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

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.

Divergent Outcomes of Direct Conspecific Pathogen Strain Interaction and Plant Co-Infection Suggest Consequences for Disease Dynamics.

Plant diseases are often caused by co-infections of multiple pathogens with the potential to aggravate disease severity. In genetically diverse pathogen species, co-infections can also be caused by multiple strains of the same species. However, the outcome of such mixed infections by different conspecific genotypes is poorly understood. The interaction among pathogen strains with complex lifestyles outside and inside of the host are likely shaped by diverse traits, including metabolic capacity and the ability to overcome host immune responses. To disentangle competitive outcomes among pathogen strains, we investigated the fungal wheat pathogen Zymoseptoria tritici. The pathogen infects wheat leaves in complex strain assemblies, and highly diverse populations persist between growing seasons. We investigated a set of 14 genetically different strains collected from the same field to assess both competitive outcomes under culture conditions and on the host. Growth kinetics of cocultured strains (~100 pairs) significantly deviated from single strain expectations, indicating competitive exclusion depending on the strain genotype. We found similarly complex outcomes of lesion development on plant leaves following co-infections by the same pairs of strains. While some pairings suppressed overall damage to the host, other combinations exceeded expectations of lesion development based on single strain outcomes. Strain competition outcomes in the absence of the host were poor predictors of outcomes on the host, suggesting that the interaction with the plant immune system adds significant complexity. Intraspecific co-infection dynamics likely make important contributions to disease outcomes in the wild. IMPORTANCE Plants are often attacked by a multitude of pathogens simultaneously, and different species can facilitate or constrain the colonization by others. To what extent simultaneous colonization by different strains of the same species matters, remains unclear. We focused on intra-specific interactions between strains of the major fungal wheat pathogen Zymoseptoria tritici. The pathogen persists in the environment before infecting plant leaves early in the growing season. Leaves are typically colonized by a multitude of strains. Strains cultured in pairs without host were growing differently compared to strains cultured alone. Wheat leaves infected either with single or pairs of strains, we found also highly variable outcomes. Interactions between strains outside of the host were only poorly explaining how strains would interact when on the host, suggesting that pathogen strains engage in complex interactions dependent on the environment. Better understanding within-species interactions will improve our ability to manage crop infections.