CN106635948A - Pollution-free preparing and culturing method for rice-false-smut-case thin-wall conidia - Google Patents
Pollution-free preparing and culturing method for rice-false-smut-case thin-wall conidia Download PDFInfo
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- 238000000034 method Methods 0.000 title claims abstract description 34
- 238000012258 culturing Methods 0.000 title abstract description 9
- HMFHBZSHGGEWLO-SOOFDHNKSA-N D-ribofuranose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H]1O HMFHBZSHGGEWLO-SOOFDHNKSA-N 0.000 claims abstract description 54
- PYMYPHUHKUWMLA-LMVFSUKVSA-N Ribose Natural products OC[C@@H](O)[C@@H](O)[C@@H](O)C=O PYMYPHUHKUWMLA-LMVFSUKVSA-N 0.000 claims abstract description 54
- HMFHBZSHGGEWLO-UHFFFAOYSA-N alpha-D-Furanose-Ribose Natural products OCC1OC(O)C(O)C1O HMFHBZSHGGEWLO-UHFFFAOYSA-N 0.000 claims abstract description 54
- 230000001954 sterilising effect Effects 0.000 claims abstract description 32
- 244000061456 Solanum tuberosum Species 0.000 claims abstract description 26
- 235000002595 Solanum tuberosum Nutrition 0.000 claims abstract description 26
- 239000007788 liquid Substances 0.000 claims abstract description 26
- 238000004659 sterilization and disinfection Methods 0.000 claims abstract description 15
- 239000001963 growth medium Substances 0.000 claims description 59
- 241001474928 Ustilaginoidea virens Species 0.000 claims description 36
- 241000894006 Bacteria Species 0.000 claims description 27
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 24
- 229920001817 Agar Polymers 0.000 claims description 20
- 239000008272 agar Substances 0.000 claims description 20
- 238000002360 preparation method Methods 0.000 claims description 12
- 238000001914 filtration Methods 0.000 claims description 6
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 claims description 5
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- 239000006185 dispersion Substances 0.000 claims 1
- 125000000548 ribosyl group Chemical group C1([C@H](O)[C@H](O)[C@H](O1)CO)* 0.000 claims 1
- 239000002609 medium Substances 0.000 abstract description 8
- 235000007164 Oryza sativa Nutrition 0.000 abstract description 6
- 235000009566 rice Nutrition 0.000 abstract description 6
- 230000001580 bacterial effect Effects 0.000 abstract description 5
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- 240000007594 Oryza sativa Species 0.000 abstract 1
- 230000008774 maternal effect Effects 0.000 abstract 1
- 239000007362 sporulation medium Substances 0.000 abstract 1
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- 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
- C12N3/00—Spore forming or isolating processes
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Abstract
The invention discloses a pollution-free preparing and culturing method forrice-false-smut-case thin-wall conidia. A triangular flask is used as a preparing and culturing tool, a potato ribose medium serves as a sporulation medium, and the high-quality rice-false-smut-case thin-wall conidia are prepared and cultured. The preparing and culturing method includes the following steps that 1) the triangular flask is cleaned ready for use; 2) a base medium is prepared; 3) ribose is subjected to non-high-temperature sterilization; 4) a potato-ribose flask-type medium is prepared; 5) maternal rice false smut cases are transplanted; 6) spore production culturing is carried out; 7) new-generation spores are collected. The preparing and culturing method has the advantages that pollution can be effectively avoided in the preparing and culturing process; a shaking culturing device is not required, the quantity of sundries of a spore liquid finished product is small, and the preparing process is rapid, high in efficiency and large in spore quantity; a spore production bacterial colony can be optionally placed for a period of time through protection of the triangular flask and is not polluted, the spore production state is kept, and joining with a working procedure requiring spores is convenient.
Description
Technical field
The present invention relates to Plant Pathology technology.Specifically a kind of conidial system of ustilaginoidea virens thin-walled for avoiding polluting
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.Indoor in experiment, 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 the conidium of two kinds of forms, 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 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 longer, whole process generally requires more than 15 days in work；Include 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
Less 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, with Solid agar culture culture spore is prepared.The method can make up aforesaid liquid culture side
The some shortcomings of method, but and another disadvantage occur, 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
Air flow inside and outside foster 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 configure the incubator with cooling/liter temperature function, and such incubator is indoor fast generally for work is realized
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
It is an object of the invention to provide a kind of ustilaginoidea virens thin-walled for avoiding polluting is conidial to prepare cultural method.
The technical scheme that the present invention solves above-mentioned technical problem is as follows：
A kind of ustilaginoidea virens thin-walled for avoiding polluting 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 basal culture medium and the sterilizing of blank triangle bottle
In parts by weight：Potato 100g, agar 20g, water 900mL, prepare basal culture medium, and are distributed into step 1 and prepare
Triangular flask；The triangular flask for separately taking step 1 preparation directly covers bottle stopper into blank triangle bottle；Then basal culture medium and blank three
Angle bottle carries out together conventional high-pressure high-temperature sterilization, standby.
3. the non high temperature sterilizing of ribose
In parts by weight：Ribose 10g, water 100mL, ribose solution is made into clear water by ribose, aseptically uses bacterium
The ribose solution is carried out filtration sterilization by filter, standby.
4. the outfit of potato ribose vial-type culture medium
Basal culture medium prepared by step 2 plus heat fusing, water-bath is balanced to 60 DEG C, while by step 3 filtration sterilization
Ribose solution is balanced in a water bath to 60 DEG C, is then simultaneously transferred to the two in superclean bench gnotobasis, by volume
Example：Basal culture medium/ribose solution=9/1, basal culture medium is mixed with ribose solution, is formed actual proportioning and is：Potato
100g, ribose 10g, agar 20g, the potato ribose culture medium of water 1000mL, after mixing shake is uniform, are distributed into step 2 accurate
Standby sterilizing blank triangle bottle, forms vial-type product spore culture medium.
5. transplanting of the mother for ustilaginoidea virens
Make female for thalline with the existing ustilaginoidea virens conidium liquid in laboratory, transplanted the mother for spore liquid with pipettor
Enter the vial-type product spore culture medium of step 4 preparation, and make spore liquid be uniformly dispersed on culture basal plane.
6. spore culture is produced
By step 5 operate it is complete after vial-type product spore culture medium proceed in the standard incubator that temperature is 28 DEG C cultivate.
7. the collection of spore of new generation
After generally step 6 is cultivated 5 days, substantial amounts of minute colony has been 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.
The present invention characteristic be with advantage：
1) triangular flask is mixed after conventional bottle stopper, can intercept passing through for common micro-organisms, protects do not received in triangular flask well
Living contaminants, thus pollution can be efficiently avoided, obtain high-quality spore finished product.
2) without the need for shaken cultivation equipment, the conidium liquid finished product of acquisition contains mycelium, culture medium and bacterial metabolism excretion
Product etc. is less.
3) prepare that process is quick, efficiency high, not only sporulation quantity is big for ustilaginoidea virens, and produce spore bacterium colony and do not form a large amount of cyclopentadienyls
Close aerial hyphae.
4) protection of triangular flask can enable produce spore bacterium colony arbitrarily place a period of time and it is not contaminated, and keep produce spore shape
State, is easy to be connected with the working procedure for needing spore.
Specific embodiment
With reference to embodiment, the invention will be further described.
The key technology of the present invention is the combination and application of triangular flask and potato ribose agar medium.
Although the everyday devices of triangular flask platymiscium PAL, its normal usage mainly contains culture medium and goes 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
Base adds after heat fusing, pours culture dish into and makes culture medium flat plate, and cultivates pathogen 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 culture technique of the culture dish with reference to 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 relies 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.
Common triangular flask is put after bottle stopper, although form the poor enclosed environment of aeration, but inventor's repetition test is sent out
It is existing, form conidium on the potato ribose agar medium that ustilaginoidea virens can be in the environment that this is more closed；And triangle
The application of bottle, then just solve the key technical problem that applied culture ware is easily caused pollution.
Potato ribose culture medium is typically rarely used in the cellar culture of pathogen, and inventor has found that the culture medium is passed through
After conventional high-pressure high-temperature sterilization, suppress growing for ustilaginoidea virens, but the culture prepared with the ribose without high-temperature process
Base, but promotes the thin-walled illumination of ustilaginoidea virens；Not only sporulation quantity is big on potato ribose culture medium for ustilaginoidea virens,
And bacterium colony does not form a large amount of dense aerial hyphaes.The present invention prepares ustilaginoidea virens thin-walled using potato ribose culture medium
Conidium, the Medium Proportion is：Potato 100g, ribose 10g, agar 20g, water 1000mL.
Potato ribose culture medium except component ribose outside, remaining component is potato agar, the potato agar group
Divide and coagulating property and basic nutrition are provided, the present invention is referred to as " basal culture medium ".Because ribose is unable to high-temperature sterilization, thus
The sterilizing of potato ribose culture medium needs the synthesis of two kinds of sterilization technologies, i.e., first use high pressure-temperature sterilizing methods by substrate culture
Base sterilizes, and ribose is sterilized with non high temperature sterilizing methods, then will it is sterilized the two mix under the conditions of non high temperature.
The mixing of basal culture medium and ribose liquid needs to take into account the coagulability of agar and ribose is molten without the need for specific ratio
The filter operation of liquid, Jing test and comparisons are with volume mixture ratio：Basal culture medium/ribose solution component=9/1, be compared with
Suitable mixed proportion.By this mixed proportion, the proportioning of basal culture medium should be：Potato 100g, agar 20g, water
900mL；And the proportioning of ribose solution should be：Ribose 10g, water 100mL.
In real work, due to becoming solid after basal culture medium sterilizing cooling, it is impossible to arbitrarily measure, so that going out
Quantitatively bottle before bacterium；Per bottle of quantitative basal culture medium for loading 90mL (or 135mL), adds the core of 10mL (or 15mL) when using
Sugar juice, such hybrid mode, it is relatively simple convenient to operate.
When using, sterilized basal culture medium plus heat fusing are taken, water-bath inner equilibrium is placed in 60 DEG C, while
The ribose solution of sterilizing is also balanced to 60 DEG C, is taken ribose solution 15mL and is added mixing in one bottle of 135mL basal culture medium, that is, obtain
Proportioning is：The potato ribose culture medium of potato 100g, ribose 10g, agar 20g, water 1000mL, is dispensed into after being well mixed
Sterilized blank triangle bottle (specification 250mL), per bottled 20～30mL, becomes the vial-type product spore culture medium of the present invention.
Special instruction is needed, in transplanting step of the mother for ustilaginoidea virens, ustilaginoidea virens mycelium is not suitable for use in this
The bright mother for being implanted into vial-type culture medium must use ustilaginoidea virens spore itself for thalline, the mother of transplanting for thalline；This female generation
Spore liquid can be from conventional liq cultural method culture gained, also can be from the inventive method culture gained, and mother is for spore
Typically without accurate quantitative analysis, general spore concentration is 10 to the spore amount of liquid6The spore liquid of individual/mL, transplanting 20～50 μ L to
On triangle vial-type culture medium, the normal biomass for preparing culture spore of new generation can be met and required.Mother is implanted into into triangle for spore
After vial-type culture medium, can gently be smeared with instruments such as sterilizing T-shaped glass rods, spore liquid is disperseed on triangle vial-type culture medium face
It is even.
Producing spore culture can implement in standard incubator, the preference temperature that temperature control grows in ustilaginoidea virens, the present invention
Cultivation temperature adopts 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 have been formed uniformly on the culture basal plane in triangular flask
Substantial amounts of conidium is formed on falling.
In the conidium of new generation operation that collection preparation culture is obtained, can be little using smooth T-shaped glass rod or sterilizing
Hairbrush scrubs bacterium colony, makes spores release to washing in spore liquid；It is intensive in piling up due to producing 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 avoiding polluting of the present invention is conidial, culture rice is prepared
The conidium of bent germ bacterial strain Uv-111, implements as follows operation：
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 basal culture medium and the sterilizing of blank triangle bottle
In parts by weight：Potato 100g, agar 20g, water 900mL, prepare basal culture medium, and are distributed into step 1 and prepare
Triangular flask, per bottled 135mL；The triangular flask for separately taking step 1 preparation directly covers bottle stopper into blank triangle bottle；Basal culture medium
Conventional high-pressure high-temperature sterilization is carried out together with blank triangle bottle, it is standby.
3. the non high temperature sterilizing of ribose
In parts by weight：Ribose 10g, water 100mL, ribose solution is made into clear water by ribose, aseptically uses bacterium
The ribose solution is carried out filtration sterilization by filter, standby.
4. the outfit of potato ribose vial-type culture medium
Basal culture medium prepared by step 2 plus heat fusing, water-bath is balanced to 60 DEG C, while by step 3 filtration sterilization
Ribose solution is balanced in a water bath to 60 DEG C, and then the two is transferred in superclean bench gnotobasis simultaneously, takes ribose molten
Liquid 15mL mixes with one bottle of basal culture medium, forms actual proportioning and is：Potato 100g, ribose 10g, agar 20g, water 1000mL
Potato ribose culture medium, after mixing shake is uniform, be distributed into the sterilizing blank triangle bottle of step 2 preparation, per bottled 30mL,
Form vial-type product spore culture medium.
5. transplanting of the mother for ustilaginoidea virens
Make female for thalline with the existing ustilaginoidea virens bacterial strain Uv-111 conidiums liquid in laboratory, with pipettor by female generation
The μ L of spore liquid 50 are implanted into the vial-type product spore culture medium of step 4 preparation, and are smeared with sterilizing T-shaped glass rod with gentle, make spore liquid exist
It is uniformly dispersed on culture basal plane.
6. spore culture is produced
By step 5 operate it is complete after vial-type product spore culture medium proceed in the standard incubator that temperature is 28 DEG C cultivate.
7. the collection of spore of new generation
After step 6 is cultivated 5 days, substantial amounts of minute colony is formed on culture basal plane, substantial amounts of conidium is formed on bacterium colony；
The spore on one bottle of bacterium colony is eluted with sterilized water 15mL, spore concentration can be obtained for 21.47 × 106Individual/mL, without living contaminants
Conidium liquid.
Embodiment 2
Cultural method is prepared using a kind of ustilaginoidea virens thin-walled for avoiding polluting of the present invention is conidial, culture rice is prepared
Bent germ bacterial strain Uv-110 conidiums, implement by the step of embodiment 11 to step 7 operation, and different is the bacterium of step 5
Strain is Uv-110；As a result the spore on one bottle of bacterium colony is eluted with sterilized water 15mL, spore concentration can be obtained for 5.74 × 106Individual/
ML, the conidium liquid without living contaminants.
Embodiment 3
Cultural method is prepared using a kind of ustilaginoidea virens thin-walled for avoiding polluting of the present invention is conidial, culture rice is prepared
Bent germ bacterial strain Uv-105 conidiums, implement by the step of embodiment 11 to step 7 operation, and different is the bacterium of step 5
Strain is Uv-105；As a result the spore on one bottle of bacterium colony is eluted with sterilized water 15mL, spore concentration can be obtained for 4.55 × 106Individual/
ML, the conidium liquid without living contaminants.
Claims (1)
1. a kind of ustilaginoidea virens thin-walled for avoiding polluting 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 basal culture medium and the sterilizing of blank triangle bottle：In parts by weight：Potato 100g, agar 20g, water 900mL,
Prepare basal culture medium, and be distributed into step 1) prepare triangular flask；Separately take step 1) prepare triangular flask directly cover bottle stopper into
Blank triangle bottle；Then basal culture medium is carried out conventional high-pressure high-temperature sterilization together with blank triangle bottle, it is standby；
3) the non high temperature sterilizing of ribose：In parts by weight：Ribose 10g, water 100mL, are made into ribose solution, in nothing with clear water by ribose
The ribose solution is carried out into filtration sterilization with biofilter under the conditions of bacterium, it is standby；
4) outfit of potato ribose vial-type culture medium：By step 2) basal culture medium for preparing adds heat fusing, water-bath balance to
60 DEG C, while by step 3) ribose solution of filtration sterilization balances in a water bath to 60 DEG C, is then simultaneously transferred to the two super
In net workbench gnotobasis, example by volume：Basal culture medium/ribose solution=9/1, by basal culture medium and ribose solution
Mixing, forming actual proportioning is：Potato 100g, ribose 10g, agar 20g, the potato ribose culture medium of water 1000mL, mix
After closing shake uniformly, the sterilizing blank triangle bottle of step 2 preparation is distributed into, forms vial-type product spore culture medium；
5) 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 into step 4 for spore liquid) the vial-type product spore culture medium for preparing, and make spore liquid dispersion on culture basal plane equal
It is even；
6) spore culture is produced：By step 5) operation it is complete after vial-type product spore culture medium proceed in the standard incubator that temperature is 28 DEG C
Culture；
7) collection of spore of new generation：Usual step 6) after culture 5 days, substantial amounts of minute colony, bacterium have been formed on culture basal plane
Fall the substantial amounts of conidium of upper 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.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710033965.4A CN106635948A (en) | 2017-01-10 | 2017-01-10 | Pollution-free preparing and culturing method for rice-false-smut-case thin-wall conidia |
Applications Claiming Priority (1)
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