A portable, quick-visualization platform for field detection of Fusarium oxysporum f. sp. cubense Tropical Race 4 (Foc TR4) in banana

Fusarium oxysporum f. sp. cubense (Foc) is a soil-borne fungal pathogen that causes Fusarium wilt of banana (FWB), leading to multiple outbreaks worldwide and resulting in severe economic losses. In the absence of any effective fungicides or soil disinfectants for this disease, rapid in-field detection of this pathogen is essential for early intervention and containment. However, current in-field detection methods still rely on instruments, making the process time-consuming and complex. Herein, we developed a portable and ultra-fast detection assay for Foc Tropical Race 4 (TR4), the most destructive Foc lineage, based on recombinase polymerase amplification (RPA) with lateral-flow dipstick (LFD) readout. We identified a TR4-specific DNA fragment as the diagnostic target and prepared crude templates using a room-temperature lysis buffer, eliminating the need for DNA purification. All RPA components, except template primer-probe mix, and magnesium acetate, were lyophilized in a single eppendorf tube to simplify the workflow and avoid step-by-step reagent addition, allowing the reaction can be conducted in the field by hand or portable metal bath. The developed RPA-LFD system offers a high specificity toward Foc TR4, showing no cross-reactivity with other Foc strains, and achieved a limit of detection of 10 fg/μL in the field. This sensitivity enables the detection of infections in asymptomatic plants at early stages. This platform provides a convenient, ultra-fast and user-friendly tool for on-site Foc TR4 surveillance and supports timely management decisions to curb the spread of FWB.