Perspective

Received: 11 March 2015 Revised: 23 April 2015 Accepted article published: 27 April 2015 Published online in Wiley Online Library: 20 May 2015

(wileyonlinelibrary.com) DOI 10.1002/ps.4029

Azole fungicides – understanding resistance

mechanisms in agricultural fungal pathogens
Claire L Price, Josie E Parker, Andrew GS Warrilow, Diane E Kelly*
and Steven L Kelly*

Abstract
Plant fungal pathogens can have devastating effects on a wide range of crops, including cereals and fruit (such as wheat and
grapes), causing losses in crop yield, which are costly to the agricultural economy and threaten food security. Azole antifungals
are the treatment of choice; however, resistance has arisen against these compounds, which could lead to devastating
consequences. Therefore, it is important to understand how these fungicides are used and how the resistance arises in order
to tackle the problem fully. Here, we give an overview of the problem and discuss the mechanisms that mediate azole resistance
in agriculture (point mutations in the CYP51 amino acid sequence, overexpression of the CYP51 enzyme and overexpression of
genes encoding efflux pump proteins).
© 2015 Society of Chemical Industry

Keywords: CYP51; azole; fungicide; resistance; cytochrome P450

1 INTRODUCTION shown to exhibit a novel spectrum not associated with inhibition,5

Plant fungal pathogens can have devastating effects on a wide
range of crops. They are responsible for a number of diseases,
including septoria leaf blotch, powdery mildews and rusts, which
can cause major losses in crop yields and in turn result in a
financial burden to the agricultural economy. Some fungi, such
as Fusarium and Penicillium, are able to synthesise mycotoxins.
These are toxic chemical compounds that, if ingested, can have
a major negative impact on the health of humans and animals.
Therefore, controlling these pathogens is paramount to ensuring
food security.
While there are a number of antimycotic compounds available to
control the spread of these agricultural fungal pathogens [i.e. ben-
zimidazoles, phenylamides, dicarboximides, anilinopyrimidines,
quinone outside inhibitors (QoIs) and carboxylic acid amides
(CAAs)],1 it is azole antifungals that are the preferred treatment
owing to their relatively low cost and effectiveness against a broad
range of fungi. They were first introduced into the clinic in 1958
with the use of the topical agent chlormidazole, but it was not
until the 1970s that azole fungicides were first used in agriculture
with the introduction of imazalil and triadimefon. Currently there
are a number of azoles available for use in agriculture, with the
most recently introduced (2002) being the triazolinethione deriva-
tive prothioconazole, which is used in the treatment of the wheat
pathogen Zymoseptoria tritici (formerly known as Mycosphaerella
graminicola). Triazoles are the most widely used class of systemic
fungicides (20% market share), with prothioconazole, epoxicona-
zole and tebuconazole being the three most used fungicides in the
United Kingdom.2,3 However, prothioconazole has recently been
shown to be a profungicide, as the antifungal effect observed in
studies with Candida albicans was found to be due to prothicona-
zole being readily converted to its desthio form rather than to
the activity of the fungicide itself.4 Furthermore, in azole binding
1054
which was further corroborated in biochemical studies where inhi-
bition of enzymatic activity was not observed.6
Azoles work by targeting the sterol 14𝛼-demethylase CYP51 (a
member of the cytochrome P450 family), which is an important
regulatory enzyme in the ergosterol biosynthetic pathway. Azole
fungicides bind through direct coordination of the triazole N-4
or the imidazole N-3 nitrogen as the sixth ligand of the haem
iron.7 The resultant CYP51–azole complex is catalytically inactive,
preventing the demethylation of lanosterol and eburicol and in
turn the production of ergosterol, which is essential to maintain-
ing fungal membrane fluidity and permeability. Treatment with
azoles depletes the amount of ergosterol in the cell, which, in
conjunction with an accumulation of 14𝛼-demethylated sterols,
disrupts the membrane structure, preventing active membrane
transport resulting in fungistasis. However, not all 14𝛼-methylated
sterols are incompatible with growth and membrane function, as
seen in mutants growing with 14𝛼-methyl fecosterol8 and even
recently lanosterol.9 These observations in medical mycology have
yet to be reproduced in plant-pathogenic fungi, although it would
be surprising were they not eventually. Possibly, fitness costs or
altered pathogenicity might mediate against them occurring.
While azole fungicides are available to control plant pathogens,
extensive use and overexposure of these compounds has led to
reduced sensitivity in the field. Therefore, it is imperative that the
effectiveness of these fungicides be maintained, as the resultant
∗
Correspondence to: Diane E Kelly and Steven L Kelly, Centre for Cytochrome
P450 Biodiversity, Institute of Life Science, College of Medicine, Swansea
University, Swansea SA2 8PP, UK. E-mail: d.kelly@swansea.ac.uk; s.l.kelly@
swansea.ac.uk
Centre for Cytochrome P450 Biodiversity, Institute of Life Science, College of

studies with the CYP51 enzyme from Z. tritici, prothioconazole was Medicine, Swansea University, Swansea, UK

Pest Manag Sci 2015; 71: 1054–1058 www.soci.org © 2015 Society of Chemical Industry
Mode of action and resistance mechanisms to azole fungicides www.soci.org

Table 1. Overview of mutations identified in the CYP51 of field isolates

Organism Crop affected Alterations in the amino acid sequence Reference

Zymoseptoria tritici Wheat L50S, D107V, D134G, V136A, V136C, V136G, Y137F, M145L, N178S, 12
S188N, S208T, N284H, H303Y, A311G, G312A, A379G, I381V, A410T,
G412A, Y459C, Y459D, Y459N, Y459P, Y459S, G460D, Y461D, Y461H,
Y461S, ΔY459 or ΔG460, ΔY459/G460, V490L, G510C, N513K, S524T
Blumeria graminis f. sp. tritici Wheat Y136F 13
Blumeria graminis f. sp. hordei Barley Y136F, K147Q 14
Erysiphe necator Grape Y136F 15
Mycosphaerella fijiensis Banana plants and plantain Y136F, A313G, Y461D, Y463D, Y463H, Y463N 16
Venturia nasicola Japanese pear Y133 17
Pyrenopeziza brassicae Oilseed rape G460S, S508T 18
Puccinia triticina Wheat Y134F 19
Penicillum digitatum Citrus fruit V55A, Y136H, M144T, K253E, Q309H, E331A, T432, I440V, K449R, G459S, 20
R462H, F506I, S507P, K508R, G511S
Oculimacula yallundae Wheat S35T, Q43H, D78Y, E106K, N244S, S505Q 21
Oculimacula acuformis Wheat A29P, V37A, Q167H, Y486H, S505Q 21

decrease in crop yields would have a detrimental effect on food
production and the economy.10 For instance, if the use of azoles
were to stop in Europe, there would be a short-term fall in wheat
production of ∼7% (9.8 million t), which would increase to ∼12%
(18.6 million t) by 2020.10 This fall in production would mean
a relative economical loss of €2.4 billion in the short term, and
€4.6 billion by 2020. This would seriously affect Europe’s ability
to produce wheat self-sufficiently.10 Therefore, it is important to
understand how these fungicides are used and how resistance
arises in order to prolong their effectiveness.
2 MECHANISMS OF RESISTANCE
Many years of exposure to azole antifungals has led to increasing
rates of resistance being seen in several plant fungal pathogens.
The first case was observed in the powdery mildew, Sphaerotheca
fuliginea (which affects cucurbits, including pumpkins and
cucumbers),11 and since then resistance has been detected in
a number of other fungal pathogens, including Puccinia triticina
(brown rust in wheat crops), Z. tritici (septoria leaf blotch) and
Penicillium digitatum (citrus fruit mould).
There are three main mechanisms of resistance in agricultural
fungi: point mutations in the CYP51 amino acid sequence; overex-
pression of the CYP51 enzyme; overexpression of genes encoding
efflux pump proteins.
2.1 Mutations in the CYP51 enzyme
The most widely reported mechanism of resistance in field iso-
lates is the presence of mutations in the CYP51 enzyme. These
mutations can cause changes in the fungicide’s affinity for the
enzyme, leading to azole tolerance. CYP51 mutations have been
documented in a number of fungal plant pathogens (see Table 1
for an overview), with the greatest number being identified in
Z. tritici. Over 30 different substitutions and deletions have been
reported in field isolates of this organism (up until 2013),12 which
have arisen owing to the extensive use of azoles (since the early
1980s) to treat septoria leaf blotch infections in wheat.
While many of the reported mutations can cause azole resis-
tance in isolation, some are lethal to the host and require other
mutations to accommodate them to restore this impairment. For
the introduction of Z. tritici CYP51 containing the I381V mutation
did not support growth. However, combining I381V with Y461H
partially restored the functionality of this CYP51.22
Combinations of mutations can also result in alterations in the
level of resistance to different azoles. During the 1990s, the most
prevalent mutation in Z. tritici was Y137F, which conferred resis-
tance to triadimenol. This was superseded by the double muta-
tion Y137F-S542T in the early 2000s and resulted in increased
resistance to the same compound. However, as triadimenol is no
longer used to treat septoria leaf blotch and has been replaced
by newer azoles, the Y137F and Y137F-S542T mutations are now
rarely seen.23
2.2 Overexpression of CYP51
Overexpression of CYP51 in fungal pathogens is not as exten-
sively documented as the mutations in the amino acid sequence.
However, it has been observed in several fungi, including Z. trit-
ici,24 P. tritici,19 Monilinia fructicola (brown rot of stone fruits),25,26
Blumeriella jaapi (cherry leaf spot),27,28 P. digitatum,29,30 Venturia
inaequalis (apple scab)31 and P. brassicae.18
Overexpression is caused by changes in the promoter region of
the Cyp51 gene by the insertion of tandem repeats or transposable
elements. It is assumed that increased mRNA levels correlate with
the resultant increase in cellular CYP51 levels and therefore drug
target levels, causing a reduction in the azole sensitivity exhibited
by the fungus. In P. digitatum, overexpression of Cyp51 (due to a
199 bp insert in the promoter region) was related to the imazalil
resistance seen in isolates.32
2.3 Reduced intracellular accumulation of azole antifungals
Although CYP51 is the main target of azole antifungals, other
non-related enzymes can be involved in causing resistance. Efflux
transporters [including ATP-binding cassette (ABC) transporters
and major facilitator superfamily (MFS) transporters] are essential
in eukaryotes to export toxins and fungicides out of the cell.
Overexpression of the genes encoding these transporters can
lead to resistance against azole antifungals as the intracellular
concentration of the fungicide is reduced.
While the link between efflux transporters and azole resistance
has been widely reported in human fungal pathogens (such as C.
1055

instance, in Saccharomyces cerevisiae complementation studies, albicans, C. glabrata and Cryptococcus neoformans), there is limited

Pest Manag Sci 2015; 71: 1054–1058 © 2015 Society of Chemical Industry wileyonlinelibrary.com/journal/ps
 www.soci.org
information available regarding this phenomenon in plant fungal
pathogens. Cases have been reported in Botrytis cinerea (grey
mould on a variety of plant species, including grapes)33,34 and P.
digitatum.35,36 It has also been suggested as a possible mechanism
of resistance in Z. tritici.37,38
In Botrytis cinerea, the ABC transporter BcatrD has been asso-
ciated with azole resistance. Overexpression of BcatrD results in
increased expression of the transporter and reduced sensitivity
to oxpoconazole. Mutants where BcatrD has been replaced have
shown increased azole sensitivity and accumulation of high lev-
els of oxpoconazole, further establishing a role for BcatrD in azole
resistance.33,34
While the above resistance mechanisms have been identi-
fied in field isolates, the true extent of these mechanisms is
not fully understood. For instance, inactivation of ERG3 (Δ7
sterol-C-5-desaturase) has been shown to lead to azole resistance
in the human pathogen C. albicans.39 However, similar mecha-
nisms have yet to be identified in field isolates.17 Recently it has
been observed, in the field, that Ascomycetes (such as Fusarium
spp., P. digitarium and Magnaporthe oryzae) carry multiple copies
of CYP51. It is believed that these extra copies contribute to the
increased resistance observed in these organisms,40 as sterol
14𝛼-demethylase activity can be retained in multiple copies.
Additionally, the re-emergence of the CYP51A paralogue in the
barley pathogen Rhynchosporium commune has been shown to
be a novel evolutionary mechanism of resistance.41
3 UNDERSTANDING THE MECHANISMS
OF RESISTANCE
There are many experimental techniques available to study the
mechanisms of azole resistance. Firstly, DNA sequencing is nec-
essary in order to determine the genotype of resistance strains.
DNA sequencing enables the identification of mutations (includ-
ing SNPs, insertions and duplications) within the coding regions
and promoter region of the CYP51 and transporters from field iso-
lates. Coupled with minimum inhibitory concentration (MIC) data,
the effect of these changes on azole affinity can be elucidated. In
the medical mycology arena, whole-genome sequencing of resis-
tant isolates has begun to be published,42 and this will occur for
plant pathogens. In genetically tractable pathogens, the correla-
tion of phenotype and genotype can be examined using genetic
manipulation.
Alternatively, biochemical studies can be undertaken using puri-
fied recombinant CYP51 proteins (ligand binding and inhibition
studies) to determine the affinity of a particular azole fungicide
for a given CYP51 protein (wild type and mutants). More recently,
in silico modelling of fungal CYP51 enzymes has been used to pre-
dict the affinity of azole antifungals for these enzymes, predomi-
nantly looking at how mutations within the structure affect azole
docking. Fungal CYP51 proteins currently modelled include Mag-
naporthe grisea CYP51,43 Ustilago maydis CYP51,44 Aspergillus fumi-
gatus CYP51,45 M. fijiensis CYP51,16 P. digitatum CYP5146 – 48 and Z.
tritici CYP51.49
The 3D structures of fungal cytochromes P450 (CYPs) have pri-
marily been based on the crystal structures of bacterial CYPs,
which are readily soluble and relatively easy to crystallise, unlike
eukaryotic CYPs, which tend to aggregate in free solution owing
to their hydrophobic N-terminal membrane anchors. While this
has allowed in silico models of fungal CYPs to be determined
using bacterial CYP templates, these models may not give a
1056
complete picture of how the fungal enzymes fold. Recently, the
wileyonlinelibrary.com/journal/ps © 2015 Society of Chemical Industry
C L Price et al.
crystal structures of a number of truncated eukaryotic CYP51s
have been elucidated. These include CYP51s from the parasites
Trypanosoma cruzi, T. brucei CYP5150 – 52 and Leishmania infan-
tum CYP51,53 as well as human CYP51.54 In addition, the first
full-length eukaryotic CYP51 crystal structure has now been solved
for S. cerevisiae.55 The advent of these eukaryotic crystal structures
will greatly improve confidence in in silico structural modelling,
with the recent S. cerevisiae structure having a modest effect on
the current models using multitemplate methodology.
4 CONCLUSIONS
Plant fungal pathogens are a blight on agricultural crops, caus-
ing devastating losses in crop yields and a financial burden on the
economy and threatening food security. Therefore, it is imperative
that these diseases be adequately controlled. Currently, the main
treatment for these pathogens is azole antifungals on account of
their broad spectrum of activity. However, time and overuse of the
fungicides in the field has led to resistance being seen in a num-
ber of fungal species. While the mechanisms of azole resistance are
still being successfully elucidated, the complex nature of the resis-
tance makes it a difficult task. Therefore, it is important to maintain
the effectiveness of these fungicides, as the resultant decrease in
crop yields would have devastating effects. Practical strategies that
could be employed to overcome this problem include alternating
between azole and non-azole fungicides, growing crop cultivars
that are inherently more resistant to fungal pathogens and devel-
oping new azole fungicides. However, continuing the challenge
of understanding how the mechanisms of azole resistance arise is
essential to tackling the problem of resistance fully.
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