Development of primers for PCR diagnosis of Xiphinema species associated with Japanese traditional ornamental trees

Abstract

Dagger nematodes (Xiphinema spp.) were detected from nine tree species in a survey conducted in three Japanese prefectures, Chiba, Saitama and Fukuoka, producing Japanese traditional ornamental trees. For PCR diagnosis of these nematodes, we designed six speciesspecific primer pairs on the basis of a known sequence of COI region of mtDNA and a known sequence of 18S small subunit to ITS1 region of rDNA. The specificity of the six designed primer pairs was confirmed from the fact that only target species or a related species was PCR amplified. As results of species-specific PCR and a BLAST search of sequences of D2–D3 expansion segments of 28S rDNA, surveyed nematodes were estimated to be X. brevicolle, X. insigne, X. parachambersi or X. hunaniense. Additionally, a primer set of multiplex PCR for comprehensive amplification of these four species and two other species (Xiphinema sp., X. bakeri) was developed.