Multiplex Amplicon Sequencing for Detecting Mutations Associated with SDHI Fungicide Resistance in Alternaria solani using Oxford Nanopore Technology

Management of potato early blight, caused by Alternaria solani, depends heavily on the application of foliar fungicides in production regions across North America. Resistance to single-site mode of action fungicides has significantly hindered early blight management for over two decades. Five missense mutations (H278Y, H278R, H134R, H133R, D123E) in the Sdh gene were identified as causing a loss in the sensitivity to SDHI fungicides. Reduction in the sensitivity to more recently registered SDHI fungicides has been observed in A. solani isolates collected across North America. Molecular characterization of these A. solani isolates resulted in the identification of four novel missense mutations (N236I, I280V, H278L, G137V) in the Sdh gene. Several PCR assays are required to detect the original five Sdh gene mutations and these assays do not detect the four novel mutations. To detect all of the mutations simultaneously, we used an Oxford Nanopore sequencing approach. Amplicons were generated through multiplex PCR assay from the three Sdh gene subunits (B, C and D) and up to 24 samples were multiplexed before sequencing on the MinION sequencer using the Flongle adapter. All nine currently known mutations were detected after analysis of the sequencing reads and results were verified through Sanger sequencing. With this approach, we can detect any Sdh gene mutations in a single assay increasing the simplicity of mutation detection at reduced costs. This approach would be broadly applicable to other pathosystem and fungicide target sites for detecting resistance.