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Molecular detection method for identifying insecticide resistance of Botrytis cinerea on zoxamide

A technology of botrytis cinerea and drug resistance, applied in the field of molecular biology, can solve the problems of long detection cycle, heavy workload, human resources and materials, etc., and achieve the effect of delaying further development

Inactive Publication Date: 2014-05-14
CHINA AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The traditional biological method, that is, by measuring the EC of the agent to the pathogenic bacteria 50 In order to distinguish sensitive and resistant strains, multiple agents are required to be treated and repeated many times. The detection cycle is long, the workload is heavy, and human resources and materials are consumed. Moreover, the detection sensitivity of this method is low, and the mutation frequency of resistant strains is required. Above 1%

Method used

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  • Molecular detection method for identifying insecticide resistance of Botrytis cinerea on zoxamide
  • Molecular detection method for identifying insecticide resistance of Botrytis cinerea on zoxamide
  • Molecular detection method for identifying insecticide resistance of Botrytis cinerea on zoxamide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Embodiment 1, Botrytis cinerea to the sensitivity determination of benzamid

[0028] 6 sensitive strains NJ11, SF2-8, FJY1-34, SQ15, LY10 and FJ1-10; 2 resistant strains RZ-BC14 and RZ-BC16.

[0029] Potato dextrose agar medium (PDA): 200g of potatoes, 18g of glucose, 15g of agar powder, distilled water to 1L, sterilized by moist heat at 121°C for 20 minutes.

[0030] 1. The experimental steps are as follows:

[0031] 1) Mix benzamid with dimethyl sulfoxide (DMSO) to make 10 5 μg / mL stock solution. The benzamid was diluted stepwise to 4×10 2 μg / mL, 6×10 2 μg / mL, 8×10 2 μg / mL, 1×10 3 μg / mL, 2×10 3μg / mL, 4×10 3 μg / mL and 5×10 3 The concentration gradient of μg / mL, 1‰DMSO was used as blank control to determine the sensitivity of 6 Botrytis cinerea sensitive strains. For the sensitivity determination of two Botrytis cinerea resistant strains, 1‰DMSO was used as a blank control, and the concentration gradient was 3×10 3 μg / mL, 5×10 3 μg / mL, 1×10 4 μg / mL, 2.5×10 ...

Embodiment 2

[0040] Example 2, Discovery of β-tubulin gene and its corresponding protein mutation site in Botrytis cinerea

[0041] 1. Strains: resistant strains RZ-BC14 and RZ-BC16; sensitive strains NJ11, SF2-8, FJY1-34, SQ15, LY10 and FJ1-10.

[0042] 2. Method:

[0043] 1) Strain cultivation: Inoculate the pre-cultured Botrytis cinerea strains on PDA medium covered with cellophane, culture in the dark at 20°C for 3 days, collect mycelium, freeze in liquid nitrogen and store at -80°C for genomic DNA extraction.

[0044] 2) Genomic DNA of resistant strains and sensitive strains were extracted respectively.

[0045] 3) PCR amplification of the complete sequence of β-tubulin gene, the primers used are shown in Table 2.

[0046] Table 2 Primers used in the amplification of the complete sequence of the cinerea cinerea β-tubulin gene

[0047]

[0048] PCR reaction system: The PCR reaction uses the reagents of Beijing Quanshijin Biotechnology Co., Ltd., and the 50 μL reaction system incl...

Embodiment 3

[0055] Embodiment 3, the method for detecting mutation site in Botrytis cinerea

[0056] 1. Primer design

[0057] The strains are the strains NJ11, SQ15, FJY1-34, RZ-BC14 and RZ-BC16 used in Example 1.

[0058] The primers are shown in Table 3. Allele specific-PCR (AS-PCR) primers were designed according to the sequence of the mutant β-tubulin gene. The second base at the 3'-end of the forward primer RZBC233T is the mutated base. To increase the specificity of the primers, a mismatched base was introduced at the first base at the 3'-end of the forward primer (primers RZBC233A, RZBC233C and RZBC233G, the mismatched bases are underlined). The reverse primer is RZBCR.

[0059] Table 3 AS-PCR detects the primers used for Botrytis cinerea showing resistance to benzamid

[0060]

[0061] The specific reaction system is the same as in Example 2. Gradient PCR with different annealing temperatures was carried out on the primers to determine the annealing temperature with high s...

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Abstract

The invention relates to cloning of a Botrytis cinerea beta-tubulin gene and an application thereof in resistance monitoring of zoxamide, particularly discloses a molecular detection method for rapidly identifying insecticide resistance of Botrytis cinerea and a special primer, and belongs to the technical field of molecular organisms. The specific primer pair is composed of deoxyribonucleic acid (DNA) molecules shown in SEQ ID NO:1 (or SEQ ID NO:1 or SEQ ID NO:3) and SEQ ID NO:5. The point mutation method for detecting the insecticide resistance on the zoxamide generated by the Botrytis cinerea, which is provided by the invention, has the characteristics of being high in sensitivity, simple and rapid, and good in stability, can rapidly and sensitively detect the development trend of the resistance on the zoxamide caused by field botrytis cinerea, and timely adjusts the disease control strategy, so as to delay and control further development of the insecticide resistance.

Description

technical field [0001] The present invention relates to the application of cloning of Botrytis cinerea (B.cinerea) β-tubulin gene in the monitoring of fungicide benzamid resistance, and specifically discloses a rapid identification of Botrytis cinerea β-tubulin gene nucleosides Molecular detection methods and special primers for acid point mutation and its resistance to benzamid. It belongs to the technical field of molecular biology. Background technique [0002] Botrytis cinerea is a worldwide disease caused by B. cinerea Pers ex Fr (anamorph of Botryotinia fuckeliana (de Bary) Whetz). The pathogen can infect not only tomatoes, but also vegetables and fruits such as cucumbers, eggplants, and grapes, and even flowers such as roses and gerberas. There are at least 235 species of hosts. The disease can generally cause a 20%-30% reduction in production, and the loss is even more severe when it is widespread in a large area. It has become a restrictive obstacle in the cultiva...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11C12N15/31C07K14/37
CPCC07K14/37C12Q1/689C12Q2600/156
Inventor 刘西莉蔡萌林东陈磊毕扬宋晰李健强刘鹏飞
Owner CHINA AGRI UNIV
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