A Rapid, Simple, Laboratory and Field-Adaptable DNA Extraction and Diagnostic Method Suitable for Insect-Transmitted Plant Pathogen and Insect Identification

Abstract

Watch a presentation about this research by first author Karolina Pusz-Bochenska. Surveillance for insect-transmitted pathogens of plants involves sampling insects in the field, followed by transport of the samples to the laboratory for DNA extraction and molecular analysis. Sample transport and DNA extraction are time consuming and can delay the implementation of measures to mitigate the effects of insect-transmitted plant pathogens. Looking for a fast and reliable method to extract DNA in the field where insects were collected, we used Flinders Technology Associates PlantSaver cards, which are designed for plant DNA extraction. Insect DNA extraction can be achieved in the field, and extracted DNA can be amplified using the field-adaptable method, loop-mediated isothermal amplification (LAMP), in less than 1 h. Additionally, we demonstrate the feasibility and accuracy of the paper extraction method for molecular identification to the species level using mitochondrial cytochrome oxidase 1 amplification and sequencing on 11 genera of insects including beetles, leafhoppers, flies, psyllids, and aphids. The method was suitable for insects collected using three common methods: live-trapped and frozen, stored in ethanol, or trapped on sticky cards. Moreover, by testing leafhoppers collected in 2018 in the field, we demonstrated that the LAMP assay using the chaperonin-60 target detects a higher proportion of samples positive for ‘Candidatus Phytoplasma asteris’ than conventional PCR targeting 16S rRNA. Lastly, the paper extraction method was used to determine the prevalence of leafhoppers carrying the plant pathogenic bacterium ‘Ca. P. asteris’, which causes aster yellows from a laboratory-reared colony using PCR-based tests (conventional PCR, qPCR, and droplet digital PCR) and the non-PCR-based LAMP assay.