Development of molecular techniques to identify mealybugs (Hemiptera: Pseudococcidae) of importance on grapevine in South Africa

Mealybugs (Hemiptera: Pseudococcidae) cause severe damage to many commercial crops, including grapevine. This is largely because of their ability to transmit various grapevine viral diseases, in particular grapevine leafroll-associated viruses (GLRaVs). Grapevine leafroll is one of the most wide-spread grapevine diseases worldwide. Managing the field-spread of grapevine leafroll disease requires, amongst others, stringent mealybug control. Mealybug monitoring and control methods rely on timely and accurate identification of the species present. However, proper identification of mealybug species is problematic, time-consuming and requires an expert taxonomist. In most cases, only adult females can be reliably identified morphologically. Immature insects, males and damaged specimens cannot be assigned to species. In this study, a molecular method was developed to rapidly and accurately distinguish three mealybug species associated with grapevine, namely the vine mealybug Planococcus ficus (Signoret), the citrus mealybug Planococcus citri (Risso) and the longtailed mealybug Pseudococcus longispinus (Targioni-Tozzetti). During the development of this identification method, a number of tasks were undertaken. Firstly, rapid and reliable DNA extraction methods were tested for mealybug DNA. Two rapid extraction methods were adapted and tested, namely the direct buffer method and the spot-PCR method. These methods reliably extracted DNA even from very small or damaged individuals, and could be performed in 15-20 minutes and three hours, respectively. Secondly, mealybug mitochondrial DNA from the cytochrome c oxidase subunit 1 (CO I) gene was amplified and sequenced. It was found that DNA from the 3’-end of CO I showed minimal intraspecific variation (