New species-specific quantitative PCR assays for <i>Liriomyza</i> leafminers: supporting biosecurity and in-field identification

The Agromyzidae are a diverse family of small flies whose larvae mine plant tissues, often with a high degree of host specificity. However, several species of Liriomyza-including the vegetable leafminer (Liriomyza sativae Blanchard), pea leafminer (Liriomyza huidobrensis [Blanchard]), American serpentine leafminer (Liriomyza trifolii [Burgess]) and tomato leafminer (Liriomyza bryoniae [Kaltenbach]) - are highly polyphagous and pose significant biosecurity risks to Australian agriculture. The stone leek leafminer (Liriomyza chinensis [Kato]), though host-specific to Allium spp., is also a concern. Effective surveillance and diagnosis are challenging due to the flies' small size, rapid life cycles, and morphological similarities. While previous quantitative PCR (qPCR) assays have enabled detection of L. sativae, L. trifolii, and L. huidobrensis, and the non-pest species Liriomyza brassicae (Riley) (cabbage leafminer), we extend this capability by developing species-specific assays for L. bryoniae and L. chinensis. The new assays were highly specific across 16 agromyzid species collected globally and demonstrated strong sensitivity. A revised assay for L. huidobrensis, targeting the mitochondrial ND5 region, eliminated cross-reactivity observed with the original qPCR assay. We also validated a simplified DNA extraction method suitable for field use and demonstrated successful in-field diagnosis using the portable FranklinTM three9 real-time PCR themocycler. These assays have already proven valuable during post-incursion surveys and general surveillance efforts. Together, they provide a flexible and rapid diagnostic toolkit to support the early detection and management of Liriomyza pests in Australia and elsewhere.