Molecular Identification and Genetic Diversity of Alternaria Isolates Causing Leaf Spot Disease in Cotton from Major Cotton Growing Areas of South Zone of India

Abstract

Background: Alternaria leaf spot caused by two species namely Alternaria macrospora and Alternaria alternata is an important foliar disease of cotton. Conidial morphology showed that most of the isolates in the study belonged to A. macrospora. Molecular confirmation is necessary to strengthen the identification of species in Alternaria. Genetic diversity study of Alternaria isolates using ISSR and hyper variable SSR primers will provide variation and grouping among the isolates collected from major areas of South Zone of India. Present study was conducted to identify Alternaria isolates at species level using molecular methods (species specific primers) and genetic diversity analysis using ITS, SSR and ISSR primers. Methods: Reported species-specific primers such as AmF and AmR as well as AaF2 and AaF3 were used for Alternaria species identification. ITS region was amplified through ITS1 and ITS4 and sequences were used for identification and clustering of isolates. Thirteen hyper variable SSR primers specific to Alternaria were designed based on the sequences retrieved from NCBI and used for diversity study. Six different ISSR primers were also used for genetic diversity study. Result: Reported species-specific primers found not suitable to identify A. macrospora and A. alternata at species level. Two SSR primers were found to be effective in showing variability among the isolates. Six clusters were formed at 71 percent genetic dissimilarity among 15 isolates of Alternaria through ISSR primers. Five clusters were formed in ITS sequence’s diversity analysis. Blasting of ITS sequences of 15 selected isolates at NCBI showed that all belong to A. alternata. This was due to absence or presence of very few sequences of A. macrospora in NCBI database itself. Further house-keeping genes like Alt a1, Plasma membrane ATPase, GAPDH and TEF -1 α sequence analysis will be useful for confirmation of A. macrospora at species level.