Development and application of a droplet digital PCR assay for the detection of Fusarium oxysporum f. sp. cubense tropical race 4 from different biological samples

Abstract

AbstractFusarium wilt of banana, caused by the pathogen Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4), is a threat to food security as it affects the global banana industry. Control of this pathogen is complex due to the lack of resistant banana or plantain genotypes, lack of effective commercial fungicides, the production of chlamydospores, late appearance of symptoms in infected plants, high adaptability of the pathogen to diverse soil conditions and the polycyclic nature of the disease. Currently, the management strategy for Foc TR4 is exclusion and eradication, which is costly for the producer and the environment because the plants are treated with glyphosate and the affected area is quarantined for a long time. Thus, the development of early detection strategies for Foc TR4 from environmental samples and asymptomatic tissues with low inoculum concentration is essential; this would enable restriction of pathogen spread and minimize the environmental impact by eradication only in areas with accurate data of the pathogen's presence. In this study, three different PCR technologies (conventional PCR, quantitative PCR and droplet digital [dd] PCR) were evaluated for Foc TR4 detection in complex environmental samples. We report the development of a sensitive and specific ddPCR primer/probe set and protocols that can be used as a tool for Foc TR4 detection from symptomatic and asymptomatic plant tissue without prior DNA extraction. The limits of detection in drainage waters, footbaths and soil samples were evaluated using artificial inoculation assays. Finally, environmental samples from Foc TR4‐affected fields were analysed.