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Single spore isolation method of Blumeria graminis

A technology for the isolation of wheat powdery mildew bacteria and single spores, applied in the direction of microorganism-based methods, separation of microorganisms, biochemical equipment and methods, etc. "Bacterial colonies and other problems, to achieve the effect of simple and easy method

Inactive Publication Date: 2017-12-08
INST OF PLANT PROTECTION & SOIL FERTILIZER HUBEI ACAD OF AGRI SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] Since wheat powdery mildew is an obligate parasite, it needs to obtain necessary nutrients from living host tissues and cannot grow on synthetic media, the above method is not suitable for the isolation of single spores of wheat powdery mildew
And in the existing literature, it is generally to obtain its "single spore heap isolate", this kind of single spore heap visible to the naked eye cannot discharge the "single" colony formed by the germination of two or more single spores, thus affecting Pathogenic mechanism, single spore sequencing, etc.

Method used

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  • Single spore isolation method of Blumeria graminis
  • Single spore isolation method of Blumeria graminis
  • Single spore isolation method of Blumeria graminis

Examples

Experimental program
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Effect test

Embodiment 1

[0022] The monospore isolation method of embodiment 1 wheat powdery mildew

[0023] (1) Put the wheat powdery mildew diseased leaves collected in the field in an ice box, blow off the old spores on the surface, put them on the agar fresh-keeping medium containing benzimidazole (50mg / L), 17°C, 18h light, Cultured in a 6-hour dark light incubator to reproduce fresh conidia.

[0024] (2) Collect the fresh conidia of step (1) on the ultra-clean workbench and collect them in a centrifuge tube, and use electronic fluorinated liquid C5-18-perfluoroalkane (FC-40) to adjust the concentration to 10 2 spores / ml.

[0025] (3) Draw the spore suspension of 100 μl of step (2), and apply it evenly on the 1.5% agarose plate with a spreader, then use the colony puncher (diameter 3mm) to beat the agarose plate into a uniform bacterial dish, See figure 1 .

[0026] (4) find oval-shaped single spores on the agarose plate of step (3) with a dissecting microscope, that is, single conidia of powd...

Embodiment 2

[0030] Embodiment 2: the efficiency of picking single spores with different spore concentrations

[0031] Cut the first leaf of the pre-prepared susceptible variety "Jinmai 47" to a length of 3 cm, select 10 leaves and put them on the agar fresh-keeping medium containing benzimidazole (50 mg / L), and put them on the ultra-clean bench. Collect the fresh conidia that have been propagated in advance into a sterile 5mL centrifuge tube, draw 4mL of C5-18-perfluoroalkane (FC-40) solution into the centrifuge tube containing spore powder, and make an inoculum suspension. Use a Vortex shaker to oscillate for 30s to suspend the spores evenly in the solution, draw 10 μL of the suspension and adjust the spore concentration to 10 with a hemocytometer under the microscope 5 、10 4 、10 3 、10 2 and 10 1 1 / mL, take 100ul of spore suspension, and evenly spread it on a 1.5% agarose plate with a spreader. After the fluoride solution evaporates, randomly punch holes on the plate. Find the mono...

Embodiment 3

[0033] Example 3: Screening of inoculation methods

[0034] According to embodiment 1, inoculate the detached leaf section of wheat, adopt upside-down respectively, two kinds of modes of inoculating with bacteria dish ( figure 2 ), see the result image 3 . Depend on image 3 It can be seen that the fungus discs upside down (the side containing spores) can be successfully morbidity, and the upside down (the side without spores) bacteria discs cannot form colonies, and the inoculation is unsuccessful. Therefore, the upside down method is all used in this single spore isolation technology system.

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Abstract

The invention provides a single spore isolation method of Blumeria graminis, and belongs to the technical field of plant pathogenic obligate fungus parasite isolation method. The single spore isolation method comprises following steps: 1, field Blumeria graminis infected samples are collected, and indoor spore production is carried out so as to obtain samples for further processing; 2, an electrically fluorinated liquid is adopted to prepare a spore suspension liquid; 3, suspension liquid coating is carried out to prepare single spore fungus dishes; 4, the single spore fungus dishes are selected and are inverted placed on wheat detached leaf segments; 5, the inoculated leaf segments are delivered into an illumination incubator for 10 days of culturing so as to obtain pure Blumeria graminis fungus colonies. The single spore isolation method is simple and convenient, and is capable of obtaining pure cultured Blumeria graminis at a relatively high success rate via separation, and solving a problem that conventional fungus separation method is not suitable for Blumeria graminis single spore isolation.

Description

technical field [0001] The invention belongs to the application technical field of a method for isolating plant pathogenic obligate parasites, and in particular relates to a method for isolating a single spore of wheat powdery mildew. Background technique [0002] The single spore isolation of pathogenic bacteria is the basic technology of fungal disease research, and it is widely used in the genetic variation, physiological and biochemical characteristics of fungi, as well as the purification and preservation of strains. Currently commonly used single spore isolation methods are: agar plate dilution method (Fang Zhongda. Plant Disease Research Methods (3rd Edition). Beijing: China Agricultural Press, 1998:137-139. Choi Y W, Hyde K D, HoW H.Single spore isolation of fungi.Fungal Diversity,1999,3:29-38.); imprint pipetting single spore isolation method (Zhao Ning. imprint pipetting single spore isolation method. Plant Protection, 1988,14(1):48-49 .); small slide single spore...

Claims

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Application Information

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IPC IPC(8): C12N1/14C12N1/02C12R1/645
CPCC12N1/02C12N1/14
Inventor 龚双军杨立军薛敏峰向礼波史文琦张学江曾凡松汪华喻大昭
Owner INST OF PLANT PROTECTION & SOIL FERTILIZER HUBEI ACAD OF AGRI SCI
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