Mycobiota and Incidence of Toxigenic Fungi in Dried Fruits from Duhok Markets, North Iraq

Thirtysamples from each of four dried fruits (apricot,fig,plum and raisins) collectedfrom local shops at Duhok governorate were surveyed for their contaminationwith fungi. Thirty eight fungalspecies belonged to 13 genera in addition to yeasts were isolated andidentified. The highest diversity of fungi were detected from raisins (35species), followed by 27 species isolated from plum, 26 species from figs and18 species on apricot. Eleven species were found common on the four types ofdried fruits. These include Alternaria alternata, Aspergillus carbonarius,A. flavus, A.fumigatus, A.niger,A.parasiticus, Penicillium citrinum, P.expansum,Cladosporium cladosporoides, Emericella nidulans and Eurotium amstelodami.Aflatoxigenic potentials of selected isolates of Aspergillussection Flavi and ochratoxigenic potential of selected isolates from Aspergillussection Nigri were detected by ELISA technique. Aflatoxin was found atlevels from 79.4 to 356ppb whereas, ochratoxin A at levels from 60-106ppb.

Aflatoxins are produced by members of Aspergillus section Flavi. A. flavus and A. parasiticus have been considered the most important aflatoxin producers (Pitt and Hocking, 2009).However, few species of Aspergillus in section Circumdati and in section Nidulans have been also found to produce aflatoxin (Cary et al. 2005).Aflatoxins are potent hepatotoxic and carcinogenic toxin causing health hazards to human and animals (Hedayati et al. 2007) Ochratoxin A was first isolated from A. ochraceus from South Africa in 1965 (Van der Merwe et al. 1965).Subsequent investigations revealed that OTA is produced by several Aspergillus species belonging to sections Circumdati, Flavi and Nigri (Frisvad et al. 2004;Samson et al. 2004).Among Penicillium species, Larsen et al. (2001) reported P. verrucosum and P. nordicum, whereas, Vega et al. (2006) reported P. brevicompactum, P. crustosum, P. olsoni and P. oxalicum as OTA producers.OTA is a potent nephrotoxic mycotoxin that has been linked to kidney problems in both livestock and human populations (Petzinger and Ziegler, 2002).
The aim of this work was to survey the fungi contaminated four types of dried fruits and to asses in vitro the aflatoxin and ochratoxin A producing potential of some fungal isolates using ELISA technique.

Dried fruit samples
Dried fruit samples (30 samples each from apricot, figs, plum and raisins) were collected randomly from local markets in Duhok governorate.The collected samples were put in paper bags and were brought into laboratory for fungal isolation.

Mycological analyses
Larger dried fruits (apricot, figs and plum) were cut aseptically into small pieces, whereas, fruits (raisins) were analyzed as whole piece.The fruit pieces were surface disinfected with 2% sodium hypochlorite for 1 min., and then rinsed with sterile distilled water.Ten pieces were placed onto Dichloran Rose Bengal Chloramphenicol (DRBC) agar medium (Fluka-Germany) and examined daily for growth and sporulation of fungi for 7 days using a stereomicroscope.

Identification of fungi
Pure colonies were established on appropriate media for identification.Majority of detected species were identified to species level based on morphological and cultural characteristics.Fungi other than the genera Aspergillus and Penicillium were identified according to the manuals of Domsch et al.,(1980) and Pitt and Hocking, (2009).
For identification of species in the genera Aspergillus and Penicillium, pure colonies were grown on four media according to Klich (2002) and Samson et al., (2000).The media are as follows: Czapeck Yeast Extract Agar incubated for seven days at 25C˚ (CYA25), Czapeck Yeast Extract Agar incubated for seven days at 37C˚ (CYA37), Czapeck Yeast Extract Agar with 20% Sucrose incubated for seven days at 25C˚ (CY20S), Malt Extract Agar (MEA) incubated for seven days at 25C˚.
Ingredients and preparation of the above five media were mentioned in Klich (2002), Pitt and Hocking (2009).Each medium was supplemented with 250mg / L chlorophenicol (SDI) to suppress bacterial growth.For each culture four plates were used, two of CYA and one each of CY20S, MEA.Each plate was inoculated at the center and incubated in the dark for seven days.One CYA was incubated at 37C˚.The rest were incubated at 25C˚.All species identifications were according to the keys and descriptions provided by Klich (2002); Samson et al., (2004); Frisvad et al., (2004); Samson et al., (2007); and Noonim et al., (2008); Pitt and Hocking (2009).
Isolation frequency of fungal species from samples was calculated by applying the following formula.Isolation frequency % = Number of samples on which a fungus appeared X 100 Total number of tested samples

Aflatoxin extraction from fungal cultures
Production of aflatoxin (AF) by randomly chosen isolates of Aspergillus section Flavi was screened according to the method of Bragulat et al., (2001) by centrally inoculating yeast extract sucrose (YES) plates and then incubated in the dark at 25 o C for 7 days.Agar plug (0.5 cm) diameter was removed from the edges of the centre of the colony and a midway between the edge and the centre of the growing colonies.The three plugs were mixed with 1 ml methanol in a small vial ,shaking vigorously and left at room temperature for 1h, mixed again and the extracts were filtered through milpoore filter (0.22 um) diameter (Millex GP Filter Unit Coringhwohill Co. Ireland ).

Ochratoxin A extraction from fungal cultures
Isolates from Aspergillus and Penicillium genera were evaluated for their ochratoxin A producing potential.The method of Bragulat et al. (2001) for extraxtion from fungal cultures was adopted.The extracts were filtered through milpoore filter (0.22 um) diameter (Millex GP Filter Unit Coringhwohill Co. Ireland).

Aflatoxin and ochratoxin A analysis
The quantitative analysis of AF was performed with the enzyme linked immunosorbent assay (ELISA).The aflatoxin assay was performed according to the instructions provided by the manufacture (Veratox Aflatoxin quantitative test, Neogen Corporation, USA).Aflatoxin produced by isolates was calculated from the standard curve derived from aflatoxin standards and expressed in ppb.OTA assay was performed according to instructions provided by the manufacture (Veratox quantitative ochratoxin test, Neogen Corporation USA).Ochratoxin produced by isolates was calculated from the standard curve derived from ochratoxin standards and expressed in ppb.

RESULTS AND DISCUSSION
The fungi contaminated four types of dried fruit samples collected from Duhok shops and their isolation frequencies were presented in Table 1.Thirty eight fungal species represented 13 genera in addition to yeasts were identified.The highest diversity of fungi were detected from raisins (35 species), followed by 27 species isolated from plum, 26 species from figs and 18 species on apricot.The majority of the recovered species were previously reported from dried fruits in many parts of the world (Zohri and Abdel-Jawad, 1993;Alghalibi and Shater, 2004;Iamnaka et al. 2005Iamnaka et al. , 2007;;Ozer et al. 2012;Sen and Nas, 2013).
Aspergillus was represented by 15 species and thus showed the widest diversity among all recovered genera.Black aspergilli (Aspergillus section Nigri) were represented by 6 species.These include A. aculeatinus.A. aculeatus, A. brasiliensis, A. carbonarius, A. japonicus and A. niger.These species were frequently isolated from soil and from different agricultural commodities in Duhok, north Iraq (Abdullah and Abdullah, 2009;Abdullah and Muhammed, 2011;Saadullah and Abdullah, 2012 a,b,c;Abdullah and Saadullah, 2013).
The most frequently isolated species from apricot was A .niger, followed by A. carbonarius and A. flavus with a percentage frequencies 78%, 40% and 36% respectively, whereas; the most frequently isolated species from plum was A. niger (66.7%), followed by A. flavus (45%) and A. carbonarius (30.3%).Iamanaka et al. (2005) stated that the most frequently detected species from dried plum in Brazil were ,A.niger, followed by Penicillium spp.and A. ochraceus, whereas; samples from dried apricot were not contaminated by any fungi.The two black aspergilli (A.niger and A. carbonarius) were the most fungal species isolated from raisins and with percentage frequency of 92.3% and 60.3% respectively.Black aspergilli were also reported as the most common species on raisins in several studies in different countries (Magnoli et al. 2004;Hakobyan et al.2010;Palumbo et al. 2011) Doster et al. (1996) from figs in California.However, on Turkish dried figs, A. flavus and A. parasiticus in general instances and in very rare cases A. niger and A. fumigatus were detected as the predominant species (Steiner et al. 1988).Javanmard (2010) reported that the most frequent species in Iranian dried figs was A. niger aggregate (90%) followed by A. flavus (63.76%).
Penicillium was second in the number of species isolated from dried fruits and was represented by six species.P. citrinum and P. expansum were the most common species and were detected from the four types of dried fruits.Zohri and Abdel-Jawad (1993) reported that Penicillium was the most predominant genus isolated from dried apricot and prunes in Egypt.The genus was represented by four species of which P. chrysogenum was the most common species in the two dried fruits.Senyuva et al. (2008) isolated P. expansum and P. chrysogenum as the most frequent species on Turkish dried figs, whereas; P. chrysogenum and P. expansum were detected in high frequency on dried figs in Yemen (Alghalibi and Shater, 2004).
The two teleomorphic ascomycetes (Emericella and Eurotium) were represented each by three species.Among them, Emericella nidulans and Eurotium amstelodami were the most frequent species and were detected from the four types of dried fruits.Cladosporium was represented by two species viz C. cladosporoides and C. herborium.The rest of genera were represented by one species each.
Table 2 showed the results of screening Aspergillus section Flavi strains for aflatoxins production abilities and isolates from Aspergillus section Nigri for Ochratoxin A production potential in culture media as detected by ELISA.Two isolates of A. flavus out of four were negative for aflatoxin production, whereas; all tested isolates of A. parasiticus showed positive abilities.The tested isolates from both A. flavus and A. parasiticus showed marked variations in their aflatoxin potential ranging from 79.4 to 334 ppb in A. flavus and from 81 to 356ppb in A. parasiticus.Abdullah and Al-Mousawy (2009) showed that out of 24and 18 isolates of A. flavus obtained from corn grains and sunflower seeds respectively, 15 isolates (62.5%) from corn and 10 isolates (55.5%) from sunflower seeds showed a positive aflatoxin activity.However, in Iraq, more recently Mohammed et al.(2010) showed that 81.8% of A. flavus isolates and 100% of A. parasiticus isolates were positive.Not all strains of Aspergillus section Flavi can produce aflatoxin and the ratio of the nonaflatoxigenic strains to aflatoxin producing strains varied according to the source and location of the isolates (Schroeder and Bolla, 1973;Abdel-Malek et al.1993).
Out of six isolates of Aspergillus section Nigri screened for their ochratoxin A potential, one isolate of A. niger and two isolates of A. carbonarius were positive for ochratoxin A production.Ochratoxin A produced by A. niger isolate 2 and A. carbonarius isolate 1 was 60 and 73 ppb respectively, whereas, A. carbonarius isolate 2 derived from apricot dried fruit showed the highest potential (106ppb).OTA production by black aspergilli isolated from dried fruits have been reported by several studies (Magnoli et al. 2004;Iamanaka et al. 2005;Leong et al. 2006;Palumbo et al. 2011;Heperkan et al. 2012b).In all of these studies, the majority of ochratoxigenic isolates were assigned to A. carbonarius, while very few isolates of A. niger aggregate were produced OTA.

CONCLUSION
The result of this study revealed that dried fruits harbor a diversity of fungal contaminants.Some of these fungi isolated are capable of producing aflatoxins and Ochratoxin A and thus there may be risk through consumption of these dried fruits.Therefore, strict hygienic mycological investigation should be done during harvest, storage and drying to minimize contamination with such fungi.

Table 1 :
Percentage occurrence of fungi on dried fruits as detected on DRBC medium . The most encountered species from dried figs were A. niger, A. flavus, A. carbonarius and A. parasiticus with a percentage frequencies of 73.4%, 66.05, 31.3,31.5% respectively.Embaby et al. (2012) recorded A. niger, A. flavus and A. parasiticus as the most frequent species on dried fig in Egypt.Similar results were reported by

Table 2 :
Quantitative production of aflatoxin and ochratoxin A by Aspergillus species in vitro by ELISA