Significant plant pests such as fruit flies that travel with fresh produce between countries as eggs or larvae pose a great economic threat to the agriculture and fruit industry worldwide. Time‐limited and expensive quarantine decisions require accurate identification of such pests. Immature stages are often impossible to identify, making them a serious concern for biosecurity agencies. Use of COI barcoding PCR, often the only molecular identification resource, is time‐consuming. We assess the suitability of the COI barcoding region for real‐time PCR assays to identify four pest fruit fly species (Family: Tephritidae), in a diagnostic framework. These species, namely Mediterranean fruit fly (Ceratitis capitata), Queensland fruit fly (Bactrocera tryoni), African invader fly (Bactrocera invadens) and Island fly (Dirioxa pornia) each provide a different set of genetic species delimitation problems. We discuss the benefits and limitations of using a single‐gene TaqMan™ real‐time approach for such species. Our results indicate that COI‐based TaqMan™ real‐time PCR assays, in particular for genetically distinct species, provide an accurate, sensitive and rapid diagnostic tool.

The tomato potato psyllid (TPP) Bactericera cockerelli (Hemiptera Triozidae) was first notified to the New Zealand Ministry of Agriculture and Forestry (MAF) in May 2006 although it has been suggested by several authors to have been present in New Zealand in 2005 MAF undertook an entry pathway analysis during the initial investigation into TPP in 2006 TPP is a vector of the bacteriumlike pathogen Candidatus Liberibacter solanacearum (liberibacter) and MAF further analysed the entry pathway of TPP during the liberibacter incursion response in 2008 This paper summarises the data and reasoning behind the conclusion that TPP was most plausibly introduced to New Zealand as a result of smuggling rather than through slippage on regulated pathways

Spotted wing drosophila (Drosophila suzukii) is an emerging pest that began spreading in 2008 and its distribution now includes 13 countries across two continents. Countries where it is established have reported significant economic losses of fresh produce, such as cherries due to this species of fly. At larval stages, it is impossible to identify due to its striking similarities with other cosmopolitan and harmless drosophilids. Molecular methods allow identification but the current technique of DNA barcoding is time consuming. We developed and validated a rapid, highly sensitive and specific assay based on real-time PCR and high resolution melt (HRM) analysis using EvaGreen DNA intercalating dye chemistry. Performance characteristics of this qualitative assay, validation and applicability in a New Zealand quarantine framework are discussed. Application of this robust and independently validated assay across the spectrum of key food production and border protection industries will allow us to reduce the further spread of this damaging species worldwide.

Spider mites of the genus Tetranychus are difficult to identify due to their limited diagnostic characters. Many of them are morphologically similar and males are needed for species-level identification. Tetranychus urticae is a common interception and non-regulated pest at New Zealand’s borders, however, most of the intercepted specimens are females and the identification was left at Tetranychus sp. Consequently, the shipments need to be fumigated. DNA sequencing and PCR-restriction fragment length polymorphism (PCR-RFLP) protocols could be used to facilitate the accurate identification. However, in the context of border security practiced in New Zealand, insect identifications are required to be provided within four hours of receiving the samples; thus, those molecular methods are not sufficient to meet this requirement. Therefore, a real-time PCR TaqMan assay was developed for identification of T. urticae by amplification of a 142 bp Internal Transcribed Spacer (ITS) 1 sequence. The developed assay is rapid, detects all life stages of T. urticae within three hours, and does not react with closely related species. Plasmid DNA containing ITS1 sequence of T. uritcae was serially diluted and used as standards in the real-time PCR assay. The quantification cycle (Cq) value of the assay depicted a strong linear relationship with T. urticae DNA content, with a regression coefficient of 0.99 and efficiency of 98%. The detection limit was estimated to be ten copies of the T. urticae target region. The assay was validated against a range of T. urticae specimens from various countries and hosts in a blind panel test. Therefore the application of the assay at New Zealand will reduce the unnecessary fumigation and be beneficial to both the importers and exporters. It is expected that the implementation of this real-time PCR assay would have wide applications in diagnostic and research agencies worldwide.

Rapid and accurate identification of organisms is crucial to many research and applied outcomes. Diagnostics is a critical first step in determining the significance of suspected biosecurity threats posed by emergency plant pests and other invasive pests and pathogens. Traditionally, the biological specimens needing identification are physically mailed to a dispersed community of taxonomic experts for determination. While effective, this is an expensive, labour-intensive and slow process, often taking days to receive a confirmed identification. Remote microscopy creates virtual, real-time networks of experts using web-based cameras mounted on microscopes that allow interactive access to real-time images of scientific specimens from anywhere in the world via the Internet. Trials conducted by the Australian Quarantine and Inspection Service and the Ministry of Agriculture and Forestry Biosecurity New Zealand to test the efficacy of remote microscopy in plant quarantine settings showed that in Australia a diagnosis to a level at which realistic biosecurity decisions could be made occurred on 77% of occasions, while in New Zealand high impact exotic pest status was determined during 92% of the diagnostic events, and regulatory status was determined during 96% of events. These positive results are leading towards the expansion of remote microscopy throughout Australia, New Zealand and into South-East Asia, as well as widening its role as part of online diagnostic frameworks.