Fungicide mode of action and resistance development in crop pathogenic fungi

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Continuous growth of the world population comes with increasing demand for food. As a consequence, agricultural practises have intensified. The large monocultures of important crop plant, such as maize and wheat, provide a rich food source for plant pathogenic fungi. In fact, fungi are the biggest challenge for our food security. Amongst the most devastating crop pathogens are the corn smut fungus (Ustilago maydis) and the Septoria tritici wheat blotch fungus (Zymoseptoria tritici/Mycosphaerella graminicola). Our farmers fight these fungi by spraying anti-fungal chemistries, so-called fungicides. These usually target the fungal cell, whilst showing little toxicity to the crop. To understand how a fungicide acts, detailed studies of the physiology of the fungal cell in the presence of the antifungal chemistry are required. The outcome is an understanding of the impact of the fungicide on the fungus, which describes is "mode of action" (MoA). In previous times, such studies were restricted by technical limitations. Consequently, the MoA of many fungicides is either not known or our knowledge is fragmentary. The recent development of live cell imaging techniques and tools for fungal pathogens allows visualisation of fungal cell in the presence of a fungicide. The PI's laboratory is world-leading in live cell imaging of fungal pathogens. The project aims to use this modern approach to monitor fungicide-induced changes in cells of U. maydis and Z. tritici. In a preliminary study, we provide a proof of principle study with the fungicide dodine, used to control fungal disease on apples. This revealed a novel MoA for dodine and illustrates the power of this approach. In the first part of the project, we will use the cell markers and live cell imaging to investigate the MoAs of 12 major fungicides that cover the most economically important fungicide groups in global use. Fungi have the ability to adapt to fungicides. Similar to bacteria, they can develop resistance, which is of high economic importance, as it renders the respective fungicide useless. Resistance can be achieved by modification of the protein that the fungicide binds to and inhibits. Alternatively, it can be achieved by other, much less understood ways, including an increased activity of cellular pumps that remove the fungicide from the fungal cell. Our understanding of the mechanism by which fungi develop resistance is limited to local changes in the genetic information of the pathogen. However, the ability to quickly sequence the entire genomic information of a resistant fungus opens the opportunity to look for all changes, accompanied by the appearance of fungicide resistance. We have developed techniques to generate fungicide resistant fungi in our laboratory (so far ~150 fungal cell lines (=strains), resistant against most of the 12 major fungicides). We have sequenced the genomic information of 15 of these fungal strains and already found strong indication for an unexpected and new mechanism conferring resistance in U. maydis. We aim to increase the number of resistant strains and extend the unbiased approach of sequencing entire genomes of resistant fungi. This, and the subsequent analysis of mutated genes, promises novel insight into the molecular adaptation of fungal pathogens to fungicides. It is increasingly difficult to control infections by the wheat pathogen Z. tritici. This is due to the appearance of resistance strains against the major fungicide classes. It was speculated that the ability to adapt to fungicides is due to the presence of 8 "dispensable" chromosomes. These are not essential for survival of the pathogen and, therefore, can be lost during cell division. In this part of the project, we will generate Z. tritici strains that are identical, but which lack individual dispensable chromosomes. We will expose these to fungicides and analyse the ability to develop resistance against the anti-fungal chemistries.