Aims: The effects of water activity (0AE90-0AE99 a w ), temperature (15-37°C), and their interaction on growth and ochratoxin A (OTA) production by eight isolates of Aspergillus carbonarius were investigated on synthetic nutrient medium (SNM) with composition similar to grapes. Methods and Results: Growth data were modelled by an multiple linear regression and response surface models were obtained. Aspergillus carbonarius grew much faster at 30°C than at the other temperature levels tested and its growth rate increased with increasing a w , maximum growth rate being between 0AE95 and 0AE99 a w . In general, isolates grew faster at 35-37°C than at 20°C, although no significant differences were found between these temperatures. OTA accumulation was also favoured by high a w levels, and although it was observed in the whole range of temperatures, maximum amounts were detected at 20°C. No OTA was found at the most unfavourable growth conditions. Conclusions: Optimum a w level for growth seems to correspond with optimum for OTA production, meanwhile the most propitious temperature for the toxin production was below the best one for growth. Significance and Impact of the Study: Prediction of A. carbonarius growth would allow estimating their presence and therefore, the OTA production, as it was found that conditions for the toxin production were more limited than those permitting growth.

Aims: The objective of this study was to determine the temporal ochratoxin A (OTA) accumulation profile of Aspergillus section Nigri at different water activity (a w ) levels. Methods and Results: Two Aspergillus carbonarius and two Aspergillus niger aggregate strains isolated from grapes were tested in vitro for OTA accumulation at 25°C on synthetic nutrient medium, over periods of 20 days at different a w levels. Results were modelled by a multiple linear regression and response surface predictive models were obtained. High levels of a w favoured OTA production by these moulds. Maximum amounts of OTA were found at the earlier growth states (5 days for A. carbonarius and 7-13 days for A. niger aggregate). Conclusions: Provided that A. section Nigri, and mainly A. carbonarius, play the main role in OTA presence in grapes, it would be critical to adjust the harvest and processing time to significantly reduce the chances for OTA accumulation. Significance and Impact of the Study: Ochratoxin A production by A. section Nigri has been shown for the first time to occur optimally after as little as 5 days on a grape-like medium.

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This paper reports the results from an extensive survey of filamentous fungi isolated from wine-producing grapes and their capacity to produce ochratoxin A (OTA) on Czapek Yeast Autolysate agar (CYA), in order to assess their potential for producing this toxin in grapes. Grapes were sampled from four Spanish wine-producing regions during 2001. The fungal infection in berries increased with time, reaching 100% at harvest. A total of 386 isolates of Aspergillus section Nigri and 10 of Aspergillus section Circumdati were isolated and tested for their ability to produce OTA in CYA. 21 strains produced OTA (18 Aspergillus section Nigri and 3 Aspergillus section Circumdati). Aspergillus section Circumdati isolates produced higher amounts of OTA than Aspergillus section Nigri ones, with means of 10.76 µg g −1 CYA and 1.42 µg g −1 CYA, respectively. Despite this, black aspergilli are believed to be highly responsible for the OTA levels found in musts and wines, as it is more widespread in grapes. Musts (n = 40) produced from the grapes collected were analysed. 15% were found to contain OTA, concentrations ranging from 0.091 to 0.813 ng ml −1 (detection limit: 0.07 ng ml −1 ), but no correlation was found with the ochratoxigenic moulds isolated from grapes.

Forty vineyards from four wine making regions of Spain were sampled at three different growth stages in 2002 and 2003. The aim was to study the fungi associated with grapes and their ability to produce ochratoxin A (OTA) on synthetic media. Among the total mycoflora, 464 (7.7%) and 648 (10.8%) Aspergillus section Nigri (black aspergilli) strains were isolated in 2002 and 2003, respectively, and were classified into three groups: isolates with uniseriate heads, A. niger aggregate and A. carbonarius. The latter presented the highest percentage of OTA-positive strains (82% in 2002 and 76% in 2003) and produced the highest levels of toxin (2.5-25 lg g )1 ). The sampling year, sampling date, the region and their interactions presented significant differences in the number of black aspergilli isolated. Most black aspergilli were found in 2003 and at harvest. A positive correlation between the number of black aspergilli found in grapes and the temperature in the field was found. Grapes from 2003, the warmest year, and from Costers del Segre, the warmest region, were significantly the most contaminated. No significant correlation between black aspergilli presence and other meteorological factors such as relative humidity or rainfall could be established. Musts from all the vineyards were also analysed in both years, although no OTA was found in either year.

A survey on the occurrence of ochratoxin A (OTA) in 240 grape-based beverages was carried out. Red and white wines from four different Spanish Designations of Origin (n = 160), musts (n = 20), grape juices (n = 10), ordinary wines (n = 20), special wines (Malaga, muscatel, sherry, vermouth, etc) (n = 20) and sparkling wines (n = 10) were assayed for OTA content using immunoaffinity column cleanup and high-performance liquid chromatography with fluorimetric detection (detection limit 0.05 µg l −1 ). Forty-three (17.9%) of the samples tested contained detectable levels of OTA. The overall mean OTA concentration in red and white wines of Designations of Origin was 0.30 and 0.18 µg l −1 respectively (ranges 0.05-3.19 and 0.05-1.13 µg l −1 respectively). The percentage of wine samples with detectable amounts of OTA was higher for red (18.3%) than for white (10%) wines. OTA was also found in two of 10 red ordinary wines (0.68 and 4.24 µg l −1 ), whereas none of 10 white ordinary wines contained OTA. The mean OTA amount detected in sparkling wines was 0.44 µg l −1 (range 0.14-0.71 µg l −1 ). Two of 20 must samples contained OTA at low levels (0.08 and 0.18 µg l −1 ), while none of 10 grape juice samples contained OTA. Highest amounts of OTA were found in special wines (45%), with a maximum of 15.25 µg l −1 in a muscatel sample.

This work gives a general overview of ochratoxin A (OTA) occurrence in wines and the methodology for OTA analysis. The results of more than two thousand samples taken from the literature have been taken into account to quite extensively describe the present situation of OTA contamination of wine. According to these data, OTA is much more commonly detected in red wines than in rosé and white wines, and OTA concentration is remarkably higher than in the latter ones. Thus OTA could be detected in 45% (median 34%) of white wine samples, whereas it was detected in 66% (median 66%) of rosé and 71% (median 90%) of red wine samples. When comparing the wines from Northern and Southern regions, the latter showed a higher contamination than those from the Northern area. It has been suggested that OTA accumulation could be due to fungi belonging to the genus Aspergillus in wines from Southern European countries because the crops are exposed to elevated temperatures, which favour growth of OTA-producing Aspergillus species over Penicillium. High performance liquid chromatography (HPLC) associated with fluorescence detection preceded by extraction of OTA using commercially available immunoaffinity columns (IAC) is currently the most applied method for OTA determination in wines.

A study was undertaken to evaluate the impact of the application of several fungicide treatments used in Spanish vines on Aspergillus carbonarius growth and ochratoxin A production. Three trials were designed in order: (1) to screen 26 fungicides at the doses recommended by manufacturers on grape-like synthetic medium at 20 and 30 degrees C; (2) to find out the minimum inhibitory concentration of each fungicide for A. carbonarius growth on synthetic medium; and (3) to investigate the effect of several fungicides on A. carbonarius-inoculated grapes. In synthetic medium nine fungicides significantly reduced A. carbonarius growth rate. Meanwhile, 13 fungicides completely inhibited its growth. In general, growth was faster at 30 degrees C than at 20 degrees C, contrary to ochratoxin A production. Fungicides that stopped fungal growth also inhibited ochratoxin A production, but not all the fungicides that reduced growth reduced the ochratoxin A synthesis. In general, fungicides that contained copper or strobilurins reduced both growth and ochratoxin A production, contrary to sulphur fungicides. At the optimum temperature for A. carbonarius growth of 30 degrees C, higher amounts of fungicide were needed to prevent fungal growth than at 20 degrees C. Among the fungicides that inhibited A. carbonarius growth on synthetic medium at the initial doses, cyprodinil seemed to be the active ingredient more effective at stopping fungal growth when testing reduced doses. The fungicide effect on grapes was similar to that on synthetic medium. Both infection and ochratoxin A production were reduced when using cyprodinil (37.5%) plus fludioxonil (25%) and azoxystrobin (25%). Penconazole (10%) also showed a clear reduction in ochratoxin A production at both temperatures, although infection was only reduced at 20 degrees C. Ochratoxin A reduction was strain and temperature-dependent. In general, fenhexamid (50%), mancozeb (80%) and copper hydroxide (80%) plus copper (50%) enhanced infection and ochratoxin A production.