International Journal of Food Microbiology 145 (2011) 195–204

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International Journal of Food Microbiology

j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / i j f o o d m i c r o

Sensitivity of Penicillium expansum field isolates to tebuconazole, iprodione,

fludioxonil and cyprodinil and characterization of fitness parameters and
patulin production
George S. Karaoglanidis a,⁎,1, Anastasios N. Markoglou b,⁎,1, George A. Bardas a, Eleftherios G. Doukas b,
Sotiris Konstantinou a, John F. Kalampokis b
a
Plant Pathology Laboratory, Faculty of Agriculture, Aristotelian University of Thessaloniki, POB 269, 54124, Thessaloniki, Greece
b
Pesticide Science Laboratory, Agricultural University of Athens, 75 Iera Odos, 118 55 Athens, Greece

a r t i c l e i n f o a b s t r a c t

Article history: A total of 236 Penicillium expansum field isolates from decayed apple fruit collected from packinghouses and
Received 20 July 2010 processing industries located in the region of Imathia, Northern Greece were tested for their sensitivity to
Received in revised form 4 December 2010 tebuconazole, fludioxonil, iprodione and cyprodinil. Preliminary fungitoxicity tests on the response of
Accepted 20 December 2010
the isolates showed several phenotypes, distinguished according to their sensitivity to fungicides tested. The
EC50 values ranged from 0.64 to 5 (average = 0.98) μg/ml for iprodione, 0.9 to 7.3 (average = 2.66) μg/ml
Keywords:
Penicillium expansum
for tebuconazole, 0.008 to 1.28 (average = 0.55) μg/ml for cyprodinil and from 0.013 to 0.47 (average =
Anilinopyrimidines 0.08) μg/ml for fludioxonil. A bimodal distribution of the EC50 values of isolates with distinct sensitive and
Phenylpyrroles resistant populations to fludioxonil and tebuconazole were observed. In the case of cyprodinil, a much
Triazoles broader, hundred-fold, range of sensitivity was found, probably indicating that some isolates are relatively
Dicarboximides insensitive to cyprodinil compared to the most sensitive ones. Isolates exhibiting simultaneously reduced
Patulin sensitivity to tebuconazole and fludioxonil or tebuconazole and iprodione or to tebuconazole and cyprodinil
were also observed at low frequencies. A small portion of the population (7.5%) showed multiple resistance to
tebuconazole, fludioxonil and iprodione. Study of fitness determining parameters showed that the resistance
to tebuconazole, fludioxonil and iprodione had a significant adverse effect on mycelial growth rate and
pathogenicity. Contrary to that, these fitness parameters were not affected in the isolates showing reduced
sensitivity to cyprodinil. Analysis of patulin production on YES-agar growth medium and on artificially
inoculated apple fruit showed that all isolates were mycotoxigenic. Most of the cyprodinil-insensitive isolates
produced patulin at concentrations similar to or relatively higher (up to 1.5-fold on growth medium) than the
sensitive ones. In contrast, a significant reduction (up to 98% of multiple resistant isolates) in patulin
production was observed in all other phenotypes, indicating an adverse effect of fitness penalties on the
mycotoxigenic ability of resistant isolates. The above mentioned data clearly show a considerable risk for the
selection of P. expansum isolates resistant to fludioxonil, iprodione, tebuconazole and cyprodinil. The potential
risk of increased patulin contamination of apples and their byproducts by the appearance and predominance
of highly mycotoxigenic isolates of P. expansum resistant to the anilinopyrimidines is discussed.
© 2010 Elsevier B.V. All rights reserved.

1. Introduction
Postharvest pathogens causing fruit decay in storage rooms
represent one of the major threats in apple fruit production. Although
more than 90 fungal species have been reported to be causal agents of
postharvest decay on apple, Penicillium expansum is considered to be
the most important, causing a disease known with the common name
⁎ Corresponding authors.
E-mail addresses: gkarao@agro.auth.gr (G.S. Karaoglanidis), markan@aua.gr
(A.N. Markoglou).
1
Contributed equal to this work.
‘blue mold’ (Morales et al., 2008a). Although evidence has been
provided for the ability of P. expansum to behave as a latent pathogen
(Biggs, 1995) most of the apple infections are mediated through
wounds caused during harvest or handling of the fruit in the field or in
the packing houses (Amiri and Bompeix, 2005; Morales et al., 2008b).
Because the fungus is able to establish infections even at temperatures
lower than 0 °C the disease symptoms appear during storage (Baraldi
et al., 2003). Losses caused by the pathogen are quantitative due to the
developing decay of the fruit, but also qualitative due to patulin-
contamination. This is particularly important because large quantities
of apples are used for juice production and usually fruit destined for
processing rather than direct consumption are of lower quality and
may have fungal infections (Morales et al., 2008a).

0168-1605/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.ijfoodmicro.2010.12.017
196 G.S. Karaoglanidis et al. / International Journal of Food Microbiology 145 (2011) 195–204
Patulin is a toxic secondary metabolite predominantly produced
by P. expansum, and is one of the best characterized and most widely
disseminated mycotoxins found in rotting pome fruit, most notably in
apples, and their byproducts. Contamination of apples has been
reported in several countries, and thus patulin contamination within
apple products poses a serious worldwide health risk (Abramson,
1997; Bullerman, 2000). Toxicological studies have shown that
patulin is cytotoxic to continuous cell lines, and is believed to exert
cytotoxic effects by forming covalent adducts with essential cellular
sulfhydryl (SH, thiol) groups of proteins and glutathione. There is no
clear evidence that patulin is carcinogenic, however, it has been
shown to cause immunotoxic and neurotoxic effects in animals (Riley
and Showker, 1991). The Joint Food and Agricultural Organization and
World Health Organization Expert Committee on Food Additives
(JECFA) has established for patulin a provisional maximum tolerable
daily intake of 0.4 μg kg− 1 body weight, which was endorsed by the
EU Scientific Committee for Food (SCF) in 2000. The maximum
permitted levels of patulin in Europe are 10 μg kg− 1 for fruit-based
baby food, 50 μg kg− 1 for fruit juice and 25 μg kg− 1 for solid apple
products (Moake et al., 2005). In 2006, the European rapid alert
system for food and feed safety (RASFF) reported seven notifications
for patulin occurrence above the regulatory level (RASFF, 2006).
The prevention of mycotoxin contamination of agricultural
products is one of the top priorities in human and animal safety.
Approaches to reduce the mycotoxin levels in contaminated foods and
feeds include various physical, chemical and biological detoxification
methods in stored and processed agricultural products. However
these techniques are still in developmental stages and have limited
efficacy (Moake et al., 2005; Morales et al., 2010).
Chemical control remains the main measure to reduce the
incidence of blue mold on apple fruit, although it is not always
effective (Morales et al., 2007, 2010). Traditionally, postharvest
applications of the benzimidazole fungicide thiabendazole and the
sterol demethylation-inhibiting (DMI) fungicide imazalil as either
drench treatments prior to storage or online spray treatment on the
packing line were used worldwide for the control of post-harvest
pathogens on apple, including P. expansum (Sholberg et al., 2005a).
However, in most of the advanced countries postharvest fungicide
applications have already been banned. Today, new fungicides from
the group of anilinopyrimidines (cyprodinil and pyrimethanil),
phenylpyrroles (fludioxonil), strobilurin-type fungicides (pyraclos-
trobin) and the new carboxamide fungicide boscalid have been shown
to be highly effective against the pathogen (Errampali, 2004; Sholberg
et al., 2005a; Smilanick et al., 2006; Xiao and Boal, 2009). These
compounds have been introduced into the spray programs applied in
the field, leading to changes in control strategies for postharvest
pathogens. Fungicide applications aimed at blue mold control are
carried out a few days before harvest, however the fungal population
existing in the orchard is exposed to a strong fungicide selection
pressure throughout the apples' growth period due the high number
of fungicide spray applications carried out against apple scab caused
by Venturia inaequalis, largely using active ingredients that are
effective against both pathogens.
Development of resistance to chemicals used against fungal
pathogens constitutes one of the most important limiting factors in
chemical control leading to a reduction and in some cases to complete
loss of control efficacy. P. expansum is a classical ‘high risk pathogen’
from the view of resistance development, and the site-specific
fungicides face this possibility. In the recent past such a resistance
risk was demonstrated for the benzimidazoles and DMIs (Baraldi
et al., 2003; Morales et al., 2008a; Rosenberger et al., 1991; Sholberg
et al., 2005b; Vinas et al., 1991, 1993). After the introduction of the
novel fungicides into the spray programs the baseline sensitivity of
P. expansum to pyrimethanil and fludioxonil was determined (Sholberg
et al., 2005a; Li and Xiao, 2008a). Furthermore, in recent studies
laboratory-induced pyrimethanil and fludioxonil resistant mutants
were characterized in terms of resistance stability, fitness parameters
(Li and Xiao, 2008b; Sholberg et al., 2005a) and mycotoxin production
(Markoglou et al., 2008b).
Although several aspects in the biology, epidemiology and control
of P. expansum have been investigated (Conway et al., 2007; Morales
et al., 2007, 2008b; Pianzzola et al., 2004), there are very limited data
about the risk for fungicide resistance development, and no
information is available regarding the influence of resistance on the
mycotoxin production. Thus, the objectives of the present study were
to: i) determine the sensitivity of P. expansum field populations
obtained from apple orchards in Greece to fludioxonil, iprodione,
tebuconazole and cyprodinil, ii) to characterize a set of fungicide-
resistant pathogen isolates selected on the basis of the revealed
resistance-phenotypes, by measuring precisely the fungicide sensi-
tivity and the fitness of the fungicide-resistant isolates and iii) to
measure patulin production capacity of fungicide-resistant field
isolates.
2. Materials and methods
2.1. Pathogen isolates and culture conditions
Isolates of P. expansum were collected during a survey conducted to
determine the causal agents of apple fruit postharvest decay in Northern
Greece. For this purpose during the 2008–2009 storage period 5 packing
houses located in the region of Imathia were sampled at 15-day intervals
through November 2008 to March 2009. Apple fruits showing decay
symptoms were collected from the packing houses during the packing
operations and transferred to the laboratory for fungal isolation.
Isolations were carried out by removing small fruit pieces at the margin
of diseased/healthy tissue and transferring to Petri dishes containing
acidified Potato Dextrose Agar (PDA), (Oxoid, Unipath Ltd., Basingstoke,
UK). After 3–4 days incubation at 20 °C, fungal colonies showing
morphological characteristics similar to Penicillium spp. were selected
and transformed to single-spore isolates using the serial dilution
technique (Tuite, 1969). A total of 236 collected isolates were identified
as P. expansum, according to Pitt (2002) description of the fungus, and
were used in the study. For the in vitro study of patulin production the
isolates were grown on Yeast Extract Sucrose medium (YES) containing
2 g yeast extract, 15 g sucrose and 1.5 g agar in 100 ml distilled water
under the same conditions. For long-term storage, the isolates were
maintained on PDA slants at 2 °C.
2.2. Fungicides, patulin and solvents
The fungicides used in this study were the commercial formulations
of cyprodinil (Chorus 50 WG, Syngenta Hellas, Greece), fludioxonil
(Medallion 50 WP, Syngenta Hellas, Greece), iprodione (Rovral 50 WP,
BASF Hellas, Greece) and tebuconazole (Folicur 25 WG, Bayer
CropScience, Greece). The fungicides were dissolved in sterilized
distilled water and stock solutions were prepared. Patulin standard
was purchased from Sigma-Aldrich (St. Louis, USA). Analytical standard
stock solutions of patulin were made in methanol at various concentra-
tions and stored at −20 °C. Ten standard solutions of patulin at
concentrations from 0.01 to 5 μg ml− 1 were prepared as calibration
standards. HPLC grade solvents, methanol and acetonitrile, were
purchased from Lab Scan (Dublin, Ireland). Ultrapure-grade HPLC
water was obtained by purification of distilled water through a Milli-Q
Gradient system (Millipore, Bedford, USA).
2.3. In vitro fungicide sensitivity and resistance frequency
An initial qualitative sensitivity assessment, using fungicide
discriminatory concentrations, was carried out with all isolates of
P. expansum. Subsequently, fungitoxicity tests were made with a
representative subset of 40 strains, from all phenotypic classes, using
 G.S. Karaoglanidis et al. / International Journal of Food Microbiology 145 (2011) 195–204
several concentrations of each fungicide to determine the EC50 values
(the concentration causing 50% reduction of mycelial growth).
The measurement of resistance frequency to cyprodinil, fludioxonil,
iprodione and tebuconazole was based on the use of a discriminatory
concentration for each fungicide tested at the minimum inhibitory
concentrations (MIC) values of the sensitive isolates. Growth media were
amended with aqueous fungicide solutions at the concentrations of
2.5 μg ml− 1 cyprodinil, 0.5 μg ml− 1 fludioxonil, 2.5 μg ml− 1 iprodione
and 5 μg ml− 1 tebuconazole. For the measurement of sensitivity to
cyprodinil, L-asparagine-based agar medium (ASP-agar) was used as
growth medium (Hilber and Schüepp, 1996), while for the remaining
fungicides the growth medium used was PDA. Control plates were not
amended with fungicide. For each isolate three replicate plates were
prepared per fungicide. Cultures were incubated at 20 °C in the dark for
7 days. Isolates exhibiting mycelial growth on the fungicide-amended
media were considered as fungicide-resistant while isolates with
completely inhibited mycelial growth were considered as fungicide-
sensitive.
To measure the EC50 values of the 40 selected isolates, ASP-agar
medium was amended with 0.01, 0.05, 0.1, 0.5, 1 and 5 μg ml− 1
cyprodinil, whereas PDA medium was amended with 0.005, 0.01, 0.05,
0.1 and 0.5 μg ml− 1 fludioxonil, 0.05, 0.1, 0.5, 1 and 5 μg ml− 1 iprodione
or 0.1, 0.5, 1, 5 and 10 μg ml− 1 tebuconazole. Control media were not
amended with fungicides. The production of the mycelial inoculum was
carried out according to Li and Xiao (2008b). Briefly, 1 ml of a 105 ml−1
conidial suspension of each isolate was mixed with approximately 15 ml
of molten PDA in a petri dish and the cultures were incubated at 20 °C
for 24 h. PDA plugs, 5-mm in diameter, were then removed using a cork-
borer and transferred upside down to fungicide-amended or unamend-
ed media. Cultures were incubated at 20 °C in the dark for 7 days. The
mean colony diameter was measured and expressed as percentage of
the mean diameter of the untreated control. Tests for each isolate were
replicated twice for each concentration of each fungicide.
2.4. Study of fitness parameters
The fitness parameters tested were the mycelial growth rate and
pathogenicity. Four 5-mm mycelial plugs of each isolate were
transferred to the centre of PDA plates for radial growth measurements.
After incubation at 20 °C in the dark for 7 days, the colony diameter of
each isolate was measured. The experiment was replicated twice.
Pathogenicity of P. expansum isolates was determined by examining
symptom severity caused by each isolate on apple fruit (Malus
domestica, cv. Granny Smith). Apple fruits, selected on a basis of size
and shape uniformity and absence of any wound, were collected from
the field at the harvest maturity stage. Prior to inoculations the fruits
were surface-disinfected for 5 min by drenching them in a 1% sodium
hypoclorite solution. After removing them from the disinfectant
solution they were rinsed three times with sterile deionized water
and subsequently air dried. The fruits were wound-inoculated at the
equator. Two wounds per fruit were made using a 2 mm diameter nail
head to a depth of 3 mm and on each wound 20 μl of a conidial
suspension containing 1 × 104 conidia per ml was placed with a pipette.
Twenty fruits for each isolate were inoculated and the experiment was
replicated twice. Uninoculated fruits treated with sterile water were used
as controls. The fruits were placed on wire mesh platforms (10 fruits
per box) in plastic boxes (23×31×10 cm [length×width×height]).
Twenty ml of water was added to each box which was then covered to
maintain high relative humidity. The inoculated fruits were incubated for
7 days at 20 °C and the infection was scored by measuring the lesion
diameter.
2.5. Patulin production, extraction and purification
Conidial suspension (approx. 108 conidia ml− 1) of each isolate was
plated on Petri dishes (three replicates for each strain) containing YES
197
agar medium. After 10 days incubation at 20 °C in the dark, 10
mycelial plugs of 10 mm diameter for each dish were harvested into a
test tube with 5 ml methanol and agitated vigorously on a rotary
shaker (150 rpm) overnight, at room temperature. The methanol
extract was collected and the remaining mycelial mass was rinsed
with another 5 ml methanol. The total mycelial extract was filtrated
and centrifuged at 10,000 rpm for 10 min. The final methanol extract,
after filtration through a Teflon filter (PVDF membrane filter, 0.2 μm
pore size, 17 mm diameter), was stored at − 20 °C until to the analysis
(Markoglou et al., 2008b).
To estimate the in vivo mycotoxigenic ability of the isolates, the
artificially inoculated fruits for the pathogenicity measurements were
further incubated for 3 additional days. Then, the fruits were ground
in a blender with 75 ml of methanol–water (60:40) for 5 min and the
supernatant was filtrated through cheese cloth. The residual extract
was partitioned with 30 ml chloroform and after evaporation to
dryness the extract was redissolved in 5 ml methanol and filtered
through a PVDF Teflon filter (Markoglou et al., 2008b).
2.6. Detection and determination of patulin production
An initial qualitative detection of patulin production by each strain
of P. expansum was performed using thin layer chromatography (TLC)
and confirmed with appropriate patulin standards. For TLC analysis
5 μl of mycelial extract was separated on TLC plates (0.2 mm silica gel
60, 20 × 20 cm, Machenery-Nagel, Germany) with toluene–ethyl
acetate–formic acid (50:40:11, v/v). Patulin was detected on TLC
plates by spraying with 3-methyl-2-benzothiazolinone hydrazone
hydrochloride (MBTH), heating at 110 °C for 15 min, and visualized
the patulin derivative with UV light at 280 nm (Markoglou et al.,
2008b).
The quantitative analysis of patulin was performed by a Shimadzu
HPLC system equipped with a binary solvent pump (LC-10ADvp), DGU-
14A degasser, CTO-10Avp column oven, SIL-10ADvp autosampler with
volume injection set at 2 μl, and a UV diode array detector (SPD-
M10Avp). Data acquisition and processing were performed with the LC/
MS solution Ver. 3.0 Workstation. The chromatographic separation was
performed on a Discovery reversed-phase C18 narrow-bore column
(250× 4.6 mm id, 5 μm particle size), thermostatted at 40 °C. The mobile
phase was water–acetonitrile (90:10, v/v), delivered isocratically at a
flow-rate of 0.7 ml min− 1 for 15 min. The calibration plot was linear for
the UV diode array detector over the range of 0.01 to 5 μg ml− 1, and the
limit of detection (LOD) of the analytical method was approximately
2.5 ng ml− 1. These results are lower than the legal limits established by
the international authorities. The obtained recoveries from spiked YES-
medium and apple samples at fortification levels of 10, 50 and 100 ng
patulin g− 1 were 95–102 and 87–98.6%, respectively, with precision
values (relative standard deviations) of 5.2–9.4 and 7.6–12.5%,
respectively.
2.7. Data analysis
The EC50 values of each isolate to cyprodinil, fludioxonil, iprodione
and tebuconazole were calculated by regressing the relative inhibition
of mycelial growth against the Log10 fungicide concentrations.
Calculations were performed using SAS (JMP, SAS Institute, Cary,
NC). Cross-resistance relationships among fungicides tested were
determined by correlating the EC50 values. Pearson correlation
coefficients between fitness components, patulin production and
sensitivities to fungicides were estimated using the mean values for
each variable per individual isolates. Data on mycelial growth rate and
pathogenicity of isolates were subjected to analysis of variance.
Overall differences in mycelial growth rates and the pathogenicity
data of phenotype-resistance group were tested with the Kruskal–
Wallis test and in case of a significant result, pair wise comparison
were performed by means of a series of Mann–Whitney tests.
198 G.S. Karaoglanidis et al. / International Journal of Food Microbiology 145 (2011) 195–204
Dunnett's multiple range test was used to assess the differences
between patulin production rating of isolates. All the statistical
analyses were made with the SPSS (SPSS Inc., Chicago, IL, USA).
3. Results
3.1. Sensitivity of P. expansum to fludioxonil, iprodione, tebuconazole
and cyprodinil
A total of 236 P. expansum single spore isolates were tested for
their sensitivity to fludioxonil, iprodione, tebuconazole and cyprodinil
based on mycelial growth inhibition at a single discriminatory dose of
each fungicide tested. The in vitro fungitoxicity bioassays showed that
only 16% of the population was sensitive (SEN phenotype) to all
fungicides tested (Fig. 1). A large portion of the population (43%)
showed reduced sensitivity to cyprodinil (ANIR phenotype) with EC50
values significantly higher (up to 100-fold) than those of the
most sensitive ones. Nine percent of the isolates showed reduced
sensitivity (up to 3-fold) to the DMI fungicide tebuconazole (DMIR
phenotype). Isolates with simultaneously reduced sensitivity to
tebuconazole and fludioxonil (DMIPHENR phenotype) or to tebuco-
nazole and iprodione (DMIDICR phenotype) or to tebuconazole and
cyprodinil (DMIANIR phenotype) were also determined at low
frequencies of 7.5%, 11% and 6%, respectively. Additionally, 7.5% of
the isolates (DMIDICPHENR phenotype) showed decreased sensitivity
to tebuconazole, iprodione and fludioxonil (Fig. 1).
As shown in Fig. 2, a bimodal distribution of distinct sensitive and
resistant subpopulations was found for fludioxonil and tebuconazole.
Based on precise sensitivity measurements, the EC50 values ranged from
0.013 to 0.47 μg ml− 1 for fludioxonil and 0.9 to 7.3 μg ml− 1 for
tebuconazole, indicating a resistance factor (Rf) from 2 to 6 and 2 to
10, respectively. Of the 236 isolates, one exhibited a reduced sensitivity
to iprodione with an EC50 value of 5 μg ml− 1. This was higher than those
of the remaining isolates, which ranged from 0.64 to 1.69 μg ml− 1. This
isolate was also resistant to fludioxonil and to tebuconazole, with EC50
values 6.7 and 0.1 μg ml− 1, respectively. A great range of sensitivity to
cyprodinil (EC50 values from 0.008 to 1.28 μg ml− 1), with up to a 100-
fold difference in EC50 between the least and the most sensitive isolates
was found. The existence of a broader range of sensitivity to cyprodinil
probably indicates a gradual, unimodal shift in sensitivity resulting from
a multi-step (polygenic) resistance in the P. expansum population.
Fig. 1. Resistance frequency of a P. expansum population from apple, to the fungicides cyprodinil (ANIR), tebuconazole (DMIR), iprodione (DICR) and fludioxonil (PHENR). Phenotype
group designated as SEN includes isolates that were sensitive to all the fungicides tested.
3.2. Cross-sensitivity to iprodione, tebuconazole, cyprodinil and fludioxonil
No significant cross-sensitivity correlation was observed between
cyprodinil, fludioxonil, iprodione and tebuconazole (Table 1). Pair-
wise comparisons of sensitivity to cyprodinil with those of fludioxonil,
iprodione and tebuconazole showed values for correlation coefficients
ranging from − 0.3 to 0.06 which were not significant (P N 0.05),
suggesting an absence of cross-resistance relationship between
cyprodinil and the other fungicides (Table 1). Correlations among
sensitivities to tebuconazole and fludioxonil and iprodione showed
significant (P b 0.05) coefficient values of 0.63 and 0.48, respectively,
suggesting that there was a tendency for selection of P. expansum
strains that they were simultaneously resistant to tebuconazole and to
either fludioxonil or iprodione (Table 1). Interestingly, no cross
resistance pattern between fludioxonil and iprodione was found
(P N 0.05).
3.3. Fitness parameters
Isolates showing reduced sensitivity either to cyprodinil or to
tebuconazole and isolates showing simultaneously reduced sensitivity
to the anilinopyrimidine and DMI fungicides showed higher mycelial
growth which was not significantly different (P N 0.05) from the mean
mycelial growth of the sensitive isolates (mean value of 46.5 mm;
Table 2). In contrast, isolates that were simultaneously resistant to DMI
and phenylpyrrole fungicides or to DMI, phenylpyrrole and dicarbox-
imide fungicides showed significantly lower (P b 0.05) mycelial growth
with mean values of 26.6 and 28.5 mm, respectively (Table 2).
Furthermore, calculations of Pearson correlation coefficients showed
that mycelial growth in P. expansum was negatively correlated (Pb 0.05)
with the level of sensitivity to tebuconazole and fludioxonil, while there
was no significant correlation between the level of sensitivity to
cyprodinil and iprodione (P N 0.05), (Fig. 3).
Data on pathogenicity showed that there was great variability even
among isolates within the same phenotypic group. Anilinopyrimidine-
resistant isolates and isolates with double resistance to anilinopyrimi-
dine and DMI fungicides were equally aggressive (PN 0.05) to the
sensitive ones (Table 2). In contrast, isolates belonging to the remaining
resistance-phenotypic groups were significantly less aggressive
(P b 0.05) than the sensitive isolates. Furthermore, calculation of
Pearson correlation coefficients showed that aggressiveness of the
isolates was positively correlated with the sensitivity to cyprodinil while
 G.S. Karaoglanidis et al. / International Journal of Food Microbiology 145 (2011) 195–204 199

Fig. 2. Distribution of EC50 values of tebuconazole (A), iprodione (B), fludioxonil (C) and cyprodinil (D) based on mycelial growth bioassays for 236 isolates of P. expansum from apple.

there was a strong (Pb 0.05) negative correlation with the sensitivity to
tebuconazole, fludioxonil and iprodione (Fig. 4).
3.4. Patulin production
Initial analyses for patulin production using TLC plates showed
that all P. expansum isolates were mycotoxigenic. Analysis of mycelial
extracts of the sensitive isolates that were grown on YES agar medium
showed that patulin production was 35 to 80 ng mg− 1 dry weight of
mycelium. In most cyprodinil-resistant isolates (approx. 93% of the
cyprodinil-resistant phenotype) patulin production was similar to the
sensitive isolates (Fig. 5). However, in some cyprodinil-resistant
isolates (F11, F16, RD83, RD86, RD118, GS19, GS20 and GS35) patulin
production on YES-agar medium was markedly higher (up to 1.5-fold)
than for the sensitive isolates. Conversely, all tebuconazole, fludiox-
onil and iprodione resistant isolates exhibited a significant reduction
in patulin production compared to the sensitive isolates, indicating
that fitness penalties may result in a reduced mycotoxigenic ability in
P. expansum (Fig. 5).
Study of the mycotoxigenic ability of isolates on apple fruit showed
that all isolates produced patulin at concentrations higher than those
obtained on YES-agar medium (Fig. 5). The patulin production by the
sensitive, the anilinopyrimidine- and the anilinopyrimidine and
triazole-resistant isolates did not significantly differ. On the contrary,
a significant reduction in the patulin production on apple fruit was
observed in isolates with reduced sensitivity to tebuconazole,
fludioxonil and/or iprodione (Fig. 5).
Calculation of Pearson correlation coefficients showed that patulin
production on decayed apple tissues was negatively correlated with
the sensitivity to tebuconazole and to fludioxonil (P b 0.05), while
there was no significant correlation between patulin production and
sensitivity to cyprodinil and iprodione (Fig. 6).
4. Discussion

Table 1 Penicillium spp. are considered high risk pathogens for resistance
Cross-sensitivity correlations between tebuconazole, cyprodinil, fludioxonil, and development to fungicides (FRAC, 2010). Factors contributing to this
iprodione in P. expansum field isolates obtained from apples.
high inherent resistance risk include the short life cycle of the pathogen
Fungicide Fungicide and the abundant sporulation on the diseased surfaces that increase the
Tebuconazole Cyprodinil Fludioxonil Iprodione possibility of development of mutant progeny and their dissemination.
Resistance development has been reported to benzimidazole and DMI
Tebuconazole – − 0.30 a
0.63⁎⁎⁎b 0.48⁎⁎⁎
Cyprodinil − 0.30 – − 0.06 0.06 fungicides in Penicillium spp. from citrus (Bus et al., 1991; Holmes and
Fludioxonil 0.63⁎⁎⁎ − 0.06 – 0.08 Eckert, 1999) and in P. expansum from apples (Baraldi et al., 2003;
Iprodione 0.48⁎⁎⁎ 0.06 0.08 – Errampali et al., 2006; Sholberg et al., 2005b; Vinas et al., 1993).
a
Pearson correlation coefficient values. The results of the current study provided evidence for widespread
b
Correlation coefficients are significant at P = 0.05. occurrence of strains of P. expansum from apples in Greece with
200 G.S. Karaoglanidis et al. / International Journal of Food Microbiology 145 (2011) 195–204

Table 2 of our knowledge, are provided for first time. In contrast, the baseline
Fitness components of field isolates of P. expansum. sensitivity to the anilinopyrimidine fungicide pyrimethanil was
Isolates Fungicide-resistance Fitness components reported in two recent studies (Sholberg et al., 2005a; Li and Xiao,
phenotypea 2008a). However, a direct comparison of the sensitivities is not
Mycelial growthb Pathogenicityc
possible since these two studies refer to a different active ingredient
RD7 SEN 48.3 32.9
to that tested in our study.
RD11 SEN 48.5 31.3
RD37 SEN 45.9 4.7 Resistance to sterol demethylation inhibitors has been reported in
RD91 SEN 46.1 28.2 the past both in P. expansum from apples (Morales et al., 2008a; Vinas
GD35 SEN 45.5 28.8 et al., 1993) and in Penicillium spp. from citrus (Eckert et al., 1994;
GS48 SEN 47.3 31.9 Kanetis et al., 2008). Interestingly, in the current study, the existence
Group mean 46.5 cd 25.6 b
F11 ANIR 45.6 28
of P. expansum isolates showing simultaneous resistance to DMIs and
F16 ANIR 43.6 26.6 to fungicides belonging to other chemical groups, such as iprodione
F20 ANIR 38.0 25.7 and fludioxonil, has been revealed. Isolates exhibiting single resis-
RD1 ANIR 33.6 31.3 tance to DMI fungicides accounted for only 9% of the population, while
RD49 ANIR 43.5 33.5
isolates showing double or triple resistance to DMIs and/or anilino-
RD60 ANIR 45.0 29.8
RD77 ANIR 45.5 31.3 pyrimidines, dicarboximides and phenylpyrroles accounted for 32% of
RD83 ANIR 45.5 25.7 the population. The absence of precise base-line sensitivity data for
RD86 ANIR 48.6 25.5 tebuconazole and fludioxonil in the sampling region do not allow
RD118 ANIR 46.4 30.3 direct comparisons. However, in a recent study carried out in
RD120 ANIR 45.0 26.7
GD5 ANIR 47.3 26.3
Washington State to determine the baseline sensitivity of P. expansum
GD20 ANIR 45.0 28 isolates from apple the EC50 values to fludioxonil were reported to
GD36 ANIR 45.1 30.8 range from 0.011 to 0.068 μg ml− 1 (Li and Xiao, 2008a). In our dataset
GD41 ANIR 44.5 27.1 there were several isolates with EC50 values significantly higher than
GS8 ANIR 46.8 31.3
the higher value of 0.068 observed by Li and Xiao (2008a).
GS15 ANIR 46.2 30.1
GS24 ANIR 43.3 27.9 Certainly, it is well accepted that the most important biochemical
GS29 ANIR 45.0 22 mechanism causing resistance of fungal pathogens to fungicides in
GS35 ANIR 44.6 31.4 practice results from a modification at the target-site of fungicidal
GS38 ANIR 46.5 27.5 action (Brent and Hollomon, 2007). However, it is difficult to envisage
Group mean 44.1 bc 28.0 b
that change in a single target site could provide a reasonable
RD22 DMIR 49.08 2.9
GS20 DMIR 48.3 30.4 explanation for the multi-drug resistance found in the present work.
Group mean 48.5 c 16.0 ab The reduction of sensitivity to fungicides affecting different cellular
RD92 DMIDICR 46.0 2.8 pathways could be explained only by a mechanism leading to a
RD96 DMIDICR 28.5 11.1
decreased accumulation of the inhibitors into the fungal mycelium by
GS7 DMIDICR 33.4 9.2
GS19 DMIDICR 48.0 28 the function of an efficient efflux pump, or by an increased binding of
Group mean 38.7 b 12.5 a the compounds in the cell wall or by degradation of the fungicides to
RD12 DMIANIR 46.0 28.6 non-fungitoxic compounds. In the recent past multi drug resistance
F3 DMIANIR 47.4 28.5 has been reported in the closely related species P. digitatum from
Group mean 46.5 c 28.0 b
citrus fruit, exhibiting azole-resistance, while simultaneously the
RD100 DMIPHENR 27.7 10.9
GD11 DMIPHENR 25.5 8.5 isolates were also cross-resistant to unrelated chemicals (Nakaune et al.,
GS49 DMIPHENR 28.3 9.3 1998, 2002). The isolates showed an up-regulation of gene expression
Group mean 26.6 a 9.0 a of ABC transporter genes.
F5 DMIPHENDICR 29.5 12.4
The development and evolution of fungicide resistance in fungal
RD3 DMIPHENDICR 28.1 7.4
Group mean 28.5 a 9.5 a populations are largely dependent on the dynamics of competition
a
between fungicide resistant and sensitive strains which is affected by
SEN: sensitive isolates, ANIR: cyprodinil-insensitive isolates, DMIR: tebuconazole-
resistant isolates, PHENR: fludioxonil-resistant isolates, and DICR: iprodione-resistant
the fitness of the strains, and this has important implications on
isolates. disease management (Parnell et al., 2005; Peever and Milgroom,
b
Mean colony diameter (mm) measurements after 7 days incubation on PDA. 1995). Fitness can be defined as the survival and reproductive success
c
Pathogenicity in terms of lesion diameter (mm) on artificially inoculated apple of an allele, individual, or group (Pringle and Taylor, 2002). The
fruits.
d development and the evolution of fungicide resistance would be
Within columns, values followed by the same letter do not differ significantly
according to Kruskal–Wallis and Mann–Whitney non parametric tests (P = 0.05). lessened if a resistant subpopulation had lower parasitic or sapro-
phytic fitness. In contrast, absence of fitness costs in the resistant
fraction of the population would lead to a stable resistance frequency
reduced sensitivity to fungicides used for their control. The fungal in the absence of fungicide selection force or to rapid development
pathogen is exposed to fungicide selection pressure from treatments and evolution of resistance under the fungicide selection force. The
targeting the control of blue mold and also heavy exposure to results of the study have shown that fungal isolates exhibiting
fungicide treatments targeting to the control of apple scab caused by simultaneously reduced sensitivity to DMI and/or dicarboximide and
V. inaequalis. The results of the study showed that phenotypes with phenylpyrrole fungicides suffer a high fitness penalty suggesting that
reduced sensitivity to cyprodinil were the most common among the in the absence of fungicide selection pressure the frequency of these
fungal population. Anilinopyrimidine fungicides were registered for isolates may decrease due to their inability to compete equally well
use in apple orchards 12 years ago and there are usually 2–3 spray with the sensitive strains. In contrast, isolates exhibiting single
applications per season. In the past, decreased sensitivity to the resistance to the anilinopyrimidine fungicide cyprodinil and the
anilinopyrimidine fungicide pyrimethanil has been reported for one DMI fungicide tebuconazole or double resistance to these two
P. expansum isolate in Canada (Sholberg et al., 2005a), however fungicides did not differ in fitness parameters from the sensitive
resistance to this fungicide group has been reported in other isolates. Such results provide evidence for the prediction made by Li
pathogens such as B. cinerea (Moyano et al., 2004; Myresiotis et al., and Xiao (2008b) that anilinopyrimidine fungicides possess a higher
2007). Sensitivity data of P. expansum isolates to cyprodinil, to the best risk for resistance development in P. expansum. They made this
 G.S. Karaoglanidis et al. / International Journal of Food Microbiology 145 (2011) 195–204 201

Fig. 3. Correlation between mycelial growth and level of sensitivity (in terms of EC50 values) to tebuconazole, cyprodinil, fludioxonil and iprodione of P. expansum isolates from apple.

Fig. 4. Correlation between pathogenicity (in terms of lesion diameter in mm) and level of sensitivity (in terms of EC50 values) to tebuconazole, cyprodinil, fludioxonil and iprodione
of P. expansum field isolates from apple.
202 G.S. Karaoglanidis et al. / International Journal of Food Microbiology 145 (2011) 195–204

Fig. 5. Patulin production by sensitive and resistant phenotypes of P. expansum on YES agar growth medium and on artificially inoculated apple fruit. Measurements were made after
10 days incubation at 20 °C. Columns with different letters indicate a significant difference according to Dunnett's multiple range test at P = 0.05.

Fig. 6. Correlation between patulin production and level of sensitivity (in terms of EC50 values) to tebuconazole, cyprodinil, fludioxonil and iprodione of P. expansum field isolates from apple.
 G.S. Karaoglanidis et al. / International Journal of Food Microbiology 145 (2011) 195–204
prediction based on the findings of their study showing that
laboratory induced fludioxonil resistant P. expansum isolates had
lower fitness compared to the sensitive isolates while laboratory-
induced pyrimethanil resistance had no effect on the fitness of the
mutant isolates. With the exception of the Li and Xiao (2008b) report
there is no any other published report on the fitness of anilinopyr-
imidine and fludioxonil-resistant P. expansum strains. The absence of a
fitness cost associated with resistance to anilinopyrimidines has also
been reported previously in other pathogens such as B. cinerea
(Bardas et al., 2008). Results on the fitness of DMI-resistant Penicillium
spp. species are rather contradictory. Morales et al. (2008a) found that
imazalil-resistant isolates of P. expansum had no fitness penalty
compared to the sensitive isolates, while Kinay et al. (2007) reported
that DMI-resistant isolates of Penicillium digitatum suffered a fitness
penalty compared to the sensitive isolates. Similarly fitness penalties
associated with resistance to phenylpyrrole fungicides have been
observed in B. cinerea (Ziogas et al., 2005) and Aspergillus parasiticus
(Markoglou et al., 2008a), while variable were the results obtained by
Kanetis et al. (2006) in P. digitatum.
The concern over the use of fungicides and in particular the possible
effect on mycotoxigenic fungal species and mycotoxin production, has
been highlighted by the European Commission Scientific Committee in
Plants (Hardy et al., 1999). Studies with laboratory and field isolates
have shown that the risk for resistance to fungicides also exists in
mycotoxin-producing fungi, and today, there is strong evidence that the
extensively and inappropriate use of fungicides in crop protection may
lead to reduced sensitivity of certain mycotoxin-producing fungi, and
thus to an increased contamination of agricultural products with
mycotoxins. Previous work by D'Mello et al. (1998) showed that
resistance to the DMI fungicide difenoconazole induced increased
persistence of the trichothecene mycotoxin 3-acetyl deoxynivalenol (3-
ADON) by Fusarium culmorum. A higher mycotoxin production (T-2
toxin, 4,15-diacetoxyscirpenol, and neosolaniol) has also been found in
a carbendazim resistant strain of Fusarium sporotrichioides (D'Mello et
al., 2000). More recently, Zhang et al. (2009) found that benzimidazole-
resistance increased the trichothecene production of Fusarium grami-
nearum. Our previous studies with laboratory mutant strains of A.
parasiticus resistant to phenylpyrrole and dicarboximide fungicides
showed that most aflatoxigenic isolates produced aflatoxins at
concentrations significantly higher than the parental wild-type strain
(Markoglou et al., 2008a). Similarly, laboratory mutant strains of A.
parasiticus, Aspergillus ochraceus and Fusarium verticillioides resistant to
the triazoles epoxiconazole and flusilazole and mutant strains of
Aspergillus carbonarius and P. expansum resistant to the phenylpyrrole
fludioxonil produced significantly higher levels of mycotoxins (ochra-
toxins, patulin and fumonisins) compared to the parental sensitive
strains (Doukas et al., 2008; Markoglou et al., 2008b, 2009). In the
present work a significant reduction in the patulin production was
found in most isolates with reduced sensitivity to tebuconazole,
fludioxonil and iprodione, probably indicating that fitness costs are
negatively correlated with the mycotoxigenic ability of P. expansum.
However, most cyprodinil-insensitive isolates produced patulin at
concentrations higher than the sensitive ones.
Our results, as well as data from laboratory and field experiments
conducted in other countries, along with the high potential of P. expansum
for quick adaptation to changes in environmental conditions, indicate
a considerable resistance risk to phenlypyrroles, dicarboximides, triazoles
and anilinopyrimidines. The reduction in pathogenicity and other fitness
characteristics of fludioxonil, iprodione and tebuconazole-resistant iso-
lates indicate that the risk for practical resistance problems may be low.
However, in the case of anilinopyrimidines, although appropriate anti-
resistance strategies already exist (limitation in the number of applica-
tions and use in mixtures with other fungicides), the commercial use of
these compounds requires careful implementation to maintain their
effectiveness and to preserve the quality and safety of the pome fruit
and their by-products.
203
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