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

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.

Keywords: Fusarium; RPB2; TEF-1α; fungal colony PCR; high-resolution melting (HRM) curves, HRM analysis; maize ear rot.

Figure 1

Workflow of high-resolution melting (HRM) curve analysis of eight major Fusarium pathogens of maize ears. (A) Identification of Fusarium species in infected maize ear samples; (B) identification or pure cultures using fungal colony PCR. sRPB2 and sTEF-1α are short and variable subsections of RPB2 and TEF-1α, suitable for HRM analysis (Table 1). Fusarium species in brackets were not well distinguishable using the sRPB2 assay.

Figure 2

Maximum likelihood analysis of the DNA sequences used for HRM analysis. (A) sRPB2 sequences (1000 bootstrap replications) (B) sTEF-1α (without bootstrapping due to a low number of sequences (n = 3)). We included partial sequences of RPB2 of Trichoderma istrianum CBS130539 (Accession KJ665281.1) and TEF-1α of F. pseudograminearum CBS131261 (Accession JX118971.1) as outgroup references. Melting curves using normalized relative fluorescence unit (RFU) data of sRPB2 (C) and sTEF-1α (D). Melting curves were generated as negative first derivative (−d(RFU)/d(T)) of relative fluorescence. Difference curves of the reference strains and a naturally infected maize ear for sRPB2 (E) and sTEF-1α (F) are shown. The difference curves were obtained by subtracting melting curve data of each reference strain or environmental sample from the mean melting curves of all reference strains (dashed horizontal line). Vertical dashed lines indicate the maximum and minimum for each reference strain. sRPB2 and sTEF-1α are short subsections of RPB2 and TEF-1α (Table 1).

Figure 3

Fungal colony PCR followed by high-resolution melting (HRM) curve analysis of (A) sRPB2 and (B) sTEF-1α. Difference curves were obtained by subtracting melting curves of each reference strain from the mean melting curve data of all reference strains (dashed horizontal line). Vertical dashed lines indicate the maximum and minimum of the curve for each reference strain. sRPB2 and sTEF-1α are short subsections of RPB2 and TEF-1α (Table 1).