CN106701653A - Pollution-reduction preparation and culture method of ustilaginoidea virens thin-wall conidia - Google Patents
Pollution-reduction preparation and culture method of ustilaginoidea virens thin-wall conidia Download PDFInfo
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- CN106701653A CN106701653A CN201710033964.XA CN201710033964A CN106701653A CN 106701653 A CN106701653 A CN 106701653A CN 201710033964 A CN201710033964 A CN 201710033964A CN 106701653 A CN106701653 A CN 106701653A
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- ustilaginoidea virens
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Abstract
The invention discloses a pollution-reduction preparation and culture method of ustilaginoidea virens thin-wall conidia. The method is characterized by adopting a triangular flask as a preparation and culture utensil, adopting a potato culture-medium as a sporulation medium, and preparing and culturing the high-quality ustilaginoidea virens thin-wall conidia. The preparation and culture method comprises the following steps of (1) cleaning the triangular flask for standby use; (2) preparing the bottle type sporulation medium of the potato culture-medium; (3) transplanting maternal ustilaginoidea virens; (4) sporulation culturing at the proper temperature; (5) eluting and collecting a new generation of thin-wall conidia. The pollution-reduction preparation and culture method of the ustilaginoidea virens thin-wall conidia provided by the invention has the advantages that (1) a shaking culture device has no need to be use; a conidia liquid finished product contains less impurities; the preparation process is fast, high in efficiency, and large in conidia quantity; (2) the pollution can be efficiently reduced during the preparation and culture process; (3) with the protection of the triangular flask, a sporulation bacterial colony can be placed at will for a period of time without being polluted, a sporulation state is kept, and a working procedure requiring the conidia can be linked conveniently.
Description
Technical field
The present invention relates to Plant Pathology technology.Specifically a kind of conidial system of ustilaginoidea virens thin-walled for reducing pollution
Standby cultural method.
Background technology
False smut is the important disease on paddy rice, can cause heavy losses to Rice Production.The disease is by ustilaginoidea virens
(Ustilaginoidea virens) infects and causes.Conidium is the vegetative propagule of ustilaginoidea virens, in the disease of false smut
Significant role is played in circulation and plant disease epidemic.In laboratory, conidium is also the indispensable material of false smut research.
Physiology of development, adaptability of existence or repellence to poor environment are formed about spore, spore is anti-with the identification interaction of host
Research should be waited, is required for using the material of spore shape；Relevant research in terms of new protective agents research and development, such as medicament is to spore
Toxic efficiency etc., it is also desirable to spore；The mutagenesis of the relevant research of biology field, such as pathogenic related gene, positioning with
Clone etc.；Also it be unable to do without spore；It can be seen that, conidium is the important thalli morphology of the daily research of false smut.And in many
In work, often require that the conidium of research institute is pure pollution-free.
Ustilaginoidea virens can form two kinds of conidiums of form, and one kind is pachypycnidium, and another kind is mitogenetic for thin-walled
Spore.In laboratory work, there is larger technical difficulty because culture prepares pachypycnidium, thus it is more thin using its
Wall conidium, " conidium " or " spore " hereinafter refers both to the thin-walled conidium of ustilaginoidea virens.
For a long time, ustilaginoidea virens thin-walled disclosed in document is conidial prepares cultural method, mainly Liquid Culture
Method, the major technique step of the method is that first culture prepares the mycelium of ustilaginoidea virens, then mycelium is implanted into liquid training
Foster base carries out agitated submerged culture, until obtaining spore；The cultural method can obtain substantial amounts of conidium, but, practice
It has also been found that some shortcomings, such as time-consuming more long, whole process is generally required more than 15 days in work；Included in cultured products mixed liquor
There are substantial amounts of nutrient media components, bacterial metabolism product and hypha body etc.；Training needs shaken cultivation equipment；Due to the spore
Smaller and cultured products are in viscous pasty state, to precipitate and obtain more simple spore liquid, generally require high speed centrifugation machine equipment.
With the progress of research and probe, the conidial solid culture method of ustilaginoidea virens thin-walled is had been set up recently,
Make culture utensil with culture dish, spore is prepared with Solid agar culture culture.The method can make up aforesaid liquid culture side
The some shortcomings of method, but occur another disadvantage again, the architectural characteristic of culture dish is just resulted from, the spore finished product for preparing culture holds
It is easily contaminated.
The culture dish that common laboratory is used is made up of ware bottom and ware lid, is limited to the Manufacturing Techniques of current culture dish,
The degree uniformly fitted is extremely difficult between ware bottom and ware lid, is existed between the ware bottom of most of culture dishes and ware lid compared with big gap, training
The air flow supported inside and outside ware can be unblocked.Therefore, the conidium product for preparing culture using Nostoc commune Vanch ware is easily received
The interference of outside contamination air, is readily obtained the spore finished product with living contaminants.Especially general Plant Pathology experiment
Room, currently prefers to incubator of the configuration with cooling/liter temperature function, and such incubator is fast in operating room generally for realizing
Fast constant temperature and temperature uniform distribution, operating room is often designed as the pattern of circulation air.Carried out using this kind of incubator
Product spore culture, culture dish surrounding air be in normal flow regime, cause the probability of material contamination in culture dish at a relatively high, generally
Culture can cause more than 50% culture plate for 5 days by living contaminants, even if can't see obvious mould on some culture plates
The bacterium colony of the contaminated bacterias such as bacterium, but the pollution probability being invisible to the naked eye is still larger.Therefore, culture false smut is prepared using culture dish
Bacterium conidium, is technically difficult the possibility for decontaminating.
The content of the invention
Conidial cultural method is prepared it is an object of the invention to provide a kind of ustilaginoidea virens thin-walled for reducing pollution.
The technical scheme that the present invention solves above-mentioned technical problem is as follows：
A kind of ustilaginoidea virens thin-walled for reducing pollution is conidial to prepare cultural method, the step of prepare cultural method such as
Under：
1. make to prepare the utensil of culture spore using common triangular flask, triangular flask cleaning is standby.
2. the preparation of vial-type product spore culture medium
Make product spore culture medium with potato culture, the proportioning of the culture medium is：Potato 100g, agar 20g, water
1000mL；Culture medium is sub-packed in the triangular flask that step 1 prepares, triangle vial-type culture medium is turned into after conventional high-pressure high-temperature sterilization,
It is standby.
3. transplanting of the mother for ustilaginoidea virens
Make female for thalline with the existing ustilaginoidea virens conidium liquid in laboratory, the mother is transplanted for spore liquid with pipettor
Enter the triangle vial-type culture medium of step 2 preparation, and spore liquid is uniformly dispersed on culture basal plane.
4. spore culture is produced
By step 3 operate it is complete after triangle vial-type culture medium be transferred in the standard incubator that temperature is 28 DEG C cultivate.
5. the collection of spore of new generation
After usual step 4 is cultivated 5 days, substantial amounts of minute colony is formed on culture basal plane, substantial amounts of point has been formed on bacterium colony
Raw spore；With the spore on aseptic water elution bacterium colony, and focus in sterilization container, that is, obtain the better quality without living contaminants
Ustilaginoidea virens thin-walled conidium liquid.
Characteristic of the invention is with advantage：
1) without shaken cultivation equipment, the conidium liquid finished product of acquisition contains mycelium, culture medium and bacterial metabolism excretion
Product etc. is less.
2) after triangular flask mixes conventional bottle stopper, passing through for common micro-organisms can be intercepted, protects do not received in triangular flask well
Living contaminants, thus pollution can be effectively reduced, obtain high-quality spore finished product.
3) produce spore bacterium colony and do not produce dense aerial hyphae typically.
4) operation of the spore than collecting spore in culture dish is collected in triangular flask more convenient.
5) protection of triangular flask may be such that product spore bacterium colony can arbitrarily place a period of time without contaminated, and keep producing spore shape
State, is easy to be connected with the working procedure of spore is needed.
Specific embodiment
With reference to embodiment, the invention will be further described.
Key technology of the invention is the combination and application of triangular flask and potato agar culture medium.
Although the everyday devices of triangular flask platymiscium PAL, its normal usage is mainly and contains culture medium and go out
Bacterium；Also being commonly used for containing fluid nutrient medium or plant tissue class culture medium (such as seed, straw) carries out Bacteria culturing.
The application of agar class culture medium, existing technical specification is all to make culture utensil using culture dish, by agar culture
After base heating fusing, pour into culture dish and be made culture medium flat plate, and pathogen is cultivated on the culture plate.
Preparation culture of the ustilaginoidea virens conidium on agar medium, is using conventional technique specification, i.e. profit
Culture is prepared with the culture technique of culture dish combination agar class culture medium；So far yet there are no and make incubator using triangular flask
Tool, carries out the spore technology of preparing that ustilaginoidea virens produces spore culture on agar class culture medium.Possible cause is the length to culture dish
Phase is relied on and custom is continued to use, and being generally considered pathogen product spore needs the good environmental condition of aeration, and culture dish is lucky
Good aeration can be met.
After common triangular flask puts bottle stopper, although form the poor enclosed environment of aeration, but inventor's repetition test is sent out
It is existing, form conidium in the potato agar culture medium in environment that ustilaginoidea virens can more be closed at this；And triangular flask
Using then just solution applied culture ware is easily caused the key technical problem of pollution.
Potato agar culture medium category is without the extra culture medium with addition of sugar substance, the battalion of ustilaginoidea virens on the culture medium
Health is long, generally fast and vigorous not as good as being grown like that in the potato sucrose culture medium containing sucrose, but inventor has found the training
Foster base is the good product spore culture medium of ustilaginoidea virens, and ustilaginoidea virens has a salient feature in culture medium product spore, just
It is that formed bacterium colony does not produce dense aerial hyphae typically.The present invention prepares culture ustilaginoidea virens thin-walled using the culture medium
Conidium, the Medium Proportion is：Potato 100g, agar 20g, water 1000mL；Boil according to a conventional method and match somebody with somebody.Culture medium is boiled matches somebody with somebody
After finishing, can direct packaging in triangular flask；Due to belonging to curved surface state triangular flask bottom, base unit weight is very few can cause bottom of bottle for culture more
Local excessively thin without culture medium or culture medium, culture medium is crossed and at most easily cause waste；Usual every bottle (250mL specifications) loading 20~
30mL culture mediums are advisable, and the triangular flask of other specifications takes the circumstances into consideration to increase and decrease liquid measure.
After packaging bottle stopper, conventional high-pressure high-temperature sterilization takes out triangular flask and is flat on straight desktop, after cooling after sterilizing
Form vial-type product spore culture medium.
Vial-type culture medium can also be dispensed alternatively, first with larger triangular flask load in the usual way liquid measure compared with
Many culture mediums are sterilized；For another example it is the same with the flat board of falling culture dish, after being melted using preceding heating, it is dispensed into the blank three of sterilizing
In the bottle of angle.
Special instruction is needed, in transplanting step of the mother for ustilaginoidea virens, ustilaginoidea virens mycelium is not suitable for use in this hair
The bright female generation transplanting thalline for being implanted into vial-type product spore culture medium, the mother of transplanting must use ustilaginoidea virens spore sheet for thalline
Body；This mother can be from conventional liq cultural method culture gained for spore, also can be female from the inventive method culture gained
For spore liquid spore amount typically without accurate quantitative analysis, general spore concentration be 106The spore liquid of individual/mL, transplants 20~50 μ L
Onto a triangle vial-type culture medium, the normal biomass requirement for preparing culture spore of new generation can be met.Mother is planted for spore
After entering triangle vial-type culture medium, can gently be smeared with the sterilizing instrument such as T-shaped glass rod, make spore liquid on triangle vial-type culture medium face
It is uniformly dispersed.
Producing spore culture can implement in standard incubator, the preference temperature that temperature control grows in ustilaginoidea virens, the present invention
Cultivation temperature uses 28 DEG C；Incubation is to illumination condition and damp condition without special demands.
After generally producing spore preparation culture 5 days, substantial amounts of minute colony, bacterium are formed uniformly on the culture basal plane in triangular flask
Substantial amounts of conidium has been formed on falling.
In the conidium of new generation operation that preparation culture is obtained is collected, can be small using smooth T-shaped glass rod or sterilizing
Hairbrush scrubs bacterium colony, makes spores release to washing in spore liquid；It is in and piles up intensive due to produces spore bacterium colony very little and spore of new generation
State, the elution efficiency for scrubbing spore with small brushes is higher.
Embodiment 1
Cultural method is prepared using a kind of ustilaginoidea virens thin-walled for reducing pollution of the present invention is conidial, culture rice is prepared
The conidium of bent germ bacterial strain Uv-105, implements operation as follows：
1. make to prepare the utensil of culture spore using the common triangular flasks of 250mL, triangular flask cleaning is standby.
2. the preparation of vial-type product spore culture medium：Make product spore culture medium with potato culture, the proportioning of the culture medium is：Horse
Bell potato 100g, agar 20g, water 1000mL；Culture medium is sub-packed in the triangular flask that step 1 prepares, per bottled 30mL, conventional high-pressure
Turn into triangle vial-type culture medium after high-temperature sterilization, it is standby.
3. transplanting of the mother for ustilaginoidea virens：In female generation, is made with the existing ustilaginoidea virens bacterial strain Uv-105 conidiums liquid in laboratory
Thalline, triangle vial-type culture medium prepared by step 2 is implanted into pipettor by the mother for the μ L of spore liquid 50, and with sterilizing T-shaped glass
Rod is gently smeared, spore liquid is uniformly dispersed on culture basal plane.
4. spore culture is produced：By step 3 operate it is complete after triangle vial-type culture medium be transferred to the Nostoc commune Vanch that temperature is 28 DEG C
Culture in case.
5. the collection of spore of new generation：After step 4 is cultivated 5 days, substantial amounts of minute colony is formed on culture basal plane, on bacterium colony
Form substantial amounts of conidium；The spore on one bottle of bacterium colony is eluted with sterilized water 15mL, it is 2.00 × 10 that can obtain spore concentration6
Individual/mL, the thin-walled conidium liquid without living contaminants.
Embodiment 2
Cultural method is prepared using a kind of ustilaginoidea virens thin-walled for reducing pollution of the present invention is conidial, culture rice is prepared
Bent germ bacterial strain Uv-110 conidiums, 1 operates to step 5 and implements the step of by embodiment 1, the bacterium of different simply step 3
Strain is Uv-110；Result elutes the spore on one bottle of bacterium colony with sterilized water 15mL, and it is 2.02 × 10 that can obtain spore concentration6Individual/
ML, the conidium liquid without living contaminants.
Embodiment 3
Cultural method is prepared using a kind of ustilaginoidea virens thin-walled for reducing pollution of the present invention is conidial, culture rice is prepared
Bent germ bacterial strain Uv-111 conidiums, 1 operates to step 5 and implements the step of by embodiment 1, the bacterium of different simply step 3
Strain is Uv-111；Result elutes the spore on one bottle of bacterium colony with sterilized water 15mL, and it is 14.51 × 10 that can obtain spore concentration6Individual/
ML, the conidium liquid without living contaminants.
Claims (1)
1. a kind of ustilaginoidea virens thin-walled for reducing pollution is conidial prepares cultural method, it is characterised in that prepare culture side
The step of method, is as follows：
1) make to prepare the utensil of culture spore using common triangular flask, triangular flask cleaning is standby；
2) preparation of vial-type product spore culture medium：Make product spore culture medium with potato culture, the proportioning of the culture medium is：Potato
100g, agar 20g, water 1000mL；Culture medium is sub-packed in step 1) prepare triangular flask, turn into after conventional high-pressure high-temperature sterilization
Triangle vial-type culture medium, it is standby；
3) female transplanting for ustilaginoidea virens：Mother is made for thalline with the existing ustilaginoidea virens conidium liquid in laboratory, pipettor is used
The mother is implanted into step 2 for spore liquid) the triangle vial-type culture medium for preparing, and make spore liquid dispersion on culture basal plane equal
It is even；
4) spore culture is produced：By step 3) operation it is complete after triangle vial-type culture medium be transferred in the standard incubator that temperature is 28 DEG C
Culture；
5) collection of spore of new generation：Usual step 4) after culture 5 days, substantial amounts of minute colony, bacterium are formed on culture basal plane
Fall the upper substantial amounts of conidium of formation；With the spore on aseptic water elution bacterium colony, and focus in sterilization container, that is, obtain without miscellaneous
The ustilaginoidea virens thin-walled conidium liquid of the better quality of bacterium pollution.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113308432A (en) * | 2021-06-15 | 2021-08-27 | 广西大学 | Transfer and dispersion method of ustilaginoidea virens thin-walled conidia |
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| CN103436480A (en) * | 2013-08-27 | 2013-12-11 | 广西大学 | Plate culture and preparation method of ustilaginoidea virens thin-wall conidium |
| CN105779372A (en) * | 2016-05-24 | 2016-07-20 | 广西大学 | Potato culture medium suitable for ustilaginoidea virens spore production and application thereof |
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- 2017-01-10 CN CN201710033964.XA patent/CN106701653A/en active Pending
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| CN103436480A (en) * | 2013-08-27 | 2013-12-11 | 广西大学 | Plate culture and preparation method of ustilaginoidea virens thin-wall conidium |
| CN105779372A (en) * | 2016-05-24 | 2016-07-20 | 广西大学 | Potato culture medium suitable for ustilaginoidea virens spore production and application thereof |
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| CN113308432A (en) * | 2021-06-15 | 2021-08-27 | 广西大学 | Transfer and dispersion method of ustilaginoidea virens thin-walled conidia |
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