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Method for fast identifying drug resistance of corynespora cassiicola on fluopyram and special primer pair

A technology for fluopyram and Corynespora multiprimum, applied in the field of molecular biology, can solve time-consuming and labor-intensive problems

Active Publication Date: 2017-10-27
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

[0005] Traditional biological methods used to detect or monitor the resistance of plant pathogens, including colony growth assays, spore germination assays, etc., are all based on the determination of the EC 50 Time-consuming and labor-intensive to distinguish sensitive and resistant strains

Method used

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  • Method for fast identifying drug resistance of corynespora cassiicola on fluopyram and special primer pair
  • Method for fast identifying drug resistance of corynespora cassiicola on fluopyram and special primer pair
  • Method for fast identifying drug resistance of corynespora cassiicola on fluopyram and special primer pair

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Example 1. Discovery of succinate dehydrogenase B subunit gene SdhB and its corresponding protein mutation sites in Corynespora polymata

[0056] 1. Determination of susceptibility of Corynespora polymata to fluopyram

[0057] 5 sensitive strains HG15050755-3, HG15041321-2, HG15041414-2, HG14102405-2 and HG14102524-4; 4 resistant strains HG15050729-5, HG15050731, HG15050737 and HG15050825-6.

[0058] Potato dextrose agar medium (PDA): 200 g of potatoes, 20 g of glucose, 15 g of agar, distilled water to 1 L, sterilized at 121° C. for 30 min.

[0059] 1) Mix fluopyram with acetone to make 5×10 4 μg / ml stock solution. For the sensitivity determination of 5 strains of Corynespora polymaine sensitive strains and 4 strains of Corynespora polymaine resistant strains, fluopyram was diluted to 10 μg / mL, 100 μg / mL, 500 μg / mL, 1000 μg / mL, 5000 μg / mL, 10000μg / mL and 25000μg / mL concentration gradient.

[0060] 2) In order from low to high, use a pipette to draw 135 μL of the dr...

Embodiment 2

[0083] Embodiment 2, the method for detecting the mutation site A1020G in Corynespora polymata

[0084] 1. Primer design, screening and amplification methods

[0085] The strains are the strains HG15050729-5, HG15050825-6, HG14102524-4, HG14102405-2, HG15041321-2, HG15041414-2, HG15050755-3 used in Example 1.

[0086] AS-PCR primers were designed according to the sequence of the mutant HG15050729-5 succinate dehydrogenase B subunit gene SdhB. The primer pair Cc1020F-Cc1020A / T / C / G.C.R was used to detect the mutation site of A1020G. Wherein, the last base of the 3'-end of the reverse primer Cc1020A.C.R is the mutated base, and the forward primer is Cc1020F (Table 2). In order to increase the specificity of the primers, mismatched bases were introduced into the 2nd and 3rd bases at the 3'-end of the reverse primer (Table 2, primers Cc1020T / C / G.C.R).

[0087] Table 2 The PCR primers used in the detection of Corynespora polymorpha strains resistant to fluopyram

[0088]

[0...

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Abstract

The invention discloses a method for fast identifying drug resistance of corynespora cassiicola on fluopyram and a special primer pair. The invention provides application of a substance for detecting succinic dehydrogenase B subunit coding gene A1020G site basic group in the corynespora cassiicola genome to the detection of the resistance of the corynespora cassiicola resistance; the A1020G site is the 1020-th site in the succinic dehydrogenase B subunit coding gene; the basic group of the A1020G site is A or G. The method for detecting the point mutation of the drug resistance of the corynespora cassiicola on fluopyram provided by the invention has the advantages that the method can be used for fast and sensitively detecting the resistance occurring and development dynamic state of the field corynespora cassiicola on fluopyram so that the prevention and control strategy on the cucumber target leaf spot diseases can be timely regulated; the further development of the drug resistance can be delayed and controlled.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to the cloning of the gene SdhB of succinate dehydrogenase B subunit of Corynespora polybasicum and its application in the detection and monitoring of fluopyram resistance, and specifically discloses a rapid identification A method and a special primer pair for Corynespora multiprimum resistance to fluopyram. Background technique [0002] In recent years, Corynespora cassiicola (Berk.&M.A.Curtis) C.T.Wei caused by Corynespora cassiicola (Berk.&M.A.Curtis) C.T.Wei has been promoted from a minor disease to a major disease in cucumber production in my country. Field disease generally reduces production by 20%, and can reach 60% to 70% in severe cases. The disease was first reported in Europe in 1906, and the occurrence of the disease was reported in North Carolina, USA in 1957. In the 1960s, Qi Peikun and others reported the occurrence of the disease in my countr...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04
CPCC12Q1/6895C12Q2600/156
Inventor 石延霞朱发娣李宝聚谢学文柴阿丽
Owner INST OF VEGETABLE & FLOWERS CHINESE ACAD OF AGRI SCI
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