Use of ITS Region for Molecular Differentiation of Exobasidium vexans Causing Blister Blight in Tea

Abstract

Blister blight caused by biotrophic fungus Exobasidium vexans Massee is the most problematic leaf disease in tea (Camellia sinensis (L.) 0. Kuntze) However, there is little information available on the genetic variation of E. vexansin Sri Lanka. Understanding the molecular make-up of the pathogen and pathogen population will be helpful for identifying virulent strains, developing and deploying cultivars with durable resistance and development of appropriate disease control strategies. This study was carried out to develop a method to collect pathogen materials for DNA analysis and to test the feasibility of using Basidiomycetes specific ITS-1F and 1TS-4B primers for studying molecular diversity of E. vexans. DNA was extracted using DNeasy® mini extraction kit from Basidiospores of E. vexanscollected by spore fall technique and pure cultures of Pestolotia theae, Cladosporium sp. (Ascomycetes) and Poria hypolateritia (Basidiomycetes) for comparisons. PCR assays were carried out using ITS-F and ITS-4B primer pairs. The primer pair preferentially amplified a prominent and reproducible region (700 bp) for both of E. vexansand P. hypolateritiabut not the two ascomycetes fungi tested. The excised bands of E. vexans collected from Talawakelle and P. hypolateritiawere then used as templates in a second PCR, using the same primers and the re-PCRed product was sequenced" The DNA sequences of both species showed higher similarity (>80% ) with the DNA sequences of Basidiomycetes fungi in the Genbank. ITS sequences of E. vexansshowed 77-89% homology with other Exobasidium spp. when subjected to BLAST analysis confirming the accuracy of the amplified region. The results of this study suggest that ITS-1F and ITS-4B primer pair can be successfully used to discriminate E. vexansfrom mixed templates and to study the genetic diversity of this pathogen in Sri Lanka.