| dc.description.abstract | Maize plants are often infected with fungal pathogens of the genus Fusarium.
Taxonomic characterization of these species bymicroscopic examination of pure cultures or assignment
to mating populations is time-consuming and requires specific expertise. Reliable taxonomic
assignment may be strengthened by the analysis of DNA sequences. Species-specific PCR assays are
available for most Fusarium pathogens, but the number of species that infect maize increases the labor
and costs required for analysis. In this work, a diagnostic assay for major Fusarium pathogens of maize
based on the analysis of melting curves of PCR amplicons was established. Short segments of genes
RPB2 and TEF-1 , which have been widely used in molecular taxonomy of Fusarium, were amplified
with universal primers in a real-time thermocycler and high-resolution melting (HRM) curves of the
products were recorded. Among major Fusarium pathogens of maize ears, F. cerealis, F. culmorum,
F. graminearum, F. equiseti, F. poae, F. temperatum, F. tricinctum, and F. verticillioides, all species except for
the pair F. culmorum/F. graminearum could be distinguished by HRM analysis of a 304 bp segment of
the RPB2 gene. The latter two species could be differentiated by HRM analysis of a 247 bp segment of
the TEF-1 gene. The assay was validated with DNA extracted from pure cultures of fungal strains,
successfully applied to total DNA extracted from infected maize ears and also to fungal mycelium that
was added directly to the PCR master mix (“colony PCR”). HRM analysis thus offers a cost-efficient
method suitable for the diagnosis of multiple fungal pathogens. | |
