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Method for detecting activity of spores of plant pathogenic fungi and plasmodiophora brassicae

A technology for plant pathogenic fungi and Brassica rhizogenes, which is applied in material excitation analysis, fluorescence/phosphorescence, etc., can solve the problems of high toxicity of dyes, toxicity of detection time and detection process, unstable detection results, etc., and achieves good repeatability. Effect

Inactive Publication Date: 2014-03-26
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

[0006] The purpose of the present invention is to provide a new detection method with a short detection time and a short detection time for the current problems such as long detection time for spore activity of plant pathogenic fungi and Plasmodium brassicae, unstable detection results, high dye toxicity in the detection process, and harmfulness to human body. Low process toxicity and economical method for the detection of spore activity of plant pathogenic fungi and Plasmodium brassicae

Method used

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  • Method for detecting activity of spores of plant pathogenic fungi and plasmodiophora brassicae
  • Method for detecting activity of spores of plant pathogenic fungi and plasmodiophora brassicae
  • Method for detecting activity of spores of plant pathogenic fungi and plasmodiophora brassicae

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Embodiment 1

[0020] Example 1: Detection of spore activity of plant pathogenic fungi and Plasmodium brassicae

[0021] 1. Materials and instruments

[0022] Hoechst33342 (SIGMA), propidium iodide (SIGMA), Olympus fluorescence microscope (DP72).

[0023] 2. Experimental method

[0024] Part of the phytopathogenic fungal spores were used as materials (Table 1), and the Hoechst33342 / propidium iodide double staining method was used to identify the dead and alive phytopathogenic fungal spores. Add 1 μl of Hoechst33342 (dissolved in PBS) with an initial concentration of 100 μg / ml to 99 μl of spore suspension, mix well, and incubate at 37°C for 10 minutes, centrifuge at 1000 r / min at 4°C, add 100 μl of sterilized Wash once with distilled water, centrifuge to remove the supernatant, add 100 μl of sterilized distilled water to resuspend, then add 500 μg / ml propidium iodide staining solution (dissolved in PBS) and mix with the spore suspension, the final concentration is 5 μg / ml, 4 Stain in the d...

Embodiment 2

[0030] Example 2: Hoechst33342 / propidium iodide staining concentration and time screening

[0031] 1. Materials and instruments

[0032] Fusarium oxysporum strain HG504281, Plasmodium brassicae strain DBC11071501, Hoechst33342 (SIGMA), propidium iodide (SIGMA), Olympus fluorescence microscope (DP72), electric thermostatic water bath (XMTD-700).

[0033] 2. Experimental method

[0034] Using P.brassicae and F.oxysporum as experimental materials, the concentration and staining time of Hoechst33342 and propidium iodide dyes were screened. Freshly prepared spore suspensions were divided into two groups: one group was untreated living spores used for germination experiments as a germination test control (group K); one group was untreated living spores used for staining experiments (UK group) ). Concentration gradient experiments with 4 different dye concentrations were designed for two dyes: stock solution 100 μg / ml, 10-fold dilution 10 μg / ml, 20-fold dilution 5 μg / ml, 50-fold d...

Embodiment 3

[0038] Combined with the effect of staining on spore activity, and the effect of fluorescent microscope observation on the distinction between dead and alive spores after staining, the staining condition that does not affect spore activity and has the best staining effect is selected: Hoechst33342100 times dilution, the corresponding dye concentration is 1 μg / ml, staining The time is to incubate at 37°C for 10 minutes, centrifuge at 4°C to remove the dye, dilute propidium iodide 20 times, the corresponding dye concentration is 5 μg / ml, and the staining time is 15 minutes. Embodiment 3: Physically treated (heat treated) Plasmodium brassica spore activity detection and quantitative analysis 1, materials and instruments

[0039] Plasmodium brassicae strain DBC11071501, Hoechst33342 (SIGMA), propidium iodide (SIGMA), Olympus fluorescence microscope (DP72), electric thermostatic water bath (XMTD-700). 2. Experimental method

[0040] Plasmodium brassicae was used as the experimenta...

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Abstract

The invention discloses a method for detecting the activity of the spores of plant pathogenic fungi and plasmodiophora brassicae woronin. According to the method, a Hochest 33342 / PI double-staining method is adopted, and a fluorescence microscope is used for detection, so that life or death of the spores of the plant pathogenic fungi and the plasmodiophora brassicae woronin can be rapidly, accurately and sensitively distinguished; a spore germination method is also used for identifying the activity of the spores of the plant pathogenic fungi and the plasmodiophora brassicae woronin, but according to the spore germination method, time-consuming and labor-consuming effects are adopted; the method can be used for replacing the traditional spore germination method and is suitable for departments such as agricultural production, plant protection, etc.

Description

technical field [0001] The invention relates to a method for detecting microbial activity, in particular to a method for detecting the activity of spores of plant pathogenic fungi and Plasmodium brassicae. Background technique [0002] Plant diseases caused by fungi have reached 30,000 species, accounting for 70-80% of plant diseases, and serious and devastating diseases such as potato late blight, wheat rust, sweet potato black spot and so on. In order to accurately predict the disease and formulate effective prevention and control strategies, it is necessary to quickly detect the existence of plant pathogenic fungi. Strong basis for disease control strategies. [0003] Plasmodiophora brassicae causes clubroot of cruciferous vegetables, and mainly parasitizes the roots of various plants of the genus Brassica. It is distributed all over the world. In recent years, its harm range has expanded from cruciferous vegetables to some flower plants, infecting more than 100 species...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/64
Inventor 李宝聚李金萍谢学文石延霞
Owner INST OF VEGETABLE & FLOWERS CHINESE ACAD OF AGRI SCI
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