Pest Management Science
Pest Manag Sci 59:1143–1151 (online: 2003)
DOI: 10.1002/ps.744
Cloning and expression analysis of the
ATP-binding cassette transporter gene
MFABC1 and the alternative oxidase gene
MfAOX1 from Monilinia fructicola†
Guido Schnabel,∗ Qun Dai‡ and Manjiri R Paradkar
Department of Plant Pathology and Physiology, 218 Long Hall, Clemson University, Clemson, SC 29634, USA
Abstract: Brown rot, caused by Monilinia fructicola (G Wint) Honey, is a serious disease of peach in all
commercial peach production areas in the USA, including South Carolina where it has been primarily
controlled by pre-harvest application of 14-α demethylation (DMI) fungicides for more than 15 years.
Recently, the Qo fungicide azoxystrobin was registered for brown rot control and is currently being
investigated for its potential as a DMI fungicide rotation partner because of its different mode of action.
In an effort to investigate molecular mechanisms of DMI and Qo fungicide resistance in M fructicola,
the ABC transporter gene MfABC1 and the alternative oxidase gene MfAOX1 were cloned to study their
potential role in conferring fungicide resistance. The MfABC1 gene was 4380 bp in length and contained
one intron of 71 bp. The gene revealed high amino acid homologies with atrB from Aspergillus nidulans
(Eidam) Winter, an ABC transporter conferring resistance to many fungicides, including DMI fungicides.
MfABC1 gene expression was induced after myclobutanil and propiconazole treatment in isolates with
low sensitivity to the same fungicides, and in an isolate with high sensitivity to propiconazole. The results
suggest that the MfABC1 gene may be a DMI fungicide resistance determinant in M fructicola.
The alternative oxidase gene MfAOX1 from M fructicola was cloned and gene expression was analyzed.
The MfAOX1 gene was 1077 bp in length and contained two introns of 54 and 67 bp. The amino acid
sequence was 63.8, 63.8 and 57.7% identical to alternative oxidases from Venturia inaequalis (Cooke)
Winter, Aspergillus niger van Teighem and A nidulans, respectively. MfAOX1 expression in some but
not all M fructicola isolates was induced in mycelia treated with azoxystrobin. Azoxystrobin at 2 µg ml−1
significantly induced MfAOX1 expression in isolates with low MfAOX1 constitutive expression levels.
 2003 Society of Chemical Industry
Keywords: strobilurin fungicides; actin; real-time PCR; drug efflux pump
1 INTRODUCTION
Brown rot caused by Monilinia fructicola (G Wint)
Honey, can be a devastating disease in commercial
stone fruit production in the USA, reducing the
yield and post-harvest quality of many stone fruits,
including peaches. For the last two decades, control
has primarily relied on pre-harvest application of
14-α demethylase inhibitor (DMI) fungicides. It is
rather surprising that, despite the sole reliance on
DMI fungicides, there have been no reports of
commercial control failure due to loss of sensitivity
to these. However, the potential of M fructicola to
develop resistance has been shown in vitro1 and in
field studies.2
Accumulating reports of DMI resistance in plant
pathogens3 – 5 have stimulated research on the
molecular basis of DMI resistance. In filamentous
fungi, mutations in the 14-α demethylase gene, the
target enzyme of DMI fungicides, over-expression of
the same gene and drug efflux pumps of the ATP
binding cassette (ABC) transporter family have been
reported to confer resistance.6 – 11 Interest in ABC
transporters from filamentous fungi has been growing
in recent years because of their possible involvement
in fungicide resistance. Examples of such transporters
are atrA and atrB from Aspergillus nidulans (Eidam)
Winter and PMR1 from Penicillium digitatum (Pers)
Sacc.10 – 12
∗ Correspondence to: Guido Schnabel, Department of Plant Pathology and Physiology, 218 Long Hall, Clemson University, Clemson,
SC 29634, USA
E-mail: schnabe@clemson.edu
† Technical Contribution No 4867 of the South Carolina Agriculture and Forestry Research System, Clemson University
‡ Current address: Department of Medicine, University of Alabama, Birmingham, AL 35294, USA
Contract/grant sponsor: South Carolina Commission on Higher Education
Contract/grant sponsor: Clemson University Research Grant Committee
Contract/grant sponsor: CSREES/USDA; contract/grant number: SC-1700159
(Received 3 July 2002; revised version received 23 January 2003; accepted 1 April 2003)
Published online 18 June 2003
 2003 Society of Chemical Industry. Pest Manag Sci 1526–498X/2003/$30.00
1143
G Schnabel, Q Dai, MR Paradkar
Recently, azoxystrobin, a fungicide with a different
mode of action, was registered for brown rot control
in the USA, and its potential as rotation partner for
DMI fungicides is being investigated. Azoxystrobin is a
strobilurin-type fungicide that specifically inhibits the
ubiquinol–cytochrome c oxidoreductase (cytochrome
bc1 ) enzyme complex at the Qo center in the
respiration chain of fungal mitochondria.13 Strobilurin
fungicides were named STAR (fungicides of the
Strobilurin Type Action and Resistance group) or QoI
(Qo) fungicides by the Fungicide Resistance Action
Committee (FRAC).
In some pathogens, resistance to Qo fungicides
evolved quickly after the first products were registered.
Point mutations in the cytochrome bc1 gene represent
a major resistance mechanism14 – 16 but other, yet
unknown, mechanisms exist.17,18 Several fungal
pathogens have been reported to be less sensitive
to Qo fungicides in vitro due to activation of the
alternative respiratory pathway,19 – 23 which branches
from the normal cytochrome-mediated pathway at
the ubiquinone-pool and terminates at an alternative
oxidase (AOX). However, when the same fungicide
was applied on plants, similar resistance mechanism
from Qo fungicide action was not apparent.24,25 It was
proposed that constitutive plant antioxidants, such as
flavones, enter fungal cells and decrease the levels of
reactive oxygen species required for the induction
of the alternative oxidase gene.19,26,27 A different
model has recently been suggested that emphasizes
the importance of the alternative respiration in
providing protection from azoxystrobin during both
saprophytic and infectious stages of the plant pathogen
Magnaporthe grisea (Hebert) Barr.28
In an effort to improve our understanding of
molecular mechanisms of fungicide resistance in
M fructicola, we cloned the ABC transporter gene
MfABC1 and the alternative oxidase gene MfAOX1
from M fructicola. The MfABC1 gene encodes a
protein with high amino acid homology to the multidrug resistance protein atrB from A nidulans, while
MfAOX1 encodes the core enzyme for alternative
respiration in M fructicola. We analyzed the expression
of the MfABC1 gene in mycelium treated and
untreated with DMI fungicides myclobutanil and
propiconazole, and the expression of the MfAOX1
gene in mycelium treated and untreated with Qo
fungicide azoxystrobin.
2 EXPERIMENTAL METHODS
2.1 Collection, isolation and sensitivity of
Monilinia fructicola isolates to myclobutanil,
propiconazole and azoxystrobin
Peaches infected with sporulating M fructicola were
randomly collected from an abandoned peach orchard
(DL) located in Anderson county, South Carolina.
The orchard had never been treated with site
specific fungicides, such as Qo or DMI fungicides.
Conidia were scraped off the fruits with a sterile
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toothpick and transferred to 1 ml of sterile water
in a micro-centrifuge tube. After vortexing, 50 µl
of the conidial suspension were evenly distributed
onto water agar in Petri dish plates (15 × 90 mm)
amended with streptomycin sulfate at 100 µg ml−1 .
After the conidia had germinated, a hyphal tip from
a germinating conidium was transferred aseptically
to potato dextrose agar (PDA). Thirty-six isolates
obtained from different fruits were collected and
named DL(number of fruit).
The sensitivity of the DL isolates to the DMI
fungicides myclobutanil and propiconazole was determined in mycelium growth assays as described
previously.2 Formulated DMI fungicides myclobutanil (Nova 40W, Rohm & Haas Co, Philadelphia,
PA) and propiconazole (Orbit 3.6E, Syngenta Crop
Protection Inc, Greensboro, NC) and Qo fungicide
azoxystrobin (Abound; Syngenta) were dissolved in
water. Salicylhydroxamic acid (SHAM) was dissolved
in methanol so that methanol concentrations did not
exceed 3 ml litre−1 in the growth medium. Methanol
at this concentration had no effect on the parameters
tested.
Six DL isolates were selected randomly to determine the sensitivity to azoxystrobin. Sensitivity to
azoxystrobin was conducted as described previously,2
except that azoxystrobin was amended to the growth
medium at 0.001, 0.003, 0.01, 0.03, 0.1, 0.3, 1, 3 and
10 µg ml−1 . SHAM was amended to the azoxystrobinamended PDA media at 100 µg ml−1 either alone or
in mixture with azoxystrobin. ED50 values (µg ml−1 )
were calculated by regressing the percentages of the
relative growth against the logarithm of inhibitor concentrations.
2.2 Isolation of DNA, the MfABC1 gene, the
MfAOX1 gene, and a large fragment of the
MfActin gene from Monilinia fructicola
The genomic DNA used to identify the ABC
transporter gene MfABC1, the alternative oxidase gene
MfAOX1, and a large fragment of the MfActin gene
was purified from mycelium of isolate DL25. The
isolate was grown for one week in potato dextrose
broth (PDB) at 28 ◦ C, 100 rev min−1 and DNA was
extracted as described previously.29
An initial fragment of the MfABC1 gene was
amplified using degenerate primer pair AtrBF2 (5 TIM MIG TIW SIT TYC ARM GIW SIG CIG
G-3 ) and AtrBR2 (5 -CRT GCC RTC RTC IAC
IGT ICC IGG IGG-3 ). An initial fragment of the
MfAOX1 gene was amplified using a nested PCR
strategy with degenerate primer pair AJ645 (5 -TGG
ATH GAR ACN YTN YT-3 ) and AJ648 (5 -ACN
GCY TCY TCY TCN ARR TA-3 ) for the first round
and primer pair AJ646 (5 -TAY AAY GAR MGN
ATG CAY YTN YT-3 ) and AJ647 (5 -ARR TAN
CCN ACR AAN CRR TG-3 ) for the second round
of PCR. For the second-round PCR amplifications, a
1 + 99 dilution of the first round product was added
to the reaction mixture as template. Reaction mixtures
Pest Manag Sci 59:1143–1151 (online: 2003)
ATP-binding cassette transporter gene from Monilinia fructicola
(50 µl) contained 1 µl (20–60 ng) of genomic DNA;
1.5 µM of each primer; 200 µM of each dNTP; 1.25 U
of Taq DNA polymerase (Gibco BRL Gaithersburg,
MD); 20 mM Tris-HCl pH 8.4; 50 mM KCl; and
1.5 mM MgCl2 . Cycling parameters were 94 ◦ C for
2 min followed by 30 cycles of 94 ◦ C for 30 s, 45 ◦ C
for 1 min, and 72 ◦ C for 1 min.
Nested sets of adaptor-specific and gene-specific
primers were used to amplify the flanking sequences
of the MfABC1 and MfAOX1 genes from PCR
libraries.30 The PCR libraries were made by digesting
genomic DNA separately with restriction enzymes
PmlI, SmaI, EcoRV and XmnI and ligating adapters
onto the ends of the digested DNA fragments.
The resulting PCR templates were named PmlI,
SmaI, EcoRV and XmnI PCR libraries. The first
PCR amplification was conducted with adapter
specific primer AP1 (5 -GGA TCC TAA TAC GAC
TCA CTA TAG GGC-3 ) combined with a genespecific primer. Nested PCR was then conducted
with adaptor-specific primer AP2 (5 -AAT AGG
GCT CGA GCG GC-3 ) combined with a genespecific primer using 1 + 99 dilution of the first
PCR product as a template. MfABC1 gene-specific
primers MfABC1-F (5 -ATG CCC CTT GTC CTG
AAT CTT-3 ) and MfABC2-F (5 -AAC CCC GCT
GAA CAC ATG ATT-3 ) as well as MfABC3-F
(5 -AGC CCG TCC TCT TCG TCA T-3 ) and
MfABC4-F (5 -ACG AAT TCA TCT ACA CCG
GCA-3 ) were used to PCR amplify DNA fragments
downstream of the initial MfABC1 gene fragment.
MfABC1 gene-specific primers MfABC1-R (5 -TGC
TCG ATA CTC AAT CCC GCT-3 ) and MfABC2R (5 -ATT TCA GTT TCT CGG CTT CGG-3 )
were used to PCR amplify DNA upstream of the
initial MFABC1 gene fragment. The MfAOX1 genespecific primers AOX-1R (5 -TTG GGG GAG AAG
AGG TAG CAG-3 ), AOX-2R (5 -AGA GGT AGC
AGA GGA AGA AGG AAT-3 ) and AOX-3R (5 CCC TCA AGC TTC TCA AGT GTC TCA-3 )
were used to PCR amplify DNA fragments upstream
of the initial MfAOX1 gene fragment and gene-specific
primer AOX-4F (5 -AAT GAA CGC ATG CAC
CTC CTA A-3 ) was used twice to PCR amplify
downstream of the internal MfAOX1 fragment.
Reaction conditions were as described previously,30
except that Expand Long Template Polymerase was
used (Roche Applied Sciences, Indianapolis, IN).
PCR products were analyzed on 1% agarose gels in
0.5 × Tris–borate–EDTA buffer. PCR reactions were
performed in an iCycler (BIO-RAD Laboratories,
Hercules, CA).
A PCR based method using degenerate primers
AJ665 (5 -AAY TGG GAY GAY ATG GAR AA3 ) and AJ666 (5 -ATC CAC ATY TGY TGR AAN
GT-3 ) was used to identify the partial MfActin gene
from M fructicola. The PCR reaction mixture and
amplification protocol were as described above for
PCR amplifying the initial MFABC1 gene fragment.
Pest Manag Sci 59:1143–1151 (online: 2003)
2.3 DNA sequencing
PCR products were purified with the QIAquick Gel
extraction kit (QIAGEN, Valencia, CA) and cloned
into the pCR4-TOPO vector using the TOPO TA
Cloning kit (Invitrogen, Carlsbad, CA) according
to the manufacturer’s instructions. The sequencing
reactions were performed at the Clemson University
Automated DNA Sequencing Facility.
2.4 RNA isolation, cDNA synthesis and
quantification of MfABC1 and MfAOX1 mRNA
Mycelia of M fructicola isolates were grown on PDA
for one week. Actively growing culture (1 cm2 ) was
ground in sterile glass grinders with sterile water (2 ml)
and transferred into 250-ml flasks containing potato
dextrose broth (PDB; 50 ml). The liquid cultures were
incubated for one week at room temperature and
100 rev min−1 , before adding 0, 0.5 or 2 µg ml−1 of
azoxystrobin, 0, 0.01, 0.1 or 1.0 µg ml−1 myclobutanil,
or 0, 0.001, 0.01 or 0.1 µg ml−1 propiconazole.
One hour (for myclobutanil and propiconazole) and
24 h (for azoxystrobin) after the fungicide treatments,
total RNA was isolated and cDNA was synthesized
as described previously,6 except that RNAse-free Oak
Ridge test tubes were used for all centrifugations.
The integrity of total RNA was checked using a
1% formaldehyde agarose gel31 before performing
the DNase treatment. Real-time PCR technology
was used to quantify mRNA levels of the MfABC1
and MfAOX1 genes in cDNA. All experiments were
performed using SYBR Green chemistry (Molecular
Probes, Inc, Eugene, OR). Fragments of the MfABC1
and MfAOX1 genes were amplified with primers
MfABC5-F (5 -CCG ATG AAA CAA GCT CGA
CCT TTT-3 ) and MfABC5-R (5 -CGG ATT TGA
TTG GGT TGT TAT-3 ), and primers AOX-2F
(5 -ATC AAA TGA GTG AAC GCA AAT GGT3 ) and AOX-3R respectively. The expression of the
MfABC1 gene was normalized with ribosomal mRNA
using a 1 + 9 dilution of 18S ribosomal internal
standards and competimers (Ambion Inc, Austin,
TX). Normalized (relative) expression is referred to as
MfABC1 expression in this study. The expression of
the MfAOX1 gene was normalized with the expression
of the MfActin gene using primers MfActin-F (5 -GTG
TTG ATA TGG CCG GTC GTG ATT-3 ) and
MfActin-R (5 -TCG GCA GTG GTG GAG AAA
GTG TAA-3 ) and normalized (relative) expression
is referred to as MfAOX1 expression in this study.
The gene fragments were PCR amplified in the same
run but in separate test tubes. Reactions (30 µl)
were performed containing 5 µl of 1 + 19 diluted
cDNA, 0.2 mM of dNTPs, 0.4 µM of each primer,
20 mM Tris-HCl (pH 8.4), 50 mM KCl, and 5 mM
MgCl2 and 0.75 units of Taq DNA Polymerase
(Invitrogen). A 1:30 000 final dilution of SYBR Green
was used in each reaction. All PCR reactions were
amplified in triplicates or quadruplicates following the
thermocycling program: one cycle of 94 ◦ C for 2 min
followed by 40 cycles of 94 ◦ C for 30 s, 52 ◦ C for 30 s,
1145
G Schnabel, Q Dai, MR Paradkar
72 ◦ C for 20 s. Reactions were performed in an Icycler
(Bio-Rad Laboratories Inc, Hercules, CA). After the
real-time assay the size of the amplified fragments was
verified using gel electrophoresis.
Data analysis for MfAOX1 gene expression was conducted following the instructions of User Bulletin #2,
Perkin-Elmer Applied Biosystems, Foster City, CA.32
The comparative CT method (separate tubes) was
chosen to determine MfAOX1 expression in fungicidetreated and non-fungicide-treated mycelia.32 Data
analysis for MfABC1 gene expression was conducted
using a mathematical model described previously.33
Constitutive MfAOX1 expression in mycelia grown
on non-fungicide-amended medium was calibrated
by subtracting an arbitrary constant, which equaled
the MfAOX1 expression from isolate DL21, from the
MfAOX1 expression values of the other five isolates.
2.5 Statistics
Expression data from fungicide-treated or untreated
mycelia were subjected to analyses of variance
(ANOVAs) with SigmaStat 2.0 statistical software
(Jandel Corporation, San Rafael, CA). Means were
separated by t-test after the data passed the normality
test and the equal variance test.
3 RESULTS
3.1 Sensitivity of Monilinia fructicola isolates to
myclobutanil, propiconazole and azoxystrobin
The range of ED50 values of 36 single spore isolates
for DMI fungicides myclobutanil and propiconazole
was 0.3192 (DL32) to 2.136 µg ml−1 (DL 73) and
0.006 (DL49) to 0.063 µg ml−1 (DL29), respectively
(data not shown). The resistance factors between the
isolates with the lowest ED50 and the highest ED50 for
myclobutanil and propiconazole were 6.7 and 10.5,
respectively.
Azoxystrobin applied at concentrations up to
10 µg ml−1 did not affect mycelium growth of
six randomly selected isolates. However, adding
100 µg ml−1 of the alternative oxidase inhibitor
SHAM21,25 to the azoxystrobin-amended medium
increased sensitivity and allowed determination of
ED50 values in the range of 0.002 (DL61) to
1.35 µg ml−1 (DL23; Table 1). Several concentrations
of SHAM were tested for direct inhibitory effects (data
not shown). The selected concentration of 100 µg ml−1
SHAM represents the highest dose that did not affect
mycelium growth of any of the six isolates tested.
3.2 Identification and characterization of the
MfABC1, MfAOX1, and MfActin genes from
Monilinia fructicola
Degenerate primers AtrBF2, and AtrBR2 were
designed based on the aligned sequences of ABC
transporter genes AtrB from A nidulans, AtrB from
Botrytis cinerea Pers and AtrA from A nidulans
(accession nos Z68905, AJ6217 and Z68904). An
initial fragment of approximately 700 bp (initial
1146
MfABC1 gene fragment) was amplified from genomic
DNA with primers AtrBF2 and AtrBR2 from
M fructicola DNA and sequenced. Fragments of
850 bp and 1 kb were amplified downstream of the
initial MFABC1 gene fragment from the PmlI library
with primer pair MfABC1-F and MfABC2-F and
primer pair MfABC3-F and MfABC4-F, respectively.
A single fragment of about 5 kb was amplified
upstream of the initial MfABC1 gene fragment from
the same library using primers pair MfABC1-R and
MfABC2-R. The start and end codons of the gene
were predicted by aligning the deduced MfABC1
amino acid sequence with sequences from other ABC
transporters available in GenBank. The predicted 5
GT and 3 AG splice site of the intron found in
the genomic sequence was in agreement with typical
fungal introns.34 The location of the N-terminal and
C-terminal ABC domain consisting of Walker A,
ABC signature and Walker B regions indicated that
MfABC1 encodes an ABC transporter of the (ABCTMR)2 type (Fig 1).
The MfABC1 gene was 4380 bp in length and
contained one intron 71 bp in length at nucleotide
position 1137. A Blast Search of the MfABC1 amino
acid sequence with others available in GenBank
revealed highest amino acid identities of 87, 71, 68,
65, 40 and 39% for ABC transporters atr5 from
Mycosphaerella graminicola (Fuckel) Schroter, atrB
from Botryotinia fuckeliana Whet, atrB from A nidulans,
PMR5 from P digitatum, atr1 from M graminicola
and atrA from B fuckeliana (nucleotide accession nos
AF364104, AJ006217 Z68905, AB59028, AJ243112
and Z68906, respectively; Fig 1).
Alignment of the alternative oxidase genes from
A niger, Ajellomyces capsulatus, Neurospora crassa
Shear & Dodge, M grisea and Podospora anserina
(Rabenh) Neissl (accession nos O74180, AAD29680,
Q01355, BAA93615 and CAC27396, respectively)
revealed several areas of high homology that were
used to design degenerate oligonucleotide primers.
Degenerate primers AJ646 and AJ647 amplified an
approximately 170-bp fragment (initial MfAOX1 gene
fragment). Adapter ligation PCR with gene-specific
primers AOX-1R in the first round and AOX-2R in the
second round allowed PCR amplification of a 399-bp
fragment from the PmlI PCR library. The same library
was used to amplify a 1.6-kb fragment containing the
upstream sequences of the initial MfAOX1 gene with
primer AOX-3R for both rounds of PCR. A 1.3-kb
fragment containing the downstream sequence of the
initial MfAOX1 gene fragment was amplified from the
EcoRV PCR library with adapter specific primers and
primer AOX-4F in both rounds of nested PCR.
The alternative oxidase gene MfAOX1 from M fructicola was 1077 bp in length. The predicted amino acid
sequence was 63.8, 63.8 and 57.7% identical to AOX
protein sequences from Venturia inaequalis (Cooke)
Winter (ViAOX1; accession number AAK61349),
A niger, and A nidulans (alxA; accession number
Pest Manag Sci 59:1143–1151 (online: 2003)
ATP-binding cassette transporter gene from Monilinia fructicola
N-terminus
Proteina
MfABC1
MgAtr5
BcAtrB
AnAtrB
PdPMR5
MgAtr1
BcatrB
Walker A
GEMLLVLGRPGAGCTTLLK
GEMLLVLGRPGAGCTSLLK
GEMLLVLGRPGAGCTTLLK
GEMLLVLGRPGSGCTTLLK
GEMLLVLGRPGSGCTTLLK
GEMMLVLGRPGSGCSTFLK
GEMLLVLGRPGSGCSTFLK
*** ******* **
**
ABC-signature
Walker B
GVSGGERKRVSIIETLATRGSVMCWDNSTRGLD
GVSGGERKRVSILETMAARATVVCWDNSTRGLD
GVSGGERKRVSIIEMLASRGSVMCWDNSTRGLD
GVSGGERKRVSIIECLGTRASVFCWDNSTRGLD
GVSGGERKRVSIIECMATRGSVFCWDQSTRGLD
GVSGGERKRVSIAETLASKSTVVCWDNSTRGLD
GVSGGERKRVSIAETLPTKKTVVSWDNSTRGLD
************ *
* ** ******
Walker A
LGALMGSSGAGKTTLLDV
LGALMGSSGAGKTTLLDV
LGALMGSSGAGKTTLLDV
LGALMGSSGAGKTTLLDV
LGALMGSSGAGKTTLLDV
MVALMGASGAGKTTLLNT
MVALMGASGAGKTTLLNT
**** *********
ABC-signature
Walker B
GLSIEQRKRLTIGVELVSKPSILIFLDEPTSGLD
GLSVEQRKRLTIGVELVSKPSILIFLDEPTSGLD
GLSVEQRKRLTIGVELVSKPSILIFLDEPTSGLD
GLSVEQRKRVTIGVELVSKPSILIFLDEPTSGLD
GLSVEQRKRVTIGVELVSKPSILIFLDEPTSGLD
SLGVEQRKRLTIGVELAAKPSLLLFLDEPTSGLD
SLSVEQRKRVTIGVELAAKPNLLLFLDEATSGLD
* ***** ****** ** * **** *****
C-terminus
Proteina
MfABC1
MgAtr5
BcAtrB
AnAtrB
PdPMR5
MgAtr1
BcatrB
aMf
= Monilinia fructicola; Mg = Mycosphaerella graminicola; Bc = Botrytis cinerea; An = Aspergillus nidulans;
Pd = Penicillium digitatum
Figure 1. Alignment of the ATP-binding domain of MfABC1 and other ABC transporters from filamentous fungi. Identical sequences are marked
with asterisks.
BAA93615) respectively. The gene possessed two predicted membrane-spanning helices, a possible surfaceexposed helix, and four conserved regions typically
found in AOX genes from plants and fungi (Fig 2).
It contained two predicted introns of 54 and 67 bp at
nucleotide positions 240 and 584, respectively.
An approximately 780-bp fragment was PCR
amplified from M fructicola genomic DNA using
degenerate primers AJ665 and AJ666. A Blast search in
GenBank revealed 96.0, 82.0, 82.0 and 83.0% amino
acid identity to the actin proteins sequences from B
cinerea, Ophiostoma novo-ulmi, Aspergillus terreus Thom
and Cladosporium fulvum Cooke, respectively.
3.3 MfABC1 and MfAOX1 gene expression
analyses in single spore isolates of Monilinia
fructicola
MFAOX1 expression was determined in azoxystrobintreated and untreated mycelia (Tables 1 and 2).
Real-time PCR was conducted with primers MfABC5F and MfABC5-R targeting a 189-bp fragment of
the MfABC1 gene, primers AOX-2F and AOX-3R
targeting a 108-bp fragment of the MfAOX1 gene,
and primers MfActin-F and MfActin-R targeting a
85-bp fragment of the MfActin gene. The efficiencies
of PCR amplifications of the target (the MfAOX1
gene) and reference (the MfActin gene) DNA were
comparable with a slope of 0.017.32
Mycelium of DL21 had the lowest constitutive
MfAOX1 expression and was used to calibrate the data
of the six isolates examined (Table 2). The MfAOX1
expression in isolates DL4, DL21, DL23 and DL61
were significantly lower than in isolates DL16 and
DL25 (P ≤ 0.05; Table 2). Azoxystrobin applied at
0.5 µg ml−1 caused a significant increase in MfAOX1
Pest Manag Sci 59:1143–1151 (online: 2003)
Table 1. Sensitivity of six Monilinia fructicola isolates to azoxystrobin
with and without the addition of SHAM and expression of the MfAOX1
gene in mycelia treated or untreated with azoxystrobin
Sensitivity to
azoxystrobina
(ED50 ; µg ml−1 )
MfAOX1 expression
(CT value) 24 h after
treatment with azoxystrobin
(µg ml−1 )b
Isolate
(−) SHAM
(+) SHAM
0.5
2
DL61
DL21
DL4
DL25
DL16
DL23
>10
>10
>10
>10
>10
>10
0.002
0.029
0.29
0.33
1.08
1.35
5.9 a
9.7 a
1.8 b
0.7 b
1.1 b
11.0 a
6.6 a
2.2 a
1.3 b
1.0 b
1.1 b
1.5 b
a
The ED50 values represent means of three different experiments.
All standard deviations were <20% of the means. In the (+) SHAM
treatment, SHAM was applied at 100 µg ml−1 .
b Means of three different reactions. All standard deviations were
<10% of the means. A CT value of 5.9 for isolate DL61 indicates
that the expression of DL61 was 5.9-fold higher in medium amended
with 0.5 µg ml−1 azoxystrobin than in non-amended medium. Different
letters within columns correspond to significant differences by LSD for
each treatment individually. Absence of letters in a row indicates a lack
of significant difference.
expression in isolates DL21, DL23 and DL61 but not
in isolates DL4, DL25 and DL16 (Table 1). MfAOX1
expression was significantly increased in mycelium of
isolates DL21 and DL61 in the 2 µg ml−1 azoxystrobin
treatment.
Single spore isolates DL31 and DL73 (high and low
sensitivity to myclobutanil, respectively) and DL31
and DL15 (high and low sensitivity to propiconazole,
respectively) were selected to determine the influence
of myclobutanil or propiconazole treatment on
1147
1148
M2
H1
H2
H3
MfAOX1
ViAOX1
AnAOX
AniALXA
AcAOX1
MgAOX1
NcAOX
PaAOX
MfAOX1
ViAOX1
AnAOX
AniALXA
AcAOX1
MgAOX1
NcAOX
PaAOX
Figure 2. Compilation of deduced alternative oxidase amino acid sequences from various fungi: MfAOX1 from Monilinia fructicola; ViAOX1 from Venturia inaequalis; AnAOX from Aspergillus niger; AniALXA from
Aspergillus nidulans; AcAOX1 from Ajellomyces capsulatus; NcAOX, Neurospora crassa; MgAOX1 from Magnaporthe grisea; PaAOX from Podospora anserina (accession nos AF420306, AAK61349, O74180,
BAA93615, AAD29680, Q01355, BAA34672 and CAC27396, respectively). Two predicted membrane-spanning helices are designated as M1 and M2, with a possible surface-exposed helix noted as S.46 H1, H2, H3
and H4 are highly conserved regions among alternative oxidase genes of plants and fungi. These sequences have been proposed to be involved in a possible di-iron active site for the alternative oxidase.45,46 Shown
is the location of primer pair AOX-2F and AOX-3R used to quantify MfAOX1 expression.
H4
YWGMPEGHRSMRDLLYYIRADEAKHREVNHTLGNLKQDEDPNPFVSVYGKEVADKPGKGIESLRPLGWEREEVI.
.YNL.....T.K...LHV.................D.NS....YA.K.DNPDVPH.R.D.KY.K.S.......-M
..K....N.T.K...L.V...................AV.V...AVEWKDPSKPH.....KH.KTT.......-V
..K....Q...K...L.V.................N.AI....YAAK.KDPTKAH.N...AD.K.T.......-I
..Q....K.TIL....................A....GV....YAAK.DNPE.PH.T.SA.IVK.T....D..-I
.........T.....L.......N..G.H......N.V........D.K---G...RPVAA.-..E.F......G
..R....K.T.K..IH.......V..G.....S..D.K........D.KE-GEGGRRPVNPA.K.T.F..A...G
..K....K.T....IL.......V..G.....S..NHK........D.KC-D..HQRP-NPA.K.T.F..S...G
NGWIETLLEEAYNERMHLLTFLKMYEPGLFMRTMILGAQGVFFNSFFLCYLFSPKTCHRFVGYLEEEAVLTYTLSIQDLENGHLPKWADPNFKAPDLAIE
H..X......S........I...L....W...LAV.........AM..S..I..R..............V...RELA...A.K..E.ETLA--...I.VD
........................A...W...L.V.........G...S..I..R..................RA.K...S.R..H.EKLE--..EI.VK
.......................LA...W...L.V.........G...S..M..RI.............I...RA.KEI.A.S..A.EKTE--..EI.VQ
...................S...LAQ..W...L.V.........G..IS..I..R..............M...HA.K...S.K..N..NQP--...I.VA
........................C...WL.KIL.I.....Y..AM.VA..I...I.............H...R..EE..R.D....S..K.QV.EI.VS
..........S..........M..C....L.K.L..........AM..S..I...IT............H...RC.REI.E......S.EK.EI.EM.VR
..........S..........M..C...W..K............AM..S..I..RIT............H...RC.REI.Q.D....S....QI....VT
S
AOX-3R
MfAOX1
ViAOX1
AnAOX
AniALXA
AcAOX1
MgAOX1
NcAOX
PaAOX
M1
QMRSKVYFAHRKPRDFSDRVALGMVRFLRWCTDFATGYKHNVEAPKTASDSNAVTATKPYQMSERKWLIRYVFLESVAGVPGMVAGMLRHLRSLRGLKRD
..N-Q.AV...DT.NW..K...IA.KL...GL.TVS....G--KAQALHAQDPQE.Q.R.G.TGKQY.V.N...X................H.M.RM...
E..-A.TVG..EAKNW..W....S..L...GM.LV.....------P.PGQEDIK---KF..T.KE..R.F.............G..........RM...
..K-Q.AI...DAKNWA.W....T..M...GM.LV...R.------PPPGREHEA---RFK.T.Q...T.FI............G..........RM...
..K-EIAI...EAKNW..W....T......A..L....R.------A.PGKQG.EVPEQF..T....V..FI...T........G..........RM...
E.VDV.P-G.....TLG.KF.WSL..IS..GM.KVS.LSSEQQQINKG.PTTSIV.A..--LT.AQ..S.FI....I.A............H...R....
E.T.V.P-E....ETVG.WL.WKL..IC..A..I...IRPEQQ-VDKHHPTT.TS.D..--LT.AQ..V.FI....I..............H...R....
E.LAV.P-Q....GSL..WL.WKL..LC..G..I...I.PEQQ-VDKSNPTT..A.Q..--LT.AQ..V.FI....I..............E...R....
AOX-2F
MfAOX1
ViAOX1
AnAOX
AniALXA
AcAOX1
MgAOX1
NcAOX
PaAOX
MYVARLSTR-PLSNPSTAQLSKAAAF---FAQSYALPSTKCTAHV-----------PSRRPFTSGAKIQVKGRDLFPEPXHG-QIKRTEPAWPHPPYSVE
..TS.TAVYCSNLTTQRSAV--TILR---TXSGT.SNGI.-SRSALTAYQQNP--SLRT.N.H.TSVV.A.--.F....-DTPH.XK.KT.....I.T..
.ST---TGPIRVAAIPKHY.QFTVRT---YTR.M.SAGLRYSNPPLVKKCYDQ--PTGK.FIS.TPQS.I.--.Y..P.-DAPK.VEVKT..A..V..E.
.NSLTATAPIR-AAIPKSYMHI.TRN---YSGVI.MSGLR.SGSLVANR--HQ--TAGK.FISTTP.S.I.--EF..P.-TAPHV.EV.T..V..V.TE.
..PTSGCA.V-.MACPAPAMLRGPLL---RPSTT.IRGLR-GSPLLYHYAATS--NSNM.Y.S.TSRRWI.--EF.AP.KETDH.VESVTT.K..VFTEK
.L.HQVN.KLC--SAKQFTHLAKVVTPA----------LSYQ.SSVYSANLPRLAA-.P.L.STTSSA.LR--.F..V-KETEH.RQ.P.T...HGLTEK
.NTPKVNI---.HA.GQ.AQLSR.LISTCHTRPLL.AGS--RVATSLHPTQTNLSS..P.N.STTSVTRL.--.F..A-KETAY.RQ.P.....HGWTE.
.ISSKT.N.IC.CS.QQTARITGIVVS---SRPAY.TGLGYPVSLRLSSAVSQSSSQHT.S.S.TRAAHL.--.F..V-KETAY.RK.P.....HG.TE.
G Schnabel, Q Dai, MR Paradkar
Pest Manag Sci 59:1143–1151 (online: 2003)
ATP-binding cassette transporter gene from Monilinia fructicola
Table 2. Constitutive expression of the MfAOX1 gene in mycelia of M
fructicola isolates
Isolate
MfAOX1 expression
(CT value)a
DL4
DL16
DL21
DL23
DL25
DL61
3.2 a
8.2 b
1.0 a
2.2 a
7.1 b
1.3 a
(Table 3). Between isolates, the increase in MfABC1
expression was significantly higher in DL73 than in
DL31 mycelium for myclobutanil treatments 0.1 and
1 µg ml−1 (Table 3). Propiconazole induced MfABC1
expression in mycelium of all treatments, as indicated
by CT values greater than one (Table 4). Between
isolates, the increase in MfABC1 expression was
significantly higher in DL31 than in DL15 mycelium
in propiconazole treatment at 0.001 µg ml−1 , but
significantly lower in the 0.1 µg ml−1 propiconazole
treatment (Table 4).
a
Means of three different reactions. All standard deviations were
<10% of the means. Different letters within columns correspond to
statistically significant differences by LSD for each isolate individually.
A CT value of 3.2 (DL4) indicates that the expression of DL4 was
3.2-fold higher than the expression of reference isolate DL21.
Table 3. Sensitivity of two Monilinia fructicola isolates to myclobutanil
and expression of the MfABC1 gene in mycelia treated or untreated
with different concentrations of myclobutanil
MfABC1 expression
(CT value) 1 h after
treatment with myclobutanil
(µg ml−1 )b
Isolate
Sensitivity to
myclobutanil
(ED50 ; µg ml−1 )a
0.01
0.1
1
DL31
DL73
0.34
2.13
6.04 a
7.34 a
2.06 a
22.9 b
2.05 a
30.73 b
a
Means of three different experiments. All standard deviations were
<20% of the means.
b Means of three different reactions. All standard deviations were
<10% of the means. Different letters within columns correspond
to statistically significant differences by LSD for each treatment
individually. A CT value of 6.04 for isolate DL31 indicates that the
expression of DL31 was 6.04-fold higher in medium amended with
0.01 µg ml−1 myclobutanil than in non-amended medium.
Table 4. Sensitivity of two Monilinia fructicola isolates to
propiconazole and expression of the MfABC1 gene in mycelia treated
or untreated with different concentrations of propiconazole
MfABC1expression
(CT value) 1 h after
treatment with propiconazole
(µg ml−1 )b
Isolate
Sensitivity to
propiconazole
(ED50; µg ml−1 )a
0.001
0.01
0.1
DL31
DL15
0.0068
0.054
23.28 a
3.97 b
23.09
22.92
7.88 a
15.7 b
a
Means of three different experiments. All standard deviations were
<20% of the means.
b Means of three different reactions. All standard deviations were
<10% of the means. Different letters within columns correspond
to statistically significant differences by LSD for each treatment
individually. Absence of letters in a column indicates a lack of
significant difference. A CT value of 23.28 for isolate DL31 indicates
that the expression of DL31 was 23.28-fold higher in medium amended
with 0.001 µg ml−1 propiconazole than in non-amended medium.
MfABC1 expression (Tables 3 and 4). Myclobutanil
induced MfABC1 expression in mycelium of all
treatments, as indicated by CT values greater than one
Pest Manag Sci 59:1143–1151 (online: 2003)
3.4 GenBank accession numbers
The nucleotide sequences of MfAOX1, MfABC1 and
the MfActin gene fragment are available in GenBank
(accession nos AF420306, AY077839 and AF420305,
respectively).
4 DISCUSSION
The development and implementation of strategies
for fungicide resistance management and monitoring
are usually initiated in response to control failure in
the field and after growers already suffered economic
losses. Fortunately, stone-fruit growers in the USA
still enjoy reliable brown rot control when using
DMI fungicides even after 15 years of perpetual use.
However, the potential of M fructicola to build up
resistance in vitro and in the field was shown by
Nunninger-Ney1 and Zehr et al 2 and it seems only
a matter of time before field resistance will develop if
DMI fungicides continue to be used as the sole control
option.
Very little is known about the molecular basis of
DMI fungicide resistance. Drug efflux pumps belonging to the ABC transporters family have been suggested to play a role in fungicide resistance.10,35 The
ABC transporter MfABC1 from M fructicola has high
amino acid homology with ABC transporters from
other fungi such as atr5 from M graminicola, a protein with yet-unknown function, atrB from B cinerea,
a protein that affects the sensitivity of B cinerea to
pytoalexins and fungicide fenpiclonil, and atrB from
A nidulans a protein conferring resistance to DMI
fungicides.12,36 This evidence suggests a similar role
for the MfABC1 protein in protection against exogenous toxic compounds, including DMI fungicides.
In support of this hypothesis is the inducibility of
MfABC1 expression with DMI fungicides myclobutanil and propiconazole. The relatively low induction
of MfABC1 expression in the myclobutanil susceptible isolate DL31 in myclobutanil treatments 0.1 and
1.0 µg ml−1 may be a result of rapid decline of the
mycelium after fungicide exposure. A more detailed
study of the MfABC1 gene is necessary but can only
be done if mutants with a disrupted MfABC1 gene
are studied. However, a transformation system for
M fructicola is not yet in place and may be difficult to
develop due to the polynucleic nature of M fructicola
cells. The constitutive expression of the MfABC1 gene
1149
G Schnabel, Q Dai, MR Paradkar
in mycelia not treated with fungicides may reflect an
additional intrinsic metabolic activity of the deduced
protein.
Recently, azoxystrobin, a Qo fungicide, was registered for the control of brown rot in commercial
peach production in the USA. Because of the different mode of action, this fungicide is now being
explored as a possible rotation partner for DMI fungicides to control pre-harvest brown rot. Qo fungicides
inhibit respiration by binding to the Qo center of
cytochrome b.37,38 Because fungi can circumvent this
pathway using alternative respiration, increasing interest has emerged in studying the alternative route of
fungal electron transfer in plant pathogenic fungi. In
a recent study, the alternative respiration provided
protection from azoxystrobin during both saprophytic
and infectious stages of the pathogen.39
We cloned and sequenced the alternative oxidase
gene MfAOX1 from M fructicola, the enzymatic core
of alternative respiration, and studied its expression
in response to azoxystrobin treatments. We observed
an interference of the alternative respiration in
actively growing mycelia of the six isolates tested
due to synergistic inhibitory effects of SHAM and
azoxystrobin treatments in mycelium growth tests.
While neither the alternative oxidase inhibitor SHAM
nor azoxystrobin was able to inhibit mycelial growth
when applied by themselves at the concentrations
tested, the combination of the two resulted in
significant growth inhibition. This result suggests that
the MfAOX1 protein allowed continued respiration in
the presence of high QoI fungicide doses in mycelium
of M fructicola.
Alternative oxidase expression is induced by certain stress stimuli and by constrictions in electron
flow through the cytochrome-mediated respiration
pathway caused by respiration inhibitors.40 – 42 Alternative oxidase activity has been induced in Pyricularia
oryzae Cavara and B cinerea using metominostrobin
and another Qo fungicide derivative, respectively.43,44
Similar induction of an alternative oxidase gene by
strobilurin fungicides was shown in M grisea and V
inaequalis.27,45 Increased levels of AOX mRNA correlated with elevated activity of alternative respiration27
and expression of the alternative oxidase was increase
in a Qo fungicide dose-dependent manner.45
MfAOX1 expression analysis indicated that the gene
is constitutively expressed in mycelia grown under
in vitro conditions, and that the level of constitutive
MfAOX1 expression may vary among isolates. It is
interesting that the isolates with lowest constitutive
MfAOX1 expression (DL21 and DL63) responded
to the 2 µg ml−1 azoxystrobin with highest increases
in MfAOX1 expression. It is possible that isolates
with constitutively high MfAOX1 expression may have
reached an expression peak earlier than isolates with
low constitutive expression levels. The induction of
MfAOX1 in mycelia of M fructicola by azoxystrobin
was limited, suggesting that the enzyme may provide
only marginal protection from Qo fungicides.
1150
5 CONCLUSIONS
The ABC transporter MfABC1 from M fructicola may
be a genetic determinant of DMI fungicide resistance
because of its high homology to multi-drug resistance
protein atrB from A nidulans and because its expression
was induced by DMI fungicides myclobutanil and
propiconazole. The induction of MfAOX1 in mycelia
of M fructicola by azoxystrobin was limited, suggesting
that the enzyme may provide only marginal protection
from Qo fungicides.
ACKNOWLEDGEMENTS
We thank Elise L Schnabel, Sarah Tennison and
Aigerim Bishanova for technical assistance and Karen
Bryson for critical review of the manuscript. We also
thank the South Carolina Commission on Higher
Education and the Clemson University Research
Grant Committee for financial support.
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