CN103834572A - Method for separating parasitic pathogenic fungus - Google Patents
Method for separating parasitic pathogenic fungus Download PDFInfo
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- CN103834572A CN103834572A CN201210479709.5A CN201210479709A CN103834572A CN 103834572 A CN103834572 A CN 103834572A CN 201210479709 A CN201210479709 A CN 201210479709A CN 103834572 A CN103834572 A CN 103834572A
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- spore
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Abstract
The invention belongs to the field of biotechnology, and provides a method for separating pathogenic fungi, in order to facilitate future research. Single spore isolation is the most effective method for separating parasitic pathogenic fungi, particularly suitable for the separation of pathogenic fungi, which are seriously polluted and difficult to separate by a traditional tissue isolation method, such as Venturia nashicola, apple Marssonina coronaria and Cercospora fungus. The traditional single spore isolation technique has complex operation, high technical requirements, and limited application. Employing capillary with diameter of 1 mm for drilling greatly simplifies the operation process, realizes strong operability of single spore isolation and higher speed; and general technical personnel can grasp the technology through simple training, so as to expand the scope of application. The method is mainly used for the separation of parasitic fungi and ascospores and gains good effect.
Description
Technical field
The invention belongs to biological technical field, relate generally to a kind of separation method of strong parasitic disease fungal pathogens.
Background technology
Strong parasitical fungi main parasitic, on live plant, is obtained necessary nutrient, poor growth on artificial medium from the host tissue of live body.Except pathogenic bacteria, in the host tissue of morbidity, also has endophyte, saprophytic microorganism etc. miscellaneous bacteria.Because these miscellaneous bacterias are grown soon on artificial medium, separate strong parasitical fungi by conventional diseased tissues partition method, often by living contaminants, Success rate of virus isolation is very low.Monospore separates the pollution that can avoid diseased tissues institute to cause with miscellaneous bacteria, and for the separation of strong parasitical fungi, success ratio is enrolled higher.
It is the basic fundamental of fungi isolation and purification that monospore separates, and is widely used in the heredity of fungi and purifying and the preservation work of variation, physio-biochemical characteristics research and bacterial classification.Special " monospore separator, and the domestic main manual operations of leaning on are invented abroad.Conventional monospore separation method has: little slide monospore partition method, impression pipettes monospore partition method, agar plate dilution method: be coated in agar plate surface by spore suspension, then direct picking monospore, zoom-stereo microscope monospore partition method and eyebrow are chosen pin picking monospore method etc.These monospore separation method complicated operations, higher to technical requirements, thus limit its application.
Summary of the invention
The object of the present invention is to provide a kind of separation method to strong parasitic disease fungal pathogens, so that research from now on.On the basis of laboratory work for many years, agar plate dilution monospore partition method is improved, improved kapillary punching monospore partition method, have easy and simple to handle, velocity of separation fast, pollute the advantages such as low.
Embodiment
The basic step that kapillary punch method monospore separates is: directly obtain spore from scab, be applied on water agar block, find under the microscope required spore, punch with kapillary, finally the agar cake that has single spore is proceeded in substratum and cultivated, concrete grammar is as follows:
(1) water agar preparation: agar powder is directly added in sterilized water, be heated to fusing in microwave oven; Or, agar powder is added in tap water, for subsequent use after sterilizing.Monospore separates conventional 2% water agar;
(2) agar block preparation: be coated on aseptic slide glass even thickness with the sterilized water agar of 1 ml sterilizing liquid-transfering gun absorption 2%;
(3) spore coating: choose the spore on pin picking scab with sterilizing, attach on water agar block, pick sterilized water, dilution spread spore with sterilizing glass rod or kapillary.Or, with the kapillary of diameter 1 mm, under spirit lamp, burn soft rear wire drawing, after fractureing, be made into fine needle (being called for short " capillary bobbin "), with capillary bobbin picking spore, be coated directly onto on water agar block;
(4) spore is chosen: microscope inspection (conventional 10 times of object lens) scribbles the water agar block of spore, finds after typical spore, moves into visual field central authorities (in the visual field only this 1 spore), then moves down Stage microscope;
(5) agar punching: get the kapillary that internal diameter is 1mm, burn soft palintrope and meet at right angles on spirit lamp, the about 1cm of galianconism length.Galianconism end is on spirit lamp after heat sterilization, and the hot spot central authorities under aligming microscope composition lens depress, and buy the cake of a diameter 1mm on water agar.Promote Stage microscope to original position, whether object observing spore on agar cake, if do not existed, chooses spore again;
(6) monospore shifts: with aseptic pin or the capillary bobbin chosen, picking, with the agar cake of target spore, proceeds in corresponding substratum, has the one side of spore to be close to substratum.Every ware can be put 3 ~ 5 monospores.
The the 2nd to the 6th step all need complete on Bechtop or in sterilisable chamber.
embodiment 1
Obtaining of dry rot germ spore: by 10ml, 1% aseptic water agar is poured in sterile petri dish, cooling.Gather the dry rot branch with pore from field, with after 70% alcohol wipe surface sterilization, cut disease skin, stick on water agar.Be inverted culture dish, under sick skin, place the agar block that a slice separates for monospore, have the accurate sick skin of facing of agar.Sealing culture dish, at room temperature moisturizing 2 ~ 12 h, the thecaspore on sick skin in ripe ascus can be ejected on water agar block, and picking monospore, proceeds in PAD and cultivates under the microscope.
embodiment 2
Dry rot germ conidium gathers the dry rot branch with pore from field, with 70% alcohol wipe surface sterilization.Cut the sick skin of sterilization, soak with sterilized water, be placed in aseptic culture dish, (20 ~ 25 ℃) moisturizing 12 h under room temperature, the elongated conidium angle of white can be extruded in spore device, uses capillary bobbin, picking cirrus, be applied on water agar block, picking monospore, proceeds in PDA and cultivates.
Claims (4)
1. the sampling of strong parasitic disease fungal pathogens, a separation method, is characterized in that: realize by following steps:
(1) sample collecting: directly obtain spore from scab, be applied on water agar block, find under the microscope required spore;
(2) sample separation: with kapillary punching, the agar cake that has single spore is proceeded in substratum and cultivated.
2. obtaining of dry rot germ spore as claimed in claim 1: by 10ml, 1% aseptic water agar is poured in sterile petri dish, cooling, gather the dry rot branch with pore from field, with after 70% alcohol wipe surface sterilization, cut disease skin, stick on water agar.
3. as claimed in claim 2ly choose the spore on pin picking scab with sterilizing, attach on water agar block, pick sterilized water with sterilizing glass rod or kapillary, dilution spread spore, or with the kapillary of diameter 1 mm, under spirit lamp, burn soft rear wire drawing, after fractureing, be made into fine needle (being called for short " capillary bobbin "), with capillary bobbin picking spore, be coated directly onto on water agar block.
4. passing through as described in claim 1,2,3 improved agar plate dilution monospore partition method, improved kapillary punching monospore partition method, have easy and simple to handle, velocity of separation fast, pollute the advantages such as low.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210479709.5A CN103834572A (en) | 2012-11-23 | 2012-11-23 | Method for separating parasitic pathogenic fungus |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210479709.5A CN103834572A (en) | 2012-11-23 | 2012-11-23 | Method for separating parasitic pathogenic fungus |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN103834572A true CN103834572A (en) | 2014-06-04 |
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| Application Number | Title | Priority Date | Filing Date |
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| CN201210479709.5A Pending CN103834572A (en) | 2012-11-23 | 2012-11-23 | Method for separating parasitic pathogenic fungus |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108359605A (en) * | 2018-02-07 | 2018-08-03 | 四川农业大学 | A kind of method for purifying and separating of phytopathogeic fungi |
| CN111471601A (en) * | 2020-06-08 | 2020-07-31 | 广东省生物工程研究所（广州甘蔗糖业研究所） | Method for culturing brown spot germs of sugarcane |
-
2012
- 2012-11-23 CN CN201210479709.5A patent/CN103834572A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108359605A (en) * | 2018-02-07 | 2018-08-03 | 四川农业大学 | A kind of method for purifying and separating of phytopathogeic fungi |
| CN111471601A (en) * | 2020-06-08 | 2020-07-31 | 广东省生物工程研究所（广州甘蔗糖业研究所） | Method for culturing brown spot germs of sugarcane |
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Application publication date: 20140604 |