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Detection of the defoliating and nondefoliating pathotypes of Verticillium dahliae in artificial and natural soils by nested PCR

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Abstract

In Spain, Verticillium wilt, caused by Verticillium dahliae, is the most important disease of cotton and olive. Isolates of V. dahliae infecting these crops can be classified into highly virulent, defoliating (D), and mildly virulent, nondefoliating (ND), pathotypes. Infested soil is the primary source of inoculum for Verticillium wilt epidemics in cotton and olive, and severity of disease relates to the prevailing V.dahliae pathotype. In this work we have adapted the use of previously developed primer pairs specific for D and ND V. dahliae for the detection of these pathotypes by nested PCR in artificial and natural soils. Success in the detection procedure depends upon efficiency in extracting PCR-quality DNA from soil samples. We developed an efficient DNA extraction method from microsclerotia infesting the soil that includes the use of acid washed sand during the grinding process and skimmed milk to avoid co-purification of Taq-polymerase inhibitors with DNA. The specific nested-PCR procedure effectively detected 10 or more microsclerotia per gram of soil. The detection procedure has proven efficient when used with a naturally infested soil, thus demonstrating usefullness of the diagnostic method for rapid and accurate assessment of soil contamination by V. dahliae pathotypes.

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Authors and Affiliations

  1. Departamento de Protección de Cultivos, Instituto de Agricultura Sostenible (IAS) Consejo Superior de Investigaciones Científicas (CSIC), Apartado 4084, 14080, Córdoba, Spain

    Encarnación Pérez-Artés, Jesús Mercado-Blanco, Ana Rosa Ruz-Carrillo, Dolores Rodríguez-Jurado & Rafael Manuel Jiménez-Díaz

  2. Escuela Técnica Superior de Ingenieros Agrónomos y de Montes, Universidad de Córdoba, Apartado 3048, 14080, Córdoba, Spain

    Rafael Manuel Jiménez-Díaz

Authors
  1. Encarnación Pérez-Artés
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  2. Jesús Mercado-Blanco
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  3. Ana Rosa Ruz-Carrillo
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  4. Dolores Rodríguez-Jurado
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  5. Rafael Manuel Jiménez-Díaz
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Correspondence to Encarnación Pérez-Artés.

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Pérez-Artés, E., Mercado-Blanco, J., Ruz-Carrillo, A.R. et al. Detection of the defoliating and nondefoliating pathotypes of Verticillium dahliae in artificial and natural soils by nested PCR. Plant Soil 268, 349–356 (2005). https://doi.org/10.1007/s11104-004-0378-1

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  • DOI: https://doi.org/10.1007/s11104-004-0378-1

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