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

Silvar, C., Duncan, J.M., Cooke, D.E.L., Williams, N.A., Diaz, J., Merino, F.
European journal of plant pathology 2005 v.112 no.1 pp. 43-52
Capsicum annuum, peppers, Phytophthora capsici, fungal diseases of plants, polymerase chain reaction, pathogen identification, disease detection, genetic markers, internal transcribed spacers, soil microorganisms
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 452 bp product from P. capsici DNA whereas CAPFW/CAPRV2 a 595 bp fragment; neither set amplifies DNA from pepper or several fungi pathogenic to pepper. In conventional (single-round) PCR, the limit of detection was 5 pg DNA for both primer sets, whereas in nested PCR the detection limit for both was of 0.5 fg. 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 8 h post-inoculation in stem samples from infected but still symptomless plants. The method was also tested to detect fungal DNA in infected soils.