Comparative-genomics-guided development of an isothermal assay for rapid detection of Fusarium oxysporum f. sp. phaseoli.

Accurate identification of closely related lineages within the Fusarium oxysporum species complex remains challenging because commonly used DNA barcode regions often provide insufficient resolution at the forma specialis level. In this study, comparative genomics approach was combined with enzyme-mediated duplex exponential amplification (EmDEA) to develop a rapid molecular assay for F. oxysporum f. sp. phaseoli (FOP). A FOP strain LM-2 was characterized by morphological, molecular, and pathogenicity analyses. A chromosome-level genome assembly of the strain comprising 14 pseudochromosomes was generated using short-read, long-read, and Hi-C sequencing. Comparative analysis with seven other F. oxysporum genomes identified 1.004 Mb of candidate diagnostic regions. A 9.82-kb region on chromosome 11 was selected, and a 1,032-bp fragment designated TC4-2 was experimentally validated and used for primer and probe design. The resulting one-tube EmDEA assay was completed at 42 °C within 30 min. Positive amplification was obtained from three tested FOP isolates, whereas no effective amplification was observed in 12 tested non-target fungi. The conservative analytical detection limit was 40 pg µL⁻¹ purified genomic DNA. The assay also detected FOP DNA in purified DNA extracted from diseased cowpea stems and field soil. These results demonstrate a comparative-genomics-guided workflow for developing rapid closed-tube assays for closely related fungal pathogens.