Detection of the defoliating and nondefoliating pathotypes of <Emphasis Type="Italic">Verticillium dahliae</Emphasis> in artificial and natural soils by nested PCR |
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Authors: | Email author" target="_blank">Encarnación?Pérez-ArtésEmail author Jesús?Mercado-Blanco Ana?Rosa?Ruz-Carrillo Dolores?Rodríguez-Jurado Rafael?Manuel?Jiménez-Díaz |
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Institution: | (1) Departamento de Protección de Cultivos, Instituto de Agricultura Sostenible (IAS) Consejo Superior de Investigaciones Científicas (CSIC), Apartado 4084, 14080 Córdoba, Spain;(2) Escuela Técnica Superior de Ingenieros Agrónomos y de Montes, Universidad de Córdoba, Apartado 3048, 14080 Córdoba, Spain |
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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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Keywords: | cotton infested-soil molecular detection olive Verticillium wilt disease |
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