A multiplex real-time PCR assay for simultaneous detection and differentiation of Ditylenchus dipsaci, D. gigas, and D. weischeri.

Abstract

The stem nematodes, Ditylenchus dipsaci, D. gigas, and D. weischeri, are closely related species that were previously considered as different races of D. dipsaci species complex. The fourth-stage (J4) of these species can survive many years of desiccation in plant tissue, seed, and soil in a state of cryptobiosis. Dry seeds of the host plants carrying these pests are an important means of dissemination from one region to another. Ditylenchus dipsaci and D. gigas are regulated quarantine pests in many countries and subject to inspection during international trade of seed and grain, whereas D. weischeri, a parasite of creeping thistle or Canada thistle Cirsium arvense, causes little damage to agricultural crops. However, the presence of D. weischeri in field pea grain contaminated with D. weischeri-infested thistle seed can cause confusion due to the morphological similarity of J4 stages among these three species. A TaqMan-based multiplex real-time PCR assay was developed in this study to simultaneously differentiate D. dipsaci, D. gigas, and D. weischeri. Primers and species-specific probes, targeting the heat shock protein (hsp90) gene, successfully detected and identified single nematode of several populations of the Ditylenchus species, alone or in mixture. This rapid, sensitive, and species-specific quantitative PCR assay presents a reliable tool for regulatory response and management programs.