Barcode-like heteroduplex DNA pattern as an aid for rapid identification of anthracnose fungi

We have shown the usefulness of the heteroduplex mobility assay (HMA) for phylogenetic analysis and for the discrimination of closely related Colletotrichum species. Because the heteroduplex mobility of a tested strain shows a unique banding pattern that is the function of the sequence of the referred strain, we further explored the potential use of heteroduplex DNA patterns (HPs) as DNA fingerprints for the identification of these fungi. The 29 Colletotrichum strains previously identified by HMA to be taxonomic members of CG, CA, CM, CC and CL species groups were re-examined with an emphasis on their unique heteroduplex banding patterns. The species attributes of these tested strains were characterized by HMA using ITS fragments amplified from six representative Colletotrichum strains as pairwise compared references. By comparing the unique homoduplex and heteroduplex banding patterns of each tested strain on a polyacrylamide gel with those of the respective reference strain, the species identity of tested strains was determined. The obtained barcode-like HPs classified these 29 Colletotrichum strains into 6 distinctive groups: CG1, CG2, CA, CM, CC and CL. Notably, the HPs differentiated strains CG1 and CG2, which differed in their ITS sequences by only six bases. The presented results revealed that the species-characteristic barcode-like HP classification of ITS regions is a relatively rapid and valuable system for species identification of Colletotrichum species. The potential use of the established barcode-like system for the identification of anthracnose fungi and other fungal pathogens is discussed.