Development of High-Throughput SNP Genotyping Assays for Rapid Detection of Strawberry Colletotrichum Species and the G143A Mutation

Abstract

Colletotrichum spp. cause major diseases of strawberry and disease management depends on the species present. However, species identification based on symptoms and spore morphology is difficult. Therefore, development of molecular techniques for trustworthy and high-throughput identification of Colletotrichum spp. is vital for the accurate diagnosis. A high-resolution melting (HRM) assay was developed for simultaneous identification and differentiation of Colletotrichum spp. from fungal colonies or from symptomatic strawberry tissue. HRM markers were designed based on the internal transcribed spacer region of Colletotrichum acutatum and C. gloeosporioides from strawberry, and accurately identified and differentiated the two species. In addition, for the rapid detection of a single-nucleotide polymorphism (SNP) in the cytochrome b (cytb) gene of C. acutatum and C. gloeosporioides associated with resistance to quinone-outside inhibitor fungicides, an endpoint SNP genotyping analysis was developed. The HRM and endpoint SNP genotyping assays are useful methods that can be implemented in plant diagnostic clinics for the rapid and accurate identification of Colletotrichum spp. and detection of the G143A mutation in the cytb gene of C. acutatum and C. gloeosporioides.

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

“…The HRM is mainly used in species identification like Alternaria and Aspergillus (Garganese et al, 2018;Xanthopoulou et al, 2019) or SNP detection for resistance like in Botrytis cinerea and Colletotrichum spp. (Chatzidimopoulos et al, 2014b;Forcelini et al, 2018). The data of the present assay suggest that HRM is most suitable for SSR genetic analysis in the haploid fungus V. inaequalis.…”

Method to detect and quantify colonization of anthracnose causal agent Colletotrichum gloeosporioides species complex in strawberry by real‐time PCR

“…gloeosporioides and C . acutatum by different melting temperatures of the amplicons (Forcelini et al, 2018). However, such methods are time‐consuming and require expensive laboratory equipment.…”

“…Metabolite changes induced by C. theobromicola infection were detected by gas chromatography-mass spectrometry (GC-MS) and considered as potential biomarkers for the diagnosis of strawberry anthracnose (Dai et al, 2019). In addition, a high-resolution melting assay targeting the internal transcribed spacer (ITS) gene was applied to differentiate C. gloeosporioides and C. acutatum by different melting temperatures of the amplicons (Forcelini et al, 2018). However, such methods are time-consuming and require expensive laboratory equipment.…”

Identification of sequence mutations in Phytophthora cactorum genome associated with mefenoxam resistance and development of a molecular assay for the mutant detection in strawberry (F. ×ananassa)

“…High-resolution melting (HRM) assay is a suitable and cost-effective method for identifying genetic variation, mutations, and SNP in DNA sequences 42 and could be deployed to distinguish between sensitive and resistant populations of P. cactorum to mefenoxam. In fact, this tool has been frequently used in plant pathology to identify and differentiate pathogens and detect mutations related to fungicide resistance [43][44][45][46][47] .…”