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A new multiplex polymerase chain reaction assay for simultaneous detection of five soil-borne fungal pathogens in winter wheat

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Abstract

Wheat (Triticum spp.) is often coinfected by several soil-borne fungal pathogens, causing serious crop yield and economic losses. Rapid diagnosis of infected fungi is critical for the application of disease control mechanisms. Therefore, we established a multiplex PCR method using specific primers for the simultaneous detection of five common soil-borne fungal pathogens affecting wheat: Rhizoctonia cerealis, Bipolaris sorokiniana, Gaeumannomyces graminis var. tritici, Fusarium pseudograminearum and F. graminearum. The specific primers were designed based on the sequences of several unique genes: ITS (internal transcribed spacer) of R. cerealis, URP-1F (universal rice primer) of B. sorokiniana, β-tubulin of G. graminis var. tritici and TEF1-α (translation elongation factor) of F. pseudograminearum and F. graminearum. During optimization, sensitivity assays showed that the detection limit of multiplex PCR was as low as 0.39 ng of mycelium DNA from a known pool of fungal DNA. To detect pathogens in wheat field, the multiplex PCR method was applied to infected wheat samples collected from wheat fields in the Fengqiu and Taikang counties in Henan Province, China. The R. cerealis and F. graminearum pathogens were identified in samples from Fengqiu, while the R. cerealis, G. graminis var. tritici, B. sorokiniana and F. graminearum pathogens were identified in samples from Taikang. In conclusion, the results indicated that our multiplex PCR method was efficient and specific for the detection of soil-borne pathogens.

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Funding

Funding was provided by Special Fund for Agro-scientific Research in the Public Interest (201503112) and “Five-twelfth” National Science and Technology Support Program of China (2015BAD26B01).

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Authors and Affiliations

  1. Department of Plant Pathology, Henan Agricultural University, Zhengzhou, 450002, China

    Bingjian Sun, Qingqing Chen, Xiaolun He, Yan Shi, Shengli Ding & Honglian Li

  2. National Key Laboratory of Wheat and Maize Crop Science, Zhengzhou, 450002, China

    Honglian Li

  3. Collaborative Innovation Center of Henan Grain Crops, Zhengzhou, 450002, China

    Honglian Li

Authors
  1. Bingjian Sun
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  2. Qingqing Chen
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  3. Xiaolun He
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  4. Yan Shi
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  5. Shengli Ding
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  6. Honglian Li
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Corresponding author

Correspondence to Honglian Li.

Ethics declarations

We collected the fungus in wheat field where they occurred and no specific permits were required in Henan Province, China. The land used as the collection area is neither privately owned nor protected, and the field studies did not involve endangered or protected species.

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The authors declared that they have no conflicts of interest to this work.

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Sun, B., Chen, Q., He, X. et al. A new multiplex polymerase chain reaction assay for simultaneous detection of five soil-borne fungal pathogens in winter wheat. J Plant Dis Prot 125, 319–324 (2018). https://doi.org/10.1007/s41348-017-0141-5

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  • DOI: https://doi.org/10.1007/s41348-017-0141-5

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