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

Area of Science:

• Plant Pathology
• Molecular Biology
• Mycology

Background:

• Maize is susceptible to infection by various *Fusarium* fungal pathogens.
• Traditional methods for *Fusarium* species identification are labor-intensive, time-consuming, and require specialized expertise.
• Molecular methods like species-specific PCR assays exist but can be costly and inefficient for diagnosing multiple pathogens.

Purpose of the Study:

• To develop a rapid, cost-efficient diagnostic assay for major *Fusarium* pathogens affecting maize.
• To utilize high-resolution melting (HRM) analysis of PCR amplicons for species identification.
• To validate the assay's effectiveness on various sample types, including infected maize ears and direct fungal material.

Main Methods:

• Amplification of short gene segments (RPB2 and TEF-1α) using universal primers in a real-time thermocycler.
• Recording and analysis of high-resolution melting (HRM) curves of the PCR amplicons.
• Validation using DNA from pure cultures, infected maize ears, and direct fungal mycelium ('colony PCR').

Main Results:

• HRM analysis of the RPB2 gene successfully distinguished most *Fusarium* species, except for *F. culmorum* and *F. graminearum*.
• The TEF-1α gene segment allowed differentiation between *F. culmorum* and *F. graminearum* using HRM.
• The developed assay demonstrated high efficiency when applied to pure cultures, infected maize, and direct fungal samples.

Conclusions:

• HRM analysis provides a powerful and cost-effective tool for the simultaneous diagnosis of multiple *Fusarium* pathogens in maize.
• This method simplifies and accelerates the identification process compared to traditional techniques.
• The assay's versatility across different sample types enhances its practical applicability in plant disease management.