International Journal of Food Microbiology 208 (2015) 84–92

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International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro

Identification and mycotoxigenic capacity of fungi associated with pre- and postharvest fruit rots of pomegranates in Greece and Cyprus Loukas Kanetis a, Stefanos Testempasis b, Vlasios Goulas a, Stylianos Samuel c, Charalampos Myresiotis d, George S. Karaoglanidis b,⁎ a

Department of Agricultural Sciences, Biotechnology and Food Science, Cyprus University of Technology, 3603 Limassol, Cyprus Plant Pathology Laboratory, Faculty of Agriculture, Forestry and Natural Environment, Aristotelian University of Thessaloniki, POB 269, 54124 Thessaloniki, Greece Ministry of Agriculture, Rural Development and Environment, Department of Agriculture, 1411 Nicosia, Cyprus d Pesticide Science Laboratory, Faculty of Agriculture, Forestry and Natural Environment, Aristotelian University of Thessaloniki, POB 269, 54124 Thessaloniki, Greece b c

a r t i c l e

i n f o

Article history: Received 24 February 2015 Received in revised form 18 May 2015 Accepted 25 May 2015 Available online 29 May 2015 Chemical compounds studied in this article: Alternariol (PubChem CID: 5359485) Alternariol monomethyl-ether (PubChem CID: 5360741) Fumonisin B2 (PubChem CID: 2733489) Ochratoxin A (PubChem CID: 442530) Tentoxin (PubChem CID: 5281143) Keywords: Alternaria Alternariol Alternariol monomethyl-ether Aspergillus Fumonisin B2 Ochratoxin A

a b s t r a c t Pre- and postharvest fruit rots of fungal origin are an important burden for the pomegranate industry worldwide, affecting the produce both quantitatively and qualitatively. During 2013, local orchards were surveyed and 280 fungal isolates from Greece (GR) and Cyprus (CY) were collected from pomegranates exhibiting preharvest rot symptoms, and additional 153 isolates were collected postharvest from cold-stored fruit in GR. Molecular identification revealed that preharvest pomegranate fruit rots were caused predominately by species of the genera Aspergillus (Aspergillus niger and Aspergillus tubingensis) and Alternaria (Alternaria alternata, Alternaria tenuissima, and Alternaria arborescens). By contrast, postharvest fruit rots were caused mainly by Botrytis spp. and to a lesser extent by isolates of Pilidiella granati and Alternaria spp. Considering that a significant quota of the fungal species found in association with pomegranate fruit rots are known for their mycotoxigenic capacity in other crop systems, their mycotoxin potential was examined. Alternariol (AOH), alternariol monomethyl-ether (AME) and tentoxin (TEN) production was estimated among Alternaria isolates, whereas ochratoxin A (OTA) and fumonisin B2 (FB2) production was assessed within the black aspergilli identified. Overall in both countries, 89% of the Alternaria isolates produced AOH and AME in vitro, while TEN was produced only by 43.9%. In vivo production of AOH and AME was restricted to 54.2% and 31.6% of the GR and CY isolates, respectively, while none of the isolates produced TEN in vivo. Among black aspergilli 21.7% of the GR and 17.8% of the CY isolates produced OTA in vitro, while in vivo OTA was detected in 8.8% of the isolates from both countries. FB2 was present in vitro in 42.0% of the GR and 22.2% of the CY isolates, while in vivo the production was limited to 27.5% and 4.5% of the GR and the CY isolates, respectively. Our data imply that mycotoxigenic Alternaria and Aspergillus species not only constitute a significant subset of the fungal population associated with pomegranate fruit rots responsible for fruit deterioration, but also pose a potential health risk factor for consumers of pomegranate-based products. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Pomegranates (Punica granatum L.) are deciduous fruit trees originating from Iran to the Himalayas and are cultivated in the Mediterranean basin, Central Asia and countries of North and South America (Fernandez et al., 2014). The intensification of the crop has been relatively new in Greece (GR) and Cyprus (CY), following an accelerating trend with promising prospects. In Greece the cultivated area during the last decade has increased to 4000 ha, with most located in the regions of the Peloponnese and Macedonia. Although the cultivated area is significantly lower in Cyprus, the acreage has increased by more than 50% the last 5 years to 2000 ha.

⁎ Corresponding author. E-mail address: [email protected] (G.S. Karaoglanidis).

http://dx.doi.org/10.1016/j.ijfoodmicro.2015.05.015 0168-1605/© 2015 Elsevier B.V. All rights reserved.

The rapid expansion of pomegranate throughout the world is due to the great nutritional value of the fruit, with health promoting and dietary benefits. Pomegranates are consumed as fresh fruit, juices, jams, sauces or they are used in food and beverage industry as flavoring and coloring agents (Gil et al., 2000). Pomegranate arils are rich in polyphenols with a high antioxidant activity and consumption of pomegranate fresh fruit or products may prevent cardiovascular diseases, diabetes and prostate cancer (Gil et al., 2000; Johanningsmeier and Harris, 2011). Pomegranate fruit rots have been shown to be one of the most important factors contributing to yield losses, along with physiological disorders such as chilling injuries, husk scald, weight loss and shrinkage (Selcuk and Erkan, 2014). Fruit decays are caused by various fungal pathogens such as Alternaria spp., Botrytis cinerea, Aspergillus niger and other Aspergillus spp., Colletotrichum gloeosporioides, Coniella spp., Nematospora spp., Pilidiella granati, Penicillium spp. and Rhizopus spp. (Palou et al., 2013). Several of these have been reported during the

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last decade as pomegranate fruit rot agents in Greece (Bardas et al., 2009a,b; Tziros et al., 2008; Tziros and Tzavella-Klonari, 2008). The aforementioned pathogens may initiate infections either through injuries and wounds of fruit exocarp or during the blooming period, via the flower parts and the crown (Palou et al., 2013). Infections may remain latent until the onset of fruit maturation and become evident during storage. Alternaria spp. and Botrytis spp. behave as latent pathogens on pomegranate fruit whereas Aspergillus spp., Penicillium spp., P. granati, etc. behave mainly as wound pathogens. Fruit decays not only reduce yields quantitatively, but may also deteriorate the quality of the fresh or processed fruit due to mycotoxin production. This is particularly important because contaminated pomegranates designated for processing, especially in concentrated juice, may compromise consumer health safety. Some of the fungal species that have been reported as decay agents of pomegranate fruit such as A. niger and Alternaria alternata are known mycotoxin producers. The most common mycotoxins produced by Alternaria spp. are alternariol (AOH), alternariol monomethyl-ether (AME), altenuene (ALT), tentoxin (TEN) and tenuazonic acid (TeA) and have been detected in several foodstuffs (Logrieco et al., 2003; Ostry, 2008). Although the acute toxicity 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). Black aspergilli (Aspergillus section Nigri) are considered the main source of ochratoxin A (OTA) contamination in a wide range of food commodities (Battilani et al., 2006a; Juan et al., 2008). OTA has been shown to be nephrotoxic, carcinogenic, genotoxic, immunotoxic and teratogenic, thus the EU has fixed maximum limits for OTA presence in several products. Black aspergilli reported to produce OTA include Aspergillus carbonarius, Aspergillus ochraceus, Aspergillus japonicus, Aspergillus aculeatus and species within the A. niger aggregate (A. niger, Aspergillus tubingensis). Apart from OTA, it was recently shown that strains of black aspergilli (A. niger and Aspergillus welwitchiae) possess putative fumonisin gene clusters and are able to produce fumonisins (B2 and B4) (Frisvad et al., 2011; Mogensen et al., 2010; Susca et al., 2014). Fumonisins are mainly associated with seed rots in cereals and maize caused by several Fusarium species and considered to be responsible for harmful effects on the health of humans or animals (Desjardins, 2006). Although pomegranate is a rapidly expanding crop, information about causal agents of fruit rots is scarce and mainly restricted to reports of the presence of individual diseases in different countries. In addition, although infections of pomegranate fruit by pathogens known for their ability to produce mycotoxins, such as Alternaria spp. and Aspergillus spp. have been reported in several countries, there is an information deficit about the mycotoxigenic ability of these fungal species on pomegranate fruit. Therefore, this study was initiated aiming to: (i) identify the causal agents and determine the prevalence and incidence of preand postharvest fruit rot diseases in two different Mediterranean countries, Greece and Cyprus and (ii) investigate the mycotoxigenic ability of Alternaria spp. and Aspergillus spp. isolates from pomegranate fruits.

samplings were conducted after harvest from packinghouses located in 4 different regions of the country (Fig. 1). In each sampling 10–15 fruits showing decay or skin discoloration were arbitrarily selected from the selection line during the packing operations from November to December 2013. All postharvest isolations were retrieved from pomegranates of the cultivar Wonderful.

2. Materials and methods

Black aspergilli and in particular those belonging in the A. niger aggregate, are difficult to identify based only on morphological differences, thus an ITS-RFLP method, developed by Martinez-Culebras and Ramon (2007), was used. The 5.8S-ITS region was amplified using the primer pair ITS5/ITS4 and the PCR products were digested with the restriction enzymes HhaI, NlaIII and RsaI (Invitrogen, Carlsbad, CA). The digested PCR products from 114 Aspergillus spp. isolates were visualized after staining with ethidium bromide under UV light, and were accordingly classified to different species based on the restriction patterns (Martinez-Culebras and Ramon, 2007). ITS-RFLP identification data were further verified on a small subset of 11 isolates using sequence analysis of their calmodulin genes. Amplification conditions and sequencing reactions were set up as previously described (O'Donnell et al., 2000; Varga et al., 2010).

2.1. Fruit sampling To investigate the etiology of preharvest fruit decays the sampling was conducted in orchards located in 6 and 3 different regions of Greece and Cyprus, respectively (Fig. 1). Fruit were sampled during August–September 2013, just before the harvest. One to seven orchards were sampled in each region depending on the presence of fruit showing rot symptoms. In Greece preharvest fungal isolations were performed from fruit of the cultivars Acco, Ermioni and Wonderful, whereas in Cyprus Acco and Wonderful cultivars were sampled. From each orchard 10–15 fruit showing decay or skin discoloration (redness) were collected and transferred to the laboratory for fungal isolations. In Greece additional

2.2. Pathogen isolation and identification Isolations were carried out from surface-disinfected fruit that had been previously drenched for 1 min in a 1% sodium hypochlorite solution. Fruit samples at the margin of diseased/healthy tissue were removed and transferred to Petri dishes containing Potato Dextrose Agar (PDA) amended with streptomycin sulfate (300 mg/L). Cultures were incubated at 22 °C in the dark for 3 to 5 days and the emerging fungal colonies were transferred to fresh PDA plates to obtain pure cultures. In total 190 and 90 isolates were obtained from fruit showing preharvest rots in Greece and Cyprus, respectively, while an additional 153 isolates came from postharvest fruit rots in Greece (Table 1). All 433 fungal isolates were preliminarily identified using morphological criteria, such as colony appearance and morphological features of fruiting bodies and spores. Known fruit rot pathogens such as B. cinerea, P. granati, Botryosphaeria dothidea and Cytospora punicae were identified to species based on fungal descriptions (Barnett and Hunter, 1998; Palou et al., 2010). 2.3. Identification of Alternaria spp. at species level Alternaria spp. isolates were identified at species level through a combination of molecular and morphological data. DNA was extracted as described previously (Ntasiou et al., 2015). The endopolygalacturonase (endoPG) gene has shown potential to delineate the closely related species within the A. alternata-complex and has been used for pathogen identification and phylogenetic analysis (Andrew et al., 2009). The endoPG gene from 82 of the collected isolates was amplified using primers PG3 and PG2b (Andrew et al., 2009). Reaction mixtures and conditions, PCR products purification, sequencing and alignment were as described previously (Ntasiou et al., 2015). The Alternaria spp. isolates were further characterized using morphological characteristics based on their sporulation patterns (Andersen et al., 2002; Pryor and Michailides, 2002). Isolates were grown on weak (0.05) PDA and incubated for 7 days at 22 °C under cool fluorescent light (60 μmol/m2/s, 10:14 h light/dark cycle) after which the sporulation apparatus was examined using a stereomicroscope at 40× magnification. Eight Alternaria spp. isolates (two of each A. alternata, Alternaria infectoria, Alternaria tenuissima and Alternaria arborescens) were kindly provided by Dr. T. J. Michailides (Kearney Agricultural Research and Extension Center, University of California) and used as reference. 2.4. Identification of Aspergillus spp. at species level

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Fig. 1. Maps of Greece and Cyprus showing the fruit sampling locations. Solid and stripped circles correspond to the locations where pomegranate fruit with pre- and postharvest rots were collected, respectively.

2.5. Extraction and determination of Alternaria toxins Analysis of Alternaria toxins in vitro was conducted as described previously by Ntasiou et al. (2015). For assessment of in vivo mycotoxin production, pomegranates (cv. Wonderful) were artificially inoculated by injecting 500 μL of a conidial suspension (2 × 105 spores/mL) directly into the arils area with a syringe and were incubated for 15 days at 22 °C. Mycotoxins were extracted from infected tissues by a modification of the QuEChERS method (Anastassiades et al., 2003). Two grams of homogenized rotten pomegranate tissue were extracted with 10 mL of acetonitrile containing 1% (v/v) acetic acid and 7.5 mL of cold water by vortexing for 4 min. Consecutively, 4 g of anhydrous magnesium sulfate and 1 g of sodium chloride were added, the mixture was shaken for 3 min and centrifuged for 6 min at 7500 rpm. Four milliliters of the upper organic phase were transferred into a tube containing 0.2 g

Table 1 Number of fungal isolates obtained from pomegranate fruit showing pre- and postharvest rot symptoms in different regions of Cyprus and Greece. Country

Region

Number of isolates Preharvest rots

Postharvest rots

Greece Greece Greece Greece Greece Greece Greece Greece total

Argolida Arta Drama Imathia Larissa Thessaloniki Xanthi

91 28 13 25 13 20 n.s. 190

n.s.a 23 29 n.s. n.s. 34 67 153

Cyprus Cyprus Cyprus Cyprus total

Limassol Nicosia Pafos

50 20 20 90

n.s. n.s. n.s. n.s.

a

n.s. = not sampled.

primary secondary amine and 0.6 g of anhydrous magnesium sulfate fine powder. Then, each extract was shaken for 2 min and centrifuged at 4000 rpm for 5 min. A volume of 2.5 mL from the supernatant was concentrated to dryness using a nitrogen stream at 30 °C. The extracts were reconstituted in 0.2 mL methanol, filtered through 0.45 μm PTFE filters and were analyzed by HPLC system (SpectraSYSTEM, Thermo Separation Products, Austin, TX, USA) consisting of a P4000 tertiary solvent pump, a UV6000LP diode array (DAD), a vacuum degasser TSP, and an AS3000 autosampler. A Hypersil BDS-C18 analytical column (250 × 4.6 mm, 5 μm) was used coupled with a 10 × 4 mm Hypersil BDS-C18 guard column, both from Thermo (Thermo Finnigan, USA). The mobile phase consisted of two eluents, namely, solvent A (water with 50 μL/L trifluoroacetic acid) and solvent B (acetonitrile with 50 μL/L trifluoroacetic acid). A gradient program with a constant flow rate of 1 mL/min was used, starting with 10% B, reaching 50% B after 25 min and 100% B after 30 min. 100% B was maintained for 1 min. Thereafter the gradient was returned to 10% B in 1 min and allowed equilibrating for 3 min before the next analysis. The injection volume was 20 μL and the column temperature was 40 °C. The maximum absorbance, resulting in the best sensitivity of the target compounds, was found to be at 256 nm (AOH, AME) and 281 nm (TEN). The mean, median and range of AOH, AME and TEN were estimated in vitro and in vivo, respectively. Two different extracts per isolate were analyzed with two replications per extract. 2.6. OTA extraction and determination In vitro OTA production was determined according to Chiotta et al. (2009). Aspergillus spp. isolates were grown on Czapek yeast extract (CYA) agar for 7 days at 25 °C. Approximately 0.5 g of agar culture material from each culture was placed in a vial with 1 mL of methanol. After 60 min the extract was filtered through a PTFE filter (0.45 μm) and injected into a HPLC system as it is described below. Pomegranate fruit were artificially inoculated with spore suspensions of all the Aspergillus spp. isolates, as previously described. The

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OTA extraction was performed according to Malandrakis et al. (2013) with slight modifications. In particular, 10 g of rotten pomegranate pulp were blended with 38 mL methanol and homogenized using Ultra Turrax Disperser. Then, the homogenized samples were centrifuged at 10,000 rpm for 15 min and the supernatants were partitioned with 15 mL chloroform and accordingly were evaporated to dryness and reconstituted in 2.5 mL methanol. After filtration, 20 μL of each extract were injected on a Spherisorb® ODS2 (Waters Corporation, Milford, Ireland) column (250 mm, 4.6 mm i.d., 5 mm). OTA was eluted isocratically by water-acetonitrile-acetic acid (99:99:2, v/v/v) at a flow rate 1 mL/min. Detection was performed by fluorescence at 330 and 460 nm as excitation and emission wavelengths, respectively (Chiotta et al., 2009). Final OTA and FB2 values for all Aspergillus isolates were estimated similarly to the Alternaria mycotoxins.

2.7. FB2 extraction and determination The extraction of FB2 from agar cultures was performed according to Frisvad et al. (2007). Aspergillus spp. isolates were grown on Czapek yeast autolysate with 20% sucrose (CY20S) agar for 7 days at 25 °C. Agar plugs (0.5 g), cut from just inside the edge of the colony were extracted with 750 μL of methanol–water (3:1, v/v) using sonication for 50 min. Then, the extracts were cleaned-up using strong anion exchange (SAX) columns (ISOLUTE SAX 500 mg/6 mL) (Jimenez et al., 2003). For in vivo FB2 determination fruit were artificially inoculated and the mycotoxin was extracted according to Perrone et al. (2013). The eluate of agar and pomegranates was evaporated to dryness at 40 °C under a stream of nitrogen. Finally the residue was re-suspended in 200 μL methanol and 800 μL of derivatization solution were added. The derivatization solution consists of 1 mL o-phthalaldehyde (40 mg/mL), 5 mL of 0.1 mol/L disodium tetraborate, and 50 μL of 2mercaptoethanol. The chromatographic separation was performed using a Spherisorb® ODS2 column (250 mm 4 mm i.d., 5 mm). The mobile phase was methanol: 0.1 mol/L sodium dihydrogen phosphate (77:23, v/v), adjusted with ortho-phosphoric acid to pH 3.35. The flow rate was 1 mL/min. Fluorescence excitation and emission wavelengths were set at 335 nm and 440 nm.

3. Results 3.1. Fungal pathogen incidence Seven and five different fungal genera were found to be associated with preharvest rots of pomegranate fruit in Greece and Cyprus, respectively (Fig. 2). In both countries Aspergillus spp. and Alternaria spp. were the major agents of preharvest rots. Aspergillus spp. was isolated at frequencies of 36.8 and 51% in Greece and Cyprus, respectively, whereas Alternaria spp. frequencies were 29.5 and 38%, respectively (Fig. 2). P. granati and Botrytis spp. were the 3rd and 4th most common causal agents of preharvest rots in Greece accounting for the 13.7 and 10.5% of the decayed pomegranate fruit, but interestingly, they were not isolated in Cyprus. The remaining fungal genera (Penicillium spp., Botryosphaeria spp. and the yeast Aureobasidium pullulans) were observed in lower frequencies ranging from 1.6 to 8%; therefore were considered of minor importance. Identification of postharvest rot agents was conducted only in Greece, where 8 different fungal genera were detected. Botrytis spp. was the major agent accounting for the 58.2% of the postharvest decays, followed by P. granati responsible for 20.2% of the decays (Fig. 2). Alternaria spp. was the 3rd most common causal agent accounting, however, for only 7.6% of the decayed fruit. The remaining fungal genera isolated included C. punicae, Penicillium spp., Aspergillus spp., Botryosphaeria spp. and A. pullulans accounting for 5.1, 2.5, 1.3, 1.3 and 0.6%, respectively (Fig. 2).

Fig. 2. Frequency of preharvest rot fungal agents in Greece (GR_PreH), Cyprus (CY_PreH) and postharvest rot fungal agents (GR_PostH) recovered from naturally infected pomegranate fruit from orchards (preharvest) or packinghouses (postharvest).

3.2. Identification of Alternaria spp. at species level Amplification and sequence analysis of the endoPG gene from the 82 Alternaria spp. isolates (48 from Greece and 34 from Cyprus) revealed that 30 isolates were A. alternata (GenBank Accession numbers KP789517-KP789546), 19 were A. tenuissima (GenBank Accession numbers KP789498-KP789516) and 14 were A. arborescens (GenBank Accession numbers KP789484-KP789497). However, the endoPG sequence analysis failed to identify the remaining 19 isolates between A. alternata and A. tenuissima. According to Woudenberg et al. (2013) A. alternata, A. tenuissima and A. arborescens belong to section Alternata, of which A. alternata is the type species. Thus, in order to confirm molecular species identification and to discriminate among the unidentified A. alternata and A. tenuissima isolates, morphological characterization was conducted based mainly on the sporulation apparatus of the tested isolates using reference isolates. Isolates that had been identified as A. arborescens were characterized by the production of a main chain of 3–7 conidia in length and abundant secondary and tertiary branching of 2–4 conidia in length. The second group of isolates included all the isolates that had been identified molecularly as A. alternata and most of the isolates that had been found to belong in the A. alternata/tenuissima species-group. These isolates showed a sporulation apparatus similar to that of A. alternata reference isolate, characterized by the formation of long conidial chains of 7 to 14 conidia in length, the presence of abundant secondary and a few tertiary chains of 2 to 7 conidia in length. The third group of isolates included all the isolates that had been identified molecularly as A. tenuissima and some of the isolates that had been found to belong in the A. alternata/ tenuissima species-group. These isolates showed a sporulation apparatus similar to that of A. tenuissima reference isolate, characterized by the formation of a single conidial chain consisting of 8–12 conidia. The combined molecular and morphological identification of Alternaria spp. showed that both in Greece and Cyprus, A. alternata was the predominant Alternaria species associated with heart rot disease of pomegranate with incidence values of 50 and 55.9%, respectively (Fig. 3A). A. tenuissima was the second most common Alternaria species with frequencies of 31.2 and 29.4% in Greece and Cyprus, respectively, whereas A. arborescens was identified in frequencies of 18.8 and 14.7% in Greece and Cyprus, respectively (Fig. 3A).

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respectively, with A. tubingensis representing only 8.7 and 2.2% of the isolates in Greece and Cyprus, respectively (Fig. 3B). Partial calmodulin gene amplification of 11 representative isolates from the pomegranate collection yielded fragments ranging from 670 to 614 bp. BLAST analysis of the acquired sequences proved confirmatory to the ITS-RFLP identification outcomes (GenBank Accession numbers KP739449-KP739459). 3.4. AOH, AME and TEN production by Alternaria spp.

Fig. 3. Frequency of (A) A. alternata, A. tenuissima and A. arborescens and (B) A. niger and A. tubingensis, associated with pomegranate fruit rots in Greece and Cyprus.

3.3. Identification of Aspergillus spp. at species level In all the 69 and 45 Aspergillus spp. isolates from Greece and Cyprus, respectively, amplification of the 5.8S-ITS region revealed, as expected, a PCR product of 650 bp. Restriction analysis led to two distinct composite types of restriction patterns, the so called type T1 restriction pattern corresponding to A. tubingensis and the type N restriction pattern, corresponding to A. niger (Martinez-Culebras and Ramon, 2007). A. niger was predominant with frequencies of 91.3 and 97.8% in Greece and Cyprus,

Irrespective of species, none of the Alternaria isolates was found to individually produce AOH, AME or TEN either in vitro or in vivo, while total absence of all three tested Alternaria mycotoxins in vitro was manifested by 16.3, 4, and 7.2% of the A. alternata, A. tenuissima, and A. arborescens isolates, respectively (Supplementary Data, Table A). Most of the A. alternata, A. arborescens and A. tenuissima isolates from both countries were able to produce AOH and AME in vitro. Isolates of all the Alternaria species originating from Greece produced AOH at average concentrations higher than the isolates of the respective species from Cyprus. Mean production of AOH by A. alternata, A. tenuissima and A. arborescens isolates from Greece had values of 360.7, 285.7 and 483.8 μg/g, respectively, while the respective values for the isolates from Cyprus were 207.4, 102.0 and 91.6 μg/g (Table 2). Similarly, isolates of A. arborescens from Greece produced higher mean AME concentrations (221.4 μg/g) than those from Cyprus (42.2 μg/g), while A. alternata and A. tenuissima isolates from Cyprus produced higher mean AME concentrations (166.6 and 147.9 μg/g), respectively (Table 2). TEN was produced in vitro by fewer isolates among all the species tested. In detail, 66.7 and 46.7% of the A. alternata and A. tenuissima isolates, respectively, from Greece were TEN producers, while the respective frequencies of the Cyprus isolates were 15.8 and 10.5% (Table 2). The mean TEN concentration produced in vitro was 34.0 and 4.4 μg/g for A. alternata and 34.0 and 6.4 μg/g for A. tenuissima. Of the A. arborescens isolates from Greece, 88.9% produced TEN with a mean concentration of 36.1 μg/g, whereas none of the Cyprus isolates produced this toxin (Table 2). Overall, concomitant in vitro production of AOH and AME was evident in 39.5, 60.0, and 35.7% of the A. alternata, A. tenuissima, and A. arborescens isolates, respectively. In addition, all three mycotoxins were simultaneously produced by 44.2, 36.0, and 57.1% of the A. alternata, A. tenuissima, and A. arborescens isolates, respectively (Supplementary Data, Table A). Analysis of mycotoxin production in artificially inoculated fruit showed that for all the Alternaria species fewer isolates were able to produce mycotoxins in vivo. More specifically, 55.8, 68.0, and 35.7% of the A. alternata, A. tenuissima, and A. arborescens isolates, respectively were found not to produce any of the tested Alternaria mycotoxins in vivo(Supplementary Data, Table A). Interestingly, none of the tested isolates was able to produce TEN in vivo and similarly none of the A. tenuissima isolates from Cyprus was able to produce any of the

Table 2 Frequency of mycotoxigenic A. alternata, A. tenuissima and A. arborescens isolates originating from pomegranate fruit showing pre- and postharvest rots in Greece (GR) and Cyprus (CY) and production of alternariol (AOH), alternariol monomethyl-ether (AME) and tentoxin (TEN) on DRYES agar medium (in vitro). Species

Country

Mycotoxins AOH Isolates (%)

A. alternata A. tenuissima A. arborescens a b c

GR CY GR CY GR CY

87.5 78.9 93.3 100.0 100 80.0

a

b

AME

TEN

Mean

Median (μg/g)

Range

Isolates (%)

Mean

Median (μg/g)

Range

Isolates (%)

Mean

Median (μg/g)

Range

360.7 207.4 285.7 102.0 483.8 91.6

309.8 112.5 170.5 137.0 349.3 97.5

19.0–1047.8 1.9–723.5 3.3–728.5 7.0–191.0 20.3–1218.5 6.7–164.9

87.5 78.9 93.3 100.0 100 80.0

147.9 166.6 79.9 147.9 221.4 42.2

135.3 87.5 78.1 166.8 110.5 35.0

4.2–460.0 2.1–614.0 5.4–221.5 8.5–287.3 16.4–946.3 14.2–84.7

66.7 15.8 46.7 10.5 88.9 0.0

34.0 4.4 34.0 6.4 36.1 n.d.c

31.6 4.2 22.9 6.4 22.3 n.d.

2.0–79.8 3.1–5.9 6.0–91.7 2.7–10.3 3.5–148.8 n.d.

Percentage of Alternaria spp. isolates exhibiting mycotoxin capacity. Mean mycotoxin is the average value of two experiments (2 replications/experiment). n.d. = not detected.

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Table 3 Frequency of mycotoxigenic A. alternata, A. tenuissima and A. arborescens isolates originating from pomegranate fruit showing pre- and postharvest rots in Greece (GR) and Cyprus (CY) and production of alternariol (AOH), alternariol monomethyl-ether (AME) and tentoxin (TEN) on artificially inoculated pomegranate fruit (in vivo). Species

Country

Mycotoxins AOH

A. alternata A. tenuissima A. arborescens a b c

a

Isolates (%)

Mean

54.2 31.6 53.4 0 88.9 20

1.1 0.5 3.1 n.d.c 6.8 0.3

GR CY GR CY GR CY

b

AME

TEN

Median (μg/g)

Range

Isolates (%)

Mean

Median (μg/g)

Range

Isolates (%)

Mean

Median (μg/g)

Range

1.0 0.4 1.3 n.d. 0.4 0.3

0.2–2.5 0.2–1.3 0.3–16.9 n.d. 0.1–50.5 0.3

54.2 31.6 53.4 0 99.9 20

0.9 0.4 1.6 n.d. 4.3 0.2

0.5 0.4 0.6 n.d. 0.3 0.2

0.1–3.4 0.2–0.8 0.1–8.6 n.d. 32.3–0.1 0.2

0 0 0 0 0 0

n.d. n.d. n.d. n.d. n.d. n.d.

n.d. n.d. n.d. n.d. n.d. n.d.

n.d. n.d. n.d. n.d. n.d. n.d.

Percentage of Alternaria spp. isolates exhibiting mycotoxin capacity. Mean mycotoxin is the average value of two experiments (2 replications/experiment). n.d. = not detected.

mycotoxins analyzed in vivo(Table 3). A. alternata isolates from Greece and Cyprus produced AOH at a mean concentration of 1.1 and 0.5 μg/ g, respectively and AME at 0.9 and 0.4 μg/g, respectively (Table 3). Similarly, A. arborescens isolates from Greece and Cyprus produced AOH at a mean concentration of 6.8 and 0.3 μg/g, respectively and AME at a mean concentration of 4.3 and 0.2 μg/g, respectively (Table 3). Of the A. tenuissima only those originating from Greece were capable of AOH and AME production in vivo at mean concentrations of 3.1 and 1.6 μg/g, respectively (Table 3). Concomitant triple-mycotoxin production of AOH and AME in vitro was evident in 44.2, 32.0, and 64.3% of the A. alternata, A. tenuissima, and A. arborescens isolates, respectively (Supplementary Data, Table A).

production. Measurements of OTA production showed that only 6.3 and 11.4% of the A. niger isolates from Greece and Cyprus, respectively were able to produce OTA with mean concentrations of 10.4 and 9.6 ng/g (Table 5). Of the A. tubingensis isolates from Greece 16.7% were able to produce OTA at a mean concentration of 13.9 ng/g, whereas none of the isolates from Cyprus was an OTA producer in vivo (Table 5). FB2 was produced by 30.2 and 4.5% of the A. niger isolates from Greece and Cyprus, respectively, with mean values of 950.8 and 5.5 ng/g, but none of the A. tubingensis isolates produced FB2 (Table 5). None of the Aspergillus isolates of the present study was able to coproduce OTA and FB2 in vivo (Supplementary Data, Table B).

3.5. OTA and FB2 production by Aspergillus spp. 4. Discussion All the A. niger and A. tubingensis isolates were evaluated for OTA and FB2 production. It is noteworthy that overall, 48.6 and 71.4% of the A. niger and A. tubingensis isolates, respectively were found not to produce any OTA and FB2 (Supplementary Data, Table B). It was found that 20.6 and 18.2% of the A. niger isolates from Greece and Cyprus were able to produce OTA in vitro at a mean concentration of 352.1 and 23.3 ng/g, respectively (Table 4). Similarly, 33.3% of the Greek A. tubingensis isolates were found to be OTA producers in vitro at a mean concentration of 37.1 ng/g, whereas none of the A. tubingensis isolates from Cyprus produced OTA (Table 4). Only A. niger isolates were capable of FB2 production. FB2 was produced by 46.0 and 22.7% of A. niger isolates from Greece and Cyprus, respectively at mean concentrations of 125.7 and 105.4 ng/g (Table 4). Dual production of OTA and FB2 was evident only in 4.7% of the A. niger isolates (Supplementary Data, Table B). Analysis of mycotoxin production in artificially inoculated fruit showed that 48.6 and 71.4% of the A. niger and A. tubingensis isolates, respectively, were found not to produce any OTA or FB 2 (Supplementary Data, Table B), and the overall frequency of the in vivo mycotoxin-producing isolates was lower than their in vitro

In the current study the main fungi responsible for pomegranate fruit rots at pre- and postharvest level were identified and their mycotoxigenic ability was determined. Fruit rots, caused mainly by species in the genera Alternaria, Aspergillus and Penicillium, are of major concern for the pomegranate industry worldwide and impact the produce, both quantitatively and qualitatively (Ezra et al., 2015; Michailides et al., 2008; Zhang and McCarthy, 2012). Our data show that among the symptomatic fruit sampled, heart rot caused by species in the genera Aspergillus and Alternaria is the predominant preharvest disease of pomegranates. The higher frequencies of Alternaria and Aspergillus spp. found in Cyprus comply with the overall restricted fungal pluralism encountered in the Cyprus isolations compared to Greek and is most likely attributed to the semi-arid climate of Cyprus, governed by extended periods of drought and high ambient temperatures. Thus, less xerophytic fungal species such as B. cinerea and P. granati were not isolated from preharvest pomegranate rots in Cyprus. Furthermore, there was no obvious relationship between sampling regions and fungal species associated with pomegranate fruit rots, therefore species frequency data were presented per country (Figs. 2 and 3).

Table 4 Frequency of mycotoxigenic A. niger and A. tubingensis isolates originating from pomegranate fruit showing pre- and postharvest rots in Greece (GR) and Cyprus (CY) and production ochratoxin A (OTA) and fumonisin B2 (FB2) on CYA and CY20S agar media, respectively (in vitro). Species

Country

Mycotoxins OTA

A. niger A. tubingensis a b c

GR CY GR CY

FB2

Isolatesa (%)

Meanb (ng/g)

Median

Range

Isolates (%)

Mean

Median (ng/g)

Range

20.6 18.2 33.3 0

352.1 23.3 37.1 n.d.c

27.3 26.3 37.1 n.d.

0.9–4303.7 2.0–37.3 26.9–47.2 n.d.

46.0 22.7 0 0

125.7 105.4 n.d. n.d.

95.5 60.3 n.d. n.d.

56.2–721.3 52.5–390.3 n.d. n.d.

Percentage of Aspergillus spp. isolates exhibiting mycotoxin capacity. Mean mycotoxin is the average value of two experiments (2 replications/experiment). n.d. = not detected.

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Table 5 Frequency of mycotoxigenic A. niger and A. tubingensis isolates originating from pomegranate fruit showing pre- and postharvest rots in Greece (GR) and Cyprus (CY) and production ochratoxin A (OTA) and fumonisin B2 (FB2) on artificially inoculated pomegranate fruit (in vivo). Species

Country

Mycotoxins OTA

A. niger A. tubingensis a b c

GR CY GR CY

b

FB2

a

Isolates (%)

Mean

Median (ng/g)

Range

Isolates (%)

Mean

Median (ng/g)

Range

6.3 11.4 16.7 0

10.4 9.6 13.9 n.d.

9.9 9.8 13.9 n.d.

9.1–12.5 7.3–12.4 13.9 n.d.

30.2 4.5 0 0

950.8 5.5 n.d.c n.d.

8.4 5.5 n.d n.d.

5.1–9725.3 5.8–5.9 n.d. n.d.

Percentage of Aspergillus spp. isolates exhibiting mycotoxin capacity. Mean mycotoxin is the average value of two experiments (2 replications/experiment). n.d. = not detected.

In a two year study in Israel, Ezra et al. (2015) reported that rot incidence of pomegranate fruit at advanced developmental stages, caused mainly by Alternaria spp. and Penicillium spp., ranged between 5% and 8.1%. Similar results were reported from California, where natural incidence of heart rot caused by Alternaria spp. was estimated at 13.5% (Michailides et al., 2008). Nevertheless, cases of substantial preharvest crop losses due to heart rot have been reported from Greece, causing fruit damage up to 50% (Tziros et al., 2008). Species in the genus Aspergillus were the leading causal agents of the disease in both countries, followed closely by small-spored Alternaria. The Aspergillus subpopulation comprised only species belonging to the section Nigri. A. niger was the predominant species, with A. tubingensis much less common in both countries. Although Ezra et al. (2015) reported that A. alternata is the predominant pathogen causing pomegranate heart rot in Israel, until our study, Alternaria heart rot of pomegranate fruit was referred to in the literature as either undefined at the species level (Palou et al., 2013; Zhang and McCarthy, 2012) or attributed to A. alternata and other Alternaria spp. (Michailides et al., 2008; Tziros et al., 2008). In the current study Alternaria heart rot was found to be caused by three distinct species, with A. alternata being the prominent, followed by A. tenuissima and A. arborescens. Considering the well documented taxonomic problems and the complexity of this collective group at the species level, we followed a polyphasic approach combining traditional morphology and endoPG sequence analysis to achieve an accurate identification (Pryor and Michailides, 2002). This combination provided the required variation to delineate the closely related species within the A. alternata-complex associated with pomegranate fruit rots into three distinct clades. Previously, the aforementioned gene has been proved adequate for the separation of Alternaria spp. in other complex diseases associated with small-spored Alternaria (Harteveld et al., 2013; Ntasiou et al., 2015). Other than fruit rots, A. alternata has been reported as the causal agent of superficial black spots on fruit and leaves in pomegranate orchards (Gat et al., 2012). Despite the ecological ubiquity of A. alternata the specialization of the black spot and the heart rot populations of the pathogen is still unclear. The incidence of Aspergillus and Alternaria rots at postharvest was dramatically reduced, rendering gray mold caused by Botrytis spp. the main postharvest disease of pomegranates, with preharvest Botrytis fruit rot rising from 10.3 to 58% at storage. Similarly, Palou et al. (2013) reported that disease caused by B. cinerea latent infections ranged from 38 to 72%. Despite the fact that B. cinerea infections occur mostly during and after the bloom period, a limited number of fruit infections will develop in the field. Most of the infections remain latent until fruit matures during storage, rendering pomegranates more susceptible and resulting in severe crop losses (Tedford et al., 2005). Part of the pomegranate gray mold developed preharvest is probably due to surface-borne inoculum infecting through rind injuries. Another type of pomegranate soft rot that contributed remarkably to the overall to pomegranate losses in Greece preharvest, as well as

postharvest, was associated with the pycnidial fungus P. granati. The disease was first reported in Cyprus in 1957 (Georghiou and Papadopoulos, 1957) and in Greece in 2007 (Tziros and TzavellaKlonari, 2008). Etiology and pathogenicity experiments suggested that the pathogen causes not only fruit rot (Palou et al., 2013), but also twig and shoot blight (Chen et al., 2014; Michailides et al., 2010). It is considered to be a host-specific pathogen and it is able to grow even at 5 °C. It is noteworthy that in our study the frequency of rotten fruit due to P. granati infections postharvest was 20%, second only after Botrytis spp. losses. Similarly, significant crop losses by P. granati infections have been observed pre- and postharvest in Spain and California (Michailides et al., 2010; Palou et al., 2010). A plethora of Penicillium species, including Penicillium adametzioides, Penicillium expansum, Penicillium glabrum, Penicillium implicatum and Penicillium purpurogenum (Bardas et al., 2009b; Ezra et al., 2015; Palou et al., 2013) have been previously reported on pomegranate. Penicillium fruit rots recorded in our study were low and similar to preharvest incidence reported in Israel (Ezra et al., 2015), ranging between 3 and 5%, while others report that conventionally cold stored pomegranates at 5 °C for 12 weeks developed Penicillium rot at 12.3% (Artes et al., 2000). Experimentation suggests that fruit wounding renders pomegranates more prone to Penicillium rots, compared to pistil inoculation (Ezra et al., 2015; Palou et al., 2013). Thus, different decay incidence due to Penicillium spp. depends primarily on rind integrity. Among the fungal population we identified responsible for pomegranate fruit rots, only Aspergillus spp., Alternaria spp. and Penicillium spp. have known history of mycotoxin production. As Penicillium fruit rot was responsible on average for only about 5% of the overall decays, we focused on the Aspergillus and Alternaria mycotoxins. The Aspergillus population consisted exclusively of A. niger and A. tubingensis, with the former being the dominant. Studies worldwide have identified section Nigri as the Aspergillus group responsible for OTA production, with A. carbonarius considered the main contamination source compared with other biseriate species of the A. niger aggregate (Garcia-Cela et al., 2014; Susca et al., 2014). In our study in vivo OTA production was evident in 8.4% and 14.3% of the A. niger and A. tubingensis isolates, respectively. Considering the Aspergillus species composition of pomegranates, there is a strong indication of low risk for OTA contamination. Although A. niger is not considered a key player in OTA contamination, numerous reports from different commodities confirm its status as an important fumonisin producer (Frisvad et al., 2011; Perrone et al., 2013; Susca et al., 2014; Varga et al., 2010). Almost 38% of the A. niger isolates produced FB2 in vitro, but in fruit inoculations only 20% of A. niger population produce FB2. Frisvad et al. (2011) reported that among all species comprising the section Nigri, only A. niger isolates were able of producing FB2. In their study, 81% of the A. niger isolates produced FB2, 17% produced OTA, and 10% were able to produce both FB2 and OTA, whereas A. tubingensis produced neither OTA nor FB 2. A recent study of raisins in Greece reported FB2 in 29% of the samples, with 20%

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co-occurring with OTA (Perrone et al., 2013). In our study coproduction of OTA and FB2 was found only in vitro in 4.67% of the isolates and in vivo co-production was absent (Supplementary Data, Table B). Although chemotaxonomy of small-spored Alternaria isolates is not a reliable method for species identification, mycotoxin profiling can be useful for sketching the toxicological potential of the isolates (Andersen et al., 2015). Our results show that at least 89% of the pomegranate strains are able to produce in vitro mycotoxins associated with Alternaria (AOH, AME and TEN), although in vivo that percentage was halved. Even though A. alternata is considered the most important mycotoxin-producing species of the genus (EFSA, 2011), our data contradict such a statement. All three Alternaria species identified in our study were capable of AOH/AME co-production in vivo (Supplementary Data, Table A). Regardless of species, in vitro AOH production was substantially higher than AME, but production of these toxins was similar in vivo. Nevertheless, A. arborescens showed the highest capacity for in vivo toxin production compared with the other two species, whereas in vitro such a differentiation was not apparent. Interestingly, exclusive production of either AOH or AME was not evident by any isolate; they were always co-produced (Supplementary Data, Table A). None of the isolates produced TEN in vivo, but in vitro 44 % of the isolates were capable of TEN production. The less frequent detection of TEN in this study is consistent with other studies (Andersen et al., 2002; Ntasiou et al., 2015). Reports regarding the TEN production capacity of A. alternata are controversial (Andersen et al., 2002, 2015; Soliman et al., 2015). Our data demonstrated in vitro TEN production from 44.2% of the A. alternata isolates tested. Geostatistical analysis performed by Battilani et al. (2006b) in an attempt to correlate climatic data to levels of contamination of wine grapes with A. section Nigri and OTA contamination suggested that geographical origin does not affect fungal growth patterns, and higher disease incidence does not necessarily imply higher OTA accumulation in grapes, as ecological conditions which favor growth and subsequent high incidence are different from those which enable optimum OTA production. In addition, climate also influences host susceptibility. Warm temperatures or drought stress may cause reduction in phytoalexin production, or compromise the integrity of physical plant or fruit barriers, resulting in cracking favoring fungal colonization of naturally senescing crop parts (Cotty and Jaime-Garcia, 2007). Bock et al. (2004) state that although aflatoxin producing fungi are native to warm arid, semi-arid and tropical regions, climate changes may result in major variations in the quantity of aflatoxin producers. In addition, Perrone et al. (2013) found that a great variability was observed in the parameters they investigated (mycotoxin contamination, fungal contamination, altitude, distance from the sea, relative humidity, temperature, and rainfall) for all regions and sometimes, within the same region. In support to the aforementioned statements the highest (4303.7 ng/g) and the lowest (0.9 ng/g) in vitro OTA production in Greece were from A. niger isolates that originated from the same region (Thessaloniki) and isolated from fruit of the same cultivar (Wonderful). The same phenomenon was found for AME production by A. alternata in Cyprus, where different isolates from Nicosia and fruit of the cultivar Acco produced 614.0 and 9.5 μg/g, respectively. Global surveys show that humans and animals are generally exposed to more than one mycotoxin (Grenier and Oswald, 2011). Although the toxicity of multi-mycotoxin exposure cannot always be predicted based upon their individual toxicities, their concurrent presence may lead to additive or synergistic effects (Creppy et al., 2004; Grenier and Oswald, 2011). Therefore, the potential co-occurrence of an array of Aspergillus and/or Alternaria toxins in pomegranate-based products could pose a higher risk for public health. It is also noteworthy to mention that the fungal population structure described in our study as responsible for pomegranate fruit rots has not been shaped by any fungicide selection pressure, since in both countries there is no registered active ingredient against fungal infections, neither

91

for pre- nor for postharvest use. Any future implementation of chemical fungicides for the management of pomegranate fungal diseases may change the population and subsequently the disease incidence and potential mycotoxin mix. In conclusion, to the best of our knowledge this is the first report on the natural occurrence of mycotoxins in pomegranates. Since it is often not easy to visually distinguish pomegranate heart rot, infected fruit may go undetected during sorting and enter the retail market, causing significant losses in trade confidence. Moreover, internally moldy, healthy-looking, pomegranates may enter the juice production process, either fresh or concentrated, resulting in multi-mycotoxin contamination. The situation worsens considering the lack of established maximum levels of Aspergillus and Alternaria toxins by EFSA for pomegranate products. The demonstration of the presence of multi-mycotoxins in pomegranate fruit should lead to mandatory scrutiny of pomegranate byproducts. Thus, in order to secure and highlight pomegranates to their full potential, appropriate initiatives should be implemented at the fruit production, product processing and the legislation level. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.ijfoodmicro.2015.05.015. Acknowledgments We are grateful to Dr. T. J. Michailides (Kearney Agricultural and Extension Center, University of California, Parlier, CA, USA) for providing reference Alternaria spp. isolates used in this study and S. Solonos for his excellent technical assistance. References Anastassiades, M., Lehotay, S.J., Stajnbaher, D., Schenck, F.J., 2003. Fast and easy multiresidue method employing acetonitrile extraction/partitioning and dispersive solid-phase extraction for the determination of pesticide residues in produce. J. AOAC Int. 86, 412–431. Andersen, B., Kroger, E., Roberts, R.G., 2002. Chemical and morphological segregation of Alternaria arborescens, A. infectoria and A. tenuissima species-groups. Mycol. Res. 106, 170–182. Andersen, B., Nielsen, K.F., Fernandez Pinto, V., Patriarca, A., 2015. Characterization of Alternaria strains from Argentinean blueberry, tomato, walnut and wheat. Int. J. Food Microbiol. 196, 1–10. Andrew, M., Peever, T.L., Pryor, B.M., 2009. An expanded multilocus phylogeny does not resolve morphological species within the small-spored Alternaria species complex. Mycologia 101, 95–109. Artes, F., Tudela, J.A., Villaescusa, R., 2000. Thermal postharvest treatments for improving pomegranate quality and shelf life. Postharvest Biol. Technol. 18, 245–251. Bardas, G.A., Tzelepis, G.D., Lotos, L., Karaoglanidis, G.S., 2009a. First report of Botrytis cinerea causing gray mold of pomegranate (Punica granatum) in Greece. Plant Dis. 93, 1346. Bardas, G.A., Tzelepis, G.D., Lotos, L., Karaoglanidis, G.S., 2009b. First report of Penicillium glabrum causing fruit rot of pomegranate (Punica granatum) in Greece. Plant Dis. 93, 1347. Barnett, H.L., Hunter, B.B., 1998. Illustrated Genera of Imperfect Fungi. 4th ed. APS, St. Paul, Minnesota. Battilani, P., Magan, N., Logrieco, A., 2006a. European research on ochratoxin A in grapes and wine. Int. J. Food Microbiol. 111, S2–S4. Battilani, P., Barbano, C., Marin, S., Sanchis, V., Kozakiewicz, Z., Magan, N., 2006b. Mapping of Aspergillus section Nigri in Southern Europe and Israel based on geostatistical analysis. Int. J. Food Microbiol. 111, S72–S82. Bock, C.H., Mackey, B., Cotty, P.J., 2004. Population dynamics of Aspergillus flavus in the air of an intensively cultivated region of south-west Arizona. Plant Pathol. 53, 422–433. Chen, Y., Shao, D.D., Zhang, A.F., Yang, X., 2014. First report of a fruit rot and twig blight on pomegranate (Punica granatum) caused by Pilidiella granati in Anhui province of China. Plant Dis. 89, 695. Chiotta, M.L., Ponsone, M.L., Combina, M., Torres, A.M., Chulze, S.N., 2009. Aspergillus section Nigri species isolated from different wine-grape growing regions in Argentina. Int. J. Food Microbiol. 136, 137–141. Cotty, P.J., Jaime-Garcia, R., 2007. Influences of climate on aflatoxin producing fungi and aflatoxin contamination. Int. J. Food Microbiol. 119, 109–115. Creppy, E.E., Chiarappa, P., Baudrimont, I., Borracci, P., Moukha, S., Carratu, M.R., 2004. Synergistic effects of fumonisin B1 and ochratoxin A: are in vitro cytotoxicity predictive of in vivo acute toxicity? Toxicology 201, 115–123. Desjardins, A.E., 2006. Fusarium Mycotoxins: Chemistry, Genetics and Biology. APS Press, St. Paul, Minnesota. EFSA on Contaminants in the Food Chain (CONTAM), 2011. Scientific opinion on the risks for animal and public health related to the presence of Alternaria toxins in feed and food. EFSA J. 9, 2407. http://dx.doi.org/10.2903/j.efsa.2011.2407.

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