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Genotyping by sequencing to identify diagnostic regions in Fusarium oxysporum f.sp. cubense Tropical Race 4 and applications in disease epidemiology

Maricar Salacinas: Wageningen University and Research

<div><strong>Genotyping by sequencing to identify diagnostic regions in <em>Fusarium oxysp</em>orum f.sp. <em>cubense</em> Tropical Race 4 and applications in disease epidemiology</strong> </p> <p>The recent damage by <em>Fusarium oxysporum </em>f.sp. <em>cubense </em>Tropical Race 4 (Foc-TR4) is alarming as it destroys banana intended for domestic consumption and international trade. Containment of the pathogen is of prime importance thus strict quarantine measures have to be implemented requiring accurate detection. Genotyping by sequencing based on Diversity Array Sequencing Technology (DArT) and subsequent <em>in silico </em>analyses were utilized to identify TR4 specific genomic sequences. A single copy and non-coding genomic region was identified as TR4 unique region. From this, primers for DNA-based diagnostics were developed for Loop Mediated Isothermal Amplification (LAMP) and TaqMan probe-based diagnostics. The low-throughput LAMP assay successfully detect TR4 in infested banana tissues within minutes. TaqMan assays were used to detect soil and water infestations, revealing that irrigation water are potential source of inoculum for further spread of TR4 in disease free areas. Mapping TR4 spatial distribution in banana and non-banana soils revealed TR4 inoculum until 100cm below the surface. Furthermore, using the developed diagnostics we showed that traffic and labor in contaminated farms contributes to the dissemination of TR4 by detection of the pathogen in the footwear used in the farm, which might contribute to regional, intra-and inter-continental dissemination of TR4. Thus, the developed diagnostics are indispensable in addressing epidemiological questions related to the spread of this pathogen and provided information for banana farmers, companies, and producers to initiate improved surveillance and management strategies.</p> <p>Keywords: banana, Foc-TR4, DArT sequencing, epidemiology</div>