Detection and Molecular Characterization of Benzimidazole Resistance Among Colletotrichum truncatum Isolates Infecting Bell Pepper in Trinidad.

Abstract

Anthracnose is an economically important disease that affects pepper (Capsicum spp.) production worldwide. Eighty-seven Colletotrichum truncatum isolates infecting bell pepper in Trinidad were isolated and screened for resistance to benomyl. All isolates were found to be highly resistant at the discriminatory dose of 10.0 μg/ml. The effective concentration required to achieve 50% colony growth inhibition values were found to be significantly higher (P ≤ 0.05) for isolates collected in South Trinidad compared with those collected in North Trinidad. Isolates with the resistant phenotype had a single amino acid substitution from glutamic acid to alanine at position 198 (E198A) within the β-tubulin 2 gene. Single-nucleotide polymorphisms that result in amino acid substitutions in the β-tubulin 2 protein are associated with high resistance to benzimidazole chemistries. There were also two other deduced amino acid changes at nucleotide positions 359 to 361 (ATA/TTG [F270Y]) and at 362 to 364 (CGC/GCC [A271S]). Genetic analysis revealed that benomyl-sensitive isolates clustered separately from the resistant isolates regardless of species, with strong bootstrap support (85%). Within the resistance cluster, there was an apparent differentiation among those isolates with the F200Y, E198A, and E198K genotypes, with moderate support (>60%) for clustering of the F200Y and E198K genotypes. C. truncatum clustered separately (97%) from the other resistant genotypes due to the additional amino acid substitutions detected. The findings also indicated that consistent benzimidazole fungicide use may explain the predominance of the C. truncatum pathogen populations in bell pepper fields in Trinidad because sensitive C. gloeosporioides sensu lato isolates were selectively minimized. This underlines the importance of accurate identification of Colletotrichum spp. associated with anthracnose disease and routine monitoring for development of fungicide resistance.