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Ochratoxin A and ochratoxigenic Aspergillus species in Argentinean wine grapes cultivated under organic and non-organic systems
International Journal of Food Microbiology 114 (2007) 131 – 135 www.elsevier.com/locate/ijfoodmicro
Ochratoxin A and ochratoxigenic Aspergillus species in Argentinean wine grapes cultivated under organic and non-organic systems Ma. Lorena Ponsone a , Mariana Combina b,1 , Ana Dalcero a,1 , Sofía Chulze a,⁎,1 a
Departamento de Microbiología e Inmunología, Facultad de Ciencias Exactas, Físico, Químicas y Naturales, Universidad Nacional de Río Cuarto, Ruta Nacional No 36 Km 601, (5800) Río Cuarto, Córdoba, Argentina b Instituto Nacional de Tecnología Agropecuaria (INTA), Luján de Cuyo, Mendoza, Argentina Received 12 October 2005; received in revised form 7 July 2006; accepted 19 July 2006
Abstract The evolution of contamination with Aspergillus section Nigri and ochratoxin A occurrence was evaluated in four vineyards located at Mendoza province, Argentina during 2003–2004. The survey included two grape varieties, one of late maturation (Bonarda) and the other of early maturation (Tempranillo). The vineyards were set under non-organic and organic cropping systems. Bunches of grapes at different growth stages were collected, and berries (50 by sample) were plated on Petri dishes containing Dichloran 18% Glycerol Agar (DG18) and Dichloran Rose Bengal Chloramphenicol Agar (DRBC) media. After an incubation period of 7 days at 25 °C ± 1 °C, the mycoflora belonging to Aspergillus section Nigri was identified. The ability to produce ochratoxin A (OTA) by the potential ochratoxigenic species was evaluated on YES (2% yeast extract, 15% sucrose) medium. The cultures were incubated at 30 °C ± 1 °C for 10 days in darkness. The OTA content of the grapes was determined by HPLC. Through the different growth stages, from setting to harvest, grape contamination by the Aspergillus species, section Nigri increased. The main species isolated belonged to the A. niger aggregate. From 246 strains evaluated 24% was ochratoxigenic. OTA was not detected in grapes during the survey. © 2006 Elsevier B.V. All rights reserved. Keywords: Aspergillus section Nigri; Ochratoxin A; Grapes
1. Introduction Ochratoxin A (OTA) is a nephrotoxic and carcinogenic mycotoxin, and showed teratogenic, immunotoxic and possibly neurotoxic and genotoxic properties. The International Agency for Research on Cancer classified OTA as a possible human carcinogen (group 2B) (IARC, 1993). The occurrence of this toxin has been reported in various vegetable products, such as cereals, grapes, coffee beans, cocoa and beverages, beer and wine (Pittet, 1998; Solfrizzo et al., 1998; MacDonald et al., 1999; Sage et al., 2002; Serra et al., 2003; Taniwaki et al., 2003). Ochratoxin A was originally described as a metabolite of Aspergillus ochraceus (Van der Merwe et al., 1965). This species and Penicillium verrucosum are considered the main OTA-producing species. P. verrucosum produces OTA in ⁎ Corresponding author. Tel.: +54 358 4676429; fax: +54 358 4676231. E-mail address: [email protected] (S. Chulze). 1 Member of the Research Career of CONICET. 0168-1605/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2006.07.001
temperate and cold climates and has been reported almost exclusively in cereal and cereal products while A. ochraceus is more commonly associated with warmer and tropical climates from stored foods mostly (Pitt and Hocking, 1997). Nevertheless, neither of those OTA-producing fungi has been reported as normal mycobiota of grapes. Aspergillus species, section Nigri are by far the most common fungi responsible for post-harvest decay of fresh fruits (Pitt and Hocking, 1997) and they are found on the surface of healthy grapes at all stages (Zahavi et al., 2000). In Europe it has been observed that the climate conditions favour the growth of ochratoxigenic Aspergillus species over Penicillium. Species within Aspergillus section Nigri were always present and they included OTA-producing strains (Abarca et al., 2001). Among these species, A. carbonarius probably plays a relevant role, because the percentage of positive strains and the amount of OTA produced in vitro were generally higher than those found in the other black Aspergilli (Teren et al., 1996; Heenan et al., 1998; Battilani et al., 2002; Cabañes et al., 2001).
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Argentina ranks in the fifth position of wine producers in the world. The central viticultural area in Argentina comprises vineyards located in Mendoza and San Juan provinces, which together represent over 90% of the Argentine wine production area. Previous studies showed that the presence of potentially OTA producer species has been detected in wine grapes (Dalcero et al., 2002; Rosa et al., 2002; Magnoli et al., 2003). No data are available on the dynamics of ochratoxigenic fungi through different stages of grape development in the field and the possible OTA contamination. In order to improve the knowledge on the epidemiology of black Aspergilli in grapes, a survey was carried out during 2003– 2004, – to evaluate the dynamic of fungal populations on grapes from the setting of the vineyard until harvest, – to evaluate the possible interactions among fungal populations, cultural practices and climatic conditions on OTA contamination. 2. Materials and methods 2.1. Grape sampling The survey was carried out in 2003/2004 in vineyards under different cropping systems (organic and non-organic) and on two grape varieties: Bonarda and Tempranillo, early and late maturation respectively. All the vineyards were settled at 85 km eastern Mendoza, 33° 16′ South Latitude 68° 09′ West Longitude. The vineyards chosen were representative of the grape-growing area as regards grape variety and farming methods. The four vineyards were treated with SO4Cu as fungicide, but only the nonorganic ones had herbicide treatment (glyphosate, three applications per year, doses 3 Lt/ha). Ten plants were marked along two major diagonal transects of each vineyard. One bunch was collected from each marked plant at setting and three further bunches were harvested in the following plants, in the same row, at one month after setting, early veraison and at harvest. Bunches collected were at 1.5 m from the ground. They were kept in paper bags and stored in portable refrigerators during transfer to the laboratory for fungal isolation and toxin analysis. Data on cropping systems and climatic conditions (mean daily air temperature, relative humidity and rainfall) were collected from a station close to the vineyards. 2.2. Fungal isolation and species identification From each vineyard and at each sampling stage fifty berries, randomly sampled from the ten different bunches were surface disinfected with sodium hypochlorite solution (1%) for 1 min; rinsed in sterile distilled water three times and plated onto a Dichloran 18% Glycerol Agar (DG18) and Dichloran Rose Bengal Chloramphenicol Agar (DRBC) media (Pitt and Hocking, 1997). After a incubation period of 7 days at 25 °C ± 1 °C the fungal isolates were identified to genus and species levels according to Pitt and Hocking (1997) and Samson et al. (2000). For Aspergillus identification, cultures were grown on Czapek Yeast Extract Agar (CYA) at 25 °C ± 1 °C and 37 °C ± 1 °C; Malt Extract Agar (MEA); 25% Glycerol Nitrate Agar (G25N); and Czapek Yeast Extract with 20% Sucrose Agar (CY20S), at
25 °C ± 1 °C. All plates were incubated for 7 days. Fungal identification was done according to Klich and Pitt (1994), Pitt and Hocking (1997) and Samson et al. (2000). 2.3. Ochratoxin A occurrence The berries from all bunches collected were manually crushed and the OTA content was determined following the methodology proposed by Zimmerly and Dick (1996)) with some modifications. A 50-g portion of berries was extracted with methanol: sodium bicarbonate at 1% (70:30, v/v) and blended at high speed for 1 min. The extract was filtered to remove particulate matter, and a 10-mL of extract was taken and diluted with 40 mL of PBS containing 0.01% Tween 20. The diluted extract was filtered through a microfibre filter. A ten-millilitre portion was taken and added to an immunoaffinity column (OchraTestTM; Vicam, Digen Ltd, Oxford, UK). The column was washed with 10 mL PBS containing 1% Tween 20 and then with 10 mL double distilled water. OTA was eluted from the column with methanol (HPLC grade), again at a flow rate of 1–2 drops per second. The HPLC apparatus used for determination of OTA was a Hewlett-Packard (Hewlett-Packard company, Palo Alto, CA, USA) chromatograph with a loop of 100 μL, equipped with a spectro-fluorescence detector (excitation, 330 nm; emission, 460 nm) and a C18 column (150 × 4.6 mm, 5 μm particle size; Supelcosil LCABZ, Supelco, Bellefonte, PA, USA), connected to a pre-column (20 × 4.6 mm, 5 μm particle size; Supelguard LC-ABZ, Supelco). The mobile phase was pumped at 1.0 mL min− 1, and consisted of an isocratic system as follows: 57% acetonitrile, 41% water and 2% acetic acid. OTA was quantified on the basis of HPLC fluorometric response compared with the OTA standard (purity N 99%; Sigma Aldrich Co., St Louis, MO, USA). The limit of detection was 1 ng g− 1. 2.4. Ochratoxin A production Ochratoxin A production was tested among 246 strains belonging to Aspergillus section Nigri (A. niger, A. awamori, A. foetidus, A. aculeatus, A. japonicus, A. carbonarius). OTA was determined following the methodology described by Téren et al. (1996). Briefly, the strains were grown in stationary cultures in 125 mL Erlenmeyer flasks containing 20 mL of YES medium (2% yeast extract, 15% sucrose) and were inoculated with a 4 mm plug from the margin of 7-day-old colonies on malt extract agar (MEA) and incubated at 30 °C ± 1 °C for 10 days in darkness. At the end of the incubation period, 1-mL portions of the culture medium were then mixed with 1 mL of chloroform and centrifuged at 4000 g for 10 min. The chloroform phase was transferred to a clean sylanized vial, evaporated to dryness and redissolved into 0.5 mL of mobile phase. OTA detection was done as described in Section 2.3. 2.5. Statistical analysis Data on contamination with Aspergillus section Nigri were analyzed previously by Arc sine plus one transformation by ANOVA followed by Tukey test in order to determine significant
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3. Results and discussion
Fig. 1. Dynamic of Aspergillus section Nigri in four vineyards (BO: Bonarda Organic; BC Bonarda Non-Organic; TO: Tempranillo Organic: TC: Tempranillo Non-Organic).
differences according to growth stage, cultural practice and grape variety. All statistical analyses were done using the Software GraphPAD InStat (Sigma St Louis, MO).
In the Bonarda variety (late maturation) settled under the non-organic system, species belonging to Aspergillus section Nigri were isolated since the setting stage. One month later these species were detected in both varieties analyzed (late and early maturation) under the two cropping systems. The number of colonized berries was low (1.3%) during the early growth stages, but the level of contamination increased up to 47% at harvest time. The percentage of samples colonized by Aspergillus section Nigri was significantly influenced by the growth stage, the grape variety or the cropping system (p b 0.05). (Fig. 1). In general, the incidence of Aspergillus species, section Nigri was higher at veraison and harvest stage in comparison with one month after setting. Most samples were colonized by A. niger and A. awamori in all growth stages. A. japonicus, A. aculeatus, A. foetidus and A. carbonarius were also present, although the distribution varied according to the growth stage, the grape variety or the cropping system (Fig. 2). These results partially agree with those reported by Battilani et al. (2006) who found that Aspergillus section Nigri species are more frequently isolated in hot and dry areas, while A. carbonarius showed
Fig. 2. Evaluation of Aspergillus section Nigri species in four Argentinean vineyards at setting ( ), 1 month setting( ), veraison and ( ) harvest (◫).
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higher incidences in hot and wetter areas. These results are supported by the findings of Mitchell et al. (2003) and Bellí et al. (2004, 2005) who showed that A. carbonarius grew optimally at 0.98–0.99 aw, while uniseriates and A. niger aggregate species showed no differences in growth in the range 0.90–0.995 aw. Although a moderate frequency of Aspergillus section Nigri was found, only 24% of the strains was OTA producers. The OTA levels produced varied depending on the species evaluated. Uniseriate species including A. japonicus and A. aculeatus produced between 1.3 and 6.3 ng/mL of the culture medium, while the biseriate species including A. niger, A. awamori and A. foetidus produced levels ranging from 1.3 to 50 ng/mL of the culture medium (Table 1). These results showed that the species within the Aspergillus section Nigri play the main role in the potential OTA contamination of grapes in the area evaluated. These results agree with previous studies from South America and Europe, since species included in the Aspergillus section Nigri has already been found as the main OTA producer species in Argentina, Brazil (Rosa et al., 2002), France (Sage et al., 2002) and Italy (Battilani et al., 2003). No evidence of contamination of grape neither by A. ochraceus nor by its closely related species P. verrucosum has been found. The ochratoxigenic fungi found in grapes are different from those found in cereals or feedstuffs in Argentina (Dalcero et al., 2002). The knowledge of the dynamics on fungal contamination, mainly by the Aspergillus species and OTA producers, is important, because these species are usually considered postharvest fungi and were also isolated from without visible symptom and damaged berries, the latter being in the risk of OTA contamination.
Table 1 Ochratoxin A production by Aspergillus section Nigri strains isolated through different grape growth stages Growth stage
Species
Positive/total a Range (ng/mL) Mean ± SE
Setting
A. niger 0/2 A. awamori 1/3 Berry A. niger 1/3 enlargement A. foetidus 0/2 A. japonicus 3/11 A. aculeatus 1/6 A. carbonarius 0/1 Veraison A. niger 3/11 A. awamori 4/8 A. japonicus 5/15 A. aculeatus 3/3 A. foetidus 2/2 Harvest A. niger 24/68
ND ND–3.09 ND–2.86 ND 3.3–8.62 ND–2.4 ND 1.3–26.9 1.2–4.8 1.3–2.4 1.2–2.1 1.56–2.41 1.4–50.61
A. awamori 2/27 A. japonicus 16/72 A. carbonarius 0/6 A. foetidus 0/6
1.44 1.31–6.13 ND ND
ND: Not detected ≤ detection limit: 1 ng/mL. SE: Standard error. a Positive OTA producer strains vs total strains isolated.
ND 3.09 ± 0 2.86 ± 0 ND 5.14 ± 0.11 2.4 ± 0 ND 10.1 ± 12.8 3.05 ± 1.75 1.6 ± 0.7 1.6± 0,3 1.98 ± 0.42 17.95 ± 16.33 1.44 ± 0 3.72 ± 2.41 ND ND
Fig. 3. Temperature (T), relative humidity (RH) and rainfall (R), computed from December to April in Santa Rosa during the 2003–2004 period.
Although all the vineyards evaluated were contaminated with OTA-producing species at harvest stage, the berries collected were OTA free (detection limit 1 ng g− 1) independent of the growth stage considered, the variety and the cropping system. These results can be explained taking into account that the presence of the toxicogenic species does not always imply OTA synthesis. According to previous studies, ecological conditions, which favour growth and subsequent high contamination, are different from those which enable optimum OTA production (Alexandre et al., 2004). Among the Aspergillus section Nigri isolates, those species belonging to A. niger aggregate produce OTA in lower concentrations, than A. carbonarius which is the dominant OTA producer (Abarca et al., 2001). In this study A. carbonarius was isolated in a lower percentage (30%) than the species belonging to A. niger aggregate and it was only present in grapes one month after setting. Climatic condition data showed that the mean daily temperature ranged between 15.3 °C and 25.2 °C from December to April and the summation of degree-days from 1 December to 31 March was 2553.1 °C and from December to April was 3012.1 °C. The total mean rainfall was 9.4 (December to March) and 9.5 mm (December to April) respectively. The rainiest month was January with a mean rainfall of 5.1 mm (Fig. 3). These conditions were quite different from those recorded by Battilani et al. (2003) in a survey carried out in Italy where the total rainfall ranged between 60 and 330 mm and the rainiest month has more than 70 mm. These data suggest that the climatic condition among grape-growing areas may be responsible for the differences in OTA contamination in grapes. This study is the first report on the dynamics of Aspergillus section Nigri in two grape varieties, under two cropping systems in Argentina. Further studies are ongoing to evaluate different potential inoculum sources of ochratoxigenic species in the vineyard. Acknowledgements This work was supported by a grant from SECyT (Secretaria de Ciencia y Técnica), Universidad Nacional de Rio Cuarto,
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(2005), ANCyPT, PICT-25522/2006. We also thank CONICET. L. Ponsone is a fellow from CONICET. References Abarca, M.L., Accensi, F., Bragulat, M.R., Cabañes, F.J., 2001. Current importance of ochratoxin A-producing Aspergillus spp. Journal of Food Protection 64, 903–906. Alexandre, E.M., Abarca, L., Bragulat, M.R., Cabañes, F.J., 2004. Effects of temperature and incubation time on production of ochratoxin A by black aspergilli. Research in Microbiology 155, 861–866. Battilani, P., Pietri, A., Bertuzzi, T., Languasco, L., Giorni, P., Kozakiewicz, Z., 2002. Occurrence of ochratoxin A producing fungi in grape grown in Italy. Journal of Food Protection 66, 633–636. Battilani, P., Giorni, P., Pietri, A., 2003. Epidemiology of toxin-producing fungi and ochratoxin A occurrence in grape. European Journal of Plant Pathology 109, 715–722. Battilani, P., Barbano, C., Marin, 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, 72–82. Bellí, N., Ramos, A.J., Sanchis, V., Marín, S., 2004. Incubation time and water activity effects on ochratoxin A production by Aspergillus section Nigri strains isolated from grapes. Letters in Applied Microbiology 38, 72–77. Bellí, N., Ramos, A.J., Coronas, I., Sanchis, V., Marín, S., 2005. Aspergillus carbonarius growth and ochratoxin A production on a synthetic grape medium in relation to environmental factors. Journal of Applied Microbiology 98, 839–844. Cabañes, F.J., Accensi, F., Bragulat, M.R., Abarca, M.L., Minguez, S., Pons, A., 2001. Aspergillus carbonarius is a source of ochratoxin A-contamination in wine. Book of Abstracts ‘Bioactive Fungal Metabolites — Impact and Exploitation’. University of Wales, Swansea, UK, p. 51. Dalcero, A., Magnoli, C., Hallak, C., Chiacchiera, S.M., Palacio, G., Da Rocha Rosa, C.A., 2002. Detection of ochratoxin A in animal feeds and capacity to produce this mycotoxin by Aspergillus section Nigri in Argentina. Food Additives and Contaminants 19, 1065–1072. Heenan, C.N., Shaw, K.J., Pitt, J.I., 1998. Ochratoxin A production by Aspergillus carbonarius and A. niger isolates and detection using coconut cream agar. Journal of Food Mycology 1, 67–72. International Agency for Research on Cancer (IARC), 1993. Ochratoxin A. En: Monographs on the Evaluation of Carcinogenic Risks to Human: Some Naturally Occurring Substances; Food Items and Constituents, Heterocyclic Aromatic amines and mycotoxins, vol. 56. IARC, Lyon, France, pp. 489–521. Klich, M.A., Pitt, J.I., 1994. A Laboratory Guide to Common Aspergillus Species and their Teleomorphs. Sydney: CSIRO, Division of Food Res. Academic Press. Australia: Commonwealth Scientific and Industrial Research.
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Ochratoxin A and ochratoxigenic Aspergillus species in Argentinean wine grapes cultivated under organic and non-organic systems Ma. Lorena Ponsone a , Mariana Combina b,1 , Ana Dalcero a,1 , Sofía Chulze a,⁎,1 a
Departamento de Microbiología e Inmunología, Facultad de Ciencias Exactas, Físico, Químicas y Naturales, Universidad Nacional de Río Cuarto, Ruta Nacional No 36 Km 601, (5800) Río Cuarto, Córdoba, Argentina b Instituto Nacional de Tecnología Agropecuaria (INTA), Luján de Cuyo, Mendoza, Argentina Received 12 October 2005; received in revised form 7 July 2006; accepted 19 July 2006
Abstract The evolution of contamination with Aspergillus section Nigri and ochratoxin A occurrence was evaluated in four vineyards located at Mendoza province, Argentina during 2003–2004. The survey included two grape varieties, one of late maturation (Bonarda) and the other of early maturation (Tempranillo). The vineyards were set under non-organic and organic cropping systems. Bunches of grapes at different growth stages were collected, and berries (50 by sample) were plated on Petri dishes containing Dichloran 18% Glycerol Agar (DG18) and Dichloran Rose Bengal Chloramphenicol Agar (DRBC) media. After an incubation period of 7 days at 25 °C ± 1 °C, the mycoflora belonging to Aspergillus section Nigri was identified. The ability to produce ochratoxin A (OTA) by the potential ochratoxigenic species was evaluated on YES (2% yeast extract, 15% sucrose) medium. The cultures were incubated at 30 °C ± 1 °C for 10 days in darkness. The OTA content of the grapes was determined by HPLC. Through the different growth stages, from setting to harvest, grape contamination by the Aspergillus species, section Nigri increased. The main species isolated belonged to the A. niger aggregate. From 246 strains evaluated 24% was ochratoxigenic. OTA was not detected in grapes during the survey. © 2006 Elsevier B.V. All rights reserved. Keywords: Aspergillus section Nigri; Ochratoxin A; Grapes
1. Introduction Ochratoxin A (OTA) is a nephrotoxic and carcinogenic mycotoxin, and showed teratogenic, immunotoxic and possibly neurotoxic and genotoxic properties. The International Agency for Research on Cancer classified OTA as a possible human carcinogen (group 2B) (IARC, 1993). The occurrence of this toxin has been reported in various vegetable products, such as cereals, grapes, coffee beans, cocoa and beverages, beer and wine (Pittet, 1998; Solfrizzo et al., 1998; MacDonald et al., 1999; Sage et al., 2002; Serra et al., 2003; Taniwaki et al., 2003). Ochratoxin A was originally described as a metabolite of Aspergillus ochraceus (Van der Merwe et al., 1965). This species and Penicillium verrucosum are considered the main OTA-producing species. P. verrucosum produces OTA in ⁎ Corresponding author. Tel.: +54 358 4676429; fax: +54 358 4676231. E-mail address: [email protected] (S. Chulze). 1 Member of the Research Career of CONICET. 0168-1605/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2006.07.001
temperate and cold climates and has been reported almost exclusively in cereal and cereal products while A. ochraceus is more commonly associated with warmer and tropical climates from stored foods mostly (Pitt and Hocking, 1997). Nevertheless, neither of those OTA-producing fungi has been reported as normal mycobiota of grapes. Aspergillus species, section Nigri are by far the most common fungi responsible for post-harvest decay of fresh fruits (Pitt and Hocking, 1997) and they are found on the surface of healthy grapes at all stages (Zahavi et al., 2000). In Europe it has been observed that the climate conditions favour the growth of ochratoxigenic Aspergillus species over Penicillium. Species within Aspergillus section Nigri were always present and they included OTA-producing strains (Abarca et al., 2001). Among these species, A. carbonarius probably plays a relevant role, because the percentage of positive strains and the amount of OTA produced in vitro were generally higher than those found in the other black Aspergilli (Teren et al., 1996; Heenan et al., 1998; Battilani et al., 2002; Cabañes et al., 2001).
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Argentina ranks in the fifth position of wine producers in the world. The central viticultural area in Argentina comprises vineyards located in Mendoza and San Juan provinces, which together represent over 90% of the Argentine wine production area. Previous studies showed that the presence of potentially OTA producer species has been detected in wine grapes (Dalcero et al., 2002; Rosa et al., 2002; Magnoli et al., 2003). No data are available on the dynamics of ochratoxigenic fungi through different stages of grape development in the field and the possible OTA contamination. In order to improve the knowledge on the epidemiology of black Aspergilli in grapes, a survey was carried out during 2003– 2004, – to evaluate the dynamic of fungal populations on grapes from the setting of the vineyard until harvest, – to evaluate the possible interactions among fungal populations, cultural practices and climatic conditions on OTA contamination. 2. Materials and methods 2.1. Grape sampling The survey was carried out in 2003/2004 in vineyards under different cropping systems (organic and non-organic) and on two grape varieties: Bonarda and Tempranillo, early and late maturation respectively. All the vineyards were settled at 85 km eastern Mendoza, 33° 16′ South Latitude 68° 09′ West Longitude. The vineyards chosen were representative of the grape-growing area as regards grape variety and farming methods. The four vineyards were treated with SO4Cu as fungicide, but only the nonorganic ones had herbicide treatment (glyphosate, three applications per year, doses 3 Lt/ha). Ten plants were marked along two major diagonal transects of each vineyard. One bunch was collected from each marked plant at setting and three further bunches were harvested in the following plants, in the same row, at one month after setting, early veraison and at harvest. Bunches collected were at 1.5 m from the ground. They were kept in paper bags and stored in portable refrigerators during transfer to the laboratory for fungal isolation and toxin analysis. Data on cropping systems and climatic conditions (mean daily air temperature, relative humidity and rainfall) were collected from a station close to the vineyards. 2.2. Fungal isolation and species identification From each vineyard and at each sampling stage fifty berries, randomly sampled from the ten different bunches were surface disinfected with sodium hypochlorite solution (1%) for 1 min; rinsed in sterile distilled water three times and plated onto a Dichloran 18% Glycerol Agar (DG18) and Dichloran Rose Bengal Chloramphenicol Agar (DRBC) media (Pitt and Hocking, 1997). After a incubation period of 7 days at 25 °C ± 1 °C the fungal isolates were identified to genus and species levels according to Pitt and Hocking (1997) and Samson et al. (2000). For Aspergillus identification, cultures were grown on Czapek Yeast Extract Agar (CYA) at 25 °C ± 1 °C and 37 °C ± 1 °C; Malt Extract Agar (MEA); 25% Glycerol Nitrate Agar (G25N); and Czapek Yeast Extract with 20% Sucrose Agar (CY20S), at
25 °C ± 1 °C. All plates were incubated for 7 days. Fungal identification was done according to Klich and Pitt (1994), Pitt and Hocking (1997) and Samson et al. (2000). 2.3. Ochratoxin A occurrence The berries from all bunches collected were manually crushed and the OTA content was determined following the methodology proposed by Zimmerly and Dick (1996)) with some modifications. A 50-g portion of berries was extracted with methanol: sodium bicarbonate at 1% (70:30, v/v) and blended at high speed for 1 min. The extract was filtered to remove particulate matter, and a 10-mL of extract was taken and diluted with 40 mL of PBS containing 0.01% Tween 20. The diluted extract was filtered through a microfibre filter. A ten-millilitre portion was taken and added to an immunoaffinity column (OchraTestTM; Vicam, Digen Ltd, Oxford, UK). The column was washed with 10 mL PBS containing 1% Tween 20 and then with 10 mL double distilled water. OTA was eluted from the column with methanol (HPLC grade), again at a flow rate of 1–2 drops per second. The HPLC apparatus used for determination of OTA was a Hewlett-Packard (Hewlett-Packard company, Palo Alto, CA, USA) chromatograph with a loop of 100 μL, equipped with a spectro-fluorescence detector (excitation, 330 nm; emission, 460 nm) and a C18 column (150 × 4.6 mm, 5 μm particle size; Supelcosil LCABZ, Supelco, Bellefonte, PA, USA), connected to a pre-column (20 × 4.6 mm, 5 μm particle size; Supelguard LC-ABZ, Supelco). The mobile phase was pumped at 1.0 mL min− 1, and consisted of an isocratic system as follows: 57% acetonitrile, 41% water and 2% acetic acid. OTA was quantified on the basis of HPLC fluorometric response compared with the OTA standard (purity N 99%; Sigma Aldrich Co., St Louis, MO, USA). The limit of detection was 1 ng g− 1. 2.4. Ochratoxin A production Ochratoxin A production was tested among 246 strains belonging to Aspergillus section Nigri (A. niger, A. awamori, A. foetidus, A. aculeatus, A. japonicus, A. carbonarius). OTA was determined following the methodology described by Téren et al. (1996). Briefly, the strains were grown in stationary cultures in 125 mL Erlenmeyer flasks containing 20 mL of YES medium (2% yeast extract, 15% sucrose) and were inoculated with a 4 mm plug from the margin of 7-day-old colonies on malt extract agar (MEA) and incubated at 30 °C ± 1 °C for 10 days in darkness. At the end of the incubation period, 1-mL portions of the culture medium were then mixed with 1 mL of chloroform and centrifuged at 4000 g for 10 min. The chloroform phase was transferred to a clean sylanized vial, evaporated to dryness and redissolved into 0.5 mL of mobile phase. OTA detection was done as described in Section 2.3. 2.5. Statistical analysis Data on contamination with Aspergillus section Nigri were analyzed previously by Arc sine plus one transformation by ANOVA followed by Tukey test in order to determine significant
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3. Results and discussion
Fig. 1. Dynamic of Aspergillus section Nigri in four vineyards (BO: Bonarda Organic; BC Bonarda Non-Organic; TO: Tempranillo Organic: TC: Tempranillo Non-Organic).
differences according to growth stage, cultural practice and grape variety. All statistical analyses were done using the Software GraphPAD InStat (Sigma St Louis, MO).
In the Bonarda variety (late maturation) settled under the non-organic system, species belonging to Aspergillus section Nigri were isolated since the setting stage. One month later these species were detected in both varieties analyzed (late and early maturation) under the two cropping systems. The number of colonized berries was low (1.3%) during the early growth stages, but the level of contamination increased up to 47% at harvest time. The percentage of samples colonized by Aspergillus section Nigri was significantly influenced by the growth stage, the grape variety or the cropping system (p b 0.05). (Fig. 1). In general, the incidence of Aspergillus species, section Nigri was higher at veraison and harvest stage in comparison with one month after setting. Most samples were colonized by A. niger and A. awamori in all growth stages. A. japonicus, A. aculeatus, A. foetidus and A. carbonarius were also present, although the distribution varied according to the growth stage, the grape variety or the cropping system (Fig. 2). These results partially agree with those reported by Battilani et al. (2006) who found that Aspergillus section Nigri species are more frequently isolated in hot and dry areas, while A. carbonarius showed
Fig. 2. Evaluation of Aspergillus section Nigri species in four Argentinean vineyards at setting ( ), 1 month setting( ), veraison and ( ) harvest (◫).
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higher incidences in hot and wetter areas. These results are supported by the findings of Mitchell et al. (2003) and Bellí et al. (2004, 2005) who showed that A. carbonarius grew optimally at 0.98–0.99 aw, while uniseriates and A. niger aggregate species showed no differences in growth in the range 0.90–0.995 aw. Although a moderate frequency of Aspergillus section Nigri was found, only 24% of the strains was OTA producers. The OTA levels produced varied depending on the species evaluated. Uniseriate species including A. japonicus and A. aculeatus produced between 1.3 and 6.3 ng/mL of the culture medium, while the biseriate species including A. niger, A. awamori and A. foetidus produced levels ranging from 1.3 to 50 ng/mL of the culture medium (Table 1). These results showed that the species within the Aspergillus section Nigri play the main role in the potential OTA contamination of grapes in the area evaluated. These results agree with previous studies from South America and Europe, since species included in the Aspergillus section Nigri has already been found as the main OTA producer species in Argentina, Brazil (Rosa et al., 2002), France (Sage et al., 2002) and Italy (Battilani et al., 2003). No evidence of contamination of grape neither by A. ochraceus nor by its closely related species P. verrucosum has been found. The ochratoxigenic fungi found in grapes are different from those found in cereals or feedstuffs in Argentina (Dalcero et al., 2002). The knowledge of the dynamics on fungal contamination, mainly by the Aspergillus species and OTA producers, is important, because these species are usually considered postharvest fungi and were also isolated from without visible symptom and damaged berries, the latter being in the risk of OTA contamination.
Table 1 Ochratoxin A production by Aspergillus section Nigri strains isolated through different grape growth stages Growth stage
Species
Positive/total a Range (ng/mL) Mean ± SE
Setting
A. niger 0/2 A. awamori 1/3 Berry A. niger 1/3 enlargement A. foetidus 0/2 A. japonicus 3/11 A. aculeatus 1/6 A. carbonarius 0/1 Veraison A. niger 3/11 A. awamori 4/8 A. japonicus 5/15 A. aculeatus 3/3 A. foetidus 2/2 Harvest A. niger 24/68
ND ND–3.09 ND–2.86 ND 3.3–8.62 ND–2.4 ND 1.3–26.9 1.2–4.8 1.3–2.4 1.2–2.1 1.56–2.41 1.4–50.61
A. awamori 2/27 A. japonicus 16/72 A. carbonarius 0/6 A. foetidus 0/6
1.44 1.31–6.13 ND ND
ND: Not detected ≤ detection limit: 1 ng/mL. SE: Standard error. a Positive OTA producer strains vs total strains isolated.
ND 3.09 ± 0 2.86 ± 0 ND 5.14 ± 0.11 2.4 ± 0 ND 10.1 ± 12.8 3.05 ± 1.75 1.6 ± 0.7 1.6± 0,3 1.98 ± 0.42 17.95 ± 16.33 1.44 ± 0 3.72 ± 2.41 ND ND
Fig. 3. Temperature (T), relative humidity (RH) and rainfall (R), computed from December to April in Santa Rosa during the 2003–2004 period.
Although all the vineyards evaluated were contaminated with OTA-producing species at harvest stage, the berries collected were OTA free (detection limit 1 ng g− 1) independent of the growth stage considered, the variety and the cropping system. These results can be explained taking into account that the presence of the toxicogenic species does not always imply OTA synthesis. According to previous studies, ecological conditions, which favour growth and subsequent high contamination, are different from those which enable optimum OTA production (Alexandre et al., 2004). Among the Aspergillus section Nigri isolates, those species belonging to A. niger aggregate produce OTA in lower concentrations, than A. carbonarius which is the dominant OTA producer (Abarca et al., 2001). In this study A. carbonarius was isolated in a lower percentage (30%) than the species belonging to A. niger aggregate and it was only present in grapes one month after setting. Climatic condition data showed that the mean daily temperature ranged between 15.3 °C and 25.2 °C from December to April and the summation of degree-days from 1 December to 31 March was 2553.1 °C and from December to April was 3012.1 °C. The total mean rainfall was 9.4 (December to March) and 9.5 mm (December to April) respectively. The rainiest month was January with a mean rainfall of 5.1 mm (Fig. 3). These conditions were quite different from those recorded by Battilani et al. (2003) in a survey carried out in Italy where the total rainfall ranged between 60 and 330 mm and the rainiest month has more than 70 mm. These data suggest that the climatic condition among grape-growing areas may be responsible for the differences in OTA contamination in grapes. This study is the first report on the dynamics of Aspergillus section Nigri in two grape varieties, under two cropping systems in Argentina. Further studies are ongoing to evaluate different potential inoculum sources of ochratoxigenic species in the vineyard. Acknowledgements This work was supported by a grant from SECyT (Secretaria de Ciencia y Técnica), Universidad Nacional de Rio Cuarto,
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