CN105861412A - Culture and preparation method for pyricularia oryza conidia - Google Patents
Culture and preparation method for pyricularia oryza conidia Download PDFInfo
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- CN105861412A CN105861412A CN201610345467.9A CN201610345467A CN105861412A CN 105861412 A CN105861412 A CN 105861412A CN 201610345467 A CN201610345467 A CN 201610345467A CN 105861412 A CN105861412 A CN 105861412A
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- 238000002360 preparation method Methods 0.000 title claims abstract description 27
- 241001330975 Magnaporthe oryzae Species 0.000 title abstract description 10
- 238000012136 culture method Methods 0.000 title abstract description 3
- 239000001963 growth medium Substances 0.000 claims abstract description 19
- 238000000034 method Methods 0.000 claims abstract description 17
- 238000005516 engineering process Methods 0.000 claims abstract description 8
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 claims description 24
- 239000007788 liquid Substances 0.000 claims description 16
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 15
- 241000894006 Bacteria Species 0.000 claims description 14
- 241000209094 Oryza Species 0.000 claims description 13
- 235000007164 Oryza sativa Nutrition 0.000 claims description 13
- 235000009566 rice Nutrition 0.000 claims description 13
- 244000052769 pathogen Species 0.000 claims description 11
- 230000001717 pathogenic effect Effects 0.000 claims description 11
- 229920001817 Agar Polymers 0.000 claims description 6
- 239000008272 agar Substances 0.000 claims description 6
- 230000001954 sterilising effect Effects 0.000 claims description 6
- 101100381997 Danio rerio tbc1d32 gene Proteins 0.000 claims description 5
- 101100381999 Mus musculus Tbc1d32 gene Proteins 0.000 claims description 5
- 238000005406 washing Methods 0.000 claims description 5
- 244000052616 bacterial pathogen Species 0.000 claims description 4
- 238000007796 conventional method Methods 0.000 claims description 3
- 239000007943 implant Substances 0.000 claims description 2
- 238000004659 sterilization and disinfection Methods 0.000 claims description 2
- 230000028070 sporulation Effects 0.000 abstract description 10
- 230000008569 process Effects 0.000 abstract description 5
- 238000004519 manufacturing process Methods 0.000 abstract description 3
- 230000008901 benefit Effects 0.000 abstract description 2
- 238000011109 contamination Methods 0.000 abstract 1
- 238000012258 culturing Methods 0.000 abstract 1
- 230000001580 bacterial effect Effects 0.000 description 7
- 238000005286 illumination Methods 0.000 description 5
- 241000196324 Embryophyta Species 0.000 description 4
- 241001344131 Magnaporthe grisea Species 0.000 description 4
- 244000144987 brood Species 0.000 description 4
- 239000012634 fragment Substances 0.000 description 4
- 239000000356 contaminant Substances 0.000 description 3
- 230000002073 mitogenetic effect Effects 0.000 description 3
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 235000011389 fruit/vegetable juice Nutrition 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 229920006395 saturated elastomer Polymers 0.000 description 2
- 239000002028 Biomass Substances 0.000 description 1
- 240000005979 Hordeum vulgare Species 0.000 description 1
- 235000007340 Hordeum vulgare Nutrition 0.000 description 1
- 241000209140 Triticum Species 0.000 description 1
- 235000021307 Triticum Nutrition 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
- 230000009471 action Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 235000005822 corn Nutrition 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 244000000004 fungal plant pathogen Species 0.000 description 1
- 230000035784 germination Effects 0.000 description 1
- 238000002513 implantation Methods 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000008595 infiltration Effects 0.000 description 1
- 238000001764 infiltration Methods 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- RSMUVYRMZCOLBH-UHFFFAOYSA-N metsulfuron methyl Chemical compound COC(=O)C1=CC=CC=C1S(=O)(=O)NC(=O)NC1=NC(C)=NC(OC)=N1 RSMUVYRMZCOLBH-UHFFFAOYSA-N 0.000 description 1
- 230000002906 microbiologic effect Effects 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 238000007747 plating Methods 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 238000005201 scrubbing Methods 0.000 description 1
- 238000011218 seed culture Methods 0.000 description 1
- 230000001568 sexual effect Effects 0.000 description 1
- 239000007787 solid 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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- Zoology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- Wood Science & Technology (AREA)
- Biomedical Technology (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a culture and preparation method for pyricularia oryza conidia. According to the method, the multi-starting-point culture technology and the aerial mycelium conidium production technology are utilized for fast culturing and preparing the pyricularia oryza conidia. The method comprises the following implementation steps that 1, a conidium production culture medium is prepared; 2, a multi-starting-point transplanting thallus is prepared; 3, the multi-starting-point transplanting thallus is planted in a culture plate; 4, the conidia are prepared and cultured; 5, the conidia are eluted and separated. The method has the advantages that fastness and high efficiency are achieved, and conidia of a new generation can reach the ideal state of a high sporulation quantity after five days of culture usually; sterile operation can be kept in the whole culture process, and the contamination probability of a finished conidium solution is greatly reduced; the operation technology is simple, and the result sporulation quantity is large.
Description
Technical field
The present invention relates to plant pathology learn a skill and microbiological technique.Specifically a kind of mitogenetic spore of Pyricularia oryzae
The cultivation preparation method of son.
Background technology
The brood body of plant pathogenic fungi expands in addition to the basic role of its population quantity except having breeding, more
It is important that have and host's interaction identification, and start to infect the function of host, therefore sick at most plants
In the research process of evil, it is required for using the brood body of pathogen to be tested material, it is therefore apparent that the most just
The cultivation preparation method of prompt brood body material, will be very beneficial for efficiently carrying out of research work.Rice blast
It is the great disease of one of China's Rice Production, the pathogen (Magnaporthe grisea) of this disease numerous
Grow body and include the ascospore of condition and conidium two type of Invisible element.At present in laboratory
Preparing sexual spore and there is bigger technical difficulty, therefore, the brood body of relevant institute so far is main
It it is the conidium using this pathogenic bacteria.
The cultivation preparation method of rice blast pathogen conidiospore can be attributed to two big classes.
One class is the culture method using plant seed to make culture medium, the most first by plant seed (such as barley corn etc.)
Culture medium is made in infiltration sterilizing, cultivates, when mycelia is covered with in then mycelium is implanted into seed culture medium
After seed, wash with water and brush off seed surface mycelia, and carry out illumination cultivation again in relaying square plate appliances,
Whole process is it is generally required to more than 18 days.The method generally can prepare substantial amounts of conidium, but, cultivates
Preparing all times longer, finished product spore liquid is difficult to avoid that living contaminants, and it is miscellaneous to there is more culture medium
Matter.
Another kind of is to use coagulator agar to make the plating method of culture medium, the most first by relevant nutrient (as
Oat juice) make solid medium with agar and change into flat board, then a little mycelium block is implanted into cultivation
Cultivate in the middle of base flat board, after covering with mycelia on flat board, strike off planar surface aerial hyphae with instrument, and turn
Being further cultured under illumination condition, whole process is it is generally required to more than 10～13 days.The method can overcome aforementioned side
The some shortcomings of method, the finished product spore liquid impurity as obtained is few, and easily avoids living contaminants.But the method consumes
Time-consuming still have long suspicion；And flat board cultivation yet suffers from higher living contaminants probability for a long time；?
Cultivate midway and need to scrape off the technical operation of aerial hyphae, and need to provide the technical supports such as illumination, practice
Application is still inconvenient.Thus the most still have and improve the space improved.
Summary of the invention
It is an object of the invention to provide the cultivation preparation method of a kind of rice blast pathogen conidiospore.
The technical scheme is that
The cultivation preparation method of a kind of rice blast pathogen conidiospore, mainly applies many starting points culture technique, with
And aerial hyphae produces spore technology, fast culture prepares rice blast pathogen conidiospore, cultivates the step of preparation method
As follows:
1. produce the preparation of spore culture medium: prepare product spore culture medium according to a conventional method, consisting of of this culture medium:
Herba bromi japonici 167g, agar 20g, water 1000ml；Conventional high temperature sterilizing, pouring 9cm culture dish into, to make cultivation flat
Plate.
2. starting point more than transplants the outfit of thalline: aseptically, with the conventional standby rice blast deposited of sterilized water washing
Pathogenic bacteria bacterium colony, obtains the broken mixed liquor of spore-mycelia, conidial many as preparation a new generation with this mixed liquor
Starting point transplants thalline.
3. by many starting points transplanting thalline implantation culture plate: many starting points step 2 being equipped with micropipettor
Transplant thalline and be implanted into the culture plate of step 1 preparation, and make thalline liquid be uniformly dispersed in platen surface.
4. spore preparation is cultivated: step 3 is operated complete after prepare culture plate proceed to temperature be 28 DEG C,
Relative humidity be 90% constant incubator in light culture.
5. the eluting of spore separates: after generally step 4 cultivates 5 days, and a new generation conidium reaches high yield spore
The state of amount；With sterilized water by spore eluting on flat board, and focus in sterilization container, i.e. obtain purer
Rice blast pathogen conidiospore liquid.
The characteristic of the present invention with advantage is:
1) after rapidly and efficiently, being generally incubated 5 days, a new generation's spore can reach the perfect condition of high sporulation quantity.
2) preparation cultivation whole process can keep sterile working, the pollution probability of finished product spore liquid to be greatly reduced.
3) operating technology is simple, and result sporulation quantity is big.
Accompanying drawing explanation
Fig. 1 is that many starting points are transplanted the Pyricularia oryzae bacterium after thalline is implanted culture plate and cultivated 2 days by the present invention
Fall.
Fig. 2 is that many starting points are transplanted the Pyricularia oryzae bacterium after thalline is implanted culture plate and cultivated 5 days by the present invention
Fall.
Detailed description of the invention
The invention will be further described with embodiment below in conjunction with the accompanying drawings.
Product spore culture medium of the present invention is the culture medium that application Herba bromi japonici makees main component, and its component is: swallow
Wheat 167g, agar 20g, water 1000ml；Component Herba bromi japonici is that after decocting in water is opened 180 minutes, extracting juice is used；Culture medium warp
After crossing conventional high-pressure high temperature sterilize standby.
It is mitogenetic that many starting points of the present invention transplanting thalline refers to that the conventional standby flat-plate bacterial colony deposited of washing is obtained
The mixed liquor that spore is broken with mycelia, this mixed liquor miospore is in the great majority, and is transplanted to this mixed liquor cultivate and puts down
Plate is also dispersed in platen surface, each spore or mycelia fragment can germination and growth, thus permissible
Forming substantial amounts of growth starting point on culture plate, therefore, the thalline liquid of this transplanting mode is claimed by the present invention
Thalline is transplanted for many starting points.Substantial amounts of growth starting point grows simultaneously, just quickly can be formed on culture plate all
Even bacterium colony.
Comparatively speaking, conventional transplanting thalline is a mycelia block, although this mycelia block may be containing more bacterium
Silk fragment, but on culture plate face, pathogenic bacteria is to radiate to surrounding blank cultures centered by this mycelia block
Growth, actual is equivalent to the most single the growth starting point.
Many starting points transplant the spore in thalline liquid and hyphae length typically without accurate quantitative analysis, take a 9cm and cultivate
The normal bacterium colony of ware adds water the thalline liquid that 10ml washing obtains, and takes its 20～50 μ l and makees to transplant thalline, can completely
The biomass requirement of a new generation's spore is cultivated in the normal preparation of foot.
Inventor is it has been observed that in producing spore incubation, a new generation's illumination is in bacterium colony surface
On aerial hyphae, the technical operation striking off bacterium colony surface mycelia can not increase sporulation quantity, drops low-yield spore on the contrary
Amount；Carry out photo-irradiation treatment equally and can not increase sporulation quantity, reduce sporulation quantity on the contrary, thus in technical operation
On, the present invention is without implementing routinely to strike off bacterium colony surface mycelia, without increasing illumination condition.
The relative humidity producing spore culture environment should maintain about 90%, and too low humidity is unfavorable for that bacterium colony produces spore,
Also it is easily caused culture medium to be dried, if real work is used without controlling the incubator of wet condition, can be in case
Place clean wet towel and increase humidity；But humidity should not be improved to being bordering on saturated humidity, because inventor
Finding, under the conditions of being bordering on saturated humidity, the product spore usefulness of this culture technique declines on the contrary.
Generally produce after spore preparation is cultivated 2 days visible significantly bacterium colony and form aerial hyphae, as it is shown in figure 1,
Just can produce a new generation's spore after cultivating 3 days, after cultivating 5 days, bacterium colony surface has formed intensive aerial hyphae,
And a new generation's spore reaches the perfect condition of high sporulation quantity, as shown in Figure 2；It addition, this high sporulation quantity shape
State can maintain to cultivation 11 days.
In the spore elution action of new generation of preparation cultivation results, can produce greatly if scrubbing with hairbrush class instrument
The mycelia fragment of amount mixes in spore liquid, and scrubs the spore liquid of acquisition with smooth glass rod, wherein mixes
Mycelia fragment is less.
Embodiment 1
The cultivation preparation method of application a kind of magnaporthe grisea spore of the present invention, Pyricularia oryzae bacterial strain is prepared in cultivation
The conidium of Mg2013-1, implements to operate as follows:
1. produce the preparation of spore culture medium
Prepare according to a conventional method and produce spore culture medium, consisting of of this culture medium: Herba bromi japonici 167g, agar 20g,
Water 1000ml；Conventional high temperature sterilizing, pours 9cm culture dish into and makes culture plate.
2. starting point more than transplants the outfit of thalline
Aseptically, with the conventional standby Pyricularia oryzae bacterial strain Mg2013-1's deposited of 10ml sterilized water washing
Bacterium colony one ware, obtains the broken mixed liquor of spore-mycelia, conidial many as preparation a new generation with this mixed liquor
Starting point transplants thalline.
3. many starting points are transplanted thalline and implant culture plate
The many starting points transplanting thalline 40 μ l taking step 2 outfit with micropipettor is implanted into training prepared by step 1
Support flat board, smear gently in platen surface with T-shaped glass rod, make thalline liquid be uniformly dispersed in platen surface.
4. spore preparation is cultivated: step 3 is operated complete after prepare culture plate proceed to temperature be 28 DEG C,
Relative humidity be 90% constant incubator in light culture.
5. the eluting of spore separates: after step 4 cultivates 5 days, and a new generation conidium reaches high sporulation quantity
State；Take sterilized water 15ml by the spore eluting of new generation of a ware culture plate, the conidium liquid obtained,
Spore concentration is up to 1.48 × 106Individual/ml.
Embodiment 2
The cultivation preparation method of application a kind of magnaporthe grisea spore of the present invention, Pyricularia oryzae bacterial strain is prepared in cultivation
The conidium of Mg2015-7, is implemented by the operation of the step 1 of embodiment 1 to step 5, simply step 2
Bacterial strain is Mg2015-7；The mitogenetic spore of a new generation that result sterilized water 15ml eluting one ware culture plate is obtained
Sub-liquid, spore concentration is up to 1.20 × 106Individual/ml.
Embodiment 3
The cultivation preparation method of application a kind of magnaporthe grisea spore of the present invention, Pyricularia oryzae bacterial strain is prepared in cultivation
The conidium of Mg2015-14, is implemented by the operation of the step 1 of embodiment 1 to step 5, simply step 2
Bacterial strain is Mg2015-14；A new generation that result sterilized water 15ml eluting one ware culture plate is obtained divides
Raw spore liquid, spore concentration is up to 1.38 × 106Individual/ml.
Claims (1)
1. the cultivation preparation method of a rice blast pathogen conidiospore, it is characterised in that utilize many starting points to cultivate
Technology, and aerial hyphae product spore technology, fast culture prepares rice blast pathogen conidiospore；The step of method
As follows:
1) preparation of spore culture medium is produced: prepare product spore culture medium according to a conventional method, consisting of of this culture medium:
Herba bromi japonici 167g, agar 20g, water 1000ml；Conventional high temperature sterilizing, pouring 9cm culture dish into, to make cultivation flat
Plate；
2) many starting points transplant the outfit of thalline: aseptically, with the conventional standby rice blast deposited of sterilized water washing
Pathogenic bacteria bacterium colony, obtains the broken mixed liquor of spore-mycelia, conidial many as preparation a new generation with this mixed liquor
Starting point transplants thalline；
3) by many starting points transplant thalline implant culture plate: with micropipettor by step 2) be equipped with a lot of
Point is transplanted thalline and is implanted into step 1) culture plate prepared, and make thalline liquid be uniformly dispersed in platen surface；
4) spore preparation is cultivated: by step 3) operate complete after prepare culture plate proceed to temperature be 28 DEG C,
Relative humidity be 90% constant incubator in light culture；
5) eluting of spore separates: generally step 4) cultivate 5 days after, a new generation conidium reaches high yield
The state of spore amount；With sterilized water by spore eluting on flat board, and focus in sterilization container, i.e. obtain purer
Clean rice blast pathogen conidiospore liquid.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610345467.9A CN105861412A (en) | 2016-05-24 | 2016-05-24 | Culture and preparation method for pyricularia oryza conidia |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610345467.9A CN105861412A (en) | 2016-05-24 | 2016-05-24 | Culture and preparation method for pyricularia oryza conidia |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN105861412A true CN105861412A (en) | 2016-08-17 |
Family
ID=56635767
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610345467.9A Pending CN105861412A (en) | 2016-05-24 | 2016-05-24 | Culture and preparation method for pyricularia oryza conidia |
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106701654A (en) * | 2017-01-10 | 2017-05-24 | 广西大学 | Regular morphology of fusarium oxysporum laboratory material and preparation and application method thereof |
| CN106754622A (en) * | 2017-01-10 | 2017-05-31 | 广西大学 | A kind of rice blast pathogen conidiospore for efficiently preventing pollution prepares cultural method |
| CN107586754A (en) * | 2017-09-08 | 2018-01-16 | 中国农业科学院果树研究所 | A kind of culture medium and its cultural method for being beneficial to improve Athelia bombacina sporulation quantities |
| CN109536432A (en) * | 2018-09-30 | 2019-03-29 | 华南农业大学 | A kind of method of the extracting method and application its secretory protein group of shotgun technical research of rice blast fungus secretory protein |
| 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 |
| CN113174335A (en) * | 2021-04-19 | 2021-07-27 | 广西大学 | Separation method of banana colletotrichum gloeosporioides |
| CN113278578A (en) * | 2021-06-15 | 2021-08-20 | 广西大学 | Transplanting method of rice blast bacterium conidia |
| CN113308431A (en) * | 2021-06-15 | 2021-08-27 | 广西大学 | Transplanting and dispersing method for botrytis cinerea conidia |
| WO2023035857A1 (en) * | 2021-09-08 | 2023-03-16 | 浙江省农业科学院 | Conidia coating method for producing large number of sexual generations of pyricularia oryzae |
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Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106701654A (en) * | 2017-01-10 | 2017-05-24 | 广西大学 | Regular morphology of fusarium oxysporum laboratory material and preparation and application method thereof |
| CN106754622A (en) * | 2017-01-10 | 2017-05-31 | 广西大学 | A kind of rice blast pathogen conidiospore for efficiently preventing pollution prepares cultural method |
| CN107586754A (en) * | 2017-09-08 | 2018-01-16 | 中国农业科学院果树研究所 | A kind of culture medium and its cultural method for being beneficial to improve Athelia bombacina sporulation quantities |
| CN109536432A (en) * | 2018-09-30 | 2019-03-29 | 华南农业大学 | A kind of method of the extracting method and application its secretory protein group of shotgun technical research of rice blast fungus secretory protein |
| CN109536432B (en) * | 2018-09-30 | 2022-05-31 | 华南农业大学 | Extraction method of rice blast fungus secretory protein and method for researching secretory proteome of rice blast fungus secretory protein by using shotgun technology |
| CN113174335A (en) * | 2021-04-19 | 2021-07-27 | 广西大学 | Separation method of banana colletotrichum gloeosporioides |
| CN113174335B (en) * | 2021-04-19 | 2022-06-17 | 广西大学 | Separation method of banana colletotrichum gloeosporioides |
| 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 |
| CN113308431A (en) * | 2021-06-15 | 2021-08-27 | 广西大学 | Transplanting and dispersing method for botrytis cinerea conidia |
| WO2023035857A1 (en) * | 2021-09-08 | 2023-03-16 | 浙江省农业科学院 | Conidia coating method for producing large number of sexual generations of pyricularia oryzae |
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