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Springer, European Journal of Plant Pathology, 1(112), p. 43-52, 2005

DOI: 10.1007/s10658-004-8232-0

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Development of specific PCR primers for identification and detection of Phytophthora capsici Leon

Journal article published in 2005 by C. Silvar ORCID, J. M. Duncan, D. E. L. Cooke, N. A. Williams, J. Díaz, F. Merino
This paper is made freely available by the publisher.
This paper is made freely available by the publisher.

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Abstract

A PCR-based method was developed for the identification and detection of Phytophthora capsici in pepper plants. Three PCR primers (CAPFW, CAPRV1 and CAPRV2) specific for P. capsiciwere designed based on the sequence of its internal transcribed spacer regions. CAPFW/CAPRV1 amplify a 452bp product from P. capsici DNA whereas CAPFW/CAPRV2 a 595bp fragment; neither set amplifies DNA from pepper or several fungi pathogenic to pepper. In conventional (single-round) PCR, the limit of detection was 5pg DNA for both primer sets, whereas in nested PCR the detection limit for both was of 0.5fg. However, when the dilution series of target DNA were spiked with plant DNA, amplification declined two-fold in both conventional and nested PCR. The CAPFW/CAPRV2 set in conventional PCR was used to detect P. capsici DNA in inoculated plants. Detection occurred as soon as 8h post-inoculation in stem samples from infected but still symptomless plants. The method was also tested to detect fungal DNA in infected soils.