CN105039491A - Method for identifying teliospores of Tilletia controversa Kuhn and Tilletia foetida - Google Patents

Abstract

The invention provides a method for identifying teliospores of Tilletia controversa Kuhn and Tilletia foetida, and relates to the technical field of cell biology. The method comprises the following steps: (1) tabletting: respectively smearing teliospores of the Tilletia controversa Kuhn and Tilletia foetida, drying, dropwise adding oil lens oil to a microscopic examination part, and then adding a cover glass; (2) and (4) observation: placing the prepared slide under a laser confocal scanning microscope, selecting a proper scanning mode and scanning parameters for scanning observation and photographing; the scanning mode is an xyz scanning mode; the scanning parameters include excitation light wavelength and emission light wavelength; the excitation wavelength of the Tilletia controversa Kuhn is 488nm, and the emission wavelength of the Tilletia controversa Kuhn is 510-550 nm; the excitation wavelength of the Tilletia foetida is 530nm, and the emission wavelength is 550-600 nm; or the wavelength of the exciting light is 488nm, and the wavelength of the emitting light is 510-550 nm. By adopting the method, the teliospores of the Tilletia controversa Kuhn and the Tilletia foetida can be rapidly identified.

Description

For differentiating the method for the winter spore of T. contraversa and Wheat canopy

Technical field

The present invention relates to technical field of cell biology, being specifically related to one utilizes laser scanning co-focusing microscope (Confocallaserscanningmicroscope, CLSM) to differentiate the method for the winter spore of T. contraversa and Wheat canopy.

Background technology

T contraversa (TilletiacontroversaK ü hn, TCK) be the short bunt (Wheatdwarfbuntdisease of wheat caused, DB) a kind of systemic infection venereal disease evil is one of domestic and international important quarantine disease endangering maximum, extremely difficult control in wheat class smut disease.This germ and its allied species T. caries (T.cariesTul., TCT), Wheat canopy (T.foeyida., TFL) very similar on morphology, be not easily distinguishable, this brings quite difficulty to the TCK of black spot from America wheat quarantine.At beginning of the nineties late 1980s, make great efforts for many years and consult through Sino-U.S. relevant expert, finally proposing TCK winter spore according to Stoekwell in 1986 and have special autofluorescence characteristic, the TCK solved in black spot from America wheat identifies problem.

Summary of the invention

The present invention attempts application laser co-focusing microscan art (CLSM), has inquired into the autofluorescence characteristic of TCK and TFL winter spore, to overcome the shortcoming that current applied optics microscopic examination TCK exists, improves import wheat TCK quarantine level.

Technical scheme of the present invention is as follows:

1, for differentiating the method for the winter spore of T. contraversa and Wheat canopy, it is characterized in that, comprising the steps:

(1) film-making: the winter spore of T. contraversa and Wheat canopy is distinguished smear, after drying, drips oily lens head oil, then adds cover glass at microscopy position;

(2) observation: under the slide made is positioned over laser confocal scanning microscope, chooses suitable scan pattern and sweep parameter is carried out scanning and takes pictures;

Described scan pattern is xyz scan pattern;

Described sweep parameter comprises excitation wavelength and wavelength of transmitted light;

Wherein, the excitation wavelength of T. contraversa is 488nm, and wavelength of transmitted light is 510 ~ 550nm;

The excitation wavelength of Wheat canopy is 530nm, and wavelength of transmitted light is 550 ~ 600nm; Or excitation wavelength is 488nm, wavelength of transmitted light is 510 ~ 550nm.

2, method according to claim 1, the excitation wavelength of Wheat canopy is 488nm, and wavelength of transmitted light is 510 ~ 550nm.

Apply the autofluorescence characteristic of Confocal laser scanning microscopy (CLSM) to dwarf bunt of wheat (TCK) and Wheat canopy (TFL) winter spore to be herein studied.Research shows: in these two kinds of winter spores, they all contain autofluorescence material separately, and TCK (exciting light 488nm/ utilizing emitted light 510 ~ 550nm); Under TFL (exciting light 530nm/ utilizing emitted light 550 ~ 600nm, exciting light 488nm/ utilizing emitted light 510 ~ 550nm, but exciting light 488nm/ utilizing emitted light 510 ~ 550nm is better), fluorescence distribution is even, and general form is clear; But layer is cut scanning discovery autofluorescence and be there is notable difference in the spatial distribution of these two kinds of winter spores: the autofluorescence material of TCK winter spore is mainly distributed on sclerine and murus, and distribution is few in protoplasma; The autofluorescence material of TFL winter spore is mainly distributed in sporoderm, and in protoplasma, distribution is few.CLSM is in actual TCK testing in application, can overcome the shortcoming that detection repeatability is poor, reliability is little that fluorescent microscope exists, improve the state of the art of TCK inspection and quarantine.

Accompanying drawing explanation

Fig. 1 is that in wheat, the autofluorescence successive layers of TCK and TFL winter spore under LeicaTCSSP5CLSM system 488nm excites cuts scanned picture: A.TCK winter spore; B.TFL winter spore (1bar=5 μm).

Embodiment

Further describe goods of the present invention below in conjunction with specific embodiment, but do not limit the scope of the invention with this.If no special instructions, all commercially available acquisition of following adopted reagent and material, the method adopted is routine operation.

the source of biomaterial

T. contraversa, Wheat canopy mycoceicidum are collected from applicant laboratory by the wheat plant infected.

key instrument equipment

LeicaTCSSP5CLSM system

Embodiment 1. utilizes the method for the invention to differentiate TCK and TFL winter spore

1 materials and methods

1.1 for examination material

For examination pathogenic bacteria: each 3 strains of T. contraversa, Wheat canopy mycoceicidum.

Slide makes: the method provided by standard GB/T/T18085-2000 makes winter spore smear, after drying, add the oily lens head oil of 1 unstressed configuration material at microscopy position, add cover glass, make the slide detected for confocal laser scanning microscope, CLSM autofluorescence.

CLSM system: adopt high performance LeicaTCSSP8, whole system primarily of 1 be inverted fluorescent phase-contrast microscope, 1 group of workstation form multiple-wavelength laser and the operation of 1 bench control system by semiconductor laser, argon ion gas laser, He-Ne ion gas laser is formed.

1.2 the successively scanning of autofluorescence (xyz scanning) observational technique

Choose xyz scan pattern, select the corresponding program that excitation wavelength 488nm/530nm exciting light and system have set, excite TCK and TFL winter spore respectively, carry out optical section, z-axis layer cuts scanning analysis.

1.3 image analysis

According to the image that test-results obtains, observe autofluorescence TCK and TFL winter spore autofluorescence characteristic separately in the respective space distribution situation of TCK and TFL winter spore and wheat.

2 results

The xyz scanning of 2.1TCK and TFL winter spore

Carry out optical section to TCK and TFL winter spore respectively with 488nm/530nm wavelength exciting light, z-axis layer cuts scanning, obtains the continuous optical section scan image of 30 different aspects separately.

2.2TCK and TFL winter spore autofluorescence characteristic separately

Through scanning display to xy λ and xyz of TCK and TCT winter spore in wheat, in two kinds of winter spores, they all contain autofluorescence material separately, and can under 488nm wavelength excitation, corresponding utilizing emitted light is obtained in 510 ~ 550nm scope, fluorescence Light distribation is even, and general form is clear, can be observed the clear structure of its different aspects under light field simultaneously.But layer is cut scanning discovery autofluorescence and be there is notable difference in the spatial distribution of these two kinds of winter spores: the autofluorescence material of TCK winter spore is mainly distributed on sclerine and murus, and distribution is few in protoplasma; The autofluorescence material of TFL winter spore is mainly distributed in sporoderm, and in protoplasma, distribution is few.

3 discuss

First Application CLSM technique means herein, scanned by laser co-focusing xyz, describe these two kinds of winter spores of TCK and TFL autofluorescence characteristic separately, further disclose as where common epifluorescence microscope (excites filter disc 485nm, barrier filter 520nm) under can observe that TCK winter spore reticulate pattern is in yellow or yellow-green colour, or in fringing phenomenon (only reticulate pattern edge tool fluorescence).And in the research of TFL, the general ordinary optical microscope that uses is distinguished, the common bunt caused by wheat light Tilletia foetida and Tilletia caries except minority be subject to the conidial cell wall of gene regulating different except, these two kinds of fungies are closely similar.It is individual that their hybridization can produce a series of morphologic variation, now observe the natural variation individuality embracing all transition between sub-form between light Tilletia foetida and net fungus tilletia winter, modal intermediate type possibility representative species species hybrid, general Morphological Identification method is difficult to them to differentiate.Adopt CLSM technology, accurately, fast can differentiate TFL.

The Confocal laser scanning microscopy that the basis of fluorescence microscopic analysis technology grows up is the modern technique in a kind of modern times, owing to using laser as light source, by spot scan and highly sensitive reception, and carry out digital imagery, its principle and imaging mode and the former have essential distinction, the suitable especially observation to weak autofluorescence of the latter.Compare with original fluorescent microscopy in the actual testing process of TCK, use high performance confocal laser scanning microscope, CLSM, can obtain Digital scan image clearly, reproducible, the reliability of result is better.Therefore along with confocal laser scanning microscope, CLSM is constantly popularized in inspection and quarantine system, in TCK testing, applying CLSM, is the qualitative leap of inspection and quarantine scientific and technological level.

Claims (2)

1. for differentiating the method for the winter spore of T. contraversa and Wheat canopy, it is characterized in that, comprising the steps:

(1) film-making: the winter spore of T. contraversa and Wheat canopy is distinguished smear, after drying, drips oily lens head oil, then adds cover glass at microscopy position;

(2) observation: under the slide made is positioned over laser confocal scanning microscope, chooses suitable scan pattern and sweep parameter is carried out scanning and takes pictures;

Described scan pattern is xyz scan pattern;

Described sweep parameter comprises excitation wavelength and wavelength of transmitted light;

Wherein, the excitation wavelength of T. contraversa is 488nm, and wavelength of transmitted light is 510 ~ 550nm;

The excitation wavelength of Wheat canopy is 530nm, and wavelength of transmitted light is 550 ~ 600nm; Or excitation wavelength is 488nm, wavelength of transmitted light is 510 ~ 550nm.

2. method according to claim 1, the excitation wavelength of Wheat canopy is 488nm, and wavelength of transmitted light is 510 ~ 550nm.