Development of species-specific primers for rapid diagnosis of Tetranychus urticae, T.kanzawai, T.phaselus and T.truncatus (Acari: Tetranychidae)

Species diagnosis is of the utmost importance to both pest management and plant quarantine services. Because of difficulties in the morphological diagnosis of spider mites, molecular techniques are of great value to rapidly and accurately diagnose closely related species. We examined four species of genus Tetranychus (the green and red forms of T.urticae, and T.kanzawai, T.phaselus and T.truncatus), which are found in Korea and are of significance to plant quarantine services. DNA samples isolated from a single egg, larva or adult weighed 64-188ng. We designed species-specific primers by performing sequence alignment for 107 sequences of the ribosomal internal transcribed spacer2 (ITS2) region, which we obtained from GenBank, and sequences generated in this study. Specific nucleotides of each species were selected for designing primers specific for each species. Each species-specific primer pair, when used to perform PCR analyses, detected only the species from which it originated. However, a T.urticae-specific primer pair did not discriminate between the green and red forms of this species. These species-specific primers can be applied in practice for the rapid and accurate diagnosis of spider mite species in plant quarantine and in agricultural fields.