CN108102932A - A kind of Antagonistic Trichoderma vitro quick-screening method - Google Patents
A kind of Antagonistic Trichoderma vitro quick-screening method Download PDFInfo
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- CN108102932A CN108102932A CN201810020739.7A CN201810020739A CN108102932A CN 108102932 A CN108102932 A CN 108102932A CN 201810020739 A CN201810020739 A CN 201810020739A CN 108102932 A CN108102932 A CN 108102932A
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- trichoderma
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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
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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
- C12N1/02—Separating microorganisms from their culture media
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/37—Assays involving biological materials from specific organisms or of a specific nature from fungi
Abstract
The invention discloses a kind of antagonistic Trichoderma vitro quick-screening methods；The screening technique includes the following steps：The switching of pathogen：Pathogen after activation is beaten, bacteria cake is taken to be cultivated in 12 orifice plates；The activation of trichoderma strain：Activation culture Trichoderma is to producing spore；Opposite culture：Reesei spores are picked, are inoculated in 12 orifice plate, is placed in 28 DEG C of constant incubators and cultivates 4 6d；The screening of antagonistic strain：Using pathogen in 12 orifice plates described in area of colony scanner scanning not by the area of Trichoderma parasitism；By the descending arrangement of pathogen area, pathogen residual area is bigger, and the i.e. corresponding trichoderma strain Antagonism in the hole is poorer.The present invention screens antagonistic Trichoderma by using porous plate combination area of colony scanner, and this method the selection result has higher correlation with classic flat-plate face-off the selection result.
Description
Technical field
The invention belongs to field of biological control, are specifically a kind of Antagonistic Trichoderma vitro quick-screening method.
Background technology
Trichoderma is that one kind can prevent the various plants such as crop botrytis cinerea, pythium spp, rhizoctonia, anthrax-bacilus, sickle-like bacteria
A kind of biocontrol bacterial strain of disease mainly passes through induction of resistance, collaboration antagonism, Competition, the biological and ecological methods to prevent plant disease, pests, and erosions such as hyperparasite and antibiosis
The factor plays a role, while plant growth can be promoted to improve disease resistance of plant.
However to obtain excellent Antagonistic Trichoderma needs to carry out the screening of multistep.During high frequency zone Trichoderma antagonistic strain
The first step of large-scale development Trichoderma resource is also an important step.The domestic and international in vitro quick sieve on trichoderma strain at present
The research report of choosing is more rare.Mostly be to continue to use traditional method, and traditional screening technique be by tablet opposite culture come
Preliminary to obtain antagonistic strain, this screening technique is less efficient.Be not suitable for the screening of extensive Trichoderma resource, constrain excellent
The utilization rate of antagonistic Trichoderma bacterial strain.
A kind of rapid screening method of suitable Antagonistic Trichoderma is studied with important practical value.
The content of the invention
The problem relatively low it is an object of the invention to be directed to traditional Antagonistic Trichoderma screening efficiency, provides a kind of antagonism antagonism
Trichoderma vitro quick-screening method solves the slow-footed problem of Antagonistic Trichoderma Large-scale Screening.The present invention is using porous
Hardened to close area of colony scanner to carry out the extensive quick primary dcreening operation of antagonistic strain, this method saves material nearly 90%.By sieving
Accuracy rate is selected up to 70% or so.Greatly improve screening efficiency.The problem of the extensive primary dcreening operation of Trichoderma is this method solve,
Easily method is provided for Large-scale Screening Trichoderma resource in the future.
The Antagonistic Trichoderma vitro quick-screening method of the present invention uses following operation scheme：By the pathogenic strains after activation
And trichoderma strain, respectively by the different time and in a manner of be seeded in 12 orifice plates.Culture after a certain period of time, is swept using area of colony
Instrument scanning pathogen is retouched not by parasitic area.Screening obtains excellent antagonistic strain.
The area of colony scanner is that pathogen by the area that trichoderma is parasitic and captures is not scanned, is calculated.
It is mainly combined by Competition, hyperparasitism and antibiosis of the trichoderma when the pathogenic fungi interaction, smaller
Region in reflect the trend of different Trichoderma Antagonism powers.
The purpose of the present invention is what is be achieved through the following technical solutions：
The present invention relates to a kind of antagonistic Trichoderma vitro quick-screening method, the screening technique includes the following steps：
S1, the switching of pathogen：Pathogen after activation is beaten, bacteria cake is taken to cultivate 24-48h in 12 orifice plates (depending on pathogen
Depending on activity)；
The activation of S2, trichoderma strain：Activation culture Trichoderma is to producing spore (more than 3d)；
S3, opposite culture：Reesei spores are picked, are inoculated in 12 orifice plate, is placed in 28 DEG C of constant incubators and cultivates
4-6d；
The screening of S4, antagonistic strain：Using pathogen in 12 orifice plates described in area of colony scanner scanning not by Trichoderma
Parasitic area；By the descending arrangement of pathogen residual area, the bigger i.e. corresponding Trichoderma in the hole of pathogen residual area
Strain Antagonism is poorer.
Preferably, in step S1, the pathogen is fungi.
Preferably, the pathogen after the activation is 25 DEG C of -28 DEG C of cultures to bacterium colony size close to half of aperture, i.e. bacterium colony
A diameter of 1.0-1.4cm.
Preferably, the bacteria cake diameter is 3-6mm.
Preferably, in step S1, the culture is using PDA culture medium.
Preferably, in step S2, the activation is to activate at least 4d in 28 DEG C of constant incubators, until bacterium colony surface production spore is rich
It is rich.
Preferably, in step S3, the reesei spores that pick are to pick reesei spores using test tube mouth, and the test tube mouth is straight
Footpath is 1.5cm.
Preferably, in step s3, the inoculation is that the test tube mouth for picking reesei spores is tipped upside down on 12 orifice bores.Trichoderma
Spore is uniformly seeded in culture medium edge in aperture as far as possible.
Preferably, in step S3, opposite culture to Trichoderma conidium carries out competition for space and hyperparasite to pathogen
After fully, the operation of the calculating growth of pathogenic bacteria area of S4 is entered step.
Preferably, test tube can between diameter 1.5cm-2.5cm any specification test tube.Only used as inoculating tool.
Compared with prior art, the present invention has the advantages that：
1st, the present invention by improving the screening technique of Antagonistic Trichoderma, using porous plate combination trichoderma difference bio-control factors pair
The growth inhibition of pathogen quickly screens excellent antagonistic strain, and this method can save material nearly 90%, screening accuracy rate is reachable
70% or so, greatly improve screening efficiency.
2nd, screening technique of the invention solves the problems, such as the extensive primary dcreening operation of Trichoderma, for Large-scale Screening Trichoderma in the future
Resource provides easily method.
Description of the drawings
Upon reading the detailed description of non-limiting embodiments with reference to the following drawings, other feature of the invention,
Objects and advantages will become more apparent upon：
Fig. 1 is the colonies schematic after the completion of Fusarium oxysporum culture in embodiment 1；
Fig. 2 is the area of colony scanning figure in embodiment 1；
Fig. 3 is the colonies schematic after the completion of cucumber anthracnose culture in embodiment 2；
Fig. 4 is the area of colony scanning figure in embodiment 2.
Specific embodiment
With reference to embodiment, the present invention is described in detail.Following embodiment will be helpful to those skilled in the art
The present invention is further understood, but the invention is not limited in any way.It should be pointed out that those of ordinary skill in the art
For, without departing from the inventive concept of the premise, it can also make certain adjustments and improvements.These belong to the guarantor of the present invention
Protect scope.
Embodiment 1
The present embodiment is related to a kind of Antagonistic Trichoderma vitro quick-screening method, comprises the following steps：
(1) switching of pathogen：5 days Fusarium oxysporums will be cultivated at 28 DEG C of constant incubator, with card punch on bacterium colony side
Edge, which is beaten, takes 3mm bacteria cakes.12 orifice plate centres are forwarded to afterwards.28 DEG C of constant incubator culture 36h, it is spare；
(2) activation of trichoderma strain：12 plants of trichoderma strains not of the same race will be chosen to pick out from glycerol tube transfer, constant temperature incubation
28 DEG C of case is cultivated 5 days；
(3) opposite culture：After all trichoderma strains start to produce spore.Trichoderma is picked using the test tube mouth that bore is 1.5cm
Spore is then tipped upside down in 12 orifice bores.It is cultivated being placed on after 12 pore plate by sealing in 28 DEG C of constant incubators；Fig. 1 is culture
Colonies schematic after finishing, as shown in Figure 1, by being inoculated with pathogen in 12 orifice plates, when making its growth to diameter 1.2cm,
All around trichoderma is inoculated with around it.Trichoderma fights for remaining growing space with pathogen and the pathogen bacteria cake to having grown is posted
Raw, parasitic ability is strong and the faster Trichoderma of growth is stronger to the inhibition of pathogen.
(4) screening of antagonistic strain：Area of colony scanner (Hangzhou Xunshu It Co., Ltd, model are used after 5d：
Shineso-Czone6 Fusarium oxysporum area of colony in 12 orifice plates) is scanned.By the descending arrangement of pathogen area, pathogen
Area is bigger, and the i.e. corresponding trichoderma strain Antagonism in the hole is poorer.Area of colony scanning figure as shown in Fig. 2, as shown in Figure 2, by
Pathogen is identical in each aperture, therefore solid colour, by area of colony analyzer to not known by parasitic area of colony
It does not scan, calculates its area.
(5) by the way that tablet face-off the selection result is ranked up comparison (table 1) with 12 orifice plate the selection results.In classic flat-plate
It is showed in face-off screening in preferable preceding 6 plants of bacterial strains, there are 4 plants to sort still at first 6 in the screening of 12 orifice plates.Illustrate that this method is sieved
Accuracy rate is selected 70% or so.And the uniform significant correlation P ＜ 0.05 of the two.
Note：Identical lowercase represents identical trichoderma strain in table.
Embodiment 2
The present embodiment is related to a kind of Antagonistic Trichoderma vitro quick-screening method, comprises the following steps：
(1) switching of pathogen：5 days cucumber anthracnoses will be cultivated at 28 DEG C of constant incubator, with card punch in bacterium colony
Edge, which is beaten, takes 3mm bacteria cakes.12 orifice plate centres are forwarded to afterwards.28 DEG C of constant incubator culture 36h, it is spare；
(2) activation of trichoderma strain：12 plants of trichoderma strains not of the same race will be chosen to pick out from glycerol tube transfer, constant temperature incubation
28 DEG C of case is cultivated 5 days；
(3) opposite culture：After all trichoderma strains start to produce spore.Trichoderma is picked using the test tube mouth that bore is 1.5cm
Spore is then tipped upside down in 12 orifice bores.It is cultivated being placed on after 12 pore plate by sealing in 28 DEG C of constant incubators；Fig. 3 is culture
Colonies schematic after finishing, from the figure 3, it may be seen that by being inoculated with pathogen in 12 orifice plates, when making its growth to diameter 1.2cm,
All around trichoderma is inoculated with around it.Trichoderma fights for remaining growing space with pathogen and the pathogen bacteria cake to having grown is posted
Raw, parasitic ability is strong and the faster Trichoderma of growth is stronger to the inhibition of pathogen.
(4) screening of antagonistic strain：After 5d 12 are scanned using area of colony scanner (Hangzhou Xunshu It Co., Ltd)
Cucumber anthracnose area of colony in orifice plate.By the descending arrangement of pathogen area, the bigger i.e. hole of pathogen area corresponds to
Trichoderma strain Antagonism it is poorer.Area of colony scanning figure is as shown in figure 4, as shown in Figure 4, due to pathogen phase in each aperture
Together, thus solid colour, by area of colony analyzer to scanning is not identified by parasitic area of colony, calculate its area.
(5) by the way that tablet face-off the selection result is ranked up comparison (table 2) with 12 orifice plate the selection results.In classic flat-plate
It is showed in face-off screening in preferable preceding 6 plants of bacterial strains, there are 4 plants to sort still at first 6 in the screening of 12 orifice plates.Illustrate that this method is sieved
Accuracy rate is selected 70% or so.And the uniform significant correlation P ＜ 0.05 of the two.
Note：Identical lowercase represents identical trichoderma strain in table.
The present invention is by improving the screening technique of Antagonistic Trichoderma, using porous plate combination trichoderma difference bio-control factors to disease
The growth inhibition of opportunistic pathogen quickly screens excellent antagonistic strain, with classic flat-plate the selection result has higher correlation.Conventional screen
Choosing method screens 12 plants of Trichodermas, and carrying out three repetitions needs PDA 720ml, and 36, culture dish, this method is to 12 plants
Trichoderma, which carries out screening, needs 3 pieces of 12 orifice plate, PDA culture medium 36ml.Save material nearly 90%.It is left up to 70% to screen accuracy rate
It is right.Greatly improve screening efficiency.
Specific embodiments of the present invention are described above.It is to be appreciated that the invention is not limited in above-mentioned
Particular implementation, those skilled in the art can make various deformations or amendments within the scope of the claims, this not shadow
Ring the substantive content of the present invention.
Claims (8)
1. a kind of antagonistic Trichoderma vitro quick-screening method, which is characterized in that the screening technique includes the following steps：
S1, the switching of pathogen：Pathogen after activation is beaten, bacteria cake is taken to cultivate 24-48h in 12 orifice plates；
The activation of S2, trichoderma strain：Activation culture Trichoderma is to producing spore；
S3, opposite culture：Reesei spores are picked, are inoculated in 12 orifice plate, is placed in 28 DEG C of constant incubators and cultivates 4-6d；
The screening of S4, antagonistic strain：It is parasitic not by Trichoderma using pathogen in 12 orifice plates described in area of colony scanner scanning
Area；By the descending arrangement of pathogen residual area, bigger pathogen residual area is that the corresponding trichoderma strain in the hole is short of money
Resistance is poorer.
2. screening technique according to claim 1, which is characterized in that in step S1, the pathogen is fungi.
3. screening technique according to claim 2, which is characterized in that the pathogen after the activation is trained for 25 DEG C -28 DEG C
It supports to bacterium colony size close to half of aperture, i.e. colony diameter is 1.0-1.4cm.
4. screening technique according to claim 2, which is characterized in that the bacteria cake diameter is 3-6mm.
5. screening technique according to claim 1, which is characterized in that in step S1, the culture is using PDA culture medium.
6. screening technique according to claim 1, which is characterized in that in step S2, the activation is to be trained in 28 DEG C of constant temperature
Case activation at least 4d is supported, until bacterium colony surface production spore enriches.
7. screening technique according to claim 1, which is characterized in that described to pick reesei spores to use in step S3
Test tube mouth picks reesei spores, a diameter of 1.5cm of test tube mouth.
8. screening technique according to claim 7, which is characterized in that test tube can appoint between diameter 1.5cm-2.5cm
Meaning specification test tube.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201810020739.7A CN108102932B (en) | 2018-01-09 | 2018-01-09 | Antagonistic trichoderma in-vitro rapid screening method |
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| CN201810020739.7A CN108102932B (en) | 2018-01-09 | 2018-01-09 | Antagonistic trichoderma in-vitro rapid screening method |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102382791A (en) * | 2011-11-21 | 2012-03-21 | 滨州职业学院 | Fermentation process of trichoderma |
| CN102533564A (en) * | 2011-11-21 | 2012-07-04 | 滨州职业学院 | Method for screening bio-control trichoderma in corn seedling stage root rot period |
-
2018
- 2018-01-09 CN CN201810020739.7A patent/CN108102932B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102382791A (en) * | 2011-11-21 | 2012-03-21 | 滨州职业学院 | Fermentation process of trichoderma |
| CN102533564A (en) * | 2011-11-21 | 2012-07-04 | 滨州职业学院 | Method for screening bio-control trichoderma in corn seedling stage root rot period |
Non-Patent Citations (2)
| Title |
|---|
| ZHANG YU-BO 等: "First step evaluation of Trichoderma antagonism against plant pathogenic fungi in dual culture", 《菌物学报》 * |
| 梁志怀: "木霉生防菌株的筛选及其发酵产物对豇豆抗病性和生长影响的研究", 《万方数据知识服务平台学位论文》 * |
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