Rapid Detection of Phytophthora infestans in Late Blight-Infected Potato and Tomato Using PCR

Abstract

Late blight caused by the oomycete pathogen Phytophthora infestans is a devastating disease of potato and tomato worldwide. A rapid and accurate method for specific detection of P. infestans is necessary for determination of late blight in infected fruit, leaves, and tubers. Ribosomal DNA (rDNA) from four isolates of P. infestans representing the four genotypes US1, US6, US7, and US8 was amplified using polymerase chain reaction (PCR) and the universal primers internal transcribed spacer (ITS) 4 and ITS5. PCR products were sequenced using an automated sequencer. Sequences were aligned with published sequences from 5 other Phytophthora species, and a region specific to P. infestans was used to construct a PCR primer (PINF). Over 140 isolates representing 14 species of Phytophthora and at least 13 other genera of fungi and bacteria were used to screen the PINF primer. PCR amplification with primers PINF and ITS5 results in amplification of an approximately 600 base pair product with only isolates of P. infestans from potato and tomato, as well as isolates of P. mirabilis and P. cactorum. P. mirabilis and P. cactorum are not pathogens of potato; however, P. cactorum is a pathogen of tomato. P. infestans and P. cactorum were differentiated by restriction digests of the amplified product. The PINF primer was used with a rapid NaOH lysis technique for direct PCR of P. infestans from infected tomato and potato field samples. The PINF primer will provide a valuable tool for detection of P. infestans in potatoes and tomatoes.

Pudrición basal causada por Phytophthora capcisi en plantas de chile tratadas con vermicompost

“…La identificación molecular se realizó en el Laboratorio de Técnicas Moleculares aplicadas a la Fitoprotección del Centro de Investigación en Protección de Cultivos (CIPROC), para lo cual se extrajo ADN a partir de micelio fresco de la cepa, utilizando el método CTAB (Trout et al, 1997). Se realizó la reacción en cadena de la polimerasa (PCR) con los cebadores específicos ITS4 e ITS5, que cubren la secuencia parcial del ARN del gen ribosomal 18S, el espaciador interno transcrito 1, el ARN del gen ribosomal 5.8S, el espaciador interno transcrito 2 y la secuencia parcial del ARN del gen ribosomal 28S.…”

The differentiation of Phytophthora species that are pathogenic on potatoes by an asymmetric PCR combined with single-strand conformation polymorphism analysis

“…The stability of ITS sequences of P. infestans, P. erythroseptica and P. nicotianae has been demonstrated in previous studies. Species-specific PCR primers have been designed from these sequences and successfully used to diagnose Phytophthora isolates diverse in fungicide sensitivity, mating type, origin and pathogenicity (Tooley et al 1997;Trout et al 1997).…”

“…Although PCR assays have been specifically developed for the identification of Phytophthora species that parasitize potatoes (Niepold and Schober-Butin 1995;Tooley et al 1997;Trout et al 1997), the focus of this laboratory has been to develop a PCR assay capable of differentiating these species using a single primer pair. Once the product of these primers has been generated, a number of approaches could then be used to differentiate fungal genera and/or species.…”

Plant pathogen diagnostics: present status and future developments

“…A key part of the reaction is the Target region amplified more than 106-fold use of a thermostable polymerase, which can withstand the application of the high temperatures used in the reaction. Many PCR assays for potato and other plant pathogens have been published (Darrasse et al, 1994;Hadidi et al, 1993;Henson & French, 1993;Schneider et al, 1993;Singh & Singh, 1996, 1998Trout et al, 1997). Yet few, if any, are in large scale use.…”