Desenvolvimento de iniciadores específicos para identificação de espécies de Macrophomina e diversidade de espécies associadas ao feijão-caupi no Nordeste brasileiro

Abstract

Macrophomina is a fungal genus belonging to the Botryosphaeriaceae family until recently considered monotypic, represented by M. phaseolina. However, after phylogenetic studies based on DNA sequences, three new species have been described: M. euphorbiicola, M. pseudophaseolina and M. vaccinii. To identify Macrophomina species on a large scale and at low cost is necessary to develop specific primers. Charcoal rot, caused by Macrophomina, is considered one of the main diseases of cowpea [Vigna unguiculata (L.) Walp.] in Northeast Brazil, mainly in semiarid regions. For many years, only M. phaseolina has been associated with this disease in Brazil and worldwide. In this context, this thesis aimed to: (i) develop specific primers and evaluate their effectiveness in identifying Macrophomina species; (ii) identify the Macrophomina species associated with the charcoal rot of cowpea in Northeastern Brazil with specific primers; (iii) characterize the prevalence of Macrophomina species associated with cowpea in Northeastern Brazil; (iv) to analyze the pathogenicity and the aggressiveness of isolates representative of the species. The specific primers MpTefF / MpTefR, MsTefF / MsTefR, MeTefF / MeTefR developed from the TEF-1α region exhibited high specificity in amplifying only the target isolates of M. phaseolina, M. pseudophaseolina and M. euphorbiicola, respectively. With these primers, were identified 263 Macrophomina isolates obtained from cowpea plants sampled in 100 growing-fields of five states in the Northeast of Brazil. Three species of Macrophomina were identified, two isolates of M. euphorbiicola (0.8%), 205 of M. phaseolina (77.1%) and 56 of M. pseudophaseolina (21.3%). Isolates of M. phaseolina and M. pseudophaseolina were found in all sampled states, with the first species registered in 92% of the fields and the second species in 41% of the fields, while M. euphorbiicola was detected in only 2% of the fields. All isolates representing the three Macrophomina species were pathogenic to cowpea, but there was a difference in aggressiveness between species. Macrophomina phaseolina and M. pseudophaseolina were the most aggressive. This is the first report of natural infection of cowpea by M. pseudophaseolina in Brazil and by M. euphorbiicola in the world.