Journal of Agronomy and Field Innovations

Background: Seed-borne fungal infecting agents such as Fusarium, and Alternari and Aspergilli species lead to pre-emergent and post-emergent losses and seed quality issues globally. Traditional detection methods are based on morphological culturing techniques and the gel-based PCR method, both of which are tedious and slow and cannot distinguish closely related species.
Objective: This study aimed to assess the utility of the High-Resolution Melting (HRM) curve analysis approach for the identification of seed-borne infecting Fungi directly from their genomic DNA.
Methods: Genomic DNA was extracted from healthy and infected tons of seeds and reference isolates and the ITS, TEF-1α, β-tubulin, and RPB2 regions were amplified in the presence of a fluorescent dye. The melting curves were obtained for the amplified samples after heating sequences at a narrow temperature range.
Results: Several fungal species generated unique melting curves, which yielded Tm values that were spaced out by between 0.8 and 5.2°C. Thus, a high rate of similarity (over 96%) with Sanger sequencing can be achieved when applying HRM analysis, which has high sensitivity and a low SD value (below 0.15°C).
Conclusion: HRM analysis allows for quick, effective, and highly specific identification of different fungal species as an alternative to traditional sequencing methods.