High-Resolution Melting (HRM) Analysis Reveals Genotypic Differentiation of Venturia inaequalis Populations in Greece

Michael Chatzidimopoulos,Olga Ntantali,Evangelos K Vellios,Ioannis Ganopoulos,Fenia Lioliopoulou,Panagiota Panagiotaki,Eleni Moraitou-Daponta

doi:10.3389/fevo.2019.00489

Michael Chatzidimopoulos, Olga Ntantali + Show 5 more

Open Access

https://doi.org/10.3389/fevo.2019.00489

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Abstract

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. Compared to the more complexed 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 populations according to the principal coordinate analysis (PCoA).

Highlights

Apple scab caused by Venturia inaequalis is the most important disease of apples in Greece
In order to efficiently protect the final product from apple scab, the growers rely on frequent fungicide applications on 5–9 day-intervals
Each diseased leaf was transferred to the laboratory in individual paper bags and from each sample a single isolate was made by streaking spores on sterilized potato dextrose agar (PDA) media amended with 100 mg l−1 chloramphenicol, after slight touching a flamed wire loop onto a freshly sporulating lesion

Summary

Introduction

Apple scab caused by Venturia inaequalis is the most important disease of apples in Greece. The polycyclic nature of the disease (MacHardy, 1996), the evolution of races and their ability to overcome the resistance genes in the host (Bus et al, 2011) and the development of resistance to fungicides (Köller and Wilcox, 2001) are the most important factors that lead to high variation in fungal genome and changes in population genetic structure. Because of the pathogen’s ability to adapt to environmental changes in order to survive (McDonald, 1997), many studies have analyzed its genetic structure among populations all over the world (Gladieux et al, 2010; Ebrahimi et al, 2016; Koopman et al, 2017).

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