Development of molecular assays to detect the G143A point mutation responsible for group 11 fungicide insensitivity in Colletotrichum lentis

Anthracnose, caused by Colletotrichum lentis, is the most important foliar disease of lentil in Canada. Management relies heavily on strobilurin fungicide active ingredients. However, insensitivity to strobilurins in C. lentis has been reported in commercial lentil fields in Saskatchewan. This study describes two polymerase chain reaction (PCR) assays to detect the G143A mutation in the cytochrome b gene of C. lentis, which confers strobilurin insensitivity. In the first assay, sensitive (S) and insensitive (I) specific custom forward primers, containing either a dideoxy cytosine (ddC) or a dideoxy guanine (ddG) at the 3′-end, and a common reverse primer were used. When non-proofreading Taq polymerase was used, neither primer combination amplified the target because the ddC or ddG at the 3′-end blocked primer extension. When a small amount of high-fidelity DNA polymerase with 3′-5′ proof-reading activity was added, the ddC and ddG primers amplified DNA templates from C. lentis carrying the allele conferring strobilurin sensitivity or insensitivity, respectively. A second assay, using mismatching of the penultimate nucleotide in the primer, yielded results consistent with the dd assay. Sequencing of the partial cytochrome b gene of eight C. lentis strains and in vitro fungicide insensitivity of C. lentis strains corroborated the molecular assays. As the methods are based on conventional PCR, these have the potential to be adapted readily to differentiate between alleles with point mutations.