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Plant pathogen activity evaluation and bactericide high throughput screening method and kit

A plant pathogenic bacteria and kit technology, which is applied to measurement devices, material analysis by optical means, instruments, etc., can solve the problems of unstable test results, labor-intensive screening, and high costs, saving manpower, rapid operation, and repeatability. Good results

Inactive Publication Date: 2015-05-20
INST OF VEGETABLE & FLOWERS CHINESE ACAD OF AGRI SCI
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  • Abstract
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The purpose of the present invention is to provide a method for evaluating the activity of plant pathogens based on double fluorescent staining and high-throughput screening of fungicides for the problems of labor-intensive and time-consuming detection of plant pathogenic bacteria and screening of fungicides, unstable test results, and high costs. The kit is used to determine the activity of pathogenic bacteria by detecting the fluorescent signals of viable and non-viable spores or mycelial cells of pathogenic bacteria, and realize the efficacy evaluation and high-throughput screening of fungicides

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  • Plant pathogen activity evaluation and bactericide high throughput screening method and kit
  • Plant pathogen activity evaluation and bactericide high throughput screening method and kit
  • Plant pathogen activity evaluation and bactericide high throughput screening method and kit

Examples

Experimental program
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Effect test

Embodiment 1

[0050] Example 1 Screening of plant pathogenic bacteria activity and agent evaluation targets based on fluorescent double staining

[0051] Use FDA (fluorescein diacetate) and PI (propidium iodide) as dyes to identify the spores and hyphae of plant pathogenic fungi and the spore viability of plant pathogenic bacteria, and screen the plant pathogenic bacteria that can be identified by fluorescent double staining target.

[0052] 1. Experimental method

[0053] Plant pathogenic fungal spores, hyphae and plant pathogenic bacterial spores were selected as targets (Table 1), and FDA / PI double staining method was used to identify the activity of plant pathogenic bacteria.

[0054] Fluorescent staining solution configuration: Weigh 0.05 g FDA, dissolve in 1 mL acetone, add 9 mL PBS 7.4 to prepare FDA stock solution with a concentration of 5 mg / mL, store in a brown bottle at -20°C for later use, and use PBS before use Dilute to a final concentration of 100 μg / mL for use. Weigh 0....

Embodiment 2

[0058] Example 2 Determination of Optimum Fluorescent Detection Time for Hoechst 33258 / PI Staining Drug Screening

[0059] A key condition for using fluorescent double staining to evaluate the activity of plant pathogens or to perform high-throughput screening of fungicides is to determine the optimal fluorescence detection time to maximize the reproducibility of the method results. Botrytis cinerea ( B. cinerea ) spore activity identification as an example, using 96-well microtiter plate and 384-well microtiter plate to conduct experiments respectively.

[0060] 1. Experimental method

[0061] Botrytis cinerea was cultured in PDA medium for 10 days, and the spore suspension was collected by the spore brush method, and divided into three groups. The spores in the first group were treated in a water bath at 95°C for 10 min to death, and the spores in the second group were treated without any treatment. Viable spores of the same group, the third group was equal mixture of h...

Embodiment 3

[0069] Example 3 Quantitative analysis of the activity of plant pathogenic bacteria detected by flow cytometry

[0070] Anthracnose melons ( C. orbitale ) spores as the target, using flow cytometry to detect the spore viability of the anthracnose melons double-stained by Annexin V–FITC / PI, and quantitatively analyze the spore survival rate. At the same time, using the spore germination method as a control, the correlation between the detection results of the fluorescent double staining method and the traditional spore germination method was compared.

[0071] 1. Experimental method

[0072] Five strains of Bacillus anthracnose melons were used as experimental materials. The freshly prepared spore suspension was treated at 30°C, 40°C, 50°C, and 60°C for three times, namely 5 minutes, 10 minutes, and 20 minutes. All samples were divided into two groups, and one group was treated with Annexin After V-FITC / PI double-staining, the flow cytometer was loaded to analyze th...

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Abstract

Disclosed are activity evaluation of plant pathogens and a high throughput screening method for microbicides based on double fluorescent staining and a kit therefor, which include using a microtiter plate as tool carrier, using two different fluorescent dyes, respectively fluorescently labelling spores or mycelia of plant pathogens having vitality and no vitality, and due to the different colouring of spores or mycelia having no vitality and vitality, quantitatively detecting the survival rate of plant pathogens through fluorescence microscopy or flow cytometry, and accordingly preparing the kit. The method and kit can be used in aspects such as microbicide screening, evaluation of food safety, and water quality evaluation using the plant pathogen survival rate as an indicator. The method and kit are rapid, objective, simple and easy in operation, and have a low cost, and both can be used in studying the mechanism of the action of microbicides and high-throughput screening and the toxicity evaluation of microbicides.

Description

technical field [0001] The invention relates to a method and kit for evaluating the activity of plant pathogenic bacteria and high-throughput screening of fungicides based on double fluorescent staining. The method and the kit can be used in the evaluation of pathogenic bacteria activity, screening of fungicides, food safety evaluation, water quality evaluation, etc. , belonging to the field of plant protection and biotechnology. Background technique [0002] Plant pathogenic bacteria are an important factor in the occurrence and prevalence of plant diseases, and the losses caused by diseases in the world are hundreds of millions of dollars every year. Rapid and accurate detection of plant pathogenic bacteria and their activity is the basis for effective prediction of disease conditions. [0003] At present, the detection of plant pathogenic bacteria mainly adopts conventional pathogen isolation and culture technology, which is time-consuming and laborious, and cannot detec...

Claims

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Application Information

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IPC IPC(8): G01N21/64
CPCG01N21/6458G01N21/6452G01N2021/6439
Inventor 李宝聚柴阿丽石延霞谢学文李金萍
Owner INST OF VEGETABLE & FLOWERS CHINESE ACAD OF AGRI SCI
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