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Species-specific DNA markers for identification of two root-knot nematodes of coffee: Meloidogyne arabicida and M. izalcoensis

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Abstract

Seven root-knot nematodes (RKN), including Meloidogyne exigua, M. incognita, M. paranaensis, M. enterolobii, M. arabicida, M. izalcoensis and M. arenaria are major pathogens of coffee crop in the Americas. Species-specific primers for their identification have been developed for five of them and constitute a fast and reliable method of identification. Here we report a PCR-based assay for specific detection of M. arabicida and M. izalcoensis. Random Amplified Polymorphic DNA fragments specific for these two species were converted into sequence characterized amplified region (SCAR) markers. PCR amplification using the SCAR primers produced a specific fragment of 300 bp and 670 bp for M. arabicida and M. izalcoensis, respectively, which were absent in other coffee-associated Meloidogyne spp. tested. SCAR primers also allowed successful amplification of DNA from single second-stage juveniles (J2), males and females. In addition, these primers were able to unambiguously detect the target species in nematode suspensions extracted from soil and roots samples, in different isolates of the same species or when used in multiplex PCR reactions containing mixtures of species. These results demonstrated the effectiveness of these SCAR markers and their multiplex use with those previously developed for M. exigua, M. incognita, M. paranaensis, M. enterolobii and M. arenaria constitute an essential detection tool. This diagnostic kit will contribute for specific J2 identification of the major RKN infecting coffee from field samples in the Americas.

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Acknowledgments

This work was supported by the Brazilian agencies Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and EMBRAPA—Recursos Genéticos e Biotecnologia. V.R. Correa and M.F.A. Santos thank CAPES for their scholarships.

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Authors and Affiliations

  1. Embrapa Recursos Genéticos e Biotecnologia, Brasília, DF, 70849-970, Brazil

    Valdir Ribeiro Correa, Marcilene Fernandes Almeida dos Santos, Maria Ritta Alves Almeida & Regina Maria Dechechi Gomes Carneiro

  2. Departamento de Fitopatologia, Universidade de Brasília, Brasília, DF, 70910-900, Brazil

    Valdir Ribeiro Correa

  3. Faculdade de Agronomia & Medicina Veterinária, Universidade de Brasília, Brasília, DF, 70910-900, Brazil

    Marcilene Fernandes Almeida dos Santos & José Ricardo Peixoto

  4. INRA-UMR1355, UNS, CNRS-UMR7254, Institut Sophia Agrobiotech, 400 route des Chappes, BP167, 06903, Sophia Antipolis, France

    Philippe Castagnone-Sereno

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  1. Valdir Ribeiro Correa
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  2. Marcilene Fernandes Almeida dos Santos
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  3. Maria Ritta Alves Almeida
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  4. José Ricardo Peixoto
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  5. Philippe Castagnone-Sereno
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  6. Regina Maria Dechechi Gomes Carneiro
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Correspondence to Regina Maria Dechechi Gomes Carneiro.

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Valdir Ribeiro Correa and Marcilene Fernandes Almeida dos Santos contributed equally to this work.

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Correa, V.R., dos Santos, M.F.A., Almeida, M.R.A. et al. Species-specific DNA markers for identification of two root-knot nematodes of coffee: Meloidogyne arabicida and M. izalcoensis . Eur J Plant Pathol 137, 305–313 (2013). https://doi.org/10.1007/s10658-013-0242-3

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