A Molecular Assay Allows the Simultaneous Detection of 12 Fungi Causing Fruit Rot in Cranberry

Abstract

Cranberry fruit rot (CFR) is arguably one of the most limiting factors of cranberry (Vaccinium macrocarpon) production throughout its growing areas. The disease is caused by a group of closely related fungi that require identification using long and cumbersome steps of isolation and microscopic observations of structural features. The objective of this study was to develop a molecular assay to simultaneously detect and discriminate 12 of the most important fungal species reported to be pathogenic on cranberry fruit to facilitate the diagnosis of CFR. As the first approach, internal transcribed spacers and large subunit regions of all fungi were sequenced and confirmed with sequences available in the NCBI database. These data were used to develop primers able to differentiate seven of the 12 species. The five remaining species, including three in the Phacidiaceae family and two in the Glomerellaceae family, were differentiated on the basis of a more discriminant marker, the translation elongation factor 1-α. Two PCR reactions were optimized to clearly delineate the 12 species. The multiplex test was first validated using pure fungal cultures; it was subsequently validated using fruit collected in cranberry beds in eastern Canada. In the latter case, the test was rigorous enough to clearly discriminate the fungal pathogens from contaminants. Within the tested samples, Physalospora vaccinii and Coleophoma empetri were most commonly found. This molecular test offers scientists, diagnosticians, and growers a powerful tool that can rapidly and precisely identify fungi causing CFR so they can implement appropriate control methods.