Abstract
Maize plants are often infected with fungal pathogens of the genus Fusarium. Taxonomic characterization of these species by microscopic 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-1α, 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-1α 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.
Highlights
Infection of crop plants with Fusarium spp. causes yield losses and leads to contamination of grains with mycotoxins [1]
Primers commonly used for the amplification of RPB2 and TEF-1α in taxonomy amplify
Primers were developed for the that are too long for new primers were developed for the identification of identification of eight major Fusarium species that cause ear rot in maize, namely F. cerealis, eight major
Summary
Infection of crop plants with Fusarium spp. causes yield losses and leads to contamination of grains with mycotoxins [1]. Fusarium ear rot and ear mold are cosmopolitan diseases of maize, caused by Fusarium species producing secondary metabolites toxic to mammals, which are called mycotoxins. The most important mycotoxins found in maize grains are trichothecenes, zearalenone, and fumonisins. These contaminants impair grain quality and pose a risk to food safety [2,3]. Pre-harvest ear rot disease of maize is characterized by the appearance of white or reddish fungal mycelium with rotting symptoms on the cob. The disease is classified into two groups: Gibberella ear rot, known as red ear rot, which is caused predominantly by F. graminearum, and Fusarium ear rot, known as Fusarium ear Pathogens 2020, 9, 270; doi:10.3390/pathogens9040270 www.mdpi.com/journal/pathogens
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