PCR Amplification of Ribosomal DNA for Species Identification in the Plant Pathogen Genus Phytophthora

Abstract

We have developed a PCR procedure to amplify DNA for quick identification of the economically important species from each of the six taxonomic groups in the plant pathogen genusPhytophthora. This procedure involves amplification of the 5.8S ribosomal DNA gene and internal transcribed spacers (ITS) with the ITS primers ITS 5 and ITS 4. Restriction digests of the amplified DNA products were conducted with the restriction enzymesRsaI, MspI, and HaeIII. Restriction fragment patterns were similar after digestions with RsaI for the following species: P. capsici andP. citricola; P. infestans,P. cactorum, and P. mirabilis;P. fragariae, P. cinnamomi, andP. megasperma from peach; P. palmivora, P. citrophthora, P. erythroseptica, and P. cryptogea; andP. megasperma from raspberry and P. sojae. Restriction digests with MspI separatedP. capsici from P. citricola and separated P. cactorum from P. infestans and P. mirabilis. Restriction digests with HaeIII separated P. citrophthorafrom P. cryptogea, P. cinnamomi fromP. fragariae and P. megasperma on peach, P. palmivora from P. citrophthora, and P. megasperma on raspberry from P. sojae. P. infestans and P. mirabilis digests were identical and P. cryptogea and P. erythroseptica digests were identical with all restriction enzymes tested. A unique DNA sequence from the ITS region I in P. capsici was used to develop a primer called PCAP. The PCAP primer was used in PCRs with ITS 1 and amplified only isolates of P. capsici,P. citricola, and P. citrophthoraand not 13 other species in the genus. Restriction digests withMspI separated P. capsici from the other two species. PCR was superior to traditional isolation methods for detection of P. capsici in infected bell pepper tissue in field samples. The techniques described will provide a powerful tool for identification of the major species in the genusPhytophthora.

Loop‐mediated isothermal amplification for detection of the tomato and potato late blight pathogen, P hytophthora infestans

“…A portion of the P. infestans 5Á8S subunit and ITS2 region of rDNA was chosen as the target for the first set of LAMP primers (ITSII primers). This region was chosen because of the sensitivity and specificity achieved with other previously developed PCR and qPCR assays (Tooley et al 1997;Trout et al 1997;Ristaino et al 1998;Lees et al 2012). The LAMP ITSII primers had sensitivity comparable to several other published LAMP assays (limit of detection of 2 pg P. infestans DNA).…”

“…Potential applications of such diagnostic repetitive sequence of P. infestans DNA. The internal transcribed spacer (ITS) regions of ribosomal DNA are commonly used in diagnostics due to their relatively high copy number and species specificity (Cassidy et al 1984;Russell et al 1984;Tooley et al 1997;Trout et al 1997;Liew et al 1998;Ristaino et al 1998;Lees et al 2012;Hussain et al 2013). Following the work of Niepold and Sch€ ober-Butin, an ITS-targeting PCR assay was developed and tested for specificity.…”

Rapid identification of Phytophthora ramorum using PCR-SSCP analysis of ribosomal DNA ITS-1

“…Further determination of other species as causal pathogens is possible by comparing SSCP profiles with the Phytophthora species examined in this study and reported previously (Kong et al 2003). These features make SSCP analysis a superior technique to other existing molecular fingerprinting methods such as restriction fragment length polymorphism (RFLP) (Förster et al 1989;Ristaino et al 1998) and isozyme analysis (Nygaard et al 1989;Oudemans and Coffey 1991); both methods usually require examining several molecular profiles to key an isolate to species.…”

“…SSCP patterns in silver-stained gels can be differentiated visually without specialized equipment normally required by other DNA fingerprinting methods for differentiation of Phytophthora spp. (Förster et al 1989;Tooley et al 1997;Ristaino et al 1998 Fig. 4 Single-strand conformation polymorphism profiles of ITS-1 for Phytophthora ramorum (Ram) and its ecologically close associates.…”

Evaluation of amplified ribosomal DNA restriction analysis as a method for the identification of Botryosphaeria species

“…The ARDRA procedure has been widely used for the identification of fungal phytopathogens, e.g. [25,26,31] and clinical fungi, e.g. [27,28].…”