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Rapid detection of Macrophomina phaseolina in common bean seeds using a visual loop-mediated isothermal amplification assay

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Abstract

Macrophomina phaseolina (Tassi) Goid is a pathogen that causes significant economic losses by infesting Phaseolus vulgaris L. (common bean). Here, we developed a rapid, visual, and sensitive loop-mediated isothermal amplification (LAMP) assay for M. phaseolina detection in common bean seeds based on sequence-characterized amplified regions (GU018142.1). The ideal conditions for detection were obtained in 45 min at 65 °C. The LAMP assay was visually inspected for the addition of hydroxynaphthol blue dye. Positive reactions turned sky blue while the negative controls remained violet under ambient light. The detection limit was 1 pg of M. phaseolina genomic DNA per reaction, and no cross reactions were observed with DNA isolated from five other seedborne pathogens. The LAMP assay detected both common bean seeds naturally infected by M. phaseolina, and seeds artificially contaminated with the pathogen. The limit of detection was one infected seed per lot of 400 seeds. This detection method was possible without prior fungal DNA extraction, by soaking seeds 2–6 h. These results suggest that the LAMP assay provides a rapid qualitative diagnostic tool for detecting M. phaseolina in bean seeds.

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Acknowledgements

Research supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), and Fundação de Amparo à Pesquisa do Estado de Goiás (FAPEG).

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Authors and Affiliations

  1. Instituto de Ciências Biológicas, Universidade Federal de Goiás, Goiânia, Goiás, Brazil

    Dhiovanna Correa Rocha, Marilia Barros Oliveira & Silvana Petrofeza

  2. JEM Análise Agrícola, Aparecida de Goiânia, Goiás, Brazil

    Marcos Augusto de Freitas

  3. Departamento de Bioquímica e Biologia Molecular, Instituto de Ciências Biológicas, Caixa Postal 131, Campus Samambaia, Goiânia, GO, 74001-970, Brazil

    Silvana Petrofeza

Authors
  1. Dhiovanna Correa Rocha
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  2. Marilia Barros Oliveira
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  3. Marcos Augusto de Freitas
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  4. Silvana Petrofeza
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Correspondence to Silvana Petrofeza.

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Rocha, D.C., Oliveira, M.B., de Freitas, M.A. et al. Rapid detection of Macrophomina phaseolina in common bean seeds using a visual loop-mediated isothermal amplification assay. Australasian Plant Pathol. 46, 205–212 (2017). https://doi.org/10.1007/s13313-017-0477-0

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  • DOI: https://doi.org/10.1007/s13313-017-0477-0

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