Rapid detection and quantification of viable potato cyst nematodes using qPCR in combination with propidium monoazide

Potato cyst nematodes (PCN), Globodera pallida and Globodera rostochiensis, are obligate parasites of solanaceous plants, causing severe losses in several potato growing areas throughout the world. To date, management of PCN is related to nematode population densities estimated as eggs per gram of soil, without considering the actual number of viable juveniles within the cysts. In classical nematology, the standard method to determine PCN viability is based on a staining assay, using Meldola's blue dye (MB) followed by microscopic visualization of MB-treated nematodes. Although MB is considered to be reliable in staining embryonated juveniles within eggs and cysts, it is a time- and labour-consuming assay. In the present work, a real-time PCR (qPCR)-based method combined with propidium monoazide (PMA), a photoreactive DNA-intercalating dye, was developed for the quantification of viable PCN. This dye renders exposed DNA of dead cells unable to be amplified by PCR, and thus only DNA from viable/intact PCN juveniles is amplified and detected. The novelty of the present method lies in the simultaneous quantitative and qualitative estimation of viable PCN inocula using species-specific primers and TaqMan probes. The PMA-qPCR viability method (v-PCR) developed for the two Globodera species successfully discriminated dead from living specimens in heat-treated samples and eggs in old and newly formed cysts. Interestingly, the detection of DNA from 34-year-old nematode cysts stored at room temperature was observed. In conclusion, the proposed v-PCR method should prove to be very useful for the routine determination of PCN viability from field samples.