquid fermentation method. The concrete preparation process and conditions are the same as the description part of the invention content.
2. Preparation of a suspension of spores of pathogenic fungi of Panax notoginseng root rot
Respectively inoculating panax notoginseng root rot pathogenic fungi fusarium oxysporum, fusarium solani, alternaria ginseng and grass stem mold to a slant of a test tube solid culture medium for culturing for 6-8 days at 25 ℃, wherein the culture medium formula is a commonly used PDA culture medium, 200g of potatoes are peeled, cut into small blocks, added with 1000ml of water, boiled for 20 minutes, filtered to obtain 1000ml of filtrate, then added with 20g of glucose, 20g of agar and natural pH, tap water is used for washing off aerial hyphae after the hyphae grow over the culture medium, the spores are cultured under the irradiation of a 400nm fluorescent lamp, and after 2-3 days, the spores are washed by distilled water, and 1% of mixed solution of potassium dihydrogen phosphate and glucose is added to prepare a pathogenic fungus spore suspension with the concentration of 30-40 spores per visual field under 10 x 10 low-fold microscope for later use.
3. Test method for inhibiting spore germination
Mixing a test sample of the pseudo-ginseng root rot resistant agent with prepared pseudo-ginseng root rot pathogenic fungi fusarium oxysporum, fusarium solani, alternaria ginseng and phoma herbarum spore suspensions in equal volume respectively, taking a drop of the drop to be dripped on a concave glass slide, repeating the treatment for 3 times, and simultaneously setting clear water as a blank control. After 24 hours of incubation at 25 ℃ in a humidified state, germination of the blank control spores was observed, and germination was observed when the length of the germ tube exceeded half the diameter of the small end of the conidia. And when the germination rate of the blank control spores reaches 95%, observing the inhibition condition of the panax notoginseng root rot disease resisting agent sample on the germination of the panax notoginseng root rot disease pathogenic fungi spores.
Calculating the spore germination inhibition rate according to the following formula:
spore germination inhibition rate (%) = (control spore germination rate-treated spore germination rate)/(control spore germination rate) × 100
Experimental results show that the test sample of the strain YIM PH20520 pseudo-ginseng root rot resistant agent has the inhibition rate of 98% on the germination of fusarium oxysporum spores, 99% on the germination of fusarium solani spores, 100% on the germination of alternaria alternate spores and 99% on the germination of phoma herbarum spores.
Example three: plate bacteriostasis test
The microbial agent for resisting pseudo-ginseng root rot is prepared from amycolatopsis YIM PH20520 (C)Amycolatopsis speibonae YIM PH20520) strain was prepared by a liquid fermentation method. The concrete preparation process and conditions are the same as the description part of the invention content. Preparation of PDA culture medium, culture of pathogenic fungi of root rot of Notoginseng radix, and method for inhibiting spore germination.
Plate bacteriostasis test: respectively taking 0.5cm of cultured pathogenic fungi of radix Notoginseng root rot, such as Fusarium oxysporum, Fusarium solani, Alternaria ginseng and phoma herbarum2Mixing large test tube slant with 10 ml PDA culture medium, pouring into sterilized culture dish with inner diameter of 9cm to obtain plate with bacteria, placing oxford cup on each plate with bacteria, adding 0.2ml of prepared preparation for resisting Notoginseng radix root rot into oxford cup, culturing in 25 deg.C incubator for 48 hr with sterile clear water as blank control for 3 times, and culturing with sterile clear waterAnd measuring the diameter of the inhibition zone by a cross method.
The experimental results show that: the diameters of inhibition zones of the strain YIM PH20520 anti-pseudo-ginseng root rot agent for pathogenic fungi of pseudo-ginseng root rot, such as fusarium oxysporum, fusarium solani, alternaria ginseng and phoma herbarum, are respectively 21.6mm, 22.5mm, 27.8mm and 22.5mm on average.
Claims (2)
1. Acidophilic pseudobranchial bacteriumAmycolatopsis speibonaeYIM PH20520, which is characterized in that the preservation number of the strain is CCTCC NO: M2017189.
2. The amycolatopsis bacterium of claim 1Amycolatopsis speibonaeApplication of YIM PH20520 strain in preparing Notoginseng radix root rot resisting agent is provided.
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| CN110885776B (en) * | 2019-12-25 | 2021-07-06 | 云南大学 | Cellulose streptomyces fermentation extract and application thereof |
| CN113892484B (en) * | 2021-05-18 | 2022-09-02 | 云南农业大学 | Application of alpha-pinene in promoting growth and inducing resistance of panax notoginseng |
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