Detection ofPhytophthoraSpecies by Oligonucleotide Hybridization to Amplified Ribosomal DNA Spacers

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

“…1 Analysis of asymmetric PCR products produced by the primer combination physscp/pProbe on a 5% nondenaturing polyacrylamide gel. Lanes 1-3: primer physscp II is used as the limiting primer in asymmetric PCR for the amplification of Phytophthora infestans isolate Mex-220A, P. nicotianae 361 and P. erythroseptica 355, respectively; in lanes 4-6: pProbe is used as the limiting primer in asymmetric PCR for the amplification of P. infestans Mex-220A, P. nicotianae 361 and P. erythroseptica 355, respectively ies of ssDNA fragments of these potato pathogens corroborated a previous report of variations in the nucleotide sequences between the 18S and 5·8S rDNA genes of Phytophthora species (Lee et al 1993).…”

“…Candidate PCR primers were chosen from sequences that flanked this site and showed the greatest similarity among the three species. The reverse primer (pProbe) used in the reaction was derived from sequences identified previously to be specific for the genus Phytophthora (Lee et al 1993). Combining pProbe with the forward primer physscpI only amplified genomic DNA products from P. infestans and P. nicotianae (Table 2).…”

“…The polymerase chain reaction (PCR) has provided the opportunity to identify rapidly some of the destructive plant pathogens of the genus Phytophthora (Lee et al 1993;Ersek et al 1994;Niepold and Schober-Butin 1995;Tooley 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. Restriction digestion of the amplicon , or hybridization with specific probes (Lee et al 1993), have been the most practical approaches to delineating the difference between genera and between species. Although the aforementioned methods are reliable, they are limited by either tedious and time-consuming protocols or costly development of probes.…”

“…Despite this homogenizing mechanism, ribosomal DNA (rDNA) from Phytophthora species has been shown to be suitable for studying these organisms, due partly to the presence of two non-coding introns or transcribed spacers Lee et al 1993;Scott et al 1997;Scott and Deahl 1998;Tooley et al , 1997. In this report, Phytophthora species pathogenic on potatoes are differentiated by amplifying ssDNA from variable regions of the internal transcribed spacer 1 (ITS I) (Lee et al 1993) using an asymmetric PCR (A-PCR) protocol and analysing the ssDNA on non-denaturing polyacrylamide gels (Scott et al 1997).…”