IAP-26-035

FUNGRASS: Fungicide resistance in fungal endophytes of grasslands

AIM: to determine fungicide resistance in wild endophytic fungal communities ranging from upland to lowland and coastal dune grasslands, to assess the impact of fungicide application on non-target fungi.

Fungal disease leads to annual crop losses of 10-20% worldwide (Steinberg and Gurr, 2020). Plant pathogens are controlled by a limited and overlapping group of chemistries including azoles, strobilurins (QoIs) and succinate dehydrogenase inhibitors (SDHIs) (Fisher et al., 2018). Azoles are also used to control animal and human fungal infections. The use of these fungicides has led to evolving fungal genomes and fungicide resistance in different fungal populations (Noel et al., 2022; Sumbula et al., 2021). Environmental studies to date have focussed on the human pathogen Aspergillus fumigatus, which is on the World Health Organisation (WHO) critical list (Fraaije et al., 2020; WHO, 2022).
However, fungicides used to control crop disease likely have adverse effects on a wide range of non-target fungi (Noel et al., 2022). For example, in tomato the richness and diversity of leaf fungal populations were negatively impacted by fungicide use (Sumbula et al., 2021). However, fungicide resistance in environmental fungal communities remains unexplored including endophytes (which are endosymbiotic colonisers of plants). Therefore, there is a need to understand how fungicide application impacts non-target environmental fungi which may have ecological and possible future agricultural impacts.
FUNGRASS will isolate endophytic fungi from samples collected from wild grassland sites ranging from upland acid to lowland (including arable field margins) and coastal dune grasslands. Sampling of wild grasses will adhere to the Scottish statutory access rights and Outdoor Access Code (NatureScot, 2005). The resulting isolate library will be screened for resistance to different fungicides including azole and Succinate Dehydrogenase Inhibitor (SDHI) fungicides to determine the percentage of fungicide resistant isolates per site (Esfehasni et al., 2025). DNA will be extracted from the fungicide resistant fungal endophytes followed by whole genome sequencing for species identification and analysis of known fungicide resistance genes including CYP51 (encoding sterol 14α-demethylase), SDH1-4 (succinate dehydrogenase inhibitors) and mitochondrial CYTB (Zulak , 2025). This will allow the prevalence of fungicide resistance and specific genetic mutations associated with different environmental fungal endophytic communities to be revealed.

Leaves of wild grasses will be collected from sites ranging from upland acid to lowland grassland (including arable field margins) and coastal dune grasslands. At each grassland site the percentage grass cover will be imaged and recorded. Grass samples from at least ten quadrats will be sampled (using a random number generator) as representative from each of the environmental grassland sites.

After site sampling a library of fungal endophytes will be collected by isolating endophytic fungi from leaves via sterilisation followed by culturing. Pure fungal cultures will be cultured and stored at -80C for subsequent fungicide screening and DNA extraction. Endophytes from each site (~100) will be screened for fungicide resistance in 96 well plates using EC50 determination (the effective concentration of fungicide required to reduce fungal growth or infection by 50%) (Esfehani et al., 2025; Fonseka et al., 2016).
Following DNA extraction the fungal samples will undergo whole genome sequencing (up to 30 per site). This will allow both species identification using the NCBI database and examination of fungicide resistance genes within the genome for allelic diversity. It will also allow future examination of additional genomic regions of interest. Fungal resistance genes will be compared to previously identified mutations present in fungal populations.

Year 1

Collect and record samples from grassland sites; upland, lowland and coastal.
Isolate and culture endophyte library from leaf samples from grassland sites.

Year 2

96 well plate screen of endophytes for fungicide resistance using EC50 determination
DNA Extraction from resistant endophytes from each grassland site.
Whole genome sequencing of fungal endophytes from grasslands

Year 3

Download publicly available sequence data from NCBI from grass-isolated fungi
Analysis of whole genome sequencing of fungal endophytes for fungicide resistance and comparison to publicly available data.

Year 3.5

Time to finalise thesis chapters and publication drafts for submission.

Skills will be developed in:
Field work collecting, recording and statistical analysis of data. .
Microbiology skills isolating and culture of endophytic fungi.
Molecular biology skills including DNA extraction and the associated bioinformatic skills for analysis of whole genome sequencing. Working with a team of agronomists and plant pathologists.
Communication and Presentation skills at seminars, conferences and to stakeholders.