Development of a real-time PCR assay to detect and quantify the tropical root-knot nematode (Meloidogyne arenaria) in latently infected potato seed

Abstract

Tropical root-knot nematodes (M. incognita, M. arenaria and M. javanica) are a serious problem to most crops, both cultivated and non-cultivated. In the recent past, accurate detection and discrimination of tropical Meloidogyne spp. has been achieved through the development of DNA-based diagnostic assays among others real-time polymerase chain reaction (qPCR) to complement results obtained using morphological and biochemical-based diagnostic methods. However, despite the developments, most of the current methods have not been optimized to quantify and characterize the tropical Meloidogyne spp. particularly in latent infections. Currently, this is a big problem in potato seed production where latently infected tubers often get distributed resulting in the distribution of root-knot nematodes to new areas. In this study, we sought to develop and validate a diagnostic method of quantifying root-knot nematode, M. arenaria in latently infected potato seed tubers. Study findings indicated that there is a high (R2 > 0.953) and significant (P < 0.05) positive correlation between target DNA concentration and Ct values, and the assays can be used to quantify as low as 1.53/100th of DNA associated with individual juvenile nematodes. Using high resolution melting curve analysis, Meloidogyne arenaria samples produced specific melting peaks (79.32 ± 0.029°C, P < 0.05) clearly distinguishing themselves from other tropical Meloidogyne species (M. incognita 79.50 ± 0.022°C and M. javanica 79.96 ± 0.046°C). The development of the high-resolution melting curve (HRMC) analysis method for quantification and characterization of M. arenaria in this study will greatly improve on accurate screening of this pathogen during latent infections to curb potato losses often associated with distribution of infected seed. Key words: Root-knot nematode, real-time PCR, high resolution melting curve analysis, South Africa, Meloidogyne spp. and potato seed