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A nested multiplex PCR for species-specific identification and detection of Botryosphaeriaceae species on mango

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Abstract

Lasiodiplodia theobromae (Pat.) Griff. & Maubl, Neofusicoccum parvum Pennycook & Samuels, N. mangiferae Syd. & P. Syd., and Fusicoccum aesculi Corda, all anamorphs of Botryosphaeriaceae species, are the causal agents of mango stem-end rot and fruit rot in Taiwan. Identification of these fungal species based on morphology has not been easy due to their extensive plasticity for some of the morphological characters. To aid reliable identification of Botryosphaeriaceae species associated with mango fruits, four pairs of species-specific primers were designed according to sequences of the ribosomal internal transcribed spacers (ITS), and a rapid method was established based on nested multiplex polymerase chain reaction (PCR) in this study. To perform the analysis, PCR was first run with ITS1 and ITS4 as the primers, followed by a second PCR with the addition of all four sets of species-specific primers. With this method, a low limit of 100 fg-1 pg of purified fungal DNA was detectable. It could also successfully detect L. theobromae, N. parvum, N. mangiferae and F. aesculi in total DNA extracted from inoculated mango fruits. This assay provides a rapid and sensitive method for the identification of Botryosphaeriaceae species and diagnosis of mango fruit rot and stem-end rot as well.

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Acknowledgements

This project was support by the Council of Agriculture, Executive Yuan, R. O. C. We also gratefully acknowledge Ms. S. L. Hsu and S. Y. Lai for their help in the isolation of fungal pathogens, pathogenicity test, and sequence analysis.

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Authors and Affiliations

  1. Department of Plant Protection, Chiayi Agricultural Experiment Station, Taiwan Agricultural Research Institute, Chiayi, Taiwan

    Hui-Fang Ni & Hong-Ren Yang

  2. Department of Biochemical Science and Technology, National Chiayi University, Chiayi, Taiwan

    Ruey-Shyang Chen

  3. Department of Plant Pathology and Microbiology, National Taiwan University, Taipei, Taiwan

    Hui-Fang Ni, Ting-Hsuan Hung & Ruey-Fen Liou

Authors
  1. Hui-Fang Ni
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  2. Hong-Ren Yang
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  3. Ruey-Shyang Chen
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  4. Ting-Hsuan Hung
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  5. Ruey-Fen Liou
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Correspondence to Ting-Hsuan Hung or Ruey-Fen Liou.

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Ni, HF., Yang, HR., Chen, RS. et al. A nested multiplex PCR for species-specific identification and detection of Botryosphaeriaceae species on mango. Eur J Plant Pathol 133, 819–828 (2012). https://doi.org/10.1007/s10658-012-0003-8

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  • DOI: https://doi.org/10.1007/s10658-012-0003-8

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