Genome Characterization and Development of Real-Time PCR Assays for Ditylenchus Dipsaci and D. Weischeri

The stem and bulb nematode Ditylenchus dipsaci is a destructive nematode pest on many crops and is internationally quarantined in many countries, whereas Ditylenchus weischeri, only known to infect a weed plant (Cirsium arvense), is an unregulated nematode species with no known economic importance. In this study, we used comparative genomics to identify multiple gene regions and developed novel real-time PCR assays for the detection of D. dipsaci and D. weischeri. We sequenced the genomes of two mixed-stage nematode populations of D. dipsaci and two mixed-stage nematode populations of D. weischeri. The assembled genomes of D. dipsaci were 228.2 Mb and 239.5 Mb, and the genomes of D. weischeri were 177.0 Mb and 196.3 Mb. Depending on the species, 21,403-27,365 gene models were predicted. Using orthologous group analysis, single-copy and species-specific genes were identified. Primers and probes were designed targeting two species-specific genes in each species. The assays detected as low as 12 pg of DNA from the target species, or as few as five nematodes, with a C_q of 31 cycles or less. Our study provides genome data for two additional D. dipsaci isolates and two D. weischeri isolates, and four new and validated molecular assays to be used for rapid detection and identification of the two species.