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.

No abstract

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.

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 pathogenZymoseptoria 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 co-cultured strains 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 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 severity and need to be integrated for a more complete understanding of host-pathogen dynamics in the environment.

Plant pathogenic fungi are very widespread and can pose severe risks to global food production through single as well as mixed infection. Crop control is based on the use of synthetic chemicals and plant resistance breeding. However, fungal plant pathogens quickly overcome both strategies. During coinfections, plant pathogens can increase disease severity and change co-infecting genotypes virulence evolution. Hence, monitoring tools improving crop pathogen detection as well as understanding the impact of co-infection events on disease severity are lacking. Additionally, we still lack information on the genetic basis shaping virulence and fungicide resistance evolution over time. In this PhD thesis I focused on developing monitoring tool to track virulence and fungicide resistance evolution in the fungal wheat pathogen Zymoseptoria tritici using different molecular and genomic tools. Here I designed a microfluidics-based amplicon sequencing assay to multiplex several loci targeting virulence and fungicide resistance genes, and randomly selected genome-wide markers. More than hundred types of samples were used to assess the performance of our assay. This later allowed an accurate amplification of all designed loci and performed well regardless of the sample type. I also explored the outcomes of mixed interactions within and outside the plant host by using a large sample size to provide a deeper picture about the consequences of such interactions on disease severity as well as virulence and growth effects. I found that the growth kinetics and virulence in single interactions deviated from those seen during mixed interactions indicating competitive exclusion between conspecific strains. Additionally, the outcomes were divergent within and outside the plant host indicating that the plant immune system plays a key role in shaping the within-plant interaction outcomes and adds significant complexity. Finally, I used a data set of worldwide populations of Z. tritici to investigate transposable elements (TEs) located nearby important virulence and fungicide resistance genes allowing to retrace the evolutionary history of the pathogen worldwide as well as a better crop protection strategies. I identified a considerable number of TEs belonging to different families and superfamilies, I also detected recent insertions represented by unique insertions called singletons. Overall, this PhD thesis allows monitoring newly evolved Z. tritici genotypes and understanding the genetic basis mediating their adaptation to their hosts and environment. Besides, the thesis provides information about the consequences of mixed interactions on disease severity and how these can shape change in virulence and growth of plant pathogens.