Development of a simple method for simultaneous detection and differentiation of Globodera pallida and G. rostochiensis

A simple, inexpensive technique has been needed to detect and distinguish the potato cyst nematodes Globodera rostochiensis and G. pallida , major pests of potatoes. We thus developed a sensitive duplex polymerase chain reaction (PCR) method using primers for the mitochondrial DNA’s cytochrome b gene region and agarose gel electrophoresis to identify G. rostochiensis and G. pallida and an efficient method to extract nematode DNA by crushing cysts in the presence of soil impurities. These methods provided the sensitivity to detect one egg of the target species, even if the extracted DNA solution was contaminated with PCR inhibitors derived from impurities. In addition, when the two species are both present in a sample at greatly different numbers, this method aided in detecting and identifying them accurately. The method will greatly benefit inspection institutions and advance the design of strategies to control these nematodes.