Rapid molecular DNA identification method for the European invasive grapevine moth Lobesia botrana
Abstract
The European Grapevine Moth (EGVM), Lobesia botrana, has been identified by the grape and wine industry as an exotic pest threat of high priority. Present throughout Europe and North America, it can cause significant harvest losses by feeding on grapevine flowers and fruit. This project developed a robust, rapid and easy-to-use molecular identification method for L. botrana, based on PCR and RFLP analysis of mitochondrial genes. In addition, a method for extracting DNA from the moth at all stages of its lifecycle (egg, larva, pupa and adult) was developed. These procedures were combined to form a new National Diagnostic Protocol for molecular identification of the European Grapevine Moth Lobesia botrana, which currently awaits review. Once nationally endorsed, this protocol will allow identification of EGVM in the event of an incursion and facilitate a rapid response by the grape and wine sector.
Summary
The aim of this project was to develop a robust, rapid and easy-to-use molecular identification method for the invasive European Grapevine Moth (EGVM) Lobesia botrana, a species which is a high priority exotic pest threat for the Australian grape and wine industry, and is difficult to identify visually. Initial work involved characterisation of the complete mitochondrial DNA (mtDNA) genome of EGVM, from which Polymerase Chain Reaction (PCR) primers were designed to amplify regions of the genes cytochrome oxidase I (COI) and cytochrome b (Cytb). Using these primers together with DNA sequencing allowed a survey of the genetic diversity of this pest moth, testing over 400 individuals sourced from both its native range across the Mediterranean basin and Asia, and from New World countries representing its recent invasive range. Extensive phylogenetic analysis based on mtDNA haplotype signatures suggests that the presence of EGVM in the Americas resulted from multiple incursion events or a single event involving at least four different maternal lineages. Use of the COI PCR primers in combination with restriction fragment length polymorphism (RFLP) analysis was able to readily distinguish individuals of L. botrana from those of other Lobesia species. However, it is recommended that DNA sequencing on the PCR amplicon be carried out to ensure accurate species identification. A DNA extraction method was refined, which can be used to obtain DNA from EGVM individuals at any stage of the life cycle (egg, larva, pupa or adult), and a simple preservation method for field-collected samples was also developed. These procedures were combined to form a National Diagnostic Protocol for molecular identification of the European Grapevine Moth Lobesia botrana, which currently awaits review. Once nationally endorsed, this protocol will allow identification of EGVM in the event of an incursion and facilitate a rapid response by the grape and wine industry.