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Visual detection of Didymella bryoniae in cucurbit seeds using a loop-mediated isothermal amplification assay

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Abstract

A method was developed using a Loop-mediated isothermal amplification assay (LAMP) for detecting Didymella bryoniae in cucurbit seeds. The LAMP primers were designed based on the DNA-dependent RNA polymerase II RPB140 gene (RPB2) from D. bryoniae. Calcein was used as an indicator for the endpoint visual detection of DNA amplification. The LAMP assay was conducted in isothermal (65 °C) conditions within 1 h. The detection threshold of the LAMP assay was 10 pg of genomic DNA and D. bryoniae was detected in 100 % of artificially infested seedlots with 0.05 % infestation or greater. With the LAMP assay, 16 of 60 watermelon and muskmelon seedlots collected from Xinjang province were determined to be positive for D. bryoniae. In contrast, a real-time PCR assay determined that 11 of the 60 seedlots from Xinjiang province were positive for the pathogen. These results showed that the LAMP technique was simple, rapid and well suited for detecting D. bryoniae DNA, especially in seed health testing.

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Acknowledgments

This research was supported by the National Natural Science Funds of China (Grant No. 31500107), China High Technology Research and Development Project (No. 2012AA101501), China Postdoctoral Science foundation (No. 2015 M570461), Science and Technology support Xinjiang project (No. 201191133) and Jiangsu Entry-Exit Inspection and Quarantine Bureau Science and Technology (No. 2015KJ52). We thank Wu Cui-ping from Jiangsu Entry-Exit Inspection and Quarantine Bureau for providing fungal isolates.

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Authors and Affiliations

  1. College of Plant Protection and Key Laboratory of Integrated Management of Crop Diseases and Pests, Ministry of Education, Nanjing Agricultural University, Nanjing, 210095, China

    Yanli Tian, Da Liu & Baishi Hu

  2. Institute of Botany, Jiangsu Province and the Chinese Academy of Sciences, Nanjing, 210014, China

    Yuqiang Zhao

  3. Nanjing Entry-Exit Inspection and Quarantine Bureau, Nanjing, 211106, China

    Jing Wu

  4. National Engineering Research Center for Cucurbits, Changji, 831100, China

    Baishi Hu

  5. Department of Plant Pathology, University of Georgia, Athens, GA, 30602, USA

    R. R. Walcott

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  1. Yanli Tian
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  2. Da Liu
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  3. Yuqiang Zhao
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  4. Jing Wu
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  5. Baishi Hu
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  6. R. R. Walcott
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Correspondence to Baishi Hu or R. R. Walcott.

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Yanli Tian and Da Liu contributed equally to this research.

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Tian, Y., Liu, D., Zhao, Y. et al. Visual detection of Didymella bryoniae in cucurbit seeds using a loop-mediated isothermal amplification assay. Eur J Plant Pathol 147, 255–263 (2017). https://doi.org/10.1007/s10658-016-0996-5

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  • DOI: https://doi.org/10.1007/s10658-016-0996-5

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