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Ochratoxin A and ochratoxigenic black Aspergillus species in Tunisian grapes cultivated in different geographic areas
Food Control 25 (2012) 75e80
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Ochratoxin A and ochratoxigenic black Aspergillus species in Tunisian grapes cultivated in different geographic areas Salma Lasram a, *, Souheib Oueslati a, Ahmed Mliki a, Abdelwahed Ghorbel a, Phillipe Silar b, Samir Chebil a a b
Laboratory of Molecular Physiology of Plants e Borj-Cedria Biotechnology Center, B.P. 901, Hammam-Lif 2050, Tunisia Institut de Génétique et Microbiologie UMR8621, Université de Paris-Sud 11, 91405 Orsay Cedex, France
a r t i c l e i n f o
a b s t r a c t
Article history: Received 6 April 2011 Received in revised form 27 September 2011 Accepted 4 October 2011
This paper summarizes the results of a large study on the occurrence of ochratoxigenic fungi and Ochratoxin A from wine and table grapes in Tunisia. Our results revealed that Aspergillus section Nigri were the unique potential OTA producing fungi isolated from grapes. Isolates belonging to Aspergillus niger aggregate were the most abundant species followed by Aspergillus carbonarius isolates, then uniseriate aspergilli. A. carbonarius presented the highest percentage of OTA-positive strains (97%) whereas only 3% of A. niger aggregate isolates were OTA positive. Grapes were analysed for their OTA content and 58% of them contained detectable levels of OTA, between 0.05 and 5.85 mg/l. Only 4 samples out of 39 exceeded the OTA limit of 2 mg/l fixed by the EU for wine and grape juices. The most contaminated grapes were those from RafeRaf region located in the North-Est and characterized by a humid climate. Grapes from the Regueb region, characterized by an arid climate, were rarely contaminated. Furthermore, A. carbonarius, which is the main OTA producer fungi on grapes, was rarely isolated in Regueb. Ó 2011 Elsevier Ltd. All rights reserved.
Keywords: Ochratoxin A Grape HPLC Black aspergilli Aspergillus carbonarius
1. Introduction Ochratoxin A (OTA) is a mycotoxin with nephrotoxic, teratogenic, and immunosuppressive properties (Pitt et al., 2001). Ochratoxin A was first detected in wine by Zimmerli and Dick (1995, 1996). Since then, presence of OTA in grape and its derived products, such as dried vine fruits, grape juices and wine has been worldwide reported (Mateo, Medina, Mateo, Mateo, & Jiménez, 2007; Varga & Kozakiewicz, 2006). The European Union legislation authorities have introduced an OTA limit of 2 mg/l in wine, must or grape juice (European Commission, 2005). Ochratoxin A is produced by species in Aspergillus sections Nigri (black aspergilli) and Circumdati, commonly found in warm and tropical climates. Penicillium verrucosum is considered as the main source in temperate climates and is more frequently associated with cereals (Pitt & Hocking, 1997). Different studies have suggested that black aspergilli are the main species producing OTA in grapes (Battilani, Barbano et al., 2006; Da Rocha Rosa et al., 2002; Leong et al., 2006). These authors found that among black aspergilli, Aspergillus carbonarius was the main species producing OTA in grapes. Some members of the Aspergillus niger aggregate have also been found to produce OTA. In the A. niger aggregate, it has always been difficult to distinguish one taxon from * Corresponding author. Tel.: þ216 79412738; fax: þ216 79412638. E-mail address: [email protected] (S. Lasram). 0956-7135/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodcont.2011.10.006
another by morphological means because the differences are very subtle. A method that differentiates the A. niger aggregate isolates into two RFLP patterns, type N and type T, corresponding to the type species of A. niger and Aspergillus tubingensis, respectively was described by Accensi, Cano, Figuera, Abarca, and Cabanes (1999). It was observed that wines from Southern Europe and North Africa, with Mediterranean climates, contained more OTA than those originating from the more temperate regions of central Europe (Otteneder & Majerus, 2000; Zimmerli & Dick, 1996). In fact, it was found that climate has an effect on the occurrence of OTA and OTAproducing fungi in grapes (Battilani, Barbano et al., 2006; Battilani, Magan, & Logdrieco, 2006; Serra, Analia, Pedro, & Venacio, 2006). The aim of this work was to survey the occurrence of OTA and to identify the ochratoxigenic moulds in Tunisian vineyards located in different viticultural regions of Tunisia with singular climate. The influence of climate and the possible interactions between ochratoxigenic species, OTA content of grapes and meteorological factors were also investigated. 2. Materials and methods 2.1. Grape sampling The survey was carried out over 3 years (2005, 2006, 2007) and involved four vineyards located in four viticultural regions of Tunisia
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S. Lasram et al. / Food Control 25 (2012) 75e80
(Belli, Baddar, Regueb, Raf-Raf) with specific climate. Six grape varieties in each vineyard, representative of their grape-growing area were selected for further study. The varieties included wine and table grapes. Grape samples were harvested at maturity stage (End of July/August) depending on the region and variety. Ten plants along diagonal transects were chosen in each vineyard and one bunch of grapes was taken from each plant. Bunches were kept in sterile bags and transported in cooled boxes (4 C) to the laboratory for analysis. During the three years of the survey, data on climatic conditions during the maturation of grape (mean temperatures, relative humidity and rainfall) were provided by the National Institute of Meteorology (INM) (Table 1).
identification of the isolates, the nucleotide sequences obtained were compared with those deposited in the data of the National Center for Biotechnology and Information (NCBI) using the BLAST search tool (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The identification of the species was carried out based on the best similarity score. The discrimination between the species A. niger and A. tubingensis was performed by rDNA-RFLP in silico. This analysis was based on the presence of the restriction site for the endonuclease RsaI (GT/AC) directly in the rDNA sequences from A. niger (Type N) and its absence in A. tubingensis (Type T) (Accensi et al., 1999). Analysis was performed with Cluster X (version 2.0). 2.3. Fungal OTA producing ability
2.2. Fungal isolation and characterisation Three berries were randomly sampled from each bunch and plated directly on Petri dishes containing Dichloran Rose Bengal Chloramphenicol medium (DRBC). Plates were incubated for 7 days at 25 C. Colonies of developing fungi belonging to Aspergillus section Nigri were transferred to Petri dishes containing Czapek Yeast Agar (CYA) and incubated at 25 C for 5 days. After the incubation period, Aspergillus isolates were examined through macroscopic and microscopic observation and classified according to the morphology of their spores and conidial heads into three groups: uniseriates, A. niger aggregate and A. carbonarius (Abarca, Accensi, Cano, & Cabanes, 2004). Ochratoxin A producing isolates belonging to the A. niger aggregate were identified by sequencing the internal transcribed spacer (ITS1-5.8S-ITS2) of ribosomal DNA (rDNA). Fungal DNA was extracted as described by Lecellier and Silar (1994). PCR amplification was performed as described by White, Bruns, Lee, and Taylor (1990) using an Eppendorf MastercyclerÒ thermal cycler (France). PCR products were used as a template for sequencing with the ITS1/ITS4 primers as prescribed by the manufacturer (Genome Express, Meylan France). The resulting sequences were aligned with Clustal X of multiple sequence alignment computer program (Thompson, Gibson, Plewniak, Jeanmougin, & Higgins, 1997). For the
The method used was adapted from that developed by Bragulat, Abarca, and Cabanes (2001). Aspergillus isolates were inoculated on three points in CYA medium and incubated for 5 days at 25 C. Three agar plugs (6 mm) were cut from the inner, middle and outer area of the colony and introduced in a vial with 1 ml of methanol. After 60 min, the extracts were filtered (MillexR SLHV 013NK, Millipore, Bedford, Massachusetts, USA) and injected into an HPLC system with a fluorescence detector (lexc 330 nm; lem 460 nm) and a C18 column (Waters Spherisorb 5 mm, ODS2, 4.6 250 mm, Milford MA, USA). The mobile phase was composed of acetonitrile:water:acetic acid (57:41:2). Detection and quantification limits (LOD and LOQ) were 0.02 mg OTA/l and 0.04 mg OTA/l, respectively. The recovery for OTA on CYA media was 90 3.5% (mean SD., n ¼ 3). 2.4. Analysis of OTA in grapes After selecting berries for fungal isolation, the ten bunches collected from each vineyard and variety were manually crushed and the resulting musts (n ¼ 72) were analyzed for OTA content according to the method of Bezzo, Maggiorotto, and Testa (2002). Musts (100 ml) were first centrifuged (2500 g, 20 min), liquid phase
Table 1 Meteorological data from the studied regions during grape maturation (06: June; 07: July; 08: August) in 2005, 2006 and 2007. Year
2005
2006
2007
Month
06
07
08
06
07
08
06
07
08
Belli
T ( C)a Tmin ( C)b Tmax ( C)c R.H. (%)d R (mm)e
24.9 19.4 30.2 69 8.0
28.2 22.3 32.7 66 2.3
27.2 21.9 31.6 69 45.7
24.2 20.1 29.1 75 35.8
26.6 18.6 28.9 68 7.8
29.7 22.4 33.5 70 19.8
28.9 22.4 32.3 68 27.1
25.1 19.7 28.5 76 83.4
29.6 19.9 30.4 ** 35.0
Baddar
T ( C) Tmin ( C) Tmax ( C) R.H. (%) R (mm)
23.2 16.1 29.8 65 6.8
26.8 20.4 32.2 62 0.6
27.3 20.6 31.6 ** 34.0
27.1 21.6 29.1 ** 30.8
27.2 19.6 31.3 67 0.0
29.8 21.0 33.5 ** 180.1
28.2 21.6 32.2 65 44.5
25.3 18.6 28.8 74 66.9
28.8 19.1 30.0 69 30.0
Raf-Raf
T ( C) Tmin ( C) Tmax ( C) R.H. (%) R (mm)
23.7 18.1 29.9 75 17.0
26.5 21.0 32.2 73 0.7
25.8 21.3 31.0 75 8.2
23.9 18.5 29.1 78 12.1
26.9 17.3 30.8 70 7.1
26.1 20.6 32.8 74 0.0
23.7 21.6 30.9 75 1.6
26.2 18.6 28.9 80 70.8
26.2 18.2 29.5 78 16.1
Regueb
T ( C) Tmin ( C) Tmax ( C) R.H. (%) R (mm)
26 18.6 33.4 55 11.1
30.2 21.6 38.4 53 1.7
28.1 20.9 35.8 52 6.6
27.2 17.2 33.9 43 4.0
29.9 21.1 36.9 49 12.2
29.2 20.8 36.2 54 13.2
28.3 18.5 35.2 50 23.6
29.6 19.3 36.8 44 0.0
29.8 21.2 37.0 53 24.0
**lack data. a T: mean daily temperature. b T max: mean daily maximum temperature. c T min: mean daily minimum temperature. d R.H.: mean daily relative humidity. e R: Rainfall
S. Lasram et al. / Food Control 25 (2012) 75e80
was, brought to a pH 7.4 (NaOH 4 M), filtered and applied to an immunoaffinity column (Ochraprep, Rhône Diagnostics Technologies Ltd, Glasgow, UK). The column was then washed with 20 ml of distilled water and dried up with an air stream. Elution was realized with 1.5 ml of methanol/acid acetic (98/2) solution. The eluate was evaporated to dryness, dissolved with 0.8 ml of mobile phase and injected into the HPLC system equipped with a fluorescence detector (lexc 230 nm; lem 458 nm) and a reversed-phase column C18 (Waters Spherisorb 5 mm, ODS 2, 4.6 250 mm, Milford, MA, USA). The mobile phase was composed by a sodium acetate 4 mM/ acetic acid (19:1: v/v) solution (52%) and acetonitrile (48%). Detection and quantification limits (LOD and LOQ) were 0.03 mg OTA/l and 0.05 mg OTA/l, respectively. The recovery for OTA on grape juice samples was 95 4% (mean SD., n ¼ 3).
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dominant group in the four regions, especially in the region of Regueb where they represent 94.5% of the isolated black aspergilli. In contrast, uniseriate isolates were recovered in small numbers in the different regions. At each sampling year, the highest level of A. carbonarius infection was observed in the region of RafeRaf where it represented a mean of 43% of black aspergilli isolated. The level of contamination by this specie was lower in the regions of Baddar and Belli with, respectively, 30.9% and 20.8% of black aspergilli. The incidence of A. carbonarius was very low in Regueb region where only four A. carbonarius (1% of black aspergilli) were isolated during the three sampling years. 3.2. Ochratoxigenic potential of black Aspergillus species
Statistic analyses were performed with Statistica software (version 7.0, StatSoft, Inc., Tulsa, OK, USA). The distribution of variables was tested for normality using the KolmogoroveSmirnov test. In order to study the significance of the considered factors (year and region), the number of Aspergilli section Nigri and the OTA content of grapes were analyzed using analysis of variance with post-hoc test (Duncan), for variables with normal distribution and KruskaleWallis test (with approximation to chi-square test) with multiple comparison test, for variables with non-normal distribution. Correlation analysis (correlation coefficients of Spearman rs and Pearson rp for variables with non-normal and normal distributions, respectively) were used to correlate the presence of A. carbonarius and the OTA content in berries.
A total of 1242 black aspergilli (270 A. carbonarius, 934 A. niger aggregate and 29 A. uniseriate) was tested for OTA production. Among the A. carbonarius isolates, OTA producers represented 99.5% (268/270); whereas, only 3.2% (29/934) of the A. niger aggregate isolates were ochratoxigenic. No OTA production by the uniseriate isolates was detected. Data on OTA production by the A. carbonarius isolates showed that 67.27% of the isolates produced less than 1.0 mg/g CYA, 29.09% between 1.0 and 5.0 mg/g CYA and 4.55% more than 5 mg/g CYA. The average OTA production was about 1.45 mg/g CYA. For the ochratoxigenic A. niger aggregate isolates, 89.47% produced less than 0.1 mg/g CYA and 10.53% between 0.1 and 1.0 mg/g CYA. The average OTA production was about 0.07 mg/g CYA. The ochratoxigenic isolates belonging to the A. niger aggregate group were classified in the two patterns N and T, corresponding to A. niger and A. tubingensis. Among the 21 isolates analyzed, 13 showed the T type pattern and 8 showed the N type pattern.
3. Results
3.3. OTA contamination of grapes
3.1. Distribution of black Aspergillus species
The data related to the OTA concentrations in musts (Table 3) showed that at maturity stage, 54% (39/72) of analyzed samples were contaminated with 10% (4/39) of OTA positive samples exceeding the EU restrictive limit (>2 mg/l). These samples came from RafeRaf (5.44 and 5.84 mg OTA/l) and Baddar (3.27 and 2.23 mg OTA/l) regions. The non parametric KruskaleWallis analysis showed that the OTA concentration in grape was not significantly different between the sampling years (P > 0.05) and significantly different between regions (P < 0.05). Eighty three percent (15/18) of grapes from RafeRaf were OTA contaminated in the range of 0.06e5.85 mg/l. In Baddar, 83% (15/18) of grape samples contained between 0.06 and 3.27 mg OTA/l. Forty four percent (8/18) of grapes from Belli contained between 0.09 and 0.96 mg OTA/l. Among the grape samples originating from Regueb, only one sample was
2.5. Statistical analysis
A total of 1242 black aspergilli were isolated along the three sampling years (2005, 2006 and 2007) distributed in the four regions from Tunisia (Belli, Baddar, Raf-Raf and Regueb). The analysis of variance revealed that the single factor region and the interaction year and region presented significant differences in the number of black aspergilli isolated (P < 0.01). The sampling year does not affect the fungal colonisation grapes (P > 0.05). Grapes from Regueb were significantly the most contaminated with black aspergilli each year. No statistical differences were found between the number of these moulds in the regions of Baddar, Belli and RafeRaf (P ¼ 0.05). The distribution of black aspergilli isolates is presented in Table 2. Isolates of A. niger aggregate formed the
Table 2 Percentage of black aspergilli isolates. Numbers in brackets are the number of black aspergilli isolates of each group isolated/the total number of black aspergilli. Group
Year
Region Baddar
Raf-Raf
Regueb
Belli
Uniseriate
2005 2006 2007 Total
2.9 (4/136) 4.8 (4/84) 0 (0/77) 2.5 (8/297)
1.0 1.1 1.6 1.2
1.9 (2/104) 10.0 (13/130) 0.7 (1/150) 4.2 (16/384)
0 (0/101) 0 (0/115) 2.3 (2/87) 0.7 (2/303)
A. carbonarius
2005 2006 2007 Total
19.9 23.8 58.4 30.9
(27/136) (20/84) (45/77) (92/297)
24.8 41.5 74.6 43.0
(25/101) (39/94) (47/63) (111/258)
2.9 (3/104) 0 (0/130) 0.7 (1/150) 1.0 (4/384)
20.8 22.6 18.4 20.8
(21/101) (26/115) (16/87) (63/303)
A. niger aggregate
2005 2006 2007 Total
76.5 60.7 36.4 61.6
(104/136) (51/84) (28/77) (183/297)
76.2 57.4 30.2 58.1
(77/101) (54/94) (19/63) (150/258)
94.2 (98/104) 90 (117/130) 98.7 (148/150) 94.5 (363/384)
79.2 77.4 79.3 78.5
(80/101) (89/115) (69/87) (238/303)
(1/101) (1/94) (1/63) (3/258)
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S. Lasram et al. / Food Control 25 (2012) 75e80
Table 3 OTA concentrations in grapes collected from the four regions in the different years (mg/l). Region
Variety
Sampling year 2005
2006
2007
Baddar (North)
Muscat Alexandrie Cabernet Sauvignon Ugni Blanc Italia Syrah Carignan
0.97 0.14 0.06 0.17 0.38
3.27 1.53 1.21 0.24 0.12 0.16
0.25
Belli (North)
Chardonnay Muscat Alexandrie Cabernet Sauvignon Italia Syrah Carignan
0.18
0.13 0.35 0.10 0.77
0.96
Raf-Raf (North-Est)
Rezeki Bezoul Khadem Akhal Raf-Raf Marsaoui Bid Hmam Muscat Raf-Raf
0.1 0.12
1.49
0.96 5.85 0.27
Italia Victoria Sultanine Superior Seedless 1 Matilde Superior Seedless 2
Regueb (Center)
a
LQ ¼ 0.05 mg/l.
contaminated with OTA at a very low concentration (0.05 mg/l). The multiple comparison test (P ¼ 0.05) showed that the OTA contamination of grape is significantly different between Regueb region and the regions of Baddar and RafeRaf. A significant difference for OTA contamination was also found between Belli and RafeRaf regions. Further to the KruskaleWallis analysis which doesn’t take into account the OTA amounts but only their ranking, the distribution of the OTA concentrations in the grapes (Fig. 1) showed that higher OTA concentrations were detected in grapes from RafeRaf with 40% of samples containing between 0.5 and 2 mg OTA/l and 13% containing more than 2 mg OTA/l. For grapes originating from Baddar, 20% of samples were contaminated in the range of 0.5e2 mg OTA/l and 13% of samples exceeded 2 m OTA/l.
Fig. 1. Number of grape samples from the different regions contaminated in different range of OTA concentrations.
This level of contamination is higher than observed in Belli were 75% of samples contained less than 0.5 mg OTA/l and 25% were contaminated with concentrations ranging between 0.5 and 1 mg OTA/l. 3.4. Correlation between the occurrence of OTA and A. carbonarius The numbers of A. carbonarius isolated from grapes were correlated to the OTA concentrations. The coefficient of correlation of Spearman was calculated for values from each variety. The incidence of A. carbonarius was positively correlated with the average OTA concentrations for each variety (rp ¼ 0.473, P < 0.0001). The coefficient of correlation of Pearson was calculated for mean values from each region and sampling year. A higher positive correlation was established (rS ¼ 0.731, P < 0.01). 4. Discussion Several surveys carried out in recent years in various countries reported that the main fungi involved in ochratoxin A contamination of grapes and derived products are black aspergilli (Aspergillus section Nigri). The A. carbonarius specie was recognized as the dominant contributor to OTA contamination of grape in Mediterranean countries (Battilani, Barbano et al., 2006) and in Australia (Leong et al., 2006). However, some authors considered that A. niger aggregate, may slightly contribute to OTA contamination of grape in Mediterranean countries; as this group is by far the most common species of Aspergillus present in vineyards (Battilani, Giorni, & Pietri, 2003; Serra, Abrunhosa, Kozakiewicz, & Venancio, 2003). Others studies conducted in South American countries concluded that A. niger aggregate group was an important OTA-producer fungi in grape (Da Rocha Rosa et al., 2002; Magnoli, Violante, Combina, Palacio, & Dalcero, 2003). In our study, the frequency of toxigenicity among A. niger aggregate, A. carbonarius and uniseriate Aspergillus isolates from Tunisian vineyards was similar to results reported from European Mediterranean countries (Battilani, Barbano et al., 2006). The highest incidence of ochratoxigenic isolates and OTA production was recorded for the A. carbonarius isolates. This fact confirmed that A. carbonarius is the main specie responsible for OTA contamination in Tunisian grapes, for both wine and table varieties. Few A. niger aggregate isolates were able to produce OTA at very low levels. The molecular characterization of these isolates showed that they were represented with both A. tubingensis and A. niger confirming the ability of A. tubingensis specie to produce OTA (Medina, Mateo, Lòpez-Ocaña, Valle-Algarra, & Jiménez, 2005; Perrone et al., 2006). The OTA analysis of grapes showed the presence of the toxin in both wine and table grapes at the three sampling years. Almost half of samples were contaminated with OTA which is similar to the level of contamination detected in Southern Italy (Lucchetta et al., 2010). Among the OTA positive samples, 4 out of 39 exceeded 2 mg/l. However, it’s important to note that grape collected at maturity will undergo several stages (transport, storage, vinification, drying) which may influence the OTA final concentration of grape product. Several factors affect the grape colonization by ochratoxigenic moulds and the OTA contamination, in particular, geographic and climatic conditions. In several Europeans countries, southern regions located in the Mediterranean basin, were found to be particularly affected (Battilani, Barbano et al., 2006; Battilani, Magan et al., 2006). Tunisia is a small country with an extended Mediterranean coast, however, areas of grape planting and production are characterized by different climatic characteristics. The vineyards selected for this study are representative of the different climatic regions existing in the country. The region of Raf-Raf is a coastal region located in the North-Est of the country. It is characterized by
S. Lasram et al. / Food Control 25 (2012) 75e80
a sub-humid climate. The grape culture in this region is characterized by the presence of an important number of local varieties used essentially for fresh consumption and for dried raisins. Baddar and Belli are located in Cap-Bon region (North) which is the main viticulture area in Tunisia and especially for wine grape. This area has a typically semi-arid Mediterranean climate. The last region studied is Regueb which is a new viticultural area in the center of the country. It’s characterized by an arid climate and grapes cultivated in this area are essentially table varieties. In the present work, the influence of the specific geographic localization and the climate of the vineyards on the occurrence of ochratoxigenic moulds and OTA contamination of grape was significant. Black aspergilli were the most abundant in Regueb region due to the highest number of A. niger aggregate isolates from this region compared to the northern regions (RafeRaf, Belli and Baddar). On the other hand, A. carbonarius was rarely isolated from Regueb vineyards. Thus, grapes collected from this region were rarely contaminated with OTA and the level detected was very low. Data on climatic characteristics of studied regions during the maturation period of grape (Table 1) showed that Regueb region has the highest values of mean temperatures (26e30.2 C), mean maximum temperatures (31.2e38.4 C) and the lowest value of mean relative humidity (43e62%). A. niger aggregate isolates are the most adapted to the heat and dryness. On the contrary, A. carbonarius was unable to grow in such climatic conditions. In a previous study (Lasram et al., 2010) the rare A. carbonarius strains isolated from Regueb region showed that under dry and hot conditions, they grew significantly faster than A. carbonarius strains those coming from northern regions. These isolates could have developed a tolerance towards arid climate. It was also found that optimal conditions for the growth of A. carbonarius isolated from Tunisian vineyards were high water activity (0.99) and moderate temperature (30 C). For toxin production optimal conditions were a high water activity (0.99) and temperatures in the range of 15e25 C. These results explain, in part, the abundance of A. carbonarius in the region of Raf-Raf and the higher OTA contamination of grapes collected from this region. Indeed, RafeRaf region is dominated by a Mediterranean influence, the temperatures in the summer are usually moderate (mean temperatures 23.7e26.9 C, mean maximum temperatures 28.9e32.2 C) and it is also very humid (HR 70e80%). These climatic conditions seem to be very favourable to A. carbonarius growth and OTA accumulation. In Baddar and Belli regions, characterized by a semi-arid climate, the incidence of A. carbonarius was continually similar (approximatively 20% of black aspegilli), except in 2007 where the fungi were particularly more frequent in Baddar. These two regions have also Mediterranean influences but with lower humidity levels and higher mean and maximum temperatures than those of Raf-Raf (Table 1). The higher incidence of A. carbonarius in 2007 cannot be correlated with any change in climatic data during this year. For OTA contamination, no statistic difference was found between the two regions, however, the distribution frequency of the OTA concentrations in the grape juices collected in the different regions (Fig. 1) showed that grapes originating from Baddar vineyards were more contaminated with OTA than those from Belli. Thus, the occurrence of ochratoxigenic fungi in the vineyard and the incidence of OTA in grapes depend not only on climatic conditions but also on other factors related to the variety and crop management, such as training system, irrigation and phytosanitary treatments, which influence on the ecosystem of the vine. However, our study revealed a strong correlation between the presence of ochratoxin producing strains on grapes and the finding of OTA in musts when considering average values for each region and sampling year (rS ¼ 0.731). In fact, OTA was not always detected in grapes, even in those colonized by ochratoxigenic fungi, but the presence of
79
A. carbonarius in a region has an impact on the level of OTA contamination in grapes cultivated in the same region. The results of the present work demonstrated the high risk of OTA and fungal contamination in Northern Tunisia, especially for coastal regions. Acknowledgement Authors are grateful to the Tunisian Ministry of Higher Education, Scientific Research and Technology for his financial support in the framework of the National Research program “Qualité du raisin et Ochratoxine A en Tunisie”. We are also thankful to the CTAA (Centre Technique de l’Agro-alimentaire) for their cooperation and assistance. References Abarca, M. L., Accensi, F., Cano, J., & Cabanes, J. F. (2004). Taxonomy of black aspergilli. Antonie Van Leeuwenhoek, 86, 33e49. Accensi, F., Cano, J., Figuera, L., Abarca, M. L., & Cabanes, F. J. (1999). New PCR method to differentiate species in the Aspergillus niger aggregate. FEMS Microbiology Letters, 180, 191e196. Battilani, P., Barbano, C., Marín, S., Sanchis, V., Kozakiewicz, Z., & Magan, N. (2006). Mapping of Aspergillus section Nigri in southern Europe and Israel based on geostatistical analysis. International Journal of Food Microbiology, 111, S72eS82. Battilani, P., Giorni, P., & Pietri, A. (2003). Epidemiology of toxin-producing fungi and ochratoxin A occurrence in grape. European Journal of Plant Pathology, 109, 715e722. Battilani, P., Magan, N., & Logrieco, A. (2006). European research on ochratoxin A in grapes and wine. International Journal of Food Microbiology, 111, 52e54. Bezzo, G., Maggiorotto, G., & Testa, F. (2002). A method for the determination of specific mycotic contaminants random occurring in wines. Paris: Office International de la Vigne et du Vin. Bragulat, M. R., Abarca, M. L., & Cabanes, F. J. (2001). An easy screening method for fungi producing ochratoxin A in pure culture. International Journal of Food Microbiology, 71, 139e144. Da Rocha Rosa, C. A., Palscios, V., Combia, M., Fraga, M. E., De Oliveira Rekson, A., Magnoli, C. E., et al. (2002). Potential ochratoxin A producers from wine grapes in Argentina and Brazil. Food Additives and Contaminants, 19, 408e414. European Commission. (2005). Commission regulation (EC) No 123/2005 of 26 January 2005 amending regulation (EC) No 466/2001 as regards ochratoxin A. Official Journal of European Union, L25, 5e36. Lasram, S., Oueslati, S., Valero, A., Marin, S., Ghorbel, A., & Sanchis, V. (2010). Water activity and temperature effects on fungal growth and ochratoxin A production by Ochratoxigenic Aspergillus carbonarius isolated from Tunisian grapes. Journal of Food Science, 75(2), M89eM97. Lecellier, G., & Silar, P. (1994). Rapid methods for nucleic acids extraction from Petri dish-grown mycelia. Current Genetics, 25(2), 122e123. Leong, S. L., Hocking, A. D., Pitt, J. I., Kazi, B. A., Emmett, R. W., & Scott, E. S. (2006). Australian research on ochratoxigenic fungi and ochratoxin A. International Journal of Food Microbiology, 111, S10eS17. Lucchetta, G., Bazzo, I., Dal Cortivo, G., Stringher, L., Bellotto, D., Borgo, M., et al. (2010). Occurrence of Black Aspergilli and Ochratoxin A on grapes in Italy. Toxins, 2, 840e855. Magnoli, C., Violante, M., Combina, M., Palacio, G., & Dalcero, A. (2003). Mycoflora and ochratoxin-producing strains of Aspergillus section Nigri in wine grapes in Argentina. Letters in Applied Microbiology, 37, 179e184. Mateo, R., Medina, A., Mateo, E. M., Mateo, F., & Jiménez, M. (2007). An overview of ochratoxin A in beer and wine. International Journal of Food Microbiology, 119, 79e83. Medina, A., Mateo, R., Lòpez-Ocaña, L., Valle-Algarra, F. M., & Jiménez, M. (2005). Study of Spanish mycobiota and ochratoxin A production by isolates of Aspergillus tubingensis and other members of Aspergillus Section Nigri. Applied and Environmental Microbiology, 71(8), 4696e4702. Otteneder, H., & Majerus, P. (2000). Occurrence of ochratoxin A in wines: influence of the type of wine and its geographical origin. Food Additives and Contaminants, 17, 793e798. Perrone, G., Mule, G., Susca, A., Battilani, P., Pietri, A., & Logrieco, A. (2006). Ochratoxin A production and Amplified Fragment Length Polymorphism analysis of Aspergillus carbonarius, Aspergillus tubingensis and Aspergillus niger strains isolated from grapes in Italy. Applied and Environmental Microbiology, 72(1), 680e685. Pitt, J. I., & Hocking, A. D. (1997). Fungi and food spoilage (2nd ed.). London: Blackie Academic and Professional. Pitt, D. B., Plestina, J. I., Shepard, R., Solfrizzo, G., Verger, M., & Walker, P. J. P. (2001). Safety evaluation of certain mycotoxins in food. In Joint FAO/WHO Expert Committee on Food Additives (JECFA) (pp. 281). Rome: Food and Agriculture Organization. Serra, R., Abrunhosa, L., Kozakiewicz, Z., & Venancio, A. (2003). Black Aspergillus species as ochratoxin A producers in Portuguese wine grapes. International Journal of Food Microbiology, 88, 63e68.
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Serra, R., Analia, L., Pedro, A., & Venacio, A. (2006). Influence of the region of origin on the mycobiota of grapes with emphasis on Aspergillus and Penicillium species. Mycological Research, 110, 971e978. Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F., & Higgins, D. G. (1997). The Clustal X windows interface: Flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acid Research, 24, 4876e4882. Varga, J., & Kozakiewicz, Z. (2006). Ochratoxin A in grapes and grape-derived products. Trends in Food Science and Technology, 17, 72e81. White, T. J., Bruns, T., Lee, S., & Taylor, J. W. (1990). Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In M. A. Innis,
D. H. Gelfand, J. J. Sninsky, & T. J. White (Eds.), PCR protocols: A guide to methods and applications (pp. 482). New York: Academic Press Inc. Zimmerli, B., & Dick, R. (1995). Determination of ochratoxin A at the ppt level in human blood, serum, milk and some foodstuffs by high-performance liquid chromatography with enhanced fluorescence detection and immunoaffinity column clean-up: methodology and Swiss data. Journal of Chromatography, B, 666, 85e99. Zimmerli, B., & Dick, R. (1996). Ochratoxin A in table wine and grapejuice: occurrence and risk assessment. Food Additives and Contaminants, 13, 655e668.
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Ochratoxin A and ochratoxigenic black Aspergillus species in Tunisian grapes cultivated in different geographic areas Salma Lasram a, *, Souheib Oueslati a, Ahmed Mliki a, Abdelwahed Ghorbel a, Phillipe Silar b, Samir Chebil a a b
Laboratory of Molecular Physiology of Plants e Borj-Cedria Biotechnology Center, B.P. 901, Hammam-Lif 2050, Tunisia Institut de Génétique et Microbiologie UMR8621, Université de Paris-Sud 11, 91405 Orsay Cedex, France
a r t i c l e i n f o
a b s t r a c t
Article history: Received 6 April 2011 Received in revised form 27 September 2011 Accepted 4 October 2011
This paper summarizes the results of a large study on the occurrence of ochratoxigenic fungi and Ochratoxin A from wine and table grapes in Tunisia. Our results revealed that Aspergillus section Nigri were the unique potential OTA producing fungi isolated from grapes. Isolates belonging to Aspergillus niger aggregate were the most abundant species followed by Aspergillus carbonarius isolates, then uniseriate aspergilli. A. carbonarius presented the highest percentage of OTA-positive strains (97%) whereas only 3% of A. niger aggregate isolates were OTA positive. Grapes were analysed for their OTA content and 58% of them contained detectable levels of OTA, between 0.05 and 5.85 mg/l. Only 4 samples out of 39 exceeded the OTA limit of 2 mg/l fixed by the EU for wine and grape juices. The most contaminated grapes were those from RafeRaf region located in the North-Est and characterized by a humid climate. Grapes from the Regueb region, characterized by an arid climate, were rarely contaminated. Furthermore, A. carbonarius, which is the main OTA producer fungi on grapes, was rarely isolated in Regueb. Ó 2011 Elsevier Ltd. All rights reserved.
Keywords: Ochratoxin A Grape HPLC Black aspergilli Aspergillus carbonarius
1. Introduction Ochratoxin A (OTA) is a mycotoxin with nephrotoxic, teratogenic, and immunosuppressive properties (Pitt et al., 2001). Ochratoxin A was first detected in wine by Zimmerli and Dick (1995, 1996). Since then, presence of OTA in grape and its derived products, such as dried vine fruits, grape juices and wine has been worldwide reported (Mateo, Medina, Mateo, Mateo, & Jiménez, 2007; Varga & Kozakiewicz, 2006). The European Union legislation authorities have introduced an OTA limit of 2 mg/l in wine, must or grape juice (European Commission, 2005). Ochratoxin A is produced by species in Aspergillus sections Nigri (black aspergilli) and Circumdati, commonly found in warm and tropical climates. Penicillium verrucosum is considered as the main source in temperate climates and is more frequently associated with cereals (Pitt & Hocking, 1997). Different studies have suggested that black aspergilli are the main species producing OTA in grapes (Battilani, Barbano et al., 2006; Da Rocha Rosa et al., 2002; Leong et al., 2006). These authors found that among black aspergilli, Aspergillus carbonarius was the main species producing OTA in grapes. Some members of the Aspergillus niger aggregate have also been found to produce OTA. In the A. niger aggregate, it has always been difficult to distinguish one taxon from * Corresponding author. Tel.: þ216 79412738; fax: þ216 79412638. E-mail address: [email protected] (S. Lasram). 0956-7135/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodcont.2011.10.006
another by morphological means because the differences are very subtle. A method that differentiates the A. niger aggregate isolates into two RFLP patterns, type N and type T, corresponding to the type species of A. niger and Aspergillus tubingensis, respectively was described by Accensi, Cano, Figuera, Abarca, and Cabanes (1999). It was observed that wines from Southern Europe and North Africa, with Mediterranean climates, contained more OTA than those originating from the more temperate regions of central Europe (Otteneder & Majerus, 2000; Zimmerli & Dick, 1996). In fact, it was found that climate has an effect on the occurrence of OTA and OTAproducing fungi in grapes (Battilani, Barbano et al., 2006; Battilani, Magan, & Logdrieco, 2006; Serra, Analia, Pedro, & Venacio, 2006). The aim of this work was to survey the occurrence of OTA and to identify the ochratoxigenic moulds in Tunisian vineyards located in different viticultural regions of Tunisia with singular climate. The influence of climate and the possible interactions between ochratoxigenic species, OTA content of grapes and meteorological factors were also investigated. 2. Materials and methods 2.1. Grape sampling The survey was carried out over 3 years (2005, 2006, 2007) and involved four vineyards located in four viticultural regions of Tunisia
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S. Lasram et al. / Food Control 25 (2012) 75e80
(Belli, Baddar, Regueb, Raf-Raf) with specific climate. Six grape varieties in each vineyard, representative of their grape-growing area were selected for further study. The varieties included wine and table grapes. Grape samples were harvested at maturity stage (End of July/August) depending on the region and variety. Ten plants along diagonal transects were chosen in each vineyard and one bunch of grapes was taken from each plant. Bunches were kept in sterile bags and transported in cooled boxes (4 C) to the laboratory for analysis. During the three years of the survey, data on climatic conditions during the maturation of grape (mean temperatures, relative humidity and rainfall) were provided by the National Institute of Meteorology (INM) (Table 1).
identification of the isolates, the nucleotide sequences obtained were compared with those deposited in the data of the National Center for Biotechnology and Information (NCBI) using the BLAST search tool (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The identification of the species was carried out based on the best similarity score. The discrimination between the species A. niger and A. tubingensis was performed by rDNA-RFLP in silico. This analysis was based on the presence of the restriction site for the endonuclease RsaI (GT/AC) directly in the rDNA sequences from A. niger (Type N) and its absence in A. tubingensis (Type T) (Accensi et al., 1999). Analysis was performed with Cluster X (version 2.0). 2.3. Fungal OTA producing ability
2.2. Fungal isolation and characterisation Three berries were randomly sampled from each bunch and plated directly on Petri dishes containing Dichloran Rose Bengal Chloramphenicol medium (DRBC). Plates were incubated for 7 days at 25 C. Colonies of developing fungi belonging to Aspergillus section Nigri were transferred to Petri dishes containing Czapek Yeast Agar (CYA) and incubated at 25 C for 5 days. After the incubation period, Aspergillus isolates were examined through macroscopic and microscopic observation and classified according to the morphology of their spores and conidial heads into three groups: uniseriates, A. niger aggregate and A. carbonarius (Abarca, Accensi, Cano, & Cabanes, 2004). Ochratoxin A producing isolates belonging to the A. niger aggregate were identified by sequencing the internal transcribed spacer (ITS1-5.8S-ITS2) of ribosomal DNA (rDNA). Fungal DNA was extracted as described by Lecellier and Silar (1994). PCR amplification was performed as described by White, Bruns, Lee, and Taylor (1990) using an Eppendorf MastercyclerÒ thermal cycler (France). PCR products were used as a template for sequencing with the ITS1/ITS4 primers as prescribed by the manufacturer (Genome Express, Meylan France). The resulting sequences were aligned with Clustal X of multiple sequence alignment computer program (Thompson, Gibson, Plewniak, Jeanmougin, & Higgins, 1997). For the
The method used was adapted from that developed by Bragulat, Abarca, and Cabanes (2001). Aspergillus isolates were inoculated on three points in CYA medium and incubated for 5 days at 25 C. Three agar plugs (6 mm) were cut from the inner, middle and outer area of the colony and introduced in a vial with 1 ml of methanol. After 60 min, the extracts were filtered (MillexR SLHV 013NK, Millipore, Bedford, Massachusetts, USA) and injected into an HPLC system with a fluorescence detector (lexc 330 nm; lem 460 nm) and a C18 column (Waters Spherisorb 5 mm, ODS2, 4.6 250 mm, Milford MA, USA). The mobile phase was composed of acetonitrile:water:acetic acid (57:41:2). Detection and quantification limits (LOD and LOQ) were 0.02 mg OTA/l and 0.04 mg OTA/l, respectively. The recovery for OTA on CYA media was 90 3.5% (mean SD., n ¼ 3). 2.4. Analysis of OTA in grapes After selecting berries for fungal isolation, the ten bunches collected from each vineyard and variety were manually crushed and the resulting musts (n ¼ 72) were analyzed for OTA content according to the method of Bezzo, Maggiorotto, and Testa (2002). Musts (100 ml) were first centrifuged (2500 g, 20 min), liquid phase
Table 1 Meteorological data from the studied regions during grape maturation (06: June; 07: July; 08: August) in 2005, 2006 and 2007. Year
2005
2006
2007
Month
06
07
08
06
07
08
06
07
08
Belli
T ( C)a Tmin ( C)b Tmax ( C)c R.H. (%)d R (mm)e
24.9 19.4 30.2 69 8.0
28.2 22.3 32.7 66 2.3
27.2 21.9 31.6 69 45.7
24.2 20.1 29.1 75 35.8
26.6 18.6 28.9 68 7.8
29.7 22.4 33.5 70 19.8
28.9 22.4 32.3 68 27.1
25.1 19.7 28.5 76 83.4
29.6 19.9 30.4 ** 35.0
Baddar
T ( C) Tmin ( C) Tmax ( C) R.H. (%) R (mm)
23.2 16.1 29.8 65 6.8
26.8 20.4 32.2 62 0.6
27.3 20.6 31.6 ** 34.0
27.1 21.6 29.1 ** 30.8
27.2 19.6 31.3 67 0.0
29.8 21.0 33.5 ** 180.1
28.2 21.6 32.2 65 44.5
25.3 18.6 28.8 74 66.9
28.8 19.1 30.0 69 30.0
Raf-Raf
T ( C) Tmin ( C) Tmax ( C) R.H. (%) R (mm)
23.7 18.1 29.9 75 17.0
26.5 21.0 32.2 73 0.7
25.8 21.3 31.0 75 8.2
23.9 18.5 29.1 78 12.1
26.9 17.3 30.8 70 7.1
26.1 20.6 32.8 74 0.0
23.7 21.6 30.9 75 1.6
26.2 18.6 28.9 80 70.8
26.2 18.2 29.5 78 16.1
Regueb
T ( C) Tmin ( C) Tmax ( C) R.H. (%) R (mm)
26 18.6 33.4 55 11.1
30.2 21.6 38.4 53 1.7
28.1 20.9 35.8 52 6.6
27.2 17.2 33.9 43 4.0
29.9 21.1 36.9 49 12.2
29.2 20.8 36.2 54 13.2
28.3 18.5 35.2 50 23.6
29.6 19.3 36.8 44 0.0
29.8 21.2 37.0 53 24.0
**lack data. a T: mean daily temperature. b T max: mean daily maximum temperature. c T min: mean daily minimum temperature. d R.H.: mean daily relative humidity. e R: Rainfall
S. Lasram et al. / Food Control 25 (2012) 75e80
was, brought to a pH 7.4 (NaOH 4 M), filtered and applied to an immunoaffinity column (Ochraprep, Rhône Diagnostics Technologies Ltd, Glasgow, UK). The column was then washed with 20 ml of distilled water and dried up with an air stream. Elution was realized with 1.5 ml of methanol/acid acetic (98/2) solution. The eluate was evaporated to dryness, dissolved with 0.8 ml of mobile phase and injected into the HPLC system equipped with a fluorescence detector (lexc 230 nm; lem 458 nm) and a reversed-phase column C18 (Waters Spherisorb 5 mm, ODS 2, 4.6 250 mm, Milford, MA, USA). The mobile phase was composed by a sodium acetate 4 mM/ acetic acid (19:1: v/v) solution (52%) and acetonitrile (48%). Detection and quantification limits (LOD and LOQ) were 0.03 mg OTA/l and 0.05 mg OTA/l, respectively. The recovery for OTA on grape juice samples was 95 4% (mean SD., n ¼ 3).
77
dominant group in the four regions, especially in the region of Regueb where they represent 94.5% of the isolated black aspergilli. In contrast, uniseriate isolates were recovered in small numbers in the different regions. At each sampling year, the highest level of A. carbonarius infection was observed in the region of RafeRaf where it represented a mean of 43% of black aspergilli isolated. The level of contamination by this specie was lower in the regions of Baddar and Belli with, respectively, 30.9% and 20.8% of black aspergilli. The incidence of A. carbonarius was very low in Regueb region where only four A. carbonarius (1% of black aspergilli) were isolated during the three sampling years. 3.2. Ochratoxigenic potential of black Aspergillus species
Statistic analyses were performed with Statistica software (version 7.0, StatSoft, Inc., Tulsa, OK, USA). The distribution of variables was tested for normality using the KolmogoroveSmirnov test. In order to study the significance of the considered factors (year and region), the number of Aspergilli section Nigri and the OTA content of grapes were analyzed using analysis of variance with post-hoc test (Duncan), for variables with normal distribution and KruskaleWallis test (with approximation to chi-square test) with multiple comparison test, for variables with non-normal distribution. Correlation analysis (correlation coefficients of Spearman rs and Pearson rp for variables with non-normal and normal distributions, respectively) were used to correlate the presence of A. carbonarius and the OTA content in berries.
A total of 1242 black aspergilli (270 A. carbonarius, 934 A. niger aggregate and 29 A. uniseriate) was tested for OTA production. Among the A. carbonarius isolates, OTA producers represented 99.5% (268/270); whereas, only 3.2% (29/934) of the A. niger aggregate isolates were ochratoxigenic. No OTA production by the uniseriate isolates was detected. Data on OTA production by the A. carbonarius isolates showed that 67.27% of the isolates produced less than 1.0 mg/g CYA, 29.09% between 1.0 and 5.0 mg/g CYA and 4.55% more than 5 mg/g CYA. The average OTA production was about 1.45 mg/g CYA. For the ochratoxigenic A. niger aggregate isolates, 89.47% produced less than 0.1 mg/g CYA and 10.53% between 0.1 and 1.0 mg/g CYA. The average OTA production was about 0.07 mg/g CYA. The ochratoxigenic isolates belonging to the A. niger aggregate group were classified in the two patterns N and T, corresponding to A. niger and A. tubingensis. Among the 21 isolates analyzed, 13 showed the T type pattern and 8 showed the N type pattern.
3. Results
3.3. OTA contamination of grapes
3.1. Distribution of black Aspergillus species
The data related to the OTA concentrations in musts (Table 3) showed that at maturity stage, 54% (39/72) of analyzed samples were contaminated with 10% (4/39) of OTA positive samples exceeding the EU restrictive limit (>2 mg/l). These samples came from RafeRaf (5.44 and 5.84 mg OTA/l) and Baddar (3.27 and 2.23 mg OTA/l) regions. The non parametric KruskaleWallis analysis showed that the OTA concentration in grape was not significantly different between the sampling years (P > 0.05) and significantly different between regions (P < 0.05). Eighty three percent (15/18) of grapes from RafeRaf were OTA contaminated in the range of 0.06e5.85 mg/l. In Baddar, 83% (15/18) of grape samples contained between 0.06 and 3.27 mg OTA/l. Forty four percent (8/18) of grapes from Belli contained between 0.09 and 0.96 mg OTA/l. Among the grape samples originating from Regueb, only one sample was
2.5. Statistical analysis
A total of 1242 black aspergilli were isolated along the three sampling years (2005, 2006 and 2007) distributed in the four regions from Tunisia (Belli, Baddar, Raf-Raf and Regueb). The analysis of variance revealed that the single factor region and the interaction year and region presented significant differences in the number of black aspergilli isolated (P < 0.01). The sampling year does not affect the fungal colonisation grapes (P > 0.05). Grapes from Regueb were significantly the most contaminated with black aspergilli each year. No statistical differences were found between the number of these moulds in the regions of Baddar, Belli and RafeRaf (P ¼ 0.05). The distribution of black aspergilli isolates is presented in Table 2. Isolates of A. niger aggregate formed the
Table 2 Percentage of black aspergilli isolates. Numbers in brackets are the number of black aspergilli isolates of each group isolated/the total number of black aspergilli. Group
Year
Region Baddar
Raf-Raf
Regueb
Belli
Uniseriate
2005 2006 2007 Total
2.9 (4/136) 4.8 (4/84) 0 (0/77) 2.5 (8/297)
1.0 1.1 1.6 1.2
1.9 (2/104) 10.0 (13/130) 0.7 (1/150) 4.2 (16/384)
0 (0/101) 0 (0/115) 2.3 (2/87) 0.7 (2/303)
A. carbonarius
2005 2006 2007 Total
19.9 23.8 58.4 30.9
(27/136) (20/84) (45/77) (92/297)
24.8 41.5 74.6 43.0
(25/101) (39/94) (47/63) (111/258)
2.9 (3/104) 0 (0/130) 0.7 (1/150) 1.0 (4/384)
20.8 22.6 18.4 20.8
(21/101) (26/115) (16/87) (63/303)
A. niger aggregate
2005 2006 2007 Total
76.5 60.7 36.4 61.6
(104/136) (51/84) (28/77) (183/297)
76.2 57.4 30.2 58.1
(77/101) (54/94) (19/63) (150/258)
94.2 (98/104) 90 (117/130) 98.7 (148/150) 94.5 (363/384)
79.2 77.4 79.3 78.5
(80/101) (89/115) (69/87) (238/303)
(1/101) (1/94) (1/63) (3/258)
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S. Lasram et al. / Food Control 25 (2012) 75e80
Table 3 OTA concentrations in grapes collected from the four regions in the different years (mg/l). Region
Variety
Sampling year 2005
2006
2007
Baddar (North)
Muscat Alexandrie Cabernet Sauvignon Ugni Blanc Italia Syrah Carignan
0.97 0.14 0.06 0.17 0.38
3.27 1.53 1.21 0.24 0.12 0.16
0.25
Belli (North)
Chardonnay Muscat Alexandrie Cabernet Sauvignon Italia Syrah Carignan
0.18
0.13 0.35 0.10 0.77
0.96
Raf-Raf (North-Est)
Rezeki Bezoul Khadem Akhal Raf-Raf Marsaoui Bid Hmam Muscat Raf-Raf
0.1 0.12
1.49
0.96 5.85 0.27
Italia Victoria Sultanine Superior Seedless 1 Matilde Superior Seedless 2
Regueb (Center)
a
LQ ¼ 0.05 mg/l.
contaminated with OTA at a very low concentration (0.05 mg/l). The multiple comparison test (P ¼ 0.05) showed that the OTA contamination of grape is significantly different between Regueb region and the regions of Baddar and RafeRaf. A significant difference for OTA contamination was also found between Belli and RafeRaf regions. Further to the KruskaleWallis analysis which doesn’t take into account the OTA amounts but only their ranking, the distribution of the OTA concentrations in the grapes (Fig. 1) showed that higher OTA concentrations were detected in grapes from RafeRaf with 40% of samples containing between 0.5 and 2 mg OTA/l and 13% containing more than 2 mg OTA/l. For grapes originating from Baddar, 20% of samples were contaminated in the range of 0.5e2 mg OTA/l and 13% of samples exceeded 2 m OTA/l.
Fig. 1. Number of grape samples from the different regions contaminated in different range of OTA concentrations.
This level of contamination is higher than observed in Belli were 75% of samples contained less than 0.5 mg OTA/l and 25% were contaminated with concentrations ranging between 0.5 and 1 mg OTA/l. 3.4. Correlation between the occurrence of OTA and A. carbonarius The numbers of A. carbonarius isolated from grapes were correlated to the OTA concentrations. The coefficient of correlation of Spearman was calculated for values from each variety. The incidence of A. carbonarius was positively correlated with the average OTA concentrations for each variety (rp ¼ 0.473, P < 0.0001). The coefficient of correlation of Pearson was calculated for mean values from each region and sampling year. A higher positive correlation was established (rS ¼ 0.731, P < 0.01). 4. Discussion Several surveys carried out in recent years in various countries reported that the main fungi involved in ochratoxin A contamination of grapes and derived products are black aspergilli (Aspergillus section Nigri). The A. carbonarius specie was recognized as the dominant contributor to OTA contamination of grape in Mediterranean countries (Battilani, Barbano et al., 2006) and in Australia (Leong et al., 2006). However, some authors considered that A. niger aggregate, may slightly contribute to OTA contamination of grape in Mediterranean countries; as this group is by far the most common species of Aspergillus present in vineyards (Battilani, Giorni, & Pietri, 2003; Serra, Abrunhosa, Kozakiewicz, & Venancio, 2003). Others studies conducted in South American countries concluded that A. niger aggregate group was an important OTA-producer fungi in grape (Da Rocha Rosa et al., 2002; Magnoli, Violante, Combina, Palacio, & Dalcero, 2003). In our study, the frequency of toxigenicity among A. niger aggregate, A. carbonarius and uniseriate Aspergillus isolates from Tunisian vineyards was similar to results reported from European Mediterranean countries (Battilani, Barbano et al., 2006). The highest incidence of ochratoxigenic isolates and OTA production was recorded for the A. carbonarius isolates. This fact confirmed that A. carbonarius is the main specie responsible for OTA contamination in Tunisian grapes, for both wine and table varieties. Few A. niger aggregate isolates were able to produce OTA at very low levels. The molecular characterization of these isolates showed that they were represented with both A. tubingensis and A. niger confirming the ability of A. tubingensis specie to produce OTA (Medina, Mateo, Lòpez-Ocaña, Valle-Algarra, & Jiménez, 2005; Perrone et al., 2006). The OTA analysis of grapes showed the presence of the toxin in both wine and table grapes at the three sampling years. Almost half of samples were contaminated with OTA which is similar to the level of contamination detected in Southern Italy (Lucchetta et al., 2010). Among the OTA positive samples, 4 out of 39 exceeded 2 mg/l. However, it’s important to note that grape collected at maturity will undergo several stages (transport, storage, vinification, drying) which may influence the OTA final concentration of grape product. Several factors affect the grape colonization by ochratoxigenic moulds and the OTA contamination, in particular, geographic and climatic conditions. In several Europeans countries, southern regions located in the Mediterranean basin, were found to be particularly affected (Battilani, Barbano et al., 2006; Battilani, Magan et al., 2006). Tunisia is a small country with an extended Mediterranean coast, however, areas of grape planting and production are characterized by different climatic characteristics. The vineyards selected for this study are representative of the different climatic regions existing in the country. The region of Raf-Raf is a coastal region located in the North-Est of the country. It is characterized by
S. Lasram et al. / Food Control 25 (2012) 75e80
a sub-humid climate. The grape culture in this region is characterized by the presence of an important number of local varieties used essentially for fresh consumption and for dried raisins. Baddar and Belli are located in Cap-Bon region (North) which is the main viticulture area in Tunisia and especially for wine grape. This area has a typically semi-arid Mediterranean climate. The last region studied is Regueb which is a new viticultural area in the center of the country. It’s characterized by an arid climate and grapes cultivated in this area are essentially table varieties. In the present work, the influence of the specific geographic localization and the climate of the vineyards on the occurrence of ochratoxigenic moulds and OTA contamination of grape was significant. Black aspergilli were the most abundant in Regueb region due to the highest number of A. niger aggregate isolates from this region compared to the northern regions (RafeRaf, Belli and Baddar). On the other hand, A. carbonarius was rarely isolated from Regueb vineyards. Thus, grapes collected from this region were rarely contaminated with OTA and the level detected was very low. Data on climatic characteristics of studied regions during the maturation period of grape (Table 1) showed that Regueb region has the highest values of mean temperatures (26e30.2 C), mean maximum temperatures (31.2e38.4 C) and the lowest value of mean relative humidity (43e62%). A. niger aggregate isolates are the most adapted to the heat and dryness. On the contrary, A. carbonarius was unable to grow in such climatic conditions. In a previous study (Lasram et al., 2010) the rare A. carbonarius strains isolated from Regueb region showed that under dry and hot conditions, they grew significantly faster than A. carbonarius strains those coming from northern regions. These isolates could have developed a tolerance towards arid climate. It was also found that optimal conditions for the growth of A. carbonarius isolated from Tunisian vineyards were high water activity (0.99) and moderate temperature (30 C). For toxin production optimal conditions were a high water activity (0.99) and temperatures in the range of 15e25 C. These results explain, in part, the abundance of A. carbonarius in the region of Raf-Raf and the higher OTA contamination of grapes collected from this region. Indeed, RafeRaf region is dominated by a Mediterranean influence, the temperatures in the summer are usually moderate (mean temperatures 23.7e26.9 C, mean maximum temperatures 28.9e32.2 C) and it is also very humid (HR 70e80%). These climatic conditions seem to be very favourable to A. carbonarius growth and OTA accumulation. In Baddar and Belli regions, characterized by a semi-arid climate, the incidence of A. carbonarius was continually similar (approximatively 20% of black aspegilli), except in 2007 where the fungi were particularly more frequent in Baddar. These two regions have also Mediterranean influences but with lower humidity levels and higher mean and maximum temperatures than those of Raf-Raf (Table 1). The higher incidence of A. carbonarius in 2007 cannot be correlated with any change in climatic data during this year. For OTA contamination, no statistic difference was found between the two regions, however, the distribution frequency of the OTA concentrations in the grape juices collected in the different regions (Fig. 1) showed that grapes originating from Baddar vineyards were more contaminated with OTA than those from Belli. Thus, the occurrence of ochratoxigenic fungi in the vineyard and the incidence of OTA in grapes depend not only on climatic conditions but also on other factors related to the variety and crop management, such as training system, irrigation and phytosanitary treatments, which influence on the ecosystem of the vine. However, our study revealed a strong correlation between the presence of ochratoxin producing strains on grapes and the finding of OTA in musts when considering average values for each region and sampling year (rS ¼ 0.731). In fact, OTA was not always detected in grapes, even in those colonized by ochratoxigenic fungi, but the presence of
79
A. carbonarius in a region has an impact on the level of OTA contamination in grapes cultivated in the same region. The results of the present work demonstrated the high risk of OTA and fungal contamination in Northern Tunisia, especially for coastal regions. Acknowledgement Authors are grateful to the Tunisian Ministry of Higher Education, Scientific Research and Technology for his financial support in the framework of the National Research program “Qualité du raisin et Ochratoxine A en Tunisie”. We are also thankful to the CTAA (Centre Technique de l’Agro-alimentaire) for their cooperation and assistance. References Abarca, M. L., Accensi, F., Cano, J., & Cabanes, J. F. (2004). Taxonomy of black aspergilli. Antonie Van Leeuwenhoek, 86, 33e49. Accensi, F., Cano, J., Figuera, L., Abarca, M. L., & Cabanes, F. J. (1999). New PCR method to differentiate species in the Aspergillus niger aggregate. FEMS Microbiology Letters, 180, 191e196. Battilani, P., Barbano, C., Marín, S., Sanchis, V., Kozakiewicz, Z., & Magan, N. (2006). Mapping of Aspergillus section Nigri in southern Europe and Israel based on geostatistical analysis. International Journal of Food Microbiology, 111, S72eS82. Battilani, P., Giorni, P., & Pietri, A. (2003). Epidemiology of toxin-producing fungi and ochratoxin A occurrence in grape. European Journal of Plant Pathology, 109, 715e722. Battilani, P., Magan, N., & Logrieco, A. (2006). European research on ochratoxin A in grapes and wine. International Journal of Food Microbiology, 111, 52e54. Bezzo, G., Maggiorotto, G., & Testa, F. (2002). A method for the determination of specific mycotic contaminants random occurring in wines. Paris: Office International de la Vigne et du Vin. Bragulat, M. R., Abarca, M. L., & Cabanes, F. J. (2001). An easy screening method for fungi producing ochratoxin A in pure culture. International Journal of Food Microbiology, 71, 139e144. Da Rocha Rosa, C. A., Palscios, V., Combia, M., Fraga, M. E., De Oliveira Rekson, A., Magnoli, C. E., et al. (2002). Potential ochratoxin A producers from wine grapes in Argentina and Brazil. Food Additives and Contaminants, 19, 408e414. European Commission. (2005). Commission regulation (EC) No 123/2005 of 26 January 2005 amending regulation (EC) No 466/2001 as regards ochratoxin A. Official Journal of European Union, L25, 5e36. Lasram, S., Oueslati, S., Valero, A., Marin, S., Ghorbel, A., & Sanchis, V. (2010). Water activity and temperature effects on fungal growth and ochratoxin A production by Ochratoxigenic Aspergillus carbonarius isolated from Tunisian grapes. Journal of Food Science, 75(2), M89eM97. Lecellier, G., & Silar, P. (1994). Rapid methods for nucleic acids extraction from Petri dish-grown mycelia. Current Genetics, 25(2), 122e123. Leong, S. L., Hocking, A. D., Pitt, J. I., Kazi, B. A., Emmett, R. W., & Scott, E. S. (2006). Australian research on ochratoxigenic fungi and ochratoxin A. International Journal of Food Microbiology, 111, S10eS17. Lucchetta, G., Bazzo, I., Dal Cortivo, G., Stringher, L., Bellotto, D., Borgo, M., et al. (2010). Occurrence of Black Aspergilli and Ochratoxin A on grapes in Italy. Toxins, 2, 840e855. Magnoli, C., Violante, M., Combina, M., Palacio, G., & Dalcero, A. (2003). Mycoflora and ochratoxin-producing strains of Aspergillus section Nigri in wine grapes in Argentina. Letters in Applied Microbiology, 37, 179e184. Mateo, R., Medina, A., Mateo, E. M., Mateo, F., & Jiménez, M. (2007). An overview of ochratoxin A in beer and wine. International Journal of Food Microbiology, 119, 79e83. Medina, A., Mateo, R., Lòpez-Ocaña, L., Valle-Algarra, F. M., & Jiménez, M. (2005). Study of Spanish mycobiota and ochratoxin A production by isolates of Aspergillus tubingensis and other members of Aspergillus Section Nigri. Applied and Environmental Microbiology, 71(8), 4696e4702. Otteneder, H., & Majerus, P. (2000). Occurrence of ochratoxin A in wines: influence of the type of wine and its geographical origin. Food Additives and Contaminants, 17, 793e798. Perrone, G., Mule, G., Susca, A., Battilani, P., Pietri, A., & Logrieco, A. (2006). Ochratoxin A production and Amplified Fragment Length Polymorphism analysis of Aspergillus carbonarius, Aspergillus tubingensis and Aspergillus niger strains isolated from grapes in Italy. Applied and Environmental Microbiology, 72(1), 680e685. Pitt, J. I., & Hocking, A. D. (1997). Fungi and food spoilage (2nd ed.). London: Blackie Academic and Professional. Pitt, D. B., Plestina, J. I., Shepard, R., Solfrizzo, G., Verger, M., & Walker, P. J. P. (2001). Safety evaluation of certain mycotoxins in food. In Joint FAO/WHO Expert Committee on Food Additives (JECFA) (pp. 281). Rome: Food and Agriculture Organization. Serra, R., Abrunhosa, L., Kozakiewicz, Z., & Venancio, A. (2003). Black Aspergillus species as ochratoxin A producers in Portuguese wine grapes. International Journal of Food Microbiology, 88, 63e68.
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