International Journal of Food Microbiology 197 (2015) 22–29

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International Journal of Food Microbiology

journal homepage: www.elsevier.com/locate/ijfoodmicro

Identification, characterization and mycotoxigenic ability of Alternaria

spp. causing core rot of apple fruit in Greece
Panagiota Ntasiou a, Charalampos Myresiotis b, Sotiris Konstantinou a,
Euphemia Papadopoulou-Mourkidou b, George S. Karaoglanidis a,⁎
a
Plant Pathology Laboratory, Faculty of Agriculture, Forestry and Natural Environment, Aristotelian University of Thessaloniki, POB 269, 54124 Thessaloniki, Greece
b
Pesticide Science Laboratory, Faculty of Agriculture, Forestry and Natural Environment, Aristotelian University of Thessaloniki, POB 269, 54124 Thessaloniki, Greece

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

Article history: Alternaria core rot is a major postharvest disease of apple fruit in several countries of the world, including Greece.
Received 1 August 2014 The study was conducted aiming to identify the disease causal agents at species level, investigate the aggressive-
Received in revised form 26 November 2014 ness of Alternaria spp. isolates and the susceptibility of different apple varieties and determine the mycotoxigenic
Accepted 9 December 2014
potential of Alternaria spp. isolates from apple fruit. Seventy-five Alternaria spp. isolates obtained from apple fruit
Available online 17 December 2014
showing core rot symptoms were identified as either Alternaria tenuissima or Alternaria arborescens at frequen-
Chemical compounds studied in this article::
cies of 89.3 and 11.7%, respectively, based on the sequence of endopolygalacturonase (EndoPG) gene. Artificial in-
Alternariol (PubChem CID: 5359485) oculations of fruit of 4 different varieties (Fuji, Golden Delicious, Granny Smith and Red Delicious) and incubation
Alternariol monomethyl-ether (PubChem CID: at two different temperatures (2 and 25 °C) showed that fruit of Fuji variety were the most susceptible and fruit of
5360741) Golden Delicious the most resistant to both pathogens. In addition, the production of 3 mycotoxins, alternariol
Tentoxin (PubChem CID: 5281143) (AOH), alternariol monomethyl ether (AME) and tentoxin (TEN) was investigated in 30 isolates of both species.
Mycotoxin determination was conducted both in vitro, on artificial nutrient medium and in vivo on artificially in-
Keywords: oculated apple fruit, using a high performance liquid chromatography with diode array detector (HPLC-DAD).
Alternaria tenuissima
The results showed that most of the isolates of both species were able to produce all the 3 metabolites both
A. arborescens
in vivo and in vitro. On apple fruit A. tenuissima isolates produced more AOH than A. arborescens isolates, whereas
Alternariol
Alternariol monomethyl ether the latter produced more TEN than the former. Such results indicate that Alternaria core rot represents a major
Endopolygalacturonase threat of apple fruit production not only due to quantitative yield losses but also for qualitative deterioration of
Tentoxin apple by-products.
© 2014 Elsevier B.V. All rights reserved.

1. Introduction that results in the appearance of the core rot. MC is considered of

Core rot of apple fruit is one of the most important postharvest
diseases of apple and is defined as a rot initiating from the loculus that
spreads into the fruit mesoderm and leads to either a dry core rot
(DCR) or wet core rot (WCR). Fruit affected by DCR are characterized
by dark brown, dry and corky tissues. DCR develops slowly and the rot
is restricted in the loculus and the mesoderm around the loculus
(Shtienberg, 2012). WCR is also characterized by dark brown tissues,
however the disease progresses more rapidly and deeply into the meso-
derm. External symptoms are not evident until harvest but during stor-
age the rot progresses and after several months, the rot may also be
evident on the fruit epidermis. However, in most cases the disease is un-
noticed until the fruit is cut open (Shtienberg, 2012). In addition to DCR
and WCR, moldy core (MC) can be another type of infection of apple
fruit loculus. It is associated with the development of fungal mycelia
within the loculus, without invasive penetration into the mesoderm
⁎ Corresponding author.
E-mail address: gkarao@agro.auth.gr (G.S. Karaoglanidis).
minor economic importance since it does not affect the fruit qualitative
characteristics (Serdani et al., 2002). Infection of the core occurs after
the establishment of the disease causal agents on the senescing parts
of the blossom and the penetration through the sinus after fruit setting
or later stages of fruit development (Combrink et al., 1984; Reuveni
et al., 2002). Yield losses due to core rot have been estimated to 3–10%
in Israel and 5–8% in South Africa (Combrink et al., 1984; Niem et al.,
2007).
The disease is caused by a complex of fungi such as Fusarium spp.,
Ulocladium spp., Cladosporium spp., Coniothyrium spp., Pezicula spp.,
Mucor spp., and Alternaria spp., with the latter being predominant
(Combrink et al., 1985b; Gao et al., 2013; Michailides et al., 1994;
Spotts, 1990; Tournas and Uppal Memon, 2009). Early studies on the
etiology of Alternaria core rot of apple fruit suggested Alternaria
alternata as the causal agent of the disease (Combrink et al., 1984; Ellis
and Barrat, 1983; Reuveni et al., 2002; Spotts, 1990). In these studies
identification was made based only on the morphological characteris-
tics of the isolates' cultures and conidia. But it is evident that morpho-
logical data are insufficient for taxonomic differentiation due to

http://dx.doi.org/10.1016/j.ijfoodmicro.2014.12.008
0168-1605/© 2014 Elsevier B.V. All rights reserved.
 P. Ntasiou et al. / International Journal of Food Microbiology 197 (2015) 22–29
overlapping morphological characters between A. alternata and other
small-spored Alternaria spp. (Pryor and Michailides, 2002; Simmons,
1992). As new molecular tools have been employed in identification
and characterization of Alternaria spp., more recent studies have
shown that several Alternaria species such as Alternaria tenuissima,
Alternaria infectoria and Alternaria arborescens may be the causal agents
of the disease (Gao et al., 2013; Kang et al., 2002; Kou et al., 2014;
Serdani et al., 2002).
Alternaria spp. are potential producers of more than 30 secondary
metabolites, mostly host-specific or non-specific phytotoxins that play
a crucial role in the development of diseases caused by the pathogens.
In addition, most of the Alternaria species are able to produce myco-
toxins with biological activity against several organisms including
mammals. Among these metabolites alternariol (AOH), alternariol
methyl ether (AME), altenuene (ALT), tentoxin (TEN) and tenuazonic
acid (TeA) are included (Logrieco et al., 2003). Although the acute tox-
icity of Alternaria mycotoxins is considered to be low in mammals,
there is strong evidence that they may be mutagenic and carcinogenic
(Logrieco et al., 2009; Ostry, 2008). Alternaria mycotoxins have been
detected in several foods such as citrus fruit, tomato, tomato products,
olives, pepper and wheat (Ostry, 2008). In addition, several Alternaria
mycotoxins have been detected in apple fruit infected by Alternaria
spp. or in apple juice concentrates (Andersen et al., 2006; Broggi et al.,
2013; Delgado et al., 1998; Prelle et al., 2013; Robiglio and Lopez,
1995; Scott and Kanhere, 2001).
In a recent study aiming to identify the causal agents of postharvest
rots of apple fruit in Greece it was found that Alternaria spp. was the
third most common pathogen after Penicillium expansum and Botrytis
cinerea, whereas in variety Fuji it was found to be the most common
pathogen (Konstantinou et al., 2011). Taking into account the increasing
significance of this disease, further research was initiated aiming to:
i) identify the causal agents of the disease at species level and character-
ize them molecularly, ii) measure variety susceptibility and aggressive-
ness of Alternaria spp. under different storage temperatures and
iii) investigate the ability of Alternaria spp. from apple fruit to produce
some common Alternaria toxins both in vitro and in vivo on artificially
inoculated apple fruit.
2. Materials and methods
2.1. Pathogen isolates
Isolates of Alternaria spp. were collected from apple fruit (cv. Golden
Delicious, Red Delicious, Fuji and Granny Smith) showing core rot
symptoms. The fruits were obtained from packinghouses located in
the region of Imathia, northern Greece, during the 2012–13 storage pe-
riod. Rotted fruit were transferred to the laboratory for isolation of the
decay agents. Isolations were carried out from surface-disinfected fruit
(they were drenched for 1 min in a 1% sodium hypoclorite solution)
by removing small fruit pieces at the margin of diseased/healthy tissue
and transferring them to Petri dishes containing acidified Potato
Dextrose Agar (Merck, Darmstadt, Germany). Petri dishes were
incubated at 22 °C in an incubator with a 12-h photoperiod provided
by fluorescent lights. After 3 to 5 days of incubation, the emerging
putative Alternaria spp. colonies were transferred to fresh PDA medium
and incubated under the above mentioned conditions for 1 additional
week to induce sporulation. In total 75 single-spore isolates of Alternaria
spp. were obtained (31, 7, 27 and 10 isolates from Fuji, Red Delicious,
Golden Delicious and Granny Smith varieties, respectively) and stored
at 4 °C until use.
2.2. DNA isolation
To extract DNA the isolates were grown in Potato Dextrose Broth
(Sigma-Aldrich, St. Louis, MO) for 5 days at 25 °C. Then, the mycelium
was harvested by filtration, dried, lyophilized, ground to a fine powder
23
using micropestles (Eppendorf International, Wesseling, Germany)
and stored at −20 °C until use. DNA was extracted using QIA Puregene
Core Kit A (Qiagen GmbH, Hilden, Germany) according to the
manufacturer's protocol. The concentration of the extracted DNA was
measured using a P330 nanophotometer (Implen GmbH, Munich,
Germany).
2.3. Species identification and phylogenetic analysis
The endopolygalacturonase (endoPG) gene, has shown potential to
delineate the closely related species within the A. alternata-complex
and was used for pathogen identification and phylogenetic analysis
(Hong et al., 2005; Andrew et al., 2009). Amplification of endoPG gene
was conducted using primers PG3 (5′-TACCATGGTTCTTTCCGA-3′) and
PG2b (5′-GAGAATTCRCARTCRTCYTGRTT-3′) (Andrew et al., 2009). Re-
action mixtures contained 3 μl genomic DNA (50 ng), 0.32 μM of PG3
primer, 1.12 μM of PG2b primer, 0.2 mM dNTPs, 2 mM MgCl2, 1× PCR
buffer [200 mM Tris–HCl (pH 8.4), 500 mM KCl], and 0.04 units Taq
DNA Polymerase and the total volume was adjusted to 25 μl with sterile
highly purified H2O. PCR amplification was conducted using the follow-
ing conditions: an initial denaturation at 94 °C for 3 min, followed by 40
cycles at 95 °C for 30 s, 53 °C for 30 s, 72 °C for 1 min and a final exten-
sion at 72 °C for 10 min. All the PCR reactions were performed in a
LabCycler thermal cycler (SensoQuest GmbH, Gottingen, Germany).
PCR products of each isolate were purified using the Qiaquick PCR
Purification Kit (Qiagen GmbH, Hilden, Germany). The purified products
were subjected to sequencing in both directions using the forward and
the reverse primers. Sequences were aligned using the computer soft-
ware package Mega 5.05. The sequences obtained were compared
with sequences in the National Center for Biotechnology Information
database using BlastN 2.2.18.
Phylogenetic analysis of EndoPG. Sequences were trimmed to 448 bp
to eliminate ambiguous sites and aligned using MAFFT version 6 (Katoh
and Toh, 2008). Models of sequence evolution were tested for each
alignment and model parameter estimates obtained for each alignment
using jModelTest version 0.1 (Posada, 2008). The K80 model was
selected for the EndoPG data with equal base frequencies, a transition/
transversion ratio of 3.05, and equal substitution rates among sites.
The rooted EndoPG phylogenies were estimated using maximum likeli-
hood with the program PhyML version 3.0 (Guindon and Gascuel,
2003). Alternaria gaisen (GenBank AY295033) was used to root the
phylogenetic tree.
2.4. Morphological characterization
For all 75 isolates used in the study the morphological characteristics
of colony and sporulation apparatus were determined using criteria as
described by Pryor and Michailides (2002). To determine the colony
characteristics the isolates were grown on PDA and incubated at 22 °C
in the darkness for 10 days. At the end of the incubation period the
colony diameter, colony color, colony texture, colony margin and the
presence of crystals in agar medium were examined. Colony diameter
was measured in mm, while the remaining characteristics were
assessed visually. For each isolate 3 replicate PDA plates were prepared
and diameter values were averaged.
Characterization of sporulation habit was conducted on weak (0.05)
PDA (3 replicate plates per isolate). The cultures were incubated for
7 days at 22 °C under cool fluorescent light (60 μmoles/m2/s, 10:14 h
light/dark cycle). At the end of the incubation period the sporulation ap-
paratus was examined using a stereomicroscope at 40× magnification.
Conidial size was measured in a ZEISS AxioImager. Z2 microscope
using a digital camera (AxioCam MRc 5). The size of 40 conidia per
isolate was measured. Eight Alternaria spp. isolates (two of each
A. alternata, A. infectoria, A. tenuissima and A. arborescens) were kindly
provided by Dr T. J. Michailides (Kearney Agricultural Research and Ex-
tension Center, University of California) and used as reference isolates.
24 P. Ntasiou et al. / International Journal of Food Microbiology 197 (2015) 22–29
2.5. Apple fruit variety susceptibility
Ten isolates of A. tenuissima and A. arborescens (5 isolates per
species) were used to assess the susceptibility of apple fruit varieties
to core rot. For conidial production the isolates were cultured on PDA
for 10 days under alternating light/dark conditions (10/14 h) at 25 °C.
Subsequently, the cultures were flooded with 5 ml of sterile distilled
water and the conidia were scraped off with a surgical blade. The
resulting conidial suspension was filtered through two layers of cheese-
cloth to remove mycelia fragments and adjusted at a concentration
of 2 × 105 spores/ml. Prior to inoculation, apple fruit (cv. Fuji,
Red Delicious, Golden Delicious and Granny Smith) were surface-
disinfected for 5 min by drenching them in a 1% NaOCl solution. The
fruit were artificially inoculated by aseptically injecting 5 ml of a conid-
ial suspension through the calyx into the fruit core with a syringe. Forty
fruit per isolate/variety combination were inoculated. The fruit were
placed on wire mesh platforms (10 fruit per box) in plastic boxes
(23 × 31 × 10 cm [length × width × height]). Twenty ml of water was
added to each box and the boxes covered to maintain high relative
humidity. Twenty of the inoculated fruit per isolate were incubated
for 21 days at 25 °C and the remaining 20 fruit were incubated for
60 days at 2 °C. At the end of the incubation period the fruit were
cut in the middle and the infections were scored by measuring lesion
diameter around the fruit core. The experiment was conducted twice.
2.6. Chemicals and reagents for mycotoxin analysis
Acetonitrile, water and ethyl acetate, methanol (all of HPLC grade)
were obtained from Merck (Darmstadt, Germany) and Chem-Lab
(Zedelgem, Belgium), respectively. Formic acid (purity 99%) was
purchased from Carlo Erba (Milano, Italy) and trifluoroacetic acid
(purity ≥ 99%) was obtained from Sigma-Aldrich (Steinheim,
Germany). Analytical standards of the Alternaria mycotoxins alternariol
(AOH, 98.5%), alternariol monomethyl ether (AME, 99.2%) and tentoxin
(TEN, 99.2%) were obtained from Sigma-Aldrich (Steinheim, Germany).
Individual stock standard solutions (100 μg/ml) of each toxin were pre-
pared in methanol and stored in darkness at −20 °C. Working standard
solutions at various concentrations were made by diluting with the
appropriate volume of methanol for the preparation of fortified samples
and for the construction of calibration curves. All working standard
solutions were stored at 4–5 °C.
2.7. Extraction procedure
Mycotoxin determination was conducted both in vitro and in vivo in
23 randomly selected A. tenuissima isolates and in all (n = 7)
A. arborescens isolates. The extraction protocol of Alternaria mycotoxins
was performed using the method described by Andersen et al. (2006),
slightly modified. For determination of mycotoxin production in vitro
the isolates were cultured in 9 cm Petri dishes (3 replicate plates per iso-
late) containing Dichloran Rose Bengal Yeast Extract (DRYES) agar and
incubated for 14 days at 25 °C, in the dark. At the end of the incubation
period 5 mm (inner diameter) mycelial agar plugs were cut from the
middle, mean and edge of each colony and were placed in a 5 ml
screw-cap vial. The plugs were extracted with 2 ml ethyl acetate con-
taining 1% formic acid by sonication for 60 min. The extracts were trans-
ferred into 10 ml glass tubes and concentrated to dryness by use of
nitrogen stream at 30 °C. The extracts were re-dissolved ultrasonically
in 0.5 ml methanol, filtered through 0.45 μm PTFE filters (Millipore, Bed-
ford, MA, USA) and transferred to autosampler vials for instrumental
analysis in HPLC system.
For the determination of mycotoxin production in vivo apple fruit
(cv. Fuji) were artificially inoculated with the same 30 Alternaria spp.
isolates following the artificial inoculation protocol described previously.
In total 5 fruit were inoculated per isolate and the fruit were incubated at
25 °C for 20 days. After the end of the incubation period the inoculated
fruit were cut into half and 1 g of apple tissue was removed from the
rotten fruit core. Then the tissue was placed in a 5 ml screw-cap vial
and extracted twice with 1 ml ethyl acetate (1% formic acid) each for
60 min in an ultrasonic bath. The final extracts were transferred to
clean glass tubes and evaporated to dryness under a stream of nitrogen.
The residues were reconstituted in 0.5 ml of methanol by sonication and
filtered through 0.45 μm PTFE filters prior to HPLC analysis
2.8. Instrumental analysis
Analyses of mycotoxins were carried out using a SpectraSYSTEM
high performance liquid chromatograph (Thermo Separation Products,
Austin, TX, USA). HPLC system consisting of a P4000 tertiary solvent
pump, a vacuum degasser TSP, an AS3000 autosampler equipped with
a 100-μl injection loop and a UV6000LP diode array detector. Chromato-
graphic separation was done on a Hypersil BDS-C18 (Thermo Finnigan,
USA) cartridge column (250 × 4.6 mm, 5 μm) with a 10 × 4 mm (inner
diameter) Hypersil BDS-C18 guard column (Thermo Finnigan, USA).
The mobile phase consisted of a linear gradient starting at 90% water
and 10% acetonitrile, reaching 50% acetonitrile after 25 min and 100%
acetonitrile after 30 min. 100% acetonitrile was maintained for 1 min.
Thereafter the gradient was returned to 10% acetonitrile in 1 min
and allowed to equilibrate for 3 min before the next analysis. Both
eluents contained 50 μl trifluoroacetic acid per liter. The flow rate was
1 ml/min, the column temperature was 40 °C and the injection volume
was 20 μl.
2.9. Method validation
The developed analytical method was evaluated for accuracy (as re-
coveries) and precision (as repeatability) by analyzing uncontaminated
blank samples (agar and apple tissue), with no presence of Alternaria
mycotoxins, fortified with known amount of mixed working standard
solutions. This procedure was done at five different concentration levels
(0.05, 0.2, 0.5, 1, 10 μg/g DRYES Agar and 0.05, 0.1, 0.5, 1, 2 μg/g apple
tissue) in triplicate. The linearity of the detector response to the analyte
was evaluated based on the correlation coefficient (r) values. The limits
of detection (LOD) and quantification (LOQ) of each method for the
three mycotoxins were determined. The LOD was calculated as the min-
imum concentration giving a response 3 times greater than the baseline
noise (S/N ≥ 3) defined from the analysis of blank (unspiked) samples.
The LOQ was defined as the lowest concentration of the toxin in which
the signal to noise ratio (S/N) was equal to 10.
2.10. Data analysis
Data from the two independent trials on core rot diameter caused by
the isolates of the two pathogens on the fruit of the tested varieties,
were tested for homogeneity of variance using the Levene's test. In
these analyses, data of the two independent trials were considered
block treatments and the replications within each trial were used as
subsamples. The data of the two independent trials fulfilled the criteria
for homogeneity of variance (P N 0.05) and therefore they were com-
bined. Mean values of core rot diameter on the different varieties caused
by each fungal species were compared using Fisher's Protected LSD Test
at P = 0.05. The pairwise Student's t-test was used to compare mean
concentrations of the 3 mycotoxins produced by A. tenuissima and
A. arborescens as isolate groups. Statistical analyses were conducted
using SPSS 17.0 (SPSS, Chicago, IL).
3. Results
3.1. Alternaria spp. identification
Amplification of the EndoPG gene produced a fragment of 448 bp,
from all the 75 Alternaria spp. isolates tested. Sequence analysis and
 P. Ntasiou et al. / International Journal of Food Microbiology 197 (2015) 22–29
Alternaria tenuissima
100
90
80
Frequency (%)
70
60
50
40
30
20
10
0
Fuji
Fig. 1. Frequency of Alternaria tenuissima (n = 67) and A. arborescens (n = 8) in 4 different varieties of apple with core rot symptoms.
comparison with already existing sequences in NCBI revealed that
67 out of 75 isolates were A. tenuissima (GenBank Accession numbers
LK054418–LK054484), while the remaining 8 were identified as
A. arborescens (GenBank Accession numbers LK054410–LK054417).
A. tenuissima was found in higher frequency in all the 4 varieties
sampled, while A. arborescens was not identified in isolates obtained
from Granny Smith fruit (Fig. 1). The analysis of the EndoPG sequences
resulted in a phylogenetic tree composed of only two major clades
(Fig. 2). All the A. tenuissima isolates were grouped together in one
clade and A. arborescens isolates were grouped in the second clade
which was subdivided into two further clades (Fig. 2).
3.2. Morphological characterization
The 67 isolates identified as A. tenuissima based on the endoPG se-
quence produced colonies of olive gray color with a thin (1–2 mm)
white margin. Most of the isolates produced white crystals in the
growth medium under the fungal mycelium, while the appearance of
all the 67 colonies was cottony. Mean colony diameter of A. tenuissima
isolates ranged from 45 to 70 mm. Sporulation apparatus of
A. tenuissima isolates produced single conidial chains with 7–12 conidia,
while secondary branching with 1–3 conidia was observed in only a lim-
ited number of isolates. Mean conidial length ranged from 11 to 26 μm.
The 8 isolates identified as A. arborescens produced colonies with a
dark olive gray color. The colonies were wavy at the margins with a
white edge of 1–2 mm in width. Crystals in the growth medium were
abundant and the appearance of the colony was cottony to wooly in
all the 8 isolates tested. Mycelial growth of A. arborescens isolates after
10 days of incubation ranged from 40 to 65 mm. Sporulation pattern
in A. arborescens isolates was characterized by the presence of conidia
chain branching. The main chain was 2 to 7 conidia in length while
there were abundant secondary and tertiary branches with 2 to 4 co-
nidia. Mean conidial length was similar to that of A. tenuissima with a
length ranging from 11 to 26 μm. Data on the morphological character-
istics of both A. tenuissima and A. arborescens isolates are summarized in
Table 1.
3.3. Apple fruit variety susceptibility
All the isolates used in the study were found to be pathogenic on
fruit of all the tested varieties. Measurements of lesion diameter on
the inoculated fruit showed that Fuji was the most susceptible to both
Alternaria species at both incubation temperatures, whereas fruit
of Golden Delicious were the most resistant at both temperatures
25
Alternaria arborescens
Red Delicious Granny Smith Golden Delicious
Variety
(Fig. 3A and B). Core rot diameter in fruits of Granny Smith and Red
Delicious was significantly lower (P b 0.05) to that of Fuji at both
incubation temperatures (Fig. 3A and B).
3.4. Validity of mycotoxin detection analytical method
Mean recovery values obtained for AOH, AME and TEN were 105,
103, 109% in PDA and 101, 96, 99% in apple samples and respective rel-
ative standard deviation values (RSDs %) of all % mean recovery values
were b 7% and b 8%. The calibration curves were linear with correlation
coefficients (r2) values N 0.9876 for AOH, N0.9874 for AME and
N0.9989 for TEN in the concentration range studied. The maximum ab-
sorbance, resulted in the best sensitivity of the target compounds, were
256 nm (AOH, AME) and 281 nm (TEN), and exhibited retention times
of 20.4 (AOH), 22.4 (TEN) and 28.4 min (AME) (Fig. 4). The LOQ and
LOD levels for all mycotoxins for both substrates (artificial nutrient me-
dium and apple fruit) were set at 0.05 μg/g and 0.02 μg/g, respectively.
3.5. Mycotoxin production
All the A. tenuissima and A. arborescens isolates included in the study
were found to produce in vitro AOH and AME. AOH was produced by
A. tenuissima and A. arborescens isolates at a range of 2.26 to 454.17 and
42.86 to 463.11 μg/g, respectively. Similarly, A. tenuissima isolates produced
AME at a range of 0.43 to 260.25 μg/g, and A. arborescens isolates at a range
of 38.75 to 216.33 μg/g (Table 2). Tentoxin was produced in vitro by most
isolates of both fungal species with an exception of 4 A. tenuissima isolates
and 1 A. arborescens isolate. No difference (P N 0.05) was observed in mean
concentrations detected for all the 3 mycotoxins analyzed, between
A. tenuissima and A. arborescens, in vitro (Table 2).
Analysis conducted on apple fruit showed that all the 3 mycotoxins
were produced in lower concentrations. AOH was produced by all but 2
A. tenuissima and by all but 2 A. arborescens isolates, at concentrations
ranging from 0.05 to 8.35 and 0.24 to 1.22 μg/g, respectively (Table 2).
Similarly AME was produced in vivo by all but 5 A. tenuissima and by
all but 2 A. arborescens isolates, at concentrations ranging from 0.02
to 2.76 and 0.11 to 1.49 μg/g, respectively (Table 2). In contrast, TEN
was produced on apple fruit by only 13 out of 23 and by 3 out of 7
A. tenuissima and A. arborescens isolates, respectively (Table 2). As a
mean, A. tenuissima isolates produced significantly higher concentra-
tions (P b 0.05) of AOH than A. arborescens, while the latter produced
significantly higher (P b 0.05) concentrations of TEN than the former
isolates (Table 2). No difference (P N 0.05) was observed between the
two species in AME production (Table 2).
26 P. Ntasiou et al. / International Journal of Food Microbiology 197 (2015) 22–29

2 Alternaria tenuissima Alternaria arborescens

a
1,8
Α
1,6
A b
1,4
1,2
B
1 c
B
0,8 c
0,6
C
0,4

Rot Diameter (cm)

0,2
0
Fuji Granny Golden Red
3 Smith Delicious Delicious
a
2,5 A Β
b
B
2 c
d C
1,5 C

0,5

0
Fuji Granny Golden Red
Smith Delicious Delicious

Apple Variety

Fig. 3. Mean diameter (cm) of core rot caused by Alternaria tenuissima and Alternaria
arborescens isolates, on apple fruit of different varieties stored at 2 °C for 60 days
(A) and at 25 °C for 20 days (B). Different letters on the columns indicate significant
differences among the different varieties for each fungal species according to a t-test for
independent samples at P = 0.05 for the core rot diameter data. Bars on the columns
indicate the standard error of the mean.

Fig. 2. Maximum likelihood phylogenetic tree using the K80 parameter model of the
sequences of the ΕndoPG genes of 75 Alternaria spp. isolates from apple fruit with core rot
symptoms. Clade 1 consists of A. tenuissima isolates and clade 2 of A. arborescens isolates.
Alternaria gaisen (GenBank accession num. AY295033) was used to root the phylogeny.
4. Discussion
The present study represents a first attempt in characterizing
Alternaria spp. isolates associated with core rot of apple fruit in
Greece. The results of the study showed that the disease is caused by
two distinct species, A. tenuissima and A. arborescens, with the former
being predominant in all apple varieties sampled. In the past Alternaria
core rot of apple fruit has been linked to A. alternata based on the
morphological characteristics of the conidia and the sporulation appara-
tus of the isolates. However, controversies and confusion have often ac-
companied the classification of small-spored species sharing common
morphological characteristics and overlapping conidial sizes (Serdani
et al., 2002). Moreover, molecular phylogenies of the A. alternata
species-group revealed little to no variation in several genetic loci that
are commonly used in fungal systematics such as nuclear ribosomal in-
ternal transcribed spacer (ITS), translation elongation factor (TEF) or
beta-tubulin (Andrew et al., 2009; Lawrence et al., 2013). In the current
study the EndoPG gene sequences provided the necessary variation to
discriminate the tested isolates into two distinct clades, one for
A. tenuissima and one for A. arborescens isolates. EndoPG gene has been
recently used to separate Alternaria spp. isolates associated with apple

Table 1
Morphological characteristics (colony appearancea and sporulation apparatusb) of Alternaria tenuissima and Alternaria arborescens isolates from apple fruit grown on PDA.

Fungal Colony Colony texture Colony margins Crystals Colony Sporulation Number of Number of Conidia
species color diameter apparatus conidia conidia in length
(mm) per chain branch chains

A. tenuissima Olive gray Cottony White color (1–2 mm) + 45–70 Mostly single chains 7–12 1–3 11–26 μm
A. arborescens Dark olive gray Cottony-wooly Wavy with white color (1–2 mm) ++ 40–65 Secondary/tertiary chains 2–7 2–4 11–26 μm
a
Colony appearance of fungal colonies grown on PDA was examined visually after 10 days of incubation at 22 °C in the dark.
b
Sporulation apparatus of fungal colonies grown on weak PDA was examined under stereomicroscope and microscope after 7 days of incubation at 22 °C and a 14/10 h light/dark cycle.
 P. Ntasiou et al. / International Journal of Food Microbiology 197 (2015) 22–29
Fig. 4. HPLC-DAD chromatograms of Alternaria mycotoxins alternariol (AOH), alternariol monomethyl ether (AME) and tentoxin (TEN) from the analysis of fortified (0.5 μg/g) apple
samples (A) and mixed standard solution (1.0 μg/ml) (B). UV absorption spectra of AOH (1), AME (2) and tentoxin (3) were taken at the apex of the target peaks.
leaf blotch and fruit spot, into A. arborescens-like, A. tenuissima-like
and A. alternata-like species-groups in Australia (Harteveld et al.,
2013). However, the sequence of the same gene failed to separate
A. tenuissima like- from A. alternata like species-group associated with
the same disease in Italy, while was able to separate both of them
from the A. arborescens-like species-group (Rotondo et al., 2012). The
molecular identification of the two fungal species was in accordance
with the morphological characterization of the isolates. Both groups of
isolates showed morphological characteristics similar to that described
for the same fungal species in previous reports (Andersen et al., 2002;
Pryor and Michailides, 2002; Rotondo et al., 2012). A. tenuissima has
been previously identified as the main causal agent of the disease in
S. Africa (Serdani et al., 2002) and very recently in China (Gao et al.,
2013) and USA (Kou et al., 2014). A. arborescens has also been identified
as a causal agent of core rot in apple fruit but only as a secondary invader
along with A. infectoria that was completely absent in our study (Serdani
et al., 2002). Interestingly, in a recent study was shown that
A. arborescens was the main agent of apple leaf blotch in Australia,
while A. tenuissima was shown to be the main agent of fruit spot
(Harteveld et al., 2013). It cannot be excluded that the same
A. tenuissima strains may cause both fruit spot and/or core rot. However,
further pathogenicity experiments are required to confirm this
hypothesis.
The artificial inoculations that we conducted on apple fruit showed
that both A. tenuissima and A. arborescens used in the study were path-
ogenic to apple fruit. Comparison of susceptibility to core rot in the 4
main apple varieties cultivated in Greece, showed that Fuji was the
most susceptible to both pathogens under both storage temperatures,
followed by Granny Smith and Red Delicious, while Golden Delicious
was found to be the most resistant variety. This is in accordance with
previous studies suggesting that core rot of apple fruit was observed
in higher frequencies in fruit of the variety Fuji or Red Delicious com-
pared to other varieties such as Golden Delicious or Granny Smith
27
(Niem et al., 2007; Serdani et al., 1998). This higher frequency has
been attributed to the fact that the pathogen requires an open sinus to
penetrate the fruit and Fuji has significantly higher number of fruit
with open sinuses compared with the remaining apple varieties
(Combrink et al., 1985a; Serdani et al., 1998; Spotts et al., 1999). Howev-
er, the observed higher susceptibility of Fuji apples in our study cannot
be attributed to the open sinuses of the Fuji fruit since for the artificial
inoculations of the fruit we followed a previously described method of
direct injection of spore suspension into the loculus (Michailides et al.,
1994). In a study aiming to investigate the factors affecting the develop-
ment of core rot in fruit of a resistant (Golden Delicious) and susceptible
(Red Delicious) variety it was found that differences in susceptibility of
the two varieties were correlated neither with the colonization of the
style or the ovary by the fungus nor with increased susceptibility of
the fruit mesoderm (Niem et al., 2007). In the same report several
lines of evidence were provided suggesting that the susceptibility of
the locule wall is a critical factor determining the development of core
rot in apple fruit. Among them the toughness of the epidermal layer,
the polyphenol concentration around the core, the lack of fungal en-
zyme activation and the acid content of the fruit had been considered
(Niem et al., 2007). In another recent study the high calcium content
in the loculus walls of the resistant variety Golden Delicious was
suggested as one factor that may contribute to the resistance of Golden
Delicious to core rot disease, by inhibiting the activity of fungal extracel-
lular pectolytic enzymes (Shtienberg, 2012). The precise mode of
infection of apple fruit by Alternaria spp. and the factors affecting fungal
development in the fruit loculus still remain unclear and further re-
search is required to elucidate them. This could aid the development
of means of the resistance of fruit loculus to fungal invasion and coloni-
zation and thus, to the successful control of the disease (Shtienberg,
2012).
Analytical methods developed for Alternaria mycotoxins determina-
tion in food and feed are based mainly on the use of high performance
28 P. Ntasiou et al. / International Journal of Food Microbiology 197 (2015) 22–29

liquid chromatography (HPLC), although gas chromatography (GC) has
also been used (EFSA, 2011). For the extraction of these mycotoxins
from solid and liquid matrices either organic solvents, such as acetoni-
trile, ethyl acetate and methanol or solvent mixtures with buffered
water are usually used. Purification of extracts usually includes liquid–
liquid extraction (LLE) and solid-phase extraction (SPE) techniques.
Detection of separated products can be done with fluorescence detec-
tion (FLD), UV detection, diode array detection (DAD) and mass spec-
trometry (MS) (EFSA, 2011; Ostry, 2008). In the present study, for the
extraction of target analytes a procedure as described by Andersen
et al. (2006) was employed. In this method, separation was carried
out with linear elution on a Hypersil BDS-C18 column and the mobile
phase consisted of mixture of water and acetonitrile containing 50 μl/l
trifluoroacetic acid, run under gradient conditions at 1.0 ml/min. In rela-
tion to the chromatographic conditions described in a previous work
(Andersen et al., 2006), the gradient profile was slightly modified to
achieve faster chromatographic elution of the target compounds in
more symmetrical and sharper peaks and at reasonably shorter reten-
tion times. Also, the solvent volume for extraction of mycelial agar
plugs was enhanced because it was observed in preliminary experi-
ments that the recovery of the target compounds increased when the
extraction volume was increased from 1 to 2 ml ethyl acetate while
higher volumes of the extractant did not affect the recovery values.
Measurements of mycotoxin production by the two Alternaria spe-
cies associated with core rot of apple in Greece showed that there is
an increased risk for mycotoxin presence in apple products. Most
A. tenuissima and A. arborescens isolates from apple were found able to
produce AOH, AME and TEN both in vitro and in vivo. Only 3 out of 23
A. tenuissima isolates were unable to produce any of the 3 mycotoxins
measured, while only 1 out of 7 A. arborescens isolates tested was unable
Table 2
Mean production of alternariol (AOH), alternariol monomethyl-ether (AME) and tentoxin
(TEN), after HPLC analysis, on DRYES agar (in vitro) and on artificially inoculated apple
fruit (in vivo) of several Alternaria tenuissima and A. arborescens isolates obtained from
apple fruit showing core rot symptoms.
Isolate code Alternaria species In vitro (μg/g) In vivo (μg/g)
to produce any of the 3 mycotoxins. The mycotoxin profile revealed for
the two fungal species is in agreement with the findings of previous re-
ports suggesting that both A. tenuissima and A. arborescens are able to
produce AOH, AME and TEN (Andersen et al., 2002; Andersen et al.,
2006). However, in contrast to findings of Somma et al. (2011) suggest-
ing that A. alternata/tenuissima isolates from tomato had a higher AME
and similar AOH production than A. arborescens isolates on tomatoes,
in our study we found that, as a group, A. tenuissima isolates showed
higher and equal production of AOH and AME, respectively, on apple
fruit. Tentoxin was the less frequently detected metabolite in both
Alternaria species tested. This is in agreement with previous findings
suggesting that tentoxin is scarcely produced by small-spored Alternaria
(Andersen et al., 2002). The detected mycotoxins may have different
toxic effects on different organisms, while the co-production of these
metabolites by the same Alternaria spp. strain can lead to strong syner-
gistic effects, and thus to increased toxicity (Bottalico and Logrieco,
1998; Logrieco et al., 2003). In the current study mycotoxins were
determined in the rotten part of the artificially inoculated fruit of Fuji
variety. However, further studies are required to investigate the possi-
ble diffusion of these mycotoxins to apparently healthy fruit tissue as
has been observed for patulin produced by P. expansum (Laidou et al.,
2011). Furthermore, future studies could focus in investigation of
Alternaria mycotoxin production in different apple varieties under
different storage conditions. Such information would be important for
managing the Alternaria mycotoxin risk in apple products.
In conclusion in this paper the etiology of core rot of apple fruit in
Greece was clarified and some of the factors that might be important
for the development of the disease were investigated. In addition, it
was shown that both A. tenuissima and A. arborescens isolates were
able to produce on infected apple fruit the 3 main Alternaria mycotoxins.
This ability emphasizes the need for monitoring in apple products such
as juices or baby foods for the presence of these toxins and the establish-
ment of maximum thresholds for the Alternaria toxins by EFSA to
ensure high quality of these products.
Acknowledgments

AOH AME TEN AOH AME TEN We are indebted to Dr. T.J. Michailides (Kearney Agricultural
F1 A. tenuissima 129.5 260.25 31.06 0.41 0.28 0.32
Research and Extension Center, University of California, Parlier, CA) for
F6 A. tenuissima 31.09 13.01 nda nd nd nd providing reference Alternaria spp. isolates for the morphological char-
F8 A. tenuissima 81.94 188.62 nd 0.27 0.88 nd acterization of the isolates. The contribution of agronomists in packing-
F9 A. tenuissima 57.25 159.24 5.99 0.22 0.19 nd houses ASEPOP Naousa, EAS Naousa, Europharm, A. Karanikolas and
F20 A. tenuissima 384.79 181.29 11.88 0.67 0.18 0.42
A.C. Pyrgon in samples collection is gratefully acknowledged.
F30 A. tenuissima 403.87 99.43 13.18 0.31 nd 0.59
F32 A. tenuissima 109.02 96.53 29.7 0.15 0.1 0.21
F33 A. tenuissima 254.35 241.4 40.27 0.19 0.19 0.37 Appendix A. Supplementary data
GS1 A. tenuissima 9.3 7.06 0.58 nd nd nd
GS4 A. tenuissima 337.55 34.81 10.84 2.13 0.89 nd Supplementary data to this article can be found online at http://dx.
GS5 A. tenuissima 146.91 120.08 72.02 0.56 0.24 0.38
GS6 A. tenuissima 218.74 128.59 43.74 0.13 0.15 0.02
doi.org/10.1016/j.ijfoodmicro.2014.12.008.
GS9 A. tenuissima 221.55 226.44 49.3 0.05 0.02 nd
GS10 A. tenuissima 181.7 71.76 42.26 0.42 nd nd References
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