CN106754396B - A kind of Chinese toon endogenetic fungus TS8 and its secondary metabolite, preparation method and application - Google Patents
A kind of Chinese toon endogenetic fungus TS8 and its secondary metabolite, preparation method and application Download PDFInfo
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Abstract
The invention belongs to microorganisms technical field, a kind of Chinese toon endogenetic fungus TS8 and its secondary metabolite, preparation method and application are disclosed.The fungi is Ascomycota, Ascomycetes, excrement shell subclass, excrement shell mesh, Mao Ke section, Chaetomium, chaetomium globosum；Depositary institution: China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica, preservation date: on September 30th, 2016, deposit number: CGMCC No.12979.Isolated one plant of endogenetic fungus from Chinese toon axis, the bacterium secondary metabolite have good inhibiting effect to soft rot Erwinia and bacterial wilt Ralstonia solanacearum to the present invention for the first time.The method of preparation Chinese toon endogenetic fungus TS8 secondary metabolite of the present invention is simple and easy, at low cost.
Description
Technical field
The invention belongs to microorganisms technical fields, and in particular to a kind of Chinese toon endogenetic fungus TS8 and its secondary metabolite,
Preparation method and application.
Background technique
China's fruits and vegetables annual output in 2014 is more than 9.4 hundred million tons.Fruits and vegetables have become second and the third-largest production after grain
Industry, and account for the specific gravity of world market 50% or more, and in gradually rising trend.Soft rot be in fruits and vegetables class agricultural production most
A kind of common bacterial disease, relevant information show that the rotten loss rate of China's fruits and vegetables circulation is up to 20-30%, cause economic damage every year
Up to more than 1,000 hundred million yuan are lost, the yield and quality of fruits and vegetables is seriously affected, causes huge economy to the numerous producers and operator
Loss.All the time, chemical pesticide is the main means of plant pest management, but can effectively prevent fruits and vegetables soft rot at present
Harmful medicament is still less, mainly uses the farm antibiotics class system such as streptomysin, kasugarnycin to the prevention and treatment of the disease both at home and abroad
The copper chemicals two major classes such as agent and Thiodiazole-copper, thiophene.But such chemical agent often exist high residue, high toxicity and
The problems such as environmental pollution, to human security there is also hidden danger, many kinds are disabled.In addition chemical agent is also easy to generate disease
The drug resistance of opportunistic pathogen, influences control efficiency.In contrast, the active material of plant and microbial source similar " pesticide " has selection
Property high and low malicious, degradable, the advantages that being not easy to produce resistance, especially protect environment in people, advocate nature and concern food
Under the new normality of safety etc., booster injection is to the novel plant and microbial source active matter Quality Research of the fruits and vegetables prevention and control of plant diseases, pest control and opens
Hair, all has significance to fruits and vegetables industry healthy and sustainable development and human health.
However due in plant natural active matter content it is lower, extraction process is complicated and plant resources often by
The influence of the factors such as geographical environment and ecological condition, so developing natural active matter from plant also has certain limitation
Property.According to symbiosis theory, plant endogenesis epiphyte can produce with the same or similar bioactive substance of host plant, plant simultaneously
Object endogenetic fungus has the characteristics that the speed of growth is fast, the fermenting and producing period is short, is easy to industrial fermentation as microorganism, thus
Plant endogenesis epiphyte is set to become the new resources for finding and finding various natural bioactivity substances.Many is derived from plant endogenesis epiphyte
Secondary metabolite identified to have provided the various biologicals such as anti-oxidant, antitumor, antibacterial, desinsection and enzyme inhibitor living
Property.The various compound of structure novel caused by plant endogenesis epiphyte, activity provides for the discovery of novel antifungal drugs
Important lead compound and new drug target, become the important sources of natural active product and new drug development.
Summary of the invention
To overcome existing fruits and vegetables soft rot to treat problem, the purpose of the present invention is intended to provide a kind of Chinese toon endogenetic fungus TS8
And its secondary metabolite, preparation method and application.
To achieve the above object, the technical solution adopted by the present invention is as follows:
A kind of Chinese toon endogenetic fungus TS8, the fungi are AscomycotaAscomycota, AscomycetesAscomycetes, excrement
Shell subclassSordariomycetidae, excrement shell meshSordariales, Mao Ke sectionChaetomiaceae, ChaetomiumChaetomium, chaetomium globosumChaetomium globosum；Depositary institution: China Committee for Culture Collection of Microorganisms
Common micro-organisms center, preservation address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica is protected
Hide the date: on September 30th, 2016, deposit number: CGMCC No.12979, classification naming: chaetomium globosumChaetomium globosum。
The method for the secondary metabolite for preparing Chinese toon endogenetic fungus 56-50 the present invention provides two kinds, specific as follows:
Method one, steps are as follows:
(1) strain fermentation:
The Chinese toon endogenetic fungus TS8 that deposit number is CGMCC No.12979 is inoculated on PDA plate culture medium, 25-28
DEG C activation culture 3-5d, is then seeded into the taper triangular flask for filling PDA liquid fermentation medium, 25-28 DEG C, 150-
180rpm shaker fermentation culture 6-8d；
(2) preparation of bacterial strain secondary metabolite:
The fermentation culture of step (1) obtained strains TS8 is first blended, refilters, obtains fermentation liquid and mycelium；Then
Fermentation liquid and mycelium are extracted respectively using ethyl acetate, repeat extracted several times, combining extraction liquid, concentration is prepared
The secondary metabolite of Chinese toon endogenetic fungus TS8；Wherein, when being extracted to fermentation liquid, the dosage and fermentation liquid of ethyl acetate
In equal volume；When extracting to mycelium, the dosage of ethyl acetate, which is subject to, impregnates mycelium.
Method two, steps are as follows:
(1) strain fermentation:
The Chinese toon endogenetic fungus TS8 that deposit number is CGMCC No.12979 is inoculated on PDA plate culture medium, 25-28
DEG C activation culture 3-5d, is then seeded into the taper triangular flask for filling PDA liquid fermentation medium, is stored at room temperature fermented and cultured
25-30d；
(2) preparation of bacterial strain secondary metabolite:
The fermentation culture of step (1) obtained strains TS8 is first blended, refilters, obtains fermentation liquid and mycelium；Fermentation
Liquid is extracted with isometric ethyl acetate, repeats extracted several times, and combining extraction liquid is concentrated in vacuo to dry, obtains the acetic acid of fermentation liquid
Methacrylate layer extract；The aqueous acetone solution soak extraction of mycelium 80v%, is extracted for several times, combined extract repeatedly, and vacuum is dense
It is reduced to after being free of acetone, then is extracted with isometric ethyl acetate, repeat extracted several times, be then combined with extract liquor and vacuum is dense
It is reduced to dry, obtains mycelial ethyl acetate layer extract；Finally merge fermentation liquid and mycelial ethyl acetate layer extract,
Up to the secondary metabolite of Chinese toon endogenetic fungus TS8.
Utilize the secondary metabolite of the Chinese toon endogenetic fungus TS8 of above two preparation method preparation.
The secondary metabolite of the Chinese toon endogenetic fungus TS8 is in degradation soft rot Erwinia and/or bacterial wilt Solanaceae
Application in Lei Er Salmonella.
Compared with prior art, the invention has the following advantages:
(1) present invention for the first time from Chinese toon axis isolated one plant of endogenetic fungus TS8 (Chaetomium globosum), which has preferable inhibit to soft rot Erwinia and bacterial wilt Ralstonia solanacearum
Effect；
(2) described in preparation Chinese toon endogenetic fungus TS8 (Chaetomium globosum) secondary metabolite method
It is simple and easy, it is at low cost.
Detailed description of the invention
Fig. 1 is 1 Chinese toon endogenetic fungus TS8 strain morphology feature of embodiment, and wherein a is bacterium colony front form, and b is that bacterium colony is anti-
Face form, c, d are respectively mycelia (× 20) and (× 100) form.
Fig. 2 is the 18S rDNA gene fragment amplification map of 1 Chinese toon endogenetic fungus TS8 of embodiment, wherein 1--18S rDNA
Gene fragment amplification product, 2 be DNA Ladder Mix Marker.
Fig. 3 is the ITS rDNA gene fragment amplification map of 1 Chinese toon endogenetic fungus TS8 of embodiment, 1--ITS rDNA gene
Fragment amplification product, 2 be DNA Ladder Mix Marker.
Fig. 4 is the systematic evolution tree that 1 Chinese toon endogenetic fungus TS8 bacterial strain of embodiment is constructed based on 18S gene order.
Fig. 5 is the systematic evolution tree that 1 Chinese toon endogenetic fungus TS8 bacterial strain of embodiment is constructed based on ITS gene order.
Specific embodiment
It is described in detail combined with specific embodiments below, it is to be understood that protection scope of the present invention is not by specific reality
Apply the limitation of example.Material as used in the following examples, reagent etc., are commercially available unless otherwise specified.
In following embodiment:
For trying Chinese toon material: picking up from Zhengzhou City Henan Province Zhongmou County country estate village Henan Academy of Agricultural Sciences Chinese toon demonstration base
Ground.
Strains tested: purchased from Chinese agriculture Microbiological Culture Collection administrative center Erwinia (Erwinia sp)
ACCC02203 and Ralstonia solanacearum (Ralstonia solanacearum) ACCC01474。
The weight percent of PDA plate culture medium and/or PDA slant medium composition are as follows: potato 20%, glucose 2%, fine jade
Rouge 2%, surplus are water, pH 6.0；The weight percent of PDA liquid fermentation medium forms are as follows: potato 20%, glucose 1%, wheat
Bud sugar 2%, mannitol 2%, peptone 0.5%, yeast extract 0.3%, monosodium glutamate 0.5%, surplus are water, pH 6.0；
The weight percent of nutrient culture medium forms are as follows: peptone 1%, beef extract 0.3%, NaCl 0.5% are remaining
Amount is water, pH 7.0.
The weight percent of nutrient agar culture medium forms are as follows: peptone 1%, beef extract 0.3%, NaCl
0.5%, agar 2%, surplus is water, pH 7.0.
Embodiment 1
1. the separation and screening of Chinese toon endogenetic fungus
Under aseptic technique, Chinese toon stem is impregnated into 1min in 75v% ethyl alcohol, in 3wt% chlorine of effective chlorine after taking-up
1min is impregnated in acid sodium solution, then impregnates 30s in 75v% ethyl alcohol, is then used rinsed with sterile water 3 times, and sterile blotting paper is dry；
It is cut into the fritter of 0.5cm or so with the scissors after sterilizing, is placed on PDA plate culture medium, every plate 4, in 28 DEG C of constant temperature
Mycelia is transferred to new PDA plate culture when inside plant tissues are to when growing mycelia around culture medium by incubator culture 5d
It crosses and cultivates on base, isolate and purify to obtain single bacterium colony after 5d, be then transferred into PDA slant medium test tube, 28 DEG C of constant temperature
After cultivating 5d, i.e. acquisition Chinese toon endogenetic fungus TS8,4 DEG C of refrigerators save, spare, and on September 30th, 2016, are preserved in China
General Microbiological Culture preservation administrative center, deposit number: CGMCC No.12979.
2. the identification of Chinese toon endogenetic fungus
2.1 strain morphology observation of characteristics
(1) the Chinese toon endogenetic fungus TS8 that 4 DEG C of refrigerators save colonial morphology feature: is seeded in PDA plate culture medium
On, 28 DEG C of continuous culture 4-5d observe the morphological features such as the color, size, quality of bacterium colony.(2) Microscope morphology feature: system
Make water logging piece, observes the mycelium of bacterial strain under an optical microscope.
2.2 bacterial strain 18S, ITS rDNA sequence and its phylogenetic analysis
2.2.1 the extraction of genomic DNA
The Chinese toon endogenetic fungus TS8 that 4 DEG C of refrigerators save is transferred on PDA plate culture medium, 28 DEG C are cultivated 5 days.Gene
The extraction of group DNA uses Ezup pillar fungal genomic DNA extraction agent box (SK8259), operates in strict accordance with specification.It mentions
The genomic DNA taken electrophoresis detection (20 min of 150 V, 100mA) in 1% Ago-Gel, 4 DEG C save backup, or in-
20 DEG C of medium-term and long-term preservations.
2.2.2 the PCR amplification of 18S rDNA and ITS genetic fragment
18S rDNA amplimer: NS1 (5 '-GTAGTCATATGCTTGTCTC-3 '), NS6 (5 '-GCAT
CACAGACCTGTTATTGCCTC-3’)。
ITS rDNA amplimer: ITS1 (5 '-TCCGTAGGTGAACCTGCGG-3 '), ITS4 (5 '-TC
CTCCGCTTATTGATATG C-3’)。
PCR reaction condition is as shown in table 1:
Pcr amplification reaction agents useful for same is purchased from Shanghai Sheng Gong Co., Ltd, and PCR is shown in reaction system such as table 2:
Pcr amplification product saves backup for 4 DEG C after the detection of 1% agarose gel electrophoresis.
2.2.3 target DNA sequence and phylogenetic analysis
Shanghai Sheng Gong Co., Ltd is transferred to be sequenced the PCR product of 18S rDNA and ITS rDNA.After obtaining sequence,
Blast comparison is carried out in NCBI, downloading utilizes ClustalX software with strain sequence similar in strains tested sequence homology
The multiple alignment for carrying out sequence, using MEGA7.0 software N-J method (Neighbor-Joining) phylogenetic tree construction, certainly
Opening up number is 1000.
3. bacterial strain ferments in a small amount
The Chinese toon endogenetic fungus TS8 that 4 DEG C of refrigerators save is inoculated on PDA plate culture medium, 28 DEG C of incubator activation trainings
4d is supported, is then seeded into the taper triangular flask for filling 200mL PDA liquid fermentation medium, 28 DEG C, 150rpm shaker fermentation
Cultivate 7d.
4. the preparation of bacterial strain secondary metabolite
After the completion of strain fermentation, the fermentation culture of Chinese toon endogenetic fungus TS8 is first blended with homogenate blender, then uses Bu Shi
Funnel filters, and obtains fermentation liquid and mycelium；Then fermentation liquid and mycelium are extracted respectively using ethyl acetate, is repeated
The secondary metabolite of Chinese toon endogenetic fungus TS8 is prepared in extraction 3 times, combining extraction liquid, concentration；Wherein, to fermentation liquid into
When row extraction, the dosage and fermentation liquid of ethyl acetate are isometric；When extracting to mycelium, the dosage of ethyl acetate is to impregnate
Firmly subject to mycelium.
5. Antifungal Activity in Vitro measures
Using the filter paper enzyme in agar diffusion method.
The secondary metabolite dehydrated alcohol dissolution of 5.1 Chinese toon endogenetic fungus TS8 is made into 20mg/mL mass concentration
Solution, as test liquid.
5.2 Erwinias (Erwinia Sp) ACCC02203 is inoculated in nutrient culture medium, 30 DEG C of culture 12h.
In the nutrient agar culture medium that the test bacteria liquid that 5.3 absorption 1mL 5.2 cultivate 12h has been heated to 100mL,
It mixes, inverted plate, is then placed on training with 4 layers of filter paper (6 mm) that three places (3 times i.e. parallel) of tweezers point will have openning hole
Primary surface is supported, and draws 15 μ L and is placed in for test liquid in the hole of each filter paper, plate is observed and measured after 30 DEG C of culture 12h
The diameter of inhibition zone.Make positive control experiment simultaneously, wherein positive control is 1mg/mL and 10mg/mL streptomysin.
6. result
6.1 strain morphology features
Bacterium colony front form and reverse side form are shown in Fig. 1 a, Fig. 1 b respectively, 20 times and 100 times forms of mycelia see respectively Fig. 1 c,
Fig. 1 d.Known to: the bacterial strain bacterium colony front TS8 is white hypha, and cotton-shaped cotton grows densely；Reverse side is then in faint yellow (Fig. 1 a, b).
Strain growth fast speed, 28 DEG C of culture 4-5d bacterium colonies are covered with culture dish.Mycelia has branch under micro- sem observation, has every smooth
Transparent (Fig. 1 c, d).
18S, ITS sequence and its phylogenetic analysis of 6.2 bacterial strains
The pcr amplification product of 18S and ITS rDNA genetic fragment is through 1% agarose gel electrophoresis, as shown in Figure 2,3, amplification
Product is respectively the specific amplification band of 1200bp and 600bp or so, and size is consistent with desired value.18S and ITS rDNA base
Because the pcr amplification product of segment is sequenced by Shanghai Sheng Gong Co., Ltd, sequencing result, respectively such as sequence table SEQ ID
Shown in No.1 and sequence table SEQ ID No.2, show: 18S the and ITS rDNA gene order length of the bacterial strain is respectively
1203bp and 553bp.
The 18S rDNA sequence (SEQ ID No.1) that PCR amplification obtains is subjected to BLAST analysis in NCBI, as a result table
It is bright, the 18S rDNA sequence and ball Chaetomium of bacterial strain TS8Chaetomium sp.18S sequence homology highest, similitude reaches
100%.Using ClastalX and Mega5.0 software, be based on 18S rDNA sequence construct phylogenetic tree, see Fig. 4, from system into
Change on tree as can be seen that each category is respectively formed an independent branch, bacterial strain TS8 withChaetomium globosum
(JN639019.1)、Chaetomium globosum(JN394588.1) andChaetomium sp.(EU826480.1) it is in
Same branch, affiliation is nearest, judges the bacterial strain for ball Chaetomium.
The ITS rDNA sequence (SEQ ID No.2) that PCR amplification obtains is subjected to BLAST comparison in NCBI, as a result table
It is bright, the sequence withChaetomium globosumITS sequence homology it is very high, similitude is up to 99%.Based on ITS rDNA sequence
Column phylogenetic tree construction, is shown in Fig. 5, from phylogenetic tree it can be seen that each kind is respectively formed an independent branch, bacterium
Strain TS8 withChaetomium globosum(GQ906953.1)With Chaetomium globosum(KJ186956.1) gather is one
Branch, value of bootstrapping are 99, show very close affiliation.
Comprehensive colonial morphology, micro-morphology and 18S and ITS rDNA gene sequencing are as a result, by raw in Chinese toon
Fungi TS8 is accredited as AscomycotaAscomycota, AscomycetesAscomycetes, excrement shell subclassSordariomycetidae, excrement shell meshSordariales, Mao Ke sectionChaetomiaceae, ChaetomiumChaetomium, ball hair
Shell bacteriumChaetomium globosum。
6.3 bacteriostatic activity
Antibacterial the results are shown in Table 3, the inhibition zone of the secondary metabolite of Chinese toon endogenetic fungal bacterial strain TS8 to Erwinia
For 19.20 ± 2.39 mm, greater than the inhibition zone (16.01 ± 0.58 mm) of 10mg/mL positive control streptomysin, so the bacterial strain
Secondary metabolite has more significant inhibiting effect to Erwinia.
Table 3:
| The secondary metabolite of bacterial strain TS8 | Streptomysin 1mg/mL | Streptomysin 10mg/mL | |
| Embodiment 1 | 19.20±2.39 | 12.16±0.19 | 16.01±0.58 |
Embodiment 2
Substantially the same manner as Example 1, difference is: the present embodiment is completed bacterial strain cometabolism according to following key step and is produced
Fermentation, preparation and the Antibacterial Activity of object.
1-2 step is the same as embodiment 1.
3. bacterial strain ferments in a small amount:
The Chinese toon endogenetic fungus TS8 that 4 DEG C of refrigerators save is inoculated on PDA plate culture medium, 28 DEG C of incubator activation trainings
4d is supported, is then seeded into the taper triangular flask for filling 200mL PDA liquid fermentation medium, is stored at room temperature fermented and cultured 30d.
4. the preparation of bacterial strain secondary metabolite:
After the completion of strain fermentation, the fermentation culture of bacterial strain TS8 is first blended with homogenate blender, and then Buchner funnel is taken out
Filter, obtains fermentation liquid and mycelium；Fermentation liquid is extracted with equivalent ethyl acetate, is repeated extraction 3 times, is merged the acetic acid second of fermentation liquid
Ester layer extract is concentrated in vacuo to dry to get the ethyl acetate layer extract for arriving fermentation liquid；Mycelium is water-soluble with the acetone of 80v%
Liquid (volume fraction that i.e. acetone accounts in aqueous solution is 80%) soak extraction, 3 times repeatedly, combined extract is concentrated in vacuo to not
It is extracted after acetone, then with isometric ethyl acetate, is repeated 3 times, is then combined with acetic acid ethyl acetate extract and is concentrated in vacuo
To dry to get mycelial ethyl acetate layer extract is arrived, finally merging obtains fermentation liquid and mycelial ethyl acetate layer extracts
Take object, the as secondary metabolite of Chinese toon endogenetic fungus TS8.
5. determination of activity
Using the filter paper enzyme in agar diffusion method.
The secondary metabolite dehydrated alcohol dissolution of 5.1 Chinese toon endogenetic fungus TS8 is made into 20mg/mL mass concentration
Solution, as test liquid.
5.2 Ralstonia solanacearums (Ralstonia solanacearum) ACCC01474 is inoculated in nutrient culture
In base, 30 DEG C of culture 12h.
In the nutrient agar culture medium that the test bacteria liquid that 5.3 absorption 1mL 5.2 cultivate 12h has been heated to 100mL,
It mixes, inverted plate, is then placed on training with 4 layers of filter paper (6 mm) that three places (3 times i.e. parallel) of tweezers point will have openning hole
Primary surface is supported, and draws 15 μ L and is placed in for test liquid in the hole of each filter paper, plate is observed and measured after 30 DEG C of culture 12h
The diameter of inhibition zone.Make positive control experiment simultaneously, wherein positive control is 1mg/mL and 10mg/mL streptomysin.
6. result:
Antibacterial the results are shown in Table 4, suppression of the secondary metabolite of Chinese toon endogenetic fungal bacterial strain TS8 to Ralstonia solanacearum
Bacterium circle is 19.59 ± 0.24 mm, suitable with the inhibition zone (19.93 ± 0.75 mm) of 1mg/mL positive control streptomysin.So
The bacterial strain secondary metabolite has good inhibiting effect to Ralstonia solanacearum.
Table 4:
| The secondary metabolite of bacterial strain TS8 | Streptomysin 1mg/mL | Streptomysin 10mg/mL | |
| Embodiment 2 | 19.59±0.24 | 19.93±0.75 | 27.75±0.84 |
SEQUENCE LISTING
<110>Henan Academy of Agricultural Sciences
<120>a kind of Chinese toon endogenetic fungus TS8 and its secondary metabolite, preparation method and application
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taagcatatc aat 553
Claims (5)
1. a kind of Chinese toon endogenetic fungus TS8, it is characterised in that: under aseptic technique, Chinese toon stem is soaked in 75v% ethyl alcohol
1min is steeped, 1min is impregnated after taking-up in effective chlorine 3wt% liquor natrii hypochloritis, then impregnate 30s in 75v% ethyl alcohol, then uses
Rinsed with sterile water 3 times, sterile blotting paper is dry；It is cut into the fritter of 0.5cm or so with the scissors after sterilizing, it is flat to be placed on PDA
On plate culture medium, every plate 4, in 28 DEG C of constant incubator culture 5d, when inside plant tissues are to growing mycelia around culture medium
When, mycelia is transferred to on new PDA plate culture medium culture of crossing, isolates and purifies to obtain single bacterium colony after 5d, then shift
Into PDA slant medium test tube, after 28 DEG C of constant temperature incubation 5d, i.e. acquisition Chinese toon endogenetic fungus TS8,4 DEG C of refrigerators are saved, standby
With；The fungi is AscomycotaAscomycota, AscomycetesAscomycetes, excrement shell subclassSordariomycetidae、
Excrement shell meshSordariales, Mao Ke sectionChaetomiaceae, ChaetomiumChaetomium, chaetomium globosumChaetomium globosum, and on September 30th, 2016, it is preserved in China General Microbiological culture presevation administrative center, deposit number: CGMCC
No.12979。
2. a kind of method for the secondary metabolite for preparing Chinese toon endogenetic fungus TS8, which is characterized in that steps are as follows:
(1) strain fermentation:
The Chinese toon endogenetic fungus TS8 that deposit number is CGMCC No.12979 is inoculated on PDA plate culture medium, 25-28 DEG C of work
Change culture 3-5d, is then seeded into the taper triangular flask for filling PDA liquid fermentation medium, 25-28 DEG C, 150-180rpm shakes
Bed fermented and cultured 6-8d；
(2) preparation of bacterial strain secondary metabolite:
The fermentation culture of step (1) obtained strains TS8 is first blended, refilters, obtains fermentation liquid and mycelium；Then it uses
Ethyl acetate respectively extracts fermentation liquid and mycelium, repeats extracted several times, combining extraction liquid, and Chinese toon is prepared in concentration
The secondary metabolite of endogenetic fungus TS8；Wherein, when being extracted to fermentation liquid, the bodies such as the dosage of ethyl acetate and fermentation liquid
Product；When extracting to mycelium, the dosage of ethyl acetate, which is subject to, impregnates mycelium.
3. a kind of method for the secondary metabolite for preparing Chinese toon endogenetic fungus TS8, which is characterized in that steps are as follows:
(1) strain fermentation:
The Chinese toon endogenetic fungus TS8 that deposit number is CGMCC No.12979 is inoculated on PDA plate culture medium, 25-28 DEG C of work
Change culture 3-5d, is then seeded into the taper triangular flask for filling PDA liquid fermentation medium, is stored at room temperature fermented and cultured 25-
30d；
(2) preparation of bacterial strain secondary metabolite:
The fermentation culture of step (1) obtained strains TS8 is first blended, refilters, obtains fermentation liquid and mycelium；Fermentation liquid is used
Isometric ethyl acetate extraction, repeats extracted several times, combining extraction liquid, is concentrated in vacuo to dry, obtains the ethyl acetate of fermentation liquid
Layer extract；The aqueous acetone solution soak extraction of mycelium 80v% extracts for several times repeatedly, and combined extract is concentrated in vacuo to
It is extracted after acetone, then with isometric ethyl acetate, repeats extracted several times, be then combined with extract liquor and be concentrated in vacuo to
It is dry, obtain mycelial ethyl acetate layer extract；Finally merge fermentation liquid and mycelial ethyl acetate layer extract to get
The secondary metabolite of Chinese toon endogenetic fungus TS8.
4. a kind of secondary metabolite of the Chinese toon endogenetic fungus TS8 prepared using the preparation method as described in Claims 2 or 3.
5. a kind of secondary metabolite of Chinese toon endogenetic fungus TS8 as claimed in claim 4 degradation soft rot Erwinia and/
Or the application in bacterial wilt Ralstonia solanacearum.
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| CN101280320A (en) * | 2008-04-30 | 2008-10-08 | 山东农业大学 | Method for preparing antibiotic substance from chaetomium globosum of plant endophytic fungi |
| CN104694397A (en) * | 2014-12-22 | 2015-06-10 | 吉林农业大学 | Chaetomium globosum and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101280320A (en) * | 2008-04-30 | 2008-10-08 | 山东农业大学 | Method for preparing antibiotic substance from chaetomium globosum of plant endophytic fungi |
| CN104694397A (en) * | 2014-12-22 | 2015-06-10 | 吉林农业大学 | Chaetomium globosum and application thereof |
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