Page 148 - Plant Canada 2024 Proceeding

PLANT CANADA 2024

*[O92] THE EVOLUTIONARY DYNAMICS OF AZOLE RESISTANCE IN FUSARIUM GRAMINEARUM.
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Kelsey Wog , Matthew. G. Bakker , and Aleeza C. Gerstein . Department of Microbiology, University of
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Manitoba, 45 Chancellors Cir, Winnipeg, MB R3T 2N2, Canada
Correspondence to: wogk@myumanitoba.ca

Fusarium graminearum is a fungal pathogen that significantly threatens wheat and barley crops in
temperate regions, causing annual global agricultural losses exceeding $1 billion. Although azoles are
generally effective fungicides, recent data suggests that resistance towards azoles may be increasing in
F. graminearum populations. To investigate the rate that F. graminearum can acquire resistance following
prolonged exposure to common azoles, we conducted a series of in vitro evolution experiments. Strain
DAOM233423, a well-characterized strain isolated in the 1980s before environmental azoles, was
exposed to 1% DMSO for 6 weeks to acclimate the strain to the solvent exposure. Four replicate lineages
were evolved to increasing concentrations of prothioconazole, tebuconazole, and a combination of both in
equal concentration until extinction. The prothioconazole and combined treatment lineages reached a
terminal MIC that exceeded the ancestor by a factor of 4, while lineages exposed to tebuconazole
exceeded the ancestral MIC by a factor of 5. Whole genome sequencing will soon be conducted to
identify putative genomic variations linked with fungicide resistance. Additionally, we are currently
assessing phenotypic changes in evolved lineages, including colony morphology, cross-resistance to
other azoles, virulence, and sexual/asexual reproduction. We hypothesize that the mutational basis of
resistance will differ between azole treatments and to find varying levels of cross-resistance to other
agricultural and clinical azoles. This research will improve our understanding of the genomic basis of
azole resistance in F. graminearum and determine how different fungicide exposures influence the
development of cross-resistance.

*[O93] THE ROLE OF HYD5 PROTEIN IN FUSARIUM-BARLEY INTERACTIONS. Anuradha U.
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Jayathissa , W. G. Dilantha Fernando , Raymond He , David N. Langelaan , and Matthew G. Bakker .
1 University of Manitoba, Department of Microbiology, Winnipeg, MB, Canada; University of Manitoba,
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Department of Plant Science, Winnipeg, MB, Canada; and Dalhousie University, Department of
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Biochemistry & Molecular Biology, Halifax, NS, Canada
Correspondence to: Matthew.Bakker@umanitoba.ca

Fusarium graminearum, a significant fungal pathogen, poses a threat to barley crops worldwide.
Hydrophobin proteins, such as Hyd5, are known to play roles in the interactions of fungi with their
environment, particularly in the colonization of hydrophobic surfaces. While the specific involvement of
Hyd5 in Fusarium-barley interactions remains poorly understood, hydrophobins have been implicated in
the excessive gushing of beer made from infected barley. In this study, a beer gushing experiment was
conducted using malt that was infected with F. graminearum wild type 233423 or a Δhyd5 knockout strain
produced via CRISPR-Cas9 gene editing. Heterologous expression of Hyd5 in E. coli was performed,
followed by purification of the hydrophobin for beer gushing experiments. Additionally, hyd5 gene
expression was examined using RT-qPCR during in planta infection (including during heading, at
flowering, and eight days post-flowering) and in malted barley infected with F. graminearum. RNA
extraction was carried out using TRIzol reagent, followed by cDNA synthesis and quantification of hyd5
transcripts, whose abundance was expressed relative to a housekeeping gene, as a unitless ratio.
Although not statistically significant, a trend for reduced beer gushing in malt infected with the knockout
strain suggested that Hyd5 may indeed play a role in beer gushing. This involvement was confirmed by a
dramatic increase in beer gushing with the addition of heterologous Hyd5 (22 times more gushing
compared to the control; T-test, P = 2.14 x 10 ). In the wildtype strain, there was a significant increase in
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hyd5 gene expression between the malt stages of steeping (0.002) and germination (0.035). However, no
significant differences in hyd5 expression were found in planta. Our results support the role of Hyd5 in
gushing, but this protein may not be heavily involved in the colonization of the plant in the field.

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