Fungicide mode of action and resistance development in crop pathogenic fungi

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.

An important feature of a fungicide is its mode of action (MoA), which describes its physiological impact onto the fungal cell. Knowledge about fungicide MoA is fragmentary, due to technical limitations of most early MoA studies. Recent advances in live cell imaging techniques provide new avenues of MoA research. This was made possible due to the development of fluorescent marker proteins in fungal plant pathogens Ustilago maydis (corn smut fungus) and Zymoseptoria tritici (Septoria tritici blotch fungus). As proof of principle, we provide preliminary results on dodine, that reveal a novel MoA on fungal membrane trafficking. We will apply this live cell imaging approach to elucidate the MoA of 13 fungicides, covering the major classes listed above.

Fungicide resistance is a major challenge to agriculture. It can be conferred by single-site mutations in the fungicide target, by changes in the expression of efflux pups and/or less well understood mechanisms. For this application, we have generated ~150 strains of U. maydis and Z. tritici, resistant to the 13 fungicides used for MoA studies. This number will be extended and whole-genome sequencing and RNAseq will be performed. This allows unbiased identification of novel resistance development mechanisms in both pathogens. As proof of principle, we have sequenced 15 genomes, which led to the identification of a novel transcription factor, likely to confer resistance to one class of fungicides. We follow this up, but also aim to understand resistance development against all other fungicide classes.

Z. tritici has recently developed resistance against azoles and strobilurins. It has been suggested that the 8 non-essential (=dispensable) chromosomes participate in such adaptation to fungicides. In the third part of the project, we will generate wild-type strains that lack individual chromosomes and test their ability of develop resistance.

The PI and Co-PI have outstanding track records for attracting industrial monies, fulfilling contract research, filing patents, exploiting data and in the delivery of high quality publications.

In this project they will exploit a unique set of fungal organelle-tagged fluorescent marker strains, generated by the PI, in state-of-the-art live cell imaging to better describe MoA in commonly used fungicides and give novel descriptors of the mode of action (MoA) of those list in FRAC as unknown or with inaccurate prose. The research will unmask mechanisms of resistance emergence (MoR) and shed light on risk associated with such emergence as well as revealing genetic markers for the development of field-based diagnostic assay (extension work, out-with this project). The work will be with two notable crop pathogens causing significant crop losses - of wheat grown in temperate climates and with considerable losses of the maize harvest. The application stems directly from our expertise in studying host pathogen interactions and from the toolkit of cellular markers we have gathered in the two fungi, Zymoseptoria tritici and Ustilago maydis, thanks respectively to a recent BBSRC initiative (published in a special edition of Fungal Genetic & Biology, 2015, edited by the PI) and with continuous BBSRC funding in support of the maize smut work. This project extends this research to utilise the cellular tools generated therein in combination with exploiting genome and transcriptome interrogation to reveal both MoA and MoR to a raft of fungicides.

Academia / Industry / Society / Policy:
The findings of this work will be of immediate interest to researchers engaged in the study of fungicides, emergence of antifungal resistance - both to plant and animal-pathogenic fungi and, more long-term, to risk modelling and management. In the short-term, the work will be published in high impact scientific journals. This will garner further interest from researchers engaged in the private sector (both in AgTech and Pharma industries). Publications will lead to ""press" interest, to farmer engagement and to public curiosity. Such releases will be orchestrated via BBSRC and Exeter University "press" offices and by interviews for the media (eg BBC "Farming Today" programme). More long-term, the findings of this study will inform practice and spray rotations to mitigate resistance development (disseminated via Home Grown Cereal Authority, HGCA). The data will be used by FRAC to provide accurate MoA descriptions for named fungicides and will be used in UK/ EU policy regarding risk management in fungicide usage.
The outcome of this project promises high societal impact - the UK government has recently invested significant amounts of funding in a new AgriTechnology strategy, aiming to develop sustainable, low input agricultural practises. Fungi are recognised as a major threat to food security. Understanding of essential and fundamental processes involved in protecting our crops from fungal disease will ultimately lead to efficient and environmentally-benign strategies of pathogen management. Democratic policy is rooted in public understanding of the challenges facing society. Therefore, the PI and co-I will use opportunities to disseminate scientific knowledge about fungal pathogenesis in a series of public presentations and MOOC as outlined in the Pathways to Impact document. This will be freely available in the internet.