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Method for separating plasmodiophora brassicae monospore on basis of methylene blue agarose method

A technique for the separation of Plasmodium brassicae and its isolation method, which is applied in the field of single-spore isolation of Plasmodium brassicae, can solve problems such as difficult picking and fuzzy targets of Pyrophylla brassicae single-spore, and improve efficiency, speed and The effect of accuracy

Active Publication Date: 2019-07-19
INST OF VEGETABLE & FLOWERS CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Using the agarose method instead of the water agar method can effectively avoid the problems of air bubbles or impurities in the water agar; adding methylene blue staining solution to the agarose, the background of the agarose is slightly blue, while the dormant spores of Plasmodium brassicae are colorless , it is easier to distinguish single spores from the agarose background, which solves the shortcomings of P. brassica single spores, such as vague targets and difficult picking

Method used

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  • Method for separating plasmodiophora brassicae monospore on basis of methylene blue agarose method
  • Method for separating plasmodiophora brassicae monospore on basis of methylene blue agarose method
  • Method for separating plasmodiophora brassicae monospore on basis of methylene blue agarose method

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Experimental program
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Effect test

Embodiment 1

[0034] Embodiment 1, the screening of optimal concentration of agarose

[0035] Dip the sterilized glass slide in different concentrations of agarose solution to form a gel on the surface of the slide. Then take 5 μL of the diluted dormant spore suspension and drop it on the surface of the agarose gel layer. Observe under a microscope, cut about 1 mm agarose gel layer containing the staining solution of single spore with a scalpel. Screen out the optimal agar block concentration. The concentration of the agarose solution was set as 1%, 1.2%, 1.3%, 1.5%, 1.8%, 2%, 2.5% and 3% for a total of 8 treatments, and the transparency, texture hardness, and individuality of the agarose gel layer were analyzed at different concentrations. The difficulty of spore picking.

[0036] Table 1 Analysis table of results of different concentrations of agarose

[0037]

[0038]

[0039] The results showed that when the concentration was 1.3%, the agarose layer had good transparency, mode...

Embodiment 2

[0040] Embodiment 2, the screening of optimal dyeing agent and dyeing solution concentration

[0041] Staining solution configuration: Weigh 0.05g acid fuchsin, 0.01g Congo red, 0.01g eosin Y sodium, 0.05g neutral red, 0.01g safranin, 0.005g methylene blue, 0.01g methylene blue, 0.02g methylene blue, 0.03 g methylene blue, 0.04g methylene blue, 0.02g bromophenol blue, 0.01g methyl green, add 100mL sterile water. The mass and volume percent concentrations are prepared to be 0.05% acid fuchsin, 0.01% Congo red, 0.01% eosin Y sodium, 0.05% neutral red, 0.01% safranin, 0.005% methylene blue, 0.01% methylene blue, 0.02% methylene blue, The staining solution of 0.03% methylene blue, 0.04% methylene blue, 0.02% bromophenol blue and 0.01% methyl green was stored in a brown bottle at 4°C for later use.

[0042] Preparation of staining solution agarose gel layer: Take 50 μL of each of the above 12 different staining solutions, add 15 mL of 1.3% agarose solution that has been melted and...

Embodiment 3

[0050] Embodiment 3, the comparison of methylene blue agarose block method and traditional water agar method single spore separation effect

[0051] The methylene blue agarose block method established by the present invention and the reported water agar method (method 1 mentioned in the background technology) were used to inoculate P. brassica monospore, and the results of P. brassica monospore isolation were compared. The chrysanthemum Chinese cabbage seedlings (purchased from Zhongshu Seed Industry Technology (Beijing) Co., Ltd.) seedlings that had been germinated for 24 hours were inoculated with two different methods, transplanted to sterile seedling trays with a diameter of 5 cm × 5 cm, and placed in a greenhouse for cultivation. 200 plants were treated with 3 repetitions. Place it in a greenhouse environment, keep the daily temperature at 20-25°C, night temperature at 11-16°C, and light at 16h·d -1 , the humidity is 100% for the first two weeks, and then watered as need...

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Abstract

The invention provides a method for separating plasmodiophora brassicae monospore on the basis of a methylene blue agarose method. The method comprises the following steps: firstly, preparing a plasmodiophora brassicae resting spore suspension with the concentration of 5*10<3> spore.mL<-1>; adding 0.01% methylene blue staining solution to a 1.3% agarose solution to prepare a mehtylene blue agaroseglass slide coagulation layer; uniformly coating the surface of the coagulation layer with 2 mu L of plasmodiophora brassicae resting spore suspension, performing observation under a microscope to determine that only one spore exists in the visual field, separating the coagulation layer containing the monospore, placing the coagulation layer at root hair of cruciferae seedlings upside down, and performing cultivation to obtain a plasmodiophora brassicae monospore system. The plasmodiophora brassicae monospore is separated by the methylene blue agarose method, the background is slightly blue,no impurities or bubbles are produced, and the plasmodiophora brassicae monospore can be clearly distinguished. The method is simple in process and easy to operate, and the separation efficiency and accuracy of the plasmodiophora brassicae monospore are greatly improved.

Description

technical field [0001] The invention belongs to the field of isolation of single spores, in particular to a method for isolation of single spores of Plasmodium brassicae based on methylene blue staining solution combined with agarose method. Background technique [0002] Clubroot disease is an obligate parasitic worldwide disease caused by Plasmodiophora brassicae Woron. It is known as "cruciferous cancer". Any cruciferous Susceptible varieties of plants in the family can be infected, causing abnormal division and enlargement of parenchyma cells, forming nodular swollen roots. The dormant spores of clubroot can survive for up to 20 years in the soil of non-susceptible host plants. Once the soil is polluted, it will no longer be suitable for the cultivation of cruciferous plants. In recent years, clubroot has spread rapidly in various regions of my country, and Southwest, Northeast, and East China have become the hardest hit areas for clubroot. According to the statistics o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N3/00C12R1/645
CPCC12N3/00
Inventor 柴阿丽李宝聚高青云石延霞谢学文
Owner INST OF VEGETABLE & FLOWERS CHINESE ACAD OF AGRI SCI
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