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Background: Bio-geographical differences in fungal infection distribution have been observed around the world, confirming that climatic conditions are decisive in colonization. This research is focused on the impact of ultraviolet radiation (UV) on Aspergillus species, based on the consideration that an increase in UV-B radiation may have large ecological effects., Results: Conidia of six mycotoxigenic Aspergillus species isolated from vineyards located in the northeast and south of Spain were incubated for 15 days under light/dark cycles and temperatures between 20 and 30 °C per day. Additionally, 6 h of exposure to UV-A or UV-B radiation per day were included in the light exposure. UV irradiance used were 1.7 ± 0.2 mW cm(-2) of UV-A (peak 365 nm) and 0.10 ± 0.2 mW cm(-2) of UV-B (peak 312 nm). The intrinsic decrease in viability of conidia over time was accentuated when they were UV irradiated. UV-B radiation was more harmful., Conclusion: Conidial sensitivity to UV light was marked in Aspergillus section Circumdati. Conidia pigmentation could be related to UV sensitivity. Different resistance was observed within species belonging to sections Flavi and Nigri. An increase in UV radiation could lead to a reduction in the Aspergillus spp. inoculum present in the field (vineyards, nuts, cereal crops). In addition, it could unbalance the spore species present in the field, leading to a higher predominance of dark-pigmented conidia., (© 2015 Society of Chemical Industry.)

Ochratoxin A (OTA) is a mycotoxin with nephrotoxic, teratogenic and immunotoxic properties which represents a serious risk for human and animal health. Aspergillus carbonarius is considered the main OTA-producing species in grapes and products such as raisins, wine or juices, although it has also been isolated from coffee, cocoa and cereals. Till now not much information is available about regulatory mechanisms of OTA production by A. carbonarius. A better understanding of how environmental factors influence OTA production and which genes are involved in its regulation could help us design new control strategies. In this study, we have evaluated the role of VeA and LaeA transcriptional factors, which have been shown to regulate secondary metabolism in response to light in A. carbonarius. To this aim, veA and laeA genes were deleted in an ochratoxigenic A. carbonarius strain by targeted gene replacement using Agrobacterium tumefaciens-mediated transformation. Loss of veA and laeA in A. carbonarius yields to an organism with slight differences in vegetative growth but a strong reduction in conidial production. A drastic decrease of OTA production that ranged from 68.5 to 99.4% in ΔveA and ΔlaeA null mutants was also observed, which was correlated with a downregulation of a nonribosomal peptide synthetase involved in OTA biosynthesis. These findings suggest that VeA and LaeA have an important role regulating conidiation and OTA biosynthesis in response to light in A. carbonarius in a similar way to other fungi where functions of VeA and LaeA have been previously described. This is the first report of a transcriptional factor governing the production of OTA by A. carbonarius., (© 2013 Elsevier B.V. All rights reserved.)

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., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The aim of this work was to model the growth of Aspergillus parasiticus and Aspergillus ochraceus, both mycotoxin producers, near to the growth/no growth boundaries and validate those models in sterile maize grain, peanuts and coffee beans. Malt extract agar was adjusted to six different water activities: 0.93, 0.91, 0.89, 0.87, 0.85 and 0.80. Plates were incubated at 10, 15, 20, 25, 30, 37 and 42 °C. For each of the 42 conditions, 10 Petri dishes were inoculated. Both kinetic and probability models were applied to colony growth data. The results of the present study indicate that the developed probability modelling approach could be satisfactorily employed to quantify the combined effect of temperature and water activity on the growth responses of A. ochraceus and A. parasiticus. However, validation of kinetic results led to poor goodness of prediction. In this study, the validation samples were placed near to the expected boundaries of the models in order to test them under the worst situation. Probability of growth prediction under extreme growth conditions was somewhat compromised, but it can be considered acceptable., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

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 a(w)/temperature conditions were tested, one optimal (0.98a(w)/25°C) and two suboptimal: 0.90a(w)/25°C and 0.98a(w)/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 0.90a(w)/25°C compared to 0.98a(w)/25°C, and a more than 4-fold higher CV at 0.98a(w)/37°C compared to 0.98a(w)/25°C. However, dispersion of lag phases was similar at 0.98a(w)/25°C and 0.90a(w)/25°C and wider at 0.98a(w)/37°C. There were significant differences (p<0.05) among OTA levels (ng/mm(2)) for the different conditions, values being lower under marginal conditions, and particularly at 0.98a(w)/37°C. Coefficients of variation (CV%) revealed a wider dispersion of OTA production at 0.90a(w)/25°C compared to 0.98a(w)/25°C, while CV at 0.98a(w)/37°C was similar to that at 0.98a(w)/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 a(w) for OTA production and temperature levels for growth., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Predictive models can be a tool to develop strategies to prevent mould development and consequently mycotoxin production. The aims of this work were to assess the impact of a) high/low levels of inoculum and b) optimal/suboptimal environmental conditions on fungal responses based on both kinetic and probabilistic models. Different levels of spore suspensions of Aspergillus carbonarius and Penicillium expansum were prepared and inoculated centrally with a needlepoint load on malt extract agar (MEA) with 50 replicates. While optimum conditions led to a colony diameter increase which followed Baranyi's function, suboptimal conditions led to different grow functions. In general, growth rate (mu) and lag phase (lambda) were normally distributed. Specifically, the growth rate (mu) showed similar distributions under optimal growth conditions, regardless of the inoculum level, while suboptimal a(w) and temperature conditions led to higher kurtosis distributions, mainly when the inoculum levels were low. Regarding lambda, more skewed distributions were observed, mainly when the inoculum levels were low. Probability models were not much affected by the inoculum size. Lower probabilities of growth were in general predicted under marginal conditions at a given time for both strains. The slopes of the probability curves were smaller under suboptimal growth conditions due to wider distributions. Results showed that a low inoculum level and suboptimal conditions lead to high variability of the estimated growth parameters and growth probability., (2010 Elsevier Ltd. All rights reserved.)

The aim of this study was to determine the effect of a natural carotenoid mixture (Capsantal FS-30-NT), containing capsanthin and capsorubin, on growth and mycotoxin production of ochratoxin A-producing A. ochraceus, A. westerdijkiae, and A. tubingensis isolates. One isolate of each species, previously isolated from paprika or chilli, was inoculated on Czapek Yeast extract Agar (CYA) medium supplemented with different amounts of capsantal (0 to 1%) and incubated at 10, 15 and 25 degrees C for 21days. Growth rates and lag phases were obtained, and OTA production was determined at 7, 14 and 21days. The taxonomically related A. ochraceus and A. westerdijkiae showed the same behavior at 15 degrees C, but A. ochraceus was able to grow at 10 degrees C and had higher growth rates at 25 degrees C. A. tubingensis had the highest growth rates and lowest OTA production capacity of the assayed isolates, and it was not able to grow at 10 degrees C. Capsantal addition resulted in increased lag phases at 15 degrees C for all the strains, while growth rates remained rather constant. At 25 degrees C capsantal reduced growth rates, with rather constant lag phases. However, the effect of capsantal on OTA production was inconclusive, because it depended on temperature or time, and mostly was not significant. Low temperature has been a crucial factor in OTA production, regardless of the capsantal concentration tested, especially for A. tubingensis and A. westerdijkiae. Industrial storage temperature for paprika and chilli is approximately 10 degrees C. If this temperature is maintained, mould growth and OTA production should be reduced., (Copyright 2010 Elsevier B.V. All rights reserved.)

A survey of the fungal contamination and occurrence of aflatoxins and ochratoxin A (OTA) in 50 pistachio nut samples collected from commercial stores around Catalonia (Spain) was carried out. Aspergillus flavus was found in 30% of samples, whereas Aspergillus section Nigri, Aspergillus ochraceus, and Penicillium verrucosum were found in 40%, 2% and 26% of samples, respectively. A total of 204 fungal isolates were obtained; 70.8% of A. flavus isolates were able to produce aflatoxin B1 and B2, whereas OTA production capacity was detected in 54.4% of the A. section Nigri biseriate isolates. Aflatoxins were detected in five samples (10% of total), all exceeding the maximum legal limit set for aflatoxin B1 (2 µg kg(-1)) or for total aflatoxins (4 µg kg(-1)), with one sample having a very high level of contamination (1134.5 µg kg(-1)). Only one sample of pistachio showed OTA contamination at 0.67 µg kg(-1). Simultaneous contamination by aflatoxins and OTA was not observed in these samples. It was concluded that the frequency of aflatoxins and OTA contamination in pistachio samples available in the Spanish market is relatively low. Thus, no significant contribution to health risk should be expected from aflatoxin consumption through pistachios among healthy consumers. However, the existence of single samples with an extremely high aflatoxin concentration reinforces the need for effective prevention strategies in pistachio processing and commercialization.

Black aspergilli are the main fungal contaminants in pistachio nuts. Ochratoxin A (OTA) production has been repeatedly reported in Aspergillus section Nigri; OTA has been occasionally detected in pistachio nuts in high concentrations. The aim of this study was to develop suitable validated models to predict the growth and OTA production boundaries by an Aspergillus carbonarius isolated from pistachios as a function of moisture content and storage temperature of pistachios. A full factorial design was used: the moisture content levels assayed were 12.5%, 17.9%, 24.0%, 29.5% and 34.8% and the incubation temperatures were 10, 15, 20, 25, 30, 37 and 42 degrees C. A probability model was built to predict the growth of the strain under the assayed conditions, which proved to be concordant in 73-91% of the cases with the observed probabilities in the validation test specifically chosen for limiting growth conditions. Similarly, the probability for the presence of OTA was correctly predicted in 90% of the cases. OTA accumulation was mainly a function of the temperature of storage, with a sharp increase at <15-20 degrees C; this value was very different from 30 to 35 degrees C, which was optimum for growth. Increasing OTA levels were found with increasing moisture content in the pistachio nuts. The impact of these results in the implementation of HACCP plans in Western Asian countries, is discussed. Probability models were applied in this work to mycotoxin accumulation for the first time, however, further research is required in the kinetics of mycotoxins accumulation to develop proper empirical models.

Aims: To evaluate the effect of preharvest grape pesticides in Aspergillus section Nigri infection in dehydrating grapes and the final ochratoxin A (OTA) content. Additionally, the effect of coinoculation of moulds frequently isolated from grapes and raisins on Aspergillus section Nigri infection was studied., Methods and Results: Fungicide-treated grapes were inoculated with Aspergillus carbonarius, Aspergillus niger aggregate, Eurotium amstelodami and Penicillium janthinellum in different combinations, then dehydrated by reducing a(w) for 20 days. The percentages of colonized grapes treated with fungicides were, in general, lower, but no differences were observed among fungicides. The untreated grapes always showed higher concentrations of OTA, regardless of the inoculum applied. In general, Chorus was the most effective antifungal treatment in reducing OTA accumulation in grapes during dehydration. Penicillium janthinellum reduced Aspergillus section Nigri colonization and OTA accumulation in grapes during dehydration., Conclusions: The four preharvest fungicides studied reduced the Aspergillus section Nigri growth and OTA production by A. carbonarius during dehydration of grapes., Significance and Impact of the Study: The success of these chemical treatments might depend on the mycobiota composition of grapes.

The effect of the application of two fungicides (cyprodinil alone and a mixture of cyprodinil and fludioxonil) on the mycoflora of grapes, especially ochratoxigenic fungi, was studied. Different doses and application times were analyzed. Grape mycobiota was isolated and identified, and the classification of black aspergilli was carried out. We found that 81.7% of the isolates belonged to Aspergillus niger aggregate and 18.3% to Aspergillus carbonarius. The ability to produce ochratoxin A (OTA) was studied on Czapek yeast extract agar (CYA) medium in 238 isolates. Most A. carbonarius (97.2%) produced detectable amounts of OTA, while only 2.9% of the A. niger aggregate were OTA producers. Most of the isolates (58%) produced less than 2.5 microg OTA per g of CYA. That, together with the highest levels of black aspergilli detected near harvest, proved the reported theory that they are primarily responsible for OTA in grapes. The fungicides studied had a significant effect on black aspergilli in three of the four vineyards sampled, as the natural increase of black aspergilli when approaching harvest was in general lower in all the fields treated with fungicides as compared to the control treatment. A mixture of cyprodinil (37.5%) and fludioxonil (25%) applied at veraison and 21 days before harvest was the most effective treatment to prevent black aspergilli in grapes, together with a single application of this mixture at veraison followed by an application of cyprodinil (50%) 21 days before harvest. No OTA was detected in musts (n=112) produced from either the control treatment or the treated grapes.

The aim of this study was to characterize Ochratoxin A (OTA) production by Aspergillus carbonarius under different environmental conditions, and to elucidate the diffusion capacity of OTA throughout synthetic medium. One strain belonging to the species A. carbonarius isolated from vine dried fruit was single-point inoculated onto triplicate synthetic nutrient medium plates at two water activities (0.92 and 0.97) and two temperatures (20 and 30 degrees C). Daily radii were measured and OTA production was tested after 4, 7, 10, 14 and 18 days of incubation at four distances from the centre of colony (1-4 cm). OTA production was detected mainly at 0.97 a(w). Earlier production was detected at 30 degrees C (optimum for growth), whereas maximum OTA concentrations were found at 20 degrees C. OTA production was detected from mycelium that was only a few days old and attained its optimum when mycelium was 4-7 days old at 0.97 a(w). OTA diffusion was observed at 0.92 a(w) and 20 degrees C. Thus OTA production is discernable in young A. carbonarius mycelium and diffusion of the toxin has been shown to occur in a solid substrate.

The effect of light and temperature regimes simulating day and night in the field in the months preceding grape harvest on Aspergillus carbonarius growth and ochratoxin A (OTA) production were investigated. Twelve-hour photoperiod affected positively A. carbonarius growth with no differences between incubating the mould at day temperature (28 degrees C) or alternating day/night temperatures (28 degrees C/20 degrees C). Slower growth, however, was observed with constant incubation at 20 degrees C. Under 12h-alternation periods of day and night temperatures, growth was faster at continuous darkness than under continuous light conditions. Light did not cause any morphological changes in the aspect of the colonies. No significant differences on OTA production were detected due to either fluctuating temperature or photoperiod. However, as photoperiod enhanced the growth of colonies, it also enhanced OTA accumulation. The ability of A. carbonarius to produce OTA reported in previous laboratory studies has been demonstrated to be stimulated in field conditions.

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.

Grapes from three different regions with a long winemaking tradition in Spain were analysed at different growth stages in order to identify the ochratoxigenic mycobiota during three consecutive seasons. The correlation between meteorological parameters and ochratoxigenic fungi was studied and revealed a significant positive correlation between black aspergilli infection and temperatures in the month preceding each sampling date. No significant correlation was found with either relative humidity or rainfall. Biodiversity indexes were also calculated in this study. Black aspergilli species were the most abundant in grapes before harvest, and among them, Aspergillus carbonarius was the main ochratoxin A (OTA) producer species and represented 78-100% of the isolates tested. The results obtained support the key role of A. carbonarius as the main source of OTA contamination in grapes.

Ochratoxin A is a well-known mycotoxin produced by species of the genera Penicillium and Aspergillus. OTA-producing species from A. section Nigri are considered the source of OTA detected in grapes, dried vine fruits and wines. Other fungi present in grapes during their maturation can grow and interact with OTA-producing Aspergillus species and affect OTA production. In this study seven fungi (Alternaria alternata, Cladosporium herbarum, Eurotium amstelodami, Trichoderma harzianum, Penicillium decumbens, P. janthinellum and Candida sp.) disolated from grapes and dried vine fruits were grown in SNM medium paired with OTA-positive A. carbonarius at two temperatures (20 and 30 degrees C) and at two water activities (0.92 and 0.97). OTA production was tested after 5, 7, 10, 14 and 18 days of incubation, at four distances (1, 2, 3 and 4 cm) from A. carbonarius inoculation point in the inter-colony axis. At 0.92 a(w) OTA production was almost negligible. At 0.97 a(w) and 30 degrees C OTA accumulation was reduced when A. carbonarius was grown in paired cultures, particularly with A. alternata, C. herbarum, P. decumbens and P. janthinellum. At 0.97 a(w) and 20 degrees C, there was no clear effect of the interacting species on OTA accumulation; in general E. amstelodami and Candida sp. seemed to stimulate OTA production, whereas T. harzianum and P. decumbens reduced it. Competing mycoflora acted as an additional control factor against OTA accumulation at 30 degrees C; but at 20 degrees C, where OTA production is optimal, this did not happen. Thus maintaining the temperature of grapes at or above 30 degrees C during dehydration may provide some control against OTA accumulation in grapes.

Mould growth was modelled on fermented bakery product analogues (FBPA) of two different pH (4.5 and 5.5), different water activity (a(w)) levels (0.80-0.90) and potassium sorbate concentrations (0-0.3%) by using seven moulds commonly causing spoilage of bakery products (Eurotium spp., Aspergillus spp. and Penicillium corylophilum). For the description of fungal growth (growth rates) as a function of a(w), potassium sorbate concentration and pH, 10-terms polynomial models were developed. Modelling enables prediction of spoilage during storage as a function of the factors affecting fungal growth. At pH 4.5 the concentration of potassium sorbate could be reduced to some extent only at low levels of a(w), whereas at pH 5.5 fungal growth was observed even by adding 0.3% of potassium sorbate. However, this preservative could be a valuable alternative as antifungal in such bakery product, of slightly acidic pH, if a long shelf life has not to be achieved.

Aims: The effects of water activity (0.90-0.99 a(w)), temperature (15-37 degrees 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 degrees C than at the other temperature levels tested and its growth rate increased with increasing a(w), maximum growth rate being between 0.95 and 0.99 a(w). In general, isolates grew faster at 35-37 degrees C than at 20 degrees 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 degrees 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.

The effects of different steady-state water activity levels (a(w), 0.93, 0.95 and 0.98) and temperature (15 and 25 degrees C) on colonisation patterns of Aspergillus and Penicillium spp., when colonising irradiated maize grain in the presence of Fusarium moniliforme and Fusarium proliferatum were assayed in terms of populations (colony forming units, CFUs g grain(-1)), seed infection and colonisation rates. The activity of F. moniliforme and F. proliferatum in grain reduced the presence of Aspergillus flavus, Aspergillus niger, Aspergillus ochraceus to some extent, particularly at 15 degrees C and higher water availabilities (0.95-0.98 a(w)). In contrast, colonisation patterns of Penicillium implicatum on maize grain were unaffected by either Fusarium spp. in terms of CFUs or seed infection. Correlations were made between CFUs, seed infection, growth rates and niche overlap indices and hyphal interactions to try and link key indicators of competitiveness and dominance by an individual species.

This study was carried out to determine the effect of water activity (aw) and temperature on the patterns of colonization of maize grain by isolates of Fusarium moniliforme and F. proliferatum in the presence of interacting spoilage fungi, such as Aspergillus flavus, A. niger, A. ochraceus, and Penicillium implicatum, over 4-week incubation periods. The impact that such interactions have on Fusarium infection of maize grain and populations and on the production of fumonisins were all evaluated. At 0.93 and 0.95 aw, interactions of the two Fusarium spp. with other species to a large extent resulted in mutual inhibition on contact or overgrowth by the Aspergillus and Penicillium spp., regardless of temperature and growth rates of the species. Seed infection by the Fusarium spp. at 25 degrees C was influenced mainly by A. flavus and A. niger, with a reduction of infection by up to 45 and 30%, respectively, after 2 weeks. At 15 degrees C and lowered aw levels, infection was reduced, and sometimes completely inhibited, by A. niger, A. ochraceus, and P. implicatum. By contrast, populations of the Fusarium spp. (CFU per gram grain) were reduced mainly by A. flavus and P. implicatum. A. niger reduced populations of the Fusarium spp. at 0.93 and 0.95 aw, particularly after 4 weeks of incubation. Interaction with A. niger, A. ochraceus, and A. flavus at 15 and 25 degrees C and 0.98 aw resulted in a significant stimulation in fumonisin production by both Fusarium spp. Interaction with P. implicatum resulted in a decrease in fumonisin production by F. moniliforme. However, interaction with F. proliferatum resulted in an enhanced production at both temperatures and 0.95 and 0.98 aw. This study has shown that fungal interactions may act as an additional control factor in Fusarium spp. development on grain. However, although interactions lead to a decreased colonization by Fusarium, mainly at lower aw, a decrease in fumonisin production accumulation does not occur at the same time. Knowledge of these interactions is essential for improving effective control procedures for preventing growth and mycotoxin production by such fungi.

This study compared the effect of temperature (5-45 degrees C), water availability (water activity, aw; 0.995-0.75) and their interactions on the temporal rates of germination and mycelial growth of three mycotoxigenic strains of Aspergillus ochraceus and one isolate each of A. flavus, A. niger, Penicillium aurantiogriseum and P. hordei in vitro on a maize extract medium. Germination was very rapid at > 0.90 aw with an almost linear increase with time for all species. However, at < 0.90 aw, the germination rates of A. flavus and P. hordei were slower. The aw minima for germination were usually lower than for growth and varied with temperature. The effect of aw x temperature interactions on the lag phases (h), prior to germination, and on the germination rates (h(-1)), were predicted for the first time for these fungi using the Gompertz model modified by Zwietering. This showed that A. flavus, A. niger and the two Penicillium spp. had very short lag times between 0.995-0.95 aw over a wide temperature range. At marginal temperatures, these were significantly higher, especially at < 10 degrees C for Aspergillus spp. and > 30 degrees C for Penicillium spp. There were also statistically significant differences between lag phases and germination rates for three different isolates of A. ochraceus. The Aspergillus spp. also germinated faster than the Penicillium spp. The temperature x aw profiles for mycelial growth varied considerably between species, both in terms of rates (mm d(-1)) and tolerances. Predictions of the effects of important environmental factors such as temperature, aw and their interactions on lag times to germination, germination rates and mycelial growth are important in the development of hurdle technology approaches to predicting fungal spoilage in agricultural and food products.

Contamination of animal feed with mycotoxins still occurs very often, despite great efforts in preventing it. Animal feeds are contaminated, at low levels, with several mycotoxins, particularly with those produced by Aspergillus and Fusarium genera (Aflatoxin B 1, Ochratoxin A, Zearalenone, Deoxynivalenol and Fumonisina B 1 ). In animal feed, to date, only Aflatoxin B 1 is limited through EU regulation. Consequently, mycotoxins cause serious disorders and diseases in farm animals. In 2009, the European Union (386/2009/EC) approved the use of mycotoxin-detoxifying agents, as feed additives, to prevent mycotoxicoses in farm animals. The present review gives an overview of the problem of multi-mycotoxin contamination of feed, and aims to classify mycotoxin adsorbing agents (minerals, organic, and synthetic) for feed decontamination, focusing on adsorbents with the ability to bind to multiple mycotoxins, which should have a more effective application in farms but they are still little studied in scientific literature. [ABSTRACT FROM AUTHOR]

Abstract: This study aimed to: (1) determine the mycobiota of Capsicum powder samples, paying a special attention to the mycotoxigenic moulds; (2) evaluate the contamination levels of aflatoxins (AF), ochratoxin A (OTA), zearalenone (ZEA), deoxynivalenol (DON), T2 and HT2 toxins in those samples. Thirty-two samples were obtained through the methods of sampling established by the European Union legislation. Aspergillus and Eurotium were the most frequently found genera. Aspergillus section Nigri had the higher relative frequency in the samples, A. niger aggregate being the most representative group of this section. Other potentially mycotoxigenic Aspergillus, Fusarium and Penicillium species were found, but in a lower frequency. Co-occurrence of mycotoxins was confirmed in the 32 Capsicum powder samples. All samples were contaminated with AF and OTA, 27% with ZEA (36% of chilli and 18% of paprika samples), 9% with DON (18% of chilli and 6% of paprika samples), 6% with T2 (18% of chilli samples) and none of the samples contained HT2. Although in the present study the most common genera found (Aspergillus and Eurotium) belong to storage moulds, some field fungi such as Fusarium spp. were also found, and their toxins were sometimes detected. This fact supports the hypothesis that mycotoxin contamination of Capsicum products may occur both in the field and/or during storage. [Copyright &y& Elsevier]

Aims: To evaluate the effect of Botrytis cinerea growth on ochratoxin A (OTA) production by Aspergillus carbonarius and degradation. Methods and Results: OTA-producing A. carbonarius and B. cinerea were grown on grape-like medium at 20°C for 7 days. Radii of colonies were daily recorded and OTA was analysed. In addition, each B. cinerea isolate was inoculated on grape-like synthetic nutrient medium (SNM) paired with each A. carbonarius isolate at a distance of 45 mm. Botrytis cinerea isolates were also grown in OTA-spiked SNM. Growth rates of B. cinerea and A. carbonarius were 20 and 7·5 mm day−1, respectively. The growth of the colonies of each species stopped when they contacted each other in paired cultures. OTA production by A. carbonarius in the contact area was affected by B. cinerea, but no clear trend was observed. All B. cinerea isolates showed to degrade between 24·2% and 26·7% of OTA from spiked SNM. Conclusions: The ecological advantage of B. cinerea, in terms of growth rate, vs. OTA-producing Aspergillus in some wine-growing regions and its ability to degrade OTA may explain the low levels of this toxin in noble wines. Significance and Impact of the Study: At determinate conditions, the presence of B. cinerea in grapes with A. carbonarius may help in reducing OTA accumulation. [ABSTRACT FROM AUTHOR]

Abstract: This study investigated the impact of skin damage on Aspergillus carbonarius colonization and ochratoxin A (OTA) production in grapes at different temperatures and relative humidity. Four ochratoxigenic A. carbonarius strains isolated from wine grapes were used to inoculate artificially damaged and undamaged table grapes. Grapes were stored at three levels of relative humidity (80%, 90% and 100%) and at two temperatures (20 and 30°C). After seven days, the infection percentage of A. carbonarius was recorded and OTA accumulation in berries was analysed. Damaged grapes were more commonly infected and development of colonies was higher than in undamaged ones; consequently more OTA was detected in the former treatment. Temperature and relative humidity had significant influences on both infection and toxin content. The amount of OTA detected at 30°C was higher than at 20°C in most of the treatments. The highest relative humidity (100%) led to maximum amounts of OTA while no significant differences were found between 90% and 80% in the OTA content. The implementation of preventive measures in order to minimise berry damage in the field by controlling pathogenic fungi and insects during grape growing and removing visibly damaged grapes at harvest may significantly reduce OTA contamination in grapes. [Copyright &y& Elsevier]

Aims: To examine how UVC affects the different genera of fungi commonly isolated from grapes, with the aim of understanding changes in mycobiota during grape ripening and possible applications for preventing grape decay during storage. Methods and Results: Spores of Aspergillus carbonarius, Aspergillus niger, Cladosporium herbarum, Penicillium janthinellum and Alternaria alternata (between 100–250 spores/plate agar) were UVC irradiated for 0 (control), 10, 20, 30, 60, 300 and 600 s. Plates were incubated at 25°C and colonies were counted daily up to 7 days. Alternaria alternata and Aspergillus carbonarius were the most resistant fungi. Conidial germination in these species was reduced by approx. 25% after 10 s of exposure, compared with greater than 70% reduction for the remaining species tested. Penicillium janthinellum spores were the most susceptible at this wavelength. UVC exposures of 300 s prevented growth of all isolates studied, except for Alternaria alternata. Conclusions: UVC irradiation plays a major role in selecting for particular fungi that dominate the mycobiota of drying grapes. Significance and Impact of the Study: The UVC irradiation of harvested grapes could prevent germination of contaminant fungi during storage or further dehydration. [ABSTRACT FROM AUTHOR]

The article presents a study which examined the effects of incubation time, especially at early growth stages, on mean ochratoxin A (OTA)-producing capacity and OTA accumulation by four Aspergillus carbonarius strains isolated from Spanish and Tunisian grapes. OTA accumulation starts earlier at high temperatures but increases slowly and becomes constant. The study concluded that OTA production and accumulation may happen in the temperature range of 15-30 degree Centigrade.

Ochratoxin A (OTA) is a secondary metabolite produced by several species of Aspergillus and Penicillium ; among them Aspergillus ochraceus and Penicillium verrucosum are two ochratoxigenic species capable of growing in different climates and thus contamination of food crops with OTA can occur worldwide. OTA can be found in a wide range of foods such as cereals, coffee, cocoa, spices, beer, wine, dried vine fruit, grapes and meat products. OTA is toxic to animals, it presents neurotoxic, immunotoxic and nephrotoxic effects. It has been implicated in a human kidney disorder known as Balkan Endemic Nephropathy. This review focuses on the ecophysiology of ochratoxin-producing Aspergillus ochraceus and Penicillium verrucosum , the effect of environmental factors on their germination, mycelial growth, and OTA production. Knowledge of environmental conditions required for sucessive stages of fungal development represent the first step towards preventing mycotoxin formation. Predictive models for different stages of fungal development are presented, which allow prediction of the time before spoilage as a function of the abiotic factors. Finally, the implications of these studies in management of barley, coffee and grapes are described. This can help to identify the critical control points in their production, storage and distribution processes. [ABSTRACT FROM AUTHOR]

Abstract: Aspergillus ochraceus is an ochratoxin A (OTA) producer mould found in grapes and this may contribute to OTA contamination in wines and grape juices. The influence of relative humidity (R.H.; 80, 90 and 100%) and temperature (10, 20 and 30°C) on visible mould growth on grapes and OTA accumulation after 14 days of incubation by this fungal species has been studied using a full factorial design with three replicates. The two abiotic factors and their interaction (R.H.×temperature) affected significantly the A. ochraceus growth in berries, which was maximum at 90–100% R.H. levels. With regard to the optimum temperature level, it occurred at 30°C at 80 and 90% R.H., whereas no significant differences were detected at 20 and 30°C when R.H. was 100%. OTA production by A. ochraceus on grapes was not significantly modified by the assayed levels of temperature and R.H, with a mean value of 3.53ngg−1. Predictive models of percentage of grapes with visible growth of A. ochraceus isolates under different relative humidity and temperature are presented. [Copyright &y& Elsevier]

n. bellí, a.j. ramos, i. coronas, v. sanchis and s. marín. 2005.The effects of water activity (0·90–0·99aw), temperature (15–37°C), and their interaction on growth and ochratoxin A (OTA) production by eight isolates ofAspergillus carbonariuswere investigated on synthetic nutrient medium (SNM) with composition similar to grapes.Growth data were modelled by an multiple linear regression and response surface models were obtained.Aspergillus carbonariusgrew much faster at 30°C than at the other temperature levels tested and its growth rate increased with increasingaw, maximum growth rate being between 0·95 and 0·99aw. 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 highaw 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.Optimumaw 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.Prediction ofA. carbonariusgrowth 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. [ABSTRACT FROM AUTHOR]

The antifungal effect of 20 essential oils against the most important moulds in terms of spoilage of bakery products (Eurotium spp., Aspergillus spp. and Penicillium spp.) was investigated. Suitable solutions of essential oils were added directly to an agar culture medium (containing 2% wheat flour) to obtain a final concentration in the range between 0 to 1,000ppm. Antifungal activity was tested at different water activity (aw) and pH conditions, and the fungal growth was followed by measuring the colony diameter during the incubation period. Only cinnamon leaf, rosemary, thyme, bay and clove essential oils exhibited some antifungal activity against all isolates. The antifungal activity depended on aw and pH levels. In general, a stronger inhibition was observed as the water availability increased, moreover, in some cases at 0.80 aw they favoured fungal growth. The interaction between essential oil concentration and pH depended mainly on the essential oil. Rosemary, thyme and bay were more effective at pH 5, loosing their activity as pH increased, while only cinnamon leaf was more effective near neutrality. These findings strengthen the possibility of using plant essential oils as an alternative to chemicals to preserve bakery products. [ABSTRACT FROM AUTHOR]

Aspergillus ochraceus as a fungal contaminant and ochratoxin A (OTA) producer plays an important role in coffee quality. Temperature and water activity (aw) significantly influence mycelial growth and OTA production by isolates of A. ochraceus on green coffee beans. Maximum mycelial growth was found at 30°C and 0.95 to 0.99 aw. A marked decrease in growth rate was observed when temperature and aw were reduced. At 0.80 aw, mycelial growth occurred only at 30 and 20°C for one isolate. Maximum OTA production was found at 20°C and 0.99 aw. At 10°C, OTA was not produced, regardless of aw. Similarly, no OTA was detected at 0.80 aw. OTA production ranged from the limit of detection (40 ng g-1 of green coffee) to 17,000 ng g-1 of green coffee. Significant intraspecific differences in mycelial growth and OTA production were found. Primary data for lag phases prior to mycelial growth under the influence of temperature and aw were modelled by multiple linear regression, and the response surface plots were obtained. [ABSTRACT FROM AUTHOR]

The effects of water activity (aw) and temperature on growth of Aspergillus section Nigri isolated from wine grapes were investigated on an agar medium with composition similar to that of grapes. Temperatures in the range of 10–37 °C were tested. Optimum temperatures for growth were between 30 and 37 °C. Water activity levels ranging from 0.90 to 0.995 were tested. Optimum aw for growth was 0.98 in most cases. Statistical differences were found among the groups tested (A. carbonarius, A. niger aggregate and A. section Nigri uniseriates). Growth rates models for the factors assayed have been obtained. [Copyright &y& Elsevier]

Ochratoxin A (OTA) is a secondary metabolite of Aspergillus and Penicillium species, including Aspergillus ochraceus, a species that can be found in stored cereal grains such as barley. The objective of this study was to determine the effects of water activity (aw, 0.80–0.99), temperature (10, 20, 30 °C), and A. ochraceus isolate differences on radial growth and OTA production in irradiated barley grains. The three isolates showed optimal conditions for growth and ochratoxin A production at 0.99 aw and 30 °C, with a marked decrease of growth rates and OTA production at the lowest levels of aw and temperature assayed. The minimum aw level for growth, observed in this study, was 0.85 and 0.90 aw for OTA production. Significant differences among the isolates were found. Lag phases prior to fungal growth and OTA production values were modelled by multiple linear regression and response surface models. These models could provide an approximate prediction of growth and OTA production. [Copyright &y& Elsevier]

Aspergillus ochraceus is an ochratoxin producing fungus that can be found on stored cereal grains such as barley. The objective of this study was to determine the effects of water activity (aw, 0.75–0.99) and temperature (10–30°C) on germination and growth on barley extract agar medium (BMEA) of three isolates of Aspergillus ochraceus. The three isolates showed an optimal aw for germination and growth of 0.99–0.95 at 20–30°C, with a marked increase of the lag phases and decrease of germination and growth rates at the marginal levels of aw and temperature assayed. Minimum level of aw for germination was 0.80 and 0.85 for growth. Data were then modeled by an MLR regression and response surface models were obtained. These models may allow a rough prediction of germination/growth as a function of the storage temperature and moisture content of barley grains. [Copyright &y& Elsevier]

Fungal infection and ochratoxin A (OTA) contamination were determined in green coffee samples from different origins, in which OTA-producing fungi were also identified. About 72% of the beans analysed by direct plating presented fungal infection, including species of Aspergillus, Penicillium and Rhizopus. The genus Aspergillus was presented in more than 90% of infected coffee beans. Aspergillus ochraceus and Aspergillus section Nigri isolates represented 2.8 and 65.4%, respectively from the total number of isolates from the coffee beans. The capacity to produce OTA was determined in 260 isolates of A. section Nigri and 19 of A. ochraceus by the agar plug method, giving positive results for 6% of the A. section Nigri isolates and 16% of the A. ochraceus. OTA production was analysed by high performance liquid chromatography. OTA contamination of green coffee beans was analysed by enzyme immunoassay. OTA levels in all samples analysed were above the limit of detection (0.6 µg/kg), with a mean OTA concentration of 6.7 µg/kg. [ABSTRACT FROM AUTHOR]

n. bellí, a.j. ramos, v. sanchis and s. marín. 2003. The objective of this study was to determine the temporal ochratoxin A (OTA) accumulation profile of Aspergillus section Nigri at different water activity ( aw) levels. 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 aw levels. Results were modelled by a multiple linear regression and response surface predictive models were obtained. High levels of aw 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). 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. 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. [ABSTRACT FROM AUTHOR]

Abstract: Aspergillus ochraceus is an important contaminant of diverse substrates, such as cereals, coffee, grapes and derivates. This fungus produce a nephrotoxic metabolite, ochratoxin A (OTA), whose presence on food and feeds may be an important risk for animal and human health. The aim of this work was to evaluate the significance of the origin of A. ochraceus isolates on their OTA production patterns on different substrates (yeast extract sucrose (YES) broth, irradiated barley grains, irradiated green coffee beans and sterilized grapes) and under different environmental conditions. Results did not show a significant influence of the isolation source on OTA-production profiles by A. ochraceus isolates on several substrates, since the isolates which produced the highest OTA amounts in vitro (YES medium) were also the isolates with the highest OTA yields on the other substrates. Abiotic factors assayed (water activity, temperature and substrate) affected significantly OTA productions by A. ochraceus. Maximum OTA amounts were detected at 25°C and 0.98 a w on all substrates tested. The highest OTA accumulations found on the different substrates were: green coffee beans (> 2mgg−1), barley grains (∼1mgg−1), YES medium (13.9μgml−1) and grape (∼3ngg−1). [Copyright &y& Elsevier]