Rapid identification method for resistance of field tobacco alternaria alternata to three types of germicides

The invention relates to a technology for the identification method of Tobacco scab, which is applied in the fields of plant pathology and molecular biology, and can solve the problems of time-consuming and labor-intensive, economic loss, control failure, etc., and achieve the effects of shortening the experiment time, accurate results and simple operation.

Inactive Publication Date: 2011-09-14
KUNMING UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method is time-consuming and labor-intensive. For example, if 100 single spores are isolated from a tissue block, it will take at least one month to finally obtain the drug resistance level of each single spore strain, and the results obtained by this method are not completely accurate. Response to the drug resistance of Tobacco Alternaria Alternaria in the field, because when the drug-resistant strains just appeared but have not yet become the dominant flora in the field, the drug-resistant strains have weaker growth competitiveness than the sensitive strains on the PDA medium plate, and finally The monospore strains obtained may all be sensitive strains, and the conclusion that "there are no drug-resistant strains in the field" is obtained, which affects the drug control strategy, and eventually makes the control ineffective, causing serious economic losses

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] 1. Collection of lesion tissue blocks and extraction of fungal genomic DNA

[0034] Tobacco leaves with typical symptoms of tobacco red spot disease were collected from the tobacco fields where pesticides were used to control tobacco brown spot disease in the field for more than 3 consecutive years, and 0.5 cm side length of diseased tobacco leaf tissue blocks were cut from the tobacco leaves, and 4 diseased tissue blocks were randomly selected , put it into a mortar, quick-freeze it with liquid nitrogen and grind it into a powder, and use E.Z.N.A. ?

[0035] 2. PCR amplification

[0036] The extracted genomic DNA was diluted with sterilized deionized water to a concentration of 10ng / μl. In the PCR amplification system, the six groups of 92 AARDs at the N-terminal of TCHK were amplified using specific primers HKFwd1 and HKRev1, and detected by electrophoresis.

[0037]HKFwd1: 5'-AGGCTGACAGACAACAGACAA-3'

[0038] HKRev1: 5'-CGAGCGTAAGTTGGGTCAT-3'

[0039] 1) PCR ampli...

Embodiment 2

[0061] 1. Collection of lesion tissue blocks and extraction of fungal genomic DNA

[0062] Tobacco leaves with typical symptoms of tobacco red spot disease were collected from the tobacco fields where pesticides were used to control tobacco red spot disease in the field for more than 3 consecutive years. From the diseased tobacco leaves, the diseased tobacco leaf tissue blocks with a side length of 1 cm were cut, and 5 diseased tobacco leaves were randomly selected. The plaque tissue block was put into a mortar, quick-frozen with liquid nitrogen and ground into a powder, and the genomic DNA of Alternaria tabacum in the lesion tissue block was extracted by using the E.Z.N.A.? HP Fungal DNA Extraction Kit.

[0063] 2. PCR amplification

[0064] The extracted genomic DNA was diluted with sterilized deionized water to a concentration of 20ng / μl. In the PCR amplification system, the six groups of 92 AARDs at the N-terminal of TCHK were amplified using specific primers HKFwd2 and HK...

Embodiment 3

[0089] 1. Collection of lesion tissue blocks and extraction of fungal genomic DNA

[0090] Tobacco leaves with typical symptoms of tobacco red spot disease were collected from the tobacco fields where pesticides were used to control tobacco red spot disease in the field for more than 3 consecutive years. From the diseased tobacco leaves, the diseased tobacco leaf tissue pieces with a side length of 0.8 cm were cut, and three diseased tobacco leaves were randomly selected. Plaque tissue blocks were put into a mortar, quick-frozen with liquid nitrogen and ground into powder, and the Genomic DNA of Alternaria tabacum in the lesion tissue blocks was extracted by using the Biospin Fungal DNA Extraction Kit.

[0091] 2. PCR amplification

[0092] The extracted genomic DNA was diluted to a concentration of 30 ng / μl with sterile deionization. In the PCR amplification system, the specific primers, PCR amplification system and amplification procedure used were the same as those in Examp...

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Abstract

The invention discloses a rapid identification method for resistance of field tobacco alternaria alternata to three types of germicides, namely dicarboximide type germicide, phenylpyrrole type germicide and aromatic hydrocarbon type germicide, based on a culture-independent and PCR (Polymerase Chain Reaction) amplification technology, belonging to the fields of plant pathology and molecular biology. In the method, tobacco alternaria alternata genome DNA (Deoxyribonucleic Acid) in a tobacco alternaria alternata scab tissue block is extracted and is used as a template, and six groups of amino acid repeating regions, 92 in total, at an end N of bi-component histidine kinase are rapidly amplified by applying a specific primer. Whether the drug resistance of the tobacco alternaria alternata to the dicarboximide type germicide, the phenylpyrrole type germicide and the aromatic hydrocarbon type germicide is generated is determined by comparing gene sequences. The method has the advantages of rapidness, easiness, convenience, accuracy and the like and has a broad application prospect in resistance monitoring of the tobacco alternaria alternata.

Description

technical field [0001] The invention relates to a method for rapid identification of resistance to three types of fungicides of the field tobacco red spot fungus, terephthalimides, pyrrolebenzenes and aromatic hydrocarbons, based on culture-free and PCR amplification techniques, belonging to plant pathology and molecular field of biology. Background technique [0002] Alternaria longifera ( Alternaria longipes ) infection caused by field tobacco red spot disease is an important tobacco fungal disease. Dicarboximide, Phenylpyrrole, and Aromatic hydrocarbon fungicides are widely used in the control of many groups of plant pathogenic fungi because of their broad bactericidal spectrum and remarkable control effect. . However, with the prolongation of the use time and the increase of the application amount of these three types of fungicides, many plant pathogenic fungi (including A. longipes ) have serious cross-resistance to the three types of fungicides, which has brought ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 罗义勇柳陈坚
Owner KUNMING UNIV OF SCI & TECH
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