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

Abstract

Crop pathogens pose severe risks to global food production due to the rapid rise of resistance to pesticides and host resistance breakdowns. Predicting future risks requires monitoring tools to identify changes in the genetic composition of pathogen populations. Here we report the design of a microfluidics-based amplicon sequencing assay to multiplex 798 loci targeting virulence and fungicide resistance genes, and randomly selected genome-wide markers for the fungal pathogen Zymoseptoria tritici. The fungus causes one of the most devastating diseases on wheat showing rapid adaptation to fungicides and host resistance. We optimized the primer design by integrating polymorphism data from 632 genomes of the same species. To test the performance of the assay, we genotyped 192 samples in two replicates. Analysis of the short-read sequence data generated by the assay showed a fairly stable success rate across samples to amplify a large number of loci. The performance was consistent between samples originating from pure genomic DNA as well as material extracted directly from infected wheat leaves. In samples with mixed genotypes, we found that the assay recovers variations in allele frequencies. We also explored the potential of the amplicon assay to recover transposable element insertion polymorphism relevant for fungicide resistance. As a proof-of-concept, we show that the assay recovers the pathogen population structure across French wheat fields. Genomic monitoring of crop pathogens contributes to more sustainable crop protection and yields.

Estimating the frequency of virulence against an Stb gene in Zymoseptoria tritici populations by bulk phenotyping on checkerboard microcanopies of wheat near‐isogenic lines

“…The BPC method could be used to monitor the frequency of several virulences at wide spatiotemporal scales or in analytical field experiments. Such monitoring is increasingly relying on molecular methods, and some such methods are currently being developed for STB (e.g., Bellah et al., 2023; Gutierrez Vazquez et al., 2022; Samils et al., 2021). These methods, based on technologies that allow deep‐sequencing of genomic DNA with short reads (i.e., Illumina) or long reads (i.e., PacBio, Nanopore), are powerful tools.…”

Estimating the frequency of virulence against anStbgene inZymoseptoria triticipopulations by bulk phenotyping on checkerboard microcanopies of wheat NILs

“…Such monitoring is increasingly relying on molecular methods, and some such methods are currently being developed for STB (e.g. Samils et al 2021; Gutierrez Vazquez et al 2022; Bellah et al 2023a). These methods, based on deep-sequencing technologies with short reads (i.e.…”

Tracking signatures of selection in natural populations of ectomycorrhizal fungi – progress, challenges, and prospects

“…Advances in genotyping-by-sequencing technologies have provided a wealth of new perspectives in population genomics research. Daniel Croll (University of Neuchâtel, Neuchâtel, Switzerland) introduced new genotyping approaches targeting short and long amplicons using Illumina and PacBio sequencing platforms, respectively, which have been applied to plant-fungal pathogen interactions (Bellah et al, 2023;Stalder et al, 2023). While the Illumina microfluidics-based pipeline is particularly powerful for monitoring new mutations arising in natural populations, the PacBio sequencing pipeline offers a wide range of applications for assessing phylogenetic relationships at the intraspecific level as well as colonisation processes in natural and human-affected environments.…”