Differentiation of Phytophthora species associated with plantation crops using PCR and high-resolution melting curve analysis

Diseases caused by closely related species of Phytophthora such as P. palmivora, P. nicotianae, P. capsici, P. meadii, P. citrophthora and P. colocasiae are major production constraints of plantation crops in India. A reliable method for the rapid identification of these Phytophthora species is required for production of disease-free planting material and promote exports. A specific PCR assay with primer pairs designed from the ITS region effectively differentiated Phytophthora species belonging to different clades viz., P. palmivora, P. nicotianae and P. capsici. In order to differentiate closely related species (P. meadii, P. citrophthora and P. colocasiae), a high-resolution melting (HRM) curve analysis, using primers designed based on single nucleotide differences in A/T and C/A alleles was developed and validated. HRM curve analysis proved to be a fast and accurate technique for differentiation of closely related species of Phytophthora.