Diagnostics of stem and bulb nematodes, Ditylenchus weischeri and D. dipsaci (Nematoda: Anguinidae), using PCR with species-specific primers

The stem and bulb nematodes of the Ditylenchus dipsaci (Kuhn) Filipjev species complex are obligate endoparasites of various agricultural plants, causing stunting and swelling and resulting in significant economic losses. Recently, a new closely related Ditylenchus species, D. weischeri Chizhov, Borisov and Subbotin, a parasite of the cosmopolitan herbaceous perennial weed, Cirsium arvense (L.) Scop., was described. Many countries impose quarantine restrictions for the presence of D. dipsaci in imported plant and soil materials. In the current study, we developed PCR with species-specific primers for the rapid and reliable separation of D. weischeri and D. dipsaci using gel electrophoresis and melting curve analysis. Species-specific primer sets were designed based on the nucleotide sequence of the heat shock protein (hsp90) gene for both nematode species. The PCR protocol was verified using samples of D. weischeri, D. dipsaci and the closely related species, D. gigas, which parasitizes broadbean (Vicia faba L.). The species-specific primer sets were able to detect D. weischeri and D. dipsaci from samples containing mixtures of Ditylenchus species. The PCR species-specific protocol should allow for more rapid identification of Ditylenchus species recovered from plant materials than previously possible.