High-Resolution Melting approaches towards plant fungal molecular diagnostics

Abstract

Reliable and early molecular detection of phytopathogenic fungi is crucial in an era where the expansion of global trade in plant material is undoubtedly increasing the risk of invasive outbreaks, with devastating effects in crop production. Genetic variation within and between fungal species or strains is also important for screening isolates regarding various resistance attributes. Until today many approaches have been employed in fungal diagnostics which are either laborand time-consuming or costly and of reduced sensitivity. Here, we demonstrate and review recent advances on high-resolution melting (HRM) analysis as a rapid, accurate and powerful tool, capable of differentiating even closely related fungal isolates. HRM technique is based on monitoring the melting of PCR amplicons, using saturating concentrations of a fluorescent intercalating dye that binds to double-stranded DNA. Additionally, we discuss the four case studies inferring applications of HRM analysis towards either genotyping of closely related fungal species or screening for fungicide resistance. We focus on the promising results of these studies, giving some technical considerations and describing the advantages of the application of this approach. Finally, we discuss current prospects and applications for research and development related to this innovative HRM technique in plant fungal diagnostics.

Quantitative real-time PCR and high-resolution melting (HRM) analysis for strain-specific monitoring of fluorescent pseudomonads used as biocontrol agents against soil-borne pathogens of food crops

“…In the plant pathology research field, HRM has been inferred in relatively few studies to discriminate pathogenic microorganisms such as viruses (Bester, Jooste, Maree, & Burger, 2012), bacteria (Gori, Cerboneschi, & Tegli, 2012) and fungi (Ganopoulos, Madesis, Zambounis, & Tsaftaris, 2012;Zambounis, Ganopoulos, Chatzidimopoulos, Tsaftaris, & Madesis, 2014).…”

Rapid and accurate identification of black aspergilli from grapes using high‐resolution melting (HRM) analysis

“…The method is specifically able to genotype and discriminate species, based on even single‐nucleotide polymorphisms (SNPs) and small insertion/deletions, through the analysis of the melting behaviors of double‐stranded DNA (dsDNA) . Gradual DNA denaturation resulting from incremental heating produces characteristic melting profiles that can be used to discriminate between species, as has been proved for various plants and fungi …”

A new high‐resolution melting assay for genotyping Alternaria species causing citrus brown spot

“…In this perspective, the aim of our study was to develop a rapid and specific assay for the characterization of Alternaria spp. based on high‐resolution melting (HRM), a technique already successfully utilized for DNA genotyping of fungal genera and species . The HRM technique is sensitive enough to allow the detection of single base changes (single nucleotide polymorphisms, SNPs) among otherwise identical nucleotide sequences, since it measures the rate of double‐stranded DNA dissociation to single‐stranded molecules with increasing temperature .…”