Invasion genetics and development of rapid diagnostics of insect pests on traded plants

Abstract

Background: Global trade of plant products is a major driving force for the unintended spread of economically harmful insect pests. This PhD thesis aimed at (i) developing and implementing molecular tools for the on-site identification of invasive insect pests at points of entry (POEs) for plant import products as a prevention measure; and (ii) investigating the invasion history of the mosaic leafhopper Orientus ishidae, a potential vector of grapevine Flavescence doree phytoplasma. Methods: To achieve the first goal, loop-mediated isothermal amplification (LAMP)-based genetic assays for the rapid on-site identification of Bemisia tabaci, Thrips palmi and several invasive fruit flies of the genera Bactrocera and Zeugodacus were developed. Using publicly available DNA sequences, LAMP primers were designed to specifically target a fragment of the mitochondrial cytochrome c oxidase subunit 1 gene. To address the second goal of this PhD thesis, the invasion genetics of O. ishidae was studied, an invasive insect species that spread from its native range from in East Asia to North America in the first half of the 20th century and only recently colonised Europe. Possible source populations and invasion pathways were investigated by assessing the genetic structure of 41 O. ishidae populations from Asia, Europe, and North America based on a mitochondrial marker and 641 single nucleotide polymorphisms (SNPs) generated by double digest restriction-site associated DNA (ddRAD) sequencing. Results: Validation performed under laboratory and on-site conditions demonstrated the robustness and reliability of the developed LAMP identification assays. Analysing 319 insect specimens, the overall diagnostic test efficiency was 98% and the overall diagnostic test specificity was 100%. The small number of false-negative results (2%) originated either from previously unknown biotypes, not included in the initial primer design, or from handling errors during LAMP preparation. The results from the molecular genetic analyses of O. ishidae revealed a clear genetic separation between a native population from Asia and the non-native populations from Europe and North America. Among the non-native populations, only faint signals of spatial genetic structuring were found. However, when comparing non-native populations from Europe and North America, elevated levels of admixture of genetically distant mitochondrial haplotypes were observed for European populations. Conclusion: Characterised by high analysis speed (<1 h) and simplicity in use (only 1 pipetting step), the validated LAMP assays were found to be suitable identification tools for on-site application by plant health inspectors. Since completion of the validation phase, the developed identification assays are routinely deployed in the phytosanitary import control process of Switzerland. The considerable genetic separation between native and non-native populations of O. ishidae together with the strikingly high genetic similarity of European and North American populations suggest an invasion scenario in which North American populations served as source for the European invasion. A slightly reduced genetic structure combined with increased admixture of genetically distant mitochondrial haplotypes furthermore indicate that the European colonisation history was shaped by multiple introductions from North America, complemented by frequent intra-European gene flow. Taken together, it is hypothesised that the overall genetic complexity of non-native populations was strongly driven by frequent international trade of plants infested by O. ishidae.