An allele-specific PCR assay for detection of mutations that confer benzimidazole resistance and zoxamide sensitivity in Phyllosticta citricarpa

Phyllosticta citricarpa is the causative agent of citrus black spot (CBS), a cosmetic fungal disease that has been reported in most of the citrus-growing regions of the world. The occurrence of CBS in orchards is predominantly controlled by the application of fungicides during the fruit susceptibility period. Benzimidazoles (benomyl and carbendazim) and toluamides (ethaboxam and zoxamide) are two systemic fungicide classes suitable for the control of CBS in South Africa. Both inhibit proper assembly of the fungal β-tubulin protein during mitosis, and resistance to these fungicides is primarily related to alterations in the binding sites on this target protein. In this study, P. citricarpa isolates with known resistance status were subjected to whole genome sequencing, the mutations conferring resistance to benomyl and zoxamide were examined and allele-specific primers targeting these mutations were developed. The allele-specific multiplex PCR assay for the detection of benomyl resistance in P. citricarpa will eliminate the need for laborious and time-consuming sensitivity assays or DNA sequencing.