Differentiation of phylogenetic lineages within the ‘Colletotrichum gloeosporioides species complex’ associated with cassava anthracnose disease by PCR-RFLP

Abstract

Different species were found associated with cassava anthracnose, considered a major disease of this crop. The correct identification of the causal agent is a first step for defining appropriate control strategies, such as resistant varieties. In silico analyses used sequences of six genomic regions of ex-type specimens from the ‘Colletotrichum gloeosporioides species complex’ (C.g.SC) and 21 restriction enzymes to identify phylogenetic lineages. The three best combinations of region/enzymes were validated in 18 Colletotrichum spp. isolates from cassava. Dendrograms for in silico PCR-RFLP for CAL, ITS and TUB2 showed considerable agreement with the phylogenetic analysis of each genomic region; however, the CAL gene presented greater discriminatory power. Since the band patterns from in gel analysis were almost the same as expected for the in silico analysis for the CAL region, a new approach was proposed based on the combined data from these two methodologies, allowing the differentiation of five phylogenetic lineages within the C.g.SC (C. tropicale; C. fructicola; C. siamense; C. gloeosporioides sensu stricto and C. theobromicola), and one outside of this complex (C. cliviae). This evaluation showed to be a reliable technique for preliminary identification of species prior to sequencing.