European Journal of Plant Pathology

, Volume 108, Issue 3, pp 197–207

Specific and Sensitive Detection of Phytophthora nicotianae By Simple and Nested-PCR

  • D. Grote
  • A. Olmos
  • A. Kofoet
  • J.J. Tuset
  • E. Bertolini
  • M. Cambra
Article

DOI: 10.1023/A:1015139410793

Cite this article as:
Grote, D., Olmos, A., Kofoet, A. et al. European Journal of Plant Pathology (2002) 108: 197. doi:10.1023/A:1015139410793

Abstract

Phytophthora nicotianae Breda de Haan is one of the most important soil-borne plant pathogens. The identification of this pathogen based on morphological or physiological characters is time-consuming and labour-intensive and requires comprehensive knowledge of fungi. Molecular analysis of the internal transcribed spacer (ITS) regions of rDNA is a novel and very effective method of species determination. Based on this concept, conventional and single closed tube nested-PCRs were developed for the specific and sensitive detection of P. nicotianae. Two new specific primers, designed from the spacer regions ITS1 and ITS2, internal to the nucleotide sequence flanked by universal primers ITS4 and ITS6, were used. To evaluate the specificity of the method, 36 morphologically characterized isolates were tested. A positive reaction, characterized by an amplification product of 737 bp, was shown by all P. nicotianae isolates and two P. nicotianae/cactorum hybrids. No amplification product was observed when other Phytophthora species and genera were assayed. The sensitivity of this method was analysed by serial dilutions of a defined amount of fungal DNA in a healthy root extract. Nested-PCR was at least 1000 times more sensitive than conventional PCR. In addition, samples from different infection sites, origins and crops, samples from nutrient solution, water and the rockwool used in hydroponic cultures, were analysed to validate this method.

DNA amplificationfungal taxonomyhydroponicsidentificationoomycetes

Copyright information

© Kluwer Academic Publishers 2002

Authors and Affiliations

  • D. Grote
    • 1
  • A. Olmos
    • 2
  • A. Kofoet
    • 1
  • J.J. Tuset
    • 2
  • E. Bertolini
    • 2
  • M. Cambra
    • 2
  1. 1.Institut für Gemüse- und Zierpflanzenbau Großbeeren/ErfurtGroßbeerenGermany
  2. 2.Departamento de Protección Vegetal y BiotecnologíaInstituto Valenciano de Investigaciones Agrarias (I.V.I.A.)Moncada, ValenciaSpain