Heteroduplex Mobility Assay for Identification and Phylogenetic Analysis of Anthracnose Fungi

Abstract

Colletotrichum spp. are casual agents of anthracnose on various economically important crops. To cope with the pitfalls of identifying the fungi by morphotaxonomic criteria, the application of heteroduplex mobility assay (HMA) of internal transcribed spacer (ITS) regions as a biochemical tool was explored. The ITS regions of 29 Colletotrichum isolates including Colletotrichum gloeosporioides, Colletotrichum acutatum, Colletotrichum musae, Colletotrichum graminicola, Colletotrichum capsici, Colletotrichum dematium, Colletotrichum lindemuthianum and three unidentified species of Colletotrichum, were PCR amplified. Comparison of the ITS sequences from 15 Colletotrichum isolates revealed a greater DNA divergence within ITS1 region than that within ITS2. The DNA distance and sequence identity within intra-species ranged from 0.0 to 1.1% and from 98.9 to 100%, respectively; whereas those within inter-species ranged from 1.46 to 13.43% and 90.02 to 98.56%, respectively. From the correlation of DNA distance and relative heteroduplex mobility observed among 15 reference isolates, a formula for estimation of distances of a tested DNA sequence was developed for estimation of DNA distances of a compared strain. The phylogenetic analysis of ITS regions of 29 Colletotrichum isolates using DNA distance inferred from relative heteroduplex mobility divided them into 5 distinctive species groups, namely CG, CA, CC, CM and CL, similar to that assembled based on DNA sequences analysis. Our results show that HMA of ITS regions is a relatively rapid and convenient method for species-specific identification of Colletotrichum spp. The potential use of the established techniques for identification of anthracnose and even other fungal diseases are discussed.