LAUSR

Abstract Septoria leaf blotch caused by Zymoseptoria tritici is a common and serious disease of winter wheat. In many wheat growing areas QoI (strobilurins) are widely used to control the disease. Due to the singlesite mode of action many plant pathogenic fungi develop QoI resistance. The most common mutation related with strobilurin resistance is glycine to alanine substitution at 143 codon position (G143A) in mitochondrial cytochrome b gene. In the study we describe a novel diagnostic protocol based on next-generation sequencing (NGS) to detect the G143A mutation in Z. tritici . In 2014, 2015 randomly chosen isolates (40) were collected from winter wheat in 26 independent locations in western Poland. Genomic DNA was extracted from fresh mycelium in 15 min procedure. The NGS library was prepared from PCR amplicons of 251 bp fragment of cytochrome b gene containing G143A mutation site. The sequencing in the paired-end (PE) mode was performed on Illumina MiSeq. Having done primer and quality trimming, paired reads were joined, resulting in a total of 722,692 paired sequences of ∼211 bp fragment. The obtained sequences were mapped to a complete genome reference sequence (EU090238.1) of the Z. tritici mitochondrion. The QoI sensitive cytochrome b variant (G143) was found in 45%, while the resistant variant (A143) was noted in 55% of the sequences analysed. The NGS assays proved to be very successful in detection and characterisation of drug resistance mutation. In conclusion, we postulate that NGS technology enables quick, accurate and large-scale monitoring, particularly in mixed populations of sensitive and resistant pathogens. To our knowledge, this is the first report describing the use of NGS technology for genotypic QoI resistance in crop pathogenic fungi.