ORIGINAL ARTICLE Differentiation of Phytophthora species associated with plantation crops using PCR and high-resolution melting curve analysis Prathibha Veerappa Hanumanthappa 1 & Vinayaka Hegde 1 & Sharadraj Kuriyathadka Mahalingeshwara 1 & Rajesh Muliyar Krishna 1 & Rachana Kaitheri Edathil 1 & Chowdappa Pallem 1 # Società Italiana di Patologia Vegetale (S.I.Pa.V.) 2018 Abstract 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. Keywords Coconut . Arecanut palm . Phytophthora . HRM analysis . Rapid detection . PCR Introduction Plantation crops play an important role in employment genera- tion as well as poverty alleviation in rural India and provide livelihood security to millions of small and marginal farmers besides earning huge foreign exchange. They blend effectively with the environment contributing to sustainability, conservation of bio-diversity and stable ecosystem. In India Phytophthora diseases pose a serious challenge to production of plantation crops like coconut (bud rot), arecanut palm (fruit rot) and cocoa (black pod) and their inter crops (Sarma et al. 2002). Various species of Phytophthora viz., P. palmivora (on coconut and cocoa), P. nicotianae (cardamom), P. capsici (black pepper and cocoa), P. meadii (arecanut palm, rubber and cardamom), P. citrophthora (cocoa) and P. colocasiae (taro) have been re- ported to cause crop losses of up to 100% (Chowdappa and Chandra Mohanan 1993, 1996; Chowdappa et al. 1993). Accurate identification of the Phytophthora species affect- ing plantation crops is a key factor for adopting eco-friendly and effective control strategies (Chowdappa et al. 2016). Identification of Phytophthora species based on morphology is time consuming, labour intensive and difficult due to over- lapping and limited number of morphological criteria and the need for mycological expertise (Chowdappa et al. 2003a). In addition to morphological approaches, PCR amplification and sequencing (Bowers et al. 2007) and restriction enzyme diges- tion patterns (Chowdappa et al. 2003b; Cooke et al. 2000; Cooke and Duncan 1997) have been used to identify, charac- terize and separate closely related Phytophthora species. Whereas conventional PCR with species-specific primers was used for the identification of Phytophthora species (Drenth et al. 2006), P. capsici in capsicum (Silvar et al. 2005; Ristaino et al. 1998) and P. citrophthora in citrus (Ersek et al. 1994), the more sensitive real time PCR with a TaqMan probes was extensively employed for detection and differentiation of many Phytophthora species (Bilodeau et al. 2007). However, the molecular detec- tion of Phytophthora species affecting plantation crops such as coconut, arecanut palm and cocoa in a single round PCR assay are limited, except for two reports on restriction digestion patterns of the ITS region of rDNA (Chowdappa et al. 2003a, b). Also, there are no reports on the use of single round PCR for differentiation of two or more Phytophthora spp. * Prathibha Veerappa Hanumanthappa prathibhavh_agri@yahoo.co.in 1 ICAR-Central Plantation Crops Research Institute, Kasaragod, Kerala 671124, India Journal of Plant Pathology https://doi.org/10.1007/s42161-018-0065-3