On-Site Amplification-Free Electrochemical Detection of Plant Pathogen Xylella fastidiosa via Cathodic Potential-Induced DNA Adsorption

Abstract

Plant diseases pose a growing threat to global food security, with invasive bacterial pathogens presenting particular challenges for early detection and containment. Xylella fastidiosa is among the most destructive of these pathogens, infecting hundreds of plant species and posing a severe biosecurity risk to agricultural systems, including those in Australia. Despite its significance, routine detection still relies on laboratory-based molecular amplification methods that are slow, costly, and poorly suited to field deployment. Here, we present an amplification-free, proof-of-concept electrochemical method for detecting X. fastidiosa DNA based on potential-induced DNA adsorption onto a screen-printed gold (SPG) electrode. Target DNA is first isolated using magnetic beads and then rapidly adsorbed onto an SPG electrode through a 30 s cathodic potential step, enabling direct differential pulse voltammetric (DPV) readout without enzymatic amplification. The method clearly discriminates the X. fastidiosa 9a5c isolate from non-specific bacterial DNA (Xanthomonas albilineans), delivering a sensitive and selective signal within 2 minutes (30 s for adsorption plus 75 s for DPV measurement). The entire assay is completed in under 30 minutes, offering approximately fourfold faster analysis than conventional molecular amplification. When applied to spiked buffer and xylem sap (i.e., a complex biological matrix) samples, the assay maintains high analytical performance, achieving a detection limit of 100 aM without compromising specificity or sensitivity. To support on-site testing, we also introduce a low-cost, 3D-printed device for rapid xylem sap extraction, allowing direct analysis with minimal handling and seamless integration into the detection workflow. Overall, the method provides a simple, rapid, and portable diagnostic strategy that advances plant pathogen detection beyond the laboratory. With further field validation, it could support earlier intervention and strengthen biosecurity surveillance for X. fastidiosa and other high-priority pathogens.