Molecule detection method of Fusarium graminearum to medium resistance level bacterial strain of carbendazim

A technology of molecular detection of Fusarium graminearum, applied in the field of epidemic prediction of drug-resistant wheat head blight, can solve problems such as failure of chemical control, achieve the effects of controlling drug resistance, reducing environmental pollution, and reducing costs

Inactive Publication Date: 2011-03-16
NANJING AGRICULTURAL UNIVERSITY
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Problems solved by technology

[0003] Due to the huge number of pathogenic bacteria in nature, when the proportion of drug-resistant individuals in the population reaches 3% to 5%, it will cause the prevalence of drug-resistant diseases and make chemical control failure

Method used

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  • Molecule detection method of Fusarium graminearum to medium resistance level bacterial strain of carbendazim
  • Molecule detection method of Fusarium graminearum to medium resistance level bacterial strain of carbendazim
  • Molecule detection method of Fusarium graminearum to medium resistance level bacterial strain of carbendazim

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Experimental program
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Effect test

Embodiment 1

[0083] Embodiment 1, be applicable to the annealing temperature in PIRA-PCR technique

[0084] During the PCR reaction, the annealing temperature Tm value is an important guarantee for the normal progress and specificity of the reaction. If the annealing temperature is too low, non-specific amplification will occur. Referring to the Tm values ​​of the inner and outer primers, we carried out gradient PCR from 50°C to 65°C, and determined that the optimal annealing temperature of the outer primers was 58°C, and the optimal annealing temperature of the inner primers was 56°C.

Embodiment 2

[0085] Example 2, detection of known resistance levels and genotype strains

[0086] Extract bacterial strain ZF43, ZF52, NT-7 (Table 1) genomic DNA, amplify with internal and external primers, obtain amplification pattern ( figure 1 ). The amplified products were digested with HindIII and TaaI respectively, see the restriction map image 3 , the test results are consistent with the theoretical results ( figure 2 ).

Embodiment 3

[0087] Embodiment 3, Jiangsu field strain detection

[0088] Fifty strains of Gibberella tritici were randomly selected from wheat fields in Jiangsu Province, and their resistance to carbendazim was detected by traditional colony diameter method and PIRA-PCR, respectively, and 3 strains were randomly selected from various genotype strains according to the results of PIRA-PCR detection. for sequencing verification.

[0089] The results are shown in Table 2. The results of the two methods for detecting drug resistance are completely consistent. Compared with the traditional colony diameter method, primer-introduced restriction endonuclease analysis polymerase chain reaction (PIRA-PCR) only takes a few hours to complete the detection, which is simple, fast and accurate; it can also detect mutations Point SNP analysis, accurate typing. However, the traditional colony diameter method takes about two weeks from isolation and culture to detection. The PIRA-PCR typing results are s...

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Abstract

The invention belongs to a molecule detection method of Fusarium graminearum to medium resistance level bacterial strain of carbendazim. The method can be used for monitoring drug resistance of Fusarium graminearum to carbendazim and prevalence warning, wherein Fusarium graminearum can cause wheat scab. The detection method includes the following three main steps: (1) nuclear genome DNA of bacterial strain to be detected is respectively extracted: (2) internal and external primer pairs are utilized to carry out nested PCR, thus obtaining a target segment; (3) the target segment is respectively restricted by restriction enzymes HindIII and TaaI (Tsp4CI), and genotype of medium resistance level bacterial strain can be accurately identified by virtue of a restriction product electrophoresis spectrum. By adopting PIRA-PCR (primer-introduced restriction analysis PCR) technology, quantity of Fusarium graminearum with medium drug resistance in field and ratio thereof in group can be rapidly and accurately detected. Detection accuracy reaches more than 95%.

Description

1. Technical field [0001] The invention belongs to a molecular detection method for strains of Fusarium graminearum with a medium level of resistance to carbendazim, which can be used for monitoring the development trend of the benzimidazole fungicide (carbendazim)-resistant Fusarium graminearum population, Prediction of the prevalence of insecticide-resistant wheat head blight. 2. Technical background [0002] Wheat scab is a worldwide disease caused by Fusarium graminearum Schwabe, 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 include carbendazim, benomyl, thiabendazole, thiophanate, etc. As a class of high-efficiency, broad-spectrum systemic fungicides used in production, it solves the problem of environmental toxicity of protective fungicides and improves the ability of humans to control the disease. Benzimidazole fu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04
Inventor 周明国陈长军王建新罗卿权
Owner NANJING AGRICULTURAL UNIVERSITY
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