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

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.