One-tube detection method for indetifying Gibberella zeae and confiming medium pesticide resistance level of Gibberella zeae to carbendazim

A technology of Fusarium graminearum and carbendazim, which is applied to the detection of drug-resistant subpopulations of plant pathogens, and in the field of Fusarium graminearum of benzimidazole fungicides, can solve the problem of short-term prediction and determination of samples that cannot be used for drug resistance Problems such as limited quantity and hard-to-resistance subgroups, etc., achieve the effect of simple method, simple detection and monitoring, and control of drug resistance

Inactive Publication Date: 2009-07-08
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method of measuring sensitivity to drugs usually takes a few weeks, and the workload is heavy. The number of samples tested is limited, and it is difficult to detect the existence of drug-resistant subpopulations early, and it cannot be used for short-term prediction of drug resistance in the current year.

Method used

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  • One-tube detection method for indetifying Gibberella zeae and confiming medium pesticide resistance level of Gibberella zeae to carbendazim
  • One-tube detection method for indetifying Gibberella zeae and confiming medium pesticide resistance level of Gibberella zeae to carbendazim
  • One-tube detection method for indetifying Gibberella zeae and confiming medium pesticide resistance level of Gibberella zeae to carbendazim

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0014] Example 1 Detection of antibacterial strains in Fusarium graminearum resistant to carbendazim by PCR method

[0015] Amplification system: dNTP 0.2 μM in 50 μL, upstream and downstream primers 1 μM, Taq (Shenergy Biotechnology Co., Ltd.) 2.5 U, template DNA 100 ng, 10×buffer (10 μM Tris-HCl, 50 mM KCl) 5 μL, use d 2 h 2 O to make up to 50 μL. Amplification conditions: 94°C 5min; 94°C 60s, 56°C 60s, 72°C 60s, 35 cycles; 72°C 15min.

[0016] Antibacterial strains (Codon 167 TTT→TAT) was detected ( figure 1 ), the probe has good specificity to the antimicrobial strains.

Embodiment 2

[0017] Example 2 Result and Analysis of Fusarium graminearum Specific Detection

[0018] Amplification system: dNTP 0.2 μM in 50 μL, upstream and downstream primers 1 μM, Taq (Shenergy Biotechnology Co., Ltd.) 2.5 U, template DNA 100 ng, 10×buffer (10 μM Tris-HCl, 50 mM KCl) 5 μL, use Make up to 50 μL with d2H2O. Amplification conditions: 94°C for 5min; 94°C for 45s, 56°C for 45s, 72°C for 45s, 35 cycles; 72°C for 15min.

[0019] The primer pair FGa / FGb has strong specificity for Fusarium graminearum. After the primer pair FGa(5'-AGTCCAAAATGTCCCGATGC-3') / FGb(5'-GCTGGGACCTGAGAAGTA-3') was used for 26 other different bacterial strains, there was no band ( figure 2 ).

Embodiment 3

[0020] Example 3 Test of Fusarium graminearum and its sensitivity to carbendazim by one-tube method

[0021] The 50 μL amplification system contains the above two pairs of primers FGa / FGb and MBCR / MBCF so that the primer concentration in the reaction system is 1 μM, dNTP 0.2 μM, Taq (Shenergy Biotechnology Co., Ltd.) 2.5U, template DNA 100ng, 10×buffer (10 μM Tris-HCl, 50 mM KCl) 5 μL, made up to 50 μL with d2H2O. The amplification conditions were 94°C for 5min; 94°C for 45s, 56°C for 45s, 72°C for 45s, 35 cycles; 72°C for 15min. Take 5 μ L of PCR product and electrophoresis on 1.5% agarose TBE gel, observe by fluorescence imaging system ( image 3 ).

[0022] After amplification by the primer pair FGa / FGb, the templates NT-21 and NT-29 had obvious bands (small electrophoresis bands), but rice bakanae disease (G.fujikuroi) had no bands, indicating that NT-21, NT-29 NT-29 belongs to Fusarium graminearum, but rice bakanae disease (G.fujikuroi) is not Fusarium graminearum. Af...

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Abstract

The present invention relates to Gibberella zeae (asexual state Fusarium graminearum) detection and a tube detection method of using the detection to confirm medium level of drug resistance produced by carbendazim, which is special for the specificity detection of Gibberella zeae strain and medium level of drug resistance produced by carbendazim. The method adopts the ASO-PCR detection to the pathogenic germs on the diseased spike, diseased tissue and diseased body collected from field, the entire process only requires 6h, and the detection accuracy rate is 99%. The method has fast, simple, accurate and sensitive features.

Description

1. Technical field [0001] The present invention is a one-tube (that is, in one PCR reaction tube) detection method for the identification of Fusarium graminearum and whether it produces moderate drug resistance (that is, intermediate resistance) to carbendazim, and belongs to the drug-resistant subgroup of plant pathogenic bacteria The detection method is dedicated to the detection of Fusarium graminearum resistant to benzimidazole fungicides such as carbendazim. 2. Technical background [0002] Wheat head blight is a worldwide disease caused by Fusarium graminearum (Gibberella zeae, anamorphic Fusarium graminearum), which seriously affects the yield and quality of wheat. For a long time, benzimidazole fungicides or compound agents based on such agents have been used for chemical control. Benzimidazole fungicides are used in production as a class of high-efficiency, broad-spectrum systemic fungicides, which solve the environmental toxicity problem of protective fungicides a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04
Inventor 陈长军周明国王建新
Owner NANJING AGRICULTURAL UNIVERSITY
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