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

Abstract

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 (<1%), but sufficient interspecific variation (7-12%) to clearly delineate species. This region was then used to develop three species-specific forward primers, which were used in conjunction with a common universal reverse primer. These primers were all used in a multiplex PCR to differentially amplify DNA from each of the three species. The primers were designed such that each yielded a DNA product of different length which could be separated by electrophoresis on an agarose gel. In this manner the identity of the species could be determined. The entire identification protocol (including extraction, PCR and electrophoresis) could be completed in approximately four hours. All amplified specimens in a blind trial were correctly identified, regardless of size or condition of the specimen. The protocol is simple enough to be implemented in any molecular laboratory. This represents a considerable improvement over currently available techniques for mealybug identification, and is certain to be of great use in diagnostic identification of mealybugs in vineyards and export consignments.