CN109182151A - The separating screening method of gingko endogenous fungus - Google Patents
The separating screening method of gingko endogenous fungus Download PDFInfo
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- CN109182151A CN109182151A CN201811289627.8A CN201811289627A CN109182151A CN 109182151 A CN109182151 A CN 109182151A CN 201811289627 A CN201811289627 A CN 201811289627A CN 109182151 A CN109182151 A CN 109182151A
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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
- C12N1/14—Fungi; Culture media therefor
Abstract
The invention discloses a kind of separating screening methods of gingko endogenous fungus, acquire Root of Ginkgoes, stem, leaf, pulp, kernel；By Ginkgo Biloba after surface sterilization, tissue of the surface without any growth is therefrom selected in culture；Sterile tissue is aseptically cut into patch-graft in PDA culture medium, setting 28 DEG C of insulating box cultures, until when growing mycelia around sample, it is spare to be transferred to PDA slant medium for the different bacterium colony of picking form after purified；Liquid fermentation medium is divided in triangular flask, is cultivated in culture bottle with the Endophytic Fungal Hyphae that inoculation hook picking isolates and purifies a little, takes fermented supernatant fluid；It takes supernatant and the PDA culture medium for being cooled to 50 DEG C in sterile petri dish, putting for trying bacteria cake, calculates each bacterial strain fermentation liquor to the inhibiting rate for trying pathogen；It is spare that the efficient inhibiting rate endogenetic fungal bacterial strain filtered out carries out preservation.
Description
Technical field
The present invention relates to microorganisms technical fields, separate more particularly to from Ginkgo Biloba, fermented to produce
The method of the raw endogenetic fungus for inhibiting fungal diseases of plants pathogen metabolite.
Background technique
Ginkgo (Ginkgo biloba L.) also known as gingko are the rare rare tree species in the distinctive world of China, are glacier fortune
The dynamic Relict Plant left, referred to as living fossil.Belong to Ginkgoaceae, is Ginkgo existing unique existence kind.It is now cultivation extensively
Tree species are worth with high research and development.Ginkgo is respectively organized to contain antibacterial substance, it was reported that Root of Ginkgoes, stem, leaf, pulp
The extract of (exosper) and kernel has inhibiting effect to the phytopathogen for examination.But plant resources amount is that limitation plant source kills
One of the principal element of microbial inoculum development scale.But the separation screening endogenetic fungus from ginkgo, and it is true to plant by fermentation acquisition
The inhibited metabolite of fungus diseases pathogen, this at home and abroad yet there are no studies have reported that.
Summary of the invention
Object of the present invention is to propose that one kind from Ginkgo Biloba, separates its metabolite and falls ill to plant mycosis
Sterilization work is extracted from plants by microbe fermentation method substitution to reach in the method that opportunistic pathogen has the endogenetic fungus of obvious inhibiting effect
The conventional method of property substance.
The purpose of the present invention is achieved by the following technical programs.
A kind of separating screening method of gingko endogenous fungus, method includes the following steps:
(1) Root of Ginkgoes, stem, leaf, pulp, kernel are acquired；
(2) by step (1) Ginkgo Biloba after surface sterilization, directly plantation is on PDA culture medium plate, setting 26 DEG C~28 DEG C perseverances
Tissue of the surface without any growth is therefrom selected in incubator culture；
(3) sterile tissue of step (2) is aseptically cut into patch-graft in PDA culture medium, setting 28 DEG C of constant temperature
Case culture, until, using Tip Splitting picking method, the different bacterium colony of picking form turns after purified when growing mycelia around sample
It is spare to be connected to PDA slant medium；
(4) its each component with every liter contained by quality be peptone 0-5.5g, corn flour 1-8g, wheat bran 0-10g, soluble starch
0-10g, glucose 1-8g, soybean cake powder 1-9g, CaCO30.5-2.5g、NH4NO30.1-1.2g、MgSO40.1-0.9g、
KH2PO40.1-0.9g；
(5) step (4) liquid fermentation medium is divided in triangular flask, is separated a little through step (3) with inoculation hook picking pure
The Endophytic Fungal Hyphae of change sets 28 DEG C of shaking tables, 200r/min, shaken cultivation 4 days, fermentation liquid was through 5000r/min in culture bottle
It is centrifuged 10min, takes fermented supernatant fluid；
(6) step (5) each supernatant is taken to mix by milliliter volume 1: 9 in sterile petri dish with the PDA culture medium for being cooled to 50 DEG C
It is interior, 1 diameter, which is put, in each culture base plane after solidification calculates each bacterium after setting 28 DEG C of incubator cultures for examination bacteria cake for 4mm
Strain fermentation liquid is to the inhibiting rate for trying pathogen；
(7) the efficient inhibiting rate endogenetic fungal bacterial strain for filtering out step (6) carries out preservation, spare.
Beneficial effects of the present invention, first is that the present invention according to " endosymbiotic theory " filtered out from ginkgo its metabolite with
Host plant extract is the same or similar, the endogenetic fungus inhibited to fungal diseases of plants pathogen, this is real
Now by extracting Fungicidal active substance in plant to the conversion of Production by Microorganism Fermentation Fungicidal active substance, effective bacterium is provided
Kind separating screening method；Second is that each endogenetic fungal bacterial strain metabolite that tracking and measuring of the present invention is separated to is to test plant disease
The inhibiting effect of opportunistic pathogen provides a batch and sets out to obtain effective strain for the further development and exploitation of microbial pesticide
Bacterial strain；Third is that the present invention has paid attention to Sterility testing early period to plant tissue, and have developed the applicable liquid of gingko endogenous fungus
Fermentation medium improves success rate and efficiency to endogenetic fungus separation screening.
Specific embodiment
The present invention will be further described below with reference to examples.
Embodiment 1:
Caulis Ginkgo tissue is acquired, carry out surface sterilization by following procedure: tap water rinses, and 75% alcohol rinses 5min, sterile water
Rinse 5 times, then with mercuric chloride impregnate 4min, aseptic water washing 4 times；Stem tissue after surface sterilization is directly planted in PDA culture medium
On plate, culture in 28 DEG C of insulating boxs is set, stem tissue of the surface without any growth is therefrom selected；By the stem through Sterility testing
(taking bast) tissue, is cut into the small pieces of about 0.5cm × 0.5cm under the conditions of superclean bench, then by small pieces plant in
On PDA culture medium plate, 28 DEG C of insulating box cultures are set；When obviously growing mycelia around sample, using Tip Splitting picking method,
The different bacterium colony of picking form is transferred to PDA slant medium after purified, spare；By it is above-mentioned separate it is gingko endogenous true
Each bacterial strain of bacterium carries out liquid fermentation culture, culture medium each component with every liter contained by quality are as follows: peptone 2.5g, corn flour
3g, wheat bran 5g, glucose 4g, soluble starch 4g, soybean cake powder 4g, CaCO3 1g、NH4NO3 0.6g、MgSO4 0.1g、
KH2PO40.6g；500mL liquid fermentation medium is divided in the triangular flask of 300mL, is 50mL per bottled liquid measure；With connecing
Kind of a little inclined-plane culture mycelium inoculation of hook picking is placed in 28 DEG C of shaking tables in culture bottle, 200r/min, and shaken cultivation 4 days；Fermentation
Liquid 5000r/min is centrifuged 10min, takes supernatant, abandons mycelium；Fermentation liquid Antibacterial Activity: it takes on above-mentioned each strain fermentation
Clear liquid 1mL is mixed rapidly in sterile petri dish with the 9mL PDA culture medium for being cooled to 50 DEG C, flat in each culture medium after cooling
1 bacteria cake (diameter 4mm) for trying bacterium Rhizoctonia solani (Rhizoctonia solani) is put in face, and bacteria cake is connected to culture dish
Center (culture dish diameter is 9cm), sets in 28 DEG C of incubators and cultivates 36h, using sterile water process as control, be repeated 3 times；It calculates
Inhibiting rate, it is spare that the efficient inhibiting rate endogenetic fungal bacterial strain filtered out carries out preservation.
Claims (1)
1. a kind of separating screening method of gingko endogenous fungus, it is characterized in that the following steps are included:
(1) Root of Ginkgoes, stem, leaf, pulp, kernel are acquired；
(2) by step (1) Ginkgo Biloba after surface sterilization, directly plantation is on PDA culture medium plate, setting 26 DEG C~28 DEG C perseverances
Tissue of the surface without any growth is therefrom selected in incubator culture；
(3) sterile tissue of step (2) is aseptically cut into patch-graft in PDA culture medium, setting 28 DEG C of constant temperature
Case culture, until, using Tip Splitting picking method, the different bacterium colony of picking form turns after purified when growing mycelia around sample
It is spare to be connected to PDA slant medium；
(4) its each component with every liter contained by quality be peptone 0-5.5g, corn flour 1-8g, wheat bran 0-10g, soluble starch
0-10g, glucose 1-8g, soybean cake powder 1-9g, CaCO30.5-2.5g、NH4NO30.1-1.2g、MgSO40.1-0.9g、
KH2PO40.1-0.9g；
(5) step (4) liquid fermentation medium is divided in triangular flask, is separated a little through step (3) with inoculation hook picking pure
The Endophytic Fungal Hyphae of change sets 28 DEG C of shaking tables, 200r/min, shaken cultivation 4 days, fermentation liquid was through 5000r/min in culture bottle
It is centrifuged 10min, takes fermented supernatant fluid；
(6) step (5) each supernatant is taken to mix by milliliter volume 1: 9 in sterile petri dish with the PDA culture medium for being cooled to 50 DEG C
It is interior, 1 diameter, which is put, in each culture base plane after solidification calculates each bacterium after setting 28 DEG C of incubator cultures for examination bacteria cake for 4mm
Strain fermentation liquid is to the inhibiting rate for trying pathogen；
(7) the efficient inhibiting rate endogenetic fungal bacterial strain for filtering out step (6) carries out preservation, spare.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811289627.8A CN109182151A (en) | 2018-10-31 | 2018-10-31 | The separating screening method of gingko endogenous fungus |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811289627.8A CN109182151A (en) | 2018-10-31 | 2018-10-31 | The separating screening method of gingko endogenous fungus |
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| Publication Number | Publication Date |
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| CN109182151A true CN109182151A (en) | 2019-01-11 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811289627.8A Withdrawn CN109182151A (en) | 2018-10-31 | 2018-10-31 | The separating screening method of gingko endogenous fungus |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112625977A (en) * | 2021-01-13 | 2021-04-09 | 重庆工贸职业技术学院 | Separation and purification method of sweet wormwood endophyte and application thereof |
| KR102308626B1 (en) * | 2020-11-26 | 2021-10-05 | 주식회사 프레코 | Coating composition for protecting tree and wood and for controlling plant disease and insect pest |
| WO2022188162A1 (en) * | 2021-03-12 | 2022-09-15 | 德州学院 | Endophytic fungus in ginkgo biloba and anti-bacterial and antitumor application thereof |
-
2018
- 2018-10-31 CN CN201811289627.8A patent/CN109182151A/en not_active Withdrawn
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR102308626B1 (en) * | 2020-11-26 | 2021-10-05 | 주식회사 프레코 | Coating composition for protecting tree and wood and for controlling plant disease and insect pest |
| CN112625977A (en) * | 2021-01-13 | 2021-04-09 | 重庆工贸职业技术学院 | Separation and purification method of sweet wormwood endophyte and application thereof |
| WO2022188162A1 (en) * | 2021-03-12 | 2022-09-15 | 德州学院 | Endophytic fungus in ginkgo biloba and anti-bacterial and antitumor application thereof |
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| Date | Code | Title | Description |
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| PB01 | Publication | ||
| PB01 | Publication | ||
| WW01 | Invention patent application withdrawn after publication |
Application publication date: 20190111 |
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| WW01 | Invention patent application withdrawn after publication |