Aspergillus Carbonarius Isolates Research Articles

Ochratoxigenic fungi and Ochratoxin A determination in dried grapes marketed in Tunisia

PurposeWith the present work, we aimed to assess the occurrence of ochratoxigenic fungi and Ochratoxin A (OTA) in dried grapes from Tunisia.MethodsDried grapes samples (n = 90) were investigated for the presence of ochratoxigenic fungi, which were further characterized at the species level through amplification of the internal transcribed spacer (ITS) region and polymerase chain reaction (PCR) product sequencing. Fungal isolates were tested for their ochratoxigenic potential by high-performance liquid chromatography with fluorescence detection (HPLC-FLD), as well as dried grapes samples after an immunoaffinity column (IAC) clean-up procedure.ResultsBlack Aspergilli isolates were the dominant genre among the filamentous fungi found in dried grapes samples and were the only OTA-producing fungi encountered. Aspergillus niger aggregate were the most frequently found isolates reaching 70%, 80%, and 85% in dried grapes samples from regions of Kelibia, Sfax, and Rafraf, respectively, while covered 100% of the relevant mycobiota found in imported samples. Aspergillus carbonarius isolates were found only in Sfax’s and Kelibia’s samples, while uniseriate Aspergilli were found between 7 and 20% in dried grapes from Kelibia, Sfax, and the imported samples. The in vitro OTA production test showed that 88.9% of OTA-producing isolates belonged to A. carbonarius with OTA levels varying from 0.06 to 1.32 μg/g of Czapek Yeast Agar (CYA). The remaining OTA-producing fungi (11.1 %) belonged to A. niger aggregate group having a maximum OTA potential of 2.88 μg/g CYA, and no uniseriate Aspergilli isolate was able to produce OTA. All dried grapes samples were free of OTA presence.ConclusionAccording to the present study’s findings, no OTA contamination was recorded in the investigated samples from Tunisian market. Nevertheless, the presence of strong OTA producers A. carbonarius in samples originated from the two out of three studied Tunisian regions, as well the high incidences of Aspergillus niger aggregate group with an attested potential for OTA production in all samples, necessitates further research on Tunisian dried grapes. Additionally, a continuous analysis of staple food of the Mediterranean diet is imperative to insure the best quality for the consumers and prevent potential health problems.

Growth And Ochratoxin A Production by Aspergillus carbonarius Isolated From Dried Figs In Aegean Region of Turkey Affected by Temperature And Water Activity

The impacts of temperature, water activity and their interactions on the growth rate and ochratoxin A (OTA) production by three Aspergillus carbonarius isolates obtained from dried figs in Agean Region of Turkey were investigated on Czapek yeast extract agar. The maximum specific radial growth rates at each set of conditions were obtained by using the primary model of Baranyi. Correlation coefficients were varied between 96% and 99%. Optimum growth of all isolates was at 30°C and 0.97 aw while optimum OTA production was observed at 15 - 20°C and 0.97 - 0.99 aw depending on the isolate. The assayed isolates showed varying abilities in growth rate and OTA production capacities as reported in the literature. The estimated optimum temperature and water activity values for growth and OTA production were in accordance with reported results for grape isolates. Temperature, water activity and their interactions significantly influenced the growth rate and ochratoxin A production by all tested isolates (P <0.05).

Competitive yeast action against Aspergillus carbonarius growth and ochratoxin A production

The aim of the present study was to investigate the interaction between 67 different yeast isolates and 3 wild isolates of Aspergillus carbonarius originated from Greek vineyards and characterized by high ochratoxigenic potential. The selected fungi were used either as single cultures or combined in a mixed culture. Yeasts and fungi were grown as mono-cultures and co-cultures in solid (MEA, CYA) and liquid (CY broth) media, grape berries and sterilized grape juice. Fungal growth was monitored by means of colony area measurements. The model of Baranyi and Roberts was further fitted to growth data to provide estimates of the colony area growth rate (cm2/day). Moreover, OTA analysis was undertaken for CY broth and agar as well as for grape berries and juice, on the 8th and 15th days of incubation at 25 °C. A significant reduction in fungal growth rate, final colony size and toxin production was observed in both liquid and solid media by the different yeast species assayed. The most competitive strains belonged to Saccharomyces, Pichia, Metschnikowia, Dekkera and Rhodotorula genera. Similar results were obtained from inoculated grape berries and grape juice. Specifically, Saccharomyces cerevisiae Y33 resulted in a decrease in fungal colony area of >90% and 93% after 3 and 4 days of co-culture, respectively. Similar results were obtained for OTA, where toxin concentration of the highest producer (A. carbonarius F3) was reduced from 14,983 and 31,565 ng/mL at 8 and 15 days, respectively, to 5 ng/mL and below detection limit (1 ng/g) when co-cultured with S. cerevisiae Y33. The results of this study could provide a pool of yeast species that must be further investigated for potential application as biological control agents at pre- and post-harvest level in wine and grape juice processing.

Influence of Saccharomyces cerevisiae and Rhotodorula mucilaginosa on the growth and ochratoxin A production of Aspergillus carbonarius

The aim of the present study was to investigate the influence of two yeast species, namely Saccharomyces cerevisiae Y33 and Rhotodorula mucilaginosa Y38 on the growth and ochratoxin A (OTA) production of four Aspergillus carbonarius isolates, grown either individually or in mixed culture. Fungal growth was assayed at five temperatures (15, 20, 25, 30, and 35 °C) and in two growth media, Czapek Yeast Extract Agar (CYA) and grape juice medium (JA). Fungal/yeast interactions were studied at different inoculum levels. Specifically, yeast species were inoculated at 103, 104, and 105 CFU/mL whereas fungal inoculum was maintained at 105 conidia/mL. Results showed that in the case of single cultures, higher values of growth rates were observed for A. carbonarius ITEM 5010 (12.79 cm2/day) on CYA at 30 °C followed by the other fungal strains with growth rates ranging from 9.74 to 10.96 cm2/day. Significantly higher values were estimated when the aspergilli were grown on JA, with A. carbonarius F80 and ITEM 5010 presenting the highest (40.11 cm2/day) and lowest (17.68 cm2/day) values, respectively. Toxin data showed that the maximum OTA production depended on the isolate and culture conditions. Specifically, A. carbonarius F71 and F80 were the highest OTA producing fungi on CYA with 91,659.6 and 74,364.2 ng/g at 20 and 15 °C, respectively. Both yeasts, but primarily S. cerevisiae Y33, were able to suppress fungal growth and reduce OTA production. A. carbonarius mixed culture produced the highest OTA amount (14,182.2 ng/g) on CYA at 20 °C. However, when co-cultured at the same conditions with S. cerevisiae Y33 and R. mucilaginosa Y38, OTA production amounted to 5.3, 11.6, 10.2 and 26.8, 3027.4, 3103.8 ng/g for 103, 104, 105 CFU/mL yeast inoculum, respectively.

English

Ochratoxin A (OTA) has been frequently found as a food contaminant, and it is considered one of the mycotoxins most harmful to human health. In this context, this study was performed aiming to evaluate mycelial growth and ochratoxin A production from three isolates of Aspergillus carbonarius and Aspergillus ochraceus. The isolates were inoculated on culture media coffee, wheat and maize as well as YES medium and cultivated at 15 and 25°C. Measurement of colony diameters was performed every 48 h for growth description. OTA quantification was made with high-performance liquid chromatography (HPLC) three times (5th, 15th and 25th day). All the isolates presented higher growth rate in YES medium at 25°C; however, growth in every culture media at both tested temperatures was observed. The maize-based medium showed the lowest growth rate. YES medium induced the most OTA production. In grain-based culture media, A. ochraceus isolates produced lower quantities of OTA, reaching a maximum of 9.95 µg/g. However, A. carbonarius isolates produced much higher quantities, showing the A. carbonarius CCDCA10608 isolate, which produced 29.83 µg/g at 15°C in YES medium and 13.06 in wheat-based medium at 15°C. Therefore, among the tested conditions, those more and less favorable to OTA production were recognized. Key words: Aspergillus, food, high performance liquid chromatography, mycotoxin, ochratoxin A.

In vitro evaluation of essential oils against Aspergillus carbonarius isolates and their effects on Ochratoxin A related gene expression in synthetic grape medium

Clove, mandarin, cinnamon and lemongrass essential oils (EOs) were tested for their efficacy against 2 wild isolates (Ac29, Ac28) and a reference strain (5010) of Aspergillus carbonarius. Two different assays were performed on Malt Extract Agar (MEA) and Czapek Yeast Extract Agar (CYA) solid cultures, as well as on synthetic grape medium (SGM) liquid cultures. The impact of EOs on fungal growth and toxin production was measured after 7 days of incubation at 25 °C. Furthermore, the effect of clove, lemongrass and mandarin EOs on A. carbonarius OTA related genes AcOTAnrps, AcOTApks and laeA was investigated by relative expression using Real Time PCR on SGM cultures. Results showed that in solid cultures, complete fungal inhibition was obtained at 100–300 μl/l of EOs revealing a dose dependant effect of clove, cinnamon and lemongrass, whereas mandarin was proved to be less effective. EOs could not inhibit OTA production but only reduce it by 15–98% depending on the type and concentration of EO, even though in certain cases of low concentrations, EOs showed to enhance toxin production. In liquid cultures, a major effect on growth inhibition and OTA reduction was observed for all EOs. Regarding gene expression, down regulation of AcOTAnrps was observed in the majority of treatments that could be associated with toxin reduction. Moreover, AcOTApks exhibited some differential expression but laeA did not show any differences in the transcriptional profile. The above gene expression results may indicate a possible mode of action of EOs in OTA biosynthetic pathway.

Modeling the effect of natamycin, pine-resin and environmental factors on the growth and OTA production by Aspergillus carbonarius using response surface methodology

The effect of two antifungal compounds (natamycin, pine-resin), temperature and water activity, on the growth rate, lag phase duration and Ochratoxin A (OTA) production by three Aspergillus carbonarius isolates (Ac-28, Ac-29, and Ac-33), was studied by means of Response Surface Methodology (RSM) based on a Central Composite Design (CCD). Two different experimental designs were performed as a function of temperature (16.6–33.4°C), water activity (0.90–0.97 aw), natamycin (0–1000ngml−1) or pine-resin (0–2.61%, w/v) on a Synthetic Grape-juice Medium (SGM). OTA production was analyzed after 5, 10 and 15days of incubation. A second-order polynomial model was fitted to each response parameter to assess the growth and OTA potential of all fungal isolates. Results showed that natamycin, aw and temperature had significant effects on the lag phase duration of all isolates, as well as on OTA accumulation after 10days of incubation for Ac-29 and 15days for Ac-28 and Ac-33 isolates. The same results were obtained for OTA production after treatment with pine-resin. However, fungal growth rates were not statistically significant in both experiments, with the exception of Ac-29 and Ac-33 after treatment with pine-resin. Overall, high natamycin concentrations (800 and 1000ngml−1) delayed fungal growth depending on the environmental factors assayed. Moreover, treatment with pine-resin at 16.6°C/0.94 aw/1.1% w/v, as well as at 25°C/0.90 aw/1.1% w/v, completely inhibited fungal growth up to 15days of incubation.

Comparative study of growth responses and screening of inter-specific OTA production kinetics by A. carbonarius isolated from grapes.

The aim of this work was to assess OchratoxinA (OTA) production of different Aspergillus carbonarius isolates, evaluate their growth profile through different growth measurements, and reveal any underlying correlation between them. Ten different isolates of A. carbonarius isolated from Greek vineyards located in different geographical regions were examined in vitro for their OTA production potential after an incubation period of up to 11 days. All fungal isolates grew on a synthetic grape juice medium (SGM) similar to grape composition at optimum conditions of temperature and water activity (25°C and 0.98 aw). Samples for OTA determination were removed at 3, 5, 7, 9, and 11 days of growth and analyzed by HPLC. Based on OTA measurements the isolates were characterized by diverse OTA production ranging from 50 to 2000 ppb at day 11. The different fungal growth responses (colony diameter, colony area, biomass, biomass dry weight, and colony density) have been measured and correlated with toxin production by means of principal components analysis (PCA), confirming satisfactory correlation and explained over 99% of data variability. Leudeking-Piret model was also used to study OTA production with time, revealing a mixed-growth associated trend and pointing a fail-safe model with slightly better prediction through colony area. This approach contributes to the assessment of correlation between mycotoxin production and different methods of fungal growth determination in relation to time.

Effect of Water Activity, Temperature, and Mixed Fungal Spore Interactions on Ochratoxin A Production by Aspergillus carbonarius

The purpose of this work was to investigate the potential of two nontoxigenic Aspergillus section Nigri species (Aspergillus tubingensis and Aspergillus japonicus) to influence the in vitro ochratoxin A (OTA) production of three toxigenic Aspergillus carbonarius isolates (Ac-28, Ac-29, and Ac-33) from Greek vineyards of different geographical areas. OTA accumulation was evaluated by inoculation of 0:100, 25:75, 50:50, 75:25, and 100:0 ratios of mixed spore suspensions on a synthetic grape juice medium for up to 28 days at different temperatures (15, 20, and 25°C), water activity (aw) levels (0.95 and 0.98 aw) and incubation time (7, 14, 21, and 28 days). Results confirmed that environmental factors and fungal species had a significant effect on OTA production. Specifically, maximum OTA concentration for Ac-28 (3.21 μg g−1) and Ac-29 (7.69 μg g−1) was observed at 20°C/0.98 aw and for Ac-33 (9.13 μg g−1) at 15°C/0.95 aw, regardless of incubation time. Moreover, A. tubingensis had no significant influence on OTA concentration of all toxigenic isolates assayed, regardless of temperature, aw, and incubation time. On the other hand, the presence of A. japonicus slightly inhibited OTA production of Ac-29 and Ac-33, while for Ac-28, stimulation of OTA was observed in some cases. Overall, lower aw levels reduced OTA accumulation for Ac-28 and Ac-29, regardless of temperature, inoculum ratio, and time. On the contrary, for Ac-33, low aw increased OTA production, regardless of the investigated parameters. The importance of this study concerns the understanding of interspecific interactions on OTA diffusion by A. carbonarius in an attempt to find ways to prevent the presence of toxins in grapes and their derivatives.

Effect of Temperature on the Growth and Ochratoxin A Production of the Aspergillus section Nigri Members Isolated from Dried Figs

AbstractThis study investigated the effects of temperature and incubation time on the growth and ochratoxin A production of three isolates of Aspergillus carbonarius and one Aspergillus niger agg. isolate obtained from dried figs. Optimum growth for all isolates were at 30C while minimum lag phase durations for A. carbonarius isolates were at 25C and for A. niger agg. at 30C. For ochratoxin A (OTA) production, lower optimum temperature values were determined for Aspergillus section Nigri members in comparison to growth temperature. Optimum OTA production was observed at 15–20C and 25C by A. carbonarius and A. niger agg., respectively. Significant amounts of OTA could be accumulated after 3 or 5 days of incubation depending on the isolates. Temperature significantly influenced the growth rate and the extension of lag phase and OTA production of all tested isolates (P < 0.05), whereas incubation time was only significant for OTA production by A. niger agg.Practical ApplicationsThis study contributes to a better understanding of the effect of temperature and incubation time on ochratoxin A (OTA) production of the Aspergillus section Nigri isolated from dried figs. Our results indicated that Aspergillus section Nigri members isolated from dried figs are able to grow and produce OTA in a wide range of temperature (15–40C), and also A. carbonarius may be the main source of OTA in dried figs because of their higher OTA production capacities. Only 3 or 5 days of incubation were required for OTA accumulation, so the length of drying and storage processes could provide sufficient time for the growth and accumulation of OTA in dried figs. Our results may help develop management strategies and drying methods at constant temperature to reduce OTA contamination on dried figs.

Effect of preharvest anti-fungal compounds on Aspergillus steynii and A. carbonarius under fluctuating and extreme environmental conditions

Ochratoxin A (OTA) has been found in pre-harvest and freshly harvested wheat. Spanish climatic conditions point to Aspergillus species as probably responsible for this OTA. In this study the effectiveness of 5 non-specific antifungal chemicals used on wheat fields (25.9% tebuconazole+60.0% N,N-capramide dimethyl; 12.70% tebuconazole+12.7% prothioconazole+59.5% N,N‐amide dimethyldecane; 12.5% epoxiconazole; 12.5% tetraconazole; and 70% thiophanate methyl) and an extract from Equisetum arvense were investigated in vitro on wheat by recording growth (colony size, fungal growth and DNA concentration) and OTA production of two ochratoxigenic isolates of Aspergillus carbonarius and three of A. steynii, simulating current and extreme climatic conditions. Inoculated wheat was incubated under two alternating temperature cycles (20/30°C and 25/35°C) with photoperiod (14/10h lightness/darkness), and two moisture levels (40 and 25%).The Aspergillus species tested seemed to be able to persist in predicted future climatic conditions, in particular, A. steynii, a high OTA producer. Azoles were effective in controlling the growth of A. carbonarius and A. steynii, and this effectiveness may not be compromised by the increase in temperature and decrease of humidity. However, azoles are not useful for the prevention of OTA accumulation, which could be only reduced in A. carbonarius under non-extreme conditions. Although some adjustment will probably be required, further studies should be conducted in the field, since the antifungals used in this study are applied at flowering and not directly on the grain. Moreover, timing of antifungal application may need to be optimized. Finally, Equisetum extract showed promising results as an antifungal, however further work to adjust the applied concentrations is required.

Optimising the number of isolates to be used to estimate growth parameters of mycotoxigenic species

The aim of the present work was to mathematically assess the minimum number of isolates that would lead to equivalent growth parameters estimates to those obtained with a high number of strains. The datasets from two previous works on 30 Aspergillus carbonarius isolates and 62 Penicillium expansum isolates were used for this purpose. First, the datasets were used to produce a global estimation of growth parameters μ (growth rate, mm/d) and λ (time to visible growth, d) under the different experimental conditions, providing also a 95% confidence interval. Second, a computational algorithm was developed in order to obtain an estimation of the growth parameters that one would obtain using a lower number of isolates and/or replicates, using a bootstrap procedure with 5000 simulations. The result of this algorithm was the probability that the obtained estimation falls in the 95% confidence interval previously produced using all sample isolates. Third, the algorithm was intensively applied to obtain these probabilities for all possible combinations of isolates and replicates. Finally, these results were used to determine the minimum number of isolates and replicates needed to obtain a reasonable estimation, i.e. inside the confidence interval, with a probability of 0.8, 0.9 and 0.95. The results revealed that increasing the number of isolates may be more effective than increasing the number of replicates, in terms of increasing the probability. In particular, 12–17 isolates of A. carbonarius led to the same growth parameters as the total 30 (p = 0.05) or 9 isolates with p = 0.20; by contrast, 25–30 isolates of P. expansum led to the same growth parameters as the total 62 (p = 0.05) or 18–21 isolates with p = 0.20. As far as we know, this is the first study that provides a systematic evaluation of the number of isolates and replicates needed when designing an experiment involving mycotoxigenic moulds responses to environmental factors, and may serve to support decision making in this kind of studies or other similar ones.

Growth and ochratoxin A production by Aspergillus carbonarius at different pHs and grape maturation stages

The objective of this work was to study the effect of some factors, linked to grape composition during ripeness process, on the growth and ochratoxin A (OTA) production of Aspergillus carbonarius isolated from grapes. Aspergillus carbonarius isolates were tested (i) in vitro, in Czapek yeast autolysate agar (CYA) at different pHs (2·5-4·5) and incubation times (2-6 days), and (ii) in situ, in fresh grapes collected at different maturation stages. Aspergillus carbonarius was able to grow with the same intensity at the different maturation stages and pH levels tested. In general, a similar trend of OTA production by A. carbonarius in response to acidity in media and in grape was observed. Low pH level seemed optimal for maximum OTA production. Aspergillus carbonarius strains can strongly grow and produce OTA on grape from the early stages of maturation. Extrinsic environmental conditions at the harvest period and skin thickness are, probably, the mains factors contributing to OTA contamination of grapes at the end of maturation. The results lead to a better understanding of the critical point during grape maturation for the growth of ochratoxigenic fungi and the toxin production.

Ochratoxigenic moulds and effectiveness of grape field antifungals in a climatic change scenario

The presence of ochratoxin A (OTA) in grapes and grape derivatives has been reported mainly in the Mediterranean area. Consequently, great efforts are being made to prevent the growth of Aspergillus on grapes. However, the European Commission suggests that climate change may result in increased temperatures and longer drought periods in southern Europe. Therefore the aim of this study was to investigate how ochratoxigenic fungal growth and the efficiency of fungicides used at present might be affected by environmental conditions predicted with climate change. The effectiveness of grape field antifungals (Switch, Flint Max and Equisetum arvense extract) under two alternating temperature, photoperiod and relative humidity (RH) scenarios (current: 20/30 °C, 16 h light/8 h darkness, 80% RH; predicted: 25/37 °C, 16 h light/8 h darkness, 75% RH) on the growth and OTA production of two Aspergillus carbonarius isolates and one Aspergillus ochraceus isolate on grapes was investigated. Predicted conditions reduced A. carbonarius and limited A. ochraceus growth. Antifungals reduced fungal infection (by 40-84%), although no correlation between climatic conditions and effectiveness of the antifungals was found. However, Switch always showed the greatest reduction and E. arvense (0.02 g mL(-1) extract) the least. Higher temperatures affected OTA production by the isolates in different ways. In general, Switch and Flint Max reduced OTA production, while E. arvense stimulated it.

Ochratoxin A and ochratoxigenic black Aspergillus species in Tunisian grapes cultivated in different geographic areas

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 μg/l. Only 4 samples out of 39 exceeded the OTA limit of 2 μg/l fixed by the EU for wine and grape juices. The most contaminated grapes were those from Raf–Raf 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.

Impact of non-selective fungicides on the growth and production of ochratoxin A by Aspergillus ochraceus and A. carbonarius in barley-based medium

The aim of this study was to assess the influence of the non-selective fungicides mancozeb, copper oxychloride, and sulfur on the growth and capability for producing ochratoxin A (OTA) of ochratoxigenic isolates of Aspergillus carbonarius and A. ochraceus in barley-based medium. Lag phases and growth rates were determined for each fungicide at different doses, at 15°C and 25°C and at 0.97 aw . Mancozeb at 40 mg l−1 inhibited fungal growth and provided lag phases >24 days at 10–20 mg l−1 and 15°C. OTA was observed only at 25°C and doses <10 mg l−1. At 15°C, copper oxychloride proved inhibitory at 800 mg l−1, while at 25°C growth was not delayed and only high doses decreased OTA levels. Sulfur was inhibitory or provided large lag phases at 5–8 g l−1 (at 15°C) while at 25°C growth took place even at 8 g l−1, although OTA levels were low or undetectable. The antifungal activity decreased in the order mancozeb > copper oxychloride > sulfur, and was lower at 25°C than at 15°C. OTA accumulation was affected by the type of fungicide, dose, temperature and time. The efficacy of these fungicides on the growth of A. carbonarius and A. ochraceus and OTA production in barley-based medium is assessed for the first time.

Is intraspecific variability of growth and mycotoxin production dependent on environmental conditions? A study with Aspergillus carbonarius isolates

The aim of this study was to assess the impact of suboptimal environmental conditions on the intraspecific variability of A. carbonarius growth and OTA production using thirty isolates of A. carbonarius.Three aw/temperature conditions were tested, one optimal (0.98aw/25°C) and two suboptimal: 0.90aw/25°C and 0.98aw/37°C as suboptimal water activity and temperature, respectively, which might take place through over ripening and dehydration of grapes. For each condition, 12 Petri dishes were inoculated, and colony growth and OTA production were measured over time.ANOVA revealed significant differences among μ and λ within the 30 assayed isolates. Coefficients of variation (CV%) revealed a wider dispersion of growth rates at 0.90aw/25°C compared to 0.98aw/25°C, and a more than 4-fold higher CV at 0.98aw/37°C compared to 0.98aw/25°C. However, dispersion of lag phases was similar at 0.98aw/25°C and 0.90aw/25°C and wider at 0.98aw/37°C.There were significant differences (p<0.05) among OTA levels (ng/mm2) for the different conditions, values being lower under marginal conditions, and particularly at 0.98aw/37°C. Coefficients of variation (CV%) revealed a wider dispersion of OTA production at 0.90aw/25°C compared to 0.98aw/25°C, while CV at 0.98aw/37°C was similar to that at 0.98aw/25°C.In order to address the strain variability in growth initiation and prove the well-established notion of reducing OTA in foods by preventing fungal growth, a greater number of strains should be included when developing models for conditions that are suboptimal both for aw for OTA production and temperature levels for growth.

Specific PCR primers for the detection of isolates of Aspergillus carbonarius producing ochratoxin A on grapevine

Aspergillus carbonarius is the main fungus responsible for ochratoxin A (OTA) production on grapes and in wine. Fungal polyketide synthases are involved in OTA biosynthesis in Aspergillus species. The ketosynthase (KS) domain of an A. carbonarius gene encoding polyketide synthase (pks) was isolated, cloned and sequenced. The nucleotide sequence showed high similarity to KS domains isolated from other Aspergillus species. The sequence was used to design a new set of primers in order to identify potential producers of OTA from among isolates of A. carbonarius. The primers amplified all the A. carbonarius strains tested specifically, and did not amplify any other species of Aspergillus or Penicillium normally found on grapes or involved in OTA biosynthesis. Further, gene expression was related to OTA production. Both gene transcription and levels of the mycotoxin were higher when A. carbonarius was grown on YES medium at 15°C and 30°C, whereas a low transcription and mycotoxin presence were observed when the fungus was grown on PDB medium at the same temperatures. No transcription or OTA production were observed when the fungus was grown on either YES or PDB at 10°C. The primers will be useful for the detection of ochratoxigenic strains of A. carbonarius both in vineyards and during wine production.

Alternating temperatures and photoperiod effects on fungal growth and Ochratoxin A production by Aspergillus carbonarius isolated from Tunisian grapes

The effect of three alternating temperatures cycles (20/30, 20/37 and 25/42 degrees C) and photoperiod on growth and Ochratoxin A (OTA) production of six isolates of Aspergillus carbonarius on synthetic nutrient medium were investigated. The different temperature regimes affected significantly both the mycelial growth and the OTA production. The best growth and OTA production were recorded at 20/30 degrees C. The isolates from the region of Baddar produced the highest OTA yields. A 24 h light cycle generally enhanced the growth of A. carbonarius. Growth rates cycles of 11 h/13 h light/darkness and 24 h darkness were often similar for individual isolates, such conditions enhanced OTA production in two of the six isolates tested.

Effect of temperature and water activity on growth and ochratoxin A production boundaries of twoAspergillus carbonariusisolates on a simulated grape juice medium

To develop and validate a logistic regression model to predict the growth and ochratoxin A (OTA) production boundaries of two Aspergillus carbonarius isolates on a synthetic grape juice medium as a function of temperature and water activity (a(w)). A full factorial design was followed between the factors considered. The a(w) levels assayed were 0.850, 0.880, 0.900, 0.920, 0.940, 0.960, 0.980 and the incubation temperatures were 10, 15, 20, 25, 30, 35 and 40 degrees C. Growth and OTA production responses were evaluated for a period of 25 days. Regarding growth boundaries, the degree of agreement between predictions and observations was >99% concordant for both isolates. The erroneously predicted growth cases were 3.4-4.1% false-positives and 0.7-1.4% false-negatives. No growth was observed at 10 degrees C and 40 degrees C for all a(w) levels assayed, with the exception of 0.980 a(w)/40 degrees C, where weak growth was observed. Similarly, OTA production was correctly predicted with a concordance rate >98% for the two isolates with 0.7-1.4% accounting for false-positives and 2.0-2.7% false-negatives. No OTA production was detected at 10 degrees C or 40 degrees C regardless of a(w), and at 0.850 a(w) at all incubation temperatures. With respect to time, the OTA production boundary shifted to lower temperatures (15-20 degrees C) as opposed to the growth boundary that shifted to higher temperature levels (25-30 degrees C). Using two literature datasets for growth and OTA production of A. carbonarius on the same growth medium, the logistic model gave one false-positive and three false-negative predictions out of 68 growth cases and 13 false-positive predictions out of 45 OTA production cases. The results of this study suggest that the logistic regression model can be successfully used to predict growth and OTA production interfaces for A. carbonarius in relation to temperature and a(w). The proposed modelling approach helps the understanding of fungal-food ecosystem relations and it could be employed in risk analysis implementation plans to predict the risk of contamination of grapes and grape products by A. carbonarius.