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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


minor economic importance since it does not affect the fruit qualitative
Core rot of apple fruit is one of the most important postharvest characteristics (Serdani et al., 2002). Infection of the core occurs after
diseases of apple and is defined as a rot initiating from the loculus that the establishment of the disease causal agents on the senescing parts
spreads into the fruit mesoderm and leads to either a dry core rot of the blossom and the penetration through the sinus after fruit setting
(DCR) or wet core rot (WCR). Fruit affected by DCR are characterized or later stages of fruit development (Combrink et al., 1984; Reuveni
by dark brown, dry and corky tissues. DCR develops slowly and the rot et al., 2002). Yield losses due to core rot have been estimated to 3–10%
is restricted in the loculus and the mesoderm around the loculus in Israel and 5–8% in South Africa (Combrink et al., 1984; Niem et al.,
(Shtienberg, 2012). WCR is also characterized by dark brown tissues, 2007).
however the disease progresses more rapidly and deeply into the meso- The disease is caused by a complex of fungi such as Fusarium spp.,
derm. External symptoms are not evident until harvest but during stor- Ulocladium spp., Cladosporium spp., Coniothyrium spp., Pezicula spp.,
age the rot progresses and after several months, the rot may also be Mucor spp., and Alternaria spp., with the latter being predominant
evident on the fruit epidermis. However, in most cases the disease is un- (Combrink et al., 1985b; Gao et al., 2013; Michailides et al., 1994;
noticed until the fruit is cut open (Shtienberg, 2012). In addition to DCR Spotts, 1990; Tournas and Uppal Memon, 2009). Early studies on the
and WCR, moldy core (MC) can be another type of infection of apple etiology of Alternaria core rot of apple fruit suggested Alternaria
fruit loculus. It is associated with the development of fungal mycelia alternata as the causal agent of the disease (Combrink et al., 1984; Ellis
within the loculus, without invasive penetration into the mesoderm and Barrat, 1983; Reuveni et al., 2002; Spotts, 1990). In these studies
identification was made based only on the morphological characteris-
⁎ Corresponding author. tics of the isolates' cultures and conidia. But it is evident that morpho-
E-mail address: gkarao@agro.auth.gr (G.S. Karaoglanidis). 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 23

overlapping morphological characters between A. alternata and other using micropestles (Eppendorf International, Wesseling, Germany)
small-spored Alternaria spp. (Pryor and Michailides, 2002; Simmons, and stored at −20 °C until use. DNA was extracted using QIA Puregene
1992). As new molecular tools have been employed in identification Core Kit A (Qiagen GmbH, Hilden, Germany) according to the
and characterization of Alternaria spp., more recent studies have manufacturer's protocol. The concentration of the extracted DNA was
shown that several Alternaria species such as Alternaria tenuissima, measured using a P330 nanophotometer (Implen GmbH, Munich,
Alternaria infectoria and Alternaria arborescens may be the causal agents Germany).
of the disease (Gao et al., 2013; Kang et al., 2002; Kou et al., 2014;
Serdani et al., 2002). 2.3. Species identification and phylogenetic analysis
Alternaria spp. are potential producers of more than 30 secondary
metabolites, mostly host-specific or non-specific phytotoxins that play The endopolygalacturonase (endoPG) gene, has shown potential to
a crucial role in the development of diseases caused by the pathogens. delineate the closely related species within the A. alternata-complex
In addition, most of the Alternaria species are able to produce myco- and was used for pathogen identification and phylogenetic analysis
toxins with biological activity against several organisms including (Hong et al., 2005; Andrew et al., 2009). Amplification of endoPG gene
mammals. Among these metabolites alternariol (AOH), alternariol was conducted using primers PG3 (5′-TACCATGGTTCTTTCCGA-3′) and
methyl ether (AME), altenuene (ALT), tentoxin (TEN) and tenuazonic PG2b (5′-GAGAATTCRCARTCRTCYTGRTT-3′) (Andrew et al., 2009). Re-
acid (TeA) are included (Logrieco et al., 2003). Although the acute tox- action mixtures contained 3 μl genomic DNA (50 ng), 0.32 μM of PG3
icity of Alternaria mycotoxins is considered to be low in mammals, primer, 1.12 μM of PG2b primer, 0.2 mM dNTPs, 2 mM MgCl2, 1× PCR
there is strong evidence that they may be mutagenic and carcinogenic buffer [200 mM Tris–HCl (pH 8.4), 500 mM KCl], and 0.04 units Taq
(Logrieco et al., 2009; Ostry, 2008). Alternaria mycotoxins have been DNA Polymerase and the total volume was adjusted to 25 μl with sterile
detected in several foods such as citrus fruit, tomato, tomato products, highly purified H2O. PCR amplification was conducted using the follow-
olives, pepper and wheat (Ostry, 2008). In addition, several Alternaria ing conditions: an initial denaturation at 94 °C for 3 min, followed by 40
mycotoxins have been detected in apple fruit infected by Alternaria cycles at 95 °C for 30 s, 53 °C for 30 s, 72 °C for 1 min and a final exten-
spp. or in apple juice concentrates (Andersen et al., 2006; Broggi et al., sion at 72 °C for 10 min. All the PCR reactions were performed in a
2013; Delgado et al., 1998; Prelle et al., 2013; Robiglio and Lopez, LabCycler thermal cycler (SensoQuest GmbH, Gottingen, Germany).
1995; Scott and Kanhere, 2001). PCR products of each isolate were purified using the Qiaquick PCR
In a recent study aiming to identify the causal agents of postharvest Purification Kit (Qiagen GmbH, Hilden, Germany). The purified products
rots of apple fruit in Greece it was found that Alternaria spp. was the were subjected to sequencing in both directions using the forward and
third most common pathogen after Penicillium expansum and Botrytis the reverse primers. Sequences were aligned using the computer soft-
cinerea, whereas in variety Fuji it was found to be the most common ware package Mega 5.05. The sequences obtained were compared
pathogen (Konstantinou et al., 2011). Taking into account the increasing with sequences in the National Center for Biotechnology Information
significance of this disease, further research was initiated aiming to: database using BlastN 2.2.18.
i) identify the causal agents of the disease at species level and character- Phylogenetic analysis of EndoPG. Sequences were trimmed to 448 bp
ize them molecularly, ii) measure variety susceptibility and aggressive- to eliminate ambiguous sites and aligned using MAFFT version 6 (Katoh
ness of Alternaria spp. under different storage temperatures and and Toh, 2008). Models of sequence evolution were tested for each
iii) investigate the ability of Alternaria spp. from apple fruit to produce alignment and model parameter estimates obtained for each alignment
some common Alternaria toxins both in vitro and in vivo on artificially using jModelTest version 0.1 (Posada, 2008). The K80 model was
inoculated apple fruit. selected for the EndoPG data with equal base frequencies, a transition/
transversion ratio of 3.05, and equal substitution rates among sites.
2. Materials and methods The rooted EndoPG phylogenies were estimated using maximum likeli-
hood with the program PhyML version 3.0 (Guindon and Gascuel,
2.1. Pathogen isolates 2003). Alternaria gaisen (GenBank AY295033) was used to root the
phylogenetic tree.
Isolates of Alternaria spp. were collected from apple fruit (cv. Golden
Delicious, Red Delicious, Fuji and Granny Smith) showing core rot 2.4. Morphological characterization
symptoms. The fruits were obtained from packinghouses located in
the region of Imathia, northern Greece, during the 2012–13 storage pe- For all 75 isolates used in the study the morphological characteristics
riod. Rotted fruit were transferred to the laboratory for isolation of the of colony and sporulation apparatus were determined using criteria as
decay agents. Isolations were carried out from surface-disinfected fruit described by Pryor and Michailides (2002). To determine the colony
(they were drenched for 1 min in a 1% sodium hypoclorite solution) characteristics the isolates were grown on PDA and incubated at 22 °C
by removing small fruit pieces at the margin of diseased/healthy tissue in the darkness for 10 days. At the end of the incubation period the
and transferring them to Petri dishes containing acidified Potato colony diameter, colony color, colony texture, colony margin and the
Dextrose Agar (Merck, Darmstadt, Germany). Petri dishes were presence of crystals in agar medium were examined. Colony diameter
incubated at 22 °C in an incubator with a 12-h photoperiod provided was measured in mm, while the remaining characteristics were
by fluorescent lights. After 3 to 5 days of incubation, the emerging assessed visually. For each isolate 3 replicate PDA plates were prepared
putative Alternaria spp. colonies were transferred to fresh PDA medium and diameter values were averaged.
and incubated under the above mentioned conditions for 1 additional Characterization of sporulation habit was conducted on weak (0.05)
week to induce sporulation. In total 75 single-spore isolates of Alternaria PDA (3 replicate plates per isolate). The cultures were incubated for
spp. were obtained (31, 7, 27 and 10 isolates from Fuji, Red Delicious, 7 days at 22 °C under cool fluorescent light (60 μmoles/m2/s, 10:14 h
Golden Delicious and Granny Smith varieties, respectively) and stored light/dark cycle). At the end of the incubation period the sporulation ap-
at 4 °C until use. paratus was examined using a stereomicroscope at 40× magnification.
Conidial size was measured in a ZEISS AxioImager. Z2 microscope
2.2. DNA isolation using a digital camera (AxioCam MRc 5). The size of 40 conidia per
isolate was measured. Eight Alternaria spp. isolates (two of each
To extract DNA the isolates were grown in Potato Dextrose Broth A. alternata, A. infectoria, A. tenuissima and A. arborescens) were kindly
(Sigma-Aldrich, St. Louis, MO) for 5 days at 25 °C. Then, the mycelium provided by Dr T. J. Michailides (Kearney Agricultural Research and Ex-
was harvested by filtration, dried, lyophilized, ground to a fine powder 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 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
Ten isolates of A. tenuissima and A. arborescens (5 isolates per and extracted twice with 1 ml ethyl acetate (1% formic acid) each for
species) were used to assess the susceptibility of apple fruit varieties 60 min in an ultrasonic bath. The final extracts were transferred to
to core rot. For conidial production the isolates were cultured on PDA clean glass tubes and evaporated to dryness under a stream of nitrogen.
for 10 days under alternating light/dark conditions (10/14 h) at 25 °C. The residues were reconstituted in 0.5 ml of methanol by sonication and
Subsequently, the cultures were flooded with 5 ml of sterile distilled filtered through 0.45 μm PTFE filters prior to HPLC analysis
water and the conidia were scraped off with a surgical blade. The
resulting conidial suspension was filtered through two layers of cheese- 2.8. Instrumental analysis
cloth to remove mycelia fragments and adjusted at a concentration
of 2 × 105 spores/ml. Prior to inoculation, apple fruit (cv. Fuji, Analyses of mycotoxins were carried out using a SpectraSYSTEM
Red Delicious, Golden Delicious and Granny Smith) were surface- high performance liquid chromatograph (Thermo Separation Products,
disinfected for 5 min by drenching them in a 1% NaOCl solution. The Austin, TX, USA). HPLC system consisting of a P4000 tertiary solvent
fruit were artificially inoculated by aseptically injecting 5 ml of a conid- pump, a vacuum degasser TSP, an AS3000 autosampler equipped with
ial suspension through the calyx into the fruit core with a syringe. Forty a 100-μl injection loop and a UV6000LP diode array detector. Chromato-
fruit per isolate/variety combination were inoculated. The fruit were graphic separation was done on a Hypersil BDS-C18 (Thermo Finnigan,
placed on wire mesh platforms (10 fruit per box) in plastic boxes USA) cartridge column (250 × 4.6 mm, 5 μm) with a 10 × 4 mm (inner
(23 × 31 × 10 cm [length × width × height]). Twenty ml of water was diameter) Hypersil BDS-C18 guard column (Thermo Finnigan, USA).
added to each box and the boxes covered to maintain high relative The mobile phase consisted of a linear gradient starting at 90% water
humidity. Twenty of the inoculated fruit per isolate were incubated and 10% acetonitrile, reaching 50% acetonitrile after 25 min and 100%
for 21 days at 25 °C and the remaining 20 fruit were incubated for acetonitrile after 30 min. 100% acetonitrile was maintained for 1 min.
60 days at 2 °C. At the end of the incubation period the fruit were Thereafter the gradient was returned to 10% acetonitrile in 1 min
cut in the middle and the infections were scored by measuring lesion and allowed to equilibrate for 3 min before the next analysis. Both
diameter around the fruit core. The experiment was conducted twice. 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
2.6. Chemicals and reagents for mycotoxin analysis was 20 μl.

Acetonitrile, water and ethyl acetate, methanol (all of HPLC grade) 2.9. Method validation
were obtained from Merck (Darmstadt, Germany) and Chem-Lab
(Zedelgem, Belgium), respectively. Formic acid (purity 99%) was The developed analytical method was evaluated for accuracy (as re-
purchased from Carlo Erba (Milano, Italy) and trifluoroacetic acid coveries) and precision (as repeatability) by analyzing uncontaminated
(purity ≥ 99%) was obtained from Sigma-Aldrich (Steinheim, blank samples (agar and apple tissue), with no presence of Alternaria
Germany). Analytical standards of the Alternaria mycotoxins alternariol mycotoxins, fortified with known amount of mixed working standard
(AOH, 98.5%), alternariol monomethyl ether (AME, 99.2%) and tentoxin solutions. This procedure was done at five different concentration levels
(TEN, 99.2%) were obtained from Sigma-Aldrich (Steinheim, Germany). (0.05, 0.2, 0.5, 1, 10 μg/g DRYES Agar and 0.05, 0.1, 0.5, 1, 2 μg/g apple
Individual stock standard solutions (100 μg/ml) of each toxin were pre- tissue) in triplicate. The linearity of the detector response to the analyte
pared in methanol and stored in darkness at −20 °C. Working standard was evaluated based on the correlation coefficient (r) values. The limits
solutions at various concentrations were made by diluting with the of detection (LOD) and quantification (LOQ) of each method for the
appropriate volume of methanol for the preparation of fortified samples three mycotoxins were determined. The LOD was calculated as the min-
and for the construction of calibration curves. All working standard imum concentration giving a response 3 times greater than the baseline
solutions were stored at 4–5 °C. 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
2.7. Extraction procedure the signal to noise ratio (S/N) was equal to 10.

Mycotoxin determination was conducted both in vitro and in vivo in 2.10. Data analysis
23 randomly selected A. tenuissima isolates and in all (n = 7)
A. arborescens isolates. The extraction protocol of Alternaria mycotoxins Data from the two independent trials on core rot diameter caused by
was performed using the method described by Andersen et al. (2006), the isolates of the two pathogens on the fruit of the tested varieties,
slightly modified. For determination of mycotoxin production in vitro were tested for homogeneity of variance using the Levene's test. In
the isolates were cultured in 9 cm Petri dishes (3 replicate plates per iso- these analyses, data of the two independent trials were considered
late) containing Dichloran Rose Bengal Yeast Extract (DRYES) agar and block treatments and the replications within each trial were used as
incubated for 14 days at 25 °C, in the dark. At the end of the incubation subsamples. The data of the two independent trials fulfilled the criteria
period 5 mm (inner diameter) mycelial agar plugs were cut from the for homogeneity of variance (P N 0.05) and therefore they were com-
middle, mean and edge of each colony and were placed in a 5 ml bined. Mean values of core rot diameter on the different varieties caused
screw-cap vial. The plugs were extracted with 2 ml ethyl acetate con- by each fungal species were compared using Fisher's Protected LSD Test
taining 1% formic acid by sonication for 60 min. The extracts were trans- at P = 0.05. The pairwise Student's t-test was used to compare mean
ferred into 10 ml glass tubes and concentrated to dryness by use of concentrations of the 3 mycotoxins produced by A. tenuissima and
nitrogen stream at 30 °C. The extracts were re-dissolved ultrasonically A. arborescens as isolate groups. Statistical analyses were conducted
in 0.5 ml methanol, filtered through 0.45 μm PTFE filters (Millipore, Bed- using SPSS 17.0 (SPSS, Chicago, IL).
ford, MA, USA) and transferred to autosampler vials for instrumental
analysis in HPLC system. 3. Results
For the determination of mycotoxin production in vivo apple fruit
(cv. Fuji) were artificially inoculated with the same 30 Alternaria spp. 3.1. Alternaria spp. identification
isolates following the artificial inoculation protocol described previously.
In total 5 fruit were inoculated per isolate and the fruit were incubated at Amplification of the EndoPG gene produced a fragment of 448 bp,
25 °C for 20 days. After the end of the incubation period the inoculated from all the 75 Alternaria spp. isolates tested. Sequence analysis and
P. Ntasiou et al. / International Journal of Food Microbiology 197 (2015) 22–29 25

Alternaria tenuissima Alternaria arborescens


100

90

80

Frequency (%)
70
60

50

40

30

20

10

0
Fuji Red Delicious Granny Smith Golden Delicious
Variety

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 (Fig. 3A and B). Core rot diameter in fruits of Granny Smith and Red
67 out of 75 isolates were A. tenuissima (GenBank Accession numbers Delicious was significantly lower (P b 0.05) to that of Fuji at both
LK054418–LK054484), while the remaining 8 were identified as incubation temperatures (Fig. 3A and B).
A. arborescens (GenBank Accession numbers LK054410–LK054417).
A. tenuissima was found in higher frequency in all the 4 varieties 3.4. Validity of mycotoxin detection analytical method
sampled, while A. arborescens was not identified in isolates obtained
from Granny Smith fruit (Fig. 1). The analysis of the EndoPG sequences Mean recovery values obtained for AOH, AME and TEN were 105,
resulted in a phylogenetic tree composed of only two major clades 103, 109% in PDA and 101, 96, 99% in apple samples and respective rel-
(Fig. 2). All the A. tenuissima isolates were grouped together in one ative standard deviation values (RSDs %) of all % mean recovery values
clade and A. arborescens isolates were grouped in the second clade were b 7% and b 8%. The calibration curves were linear with correlation
which was subdivided into two further clades (Fig. 2). 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-
3.2. Morphological characterization sorbance, resulted in the best sensitivity of the target compounds, were
256 nm (AOH, AME) and 281 nm (TEN), and exhibited retention times
The 67 isolates identified as A. tenuissima based on the endoPG se- of 20.4 (AOH), 22.4 (TEN) and 28.4 min (AME) (Fig. 4). The LOQ and
quence produced colonies of olive gray color with a thin (1–2 mm) LOD levels for all mycotoxins for both substrates (artificial nutrient me-
white margin. Most of the isolates produced white crystals in the dium and apple fruit) were set at 0.05 μg/g and 0.02 μg/g, respectively.
growth medium under the fungal mycelium, while the appearance of
all the 67 colonies was cottony. Mean colony diameter of A. tenuissima 3.5. Mycotoxin production
isolates ranged from 45 to 70 mm. Sporulation apparatus of
A. tenuissima isolates produced single conidial chains with 7–12 conidia, All the A. tenuissima and A. arborescens isolates included in the study
while secondary branching with 1–3 conidia was observed in only a lim- were found to produce in vitro AOH and AME. AOH was produced by
ited number of isolates. Mean conidial length ranged from 11 to 26 μm. A. tenuissima and A. arborescens isolates at a range of 2.26 to 454.17 and
The 8 isolates identified as A. arborescens produced colonies with a 42.86 to 463.11 μg/g, respectively. Similarly, A. tenuissima isolates produced
dark olive gray color. The colonies were wavy at the margins with a AME at a range of 0.43 to 260.25 μg/g, and A. arborescens isolates at a range
white edge of 1–2 mm in width. Crystals in the growth medium were of 38.75 to 216.33 μg/g (Table 2). Tentoxin was produced in vitro by most
abundant and the appearance of the colony was cottony to wooly in isolates of both fungal species with an exception of 4 A. tenuissima isolates
all the 8 isolates tested. Mycelial growth of A. arborescens isolates after and 1 A. arborescens isolate. No difference (P N 0.05) was observed in mean
10 days of incubation ranged from 40 to 65 mm. Sporulation pattern concentrations detected for all the 3 mycotoxins analyzed, between
in A. arborescens isolates was characterized by the presence of conidia A. tenuissima and A. arborescens, in vitro (Table 2).
chain branching. The main chain was 2 to 7 conidia in length while Analysis conducted on apple fruit showed that all the 3 mycotoxins
there were abundant secondary and tertiary branches with 2 to 4 co- were produced in lower concentrations. AOH was produced by all but 2
nidia. Mean conidial length was similar to that of A. tenuissima with a A. tenuissima and by all but 2 A. arborescens isolates, at concentrations
length ranging from 11 to 26 μm. Data on the morphological character- ranging from 0.05 to 8.35 and 0.24 to 1.22 μg/g, respectively (Table 2).
istics of both A. tenuissima and A. arborescens isolates are summarized in Similarly AME was produced in vivo by all but 5 A. tenuissima and by
Table 1. 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
3.3. Apple fruit variety susceptibility 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
All the isolates used in the study were found to be pathogenic on mean, A. tenuissima isolates produced significantly higher concentra-
fruit of all the tested varieties. Measurements of lesion diameter on tions (P b 0.05) of AOH than A. arborescens, while the latter produced
the inoculated fruit showed that Fuji was the most susceptible to both significantly higher (P b 0.05) concentrations of TEN than the former
Alternaria species at both incubation temperatures, whereas fruit isolates (Table 2). No difference (P N 0.05) was observed between the
of Golden Delicious were the most resistant at both temperatures 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 morphological characteristics of the conidia and the sporulation appara-
sequences of the ΕndoPG genes of 75 Alternaria spp. isolates from apple fruit with core rot tus of the isolates. However, controversies and confusion have often ac-
symptoms. Clade 1 consists of A. tenuissima isolates and clade 2 of A. arborescens isolates. companied the classification of small-spored species sharing common
Alternaria gaisen (GenBank accession num. AY295033) was used to root the phylogeny.
morphological characteristics and overlapping conidial sizes (Serdani
et al., 2002). Moreover, molecular phylogenies of the A. alternata
4. Discussion species-group revealed little to no variation in several genetic loci that
are commonly used in fungal systematics such as nuclear ribosomal in-
The present study represents a first attempt in characterizing ternal transcribed spacer (ITS), translation elongation factor (TEF) or
Alternaria spp. isolates associated with core rot of apple fruit in beta-tubulin (Andrew et al., 2009; Lawrence et al., 2013). In the current
Greece. The results of the study showed that the disease is caused by study the EndoPG gene sequences provided the necessary variation to
two distinct species, A. tenuissima and A. arborescens, with the former discriminate the tested isolates into two distinct clades, one for
being predominant in all apple varieties sampled. In the past Alternaria A. tenuissima and one for A. arborescens isolates. EndoPG gene has been
core rot of apple fruit has been linked to A. alternata based on the 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 27

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 (Niem et al., 2007; Serdani et al., 1998). This higher frequency has
and A. alternata-like species-groups in Australia (Harteveld et al., been attributed to the fact that the pathogen requires an open sinus to
2013). However, the sequence of the same gene failed to separate penetrate the fruit and Fuji has significantly higher number of fruit
A. tenuissima like- from A. alternata like species-group associated with with open sinuses compared with the remaining apple varieties
the same disease in Italy, while was able to separate both of them (Combrink et al., 1985a; Serdani et al., 1998; Spotts et al., 1999). Howev-
from the A. arborescens-like species-group (Rotondo et al., 2012). The er, the observed higher susceptibility of Fuji apples in our study cannot
molecular identification of the two fungal species was in accordance be attributed to the open sinuses of the Fuji fruit since for the artificial
with the morphological characterization of the isolates. Both groups of inoculations of the fruit we followed a previously described method of
isolates showed morphological characteristics similar to that described direct injection of spore suspension into the loculus (Michailides et al.,
for the same fungal species in previous reports (Andersen et al., 2002; 1994). In a study aiming to investigate the factors affecting the develop-
Pryor and Michailides, 2002; Rotondo et al., 2012). A. tenuissima has ment of core rot in fruit of a resistant (Golden Delicious) and susceptible
been previously identified as the main causal agent of the disease in (Red Delicious) variety it was found that differences in susceptibility of
S. Africa (Serdani et al., 2002) and very recently in China (Gao et al., the two varieties were correlated neither with the colonization of the
2013) and USA (Kou et al., 2014). A. arborescens has also been identified style or the ovary by the fungus nor with increased susceptibility of
as a causal agent of core rot in apple fruit but only as a secondary invader the fruit mesoderm (Niem et al., 2007). In the same report several
along with A. infectoria that was completely absent in our study (Serdani lines of evidence were provided suggesting that the susceptibility of
et al., 2002). Interestingly, in a recent study was shown that the locule wall is a critical factor determining the development of core
A. arborescens was the main agent of apple leaf blotch in Australia, rot in apple fruit. Among them the toughness of the epidermal layer,
while A. tenuissima was shown to be the main agent of fruit spot the polyphenol concentration around the core, the lack of fungal en-
(Harteveld et al., 2013). It cannot be excluded that the same zyme activation and the acid content of the fruit had been considered
A. tenuissima strains may cause both fruit spot and/or core rot. However, (Niem et al., 2007). In another recent study the high calcium content
further pathogenicity experiments are required to confirm this in the loculus walls of the resistant variety Golden Delicious was
hypothesis. suggested as one factor that may contribute to the resistance of Golden
The artificial inoculations that we conducted on apple fruit showed Delicious to core rot disease, by inhibiting the activity of fungal extracel-
that both A. tenuissima and A. arborescens used in the study were path- lular pectolytic enzymes (Shtienberg, 2012). The precise mode of
ogenic to apple fruit. Comparison of susceptibility to core rot in the 4 infection of apple fruit by Alternaria spp. and the factors affecting fungal
main apple varieties cultivated in Greece, showed that Fuji was the development in the fruit loculus still remain unclear and further re-
most susceptible to both pathogens under both storage temperatures, search is required to elucidate them. This could aid the development
followed by Granny Smith and Red Delicious, while Golden Delicious of means of the resistance of fruit loculus to fungal invasion and coloni-
was found to be the most resistant variety. This is in accordance with zation and thus, to the successful control of the disease (Shtienberg,
previous studies suggesting that core rot of apple fruit was observed 2012).
in higher frequencies in fruit of the variety Fuji or Red Delicious com- Analytical methods developed for Alternaria mycotoxins determina-
pared to other varieties such as Golden Delicious or Granny Smith 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 to produce any of the 3 mycotoxins. The mycotoxin profile revealed for
also been used (EFSA, 2011). For the extraction of these mycotoxins the two fungal species is in agreement with the findings of previous re-
from solid and liquid matrices either organic solvents, such as acetoni- ports suggesting that both A. tenuissima and A. arborescens are able to
trile, ethyl acetate and methanol or solvent mixtures with buffered produce AOH, AME and TEN (Andersen et al., 2002; Andersen et al.,
water are usually used. Purification of extracts usually includes liquid– 2006). However, in contrast to findings of Somma et al. (2011) suggest-
liquid extraction (LLE) and solid-phase extraction (SPE) techniques. ing that A. alternata/tenuissima isolates from tomato had a higher AME
Detection of separated products can be done with fluorescence detec- and similar AOH production than A. arborescens isolates on tomatoes,
tion (FLD), UV detection, diode array detection (DAD) and mass spec- in our study we found that, as a group, A. tenuissima isolates showed
trometry (MS) (EFSA, 2011; Ostry, 2008). In the present study, for the higher and equal production of AOH and AME, respectively, on apple
extraction of target analytes a procedure as described by Andersen fruit. Tentoxin was the less frequently detected metabolite in both
et al. (2006) was employed. In this method, separation was carried Alternaria species tested. This is in agreement with previous findings
out with linear elution on a Hypersil BDS-C18 column and the mobile suggesting that tentoxin is scarcely produced by small-spored Alternaria
phase consisted of mixture of water and acetonitrile containing 50 μl/l (Andersen et al., 2002). The detected mycotoxins may have different
trifluoroacetic acid, run under gradient conditions at 1.0 ml/min. In rela- toxic effects on different organisms, while the co-production of these
tion to the chromatographic conditions described in a previous work metabolites by the same Alternaria spp. strain can lead to strong syner-
(Andersen et al., 2006), the gradient profile was slightly modified to gistic effects, and thus to increased toxicity (Bottalico and Logrieco,
achieve faster chromatographic elution of the target compounds in 1998; Logrieco et al., 2003). In the current study mycotoxins were
more symmetrical and sharper peaks and at reasonably shorter reten- determined in the rotten part of the artificially inoculated fruit of Fuji
tion times. Also, the solvent volume for extraction of mycelial agar variety. However, further studies are required to investigate the possi-
plugs was enhanced because it was observed in preliminary experi- ble diffusion of these mycotoxins to apparently healthy fruit tissue as
ments that the recovery of the target compounds increased when the has been observed for patulin produced by P. expansum (Laidou et al.,
extraction volume was increased from 1 to 2 ml ethyl acetate while 2011). Furthermore, future studies could focus in investigation of
higher volumes of the extractant did not affect the recovery values. Alternaria mycotoxin production in different apple varieties under
Measurements of mycotoxin production by the two Alternaria spe- different storage conditions. Such information would be important for
cies associated with core rot of apple in Greece showed that there is managing the Alternaria mycotoxin risk in apple products.
an increased risk for mycotoxin presence in apple products. Most In conclusion in this paper the etiology of core rot of apple fruit in
A. tenuissima and A. arborescens isolates from apple were found able to Greece was clarified and some of the factors that might be important
produce AOH, AME and TEN both in vitro and in vivo. Only 3 out of 23 for the development of the disease were investigated. In addition, it
A. tenuissima isolates were unable to produce any of the 3 mycotoxins was shown that both A. tenuissima and A. arborescens isolates were
measured, while only 1 out of 7 A. arborescens isolates tested was unable able to produce on infected apple fruit the 3 main Alternaria mycotoxins.
This ability emphasizes the need for monitoring in apple products such
Table 2 as juices or baby foods for the presence of these toxins and the establish-
Mean production of alternariol (AOH), alternariol monomethyl-ether (AME) and tentoxin ment of maximum thresholds for the Alternaria toxins by EFSA to
(TEN), after HPLC analysis, on DRYES agar (in vitro) and on artificially inoculated apple ensure high quality of these products.
fruit (in vivo) of several Alternaria tenuissima and A. arborescens isolates obtained from
apple fruit showing core rot symptoms.
Acknowledgments
Isolate code Alternaria species In vitro (μg/g) In vivo (μg/g)

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
RD1 A. tenuissima 454.17 178.36 6.4 2.07 0.65 0.07
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