Ferramentas moleculares para a caracterização de Colletotrichum spp. associados à antracnose da mandioca

Abstract

Anthracnose caused by Colletotrichum gloeosporioides sensu lato is an important disease in different cultures, among them cassava. Because the pathogen identification difficulty through morphological characteristics, molecular methods have been used in supporting differentiation and identification of the pathogen. This study was conducted in order to test methods of molecular species differentiation of C. gloeosporioides sensu lato which can be used which can be used prior to sequencing. Plant material with symptoms of the disease were collected in 17 cities belonging to the Recôncavo da Bahia to perform the isolation and obtaining monosporic cultures of the pathogen. Healthy detached cassava leaves were used for inoculation of the pathogen and pathogenicity testing. Two strategies were used, the first based on the grouping of isolated by the repetitive regions BOX and ERIC; and second techniques based on PCR-RFLP performed in silico and in gel. For the analysis of repetitive regions, PCR products were run on 1.5% agarose gel, followed by evaluation of the bands for each isolate. For data analysis, the band patterns shown in the gels have been converted into a matrix with '0' the absence of the band and '1' presence. The obtained binary matrices were used to estimate gnetic distance and to build the dendrograms. Regarding to PCR-RFLP, in silico analyzes were performed for determination of gene regions of greatest potential for separation of species of the complex as well as the best combinations of restriction enzymes to be used. After selecting the region in gel assays were performed (amplified by PCR and digestion with restriction enzymes). The cleavage products were observed in 2% agarose gel. For repetitives regions, the BOX-PCR showed a low polymorphism for the isolate tested, whereas the amplification based on ERIC-CRP showed a greater degree of polymorphism, and allowed a better grouping individuals and separation of some lineages within C. gloeosporioides complex. The combined use of data BOX and ERIC-PCR reduced the discriminatory capacity presented only by ERIC. In relation to PCR-RFLP technique, the combination (region of interest + restriction enzyme) that showed the highest potential discriminatory phylogenetic lineages was Calmodulin (CAL), in the in silico analysis. Given that the enzymes that showed better results were AluI, BsuRI, HhaI, HinfI, MspI and TaqI. The validation of these analyzes in gel allowed to observe the same expected patterns of in silico analysis, including allowing the use of data "hybrid", ie, estimation of probable lineage phylogenetic of individuals based on the expected pattern for each species of the C. gloeosporioides complex generated by in silico analysis, comparing them with the band patterns obtained in the in gel analysis. The use of ERIC-PCR and PCR-RFLP CAL was effective in differentiating phylogenetic lineages belonging to the C. gloeosporioides complex and can be used as a tool in identifying and alternative to sequencing.