A novel, high-resolution melting (HRM) analysis was developed to detect single nucleotide polymorphisms (SNPs) associated with resistance to fenhexamid (hydroxyanilides) and boscalid (succinate dehydrogenase inhibitors) in Botrytis cinerea isolates. Thirty-six single-spore isolates arising from 13 phenotypes were selected and tested for fungicide sensitivity. Germ tube elongation assays showed two distinct sensitivity levels for each fungicide. Sequencing revealed that resistance to fenhexamid was due to a nucleotide change in the erg27 gene, resulting in an amino acid replacement of phenylalanine (F) with serine (S) or valine (V) at position 412 of the protein, whereas in isolates resistant to boscalid, a nucleotide change in the sdhB gene resulted in the replacement of histidine (H) with arginine (R) or tyrosine (Y) at position 272 of the respective protein. In each case, melting curve analysis generated three distinct profiles corresponding to the presence of each nucleotide in the targeted areas. HRM analysis successfully detected and differentiated the substitutions associated with resistance to both fungicides. In vitro bioassays, direct sequencing and high-resolution melting analysis showed a 100% correlation with detection of resistance. The results demonstrate the utility of HRM analysis as a potential molecular tool for routine detection of fungicide resistance using known polymorphic genes of B. cinerea populations.

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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.

For two consecutive growing seasons (2017 and 2018), three different fungicide spray programs, each with five sprays from unrelated chemical groups, were evaluated for their effectiveness against apple scab (causal agent: Venturia inaequalis) in an experimental trial in Greece. The targeted application programs consisted of five sprays with protective and systemic fungicides from unrelated chemical groups, in alternation. The applications were started at the pink bud stage (a copper-based fungicide had previously been applied at the green bud stage) and completed at the second fruit fall to arrest the primary infections by ascospores. These five-spray programs were compared to the standard farmer practice (12 sprays per season), whereas untreated plots were used as controls. The timing of the applications was based: a) on the critical growth stage of the crop, and b) on the risk analysis for infection calculated by the software Field Climate, which incorporated meteorological data from the trial site. All the five-spray programs were of very high efficacy against apple scab, showing disease severity ratings on leaves and fruits below 1.88%. In both years, in the untreated control, the disease incidence and severity on leaves ranged from 96.5% to 99.3% and from 65.2% to 75.93%, respectively. The five-spray programs showed similar efficacy to the standard 12-application program in all cases. From the results, it becomes apparent that apple scab can be controlled effectively by five targeted applications with selected fungicides at critical growth stages of the crop.

A rapid, high-resolution melting (HRM) analysis protocol was developed to detect sequence variations associated with resistance to the QoIs, benzimidazoles and dicarboximides in Botrytis cinerea airborne inoculum. HRM analysis was applied directly in fungal DNA collected from air samplers with selective medium. Three and five different genotypes were detected and classified according to their melting profiles in BenA and bos1 genes associated with resistance to benzimidazoles and dicarboximides, respectively. The sensitivity of the methodology was evident in the case of the QoIs, where genotypes varying either by a single nucleotide polymorphism or an additional 1205-bp intron were separated accurately with a single pair of primers. The developed two-step protocol was completed in 82 min and showed reduced variation in the melting curves' formation. HRM analysis rapidly detected the major mutations found in greenhouse strains providing accurate data for successfully controlling grey mould.

During the recent years, High Resolution Melting (HRM) analysis has been developed as a rapid and accurate tool in plant disease diagnostics, species identification and SNP genotyping. This approach has been applied to analyze the genetic diversity in several plant species with molecular markers, including single sequence repeats (SSR). However, no studies have been carried out to investigate the variation of SSR in plant pathogenic fungi by using the HRM technology. In this report, the genetic structure of Venturia inaequalis populations in Greece was investigated for the first time by using six microsatellite markers. The developed HRM protocol was able to generate genotype-specific melting curves, consistent with the haploid nature of the fungus. Unknown samples were genotyped using standard samples as reference controls among multiple runs. Compared to the more complex genome of diploid plants, several limitations were avoided. The shape of the melting curves revealed differences between the genotypes in each SSR marker and showed that all the genotypes could be easily distinguished. The genetic analysis of apple scab populations revealed high genetic variation within the populations (96%), while only 4% of the total gene diversity was attributed to among-population variation. The isolates were grouped into three higher-level populations according to the principal coordinate analysis (PCoA).

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The excessive use of fungicides against Venturia inaequalis, the causal agent of apple scab, has led to the emergence of resistant populations to multiple fungicides over the years. In Greece, there is no available information on fungicide resistance, despite the fact that control failures have been reported on certain areas. An amount of 418 single-spore isolates were collected from three major apple production areas and tested for their sensitivity to eight commonly used fungicides from unrelated chemical groups. The isolates were tested on malt extract agar media enriched with the discriminatory dose of each fungicide using the point inoculation method. To define the discriminatory dose for assessing the levels of resistance, EC50 values on both spore germination and mycelial growth assays were previously determined. Isolates exhibiting high resistance to trifloxystrobin (92% in total) and difenoconazole (3%); and moderate resistance to cyprodinil (75%), dodine (28%), difenoconazole (36%), boscalid (5%), and fludioxonil (7%) were found for the first time in Greece. A small percentage of the isolates were also found less sensitive to captan (8%) and dithianon (6%). Two isolates showed various levels of resistance to all eight fungicides. Despite the occurrence of strains with multiple resistances to many fungicides, we concluded that this practical resistance in the field arose mainly due to the poor control of apple scab with trifloxystrobin and difenoconazole.