CN106033058A - Histology observation method of rot pathogen in and out of branches of apple trees and pear trees - Google Patents
Histology observation method of rot pathogen in and out of branches of apple trees and pear trees Download PDFInfo
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Abstract
The invention discloses a histology observation method of rot pathogen in and out of branches of apple trees and pear trees, and relates to the field of observation of pathogen in plant tissues. The method is suitable for observation of fresh samples or samples that have been fixed in the early period. According to the method, a sample is sliced by a slicing machine or hands, the thickness of slices is not more than 100 micrometers; the tissue slices are processed by a sodium hypochlorite solution (1 to 15%) so that the tissue slices become transparent; then the tissue slices are soaked in a potassium hydroxide solution (1 to 30%) or a sodium hydroxide solution (1 to 30%) for 3 to 5 minutes; finally the tissue slices are dyed for 1 to 3 times by an aniline blue/methyl blue water solution (0.1 to 3%), dyed slices are triggered by UV rays under a fluorescence microscope, and the hyphae give off yellow-green fluorescence. The method has the advantages that the slice preparation time is short, the method is convenient and does not need special equipment and reagent; all hyphae in tissue slices can be dyed, thus the tissue slices are different from the host tissues; after UV excitation, the tissues are clear and visible, the shortage that in the prior art, the latent hyphae, latent parts, and overall distribution of rot pathogen cannot be directly observed is overcome; and a novel technology is provided for the researches on rot disease.
Description
Technical field
The present invention relates to the observation technology of pathogen in plant tissue, be particularly suited for utilizing the mycelia of pathogen in the observation of plant tissue of fluorescence microscopy border and in in-house parasitic site and distribution.
Background technology
Canker is the important disease of Fructus Mali pumilae and pear tree, mainly causes dead arm, Si Shuhehui garden.On apple tree, canker is the first big disease, and is difficult to prevent and treat, and is considered " cancer " of apple tree.From the fifties in last century, apple tree canker the most popular 4 times in China, almost destroy all apple trees newly planted, currently face the 5th pandemic threat.Canker is also the important disease on pear tree, and in the susceptible variety such as Pyrus communis, Kuerle, Xinjiang pears, harm is serious.
Rotten pathogenic bacteria has latent infection phenomenon.Research at present is thought, rotten pathogenic bacteria mainly infects from positions such as cutting saw kerf, wound, hole skin, eye, can not cause a disease after infection process at once, but hide extremely organize in wound, the position such as branch top layer is extremely organized, xylem, when tree vigo(u)r is weak, or after tree body suffers freeze injury, the pathogenic bacteria that hides could extend causes a disease.The latent infection of pathogenic bacteria result in the uncertainty of canker morbidity, and researcher is difficult to the time of origin according to disease and occurrence degree deduces time of infection and the amount of infecting in a certain period of pathogenic bacteria, thus restricts the research of canker.At present, regularity of infection, morbidity to canker are the most extremely limited with the understanding of the condition of prevalence, the mechanism of causing a disease etc. of pathogenic bacteria, it is more difficult to propose to prevent and treat effectively arranging of canker, therefore, the important disease that canker is always on apple tree.
Inside and outside branch, the observation technology of rotten pathogenic bacteria is research and the guardian technique of understanding canker pests occurrence rule.If the rotten pathogenic bacteria hiding in host tissue can be observed, just can pass through the infection condition of the technique study pathogenic bacterias such as artificial vaccination, infect position and pathogenic conditions；Understand latency site and the growth extended dynamic of pathogenic bacteria of pathogenic bacteria；Understanding pathogenic bacteria tissue in hide reason and mechanism of causing a disease mechanism；The antibacterial that assessment the is used prevention effect to disease.
Traditional tissue microtechnique is the main method of pathogenic bacteria in current tissues observed.Traditional tissue slice, such as paraffin section, mainly by dyestuffs such as aniline blue, methyl blue, bent profit benzene indigo plants, by counter for mycelia in plant tissue blueness of dying, is examined under a microscope by transmitted light source.But, traditional colouring method poor specificity, under the microscope, it is difficult to offer an explanation pathogenic bacteria mycelia and host tissue, the obvious mycelia of morphological characteristic can only be offered an explanation, it is more difficult to observe mycelia in in-house overall distribution situation, and prior paraffin tabletting technology complex steps, cycle is long, it is impossible to meet the observation requirements of a large amount of sample.
Electron Microscopic Observation also can observe the canker mycelia in host tissue.Ultrathin section is mainly used in observing the ultrastructure posting tissue and mycelia, and the requirement to sample is high, and microsection manufacture process is complicated, and needs to thank ultramicroscope, and general laboratory condition has been difficult to, and more cannot be used for the observation of a large amount of sample.
Whether Protocols in Molecular Biology detection exists rotten pathogenic bacteria in also detecting host tissue.Molecular biology method mainly by specific probe, the DNA fragmentation of amplification host tissue pathogenic bacteria, according to the presence or absence of amplification of DNA fragments, infers whether host exists rotten pathogenic bacteria in organizing.The greatest drawback of molecular Biological Detection is the accurate location being difficult to observe pathogenic bacteria intuitively in host tissue.
Summary of the invention
Defect for prior art, it is an object of the invention to provide a set of rotten pathogenic bacteria mycelia latency site and the transparency of organization of overall distribution, dyeing and observational technique inside and outside observation Fructus Mali pumilae directly perceived and pear tree branch, provide a set of succinctly and intuitively observation technology for canker occurrence regularity, the morbidity condition of prevalence, mechanism of causing a disease, pharmacodynamic assessment and field Pathogen detection.
The following technical scheme that this invention uses: the specimen material section to observation with freezing microtome or blade, it is thus achieved that the thickness tissue slice less than 100 microns；With sodium hypochlorite, tissue slice is carried out transparent processing, to increase the light transmittance of tissue slice；After tissue slice is transparent, then soaking 3 ~ 5 minutes with highly basic, it is therefore an objective to increase the permeability to dyestuff of observation material, in making tissue, all mycelia can be colored agent dyeing；Tissue slice carries out specificity fluorescent dyeing after highly basic processes again, makes whole mycelia to colour；Dyestuff in the tissue slice ultraviolet excitation mycelia made, makes dyestuff send yellow-green fluorescence, the most just can clearly tissue visualization section in all mycelia.
The concrete step making tissue slice and histology's observation is poly-as follows:
(1) sampling: Fructus Mali pumilae that clip is fresh or pear tree branch, cuts position to be observed, accomplishes 3 ~ 5 millimeters of square piece of tissue with blade, be directly used in section；Or proceed to fixative preserves, for section later；
( 2 )Section: cut into slices with freezing microtome, or ordinary blade freehand section, it is thus achieved that the thickness tissue slice less than 100 microns；Optimal cutting layer thickness is 10 ~ 20 microns.Tissue slice is proceeded on microscope slide, or in little culture dish, carries out transparent and dyeing；
( 3 )Sodium hypochlorite is transparent: takes chlorinty 1 ~ 15% liquor natrii hypochloritis 2 ~ 3 with dropper, is added drop-wise on tissue slice, section is carried out transparent processing, until section is fully transparent.Clearing time is unsuitable long.Rinse 2 ~ 3 times with distilled water after transparent.Clearing time is 1 ~ 20 minute, and the concentration of clearing time sodium chlorate successively, slice thickness, the quality of section determine.Transparency of organization commonly uses the liquor natrii hypochloritis that chlorinty is 10%；
( 4) highly basic process: prepare the aqueous solution of 1% ~ 30% with potassium hydroxide or sodium hydroxide, take strong base solution with dropper and be added drop-wise on tissue slice, soak 3 ~ 5 minutes, rinse 2 ~ 3 times with distilled water after process.The concentration of conventional strong base solution is 10%；
( 5 )Hallmark dyeing: be configured to the aqueous solution of 0.1% ~ 3% with aniline blue, or methyl blue preparation 0.1% ~ 5% aqueous solution makees stain, take 2 ~ 3 stains with dropper to be added drop-wise on tissue slice, dye 3 ~ 5 minutes, rinse 1 time with distilled water, continuous dyeing 2 ~ 3 times, until dyeing liquor no longer becomes yellow green.Conventional dyeing liquor is the aniline blue aqueous solution of 0.2%；
( 6 )Make interim slide: become the aqueous solution of 20% ~ 80% to make floating supporting agent with glycerol, proceed on microscope slide by the tissue slice of dyeing, put whole dose, drip 1 ~ 2 floating supporting agent, covered, make interim slide.Conventional floating supporting agent is the glycerine water solution of 50%；
( 7 )Upper sem observation: be placed under fluorescence microscope by interim slide, with ultraviolet excitation, ultraviolet wavelength range 315 ~ 400nm, in making tissue, mycelia can send strong yellow-green fluorescence, the most just can clearly observe mycelia.
It is an advantage of the current invention that: application this invention technology just can make a set of tissue slice in 20 ~ 30 minutes, it is not necessary to special instrument or equipment, freehand section also can reach ideal effect, and agents useful for same is all laboratory conventional reagent.
One of application example, wood wants the observation of matter portion mycelia: takes the rotten pathogenic bacteria xylem from new clip inoculation Fuji apple annotinous branch, takes the branch inoculating 2 centimetres, clip bottom, be cut into the tissue slice of thickness 30 microns with freezing microtome after 3 months；Being 10% sodium hypochlorite transparent 5 minutes with chlorinity, distilled water rinses 2 times；Potassium hydroxide aqueous solution with 10% soaks 3 minutes, and distilled water rinses 2 times；Dye 3 times with 0.2% aniline blue aqueous solution, each 2 minutes；Make floating supporting agent with 50% glycerine water solution and make interim slide；With wavelength 365 nm ultraviolet excitation under fluorescence microscope (Lycra DM2500), having the mycelia of a large amount of green glow that turns to be yellow in the object lens of 20 times can observe tissue slice, mycelial structure is obvious, as shown in Fig. 1 and Fig. 2；Under 60 times of object lens, the morphosis that vessel growth extends can be passed, as shown in Figure 3 by mycelia visible in detail.
The two of application example, wound extremely organizes interior mycelia to observe: in JIUYUE, 2014, with little hacksaw by 1 year raw branch sawing injury of Fuji apple, and spray spore suspension inoculation immediately, with plastic foil moisturizing 24 hours after inoculation；The wound sections observation of inoculated pathogenic bacteria, slice thickness 50 microns is taken after 3 months.Transparent, highly basic processes, dyeing, film-making and observational technique be with application example one.Under 5 times of object lens, can observe to inoculation wound formed phellem layer, phellem layer protects live body cortical tissue, the tissue necrosis that phellem layer isolates, and phellem layer sends strong blue-fluorescence after ultraviolet excitation, micro-micro-under high-visible；The fluorescence that dead tissue outside phellem layer finds is the most weak, but has a large amount of mycelia in dead tissue, sends strong yellow-green fluorescence, high-visible, as shown in Figure 4；Under the object lens of 20 times, it is seen that typical mycelial structure, as shown in Figure 5.
Accompanying drawing illustrates: Fig. 1 is the longitudinal section of the apple branch xylem of catching an illness under 20 times of object lens, and after ultraviolet excitation, in xylem, mycelial structure is obvious, and turn to be yellow green fluorescence.Fig. 2 is the wooden cross section of apple branch of catching an illness under 20 times of materials, and the mycelial structure in conduit is obvious, and mycelia is high-visible.Fig. 3 is the mycelia in apple branch xylem that catches an illness under 60 times of object lens, and mycelia can enter another conduit from penetrating catheter wall.Fig. 4 is the wound under 5 times of object lens, and 3 months wound ages, time injured, spray inoculation crosses the spore of rotten pathogenic bacteria, and this wound has formed phellem layer, finds strong blue-fluorescence after ultraviolet excitation, and the dead in-house canker mycelia of wound sends yellow-green fluorescence.Fig. 5 is the dead in-house rotten pathogenic bacteria mycelia of wound under 20 times of object lens, and mycelial structure is high-visible, phellem layer checkmate tissue and living tissue isolation.
Claims (2)
- Rotten pathogenic bacteria histology's observation procedure inside and outside the most a set of Fructus Mali pumilae, pear tree branch, it is characterised in that: being suitable for observing fresh and early stage and fix sample, detailed process comprises section, transparent, highly basic immersion, dyeing, film-making, six steps of upper sem observation；Described fresh sample is from apple tree or the fresh braches of pear tree clip, cuts position to be observed, accomplishes 3 ~ 5 millimeters of square piece of tissue；It is early stage clip branch from apple tree or pear tree that described early stage fixes sample, cuts position to be observed, accomplishes 3 ~ 5 millimeters of square piece of tissue, is placed in fixative the sample of fixing preservation；Described section is that sample to be observed is carried out tissue slice, and tissue slice can use freezing microtome to cut into slices, it is also possible to ordinary blade carries out free-hand cutting and levies, and slice thickness is less than 100 microns；Section optimum thickness is 10 ~ 20 microns；Described transparent be to drip 2 ~ 3 transparent liquid on tissue sections, process 3 ~ 10 minutes, until tissue slice is fully transparent；Transparency of organization can be carried out on microscope slide, it is also possible to carries out in other small containers, transparent liquid sodium hypochlorite (NaClO3) preparation, chlorinty is 1 ~ 15%, and conventional transparent liquid chlorinty is 10%, rinses 2 ~ 3 times with distilled water after transparency of organization；It is 2 ~ 3 strong alkali aqueous solutions of dropping on transparent tissue slice that described highly basic soaks, and soaks 3 ~ 5 minutes；Strong alkali aqueous solution potassium hydroxide (KOH) or sodium hydroxide (NaOH) are prepared, and concentration is 1% ~ 30%, and typical concentrations is 10%；After highly basic soaks, rinse 2 ~ 3 times with distilled water；Described dyeing is to drip 2 ~ 3 aniline blues on tissue sections, or methyl blue dyeing liquor, dyes 2 ~ 3 minutes, rinses 1 time with distilled water, dye altogether 2 ~ 3 times after dyeing, until dyeing liquor no longer variable color；Aniline blue aqueous solution aniline blue (Aniline Blue) preparation, concentration is 0.1% ~ 3%, and typical concentrations is 0.2%；Methyl blue aqueous solution methyl blue (Methyl blue) is prepared, and concentration is 0.1% ~ 5%, and typical concentrations is 0.5%；Conventional stain is 0.2% aniline blue aqueous solution；Described film-making is that the tissue slice after dyeing proceeds on microscope slide, puts whole dose, drips 2 ~ 3 glycerine water solutions and makees floating supporting agents, makes interim slide；The concentration of glycerine water solution is 20% ~ 80%, and typical concentrations is 50%；Described upper sem observation is the interim slide made, and is placed under fluorescence microscope, after ultraviolet excitation, observes the mycelia with yellow-green fluorescence, and the wavelength of ultraviolet light is between 315 ~ 400nm.
- 2. rotten pathogenic bacteria histology's observation procedure inside and outside a set of Fructus Mali pumilae described in claim 1, pear tree branch, is characterized in that: before tissue section strain, need to process and highly basic soaks through transparency of organization.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2017177870A1 (en) * | 2016-04-13 | 2017-10-19 | 武汉沃亿生物有限公司 | Automated batch method for staining and making tissue slices |
| CN107446985A (en) * | 2017-08-30 | 2017-12-08 | 李保华 | A set of Apple top layer colonizes and infected the rapid examination method of fungi |
| CN113109327A (en) * | 2021-03-09 | 2021-07-13 | 杭州市林业科学研究院 | Prediction method of dry rot of hickory nut |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101071102A (en) * | 2007-04-28 | 2007-11-14 | 天津出入境检验检疫局动植物与食品检测中心 | Reed fescue and lolium perenne endophytic fungus real-time fluorescent PCR detecting method |
| CN102495037A (en) * | 2011-12-07 | 2012-06-13 | 南京农业大学 | Fluorescent tracing method of assimilation substance unloading path of phloem in pear fruit |
| US20130137119A1 (en) * | 2011-11-30 | 2013-05-30 | The United States Of America, As Represented By The Secretary, Department Of Health And Human | Detection of bacterial contamination in a sample |
| CN103323441A (en) * | 2013-06-14 | 2013-09-25 | 中南林业科技大学 | Fluorescence microscopy method for rapidly and efficiently observing camellia plant pollen tube |
-
2015
- 2015-03-11 CN CN201510105934.6A patent/CN106033058A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101071102A (en) * | 2007-04-28 | 2007-11-14 | 天津出入境检验检疫局动植物与食品检测中心 | Reed fescue and lolium perenne endophytic fungus real-time fluorescent PCR detecting method |
| US20130137119A1 (en) * | 2011-11-30 | 2013-05-30 | The United States Of America, As Represented By The Secretary, Department Of Health And Human | Detection of bacterial contamination in a sample |
| CN102495037A (en) * | 2011-12-07 | 2012-06-13 | 南京农业大学 | Fluorescent tracing method of assimilation substance unloading path of phloem in pear fruit |
| CN103323441A (en) * | 2013-06-14 | 2013-09-25 | 中南林业科技大学 | Fluorescence microscopy method for rapidly and efficiently observing camellia plant pollen tube |
Non-Patent Citations (3)
| Title |
|---|
| 张高雷等: "苹果轮纹病瘤组织形态研究", 《植物病理学报》 * |
| 李保华等: "苹果黑星病菌在叶组织内的扩展形态", 《莱阳农学报》 * |
| 王萍等: "4 种染色方法对甜瓜白粉病菌染色效果的观察比较", 《菌物学报》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2017177870A1 (en) * | 2016-04-13 | 2017-10-19 | 武汉沃亿生物有限公司 | Automated batch method for staining and making tissue slices |
| CN107446985A (en) * | 2017-08-30 | 2017-12-08 | 李保华 | A set of Apple top layer colonizes and infected the rapid examination method of fungi |
| CN113109327A (en) * | 2021-03-09 | 2021-07-13 | 杭州市林业科学研究院 | Prediction method of dry rot of hickory nut |
| CN113109327B (en) * | 2021-03-09 | 2023-11-17 | 杭州市林业科学研究院 | Method for predicting dry rot of hickory |
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Application publication date: 20161019 |