Differentiation of phylogenetic lineages within the ‘Colletotrichum gloeosporioides species complex’ associated with cassava anthracnose disease by PCR-RFLP
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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.
KeywordsColletotrichum spp. DNA fingerprinting DNA polymorphism in silico analysis
The authors would like to thank the Empresa Brasileira de Pesquisa Agropecuária (Embrapa) for the financial support of this project and the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for the fellowship (Master Degree) of Leandro Lopes da Silva.
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