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Detection method of colletotrichum gloeosporioides penz drug-resistant strain to benzimidazoles bactericide

A technology for benzimidazoles and anthracnose bacteria is applied in the detection of drug resistance of bacteria and the field of drug resistance detection of bacteria, and can solve the problems of failure, high resistance level of drug-resistant strains, and easy generation of drug resistance.

Inactive Publication Date: 2010-12-22
SOUTH SUBTROPICAL CROPS RES INST CHINESE ACAD OF TROPICAL AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, benzimidazole fungicides are systemic fungicides with a single site of action, and are prone to drug resistance. In some mango producing areas where benzimidazole fungicides are frequently used, drug resistance has appeared. The resistance level of the disease is high, reaching the level of carbendazim failure, leading to the outbreak and epidemic of the disease

Method used

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  • Detection method of colletotrichum gloeosporioides penz drug-resistant strain to benzimidazoles bactericide
  • Detection method of colletotrichum gloeosporioides penz drug-resistant strain to benzimidazoles bactericide

Examples

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Effect test

example 1

[0024] A kind of detection method of mango anthracnose bacterium to benzimidazoles fungicide resistant bacterial strain, it comprises the steps:

[0025] (1) Extraction of Genomic DNA of Mango Anthracnose Pathogen:

[0026] (a) Get pathogenic bacteria and inoculate in potato dextrose liquid culture medium, vibrate and cultivate for 6 days, take culture solution and filter to obtain mycelium, and mycelium freeze-dry for subsequent use;

[0027] (b) Take 20mg of freeze-dried mycelium and grind it with liquid nitrogen, transfer it to a 1.5mL centrifuge tube, add 400μL of SDS extraction buffer in a 65°C water bath, the preparation of SDS extraction buffer: 200mmol / L Tris-HCl, 250mmol / L NaCl, 25mmol / LEDTA, 5% SDS, mix well and put in a water bath at 65°C for 30min, shake well three times at intervals;

[0028] (c) Centrifuge at 13000r / min for 10min, take 300μL supernatant, add 300μL mixed solution of phenol, chloroform and isoamyl alcohol, phenol: chloroform: isoamyl alcohol is 25...

example 2

[0038] Collect 64 anthracnose pathogenic bacteria strains located in the mango orchard where carbendazim and thiophanate-methyl have been used many times in Zhanjiang, Guangdong, and test according to the method of the present invention. There are 2 electrophoresis strips of 25 bacterial strains and they are respectively 107bp and 222bp , the other 39 strains had one electrophoresis band of 329bp, and the percentage of resistant strains was 39%. At the same time, it was verified by traditional biological assay methods, and the assay results were consistent with this test.

example 3

[0040] Collect 23 anthracnose pathogenic bacteria strains located in the mango orchards of Sanya, Hainan, where carbendazim, thiophanate-methyl, Shibaoke, and Dasheng have been used many times, and test according to the method of the present invention. There are 2 electrophoresis strips of 2 bacterial strains with 2 and respectively 107bp and 222bp, and one of the other 21 strains had 329bp in the electrophoresis band, and the percentage of resistant strains was 9%. At the same time, it was verified by traditional biological assay methods, and the assay results were consistent with this test.

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Abstract

The invention relates to a detection method of a colletotrichum gloeosporioides penz drug-resistant strain to benzimidazoles bactericide, comprising the following four steps of: (1) extracting a colletotrichum gloeosporioides penz genomic DNA; (2) carrying out PCR (Polymerase Chain Reaction) amplification on a specific primer under certain reaction conditions; (3) carrying out enzyme cutting on a PCR amplified product; and (4) analyzing an enzyme-cutting product, wherein the detection method has 100 percent of detectable rate on the colletotrichum gloeosporioides drug-resistant strain. In the invention, the DNA segment (includes a specific restriction enzyme cutting site) amplification is carried out on a specific primer pair selected according to the specific restriction enzyme cutting site of a pathogenic bacteria resistant strain, the enzyme cutting is carried out on the amplified DNA segment by using a restriction endonuclease, and the enzyme-cutting is subjected to electrophoresis and gel imaging so as to accurately judge whether the pathogenic bacterium is a resistant strain or a sensitive strain. The method is proved to be stable and reliable through multiple tests.

Description

technical field [0001] The invention relates to a method for detecting drug resistance of pathogens, in particular to a method for detecting bacterial strains resistant to benzimidazole fungicides caused by mango anthracnose, and belongs to the field of detection methods for drug resistance of pathogens. Background technique [0002] Benzimidazole fungicides are an important class of fungicides developed in the late 1960s, which can prevent and control most plant diseases caused by Ascomycetes, Deuteromycetes and Basidiomycetes. Such compounds mainly include carbendazim, benomyl, thiabendazole, fuberidazole, thiophanate-methyl, etc. After the application of such agents, In plants, the common derivative carbendazim or its ethyl homologue ethyl carbendazim will be produced, which is the final product of interaction with pathogens. This type of agent has good systemic properties, so it is widely used in the prevention and control of some important crop diseases. Its main role ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12R1/645
Inventor 詹儒林黄俊生姚全胜何衍彪雷新涛赵艳龙常金梅王松标
Owner SOUTH SUBTROPICAL CROPS RES INST CHINESE ACAD OF TROPICAL AGRI SCI
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