Species-specific detection of viable Globodera pallida using real-time reverse transcription PCR

Abstract

Summary Globodera pallida is a major pest of potatoes worldwide. In Japan, aiming at eradication of G. pallida, control measures have been implemented in infested fields. To determine the necessity of control measures, the detection of viable G. pallida is required. However, the conventional inoculation test performed in Japan, named the ‘cup test,’ is time-consuming, and conventional PCR methods targeting DNA can detect dead individuals. In this study, we developed an intercalator-based RT-qPCR method for the rapid detection of viable G. pallida. We designed a primer set for the partial cDNA sequence of the Y45F10D.4 gene of G. pallida. This primer set successfully amplified Y45F10D.4 mRNA of all tested G. pallida populations without any cross-reactions with other species. The RT-qPCR method detected RNA corresponding to a minimum of 3.9 G. pallida eggs, and a significant negative correlation was observed between the concentrations of RNA extracted from viable eggs and the Ct values. In addition, no amplification by RT-qPCR was observed in G. pallida treated with 1,3-Dichloropropene, indicating that this method detected viable G. pallida specifically. We then compared the detection sensitivity between the cup test and RT-qPCR method using 24 soil samples, and the results showed that the detection sensitivity of the RT-qPCR method was higher than that of the cup test. The RT-qPCR method enabled the rapid and reliable detection of viable G. pallida.