Identification and differentiation ofPhyllostictaspecies causing freckle disease of banana using high resolution melting (HRM) analysis

Freckle disease of banana is caused by three closely related species of Phyllosticta, namely P. musarum, P. maculata and P. cavendishii. In this study, a high resolution melting (HRM) analysis assay was developed and its potential to identify these three fungal species is reported. The assay, which targets the ITS of the nuclear rDNA of the fungal species, generates three distinct melt profiles for the three Phyllosticta species. It is also able to distinguish a combination of up to three co-infecting species by generating a deviant melt curve. Thirty-five fungal cultures and infected herbarium leaf specimens, previously characterized using nucleotide sequencing as belonging to one of the three Phyllosticta species, were used for validation of the HRM analysis assay. The normalized curves generated differentiated all samples, with samples from each species correctly identified. The assay was further evaluated against 18 uncharacterized infected leaf specimens from various geographic locations and the results were verified by subsequent nucleotide sequencing. This HRM analysis assay allows rapid identification and differentiation of the three Phyllosticta species using a single primer pair in a one-step closed-tube system without labelled fluorescence probes. This novel assay format has potential for simultaneously identifying and differentiating other closely related species of plant pathogens, as well as the classification of infected historic specimens.