Molecular species identification has become more ubiquitous in diagnostics of insects, particularly in situations where morphological identification is a laborious or time-consuming process. Tetranychid mites are serious agricultural pests. Identification of tetranychid mites is usually arduous and requires a high level of taxonomic expertise because of their minute size, their close morphological similarities as well as limited number of diagnostic characters. Most species of the spider mites of the genus Tetranychus Dufour in Iran are morphologically similar, differing only in the diameter of the aedeagal knob in males. Because this genus contains many important pests, the unambiguous identification of species is crucial for effective pest management. In this study, a single-step multiplex Polymerase Chain Reaction (multiplex PCR) was used to discriminate two predominant spider mite species occurring in greenhouses in Iran: Tetranychus urticae Koch and Tetranychus turkestani (Ugarov and Nikolskii). The single-step multiplex PCR developed here, based on ITS regions, is rapid, reliable, sensitive and relatively simple. The entire identification protocol from DNA extraction to electrophoresis could be completed in four hours. Moreover, it is adequately simple to be implemented in any molecular laboratory.

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