Rapid diagnosis of the invasive wax scale, Ceroplastes rusci Linnaeus (Hemiptera: Coccoidea: Coccidae) using nested PCR

The fig wax scale, Ceroplastes rusci (Linnaeus) (Hemiptera: Coccoidea: Coccidae), is an invasive fruit pest of Afrotropical origin and potentially could become a serious threat to commercial fruit crops in China. C.rusci is difficult to identify owing to the shortage of easily distinguishable morphological characters. A rapid, accurate and reliable method to identify C.rusci in quarantine work is needed to detect further spread. In the present study, we describe a nested PCR method for the molecular identification of C.rusci. The nested PCR primers were designed based on variations in the barcode region of COI sequences between C.rusci and five other Ceroplastes species. A 200-bp fragment was successfully amplified from 96 C.rusci individuals of seven geographical populations in China and Vietnam, and 13 individuals of two populations in Italy (the type country for C.rusci). These provided diagnostic bands that were not observed in any of five other Ceroplastes species widely distributed in China, namely, C.ceriferus (Fabricius), C.floridensis Comstock, C.japonicus Green, C.pseudoceriferus Green and C.rubens Maskell. Sensitivity tests revealed that diagnostic bands were generated even with a DNA template concentration of 1.5x10(-5)ng/l, and with average DNA template concentrations for adult females, single first-instar nymphs and eggs of 14.7, 6.3 and 3.0ng/l, respectively. Our study demonstrates that the molecular diagnosis of C.rusci using nested PCR is rapid and accurate and shows potential in plant quarantine programmes.