CN105548091A - Laser confocal microscope detection method for Tilletia foetida - Google Patents
Laser confocal microscope detection method for Tilletia foetida Download PDFInfo
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- CN105548091A CN105548091A CN201510909600.4A CN201510909600A CN105548091A CN 105548091 A CN105548091 A CN 105548091A CN 201510909600 A CN201510909600 A CN 201510909600A CN 105548091 A CN105548091 A CN 105548091A
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- wheat
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- 241000031845 Tilletia laevis Species 0.000 title claims abstract description 29
- 238000001514 detection method Methods 0.000 title abstract description 3
- 241000209140 Triticum Species 0.000 claims abstract description 48
- 235000021307 Triticum Nutrition 0.000 claims abstract description 48
- 238000004043 dyeing Methods 0.000 claims abstract description 19
- 238000000034 method Methods 0.000 claims abstract description 18
- 238000012360 testing method Methods 0.000 claims abstract description 8
- 230000032683 aging Effects 0.000 claims description 9
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 7
- 229920001213 Polysorbate 20 Polymers 0.000 claims description 3
- 238000000386 microscopy Methods 0.000 claims description 3
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 claims description 3
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 claims description 3
- 238000011160 research Methods 0.000 abstract description 8
- 241000196324 Embryophyta Species 0.000 abstract description 6
- 201000010099 disease Diseases 0.000 abstract description 5
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 abstract description 5
- 238000004624 confocal microscopy Methods 0.000 abstract 1
- ZDWVWKDAWBGPDN-UHFFFAOYSA-O propidium Chemical compound C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CCC[N+](C)(CC)CC)=C1C1=CC=CC=C1 ZDWVWKDAWBGPDN-UHFFFAOYSA-O 0.000 abstract 1
- 238000010186 staining Methods 0.000 abstract 1
- 238000005406 washing Methods 0.000 abstract 1
- 239000000975 dye Substances 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000011081 inoculation Methods 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 239000005418 vegetable material Substances 0.000 description 2
- 241000251468 Actinopterygii Species 0.000 description 1
- 241000221198 Basidiomycota Species 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 206010028813 Nausea Diseases 0.000 description 1
- 208000010513 Stupor Diseases 0.000 description 1
- 241000722133 Tilletia Species 0.000 description 1
- 241000221484 Tilletiaceae Species 0.000 description 1
- 241000221561 Ustilaginales Species 0.000 description 1
- 244000052616 bacterial pathogen Species 0.000 description 1
- 235000013339 cereals Nutrition 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 239000006059 cover glass Substances 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 230000001066 destructive effect Effects 0.000 description 1
- 235000019441 ethanol Nutrition 0.000 description 1
- 239000011888 foil Substances 0.000 description 1
- 238000007429 general method Methods 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 231100000567 intoxicating Toxicity 0.000 description 1
- 230000002673 intoxicating effect Effects 0.000 description 1
- 230000000873 masking effect Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000008693 nausea Effects 0.000 description 1
- 210000001672 ovary Anatomy 0.000 description 1
- 230000003071 parasitic effect Effects 0.000 description 1
- 239000000575 pesticide Substances 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 210000004916 vomit Anatomy 0.000 description 1
- 230000008673 vomiting Effects 0.000 description 1
Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Physics & Mathematics (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Engineering & Computer Science (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention belongs to the technology of plant disease research, and particularly relates to a laser confocal microscope detection method of Tilletia foetida. The laser confocal microscopy method for detecting the Tilletia foetida is characterized by comprising the following steps of: (1) dyeing a sample: sequentially using Propidium to test samples? idode and WGA-AF? 488 staining followed by washing with PBS; (2) microscopic examination: observing the dyed sample under a laser confocal microscope; if the green hypha appears under a microscope, the sample to be detected contains the Tilletia foetida. The method can simply judge whether the wheat for research is successfully inoculated with the Tilletia foetida.
Description
Technical field
The invention belongs to plant disease detection technique, particularly relate to the laser confocal microscope detecting method of wheat light Tilletia foetida.
Background technology
By wheat light Tilletia foetida (Tilletiafoetida (Wallr.) Liro, TFL) the wheat light bunt caused is one of destructive wheat diseases of most in the world, occurring more at northern China Mai Qu, is that Beijing supplements agricultural plant quarantine harmful organisms.This germ belongs to Basidiomycetes, Hemibasidii, Ustilaginales, Tilletiaceae, Tilletia, can by soil and seed dispersal.Had strong fish bad smell by the wheat seed of this infection process, this germ spore content can cause serious intoxicating phenomenon more than 0.6%, and people and animals eat rear severe one and can cause nausea, vomit even toxicity symptom (Goats, 1999 such as stupor; Hoffman, 1982), have a strong impact on wheat quality and commercial value.China once strict regulations bunt of wheat grain more than 6/kilogram, will not purchase to the wheat polluted by the gross (Zhang Jinliang etc., 2000).This disease all has generation and throughout world wheat planting district on spring wheat and winter wheat, and bunt can infect the wheat head of more than 70%, causes the underproduction of 25% ~ 50%, serious even total crop failure (Holton, 1947).Bunt once reached 28% ~ 38% (Gan Guofu etc., 1995) at Gansu Province's average attack rate.Therefore, only have and early warning is carried out to wheat light bunt, the economic loss that this disease is brought could be reduced to greatest extent.
At present, adopting for the doubtful scanning electron microscopic observation being infected wheat by wheat light Tilletia foetida is general method, the method that " ultrastructural studies of Germinating Teliospores of Tilletia foetida " that the people such as such as Ma Qing deliver 1999 is recorded in Northwest Agricultural University's journal and the picture observed, mycelia can not be seen at a glance, owing to not passing through dyeing, the mycelia of wheat light Tilletia foetida and Wheat Tissue do not have difference feature clearly, cannot judge in research process, whether the wheat light Tilletia foetida of inoculation is successfully inoculated.Accurately judge and the seed of the inoculation that succeeds if can give, use it for next step research best, can the efficiency improving next step research be given.
Summary of the invention
According to deficiency and the demand in above-mentioned field, the invention provides the microscopic detecting method of a grow wheat light Tilletia foetida, efficiently simply, Color is good, contrast is strong under the microscope to make mycelia and vegetable material, clearly can observe the degree that mycelia in vegetable material is infected.
The technical scheme of application claims protection is as follows:
The laser confocal microscope detecting method of wheat light Tilletia foetida, is characterized in that, comprise the steps:
(1) sample dyeing: dye testing sample dyeing liquor I 10-30 minute, then with 10 μ g/mLWGA-AF488 dyeing 10-30 minute, finally uses the 10 × PBS of pH7.4 to rinse 3-5 time;
Institute's dyeing liquor I is that 5 μ g/mLPropidiumidodide and 0.02%Tween20 mix with volume ratio 1:1;
(2) microscopy: observe under the sample after dyeing is placed in laser confocal microscope, the parameter of employing is as follows:
Propidiumidodide excites green wavelength to be 561nm, and wavelength of transmitted light is 590 ~ 640nm; WGA-AF488 excites blue light wavelength to be 488nm, and wavelength of transmitted light is 500 ~ 520nm,
If there is green mycelia under microscope, illustrate that testing sample contains wheat light Tilletia foetida.
Described testing sample is the tissue taking from wheat aging time root, stem stalk portion or blade part.
Ethanol is adopted to fix before described testing sample dyeing.
Experimental result shows, adopt the plant tissue that the inventive method observation wheat light Tilletia foetida infects, have the advantage that dyeing is clear, contrast is strong, what clearly can judge wheat light Tilletia foetida infects degree.
Method of the present invention is simply efficient, is easy to operation.In the process that research wheat and parasitic pathogenic bacteria are done mutually, can know whether wheat light Tilletia foetida is successfully inoculated concisely.Thus accurately judge and obtain the seedling being used for next step research, the efficiency of research work can be improved.
Accompanying drawing explanation
Fig. 1. adopt the wheat linked groups that the inventive method observation wheat light Tilletia foetida infects,
Wherein, A, B, C (Merge), what D (light field) showed is that wheat aging time root is infected by TFL mycelia; E, F, G (Merge), what H (light field) showed is that wheat aging time stem stalk portion is infected by TFL mycelia; I, J, K (Merge), what L (light field) showed is that wheat aging time blade part is infected by TFL mycelia.
Embodiment
Be described in further detail below by specific embodiment, it is to be appreciated that following examples only illustratively and illustrate, and the scope do not limited the present invention in any way.
Wheat light Tilletia foetida: gather from field, Henan
Wheat: the winter selects No. 3 Plant Protection institute, Chinese Academy of Agricultral Sciences
Biological chemical reagent not specified in following embodiment is this area conventional reagent, and can be prepared by commercially available or employing this area conventional method and be obtained, specification be the pure level in laboratory.
The Wheat Tissue that embodiment 1. adopts the inventive method observation wheat light Tilletia foetida to infect
Draw materials: the wheat aging time root that clip wheat light Tilletia foetida infects, stem stalk portion and blade part, the blade section of cutting becomes the square of about 5mm, removing edge makes it fully be immersed in absolute ethyl alcohol immobile liquid, and ovary and flower pesticide are directly fully immersed in absolute ethyl alcohol immobile liquid
Dyeing: sample is placed in centrifuge tube, add 0.5mL5 μ g/mLPropidiumidodide, 0.5mL0.02%Tween20 dyes 20min, remove dyestuff, then add 0.5mL10 μ g/mLWGA-AF488 dyeing 20min, remove dyestuff, rinse 3 to 5 times with the 10 × PBS of 1mLpH7.4, then, after completely cutting off light with masking foil parcel centrifuge tube, 4 degree of refrigerators are placed in for subsequent use.
Microscopy: be placed on microslide by the sample after above-mentioned dyeing, adds cover glass and is inverted, and observes under being placed on laser confocal microscope (SP8, LEICA, Germany).WGA-AF488 excites blue light wavelength to be 488nm, and wavelength of transmitted light is 500 ~ 520nm; Propidiumidodide excites green wavelength to be 561nm, and wavelength of transmitted light is 590 ~ 640nm.
Result as shown in Figure 1, A, B, C (Merge), the wheat aging time root infected by TFL mycelia that D (light field) shows; E, F, G (Merge), the wheat aging time stem stalk portion of being infected by TFL mycelia that H (light field) shows; I, J, K (Merge), the wheat aging time blade part infected by TFL mycelia that L (light field) shows.
Wheat light Tilletia foetida mycelia green as can be observed from Figure and red Wheat Tissue, show thus, adopt the plant tissue that the inventive method observation wheat light Tilletia foetida infects, have the advantage that dyeing is clear, contrast is strong, what clearly can judge wheat light Tilletia foetida infects degree.
Claims (3)
1. the laser confocal microscope detecting method of wheat light Tilletia foetida, is characterized in that, comprise the steps:
(1) sample dyeing: dye testing sample dyeing liquor I 10-30 minute, then with 10 μ g/mLWGA-AF488 dyeing 10-30 minute, finally uses the 10 × PBS of pH7.4 to rinse 3-5 time;
Institute's dyeing liquor I is that 5 μ g/mLPropidiumidodide and 0.02%Tween20 mix with volume ratio 1:1;
(2) microscopy: observe under the sample after dyeing is placed in laser confocal microscope, the parameter of employing is as follows:
Propidiumidodide excites green wavelength to be 561nm, and wavelength of transmitted light is 590 ~ 640nm; WGA-AF488 excites blue light wavelength to be 488nm, and wavelength of transmitted light is 500 ~ 520nm,
If there is green mycelia under microscope, illustrate that testing sample contains wheat light Tilletia foetida.
2. method according to claim 1, is characterized in that, described testing sample is the tissue taking from wheat aging time root, stem stalk portion or blade part.
3. method according to claim 1 and 2, is characterized in that, adopts ethanol to fix before sample dyeing.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510909600.4A CN105548091A (en) | 2015-11-19 | 2015-12-10 | Laser confocal microscope detection method for Tilletia foetida |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2015108086630 | 2015-11-19 | ||
| CN201510808663 | 2015-11-19 | ||
| CN201510909600.4A CN105548091A (en) | 2015-11-19 | 2015-12-10 | Laser confocal microscope detection method for Tilletia foetida |
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| CN105548091A true CN105548091A (en) | 2016-05-04 |
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| CN201510909600.4A Pending CN105548091A (en) | 2015-11-19 | 2015-12-10 | Laser confocal microscope detection method for Tilletia foetida |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106990076A (en) * | 2017-05-05 | 2017-07-28 | 中国农业科学院植物保护研究所 | The method of wheat ovary is infected for observing T contraversa |
| CN110132912A (en) * | 2019-04-16 | 2019-08-16 | 中国农业科学院植物保护研究所 | A method of identifying T. contraversa teleutospore and Wheat canopy teleutospore |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105043842A (en) * | 2015-07-10 | 2015-11-11 | 中国农业科学院植物保护研究所 | Dyeing method for determining wheat smut (Tilletia contrversasa Kuhn) infected wheat tissue |
| CN105039491A (en) * | 2015-07-10 | 2015-11-11 | 中国农业科学院植物保护研究所 | Method for identifying teliospores of Tilletia controversa Kuhn and Tilletia foetida |
-
2015
- 2015-12-10 CN CN201510909600.4A patent/CN105548091A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105043842A (en) * | 2015-07-10 | 2015-11-11 | 中国农业科学院植物保护研究所 | Dyeing method for determining wheat smut (Tilletia contrversasa Kuhn) infected wheat tissue |
| CN105039491A (en) * | 2015-07-10 | 2015-11-11 | 中国农业科学院植物保护研究所 | Method for identifying teliospores of Tilletia controversa Kuhn and Tilletia foetida |
Non-Patent Citations (3)
| Title |
|---|
| M. ZHANGA, ET AL.: "Identification of a Specific SCAR Marker for Detection of Tilletia foetida (Wall) Liro Pathogen of Wheat", 《RUSSIAN JOURNAL OF GENETICS》 * |
| STEFFI TREITSCHKE,ET AL.: "The Myosin Motor Domain of Fungal Chitin Synthase V Is Dispensable for vesicle Motility but Required for Virulence of the Maize Pathogen Ustilago maydis", 《THE PLANT CELL》 * |
| 王宏毅 等: "应用激光共聚焦显微扫描术研究小麦矮腥黑穗病菌和小麦网腥黑穗病菌冬孢子的自发荧光特性", 《植物检疫》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106990076A (en) * | 2017-05-05 | 2017-07-28 | 中国农业科学院植物保护研究所 | The method of wheat ovary is infected for observing T contraversa |
| CN110132912A (en) * | 2019-04-16 | 2019-08-16 | 中国农业科学院植物保护研究所 | A method of identifying T. contraversa teleutospore and Wheat canopy teleutospore |
| CN110132912B (en) * | 2019-04-16 | 2021-01-22 | 中国农业科学院植物保护研究所 | Method for identifying Tilletia controversa Kuhn spores and Tilletia controversa Kuhn spores |
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Application publication date: 20160504 |