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Open AccessArticle

High-Resolution Melting (HRM) Curve Assay for the Identification of Eight Fusarium Species Causing Ear Rot in Maize

1
Molecular Phytopathology and Mycotoxin Research, University of Goettingen, 37077 Goettingen, Germany
2
Centro para Investigaciones en Granos y Semillas, University of Costa Rica, 2060 San Jose, Costa Rica
3
Plant Pathology and Crop Protection, University of Goettingen, 37077 Goettingen, Germany
*
Authors to whom correspondence should be addressed.
Pathogens 2020, 9(4), 270; https://doi.org/10.3390/pathogens9040270
Received: 12 March 2020 / Revised: 3 April 2020 / Accepted: 5 April 2020 / Published: 7 April 2020
(This article belongs to the Section Plant Pathogens)
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. View Full-Text
Keywords: Fusarium; high-resolution melting (HRM) curves, HRM analysis; maize ear rot; fungal colony PCR; RPB2; TEF-1α Fusarium; high-resolution melting (HRM) curves, HRM analysis; maize ear rot; fungal colony PCR; RPB2; TEF-1α
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Schiwek, S.; Beule, L.; Vinas, M.; Pfordt, A.; von Tiedemann, A.; Karlovsky, P. High-Resolution Melting (HRM) Curve Assay for the Identification of Eight Fusarium Species Causing Ear Rot in Maize. Pathogens 2020, 9, 270.

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