CN106754622A - A kind of rice blast pathogen conidiospore for efficiently preventing pollution prepares cultural method - Google Patents
A kind of rice blast pathogen conidiospore for efficiently preventing pollution prepares cultural method Download PDFInfo
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- CN106754622A CN106754622A CN201710033997.4A CN201710033997A CN106754622A CN 106754622 A CN106754622 A CN 106754622A CN 201710033997 A CN201710033997 A CN 201710033997A CN 106754622 A CN106754622 A CN 106754622A
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- 235000007164 Oryza sativa Nutrition 0.000 title claims abstract description 24
- 235000009566 rice Nutrition 0.000 title claims abstract description 24
- 238000000034 method Methods 0.000 title claims abstract description 20
- 230000001717 pathogenic effect Effects 0.000 title claims abstract description 19
- 244000052769 pathogen Species 0.000 title claims abstract description 18
- 240000007594 Oryza sativa Species 0.000 title 1
- 239000001963 growth medium Substances 0.000 claims abstract description 38
- 241000894006 Bacteria Species 0.000 claims abstract description 24
- 241000209094 Oryza Species 0.000 claims abstract description 23
- 241001330975 Magnaporthe oryzae Species 0.000 claims abstract description 15
- 239000002609 medium Substances 0.000 claims abstract description 9
- 238000004140 cleaning Methods 0.000 claims abstract description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 10
- 239000000356 contaminant Substances 0.000 claims description 7
- 239000007788 liquid Substances 0.000 claims description 7
- 230000001954 sterilising effect Effects 0.000 claims description 7
- 238000002360 preparation method Methods 0.000 claims description 5
- 238000004659 sterilization and disinfection Methods 0.000 claims description 4
- 238000010828 elution Methods 0.000 claims description 2
- 239000010977 jade Substances 0.000 claims 1
- 239000000047 product Substances 0.000 abstract description 9
- 238000005516 engineering process Methods 0.000 abstract description 6
- 239000012467 final product Substances 0.000 abstract 1
- 229920001817 Agar Polymers 0.000 description 13
- 239000008272 agar Substances 0.000 description 13
- 241000196324 Embryophyta Species 0.000 description 8
- 238000011160 research Methods 0.000 description 7
- 201000010099 disease Diseases 0.000 description 6
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 6
- 239000000463 material Substances 0.000 description 6
- 241001411320 Eriogonum inflatum Species 0.000 description 3
- 238000005273 aeration Methods 0.000 description 3
- 238000005286 illumination Methods 0.000 description 3
- 208000015220 Febrile disease Diseases 0.000 description 2
- 241001344131 Magnaporthe grisea Species 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 235000011389 fruit/vegetable juice Nutrition 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 238000002513 implantation Methods 0.000 description 2
- 239000012535 impurity Substances 0.000 description 2
- 230000002073 mitogenetic effect Effects 0.000 description 2
- 235000015097 nutrients Nutrition 0.000 description 2
- 238000004806 packaging method and process Methods 0.000 description 2
- 230000007170 pathology Effects 0.000 description 2
- 230000001932 seasonal effect Effects 0.000 description 2
- 240000005979 Hordeum vulgare Species 0.000 description 1
- 235000007340 Hordeum vulgare Nutrition 0.000 description 1
- 241001344133 Magnaporthe Species 0.000 description 1
- 241001529246 Platymiscium Species 0.000 description 1
- 101100505672 Podospora anserina grisea gene Proteins 0.000 description 1
- 241001052560 Thallis Species 0.000 description 1
- 240000008042 Zea mays Species 0.000 description 1
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 1
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 244000052616 bacterial pathogen Species 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 235000005822 corn Nutrition 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000002354 daily effect Effects 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 230000003203 everyday effect Effects 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000002703 mutagenesis Methods 0.000 description 1
- 231100000350 mutagenesis Toxicity 0.000 description 1
- 239000003223 protective agent Substances 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 238000011218 seed culture Methods 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 230000028070 sporulation Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
Classifications
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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
- C12N3/00—Spore forming or isolating processes
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- Bioinformatics & Cheminformatics (AREA)
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- Chemical & Material Sciences (AREA)
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Abstract
Cultural method is prepared the invention discloses a kind of rice blast pathogen conidiospore for efficiently preventing and polluting, makees to prepare the utensil of culture spore using common triangular flask, prepared and cultivate high-quality rice blast pathogen conidiospore, prepare cultural method step as follows：1) it is common triangular flask cleaning is standby；2) vial-type oat medium is prepared；3) Pyricularia oryzae mycelium is implanted into vial-type culture medium；4) spore culture is produced under 28 DEG C of temperature conditionss；5) wash-out collects conidium.It is an advantage of the invention that：1) pollution is efficiently prevented, spore final product quality is higher；2) operating technology is simple；3) operation of the spore than collecting spore in culture dish is collected in triangular flask more convenient；The 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 state, be easy to be connected with the working procedure of spore is needed.
Description
Technical field
The present invention relates to Plant Pathology technology, specifically a kind of system of the rice blast pathogen conidiospore for efficiently preventing from polluting
Standby cultural method.
Background technology
Rice blast is the great disease of global Rice Production, the pathogenic bacteria Pyricularia oryzae (Magnaporthe of the disease
Grisea) be Plant Pathology pattern pathogen, be all expected that by both at home and abroad to Pyricularia oryzae carry out comprehensively and deep phase
Research is closed, to disclose the universal law that phytopathogen causes a disease to host plant, and then is sought and is set up more effectively feasible
The anti-approach of plant disease control.
Conidium is the vegetative propagule of Pyricularia oryzae, plays great in the Disease Cycle and plant disease epidemic of rice blast
Effect.In laboratory, conidium is also that rice blast studies indispensable material.Relevant conidial formation develops life
Reason, to the adaptability of existence or repellence of poor environment, the research such as spore and the identification interaction of host are reacted is required for using spore
The material of sub- form；Relevant research in terms of new protective agents research and development, such as Toxic efficiency of the medicament to spore, it is also desirable to spore
Son；The mutagenesis of the relevant research of biology field, such as pathogenic related gene, positioning and clone；Also it be unable to do without spore；Can
See, conidium is the important thalli morphology of the daily research of rice blast, therefore, fast and efficiently culture prepares high-quality point
Raw spore material, is very beneficial for smooth implementation of the rice blast about research work.
The culture preparation method of current rice blast pathogen conidiospore, mainly there is two major class technologies.
One class is to make the cultivation of culture medium using plant seed, i.e., first plant seed (such as barley corn) is infiltrated and gone out
Bacterium is made culture medium, then mycelium is implanted into seed culture medium and is cultivated, and after mycelia covers with seed, is washed off with clear water
Seed surface mycelia, and relay and carry out illumination cultivation in square plate appliances again；The method generally can prepare substantial amounts of mitogenetic spore
Son, but, culture prepares that all times are (generally requiring more than 18 days) more long, and finished product spore liquid is difficult to avoid that living contaminants, and
And there is more culture medium impurity.
The another kind of culture dish cultivation for being to make using agar coagulator, i.e., first by about nutrient (such as rice bran cooks juice) and
Agar is made solid medium and changes into flat board with culture dish, and then Pyricularia oryzae is implanted into the culture basal plane in culture dish
On, return again to be cultivated under appropraite condition；Until bacterium colony is formed, the aerial hyphae of bacterium colony is then struck off, increases particular light culture,
Until forming spore.The method can overcome some shortcomings of preceding method, and such as pollution level is low, be readily available few pure of impurity
Spore finished product；But culture crucial utensil used is culture dish, and the appliances have individual inadequate natural endowment, and it is straight inside and outside culture dish to be exactly
Logical, the contaminated air outside culture dish is easy to be exchanged with ware inner air flow, thus is difficult to avoid culture material in culture dish
Material is contaminated, to obtain the conidium material completely without living contaminants, and technical difficulty is big.This to it is many need high-quality,
The research work of the spore of non-contaminated state is very unfavorable.
The content of the invention
Cultural method is prepared it is an object of the invention to provide a kind of rice blast pathogen conidiospore for efficiently preventing and polluting.
The technical scheme that the present invention solves above-mentioned technical problem is as follows：
A kind of rice blast pathogen conidiospore for efficiently preventing pollution prepares 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 culture medium：Make product spore culture medium with oat medium, the proportioning of the culture medium is：Oat
167g, agar 20g, water 1000mL；Culture medium is sub-packed in the triangular flask that step 1 prepares, is turned into after conventional high-pressure high-temperature sterilization
Vial-type culture medium, it is standby.
3. the transplanting of Pyricularia oryzae：With transplanting tool by the standing Pyricularia oryzae mycelium in laboratory, it is implanted into step 2 and makes
On standby vial-type culture medium.
4. spore is produced in culture：By step 3 operate it is complete after vial-type culture medium be transferred in the standard incubator that temperature is 28 DEG C
Culture；After being generally incubated 7 days, the intensive bacterium colony of mycelia is formed on vial-type culture basal plane, substantial amounts of mitogenetic spore has been formed on bacterium colony
Son.
5. conidial collection：The spore on bacterium colony obtained with sterilized water elution step 4, and focuses on sterilization container
It is interior, that is, obtain the rice blast pathogen conidiospore liquid of the better quality without living contaminants.
It is an advantage of the invention that：
1) 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, obtains high-quality spore finished product.
2) extra illumination required for without conventional formulation techniques during cultivating, halfway without mycelia is struck off, operate skill
Art is simple.
3) operation of the spore than collecting spore in culture dish is collected in triangular flask more convenient.
4) 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 agar class 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, stalk) 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 rice blast pathogen conidiospore 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 of Involved in Sporulation in Magnaporthe grisea 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 on the agar medium in environment that Pyricularia oryzae can more be closed at this；And the application of triangular flask, then
Just solve the key technical problem that applied culture ware is easily caused pollution.
Pyricularia oryzae can form spore on various agar mediums, and the present invention makees to produce spore culture using oat medium
Base, its proportioning is：Oat 167g, agar 20g, water 1000mL；Component oat is that juice use is taken after being boiled 180 minutes with water.Culture
Base is boiled with 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 make for culture more
Local excessively thin without culture medium or culture medium into bottom of bottle, culture medium is crossed and at most easily cause waste；Usual every bottle (250mL specifications) dress
Enter 20~30mL culture mediums to be advisable, 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 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.
The transplanting of Pyricularia oryzae can be transplanted in the usual way, and the parent bacterium of transplanting is used as with the mycelium on common bacterium colony
Body；To accelerate culture process, polylith mycelia block can be implanted on a vial-type culture medium.
Produce spore culture can implement in standard incubator, temperature control Growth of Magnaporthe Grisea preference temperature, with 28 DEG C
It is preferred；Without routinely implementing to strike off bacterium colony surface mycelia in incubation, without the special illumination condition of increase.
After generally product spore prepares culture 7 days, intensive aerial hyphae can be formed with a large amount of points on culture basal plane in triangular flask
Raw spore.
The conidium for obtaining is collected with sterilized water, practical operation can scrub bacterium colony, make spores release to washing with T-shaped glass rod
In spore aqueous.
Embodiment 1
Cultural method is prepared using a kind of rice blast pathogen conidiospore for efficiently preventing from polluting of the present invention, culture rice is prepared
The conidium of seasonal febrile diseases bacteria strain Mg2013-1, implements operation as follows：
1. make to prepare the utensil of culture spore using 250mL triangular flasks, triangular flask cleaning is standby.
2. the preparation of vial-type culture medium：Make product spore culture medium with oat medium, the proportioning of the culture medium is：Oat
167g, agar 20g, water 1000mL；Culture medium is sub-packed in the triangular flask that step 1 prepares, every bottle of 30mL；Conventional high-pressure high temperature goes out
Turn into vial-type culture medium after bacterium, it is standby.
3. the transplanting of Pyricularia oryzae：With transplanting pin by the standing Pyricularia oryzae bacterial strain Mg2013-1 mycelium in laboratory, move
On vial-type culture medium prepared by implantation step 2.
4. spore is produced in culture：By step 3 operate it is complete after vial-type culture medium be transferred in the standard incubator that temperature is 28 DEG C
Culture；The intensive bacterium colony of mycelia is formed after 7 days on vial-type culture basal plane, conidium is produced on bacterium colony.
5. conidial collection：Take on one bottle of bacterium colony of the acquisition of sterilized water implantation step 4, bacterium colony is scrubbed with T-shaped glass rod
Spore, spore concentration can be obtained for 105The rice blast pathogen conidiospore liquid 75mL of individual/mL, the better quality without living contaminants.
Embodiment 2
Cultural method is prepared using a kind of rice blast pathogen conidiospore for efficiently preventing from polluting of the present invention, culture rice is prepared
The conidium of seasonal febrile diseases bacteria strain Mg2015-15,1 operates to step 5 and implements the step of by embodiment 1, different simply steps 3
Bacterial strain be Mg2015-15；It is 10 that result can obtain spore concentration5The Pyricularia oryzae of individual/mL, the better quality without living contaminants
Conidium liquid 36mL.
Claims (1)
1. a kind of rice blast pathogen conidiospore for efficiently preventing pollution prepares cultural method, it is characterised in that prepare culture side
The step of method, is as follows：
1) made to prepare the utensil of culture spore with common triangular flask, triangular flask cleaning is standby；
2) preparation of vial-type culture medium：Make product spore culture medium with oat medium, the proportioning of the culture medium is：Oat 167g, fine jade
Fat 20g, water 1000mL；Culture medium is sub-packed in step 1) prepare triangular flask, after conventional high-pressure high-temperature sterilization as vial-type train
Base is supported, it is standby；
3) transplanting of Pyricularia oryzae：With transplanting tool by the standing Pyricularia oryzae mycelium in laboratory, step 2 is implanted into) prepare
Vial-type culture medium on；
4) spore is produced in culture：By step 3) the vial-type culture medium after operation is complete is transferred to training in standard incubator that temperature is 28 DEG C
Support；After being generally incubated 7 days, the intensive bacterium colony of mycelia is formed on vial-type culture basal plane, substantial amounts of conidium has been formed on bacterium colony：
5) conidial collection：With sterilized water elution step 4) obtain bacterium colony on conidium, and focus on sterilizing hold
In device, that is, obtain the rice blast pathogen conidiospore liquid of the better quality without living contaminants.
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| CN201710033997.4A CN106754622A (en) | 2017-01-10 | 2017-01-10 | A kind of rice blast pathogen conidiospore for efficiently preventing pollution prepares cultural method |
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| CN201710033997.4A CN106754622A (en) | 2017-01-10 | 2017-01-10 | A kind of rice blast pathogen conidiospore for efficiently preventing pollution prepares cultural method |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107517788A (en) * | 2017-09-25 | 2017-12-29 | 安徽省中日农业环保科技有限公司 | A kind of method for preventing and treating rice blast |
| CN107593196A (en) * | 2017-09-25 | 2018-01-19 | 安徽省中日农业环保科技有限公司 | A kind of biological Control Technology of rice blast |
| CN107593336A (en) * | 2017-09-25 | 2018-01-19 | 安徽省中日农业环保科技有限公司 | A kind of prevention and controls of rice blast |
| CN113005074A (en) * | 2021-05-14 | 2021-06-22 | 浙江省农业科学院 | Large-scale culture method of rice blast bacterium conidia |
| CN113278578A (en) * | 2021-06-15 | 2021-08-20 | 广西大学 | Transplanting method of rice blast bacterium conidia |
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| CN107593336A (en) * | 2017-09-25 | 2018-01-19 | 安徽省中日农业环保科技有限公司 | A kind of prevention and controls of rice blast |
| CN113005074A (en) * | 2021-05-14 | 2021-06-22 | 浙江省农业科学院 | Large-scale culture method of rice blast bacterium conidia |
| CN113278578A (en) * | 2021-06-15 | 2021-08-20 | 广西大学 | Transplanting method of rice blast bacterium conidia |
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Application publication date: 20170531 |
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| RJ01 | Rejection of invention patent application after publication |