A qRT-PCR method to evaluate viability of potato cyst nematode (Globodera spp.)

Potato cyst nematodes (PCN)–Globodera rostochiensis and G. pallida–cause significant yield losses on potato worldwide. One of the main challenges to PCN management is the ability of PCN to remain dormant in the soil for several decades. For that reason, many countries have strict quarantine regulations for PCN. These regulations, although expensive and restrictive for growers, are necessary to prevent further spread of PCN but should be lifted when no more viable cysts are found. Here, we report a promising qRT-PCR method for the quantification of viable eggs and propose that this method be included in routine testing. The method was successful for quantifying G. ellingtonae, G. rostochiensis and G. pallida and was found to be very sensitive with the systematic detection of a single larva. Intron-flanking probes were used to eliminate the possibility of false positives due to genomic DNA, and an internal control was added to detect failure in PCR due to inhibitors. No amplification occurred during the testing of eggs that had previously received heat treatments or fumigation with methyl bromide. This qRT-PCR assay was used to evaluate the viability of field populations of G. rostochiensis 10 years after the establishment of a quarantine area in Saint-Amable, Quebec, Canada. The number of viable eggs after a decade of regulation was found to be very low and confirmed the effectiveness of the measures put in place. Egg viability was also monitored in microplots following five continuous years of planting resistant potatoes, and no signs of resistance-breaking genotypes were observed.