Development of PCR primers from internal transcribed spacer region 2 for detection of Phytophthora species infecting potatoes

Abstract

We developed PCR primers and assay methods to detect and differentiate three Phytophthora species which infect potatoes and cause late blight (Phytophthora infestans) and pink rot (P. erythroseptica and P. nicotianae) diseases. Primers based on sequence analysis of internal transcribed spacer region 2 of ribosomal DNA produced PCR products of 456 bp (P. infestans), 136 bp (P. erythroseptica), and 455 bp (P. nicotianae) and were used to detect the pathogens in potato leaf (P. infestans) and tuber (P. infestans, P. erythroseptica, and P. nicotianae) tissue with a sensitivity limit of 1 to 10 pg of DNA. Leaf and tuber tissue were processed for PCR by a rapid NaOH method as well as a method based on the use of commercially available ion-exchange columns. P. infestans primers and the rapid NaOH extraction method were used to detect late blight in artificially and naturally infected tubers of potato cultivar Red LaSoda. In sampling studies, P. infestans was detected by PCR from artificially infected tubers at 4 days postinoculation, before any visible symptoms were present. The PCR assay and direct tissue extraction methods provide tools which may be used to detect Phytophthora pathogens in potato seedlots and storages and thus limit the transmission and spread of new, aggressive strains of P. infestans in U.S. potato-growing regions.

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).…”

“…Similarly, the ITS-based PCR assay developed by Trout et al (1997) failed to differentiate two clade 1 species, although it could differentiate P. infestans and P. nicotianae. Phytophthora nicotianae has a wide host range and geographic distribution, and notably causes buckeye rot and root rot of tomato and tuber rot, pink rot and leaf and stem blight of potato (Solanum tuberosum L.) (Erwin and Ribeiro 1996;Tooley et al 1997). Although P. nicotianae is less common than late blight as a foliar disease of either tomato or potato, it is occasionally observed as a foliar disease resembling late blight (Everts 2013).…”

“…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.…”

“…This assay was found to be specific for P. infestans and the closely related species P. mirabilis and P. cactorum (Trout et al 1997). Another assay targeting the ITS region was developed for the specific purpose of differentiating three Phytophthora potato pathogens (P. infestans, P. erythroseptica and Phytophthora nicotianae) (Tooley et al 1997). Highly repetitive DNA sequences have also been targeted to improve detection sensitivity over ITS-based assays (Judelson and Tooley 2000;Llorente et al 2010).…”

“…These assays have potential for immediate utility in plant disease research and diagnostic laboratories. tools have previously been discussed, and include potato seed lot and tomato seedling testing (Tooley et al 1997;Keil et al 2010), potato and tomato late blight disease diagnostics (Trout et al 1997) and various experimental applications (Llorente et al 2010;Lees et al 2012). The numerous P. infestans diagnostic tools developed to date are not redundant; rather, each offers a unique capability relative to previously described methods.…”