Pest Management Science Pest Manag Sci 59:1333–1343 (online: 2003)DOI: 10.1002/ps.766

Multiple mechanisms account for variation inbase-line sensitivity to azole fungicides in fieldisolates of Mycosphaerella graminicolaIoannis Stergiopoulos,1 Johannes GM van Nistelrooy,1 Gert HJ Kema2 andMaarten A De Waard1∗1Laboratory of Phytopathology, Department of Plant Sciences, Wageningen University, PO Box 8025, Binnenhaven 5, 6709 PDWageningen, The Netherlands2Plant Research International, PO Box 16, 6700 AA Wageningen, The Netherlands

Abstract: Molecular mechanisms that account for variation in base-line sensitivity to azole fungicideswere examined in a collection of twenty field isolates, collected in France and Germany, of the wheatpathogen Mycosphaerella graminicola (Fuckel) Schroeter. The isolates tested represent the wide base-line sensitivity to the azole fungicide tebuconazole described previously. The isolates were cross-sensitiveto other azoles tested, such as cyproconazole and ketoconazole, but not to unrelated chemicals likecycloheximide, kresoxim-methyl or rhodamine 6G. Progenies from a genetic cross between an isolatewith an intermediate and a high sensitivity to azoles displayed a continuous range of phenotypes withrespect to cyproconazole sensitivity, indicating that variation in azole sensitivity in this haploid organismis polygenic. The basal level of expression of the ATP-binding cassette transporter genes MgAtr1-MgAtr5from M graminicola significantly varied amongst the isolates tested, but no clear increase in the transcriptlevel of a particular MgAtr gene was found in the less sensitive isolates. Cyproconazole strongly inducedexpression of MgAtr4, but no correlation between expression levels of this gene and azole sensitivity wasobserved. One isolate with intermediate sensitivity to azoles over-expressed CYP51, encoding cytochromeP450 sterol 14α-demethylase from M graminicola. Isolates with a low or high sensitivity to azoles weretested for accumulation of cyproconazole, but no clear correlation between reduced accumulation of thefungicide in mycelium and sensitivity to azoles was observed. Therefore, differences in accumulationcannot account exclusively for the variation in base-line sensitivity of the isolates to azoles. The resultsindicate that multiple mechanisms account for differences in base-line sensitivity to azoles in field isolatesof M graminicola. 2003 Society of Chemical Industry

Keywords: ATP-binding cassette (ABC) transporters; azoles; base-line sensitivity; CYP51; DMIs; fungicideresistance; Septoria tritici

1 INTRODUCTIONAzoles represent a major class of fungicides that hasbeen extensively used over the past three decadesin the control of fungal pathogens of medicaland agricultural importance. They are systemicfungicides with both protective and curative activityin disease control.1 Their mode of action is basedon inhibition of cytochrome P450 sterol 14α-demethylase (P45014DM), a key enzyme involved in thebiosynthesis of ergosterol in fungi. For this reason azolefungicides are also described as sterol demethylationinhibitors (DMIs).2

Because of their site-specific mode of action,the risk of resistance developing to DMIs is

significant. However, laboratory-generated azole-resistant mutants often display reduced fitness withrespect to spore germination, mycelial growth and vir-ulence, and their levels of resistance are low. For thesereasons, resistance development under field condi-tions was considered unlikely.3–5 Nevertheless, DMIresistance emerged in several pathogen populations,although relatively slowly as compared to other classesof fungicide.6 One of the first cases of field resistancewas reported for Sphaerotheca fuliginea (Schlecht ex Fr)Poll, the causal agent of cucumber powdery mildew.7

Since then, DMI resistance is also reported in otherfungal pathogens, such as Penicillium digitatum (Pers)Sacc,8 Erysiphe graminis DC f sp hordei Marchal,9

∗ Correspondence to: Maarten A De Waard, Laboratory of Phytopathology, Department of Plant Sciences, Wageningen University, PO Box8025, Binnenhaven 5, 6709 PD Wageningen, The NetherlandsE-mail: maarten.dewaard@wur.nlContract/grant sponsor: The European Commission, Mobility of Researchers (TMR) Programme—Marie Curie Research Training Grants;contract/grant number: ERBFMBICT983558(Received 5 September 2002; revised version received 25 March 2003; accepted 29 May 2003)Published online 5 August 2003

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Venturia inaequalis (Cooke) Winter10 and Rhynchospo-rium secalis (Oudem) Davis.11

Analysis of azole resistance in fungi revealed that dif-ferent molecular mechanisms may operate in resistantisolates. In Candida albicans (Robin) Berkhout, sev-eral alterations in CYP51 (also described as ERG11),that encodes P45014DM, have been identified. Theseinclude point mutations in the coding region of CYP51that result in reduced affinity of azoles for the targetenzyme12 or over-expression of this gene resulting inhigher intracellular levels of P45014DM.13–17 Similaralterations in CYP51 have been reported in fungalpathogens of agricultural importance. Point mutationsin CYP51 associated with decreased azole sensitivityhave been described in laboratory-generated mutantsof Ustilago maydis (DC) Corda18 and P digitatum,19

and in field isolates of Uncinula necator (Schw) Burr20

and E graminis f sp hordei.21 Resistance to azoles inP digitatum and V inaequalis correlates positively withincreased expression of CYP51 caused by the pres-ence of tandem repeats in the promoter region ofthis gene.22,23

Reduced accumulation of azoles in cells as aresult of active efflux mechanisms constitutes anotherwell-characterised mechanism of resistance. Earlyreports indicated that fenarimol-resistant strains of Anidulans accumulate less fungicide than the wild-typecontrol.24,25 This reduced accumulation is ascribedto an increased energy-dependent efflux of thecompounds from cells, mediated by ATP-bindingcassette (ABC) transporters.26,27 ABC transporterscomprise one of the best-characterised protein familiesassociated with active excretion of drugs. They utilisethe energy derived from hydrolysis of ATP to drivethe transport of a wide variety of cytotoxic agentsover biological membranes.28 In particular, ABCtransporters of filamentous fungi are known to exhibitan important function in protection against synthetictoxic compounds such as antibiotics, fungicidesand other xenobiotics.29 Overproduction of ABCtransporters can result in pleiotropic effects suchas simultaneous resistance to structurally unrelatedcompounds, a phenomenon described as multi-drugresistance (MDR).30

Despite the extensive knowledge of molecular mech-anisms of azole resistance in laboratory-generatedmutants, the relevance and frequency of such mech-anisms in field populations of fungal pathogens isstill unclear. In P digitatum increased expression lev-els of ABC transporters genes such as PMR1 andPMR5, as well as increased expression of CYP51, weredetected in DMI-resistant isolates.22 In this paperwe studied mechanisms that could potentially explainthe wide variation in base-line sensitivity to azolesin field isolates of the fungus Mycosphaerella gramini-cola (Fuckel) J Schroeter in Cohn (anamorph: Septoriatritici Roberge in Desmaz), the causal agent of septo-ria tritici blotch of wheat. The economic importanceof this disease has dramatically increased over thepast few decades and control management of this

pathogen has widely involved the use of azole fungi-cides. In laboratory-generated azole-resistant mutantsof M graminicola, different mechanisms of resistance toazoles may operate, amongst others, over-expressionof specific ABC transporter genes.31 Resistance devel-opment in M graminicola to azole fungicides underfield conditions has not been reported.32 However,field populations of M graminicola consist of iso-lates with a broad range in sensitivity levels to thesefungicides.33,34 Here we report studies on twenty fieldisolates of M graminicola, sampled in France and Ger-many, that possess a significant difference in base-linesensitivity to the azole fungicide tebuconazole. Anal-ysis of the progeny of a genetic cross between twoisolates with a 100-fold difference in sensitivity tocyproconazole indicated that azole sensitivity in thisfungus is polygenic. Expression studies of ABC trans-porter genes and CYP51 showed that isolates displaylarge differences in basal level of expression of thesegenes. Accumulation of cyproconazole did not cor-relate with azole sensitivity. The results indicate thatmultiple mechanisms may operate in field isolates ofM graminicola that determine base-line sensitivity toazole fungicides.

2 MATERIALS AND METHODS2.1 Fungal strainsEleven field isolates of M graminicola originating fromFrance and nine field isolates derived from Germany(see Table 1) were kindly provided by Dr JM Seng(Biotransfer, Montreuil Cedex, France) and Dr ASuty (Bayer AG, Landwirtschaftszentrum Monheim,Leverkusen, Germany), respectively. The isolates weresampled from wheat fields treated with tebuconazoleand selected for differential sensitivity to this fungicide.Single spore isolates were prepared from bulk samplesand used throughout the whole study. A genetic crossbetween isolates M1 and M3 was made accordingto Kema et al35 and 40 progeny isolates (M1M31–M1M3 40) were obtained. Stock cultures of theisolates were kept as yeast-like cells at −80 ◦C.

2.2 Culture conditionsYeast-like cells of the isolates were grown in liq-uid Yeast Sucrose Medium (YSM; yeast extract10 g litre−1, sucrose 10 g litre−1) at 18 ◦C and140 rev min−1. Mycelium was grown in liquid Cza-pek Dox-Mycological Peptone medium (CzD-MP;Czapek Dox 33.4 g litre−1, mycological peptone5 g litre−1) at 25 ◦C and 140 rev min−1. Toxicity assayswere performed in Potato Dextrose Broth (PDB;24 g litre−1) at 25 ◦C.

2.3 CompoundsThe chemicals used in this study were the azolefungicides cyproconazole (Syngenta, Basel, Switzer-land), ketoconazole (Janssen Research Founda-tion, Beerse, Belgium), and tebuconazole (BayerAG, Landwirtschaftszentrum Monheim, Leverkusen,

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Germany), the antibiotic cycloheximide (Sigma–Aldrich Chemie BV, Zwijndrecht, The Netherlands),the Qo-inhibitor (QoI) kresoxim-methyl (BASF AG,Limburgerhof, Germany) and the dye rhodamine6G (Sigma–Aldrich Chemie BV, Zwijndrecht, TheNetherlands). All compounds were of technical gradeand used as 100× concentrated stock solutions of1000, 562, 316, 178, 100, 56.2, 31.6, 17.8, 10, 5.62,3.16, 1.78, 1, 0.562, 0.316, 0.178 and 0.1 mg litre−1

in methanol.

2.4 Fungicide activity testSensitivity of isolates to compounds was assessed ina microtitre plate test, adapted from Pijls et al36 Cellsuspensions (105 ml−1) of the isolates were preparedin PDB from 3-day-old cultures. Wells of sterileflat-bottomed microtitre plates (Greiner BV, Alphenaan de Rijn, The Netherlands) were filled with cellsuspension (50 µl) and mixed’ with PDB (150 µl)amended with fungicides at different concentrations.PDB with methanol (1%) was used as a control. Alltreatments were tested in fourfold per microtitre plateand tests were repeated at least once. Plates were sealedto prevent evaporation from wells and incubated for5 days as still cultures at 25 ◦C in the presence oflight. Growth was assessed with a microtitre platereader (Bio-Tek Instruments Inc, Vermont, U.S.A.)by measuring cell density at 630 nm. Sensitivities of theisolates to the compounds were expressed as minimuminhibitory concentration (MIC) values. These valueswere the lowest concentration of compound in thescale used that did not allow growth. The variationfactor of isolates for sensitivity to compounds wasdefined as the ratio between the MIC value of themost and least sensitive isolate.

2.5 Expression analysis2.5.1 RNA isolation and northern blot analysisYSM or CzD-MP (20 ml) was inoculated withyeast-like cells derived from stock cultures of theisolates tested. After 3 days of culturing at 18 or25 ◦C and 140 rev min−1, the optical density of thecultures was determined at 600 nm (OD600) andnew cultures (20 ml) with a starting density ofOD600 0.1 were initiated. Three days later, theOD600 of these cultures was adjusted to 0.5 in freshmedium (20 ml) and the cultures were incubatedagain overnight under similar conditions as describedabove. Cyproconazole treatments (10 mg litre−1, 0.1%methanol) were performed at 140 rev min−1 for 1 h.Yeast-like cells or mycelium were grown in YSMat 18 ◦C or CzD-MP at 25 ◦C, respectively. Controltreatments were performed with methanol (0.1%).Fungal biomass was harvested by centrifugation at10 000 rev min−1 and 0 ◦C for 10 min, and instantlyfrozen in liquid nitrogen.

RNA was isolated from frozen biomass with theTRIzol reagent (Life Technologies Inc, Maryland,USA) according to the manufacturer’s instructions.RNA (10 µg, 4.5 µl) was denatured in a mixture

of glyoxal (6 M; 4.5 µl), sodium phosphate (0.1 M;3.0 µl), and dimethyl sulfoxide (13.3 µl) at 50 ◦Cfor 1 h, and subjected to electrophoresis on 1.6%agarose gel in sodium phosphate buffer (10 mM;pH 7.0). Blotting was carried out on Hybond-N+

membranes (Amersham Pharmacia Biotech, LittleChalfont Buckinghamshire, UK) by capillary transferwith 10× SSC solution. Homologous hybridisationswere performed overnight at 65 ◦C in ‘Nasmyth’ssolution’ buffer (dextran sulfate 185 g litre−1, sarcosyl18.5 g litre−1, EDTA 0.011 M, Na2HPO4 0.3 M, NaCl1.1 M; pH 6.2). This solution (5.4 ml) was mixedwith distilled water (4.6 ml) just before use to obtainthe hybridisation buffer (10 ml) while sheared herringsperm DNA (100 µg ml−1) was included as blockingagent. Blots were washed twice in 2× SSC, 0.1× SDSand twice in 0.1× SSC, 0.1× SDS at 65 ◦C for 15 min.

2.5.2 ProbesGene-specific probes of the ABC transporter genesMgAtr1 (Acc AJ243112), MgAtr2 (Acc AJ243113),MgAtr3 (Acc AF364105), MgAtr4 (Acc AF329852)and MgAtr5 (Acc AF364104) used in northern blotanalysis represented a 0.84-kb EcoRI fragment ofMgAtr1, a 0.75-kb SalI fragment of MgAtr2, a 0.85-kb SalI fragment of MgAtr3, an 1.14-kb BamHI/PstIfragment of MgAtr4, and a 0.6-kb EcoRI fragmentof MgAtr5. A 0.65-kb DNA fragment of CYP51 (AccAF263470), encoding P45014DM of M graminicola, wasobtained by PCR using genomic DNA from isolateIPO32337 as template. Primers used for amplificationwere CYPinternal4 (5′-CAG CAC TCT TCA TCTGCG AC-3′) and CYP3′anchor (5′-TCC CTC CTCTCC CAC TTT AC-3′). PCR conditions consistedof 30 cycles of 1 min denaturation at 94 ◦C, 1 minof annealing at 58 ◦C, and 1 min of extension at72 ◦C with an additional extension step of 10 minat 72 ◦C at the end of the reaction. Equal loadingof samples on blots was examined with a 0.6-kb EcoRI fragment of the 18S rRNA gene fromAspergillus niger.

Randomly primed DNA isotopic probes wereprepared by enzymatic incorporation of [α32P]dATP.In each labelling reaction, 50 ng of a probe templatewas used. Probes were purified using the QIAquickNucleotide Removal kit (QIAGEN, Westburg BV,Leusden, The Netherlands) before adding to thehybridisation solution.

2.6 Accumulation of [14C]cyproconazoleThree-day-old mycelial cultures were harvested byfiltering through a 0.85-mm pore sieve and by col-lection of the mycelium on a 0.055-mm pore sieve,washed with phosphate buffer (50 mM; pH 6.0)and re-suspended in the same buffer at a net wetweight of 6 mg litre−1. Cultures were incubated at25 ◦C and 140 rev min−1 for 30 min before addi-tion of [14C]cyproconazole, at an initial externalconcentration of 100 µM. Mycelium from cultures

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(5 ml) was collected by vacuum filtration at inter-vals of 5, 15, 25, 35, 45, 55, 95 and 105 min afteraddition of [14C]cyproconazole, washed (5×) withphosphate buffer (pH 6.0; 5 ml) and stored in vialscontaining LUMASAFE PLUS (3 ml; Groningen,The Netherlands). Energy-dependency of cyprocona-zole accumulation was examined by addition ofcarbonyl-cyanide 3-chlorophenylhydrazone (CCCP,20 µM) 55 min after addition of [14C]cyproconazole.Radioactivity in the samples was measured witha Beckman LS6000TA liquid scintillation counter(Beckman Instruments Inc, Fullerton, USA). Accu-mulation of [14C]cyproconazole was calculatedas nmol of cyproconazole per mg dry weightof biomass.

2.7 Statistical analysisStatistical analysis of the results was performed usingSPSS for Windows release 10.0.5 (SPSS Inc, Chicago,Illinois, USA) and MicrocalOrigin version 5.0(Microcal Software Inc, Northampton, USA) statisti-cal software. Linear association between sensitivityto different compounds or between sensitivity toazoles and accumulation of [14C]cyproconazole wasexamined using the Pearson’s correlation coefficient.Statistically significant differences between mean val-ues of [14C]cyproconazole accumulation were testedusing the Student–Newman–Keuls (S-N-K) analy-sis at a significance level of 0.05 using 11 degreesof freedom.

3 RESULTS3.1 Sensitivity assaysThe sensitivity of the field isolates to the azole fungi-cides cyproconazole, ketoconazole, and tebuconazolevaried significantly (Table 1). Mean MIC values ofcyproconazole, ketoconazole and tebuconazole were44.8, 15.5 and 24.3 mg litre−1, respectively, indi-cating that ketoconazole was the most active azoletested (Table 2). MIC values of tebuconazole rangedbetween 0.316 and 56.2 mg litre−1, indicating that thevariation factor (defined as the ratio between the MICvalue of the most and least sensitive isolates) amounted178. MIC values for cyproconazole and ketoconazoleranged between 0.0562 and 100 mg litre−1, resultingin a variation factor of 1779. Box-and-whiskers plotsshowed that the variation in MIC values of the isolatesinside the boxes (50% of population) was lower fortebuconazole than for cyproconazole and ketoconazole(Fig 1).

Mean MIC values for the non-azole compoundscycloheximide, kresoxim-methyl and rhodamine 6Gwere 63.0, 0.87 and 2.57 mg litre−1, respectively(Table 2). The data indicate that the Qo-inhibitorkresoxim-methyl was the most active compound ofall the chemicals tested. Sensitivities of the isolatesto cycloheximide, kresoxim-methyl and rhodamine6G showed variation factors of 10, 56.2 and 5.62,respectively (Table 1). Hence, the variation in MICvalues observed for non-azole compounds was muchsmaller than the variation observed for azoles.

Table 1. Sensitivity of Mycosphaerella graminicola field isolates to the azole fungicides cyproconazole, ketoconazole and tebuconazole, the

antibiotic cycloheximide, the Qo-inhibitor kresoxim-methyl and the dye rhodamine 6G

Minimum inhibitory concentrations (mg litre−1)

Azole fungicidesa Non-azole compounds

Isolates Year Origin

Cyproconazolen = 3 (M)/n = 2 (S)

Ketoconazolen = 3 (M)/

n = 1 (S)

Tebuconazolen = 2 (M)/

n = 1 (S)

Cycloheximiden = 1

Kresoxim-methyln = 1

Rhodamine 6Gn = 1

M1 1993 France 0.1 0.178 1.78 100 3.16 3.16M2 1993 France 1 10 17.8 5.62 0.1 3.16M3 1993 France 17.8 5.62 31.6 100 0.0562 1.78M4 1995 France 100 100 17.8 100 1 1.78M5 1994 France 56.2 0.562 31.6 56.2 0.178 3.16M6 1995 France 100 100 31.6 31.6 3.16 3.16M7 1994 France 0.0562 0.1 0.316 56.2 0.562 1M8 1995 France 0.178 0.1 0.562 56.2 3.16 3.16M9 1995 France 56.2 10 17.8 56.2 0.178 3.16M10 1994 France 56.2 31.6 31.6 56.2 0.0562 3.16M11 1995 France 10 0.562 17.8 56.2 0.562 3.16S006 1996 Germany 1 0.0562 1.78 10 1 0.562S009 1996 Germany 31.6 5.62 31.6 100 3.16 3.16S030 1998 Germany 56.2 1.78 17.8 100 1 3.16S042 1998 Germany 100 1 31.6 100 0.562 1.78S043 1998 Germany 0.1 0.1 17.8 56.2 0.178 3.16S054 1996 Germany 100 0.562 56.2 31.6 1 3.16S175 1997 Germany 10 0.562 17.8 100 0.178 1.78S176 1997 Germany 100 31.6 56.2 56.2 0.562 1.78S190 1997 Germany 100 10 56.2 31.6 0.562 3.16

a Number of repetitions (n) for French (M) and German (S) isolates.

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1000

100

10

1

0.1

0.01

MIC

s (m

g lit

re-1

)

Cyp

roco

nazo

le

Ket

ocon

azol

e

Tebu

cona

zole

Cyc

lohe

xim

ide

Kre

soxi

m-

met

hyl

Rho

dam

ine

6G

Mean50 % of population

Median outliers

Figure 1. Box-and-whiskers plots of MIC values of 20 field isolates ofMycosphaerella graminicola to the azole fungicides cyproconazole,ketoconazole, and tebuconazole, the antibiotic cycloheximide, theQo-inhibitor fungicide kresoxim-methyl, and the dye rhodamine 6G.

Table 2. Sensitivity of field isolates of Mycosphaerella graminicola to

the azole fungicides cyproconazole, ketoconazole and tebuconazole,

the antibiotic cycloheximide, the Qo-inhibitor kresoxim-methyl and

the dye rhodamine 6G

Minimum inhibitory concentrations(mg litre−1)a

Compounds Minimum Maximum Mean Median

Cyproconazole 0.0562 100 44.83 44.25Ketoconazole 0.0562 100 15.50 11.66Tebuconazole 0.316 56.2 24.36 17.80Cycloheximide 5.62 100 63.00 56.20Kresoxim-methyl 0.0562 3.16 0.87 0.79Rhodamine 6G 0.562 3.16 2.57 3.16

a Average of 20 isolates tested.

3.2 Cross-sensitivitySensitivity of the isolates to cyproconazole and tebu-conazole (r = 0.82, P < 0.0001), cyproconazole and

ketoconazole (r = 0.71, P < 0.0001), and tebucona-zole and ketoconazole (r = 0.64, P < 0.003) corre-lated positively, indicating cross-sensitivity to theazoles tested. In contrast, no correlation was observedbetween sensitivity to azole fungicides and the non-azole compounds tested (Table 3, Fig 2). Similarresults were obtained when cross-sensitivity was exam-ined for French and German isolates, separately (datanot shown).

3.3 Analysis of the progeny isolatesThe sensitivity to cyproconazole of 40 progeny iso-lates obtained from a genetic cross between iso-lates M1 (MIC: 0.178 mg litre−1) and M3 (MIC:17.8 mg litre−1) was tested. MIC values of the progenyisolates ranged between 0.316 and 17.8 mg litre−1,indicating a continuous distribution in sensitivity tocyproconazole between the MIC values of the parents(Fig 3).

3.4 Expression analysisExpression of the ABC transporter genes MgAtr1–MgAtr5 was examined in yeast-like cells and myceliumof isolates with high (M1, M7, M8, S043), medium(M3), and low (M4, M6, S054, S190) sensitivityto cyproconazole (Fig 4). Transcripts of MgAtr2 andMgAtr3 could not be detected in any of the isolatesunder the conditions tested (data not shown). Incontrast, basal transcript levels of MgAtr1, MgAtr4 andMgAtr5 varied significantly. Some isolates displayed ahigh basal level of expression for one specific geneonly (eg MgAtr1 in isolate S054 in yeast-like cells) butnot for the others. Such differences occurred in bothyeast-like cells and the mycelium form of the fungus.Obvious basal transcript levels of MgAtr1 were foundin yeast-like cells of isolates M1, M3, M7 and S054,and of MgAtr4 and MgAtr5 in all isolates tested, but forMgAtr5 particularly in isolate M1. In mycelium, basalexpression of MgAtr1 was observed in isolates M3and M7 and of MgAtr4 in most of the isolates tested(except for M1 and S054). Basal expression of MgAtr5

Table 3. Patterns of cross sensitivity of 20 field isolates of Mycosphaerella graminicola to the azole fungicides cyproconazole, ketoconazole and

tebuconazole, the antibiotic cycloheximide, the Qo-inhibitor kresoxim-methyl and the dye rhodamine 6G

Compounds Correlation Cyproconazole Ketoconazole Tebuconazole Cycloheximide Kresoxim-methyl Rhodamine 6G

Cyproconazole ra — 0.71∗ 0.82∗ 0.18 0.04 0.22Pb — <0.001 <0.001 0.45 0.87 0.35

Ketoconazole r 0.71∗ — 0.64∗ 0.02 −0.03 0.29P <0.0001 — 0.003 0.92 0.90 0.22

Tebuconazole r 0.82∗ 0.64∗ — 0.07 −0.14 0.43P <0.0001 0.003 — 0.77 0.55 0.06

Cycloheximide r 0.18 0.02 0.07 — 0.11 0.17P 0.45 0.92 0.77 — 0.65 0.49

Kresoxim-methyl r 0.04 −0.03 −0.14 0.11 — −0.05P 0.87 0.90 0.55 0.65 — 0.85

Rhodamine 6G r 0.22 0.29 0.43 0.17 −0.05 —P 0.35 0.22 0.06 0.49 0.85 —

a Pearson correlation coefficient.b∗Significant correlation at P = 0.01 level.

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2.5

2.0

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-0.5

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MIC

s te

buco

nazo

le (

mg

litre

-1) 2.5

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-1.5Log

MIC

s ke

toco

nazo

le (

mg

litre

-1)

0.6

0.0

0.4

0.2

-0.2

-0.4

-0.6

-0.8

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-1.2

-1.4-1.5 -1.0 -0.5 0.0 0.5 1.0 1.5 2.0 2.5L

og M

ICs

kres

oxim

-met

hyl (

mg

litre

-1)

Log MICs cyproconazole (mg litre-1)

-1.5 -1.0 -0.5 0.0 0.5 1.0 1.5 2.0 2.5

Log MICs cyproconazole (mg litre-1)

Log MICs cyproconazole (mg litre-1)

-0.5 0.0 0.5 1.0 1.5 2.0

Log MICs tebuconazole (mg litre-1)

2.5

2.0

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0.5

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-1.5Log

MIC

s ke

toco

nazo

le (

mg

litre

-1)

r = 82P < 0.0001

r = 71P < 0.0001

r = 64P = 0.003

r = 0.04P = 0.87

Figure 2. Correlation between sensitivity of 20 field isolates of Mycosphaerella graminicola to the azole fungicides cyproconazole, ketoconazole,and tebuconazole, and the Qo-inhibitor fungicide kresoxim-methyl.

in mycelium was limited to isolates M3, M7 and S190.The basal expression level of MgAtr5 significantlydiffered between yeast-like cells and mycelium forall the isolates tested.

Treatment with cyproconazole strongly inducedtranscription of MgAtr4 in most of the isolates tested,especially in yeast-like cells. Induced levels of MgAtr1and MgAtr5 were also observed but only for a limitednumber of isolates such as M3 and M7 for MgAtr1 and

100

10

1

01

MIC

cyp

roco

nazo

le (

mg

litre

-1)

Progenies of cross M1 × M3 (n = 40)

Figure 3. Sensitivity of 40 progeny isolates of Mycosphaerellagraminicola obtained from a cross between isolates M1 (MIC0.178 mg litre−1) and M3 (MIC 17.8 mg litre−1) to cyproconazole.Median value is indicated as a continuous black line and mean valueas a dashed line. Sensitivity of the parent isolates M1 and M3 areindicated as dotted and dash-dotted lines, respectively.

M1 for MgAtr5, in both yeast-like cells and mycelium.Differences in induced level of expression among theisolates were also detected. For example, inductionof MgAtr4 in mycelium was much higher in isolatesM3 and M8 than in isolates M6, M7 and S190.Nevertheless, a correlation between expression of aspecific ABC gene and sensitivity of the isolates tocyproconazole was not clear.

CYP51 displayed basal transcripts in all isolates.However, in isolate M3 a relatively high basal level ofexpression was observed, especially in mycelium of thefungus. Treatment with cyproconazole did not effectthe expression level of this gene (Fig 4).

3.5 Accumulation studiesAccumulation of [14C]cyproconazole in thirteen fieldisolates tested proved to be constant in time. Meanaccumulation levels measured ranged between 0.35and 1 nmol cyproconazole mg−1 dry weight (Table 4).Addition of CCCP induced higher cyproconazoleaccumulation levels in all isolates, indicating thataccumulation was energy-dependent. No positive cor-relation between accumulation of [14C]cyproconazoleand sensitivity to cyproconazole (r = 0.39, P = 0.19),ketoconazole (r = 0.29, P = 0.34), or tebuconazole(r = 0.31, P = 0.31) was present. Statistical anal-ysis for significant differences in cyproconazoleaccumulation identified four overlapping groups ofisolates. Each group consisted of isolates with low

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Figure 4. Northern blot analysis of the ABC transporter genes MgAtr1, MgAtr4, and MgAtr5 and CYP51 in yeast-like cells and mycelium ofMycosphaerella graminicola after treatment with cyproconazole (C). Control samples are treated with 0.1% methanol (M). Isolates tested had low(M1, M7, M8, S043), medium (M3) and high (M4, M6, S054, S190) MIC values of cyproconazole. Hybridisation of the blots with an 18S rRNA probefrom Aspergillus niger served as a loading control.

and high sensitivity to azoles (Table 4). IsolatesM4 and S190, both with a low sensitivity tocyproconazole (MIC 100 mg litre−1) displayed thehighest (1.08 nmol mg−1 dry weight) and lowest(0.36 nmol mg−1 dry weight) accumulation level ofcyproconazole as compared to the other isolates tested(Table 4; Fig 5).

2.0

1.8

1.6

1.4

1.2

1.0

0.8

0.6

0.4

0.2

0.00 5 15 25 35 45 55 65

Acc

umul

atio

n(n

mol

cyp

roco

nazo

le m

g dr

y w

eigh

t-1)

Time (min)

Figure 5. Accumulation of [14C]cyproconazole by field isolates ofMycosphaerella graminicola with different levels of sensitivity to azolefungicides. MIC values of cyproconazole for isolates (�) M4, (�) M7,(♦) S043 and (ž) S190 are 100, 0.0562, 0.1 and 100 mg litre−1,respectively.

4 DISCUSSIONIn this study, we set out to investigate the physiologicalbasis of variation in sensitivity to azole fungicides infield isolates of M graminicola. The results obtainedindicate that the field isolates of M graminicola testedvary significantly in sensitivity to azole fungicides.The variation factor for sensitivity of the isolatesfor tebuconazole was 178. This value is in thesame range as described by Suty and Kuck,34 whoreported a variation factor of 100 in a collection ofmore than 1500 isolates. The variation factors forthe azole fungicides cyproconazole and ketoconazoleamounted to 1779 and this value is significantly higherthan for tebuconazole. Such a broad variation insensitivity for these compounds is intriguing, especiallysince the isolates were previously never exposed toketoconazole, an azole antimycotic used in treatmentsof human fungal pathogens, or to cyproconazole,which was not used in the fields from which theisolates were collected. Significant variation in base-line sensitivity to fungicides has been reported beforeand is ascribed to naturally tolerant genotypes thatalready exist in fungal populations prior to fungicidetreatment.38 Studies monitoring the sensitivity of fieldpopulations of M graminicola to azoles over recentdecades have not yet detected the evolution of resistantphenotypes.32 Hence, the significant difference insensitivity to the azoles tested is regarded as naturalvariation in base-line sensitivity to azole fungicides.

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Table 4. Accumulation of [14C]cyproconazole by 13 field isolates of Mycosphaerella graminicola with different sensitivity levels to azole fungicides

Isolates

Sensitivity tocyproconazole(mg litre−1)

Number ofreplicates

(n)

Mean accumulation(nmol cyproconazole

mg−1 dry weight)

Statisticalsignificance ofaccumulationa

Mean accumulationafter CCCP addition(nmol cyproconazole

mg−1 dry weight)

M7 0.0562 5 0.59 (0.14)b a, b, c 2.08 (0.49)M1 0.1 4 0.41 (0.09) a, b 1.81 (0.42)S043 0.1 8 0.51 (0.13) a, b, c 1.76 (0.38)M8 0.178 6 0.42 (0.06) a, b 1.55 (0.50)M2 1 5 0.80 (0.37) b, c, d 2.11 (0.77)S175 10 1 0.51 (—) — 2.03 (—)M3 17.8 6 0.58 (0.17) a, b, c 2.08 (0.40)M5 56.2 2 0.88 (0.01) c, d 2.58 (0.05)M4 100 9 1.08 (0.31) d 2.59 (0.74)M6 100 6 0.41 (0.09) a, b 1.23 (0.28)S054 100 6 0.88 (0.14) c, d 2.40 (0.55)S176 100 2 0.52 (0.12) a, b, c 2.02 (0.37)S190 100 3 0.36 (0.12) a 1.43 (0.23)

a Statistical significant differences between mean values. Letters represent groups of homogeneous subsets calculated using theStudent–Newman–Keuls analysis. Sigma values for groups a, b, c and d are 0.65, 0.09, 0.11 and 0.18, respectively. Differences were calculated ata significance level of P = 0.05.b Standard deviation.

The field isolates tested showed cross-sensitivity toazole fungicides. Cross-sensitivity to azoles had beenreported earlier for other fungal pathogens of bothmedical14 and agricultural importance,39 includingM graminicola.32 A correlation in sensitivity betweenthe azole fungicides and cycloheximide, kresoxim-methyl or rhodamine 6G was not observed. Theseresults indicate that field isolates with a relativelylow sensitivity to azoles do not have a multi-drugresistance phenotype, as described for laboratory-generated azole-resistant mutants of M graminicola.31

This discrepancy indicates that different mechanismsof resistance operate in laboratory-generated mutantsand in field isolates.

The sensitivity of progeny isolates obtained froma genetic cross between isolates with high andintermediate sensitivity to cyproconazole rangedbetween the sensitivity of both parents. The frequencydistribution of MIC values among the progeny wascontinuous and, therefore, progeny isolates couldnot be classified in distinct sensitivity groups. Weconclude that sensitivity to cyproconazole in thehaploid organism M graminicola is polygenic. Thisobservation may, at least in part, explain the extremelyhigh variation factor in base-line sensitivity of theisolates to the azoles tested and may imply thatdifferent mechanisms are involved in azole tolerancein this fungus. This corroborates the observationthat multiple resistance mechanisms may operate inlaboratory-generated mutants of M graminicola withdecreased sensitivity to azoles.31 Polygenic control ofresistance to azole fungicides has been reported for Anidulans40 and Nectria haematococca Berk & Broomevar cucurbitae.41

None of the M graminicola progeny isolates wereless sensitive to cyproconazole than the parent isolateM3. This observation indicates that recombination of

putative azole-resistance genes in the progeny isolatesdid not result in an additive effect that exceeded thetolerance level of the parent isolate M3. Therefore,genetic recombination in M graminicola may noteasily result in the generation of progeny isolates withazole sensitivity that exceeds the base-line sensitivityobserved. Instead, a gradual shift in frequency of theleast sensitive genotypes in the population may beexpected as a result of the polygenic inheritance.42

Expression of the ABC genes MgAtr1–MgAtr5was examined in northern blot experiments inboth yeast-like cells and mycelium of the fungus.Differences in basal level of expression amongthe field isolates were observed, but no obviousincrease in transcript levels of a specific MgAtr genewas found exclusively in the less sensitive isolates,either in yeast-like cells or in mycelium, except forMgAtr1 in isolate M3 (yeast-like cells and mycelium)and S054 (yeast-like cells). Cyproconazole-inducedexpression was observed in particular for MgAtr4 inyeast-like cells, but a correlation between inducedtranscript levels and azole sensitivity was not present.Increased expression of ABC transporter genes hasbeen described as a resistance mechanism to azoleantifungals in azole-resistant isolates of P digitatum,43

A nidulans44 and B cinerea.45 Hence, we do notexclude that increased transcript levels of MgAtr1might play a role in the increased azole toleranceof isolates M3 and S054. This would corroboratethe finding that overexpression of MgAtr1 leads toazole-resistance in some laboratory-generated mutantsof M graminicola.31 It is also possible that not-yet-identified ABC transporter genes play a role in theazole sensitivity of field isolates as well. This hypothesisis supported by the observation that, from a singleazole-sensitive cell line of C albicans, multiple strainswith different levels of resistance were obtained that

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displayed distinct over-expression patterns of at leastfour genes involved in azole resistance. These includedthe ABC transporter genes CDR1 and CDR2, themajor facilitator gene MDR1, and ERG11, the geneencoding the target enzyme of the azoles in theergosterol biosynthetic pathway.46 The differentialexpression pattern of MgAtr1–MgAtr5 in the twodimorphic states of the fungus, yeast-like cells andmycelium, has been described previously,47,48 andmay reflect differences in cell wall and membranecomposition that can influence ABC transporteractivity.49–51

The expression studies demonstrate a high basaltranscript level of CYP51 in isolate M3. This isolatehas an intermediate sensitivity to azoles as compared toother isolates tested. Increased expression of CYP51is described as a mechanism of resistance to azolesin many fungi.52 In C albicans and S cerevisiae,azole tolerance as a result of enhanced transcriptionor amplification of the CYP51 (ERG11) gene hasalso been observed.13,14,16,17 In P digitatum and Vinaequalis, tandem repeats in the promoter region ofCYP51 enhanced transcription of this gene and thecopy number of these repeats correlated positivelywith CYP51 expression and tolerance to azoles.22,23

It is possible that a similar mechanism is operating inisolate M3.

Point mutations in the open reading frame of theCYP51 gene of filamentous fungi and yeasts can alsoconfer resistance to azoles.12 However, the sequenceof the CYP51 gene from laboratory-generated azole-resistant mutants of M graminicola and their parentalstrains was the same and did not show any ofthe point mutations involved in resistance to azoleantifungal agents in C albicans,12 laboratory mutantsof U maydis18 and P italicum,19 and in field isolatesof U necator20 and E graminis f sp hordei.21,31 Allelicvariations in the CYP51 sequence of field isolates ofM graminicola, make it difficult to assess whether thebase-line sensitivity is affected by point mutations.

Uptake experiments demonstrated that accumula-tion levels of [14C]cyproconazole by field isolates ofM graminicola did not correlate with sensitivity toazoles. Individual isolates with decreased sensitiv-ity to azoles, such as S190, showed a relatively lowaccumulation level of [14C]cyproconazole, but otherisolates with decreased sensitivity to azoles, such asM4, display a relatively high accumulation level ofthe fungicide. Hence, reduced accumulation of azolesfrom mycelium cannot account exclusively for the dif-ferences in base-line sensitivity in the isolates tested.The absence of a correlation between accumulation ofcyproconazole and azole sensitivity may reflect the highgenetic diversity of M graminicola field populations53

that accounts for morphological and physiological dif-ferences. These differences can have a significant effecton accumulation of [14C]cyproconazole and may maskthe variations in accumulation mediated by changesin ABC transporter activity. Studies on laboratory-generated azole-resistant mutants of M graminicola

showed that mutants occur with either a decreased oran increased accumulation of azoles as compared totheir parent isolates.31 Such a relation cannot easily bemade for field isolates, since the genetic relationshipbetween field isolates tested is virtually unknown. Forthese reasons it is still possible that the mechanism ofresistance in the least-sensitive field isolates is medi-ated by changes in ABC transporter activity, resultingin either a decreased or increased accumulation of thefungicide. Active drug disposal in vacuoles or in lipidbodies could possibly explain the relatively high accu-mulation level observed for some of the least sensitivefield isolates and might relate to overexpression ofABC transporters localised in vacuolar membranes.54

This has been described for azole-resistant strains of Umaydis that accumulated triadimenol to higher levelsas compared to sensitive strains.55 Alternative mecha-nisms of resistance, such as alterations in the target-siteof azoles can also not be excluded. For these reasonswe anticipate that a high correlation between azolesensitivity and accumulation in field isolates of Mgraminicola can not easily be established. However,previous literature data suggest that such a correla-tion is present.56,57 The discrepancy with the work ofJoseph-Horne et al56 can be ascribed to the low num-ber of isolates tested. The work of Gisi et al57 is difficultto judge since data that support their conclusion werenot published.

In summary, our studies suggest that variation insensitivity to azoles in field isolates of M graminicola isbased on multiple mechanisms. These may includedecreased accumulation of the fungicide in thecytoplasm, increased accumulation of the fungicide asa result of sequestration of the fungicide in cellularcompartments and overproduction of P45014DM.These multiple mechanisms would corroborate thepolygenic nature of azole sensitivity as shown by theanalysis of a genetic cross of M graminicola isolates.

ACKNOWLEDGEMENTSWe kindly acknowledge Dr JM Seng (Biotransfer,Montreuil Cedex, France) and Dr A Suty (Bayer AG,Landwirtschaftszentrum Monheim, Leverkusen, Ger-many) for providing the field isolates of Mycosphaerellagraminicola and ECP Verstappen for technical assis-tance with the crosses of M graminicola. We are gratefulto Professor Dr PJGM De Wit (WUR) and Dr L-HZwiers for critical reading of the manuscript and theWageningen Mycosphaerella group for key discussionsand suggestions during this work. Ioannis Stergiopou-los was financially supported by the Training andMobility of Researchers (TMR) Programme—MarieCurie Research Training Grants, The European Com-mission (Contract No ERBFMBICT983558).

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