A search for novel synergistic combinations against prevalent fungal phytopathogens

Young, Taran (2023) A search for novel synergistic combinations against prevalent fungal phytopathogens. PhD thesis, University of Nottingham.

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With the global population rising, the emergence of new types or variants of crop pathogens (phytopathogens), and the increasing resistance of these organisms to current crop treatments, there is an urgent need to improve global food security. Each year, fungi destroy crops that could feed up to 600 million people annually. Wheat, the most widely grown crop worldwide, has the second highest yield of all crops, with a global production of 778.5 million metric tonnes in 2021/2022. Zymoseptoria tritici, a major pathogen of wheat, causes Septoria Tritici Blotch (STB). This organism is responsible for around 70% of annual fungicide usage in the EU and up to 20% of annual yield losses in the UK. Novel treatments involving synergistic combinations of inhibitory compounds could improve efficacy while decreasing cost and toxicity to both non-target organisms and the wider environment. In this thesis, novel synergistic combinations of agents, primarily targeting Z. tritici, were sought. The initial focus was on protein translation as a target. Specifically, it was hypothesised that pairs of agents disrupting the fidelity of protein translation could be effective at synergistically inhibiting phytopathogenic fungi. To examine this, rational combinations of compounds were tested against fungi of interest.

Several synergies against Botrytis cinerea and Z. tritici were found among compounds of interest. A series of different combinations involving the dual targeting of aminoacylation (attachment of an amino acid to a tRNA), with one compound inhibiting a tRNA synthetase, and a second compound targeting the biosynthesis of the corresponding amino acid, yielded several synergies. One such synergy was between tavaborole, an inhibitor of the leucyl-tRNA synthetase, and chlorimuron ethyl, an inhibitor of the non-cognate branched-chain amino acids. This synergy decreased the minimum inhibitory concentrations (MIC) for each compound by four-fold when in combination, compared to individual application. This proof of principle for dual targeting – inhibiting both biosynthesis and aminoacyl tRNA synthetase function for particular amino acids – opens up the possibility for dozens of potential synergistic combinations targeting the availability of different amino acids for protein translation, and hence growth, of undesirable fungi. For fungi pathogenic to humans, one or both targets should be the availability of essential amino acids to decrease cytotoxicity risks.

Furthermore, a powerful synergy against Z. tritici was discovered. This synergy decreased the MICs of the two compounds involved, cyprodinil and diphenyleneiodonium, by 16- and 32-fold when in combination, compared to individual application. However, this synergy was less potent in B. cinerea and Saccharomyces cerevisiae. The mechanism behind this synergy was investigated, initially testing for synergistic mechanisms based around the NADH kinase Pos5p and the electron transport chain, as these were reported as potential targets for the compounds. Experiments suggested that the mechanism most likely involves the synergistic increase of oxidative damage within mitochondria, although some experiments contradict this. Diphenyleneiodonium, an inhibitor of NAD(P)H oxidases, was evidenced to confer increased sensitivity to the pro-oxidant H2O2. The specific action of cyprodinil in the synergy remains unclear while characteristics of increased oxidative stress were seen.

Finally, two synergies of interest were tested on live wheat plants to determine if they could protect crops from Z. tritici infection. Experiments showed that at the concentrations used, these synergistic combinations were unable to effectively inhibit the pathogenicity of Z. tritici. Nevertheless, this poor reproduction of laboratory findings in real infections highlights one difficulty with the discovery of novel treatments against fungal phytopathogens, viable for real-world applications.

Despite the poor success in preventing Z. tritici infection in the small trial with wheat at the end of this study, the potency of inhibitory-agents acting synergistically, demonstrated in the laboratory here, remains a strategy of high potential. The synergies discovered in this research could be tested against other undesirable fungi with the possibility of successfully controlling infection, and the mechanisms of synergistic action proposed here may provide a foundation to inform future combinatorial testing.

Item Type: Thesis (University of Nottingham only) (PhD)
Supervisors: Avery, Simon
Dickinson, Matthew
Corran, Andy
Keywords: Phytopathogenic fungi; Inhibitory compounds; Z. tritici; Protein translation; Crop pathogens
Subjects: Q Science > QK Botany > QK710 Plant physiology
Faculties/Schools: UK Campuses > Faculty of Medicine and Health Sciences > School of Life Sciences
Item ID: 76530
Depositing User: Young, Taran
Date Deposited: 26 Feb 2024 10:05
Last Modified: 26 Feb 2024 10:05
URI: https://eprints.nottingham.ac.uk/id/eprint/76530

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