Development of a PacBio Long-Read Sequencing Assay for High Throughput Detection of Fungicide Resistance in Zymoseptoria tritici

Abstract

Fungicide resistance has become a challenging problem in management of Septoria tritici blotch (STB), caused by Zymoseptoria tritici, the most destructive disease of winter wheat throughout western and northern Europe. To ensure the continued effectiveness of those fungicides currently used, it is essential to monitor the development and spread of such resistance in field populations of the pathogen. Since resistance to the key families of fungicides used for STB control (demethyalation inhibitors or azoles, succinate dehydrogenase inhibitors or SDHIs and Quinone outside Inhibitors or QoIs) is conferred through target-site mutations, the potential exists to monitor resistance through the molecular detection of alterations in the target site genes. As more efficient fungicides were developed and applied, the pathogen has continuously adapted through accumulating multiple target-site alterations. In order to accurately monitor these changes in field populations, it is therefore becoming increasingly important to completely sequence the targeted genes. Here we report the development of a PacBio assay that facilitates the multiplex amplification and long-read sequencing of the target gene(s) for the azole (CYP51), SDHI (Sdh B, C, and D), and QoI (cytochrome b) fungicides. The assay was developed and optimised using three Irish Z. tritici collections established in spring 2017, which capture the range of fungicide resistance present in modern European populations of Z. tritici. The sequences obtained through the PacBio assay were validated using traditional Sanger sequencing and in vitro sensitivity screenings. To further exploit the long-read and high throughput potential of PacBio sequencing, an additional nine housekeeping genes (act, BTUB, cal, cyp, EF1, GAPDH, hsp80-1, PKC, TFC1) were sequenced and used to provide comprehensive Z. tritici strain genotyping.

New insights from short and long reads sequencing to explore cytochrome b variants of Plasmopara viticola populations collected in vineyard and related to resistance to complex III inhibitors

“…To go further and analyse cyt b polymorphism at a natural population scale, NGS technologies were first benchmarked to sequence the complete cyt b from single-sporangia strains and then applied to analyse field populations of P. viticola . Still relatively poorly present in studies associated to plant pathogen resistance to fungicide and targeting usually full pathogen genomes [23,24], both short-read (Ion Torrent and Illumina) and long-read (Oxford Nanopore Technologies, ONT) technologies were tested and compared in our study. They revealed to be efficient to identify and monitor cyt b variants in natural populations but also promising to become a robust tool for decision-support in management of fungicide resistance, potentially directly in the field, in near future.…”

A highly multiplexed assay to monitor pathogenicity, fungicide resistance and gene flow in the fungal wheat pathogenZymoseptoria tritici

“…The main advantage is the ability to generate long-reads capturing significant haplotype information of individual strains revealing a series of alterations conferring increased resistance in response to different commercial fungicides. However, due to the varying amplicon sizes generated by this assay two separate multiplex PCRs were required to separate shorter and longer amplicons (Samils et al 2021). High degrees of multiplexing for amplicons and samples were recently achieved using two parallel approaches for animal and plant species.…”

“…Monitoring of fungicide resistance mutations is mainly achieved through the sequencing of target genes including the recent development of long-read sequencing assays (Samils et al 2021). A joint monitoring of virulence mutations and genetic diversity is lacking though.…”

“…Similarly, pathogen populations virulent on previously resistant crop varieties have often mutated or deleted a specific set {Citation}of genes that encode proteins recognized by the plant immune system (Kashiwa et al 2016; Hartmann et al 2017; Hartmann 2022; Olivera et al 2022). Fungicide resistance has traditionally been detected using in vitro fungicide sensitivity assays (Saville et al 2015; Samils et al 2021). Such analyses require the isolation and culturing of individual fungal strains that can then be tested for growth on media containing different fungicide concentrations.…”

“…Gene flow among Z. tritici populations is leading to significant weak differentiation at the continental scale and high local diversity (Linde et al 2002; Zhan et al 2003; Singh et al 2021). Monitoring of fungicide resistance mutations is mainly achieved through the sequencing of target genes including the recent development of long-read sequencing assays (Samils et al 2021). A joint monitoring of virulence mutations and genetic diversity is lacking though.…”