Exploring Oxford Nanopore technology to detect SDHI fungicide resistance in Alternaria solani populations through amplicon sequencing

Fungicide resistance has challenged the effective management of potato early blight, caused by Alternaria solani, for over twenty years. Succinate dehydrogenase inhibitor (SDHI) fungicides are among the most effective for managing early blight. Selection pressure on the pathogen population has resulted in resistance and reduced fungicide efficacy. Our previous studies identified nine missense mutations across three Sdh gene subunits (B, C, and D), along with the co-occurrence of two mutations in different Sdh subunits of single isolates. For effective resistance management, mutation detection methods should be effective, quick, and flexible. Previous PCR-based assays are unable to detect the new mutations. The advancement in third-generation sequencing technology, especially Oxford Nanopore sequencing, has brought more accessible and affordable options for in-house sequencing and yielded high-throughput data in a few hours. Nanopore sequencing has been used to detect mutations in the medical field. It has gained less attention in fungicide resistance research; however, its use has increased in recent years due to its effectiveness. In this study, we employed nanopore sequencing using single-use flow cells with a Flongle adapter to produce real-time DNA sequencing data to detect mutations associated with SDHI fungicide resistance in A. solani. Libraries were prepared using multiplex PCR amplicons of each Sdh gene subunit (B, C, and D). Amplicons were sequenced using the Flongle adapter in the MinION sequencing device. Through this approach, we detected all previously identified mutations and the co-occurrence of two mutations, with results verified by standard Sanger sequencing. This cost-effective approach developed in this study is fast, easy to use, and flexible in detecting any known and novel mutations in the Sdh gene.