Phytoparasitica
https://doi.org/10.1007/s12600-023-01063-0
REVIEW
Mutations associated with fungicide resistance
in Colletotrichum species: A Review
Cris Q. Cortaga · Benjamine William P. Cordez
Mark Angelo O. Balendres · Fe M. Dela Cueva
Received: 25 September 2022 / Accepted: 2 March 2023
© The Author(s), under exclusive licence to Springer Nature B.V. 2023
Abstract Anthracnose is an important plant disease
often caused by Colletotrichum spp. Fungicide resist-
ance is a major challenge in controlling anthracnose.
The Fungicide Resistance Action Committee (FRAC)
reports that Colletotrichum species develop resistance
against methyl benzimidazole carbamates (MBC),
quinone-outside inhibitors (QoI), and demethylation
inhibitors (DMI) fungicide classes. The high resist-
ance of Colletotrichum species to MBC fungicides
is attributed to the substitution of glutamic acid (E)
with alanine (A) or lysine (K) in codon 198 of the
nuclear β-tubulin (TUB2) gene (E198A/K mutation).
Meanwhile, moderate MBC resistance is attributed
to the substitution of phenylalanine (F) with tyrosine
Supplementary Information The online version
contains supplementary material available at https://​doi.​
org/​10.​1007/​s12600-​023-​01063-0.
C. Q. Cortaga (*) · B. W. P. Cordez ·
M. A. O. Balendres · F. M. Dela Cueva
Institute of Plant Breeding, College of Agriculture
and Food Science, University of the Philippines Los
Baños, 4031 College, Laguna, Philippines
e-mail: cqcortaga@up.edu.ph
L. S. Dacones
Institute of Biology, College of Science, National Science
Complex, University of the Philippines Diliman, NCR,
1101 Quezon City, Philippines
M. A. O. Balendres
Department of Biology, College of Science, De La Salle
University, Manila, Philippines
· Leilani S. Dacones ·
(Y) in codon 200 of TUB2 (F200Y mutation). High
QoI-resistance is associated with the substitution
of glycine (G) with alanine (A) in codon 143 of the
mitochondrial cytochrome b (CYTB) gene (G143A
mutation). On the other hand, moderate QoI resist-
ance is attributed to the substitution of phenylalanine
(F) with leucine (L) in codon 129 of CYTB (F129L
mutation). In contrast to MBC- and QoI-resistance,
which are associated with point mutations in the tar-
get genes, complex nucleotide mutations for DMI-
resistance occur in the nuclear sterol 14α-demethylase
(CYP51) gene. Taken together, these mutations
in nucleotide/codon sequences cause changes in
the translated amino acid residues, resulting in
altered structure of proteins targeted by fungicides,
thus affecting fungicide binding and effectivity. The
underlying molecular mechanisms of fungicide resist-
ance caused by these mutations, including the other
resistance mechanisms independent from genetic
mutations, are also reviewed. The distribution and
phylogeography of reported Colletotrichum species
that exhibit fungicide resistance are also discussed.
Keywords Fungicide resistance · Codon · Amino
acid · Mutation · Colletotrichum
Introduction
The genus Colletotrichum is composed of about 189
species (Jayawardena et al., 2016) belonging to 11
Vol.: (0123456789)
13

complexes (caudatum, graminicola, spaethianum,
destructivum, acutatum, dematium, gigasporum, gloe-
osporioides, boninense, truncatum, and orbiculare)
(Marin-Felix et al., 2017). These fungal species are
known causative agents of anthracnose in many plant
species, including crops with economic and agricul-
tural significance. Among the control measures to
manage anthracnose in crops are fungicides applied
at different crop growth and development stages. The
use of a fungicide is a critical component of IPM
(Integrated Pest Management) for disease manage-
ment. However, frequent application of site-specific
fungicides can lead to increased selection pressure for
resistance. Further, the rotational application of dif-
ferent fungicide classes can result in multiple fungi-
cide resistance (Chechi et al., 2019; Hu et al., 2015).
The Fungicide Resistance Action Committee (FRAC)
reports field and laboratory evidence of Colletotri-
chum species developing resistance against methyl
benzimidazole carbamates (MBC), quinone-outside
inhibitors (QoI), and demethylation inhibitors (DMI)
fungicide classes (FRAC, 2020). Farmers heavily
depend on these fungicides to limit Colletotrichum
infection in the field, thus, if active resistance man-
agement is not done, there is high risk of Colletotri-
chum subpopulations developing resistance against
such fungicides (Fernández-Ortuño et al., 2014).
The resistance phenotype observed among Colle-
totrichum isolates often reflects mutations in the
genes coding for the protein targeted by the fungi-
cide. For instance, MBC resistance has been associ-
ated with point mutations in the β-tubulin (TUB2)
gene, which codes for the protein targeted by MBC
fungicides (Nalumpang et al., 2010; Vieira et al.,
2017; Wong et al., 2008). Similarly, QoI resistance
is also associated with point mutations in the mito-
chondrial cytochrome b (CYTB) gene (Forcelini et al.,
2016; Ren et al., 2020). In contrast, DMI resistance is
associated with multiple mutations at the cytochrome
P450 sterol 14α-demethylase (CYP51) gene (Chen
et al., 2018a; Wang et al., 2020; Wei et al., 2020;
Zhang et al., 2017). In general, these mutations in
nucleotide/codon sequences and translated amino acid
residues result in altered structure of proteins targeted
by fungicides which affect fungicide binding and
efficacy, leading to resistance. The position of codon
mutation and amino acid substitution may determine
the fungicide resistance of a fungal isolate (e.g.,
high or moderate resistance). This review compiles
Vol:. (1234567890)
13
Phytoparasitica
and provides an overview of the known mutations in
Colletotrichum species (infecting various host plants)
that mediate resistance to major fungicides used to
control anthracnose. Also reviewed are the underlying
molecular mechanisms of mutation-mediated fungi-
cide resistance, the other resistance mechanisms inde-
pendent from genetic mutations, and the distribution
and phylogeography of Colletotrichum isolates bear-
ing resistance.
Point mutations in Colletotrichum species associated
with MBC fungicide resistance
The MBC fungicides belonging to FRAC code 1
include benzimidazoles (benomyl, carbendazim,
thiabendazole, and fuberidazole) and thiophanates
(thiophanate-methyl and thiophanate) (FRAC, 2020).
These fungicides distort and inhibit the β-tubulin
polymerization required for microtubule formation
during cell division, thereby killing the fungal cells
due to mitotic failure (Davidse, 1973). Thus, muta-
tions in the nuclear β-tubulin (TUB2) gene usually
confers resistance against this class of fungicides
(Forcelini et al., 2016; Takushi et al., 2014). In TUB2,
high resistance of Colletotrichum species to MBC
fungicides is usually attributed to E198A/K mutation
or substitution of glutamic acid (E) with alanine (A)
or lysine (K) in codon 198 (GAG to GCG or AAG,
respectively) (Table 1). In addition, multiple point
mutations in this gene have also been associated with
high resistance (Ramdial et al., 2016). However, the
definition of “high resistance” differs greatly depend-
ing on the fungicide used and the scale adopted
by the researchers (Table 1). For instance, isolates
that have high resistance towards carbendazim are
often defined as those that have > 100 µg ­mL−1 to
> 500 µg ­mL−1 ­EC50, > 1,000 µg ­mL−1 MIC, or
exhibit uninhibited growth at 50 µg m ­ L-1 or 500 µg
−1
­L carbendazim. For benomyl, highly resistant iso-
lates have > 500 µg ­mL−1 ­EC50 or uninhibited growth
at 10 µg m­ L-1. For thiabendazole, isolates with high
resistance have > 100 µg ­mL−1 ­ED50. Lastly, for thi-
ophanate-methyl, high resistance is often associated
with > 100 µg ­L−1 ­EC50.
Resistance and moderate resistance are usu-
ally attributed to the F200Y mutation or substi-
tution of phenylalanine (F) with tyrosine (Y) in
codon 200 (TTC to TAC) in TUB2 (Table 1). Like
high resistance, the definition of resistance and
 Table 1  Point mutations in β-tubulin gene of Colletotrichum species associated with MBC fungicide resistance

Phytoparasitica
Change in codon Code Species Reduced sensitivity to Level of Resistance a
(Amino acid)

Wild type Mutated

(Susceptible) (Resistant)

GAG​ GCG​ E198A C. gloeosporioides Carbendazim HR

HR
HR
HR
HR
R
Benomyl HR
R
HR
R
Thiabendazole R
R
Thiophanate-methyl HR
Benomyl NR
Benomyl R
C. siamense Thiophanate-methyl HR
HR
HR
HR
Carbendazim HR
HR
HR
C. truncatum Thiabendazole HR
HR
HR
IR
C. cereale Carbendazim HR
C. fructicola Thiophanate-methyl HR
Carbendazim HR
HR
Vol.: (0123456789)

HR
13

C. asianum Carbendazim HR
GAG​ AAG​ E198K C. acutatum Thiophanate-methyl, Benomyl R
Benzimidazole + Diethofencarb R
 Vol:. (1234567890)
13

Table 1  (continued)

Change in codon Code Species Reduced sensitivity to Level of Resistance a

(Amino acid)

Wild type Mutated

(Susceptible) (Resistant)

TTC​ TAC​ F200Y C. gloeosporioides Carbendazim R

Carbendazim + benomyl R

Benomyl MR

Thiabendazole MR

Carbendazim MR

Diethofencarb & Benzimidazole R

Carbendazim R

C. siamense Carbendazim MR

MR

C. cereale Thiophanate-methyl R

C. musae Thiabendazole LS

Thiophanate-methyl MR

C. truncatum Carbendazim HR

C. fructicola Carbendazim MR
GAG​ GCG​ E198A C. truncatum Carbendazim HR
GAC​ AAC​ D209N
GAG​ GCG​ E198A C. truncatum Benomyl HR
ATA​ TTG​ F270Y
CGC​ GCC​ A271S

Phytoparasitica
 Table 1  (continued)

Phytoparasitica
Change in codon EC50/ED50/MIC b Scale Host Reference
(Amino acid)
Wild type Mutated
(Susceptible) (Resistant)

GAG​ GCG​ > 1000 µg ­mL−1 MIC > 1000 µg ­L−1 MIC Mango Bincader et al., 2021
- Uninhibited growth Mango Nalumpang et al., 2010
at ≥ 500 mg ­L−1
- Uninhibited growth Mango Kongtragoul et al., 2011
at ≥ 500 mg ­L−1
> 500 µg ­mL−1 ­EC50 EC50 > 500 µg ­mL−1 Strawberry Chung et al., 2010
> 500 µg ­mL−1 ­EC50 EC50 > 500 µg ­mL−1 Mango
100–500 µg ­mL−1 ­EC50 EC50 100-500 µg ­mL−1 Mango
> 500 µg ­mL−1 ­EC50 EC50 > 500 µg ­mL−1 Strawberry
100–500 µg ­mL−1 ­EC50 EC50 100-500 µg ­mL−1 Strawberry
> 500 µg ­mL−1 ­EC50 EC50 > 500 µg ­mL−1 Mango
100–500 µg ­mL−1 ­EC50 EC50 100-500 µg ­mL−1 Mango
100–500 µg ­mL−1 ­EC50 EC50 100-500 µg ­mL−1 Strawberry
100–500 µg ­mL−1 ­EC50 EC50 100-500 µg ­mL−1 Mango
> 100 µg ­mL−1 ­EC50 EC50 > 100 mg ­L−1 Japanese pear Chung et al., 2006
- Uninhibited growth at Blueberry Peres et al., 2004
10 µg ­mL−1
- Uninhibited growth at Statice Maymon et al., 2006
10 µg ­mL−1
> 100 µg ­mL−1 ­EC50 - Blueberry Hu et al., 2015
> 100 µg ­mL−1 ­EC50 - Peach
> 100 µg ­mL−1 ­EC50 EC50 > 100 mg ­L−1 Apple Chechi et al., 2019
> 100 µg ­mL−1 MIC Growth was inhibited Strawberry Luo et al., 2020
­ L−1
by ≤ 30% at 100 µg m
- Growth uninhibited by Strawberry Han et al., 2018
50 µg ­mL−1
> 1000 µg ­mL−1 MIC > 1000 µg ­L−1 MIC Mango Bincader et al., 2021
- Growth uninhibited by Strawberry Zhong et al., 2020
500 µg ­L−1
Vol.: (0123456789)
13
 Vol:. (1234567890) Table 1  (continued)
13
Change in codon EC50/ED50/MIC b Scale Host Reference
(Amino acid)
Wild type Mutated
(Susceptible) (Resistant)
- ED50 > 100 µg ­mL−1 Papaya Torres-Calzada et al., 2015
- ED50 > 100 µg ­mL−1 Capsicum anuum
- ED50 > 100 µg ­mL−1 Jatropha curcas
- ED50 10-100 µg ­mL−1 Papaya
> 2000 µg ­mL−1 ­EC50 EC50 > 100 µg ­mL−1 Soybean Poti et al., 2020
−1 −1
> 10 µg ­mL ­EC50 EC50 > 10 µg ­mL turfgrass Young et al., 2010a
- Growth uninhibited by Strawberry Han et al., 2018
50 µg ­mL−1
- Growth uninhibited by Yam
50 µg ­mL−1
- Uninhibited growth at Strawberry Zhong et al., 2020
500 µg ­mL−1
> 1000 µg ­L−1 MIC > 1000 µg ­L−1 MIC Mango Bincader et al., 2021
GAG​ AAG​ > 30 µg ­mL−1 ­ED50 > 30 µg ­mL−1 ­ED50 Turfgrass Wong et al., 2008
- - Strawberry Ishii et al. 2002
TTC​ TAC​ > 10 µg ­mL−1 MIC > 10 µg ­mL−1 MIC Grape Hwang et al., 2010
> 10 µg ­mL−1 MIC > 10 µg ­mL−1 MIC Grape
10–100 µg ­mL−1 ­EC50 10–100 µg ­mL−1 ­EC50 Mango Chung et al., 2010
10–100 µg ­mL−1 ­EC50 10–100 µg ­mL−1 ­EC50 Mango
10–100 µg ­mL−1 ­EC50 10–100 µg ­mL−1 ­EC50 Mango
- - Japanese Pear Yano et al., 2004
100 µg ­L−1 MIC 100 µg ­L−1 MIC Mango Bincader et al., 2021
- Uninhibited growth at Strawberry Han et al., 2018
1 µg ­mL−1 but inhibited at
50 µg ­mL−1
100 µg ­L−1 MIC 100 µg ­L−1 MIC Mango Bincader et al., 2021
Growth was uninhibited at Strawberry Zhong et al., 2020
100 µg ­mL−1
> 10 µg ­mL−1 ­EC50 EC50 > 10 µg ­mL−1 Turfgrass Young et al., 2010a

Phytoparasitica
51.51–53.74 µg mL −1EC50 N/A Banana Leite et al., 2020
- 10–100 µg ­mL−1 ­EC50 Banana Vieira et al., 2017
 Phytoparasitica

moderate resistance may greatly vary from one study

to another (Table 1). As noted, resistance towards
carbendazim is defined as 100–500 µg ­ mL−1 ­EC50

Ramdial et al., 2016

−1
or > 10 µg ­mL MIC. Moderate resistance towards

Zhong et al., 2020

Poti et al., 2020 carbendazim, on the other hand, can be defined as

Poti et al., 2020

isolates with 10-100 µg ­ mL−1 ­EC50 or uninhibited
Reference

−1
growth at 100 µg ­mL . Resistance towards benomyl
is defined as uninhibited growth at 10 µg ­mL−1 or
100–500 µg ­mL−1 ­EC50, while moderate resistance is
defined as 10–100 µg ­mL−1 ­EC50. For thiabendazole,
resistance is defined as 100–500 µg ­mL−1 ­EC50 while

HR – Highly Resistant; NR – Naturally resistant; R – Resistant; MR – Moderately resistant; IR – Intermediate resistance; LS – Less sensitive
moderate (or intermediate) resistance is defined as
10–100 µg ­mL−1 ­EC50. Lastly, for thiophanate methyl,
resistance is defined as > 10 µg ­mL−1 ­EC50 while mod-
Bell pepper
Strawberry

erate resistance is defined as 10–100 µg ­mL−1 ­EC50.

Soybean

Soybean

Among the Colletotrichum species reported with

Host

point mutations in TUB2 and resistance to MBC fun-

gicides include C. gloeosporioides, C. siamense, C.
Growth was uninhibited at

truncatum, C. cereale, C. fructicola, C. acutatum, and

Uninhibited growth at
EC50 > 100 µg ­mL−1

EC50 > 100 µg ­mL−1

C. musae.
100 µg ­mL−1

10 µg ­mL−1

Point mutations in Colletotrichum species associated

with QoI fungicide resistance
­ D50 – Effective dose
Scale

QoI fungicides are respiration-inhibitor fungicides

that belong to FRAC code 11 (FRAC, 2020) and have
a broad-spectrum and potent antifungal activity. Among
> 2000 µg ­mL−1 ­EC50

> 2000 µg ­mL−1 ­EC50

> 1000 µg ­mL−1 ­EC50

the common QoI fungicides used to control Colle-

MIC – Minimum inhibitory concentration; ­EC50 – Effective concentration; E
EC50/ED50/MIC b

totrichum are strobilurins such as azoxystrobin, picox-

ystrobin, pyraclostrobin, trifloxystrobin, etc. (FRAC,
2020). QoIs block the electron transfer by binding to the
quinone-outside (Qo) site of the cytochrome b (a com-
ponent of respiratory chain complex III), thus, inhibiting
-

mitochondrial respiration and ATP production (Vin-

celli, 2002; Wood & Hollomon, 2003). As a result, the
energy cycle is disrupted, which leads to fungal death.
Hence, mutations in the mitochondrial cytochrome b
(CYTB) gene usually confers resistance against this
(Resistant)

fungicide. In Colletotrichum, the substitution of gly-

Mutated

cine (G) with alanine (A) in codon 143 (GGT to GCT)

GCG​

GCG​
AAC​

GCC​
TTG​

or G143A mutation in the mitochondrial cytochrome

b (CYTB) gene is usually associated with high levels
of QoI-resistance (Table 2). High resistance, complete
resistance, and resistance towards azoxystrobin, thi-
Table 1  (continued)

ophanate-methyl, and pyraclostrobin is often defined as

Change in codon

the ability of the fungi to grow at concentrations greater

(Susceptible)
(Amino acid)

than > 100 µg ­mL−1 or the E ­ C50 is > 100 µg ­mL−1. In

Wild type

comparison, high resistance towards picoxystrobin can

GAG​

GAG​
GAC​

CGC​
ATA​

be defined as the ability of the fungi to grow uninhibited

b
a

Vol.: (0123456789)
13

at > 9.52 µg ­mL−1 (Table 2). Meanwhile, moderate
resistance is attributed to the substitution of phenylala-
nine (F) with leucine (L) in codon 129 (TTC to TTG) of
CYTB (F129L mutation) (Table 2). Moderate (or inter-
mediate) resistance towards azoxystrobin can be defined
as isolates with ­EC50 of 1.03–100 µg ­mL−1 or inhibi-
tion at 8 µg ­mL−1, while moderate resistance towards
pyraclostrobin can be defined as isolates with E ­ C50 of
0.11–10 µg ­mL−1 or 15–50% growth inhibition at 100
µg ­mL−1 (Table 2). Aside from these mutations, it has
been observed that the absence of introns in the CYTB
gene may have been linked to increased resistance to
QoI fungicides in Colletotrichum siamense (Hu et al.,
2015). The two genotypes observed were those with no
introns and those with two introns in the CYTB gene.
Although the group with two introns also contained
resistant isolates, all intronless C. siamense were G143A
mutants and were highly resistant to azoxystrobin fungi-
cide (Hu et al., 2015). However, due to the limited sam-
ple size, caution must be observed to conclude that this
genotype is also linked to high resistance. Among the
Colletotrichum species reported with point mutations in
CYTB and resistance to QoI fungicides include C. gloe-
osporioides, C. acutatum, C. siamense, C. cereale, C.
nymphaeae, C. fructicola, and C. graminicola.
Complex mutations in Colletotrichum species
associated with DMI fungicide resistance
DMI fungicides belong to FRAC code 3 and some of
its known fungicides against Colletotrichum include
difenoconazole, tebuconazole, propiconazole, met-
conazole, flutriafol, fenbuconazole, myclobutanil,
among others (FRAC, 2020). DMI fungicides target
the sterol 14α-demethylase, an enzyme encoded by
the CYP51 gene, which is involved in the ergosterol
biosynthetic pathway to synthesize important sterol
components essential for the permeability and rigidity
of fungal plasma membranes (Becher & Wirsel, 2012;
Wang et al., 2020). Thus, mutations in the nuclear
sterol 14α-demethylase (CYP51) gene have been
associated with DMI fungicide resistance. Unlike
MBC and QoI resistance, which are associated with
point mutations in the target genes, multiple and com-
plex nucleotide mutations for DMI resistance occur
in the two paralogous genes Cyp51A and Cyp51B of
Colletotrichum species (Table 3). The mutations also
seem to vary in different species or isolates.
Vol:. (1234567890)
13
Phytoparasitica
In C. gloeosporioides infecting grapes, Wang
et al. (2020) identified field-resistant isolates
whose ­EC50 towards difenoconazole ranged from
0.34–2.11 µg ­mL−1. The isolates also exhibited cross-
resistance towards propiconazole and prochloraz with
­EC50 of 0.85–2.10 µg ­mL−1 and 0.1–0.3 µg ­mL−1,
respectively. Sequencing of the Cyp51A and Cyp51B
led to the identification of six resistant genotypes
(RG) that may be associated with different levels
of resistance toward DMI fungicide (Table 3). RG I
showed a low resistant (LR) phenotype with L58V,
S175P, and P283S mutations at CgCYP51A and
T261A mutation at CgCYP51B. RG II showed LR
phenotype with L24V, L58V, A73V, I94L, S175P,
V387I, and E423K mutations, including a stop
codon mutation leading to the addition of five amino
acid residues (514G, 515N, 516E, 517 T, 518I)
extended at the C-terminal of the last open read-
ing frame (ORF) of CgCYP51A (Table 3); while in
CgCYP51B, three mutations (L189M, Q359H, and
T397I) were observed. RG III showed a moderately
resistant (MR) phenotype with the same mutations as
RGII. However, the Q359H mutation is replaced with
Q359N at CgCYP51B. RG IV showed MR pheno-
type with mutations identical to RG III and an addi-
tional synonymous mutation at codon ten wherein
CCG is replaced with CCT. However, both produce
the same amino acid Proline [P(CCG)10P(CCT)]
at CgCYP51A. RG V showed MR phenotype with
identical mutations to RG IV, but the Q359N muta-
tion is replaced with Q359H at CgCYP51B. RG VI
showed MR phenotype with similar mutations to RG
V but with two extra mutations (I328V and N518K)
at CgCYP51B. Reverse genetics showed that the
function of CgCYP51A and CgCYP51B acts like a
fulcrum. For instance, deletion of CgCYP51A led to
decreased resistance to difenoconazole, while dele-
tion of CgCYP51B led to increased resistance to
difenoconazole. However, deletion of CgCYP51A led
to increased resistance against prochloraz, while dele-
tion of CgCYP51B led to decreased resistance against
prochloraz. Meanwhile, in C. gloeosporioides isolated
from chili, Wei et al. (2020) reported six DMI fungi-
cide-resistant isolates whose growth was uninhibited
at 10 µg ­mL−1 of tebuconazole (with positive cross-
resistance with difenoconazole or propiconazole)
(Table 3). These isolates exhibited three different gen-
otypes, Genotype I (4 isolates) harbors V18F, L58V,
S175P, and P341A mutations at CgCYP51A and no
 Table 2  Point mutations in cytochrome b gene of Colletotrichum species associated with QoI fungicide resistance

Phytoparasitica
Change in codon Code Species Reduced sensitivity to Level of Resistancea EC50/ ED50/MICb Scale Host Reference
(amino acid)

Wild type Mutated

(Susceptible) (Resistant)

GGT​ GCT​ G143A C. gloeosporioides Picoxystrobin HR 9.52–18.9 µg ­mL−1 ­EC50 - Pepper Ren et al.,
2020
Pyraclostrobin HR - ­ L−1
Slightly inhibited by 100 g m Blueberry Ali et al., 2019

Azoxystrobin HR > 3000 ppm MIC - Mango Takushi et al.,

2014
HR 4.68–8.64 µg ­mL−1 ­EC50 - Grape Wei et al. 2021

Azoxystrobin & Kresoxim-methyl R - - Strawberry Inada et al.,

2008
C. forcelini Azoxystrobin CR - ­ L−1
Uninhibited growth at 100 µg m Strawberry Forcelini et al.,
2018a
R > 40 µg ­mL−1 ­EC50 - Strawberry Haack et al.,
2018
Azoxystrobin Pyraclostrobin R > 100 µg ­mL−1 ­EC50 - Strawberry Forcelini et al.,
> 10 µg ­mL−1 ­EC50 2016
Pyraclostrobin R - none to < 15% growth inhibition at Strawberry Ali et al., 2020
100 µg ­mL−1
MR - 15–50% growth inhibition at Strawberry
100 µg ­mL−1
C. siamense Azoxystrobin HR ­ L−1 ­EC50
> 100 µg m - Apple Chechi et al.,
2019
R > 100 µg ­mL−1 ­EC50 < 30% growth inhibition at Strawberry Luo et al.,
100 µg ­mL−1 2020
Azoxystrobin Thiophanate-methyl R > 100 µg m­ L−1 ­EC50 - Blueberry Hu et al., 2015
> 100 µg ­mL−1 ­EC50
R > 100 µg ­mL−1 ­EC50 - Peach
> 100 µg ­mL−1 ­EC50
C. cereale Azoxystrobin HR > 8.0 µg ­mL−1 - Annual bluegrass Wong et al.,
Trifloxystrobin Pyraclostrobin 0.66 µg ­mL−1 2007
3.3 µg ­mL−1 ­ED50
Azoxystrobin R - Minimal growth inhibition at 8 Annual bluegrass Young et al.,
µg ­mL−1 2010b
R - Minimal growth inhibition Creeping bentgrass
at 8 µg ­mL−1
C. nymphaea Azoxystrobin R ­ L−1 ­EC50
> 100 µg m < 30% growth inhibition at Strawberry Luo et al.,
100 µg ­mL−1 2020
C. fructicola Azoxystrobin R - Growth > 50% at 100 ppm Apple Yokosawa
Vol.: (0123456789)

et al., 2017
13

C. graminicola Azoxystrobin HR > 10 µg ­mL−1 ­ED50 ED50 > 10 µg ­mL−1 Bent grass Avila-Adame
et al., 2003
HR > 10 µg ­mL−1 ­ED50 ­ L−1
ED50 > 10 µg m Blue grass
 Phytoparasitica

mutation at CgCYP51B; Genotype II (1 isolate) har-

Wei et al. 2021

Forcelini et al.,

Young et al.,
bors L58V, S175P, A340S, T379A and N476T muta-
Reference

Luo et al.,
2010b
2016

2020
tions at the CgCYP51A including D121N, T132A
and F391Y mutations at CgCYP51B. Genotype III
(1 isolate) harbors L58V and S175P mutations at
Annual bluegrass
the CgCYP51A and T262A at CgCYP51B. For C.
Strawberry
Strawberry

Strawberry

Strawberry
truncatum isolated from peach, soybean, citrus, and

Grape
Host

begonia (Chen et al., 2018a), resistance to DMI fun-

gicides was associated with L208Y, H238R, S302A,
­ L−1

I366L mutations in CtCYP51A and H373N, M376L,

Minimal growth at 0.031 µg m
and significant inhibition at

30–90% growth inhibition at

30–90% growth inhibition at

S511T mutations in CtCYP51B. Isolates bearing

these mutations had high resistance towards flutriafol
and fenbuconazole ­(EC50 > 50 µg ­mL−1) and 27- and
100 µg ­mL−1

100 µg ­mL−1
8 µg ­mL−1

96-fold less sensitivity against metconazole and tebu-

conazole, respectively, as compared to the sensitive
Scale

isolates. These isolates are also fivefold less sensitive

-
-

CR – Completely resistant; HR – Highly resistant; MR – Moderately resistant; IR – Intermediate resistance; R – Resistant

towards difenoconazole and propiconazole than the

0.11-10 µg ­mL−1 ­EC50

­ L−1 ­EC50

­ L−1 ­EC50

sensitive isolates. Chen et al. (2018a) also noted that

3-100 µg ­mL−1 ­EC50

1.03 µg ­mL−1 ­EC50

EC50/ ED50/MICb

expression of CYP51a and CYP51b did not correlate

> 100 µg m

> 100 µg m

to DMI fungicide resistance.

-

Effects of target gene mutations on fungicide binding

MIC – Minimum inhibitory concentration; ­EC50 – Effective concentration; ED – Effective dose
Level of Resistancea

The capability of fungicides to bind effectively to

the target proteins is essential to manifest its effects.
Therefore, modifications in the target proteins could
potentially hinder the effective binding of the fun-
MR
MR

MR

MR

MR
IR

gicide, which may then confer fungicide resistance.

Molecular docking analysis has been used extensively
to elucidate the impact of genetic mutations on fungi-
Reduced sensitivity to

cide binding.
Molecular docking analysis has revealed that the
Pyraclostrobin
Azoxystrobin

Azoxystrobin

Azoxystrobin

Azoxystrobin

Azoxystrobin

E198K and F200Y mutations could potentially be

involved in the decreased fungicide binding affinity to
beta-tubulin (Cai et al., 2015). Meanwhile, Vela-Cor-
cia et al. (2018) showed that the amino acid residues
C. gloeosporioides

198 and 200 are not directly involved in decreased

C. nymphaea
C. acutatum

C. siamense
C. cereale

MBC fungicide affinity but instead cause an introduc-

Species

tion of additional β-strands, i.e., β8, β9, β10, and β11,

which, in turn, may be responsible for the decreased
F129L

MBC fungicide binding affinity. The mutation M233I

Code

was also reported to decrease fungicide binding

affinity to beta-tubulin (Cai et al., 2015). However,
(Resistant)
Mutated

the M233I mutation has not been observed in Colle-

Table 2  (continued)

TTG​

totrichum. The effect of multiple point mutations

in the β-tubulin gene, as observed by Ramdial et al.
Change in codon

(2016), should also be elucidated.

(Susceptible)
(amino acid)

Wild type

Similarly, the G143A and F129L mutations also

TTC​

caused reduced binding affinity of azoxystrobin

b
a

Vol:. (1234567890)
13
 Table 3  Multiple mutations in sterol 14α-demethylase (CYP51) gene of Colletotrichum species associated with DMI resistance

Phytoparasitica
Species Gene Missense mutation Genotype Reduced Level of Resistancea Scale EC50/ED50/MICb Host Reference
sensitiv-
ity to

C. gloeosporioides CYP51A L58V, S175P, P285S RG I Difenoconazole LR Uninhibited growth at 0.64ug ­mL−1 ­EC50/32ug Grape Wang et al., 2020
32 µg ­mL−1 ­mL−1 MIC
CYP51B T261A
CYP51A L24V, L58V, A73V, I94L, RGII LR Uninhibited growth at 0.51–0.86 µg ­ml−1 Grape
S175P, V387I, E423K; 32 µg ­mL−1 ­EC50/32 µg ­ml−1 MIC
one mutation at stop
codon that led to addi-
tion of 514G, 515N,
516E, 517 T and 518I
CYP51B L189M, Q359H, T397I
CYP51A L24V, L58V, A73V, I94L, RGIII MR Uninhibited growth at 0.34 µg ­ml−1 ­EC50 Grape
S175P, V387I, E423K; 64 µg ­mL−1 /32 µg ­ml−1 MIC
one mutation at stop
codon that led to addi-
tion of 514G, 515N,
516E, 517 T and 518I
CYP51B L189M, Q359N, T397I
CYP51A L24V, L58V, A73V, I94L, RGIV MR Uninhibited growth at 1.10–1.52 µg ­ml−1 Grape
S175P, V387I, and 64 µg ­mL−1 ­EC50/64 µg ­ml−1 MIC
E423K; one mutation
at stop codon that led
to addition of 514G,
515N, 516E, 517 T and
518I; one synonymous
mutation at codon 10
[P(CCG)10P(CCT)]
CYP51B L189M, Q359N, T397I
CYP51A L24V, L58V, A73V, I94L, RGV MR Uninhibited growth at 1.15 µg ­ml−1 Grape
S175P, V387I, and 64 µg ­mL−1 ­EC50/64 µg ­ml−1 MIC
E423K; one mutation
at stop codon that led
to addition of 514G,
515N, 516E, 517 T and
518I; one synonymous
mutation at codon 10
[P(CCG)10P(CCT)]
CYP51B L189M, Q359H, T397I
CYP51A L24V, L58V, A73V, I94L, RGVI MR Uninhibited growth at 2.11 µg ­ml−1 Grape
S175P, V387I, and 64 µg ­mL−1 ­EC50/64 µg ­ml−1 MIC
E423K; one mutation
at stop codon that led
to addition of 514G,
515N, 516E, 517 T
and 518I; one mutation
at stop codon 514G,
Vol.: (0123456789)

515N, 516E, 517 T

13

and 518I
CYP51B L189M, I328V, Q359H,
T397I, N518K
 Phytoparasitica

to cytb protein, with the G143A mutation causing

Chen et al., 2018a

a higher reduction in binding affinity than F129L

Wei et al., 2020

(Wei et al., 2021; Fisher & Meunier, 2005). This is
Reference

in line with the observation that the G143A mutation

causes a higher resistance level than F129L mutations
(Table 2).

soybean,

begonia
citrus,
The complex mutations in the CYP51 genes can

Peach,
Chili

Chili

Chili
Host

also cause reduced affinity toward DMI fungicides

­ l−1 ­EC50/

(Chen et al., 2018a; Wei et al., 2020); and confor-

­ l−1 ­EC50/

­ l−1 ­EC50/

­ l-1EC50
mational changes of DMI fungicides in the binding

­ l-1EC50
­ l-1EC50
­ l-1EC50
0.76 ± 0.02 µg m
60 µg ­ml−1 MIC

90 µg ­ml−1 MIC

70 µg ­ml−1 MIC
EC50/ED50/MICb

­ l-1EC50
­ l-1EC50
pocket, which prevent the formation of the Fe–N
0.77 ± 0.03 µg m

1.42 ± 0.09 µg m

62.4 ± 28.4 µg m
13.2 ± 0.3 µg m
2.4 ± 0.3 µg m
2.5 ± 0.7 µg m
0.49 ± 0.02 –

coordinate bond between the heme iron active site and

> 50 µg m
> 50 µg m

some DMIs (Zhang et al., 2017). Besides, deletion of

Cyp51A or Cyp51B genes in two Colletotrichum spe-
cies (C. nymphaeae and C. fioriniae) showed species-
Uninhibited growth at

Uninhibited growth at

Uninhibited growth at

specific, differential binding of DMI fungicides onto

10 µg ­ml−1

10 µg ­ml−1

10 µg ­ml−1

the two CYP51 proteins, indicating that the use of

DMI mixture can have a synergistic effect against
Colletotrichum (Chen et al., 2020).
-

The major point mutations characterized in the tar-

Scale

get genes TUB2 and CYTB are adequate to provide

fungicide resistance (against MBCs and QoIs, respec-
tively), though, in some cases, additional mutations
MIC – Minimum inhibitory concentration; ­EC50 – Effective concentration; ED – Effective dose

in the target genes may accompany these. Due to the

MR
MR

high association to fungicide resistance, these major

R

R
R
R
R
Level of Resistancea

point mutations in target genes are being utilized as

markers for developing molecular detection systems
such as loop-mediated isothermal amplification assay
(LAMP), polymerase chain reaction (PCR)-based
Difenoconazole
Fenbuconazole

Propiconazole
Tebuconazole

Tebuconazole
Metconazole

assays, high-throughput SNP genotyping assays,

Flutriafol
Reduced
sensitiv-

etc. (Chung et al., 2010; Fan et al., 2019; Forcelini

ity to

et al., 2018a, 2018b; Wu et al., 2019). For example,

R – Resistant; MR – Moderately resistant; LR – Low resistance

in the plant pathogen Monilinia fructicola, a unique

Genotype III

65-bp repetitive element upstream of CYP51 (in the

Genotype II
Genotype I
Genotype

promoter region) called ‘Mona’ is used as a molecu-

lar marker for detecting DMI-resistance (Chen et al.,
-

2018b; Luo & Schnabel, 2008).

L208Y, H238R, S302A,
V18F, L58V, S17P, and

L58V, S175P, A340S,

T379A and N476T

H373N, M376L, and

D121N, T132A, and
Missense mutation

L58V and S175P

Fungicide resistance in non‑mutated Colletotrichum

and I366L

genotypes
P341A

F391Y

S511T
T262A

Fungicide resistance is not solely caused by point

mutations in the genes that code for proteins targeted
by fungicides. This is because resistant Colletotri-
CYP51A

CYP51A

CYP51A

CYP51A
CYP51B

CYP51B

CYP51B
Table 3  (continued)
Gene

chum isolates that do not harbor mutations in any

of the genes targeted by fungicides have been well
C. gloeosporioides

documented. In addition, such a phenomenon has

C. truncatum

been associated with various biological activities in

Species

the fungal cell. This section discusses the molecular

b
a

Vol:. (1234567890)
13
Phytoparasitica
mechanisms of fungicide resistance that are not gov-
erned by mutations in the fungicide’s target protein.
Species of Colletotrichum reported to have resist-
ance towards MBC fungicides but do not bear point
mutation are C. gloeosporioides (Chung et al., 2006),
C. acutatum (Chung et al., 2006; Hwang et al., 2010;
Peres et al., 2004), C. aenigma (Yokosawa et al.,
2017), C. siamense (Yokosawa et al., 2017), C. kaha-
wae (Yokosawa et al., 2017), and C. truncatum (Tor-
res-Calzada et al., 2015). Similarly, QoI-resistant C.
acutatum (Ali et al., 2020) and DMI-resistant C. trun-
catum (Zhang et al., 2017) that do not bear any point
mutations at the gene targeted by the fungicides have
also been reported.
There are many possible reasons for the occur-
rence of these non-target site mechanisms of fungi-
cide resistance. One is the presence of efflux systems
that pump out toxic compounds from the fungal cells.
The major efflux pumps are the ATP-binding cas-
sette (ABC) transporters located at the outer plasma
membrane. These transporters can transport differ-
ent macromolecules, which include alkaloids, lipids,
peptides, steroids, terpenoids, flavonoids, sugars,
inorganic anions, fungicides, and other drugs (de
Waard et al., 2006; de Waard, 1997). ABC transport-
ers’ exact molecular mechanism for removing toxins
from the fungal cell is unknown (de Waard et al.,
2006). There have been models describing how these
proteins transport their substrates. However, further
investigation is suggested (de Waard et al., 2006).
Despite this, its importance to fungicide resistance
has been shown by many. For example, the ABC
transporter of C. acutatum, CaABC1, exhibited ≥ 1.5-
fold increased expression when exposed to the fun-
gicides iprobenfos, kresoxim-methyl, thiophanate-
methyl, and the antifungal drug hygromycin, thus
indicating the role of this gene to fungicide resistance
(Kim et al., 2014). However, mutational analysis of
the genes targeted by fungicides is necessary to pro-
vide circumstantial evidence on the importance of
this protein to the decreased fungicide sensitivity
in other Colletotrichum spp. Similarly, in Botrytis
cinerea, Hayashi et al. (2002) observed that exposure
of isolates to fungicides led to the increased expres-
sion of BcatrD, an ABC transporter. The authors also
noted that overexpression of BcatrD led to significant
fungicide resistance. Furthermore, exposure to efflux
pump inhibitors increased the sensitivity of Pyre-
nophora tritici-repentis isolates to fungicides.
Another reason for decreased fungicide sensitivity
is the enhanced expression of proteins targeted by the
fungicides. This has been observed in the enhanced
resistance of C. acutatum isolates to MBC fungi-
cides. Through gene disruption studies, Nakaune
and Nakano (2007) revealed that the inherent resist-
ance of C. acutatum to benomyl could be caused by
the enhanced expression of β-tubulin 1 (CaTUB1)
gene regulated by CaBEN1 in the presence of beno-
myl. CaBEN1 was predicted to have a leucine zip-
per motif and different sites for protein modification.
However, the exact mechanism of CaBEN1 inducing
CaTUB1 in the presence of benomyl is yet to be elu-
cidated. Interestingly, for DMI resistance, the impact
of overexpression of CYP51 genes appears to vary. In
the study of Wei et al. (2020), overexpression of C.
gloeosporioides CgCYP51 genes in response to tebu-
conazole was observed and associated with decreased
sensitivity of the isolates to DMI fungicides. For C.
gloeosporioides, the mechanism of how the CYP51
overexpression occurs remains to be elucidated. How-
ever, for other fungi, CYP51 overexpression is caused
by promoter insertions (Luo & Schnabel, 2008) or
retrotransposons (Ma et al., 2006). In contrast to the
findings of Wei et al. (2020), Chen et al. (2018a)
was not able to find a correlation between enhanced
CYP51 expression to DMI sensitivity. However, the
authors compared resistant and sensitive isolates
of different species. Thus, there could have been
interspecies variations that could have affected the
conclusion.
The activation of metabolic pathways that act
as alternative pathways when the main pathway is
deactivated due to fungicide-induced stress could
also influence fungicide resistance. For instance, the
decreased supply of cellular ATP due to QoI fungi-
cide-mediated mitochondrial respiration inhibition
could be alleviated by the alternative oxidase (AOX)
pathway (Wood & Hollomon, 2003). Since this oxi-
dase is resistant to QoI fungicide, it can mediate the
electron transport chain in the mitochondrion, thus
aiding in the continuation of ATP synthesis when
the mitochondrial complex III is inhibited. Using
SHAM, an inhibitor of AOX, the effect of the alterna-
tive pathway has been evident due to the substantial
increase in ­EC50 among isolates exposed to the QoI
fungicides but not to SHAM (Piccirillo et al., 2018;
Wood & Hollomon, 2003). However, evidence shows
that SHAM could also be deleterious to fungi as it
Vol.: (0123456789)
13

may inhibit fungal growth (Pang et al., 2010; Gale,
1966; Khozin-Goldberg et al., 1999). Therefore, a
thorough evaluation of the role of SHAM in assess-
ing QoI fungicide sensitivity among fungi is still
required. Further, some fungi do not need AOX to
mitigate QoI-mediated fungicide inhibition (Chechi
et al., 2019; Forcelini et al., 2018a; Hu et al., 2015).
Interestingly, the AOX pathway has been observed to
be active only in vitro and not in vivo, possibly due
to the presence of plant metabolites that can suppress
AOX activity (Olaya et al., 1998; Olaya & Köller,
1999; Wood and Hollomon, 2003). In addition to the
AOX pathway, other proteins that aid in alleviating
mitochondrial respiration have been postulated. How-
ever, further investigation is needed to establish their
role in fungicide resistance (reviewed in Wood &
Hollomon, 2003). Regarding the inhibition of sterol
biosynthesis mediated by DMI fungicides, other pro-
teins and genes involved in sterol synthesis could also
play a role in DMI resistance. For instance, Martel
et al. (2010) observed that Candida albicans isolates
having mutations at erg3 gene (coding for Sterol
∆5,6-desaturase) and not erg11 gene (coding for sterol
14α-demethylase) exhibit resistance to DMI fungi-
cides. Deletions or mutations in the genes involved
in sterol biosynthesis led to enhanced DMI fungicide
sensitivity indicating the role of such genes in DMI
resistance (Martel et al., 2010; Yang et al., 2015).
Other non-target site mechanisms of fungicide
resistance reported are mitochondrial heteroplasmy,
removal of toxic compounds, activation of fungal
stress response pathways, alteration of sterol metabo-
lism (for DMI fungicide resistance), and presence of
multiple genetic loci (reviewed in Hu et al., 2015).
Therefore, due to other unknown resistance mecha-
nisms apart from genetic mutations, the employment
of molecular-based approaches to detect resistant
strains may provide incomplete results (Chechi et al.,
2019). This also emphasizes the importance of resist-
ance monitoring tests (e.g., in vitro sensitivity assays;
field studies) for Colletotrichum species rather than
depending on molecular-based detection approaches.
Distribution and phylogeographic analysis of
fungicide‑resistant Colletotrichum isolates
With the availability of the published reports and
gene sequences (i.e., in NCBI; Online Resource 1)
Vol:. (1234567890)
13
Phytoparasitica
associated with fungicide resistance, the distribution
and phylogeography of fungicide-resistant Colletotri-
chum isolates were also determined. Based on the
literature, most of the reports of Colletotrichum iso-
lates harboring gene mutations were from the USA,
followed by countries in Asia such as China, Japan,
and Thailand (Fig. 1; Fig. 2). Conversely, the least
reported Colletotrichum strains harboring gene muta-
tions were from South Korea, Taiwan, South Africa,
Brazil, Mexico, and Israel (Fig. 1; Fig. 2). In addition,
most of the reported Colletotrichum strains in the
USA and China had mutations in the cytochrome b
gene, which confers QoI fungicide resistance (Fig. 2).
Meanwhile, in Thailand, Japan, South Korea, Taiwan,
South Africa, Brazil, Mexico, and Israel, the major-
ity of Colletotrichum isolates reported had a mutation
in the β-tubulin gene, which confers MBC fungicide
resistance (Fig. 2). With regards to the host plant, the
majority of the Colletotrichum strains bearing muta-
tions were isolated from strawberry, followed by
grass, mango, grape, Capsicum, apple, peach, soy-
bean, statice, blueberry, banana, yam, citrus, Japanese
pear, begonia, papaya, and jatropha (Fig. 3).
Consistent exposure to fungicides applied in the
field appears to drive pathogen population shifts,
acting as selection pressure associated with the
increased prevalence of fungicide-resistant strains
and their progenies. Multigene phylogenetic analy-
sis of azoxystrobin-resistant C. siamense isolated
from either blueberry or peach revealed that resist-
ant strains were closely related to each other and thus
may have evolved from the same ancestor (Hu et al.,
2015). Therefore, it is evident that growers need to
employ disease management strategies without reli-
ance on chemical fungicides. Management strategies
that can be employed to reduce fungicide resistance
risk are alternation or combined application of fun-
gicides with different mode of actions, and adoption
of integrated disease management strategies (Brent
& Hollomon, 1998). Interestingly, there have been
reports of decreased biological fitness among resist-
ant isolates (Wei et al., 2021; Avila-Adame et al.,
2003), hence, implying that fungicide resistant strains
may not persist in natural environment if it were not
for the consistent selection pressure. However, more
studies must be done to support this claim.
In this paper, phylogenetic and haplotype analy-
ses using CYTB and TUB2 genes showed that, gen-
erally, the resistant and sensitive Colletotrichum sp.
 Phytoparasitica
Fig. 1  Global distribution of reported fungicide-resistant Colletotrichum species bearing point mutations
Vol.: (0123456789)
13
 Vol:. (1234567890)
13

Fig. 2  Global distribution of gene mutations conferring fungicide resistance in Colletotrichum species

Phytoparasitica
Phytoparasitica
Fig. 3  Number of fungicide-resistant Colletotrichum isolates reported globally per crop
isolates (C. cereale, C. fructicola, C. siamense, C.
musae, C. acutatum) are clustered separately (Figs. 4
and 5; Online Resource 2; Online Resource 3). The
separation in clusters and haplotype groups (haplo-
groups) is largely dictated by the presence/ absence
of point mutations conferring fungicide resistance
in the target genes. With regards to TUB2, E198A
mutants of C. fructicola from China formed a sin-
gle haplogroup and clade, while the sensitive iso-
lates from China and USA formed two haplogroups
and clades (Online Resource 2; Online Resource 3).
E198A mutants of C. siamense (also from China and
the USA) formed two haplogroups and clades (Online
Resource 2; Online Resource 3). E198A mutants of
C. cereale from the USA displayed six haplogroups
which can be formed into two major groups/clades:
group 1 (H1-H2) and group 2 (H7-H10) (Fig. 4;
Online Resource 3). The sensitive haplotypes H3
and H6 appear to be the closely related isolates to the
group 1 and 2 resistant isolates, respectively (Fig. 4).
However, all sensitive isolates seem genetically dis-
tant from the two major groups, as shown in the hap-
lotype network (Fig. 4). E198K mutants of C. cereale
from the USA formed three haplogroups and clades
(Fig. 4; Online Resource 3). Here, the sensitive hap-
lotype H4 appears to be their closely related isolate
(Fig. 4). F200Y mutants of C. musae from Brazil
formed two haplogroups and clades (Online Resource
2; Online Resource 3). In C. siamense from China,
only one haplogroup was formed (Online Resource
2; Online Resource 3). F200Y mutants of C. cere-
ale from the USA formed four haplogroups, with
H5 as the closely related haplotype among the sensi-
tive isolates (Fig. 5). With regards to CYTB, G143A
mutants of C. siamense formed two haplogroups
and clades (Online Resource 2; Online Resource 3).
Here, resistant isolates remain closely related to the
sensitive isolate since QoI resistance was observed
Vol.: (0123456789)
13

H3
H2
H1
H4
H5
H2
H3
H1 H4
E198K
Fig. 4  Median-joining haplotype networks of sensitive and
fungicide resistant C. cereale isolates harboring E198A/K
mutations analyzed using PopART (Leigh and Bryant, 2015).
despite the lack of mutations due to the expression of
the alternative pathway, as discussed earlier. G143A
mutants of C. cereale from the USA formed seven
haplogroups (Fig. 5). Like E198A mutants, these hap-
logroups can also be formed into two larger, major
groups/clades: group 1 (H1-H3) and group 2 (H6-
H9) (Fig. 5; Online Resource 3). However, unlike in
MBC-sensitive isolates, the QoI-sensitive isolates
are more related to the fungicide-resistant isolates in
group 1 (Fig. 5; Online Resource 3). Meanwhile, the
haplotype network of C. acutatum separated the com-
pletely and moderately resistant isolates (G143A and
Vol:. (1234567890)
13
Phytoparasitica
H6 H8
H10
H7
H9
E198A
H6
H5
H7
H8
The genetic distance is presented as line. The nucleotide differ-
ences are presented as hatch marks. The size of each haplotype
corresponds to number of samples/sequences
F129L mutants, respectively) (Online Resource 2).
F129L mutants of C. cereale formed a single major
haplogroup, while QoI-sensitive isolates formed two
haplogroups (Online Resource 2).
Interestingly, these findings show that the sepa-
ration between fungicide-resistant and sensitive
isolates in C. cereale does not seem to be dic-
tated entirely by the point mutations. The sensi-
tive and mutated isolates (with E198A/K, F200Y,
and G143A mutations) appear to have different
evolutionary histories, as observed in the distant,
diverse, and distinct phylogenetic and haplotype
Phytoparasitica

H5
H1 H6

H2
H3

H4 H7
F200Y H8

H5

H4 H9

H8
H6

H2 H7
H1

G143A
H3

Fig. 5  Median-joining haplotype networks of sensitive and differences are presented as hatch marks. The size of each hap-
fungicide resistant C. cereale isolates harboring F200Y and lotype corresponds to number of samples/sequences. The loop
G143A mutations analyzed using PopART (Leigh and Bryant, in the G143A network indicate haplotype relatedness
2015). The genetic distance is presented as line. The nucleotide

groupings of resistant and sensitive isolates (Figs. 4 These results indicate the immense and long history
and 5; Online Resource 3). High haplotype number of selection pressure for fungicide resistance in C.
and haplotype diversity can also be observed in the cereale. Meanwhile, no significant and conclusive
resistant C. cereale isolates (Online Resource 4). results were established in the neutrality tests (i.e.,

Table 4  Neutrality tests Gene Mutation Species na Fu’s Fs Tajima’s D

of fungicide-resistant
b
Colletotrichum sp. isolates Fs P value D Significance
analyzed using DnaSP
(Rozas et al., 2017) CYTB G143A C. cereale 26 2.8540 0.8940 1.6611 P > 0.10
C. siamense 6 0.6250 0.7830 0.8506 P > 0.10
F129L C. cereale 8 no polymorphism
TUB2 E198A C. siamense 5 1.0400 0.7920 -0.9726 P > 0.10
C. cereale 15 2.0910 0.8330 1.1391 P > 0.10
E198K C. cereale 12 -1.3250 0.0452 -1.4514 P > 0.10
a
n = no. of sequences F200Y C. musae 7 -0.0950 0.1409 -1.0062 P > 0.10
b
Significant (5% level) if C. cereale 9 -3.0840 0.0131 -1.1278 P > 0.10
P < 0.02

Vol.: (0123456789)
13

Fu’s Fs and Tajima’s D) of fungicide-resistant Colle-
totrichum sp. isolates (i.e., C. cereale, C. siamense,
and C. musae), though some insights could be drawn
(Table 4). The TUB2 gene appears to display signa-
tures of positive selection for MBC resistance, as
shown by predominant negative values in the tests
(Table 4). Meanwhile, for the CYTB gene, a bal-
ancing selection for QoI resistance may be inferred
from the positive values in the neutrality tests
(Table 4). Neutrality tests were not analyzed in C.
fructicola and C. acutatum due to very low sequence
data available from fungicide-resistant isolates.
Given the global distribution of this plant patho-
gen, it is plausible to assume that there could be
underrepresentation or underreporting of fungicide-
resistant Colletotrichum strains. Still, this review
provides insights into the diversity and distribution
of Colletotrichum species resistant to QoI, MBC, and
DMI fungicide classes. However, the underreporting
of fungicide-resistant Colletotrichum strains should
be addressed immediately to prevent the uncontrolled
rise of fungicide-resistant subpopulations.
Conclusions
Fungicide resistance in Colletotrichum species is a
major challenge in controlling anthracnose. Though
Colletotrichum comprises a diverse group of plant
pathogens, the molecular mechanisms of fungicide
resistance have been elucidated only in a few species,
suggesting that more investigations should be con-
ducted. The molecular bases for MBC and QoI resist-
ance are well-studied. They are usually associated
with point mutations in the target genes. In contrast,
the molecular bases for DMI resistance remain largely
unexplored due to the complex mutations in the target
gene. For resistant Colletotrichum species or isolates
lacking mutations in the target genes, further research
must be conducted to unravel other fungicide resist-
ance mechanisms. Lastly, insights into the distribu-
tion and phylogeography of Colletotrichum isolates
with decreased sensitivity to well-known fungicides
can aid in addressing and formulating effective man-
agement strategies to prevent future epidemics.
Acknowledgements The authors thank the Institute of Plant
Breeding, College of Agriculture and Food Science, University
of the Philippines Los Baños and the Department of Science
Vol:. (1234567890)
13
Phytoparasitica
and Technology-Philippine Council for Agriculture, Aquatic
and Natural Resources Research and Development (DOST-
PCAARRD) for continued support. The authors also thank
Dr. Maria Luz J. Sison for the assistance in proofreading the
manuscript.
Authors’ contributions Cris Q. Cortaga and Benjamine
William P. Cordez- conceptualization, design, data analysis,
and manuscript drafting; Leilani S. Dacones- design, supervi-
sion, review, and editing; Mark Angelo O. Balendres and Fe M.
Dela Cueva- supervision, review, and editing. The authors have
read and approved the final manuscript for publication.
Data availability All data generated or analyzed during this
study are included in this published article and its supplemen-
tary information files.
Declarations
Ethical approval Not applicable.
Consent for publication Not applicable.
Competing interests The authors have no competing inter-
ests to declare that are relevant to the content of this article.
References
Adaskaveg, J. E., & Hartin, R. J. (1997). Characterization of
Colletotrichum acutatum isolates causing anthracnose of
almond and peach in California. Phytopathology, 87(9),
979–987. https://​doi.​org/​10.​1094/​PHYTO.​1997.​87.9.​979
Ali, M. E., Hudson, O., Hemphill, W. H., Brenneman, T. B.,
& Oliver, J. E. (2019). First report of resistance to pyra-
clostrobin, boscalid, and thiophanate-methyl in Colletotri-
chum gloeosporioides from blueberry in Georgia. Plant
Health Progress, 20(4), 261–262. https://​doi.​org/​10.​1094/​
PHP-​08-​19-​0058-​BR
Ali, M. E., Hudson, O., Waliullah, S., Cook, J., & Brannen,
P. M. (2020). Sensitivity of Colletotrichum isolates col-
lected from strawberries in Georgia to pyraclostrobin,
a quinone outside inhibitor (QoI) fungicide. Plant
Health Progress, 21(1), 69–70. https://​doi.​org/​10.​1094/​
PHP-​12-​19-​0090-​BR
Avila-Adame, C., Olaya, G., & Köller, W. (2003). Characteri-
zation of Colletotrichum graminicola isolates resistant to
strobilurin-related QoI fungicides. Plant Disease, 87(12),
1426–1432. https://​doi.​org/​10.​1094/​PDIS.​2003.​87.​12.​
1426
Baggio, J. S., Wang, N. Y., Peres, N. A., & Amorim, L. (2018).
Baseline sensitivity of Colletotrichum acutatum iso-
lates from Brazilian strawberry fields to azoxystrobin,
difenoconazole, and thiophanate-methyl. Tropical
Plant Pathology, 43, 533–542. https://​doi.​org/​10.​1007/​
s40858-​018-​0232-2
Phytoparasitica
Becher, R., & Wirsel, S. G. R. (2012). Fungal cytochrome
P450 sterol 14α-demethylase (CYP51) and azole resist-
ance in plant and human pathogens. Applied Microbiol-
ogy and Biotechnology, 95, 825–840. https://​doi.​org/​10.​
1007/​s00253-​012-​4195-9
Bincader, S., Pongpisutta, R., & Rattanakreetakul, C. (2021).
Colletotrichum species related to “Nam Dork Mai See
Tong” mango in Central Thailand and risk detection
of carbendazim-resistant strains. Research Square.
Retrieved September 1, 2021, from https://​doi.​org/​10.​
21203/​rs.3.​rs-​782870/​v1
Brent, K. J., & Hollomon, D. W. (1998). Fungicide resist-
ance: the assessment of risk (Vol. 2). Brussels, Belgium:
Global Crop Protection Federation.
Cai, M., Lin, D., Chen, L., Bi, Y., Xiao, L., & Liu, X. L.
(2015). M233I mutation in the β-tubulin of Botry-
tis cinerea confers resistance to zoxamide. Scientific
Reports, 5, 16881. https://​doi.​org/​10.​1038/​srep1​6881
Chechi, A., Lewis Ivey, M. L., Kaufman, R. R., Bryson,
K. P., & Schnabel, G. (2020). Quinone outside inhibi-
tor-resistant Colletotrichum nymphaeae isolates from
strawberry lack mutations in cytb gene. Journal of
Plant Pathology, 102, 681–683. https://​doi.​org/​10.​1007/​
s42161-​020-​00565-8
Chechi, A., Stahlecker, J., Dowling, M. E., & Schnabel, G.
(2019). Diversity in species composition and fungicide
resistance profiles in Colletotrichum isolates from apples.
Pesticide Biochemistry and Physiology, 158, 18–24.
https://​doi.​org/​10.​1016/j.​pestbp.​2019.​04.​002
Chen S., Hu M., Schnabel G., Yang D., Yan X., Yuan H. (2020)
Paralogous CYP51 genes of Colletotrichum spp. medi-
ate differential sensitivity to sterol demethylation inhibi-
tors. Phytopathology, 110(3), 615–625. https://​doi.​org/​10.​
1094/​PHYTO-​10-​19-​0385-R
Chen, S., Schnabel, G., Yuan, H., & Luo, C. (2018a). LAMP
detection of the genetic element ‘Mona’ associated with
DMI resistance in Monilinia fructicola. Pest Management
Science, 75(3), 779–786. https://​doi.​org/​10.​1002/​ps.​5178
Chen, S., Wang, Y., Schnabel, G., Peng, C. A., Lagishetty, S.,
Smith, K., Luo, C., & Yuan, H. (2018b). Inherent resist-
ance to 14α-demethylation inhibitor fungicides in Colle-
totrichum truncatum is likely linked to CYP51A and/or
CYP51B gene variants. Phytopathology, 108(11), 1263–
1275. https://​doi.​org/​10.​1094/​PHYTO-​02-​18-​0054-R
Chen S.N., Luo C.X., Hu M.J., Schnabel G. (2016) Sensitivity
of Colletotrichum species, including C. fioriniae and C.
nymphaeae, from peach to demethylation inhibitor fungi-
cides. Plant Disease, 100(12), 2434–2441. https://​doi.​org/​
10.​1094/​PDIS-​04-​16-​0574-​RE
Chung, W. H., Chung, W. C., Peng, M. T., Yang, H. R., &
Huang, J. W. (2010). Specific detection of benzimidazole
resistance in Colletotrichum gloeosporioides from fruit
crops by PCR-RFLP. New Biotechnology, 27(1), 17–24.
https://​doi.​org/​10.​1016/j.​nbt.​2009.​10.​004
Chung, W. H., Ishii, H., Nishimura, K., Fukaya, M., Yano, K.,
& Kajitani, Y. (2006). Fungicide sensitivity and phyloge-
netic relationship of anthracnose fungi isolated from vari-
ous fruit crops in Japan. Plant Disease, 90(4), 506–512.
https://​doi.​org/​10.​1094/​PD-​90-​0506
Davidse, L. C. (1973). Antimitotic activity of methyl benzi-
midazole-2-yl carbamate (MBC) in Aspergillus nidulans.
Pesticide Biochemistry and Physiology, 3, 317–325.
https://​doi.​org/​10.​1016/​0048-​3575(73)​90030-8
de Waard, M. A., Andrade, A. C., Hayashi, K., Schoonbeek,
H.-j., Stergiopoulos, I., & Zwiers, L.-H. (2006). Impact of
fungal drug transporters on fungicide sensitivity, multid-
rug resistance and virulence. Pest Management Science,
62, 195–207. https://​doi.​org/​10.​1002/​ps.​1150
de Waard, M. A. (1997). Significance of ABC transporters in
fungicide sensitivity and resistance. Pesticide Science,
51(3), 271–275. https://​doi.​org/​10.​1002/​(SICI)​1096-​
9063(199711)​51:3%​3c271::​AID-​PS642%​3e3.0.​CO;​2-%​23
Fan, F., Hahn, M., Li, G. Q., Lin, Y., & Luo, C. X. (2019).
Rapid detection of benzimidazole resistance in Botry-
tis cinerea by loop-mediated isothermal amplification.
Phytopathology Research, 1, 10. https://​doi.​org/​10.​1186/​
s42483-​019-​0016-8
Fernández-Ortuño, D., Grabke, A., Bryson, P. K., Amiri, A.,
Peres, N. A., & Schnabel, G. (2014). Fungicide resistance
profiles in Botrytis cinerea from strawberry fields of seven
southern U.S. states. Plant Disease, 98, 825–833. https://​
doi.​org/​10.​1094/​PDIS-​09-​13-​0970-​RE
Fernández-Ortuño, D., Torés, J. A., De Vicente, A., & Pérez-
García, A. (2008). Mechanisms of resistance to QoI fungi-
cides in phytopathogenic fungi. International Microbiol-
ogy, 11(1), 1. https://​doi.​org/​10.​2436/​20.​1501.​01.​38
Fisher, N., & Meunier, B. (2005). Re-examination of inhibitor
resistance conferred by Qo-site mutations in cytochrome b
using yeast as a model system. Pest Management Science,
61(10), 973–978. https://​doi.​org/​10.​1002/​ps.​1066
Forcelini, B. B., Lee, S., Oliveira, M. S., & Peres, N. A.
(2018a). Development of high-throughput SNP geno-
typing assays for rapid detection of strawberry Colle-
totrichum species and the G143A mutation. Phytopa-
thology, 108(12), 1501–1508. https://​doi.​org/​10.​1094/​
PHYTO-​04-​18-​0128-R
Forcelini, B. B., Rebello, C. S., Wang, N. Y., & Peres, N. A.
(2018b). Fitness, competitive ability, and mutation stabil-
ity of isolates of Colletotrichum acutatum from strawberry
resistant to QoI fungicides. Phytopathology, 108(4), 462–
468. https://​doi.​org/​10.​1094/​PHYTO-​09-​17-​0296-R
Forcelini, B. B., Seijo, T. E., Amiri, A., & Peres, N. A. (2016).
Resistance in strawberry isolates of Colletotrichum acu-
tatum from Florida to quinone-outside inhibitor fungi-
cides. Plant Disease, 100(10), 2050–2056. https://​doi.​org/​
10.​1094/​PDIS-​01-​16-​0118-​RE
FRAC (Fungicide Resistance Action Committee). (2020, May).
List of first confirmed cases of plant pathogenic organ-
isms resistant to disease control agents. Retrieved August
1, 2022, from https://​www.​frac.​info/​docs/​defau​lt-​source/​
publi​catio​ns/​list-​of-​resis​tant-​plant-​patho​gens/​list-​of-​first-​
confi​rmed-​cases-​of-​plant-​patho​genic-​organ​isms-​resis​tant-​
to-​disea​se-​contr​ol-​agents_​05_​2020.​pdf
Fuentes-Aragón, D., Guarnaccia, V., Rebollar-Alviter, A.,
Juárez-Vázquez, S. B., Aguirre-Rayo, F., & Silva-Rojas,
H. V. (2020). Multilocus identification and thiophanate-
methyl sensitivity of Colletotrichum gloeosporioides spe-
cies complex associated with fruit with symptoms and
symptomless leaves of mango. Plant Pathology, 69(6),
1125–1138. https://​doi.​org/​10.​1111/​ppa.​13195
Gale, G. R. (1966). Selective inhibition of deoxyribonucleic
acid synthesis by salicylhydroxamic acid. Proceedings
Vol.: (0123456789)
13

of the Society for Experimental Biology and Medi-
cine, 122(4), 1236–1240. https://​doi.​org/​10.​3181/​00379​
727-​122-​3136
Haack, S. E., Ivors, K. L., Holmes, G. J., Förster, H., &
Adaskaveg, J. E. (2018). Natamycin, a new biofungicide
for managing crown rot of strawberry caused by QoI-
Resistant Colletotrichum acutatum. Plant Disease, 102(9),
1687–1695. https://​doi.​org/​10.​1094/​PDIS-​12-​17-​2033-​RE
Han, Y. C., Zeng, X. G., Xiang, F. Y., Zhang, Q. H., Guo, C.,
Chen, F. Y., & Gu, Y. C. (2018). Carbendazim sensitiv-
ity in populations of Colletotrichum gloeosporioides com-
plex infecting strawberry and yams in Hubei Province of
China. Journal of Integrative Agriculture, 17(6), 1391–
1400. https://​doi.​org/​10.​1016/​S2095-​3119(17)​61854-9
Hayashi, K., Schoonbeek, H. J., & De Waard, M. A. (2002).
Expression of the ABC transporter BcatrD from Botrytis
cinerea reduces sensitivity to sterol demethylation inhibi-
tor fungicides. Pesticide Biochemistry and Physiology,
73(2), 110–121. https://​doi.​org/​10.​1016/​S0048-​3575(02)​
00015-9
Hu, M. J., Grabke, A., Dowling, M. E., Holstein, H. J., & Sch-
nabel, G. (2015). Resistance in Colletotrichum siamense
from peach and blueberry to thiophanate-methyl and
azoxystrobin. Plant Disease, 99(6), 806–814. https://​doi.​
org/​10.​1094/​PDIS-​10-​14-​1077-​RE
Hu, M., & Chen, S. (2021). Non-target site mechanisms of
fungicide resistance in crop pathogens: A review. Micro-
organisms, 9(3), 502. https://​doi.​org/​10.​3390/​micro​organ​
isms9​030502
Hwang S., Kim H. R., Kim J., Park J. H., Lee S. B., Cheong
S. R., & Kim, H. T. (2010). Sensitivity of Colletotrichum
spp. isolated from grapes in Korea to carbendazim and the
mixture of carbendazim plus diethofencarb. Plant Pathol-
ogy Journal, 26(1), 49–56. https://​doi.​org/​10.​5423/​PPJ.​
2010.​26.1.​049
Inada, M., Ishii, H., Chung, W. H., Yamada, T., Yamaguchi, J.,
& Furuta, A. (2008). Occurrence of strobilurin-resistant
strains of Colletotrichum gloeosporioides (Glomerella
cingulata), the causal fungus of strawberry anthrac-
nose. Japanese Journal of Phytopathology, 74, 114–117.
https://​doi.​org/​10.​3186/​jjphy​topath.​74.​114
Ishii H. (2002) DNA-based approaches for diagnosis of fun-
gicide resistance. In Clark J.M., & Yamaguchi I. (Eds.),
Agrochemical Resistance: Extent, Mechanism, and Detec-
tion ­(808th vol., pp.242–259) American Chemical Society.
Ishii, H., Fraaije, B. A., Sugiyama, T., Noguchi, K., Nishimura,
K., Takeda, T., Amano, T., & Hollomon, D. W. (2001).
Occurrence and molecular characterization of strobilurin
resistance in cucumber powdery mildew and downy mil-
dew. Phytopathology, 91(12), 1166–1171. https://​doi.​org/​
10.​1094/​PHYTO.​2001.​91.​12.​1166
Jayawardena, R. S., Hyde, K. D., Damm, U., Cai, L., Liu, M.,
Li, X. H., Zhang, W., Zhao, W. S., & Yan, J. Y. (2016).
Notes on currently accepted species of Colletotrichum.
Mycosphere, 7(8), 1192–1260. https://​doi.​org/​10.​5943/​
mycos​phere/​si/​2c/9
Khozin-Goldberg, I., Bigogno, C., & Cohen, Z. (1999). Salicyl-
hydroxamic acid inhibits Δ6 desaturation in the microalga
Porphyridium cruentum. Biochimica et Biophysica Acta
(BBA)-Molecular and Cell Biology of Lipids, 1439(3),
384–394. https://​doi.​org/​10.​1016/​S1388-​1981(99)​00107-9
Vol:. (1234567890)
13
Phytoparasitica
Kim, S., Park, S. Y., Kim, H., Kim, D., Lee, S. W., Kim, H.
T., Lee, S., Kim, H. T., & Choi, W. (2014). Isolation and
characterization of the Colletotrichum acutatum ABC
transporter CaABC1. The Plant Pathology Journal,
30(4), 375. https://​doi.​org/​10.​5423/​PPJ.​OA.​08.​2014.​
0077
Kongtragoul, P., Nalumpang, S., Miyamoto, Y., Izumi, Y., &
Akimitsu, K. (2011). Mutation at codon 198 of TUB2
gene for carbendazim resistance in Colletotrichum gloe-
osporioides causing mango anthracnose in Thailand.
Journal of Plant Protection Research, 51(4), 377–384.
https://​doi.​org/​10.​2478/​v10045-​011-​0061-5
Leite, I. C. H. L., Silva, R. A., Santos, J. E. C. C., Freitas-
Lopes, R. L., Câmara, M. P. S., Michereff, S. J., &
Lopes, U. P. (2020). Analysis of Colletotrichum musae
populations from Brazil reveals the presence of isolates
with high competitive ability and reduced sensitivity to
postharvest fungicides. Plant Pathology, 69(8), 1529–
1539. https://​doi.​org/​10.​1111/​ppa.​13229
Liu, Z., Jian, Y., Chen, Y., Corby, K. H., He, P., Ma, Z., &
Yin, Y. (2019). A phosphorylated transcription factor
regulates sterol biosynthesis in Fusarium graminearum.
Nature Communications, 10, 1228. https://​doi.​org/​10.​
1038/​s41467-​019-​09145-6
Luo, C. X., & Schnabel, G. (2008). The cytochrome P450
lanosterol 14α-demethylase gene is a demethylation
inhibitor fungicide resistance determinant in Monilinia
fructicola field isolates from Georgia. Applied and Envi-
ronmental Microbiology, 74(2), 359–366. https://​doi.​
org/​10.​1128/​AEM.​02159-​07
Luo, Q., Schoeneberg, A., & Hu, M. (2020). Resistance to
azoxystrobin and thiophanate-methyl is widespread
in Colletotrichum spp. isolates from the Mid-Atlantic
Strawberry Fields. Plant Disease, 105, 2202–2208.
https://​doi.​org/​10.​1094/​PDIS-​09-​20-​2048-​RE
Ma, Z., Proffer, T. J., Jacobs, J. L., & Sundin, G. W. (2006).
Overexpression of the 14α-demethylase target gene
(CYP51) mediates fungicide resistance in Blumeriella
jaapii. Applied and Environmental Microbiology, 72(4),
2581–2585. https://​doi.​org/​10.​1128/​AEM.​72.4.​2581-​
2585.​2006
Marin-Felix, Y., Groenewald, J. Z., Cai, L., Chen, Q., Marin-
cowitz, S., Barnes, I., Bensch, K., Braun, U., Camporesi,
E., Damm, U., de Restrepo, Z. W., Hyde, K. D., Jaya-
wardena, R. S., Lombard, L., & Luangsa-ard., J., … &
De Beer Z.W. (2017). Genera of phytopathogenic fungi:
GOPHY 1. Studies in Mycology, 86, 99–216. https://​doi.​
org/​10.​1016/j.​simyco.​2017.​04.​002
Martel, C. M., Parker, J. E., Bader, O., Weig, M., Gross, U.,
Warrilow, A. G. S., Rolley, N., Kelly, D. E., & Kelly,
S. L. (2010). Identification and characterization of four
azole-resistant erg3 mutants of Candida albicans. Anti-
microbial Agents and Chemotherapy, 54(11), 4527–
4533. https://​doi.​org/​10.​1128/​AAC.​00348-​10
Maymon, M., Zveibil, A., Pivonia, S., Minz, D., & Free-
man, S. (2006). Identification and characterization of
benomyl-resistant and-sensitive populations of Colle-
totrichum gloeosporioides from statice (Limonium spp.).
Phytopathology, 96(5), 542–548. https://​doi.​org/​10.​
1094/​PHYTO-​96-​0542
Phytoparasitica
Moreira, R. R., Hamada, N. A., Peres, N. A., & De Mio, L.
L. M. (2019). Sensitivity of the Colletotrichum acutatum
species complex from apple trees in Brazil to dithiocar-
bamates, methyl benzimidazole carbamates, and quinone
outside inhibitor fungicides. Plant Disease, 103(10),
2569–2576. https://​doi.​org/​10.​1094/​PDIS-​07-​18-​1144-​RE
Nakaune, R., & Nakano, M. (2007). Benomyl resistance of
Colletotrichum acutatum is caused by enhanced expres-
sion of β-tubulin 1 gene regulated by putative leucine
zipper protein CaBEN1. Fungal Genetics and Biology,
44(12), 1324–1335. https://​doi.​org/​10.​1016/j.​fgb.​2007.​03.​
007
Nakaune, R., Adachi, K., Nawata, O., Tomiyama, M., Akutsu,
K., & Hibi, T. (1998). A novel ATP-binding cassette
transporter involved in multidrug resistance in the phy-
topathogenic fungus Penicillium digitatum. Applied and
Environmental Microbiology, 64(10), 3983–3988. https://​
doi.​org/​10.​1128/​AEM.​64.​10.​3983-​3988.​1998
Nalumpang, S., Miyamoto, Y., Miyake, C., Izumi, Y., Akit-
mitsu, K., & Kongtragoul, P. (2010). Point mutations in
the beta-tubulin gene conferred carbendazim-resistant
phenotypes of Colletotrichum gloeosporioides causing
‘Nam Dok Mai’ mango anthracnose. International Jour-
nal of Agricultural Technology, 6(2), 365–378.
Olaya, G., & Köller, W. (1999). Baseline sensitivities of Ven-
turia inaequalis populations to the strobilurin fungicide
kresoxim-methyl. Plant Disease, 83, 274–278. https://​doi.​
org/​10.​1094/​PDIS.​1999.​83.3.​274
Olaya, G., Zheng, D., & Köller, W. (1998). Differential
responses of germinating Venturia inaequalis conidia to
kresoxim-methyl. Pesticide Science, 54, 230–236. https://​
doi.​org/​10.​1002/​(SICI)​1096-​9063(19981​10)​54:3%​3c230::​
AID-​PS815%​3e3.0.​CO;2-O
Pang, S. Y. M., Tristram, S., & Brown, S. (2010). Salicylhy-
droxamic acid inhibits the growth of Candida albicans.
International Journal of Biological and Life Sciences, 6,
40–46.
Peres, N. A. R., Souza, N. L., Peever, T. L., & Timmer, L. W.
(2004). Benomyl sensitivity of isolates of Colletotrichum
acutatum and C. gloeosporioides from citrus. Plant Dis-
ease, 88, 125–130. https://​doi.​org/​10.​1094/​PDIS.​2004.​
88.2.​125
Piccirillo, G., Carrieri, R., Polizzi, G., Azzaro, A., Lahoz, E.,
Fernández-Ortuño, D., & Vitale, A. (2018). In vitro and
in vivo activity of QoI fungicides against Colletotrichum
gloeosporioides causing fruit anthracnose in Citrus sinen-
sis. Scientia Horticulturae, 236, 90–95. https://​doi.​org/​10.​
1016/j.​scien​ta.​2018.​03.​044
Poti, T., Mahawan, K., Cheewangkoon, R., Arunothayanan,
H., Akimitsu, K., & Nalumpang, S. (2020). Detection
and molecular characterization of carbendazim-resistant
Colletotrichum truncatum isolates causing anthracnose of
soybean in Thailand. Journal of Phytopathology, 168(5),
267–278. https://​doi.​org/​10.​1111/​jph.​12888
Ramdial, H., Hosein, F. N., & Rampersad, S. N. (2016).
Detection and molecular characterization of benzimida-
zole resistance among Colletotrichum truncatum isolates
infecting bell pepper in Trinidad. Plant Disease, 100,
1146–1152. https://​doi.​org/​10.​1094/​PDIS-​09-​15-​0995-​RE
Reimann, S., & Deising, H. B. (2005). Inhibition of efflux
transporter-mediated fungicide resistance in Pyrenophora
tritici-repentis by a derivative of 4′-hydroxyflavone and
enhancement of fungicide activity. Applied and Environ-
mental Microbiology, 71(6), 3269–3275. https://​doi.​org/​
10.​1128/​AEM.​71.6.​3269-​3275.​2005
Ren, L., Wang, S. F., Shi, X. J., Cao, J. Y., Zhou, J. B., & Zhao,
X. J. (2020). Characterisation of sensitivity of Colletotri-
chum gloeosporioides and Colletotrichum capsici, causing
pepper anthracnose, to picoxystrobin. Journal of Plant
Diseases and Protection, 127, 657–666. https://​doi.​org/​10.​
1007/​s41348-​020-​00316-y
Rozas, J., Ferrer-Mata, A., Sánchez-DelBarrio, J. C., Guirao-
Rico, S., Librado, P., Ramos-Onsins, S. E., & Sánchez-
Gracia, A. (2017). DnaSP 6: DNA sequence polymor-
phism analysis of large data sets. Molecular Biology and
Evolution, 34(12), 3299–3302. https://​doi.​org/​10.​1093/​
molbev/​msx248
Sato, T. (1998). Occurrence of apple bitter rot by grayish col-
ony form of Colletotrichum acutatum in Japan and path-
ogenicity to apple fruits of C. acutatum and Glomerella
cingulata isolated from other plants. Transactions of the
Mycological Society of Japan, 39, 35–44.
Seyran, M., Brenneman, T. B., & Stevenson, K. L. (2010). In
vitro toxicity of alternative oxidase inhibitors salicylhy-
droxamic acid and propyl gallate on Fusicladium effusum.
Journal of Pest Science, 83(4), 421–427. https://​doi.​org/​
10.​1007/​s10340-​010-​0312-7
Takushi, T., Kadekaru, K., Arasaki, C., & Taba, S. (2014).
Occurrence of strobilurin-resistant strains of Colle-
totrichum gloeosporioides, the causal fungus of mango
anthracnose. Japanese Journal of Phytopathology, 80(2),
119–123. https://​doi.​org/​10.​3186/​jjphy​topath.​80.​119
Torres-Calzada, C., Tapia-Tussell, R., Higuera-Ciapara, I.,
Martin-Mex, R., Nexticapan-Garcez, A., & Perez-Brito,
D. (2015). Sensitivity of Colletotrichum truncatum to four
fungicides and characterization of thiabendazole-resistant
isolates. Plant Disease, 99(11), 1590–1595. https://​doi.​
org/​10.​1094/​PDIS-​11-​14-​1183-​RE
Vela-Corcía, D., Romero, D., de Vicente, A., & Pérez-García,
A. (2018). Analysis of β-tubulin-carbendazim interac-
tion reveals that binding site for MBC fungicides does
not include residues involved in fungicide resistance.
Scientific Reports, 8, 7161. https://​doi.​org/​10.​1038/​
s41598-​018-​25336-5
Vieira, W. A. S., Lima, W. G., Nascimento, E. S., Michereff,
S. J., Reis, A., Doyle, V. P., & Câmara, M. P. S. (2017).
Thiophanate-methyl resistance and fitness components
of Colletotrichum musae isolates from banana in Brazil.
Plant Disease, 101(9), 1659–1665. https://​doi.​org/​10.​
1094/​PDIS-​11-​16-​1594-​RE
Vincelli, P. (2002). QoI (Strobilurin) Fungicides: Benefits and
Risks. The Plant Health Instructor. https://​doi.​org/​10.​
1094/​PHI-I-​2002-​0809-​02
Wang, J., Shi, D., Wei, L., Chen, W., Ma, W., Chen, C., &
Wang, K. (2020). Mutations at sterol 14α-demethylases
(CYP51A&B) confer the DMI resistance in Colletotri-
chum gloeosporioides from grape. Pest Management Sci-
ence, 76(12), 4093–4103. https://​doi.​org/​10.​1002/​ps.​5964
Wei, L. L., Chen, W. C., Zhao, W. C., Wang, J., Wang, B.
R., Li, F. J., Wei, M.-d, Guo, J., Chen, C., Zheng, J.,
& Wang, K. (2020). Mutations and overexpression of
CYP51 associated with DMI-resistance in Colletotrichum
Vol.: (0123456789)
13

gloeosporioides from Chili. Plant Disease, 104(3), 668–
676. https://​doi.​org/​10.​1094/​PDIS-​08-​19-​1628-​RE
Wei, L., Zheng, H., Zhang, P., Chen, W., Zheng, J., Chen, C.,
& Cao, A. (2021). Molecular and biochemical characteri-
zation of Colletotrichum gloeosporioides isolates resistant
to azoxystrobin from grape in China. Plant Pathology, 70,
1300–1309. https://​doi.​org/​10.​1111/​ppa.​13372
Wong, F. P., de la Cerda, K. A., Hernandez-Martinez, R., &
Midland, S. L. (2008). Detection and characterization of
benzimidazole resistance in California populations of
Colletotrichum cereale. Plant Disease, 92(2), 239–246.
https://​doi.​org/​10.​1094/​PDIS-​92-2-​0239
Wong, F. P., Midland, S. L., & de la Cerda, K. A. (2007).
Occurrence and distribution of QoI-resistant isolates of
Colletotrichum cereale from annual bluegrass in Califor-
nia. Plant Disease, 91(12), 1536–1546. https://​doi.​org/​10.​
1094/​PDIS-​91-​12-​1536
Wood, P. M., & Hollomon, D. W. (2003). A critical evaluation
of the role of alternative oxidase in the performance of
strobilurin and related fungicides acting at the Qo site of
complex III. Pest Management Science, 59(5), 499–511.
https://​doi.​org/​10.​1002/​ps.​655
Wu, J. Y., Hu, X. R., & Zhang, C. Q. (2019). Molecular
detection of QoI resistance in Colletotrichum gloe-
osporioides causing strawberry anthracnose based on
loop-mediated isothermal amplification assay. Plant
Disease, 103(6), 1319–1325. https://​doi.​org/​10.​1094/​
PDIS-​09-​18-​1593-​RE
Yang, H., Tong, J., Lee, C. W., Ha, S., Eom, S. H., & Im, Y. J.
(2015). Structural mechanism of ergosterol regulation by
fungal sterol transcription factor Upc2. Nature Communi-
cations, 6(1), 1–13. https://​doi.​org/​10.​1038/​ncomm​s7129
Yano, K., Ishii, H., Fukaya, M., Kawada, Y., & Sato, T. (2004).
Anthracnose on Japanese pear caused by intermediately
benzimidazole-resistant strains of Colletotrichum gloe-
osporioides (Glomerella cingulata). Japanese Journal
of Phytopathology, 70, 314–319. https://​doi.​org/​10.​3186/​
jjphy​topath.​70.​314
Yokosawa, S., Eguchi, N., Kondo, K. I., & Sato, T. (2017).
Phylogenetic relationship and fungicide sensitivity of
Vol:. (1234567890)
13
Phytoparasitica
members of the Colletotrichum gloeosporioides species
complex from apple. Journal of General Plant Pathology,
83, 291–298. https://​doi.​org/​10.​1007/​s10327-​017-​0732-9
Young, J. R., Tomaso-Peterson, M., de la Cerda, K., & Wong,
F. P. (2010a). Two mutations in β-tubulin 2 gene associ-
ated with thiophanate-methyl resistance in Colletotrichum
cereale isolates from creeping bentgrass in Mississippi
and Alabama. Plant Disease, 94(2), 207–212. https://​doi.​
org/​10.​1094/​PDIS-​94-2-​0207
Young, J. R., Tomaso-Peterson, M., Tredway, L. P., & de la
Cerda, K. (2010b). Occurrence and molecular identifi-
cation of azoxystrobin-resistant Colletotrichum cereale
isolates from golf course putting greens in the southern
United States. Plant Disease, 94(6), 751–757. https://​doi.​
org/​10.​1094/​PDIS-​94-6-​0751
Zhang, C., Diao, Y., Wang, W., Hao, J., Imran, M., Duan, H.,
& Liu, X. (2017). Assessing the risk for resistance and
elucidating the genetics of Colletotrichum truncatum
that is only sensitive to some DMI fungicides. Frontiers
in Microbiology, 8, 1779. https://​doi.​org/​10.​3389/​fmicb.​
2017.​01779
Zhong, S., Miao, J., Liu, X., & Zhang, G. (2020). Characteri-
zation of Colletotrichum spp. sensitivity to carbendazim
for isolates causing strawberry anthracnose in China.
Plant Disease, 105, 87–95. https://​doi.​org/​10.​1094/​
PDIS-​04-​20-​0875-​RE
Publisher’s Note Springer Nature remains neutral with regard
to jurisdictional claims in published maps and institutional
affiliations.
Springer Nature or its licensor (e.g. a society or other partner)
holds exclusive rights to this article under a publishing
agreement with the author(s) or other rightsholder(s); author
self-archiving of the accepted manuscript version of this article
is solely governed by the terms of such publishing agreement
and applicable law.