TaqMan REAL-Time PCR-based approach for differentiation between Globodera rostochiensis (golden nematode) and Globodera artemisiae species

Abstract

Cyst nematodes from the genus Globodera are common, and widely distributed parasites of Solaraceae. Intact cysts persist in soil even up to 10 years without detriment. Out of two Globodera species occurring in Poland, Globodera rostochiensis is considered by European and Mediterranean Plant Protection Organization (EPPO) as a quarantine pest, while Globodera artemisiae is not. Therefore, the distinction between these two species is crucial. Classic methods of detection and differentiation are laborious and time-consuming. Instead, application of molecular biology techniques allows obtaining of rapid and reliable results. The aim of this study was to establish detection and differentiation method of two species, G. rostochiensis and G. artemisiae, based upon real-time polymerase chain reaction with the use of TaqMan probes. In reaction with primers and probes specific for the nematodes' ribosomal DNA (rDNA), the samples used were DNAs isolated from the two species, alone or in mixture, as well as crushed single cysts. Applied probes enable not only to identify the species in DNA mixtures but also in a single cyst. The use of a crushed cyst eliminates long-lasting procedure of DNA isolation and reduces costs of analysis.