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

Maize plants are often infected with fungal pathogens of the genus . 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...

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Bibliographic Details
Published in:Pathogens (Basel) 2020-04, Vol.9 (4), p.270
Main Authors: Schiwek, Simon, Beule, Lukas, Vinas, Maria, Pfordt, Annette, von Tiedemann, Andreas, Karlovsky, Petr
Format: Article
Language:English
Subjects:
Assaying
Binding sites
Corn
Cost analysis
Deoxyribonucleic acid
Diagnostic systems
DNA
Ear rot
fungal colony PCR
Fungi
Fusarium
Gene sequencing
High resolution
high-resolution melting (HRM) curves, HRM analysis
Identification
Infections
maize ear rot
Melting
Metabolites
Nucleotide sequence
Pathogens
Polymerase chain reaction
RNA polymerase
RPB2
Species
Taxonomy
TEF-1α
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Summary:Maize plants are often infected with fungal pathogens of the genus . 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 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 pathogens of maize based on the analysis of melting curves of PCR amplicons was established. Short segments of genes and , which have been widely used in molecular taxonomy of , were amplified with universal primers in a real-time thermocycler and high-resolution melting (HRM) curves of the products were recorded. Among major pathogens of maize ears, , and , all species except for the pair could be distinguished by HRM analysis of a 304 bp segment of the gene. The latter two species could be differentiated by HRM analysis of a 247 bp segment of the 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.
ISSN:2076-0817
2076-0817
DOI:10.3390/pathogens9040270