Molecular Characterization of Benomyl-resistant Isolates of Mycosphaerella fijiensis, Collected in Colombia

Benzimidazoles are systemic fungicides widely used for the control of Black Sigatoka. They exert their antifungal activity by binding to b-tubulin and disrupting fungal cell division. The specific mode of action of benzimidazoles, along with their intensive use, has favoured the appearance of resistance in Mycosphaerella fijiensis, a situation which has precluded their use in several countries. Studies made in different fungi have identified several single nucleotide polymorphisms (SNP) associated with resistance to these fungicides. Despite the economic importance of this disease and the appearance of resistance to benzimidazoles, studies of the molecular mechanisms involved in resistance of this fungus have not been carried out. In this study, a fragment of the b-tubulin gene of 10M. fijiensis isolates with three different levels of resistance to benomyl, was amplified and sequenced. The analysis of the sequences showed a change of adenine instead of cytosine in codon 198 of all the b-tubulin genes obtained from the isolates classified as having medium or high resistance. The single nucleotide change found, allowed the development of a molecular method based on polymerase chain reaction amplification and restriction digestion, which is able to differentiate resistant from susceptible isolates. The method was validated on a total of 44 isolates collected from different regions from Colombia, showing a 100% correlation between the presence of the resistant allele and the ability of the fungal isolate to grow in at least 10 lg a.i./ml benomyl. This method will be a valuable tool for monitoring benomyl resistance in M. fijiensis.