Effect of water activity and temperature on growth of ochratoxigenic strains of Aspergillus carbonarius isolated from Argentinean dried vine fruits

Aspergillus carbonarius is an ochratoxin A (OTA) producing fungus, predominantly responsible for the production of this mycotoxin in grapes, wine and dried vine fruits. The objective of this study was to determine the in vitro effects of water activity (a(w), 0.80-0.95) and temperature (15-35 degrees C) on lag phase extension and radial growth rate of a cocktail inoculum of four strains of A. carbonarius. The maximum growth rate was observed at 0.95 a(w) and 30 degrees C (17.46 mm day(-1)). In general, growth rates increased with the increment of a(w). No growth was observed at a(w) below 0.85. For all a(w) levels tested the highest growth rate was detected at 30 degrees C. At 15 degrees C growth only occurred at the higher a(w) levels evaluated (0.925 and 0.95) at a growth rate of 3.82 and 5.57 mm day(-1) respectively. The shortest lag phase (0.26 days) was found at 0.925 a(w) and 35 degrees C. At marginal conditions of a(w) and temperature the lag phases increased, being the highest registered at 20 degrees C and 0.89 a(w) (33.7 days). The pattern of effects of environmental factors on growth was similar among Argentinean A. carbonarius strains and those from several European countries, Israel and Australia.     

Water activity and temperature effects on growth of Aspergillus niger, A. awamori and A. carbonarius isolated from different substrates in Argentina

The objectives of this study were to determine the effect of water activity, temperature, and their interactions on a) mycelial growth rate and b) the lag phase prior to grow of seven isolates of Aspergillus section Nigri isolated from peanuts, maize kernels, dried grapes and coffee cherries from Argentina. Three Aspergillus niger, three A. awamori and one A. carbonarius isolates examined showed optimum a(W) level for growth at 0.97 with optimal temperature of 30 degrees C. for most of the isolates and 25 degrees C for only one (A. awamori RCP176). Minimal a(W) for growth was 0.85 at the highest temperature tested. Overall growth was reduced up to 50% at 0.93 a(W). Growth was also to a large extend inhibited at 0.85 a(W) for most isolates even after 21 days of incubation at temperatures lower than 30 degrees C. The analysis of variance of the effect of single (isolate, a(W) and temperature), two- and three-way interaction showed that all factors alone and all interactions were statistically significant (P<0.001) in relation to growth rates and lag phase for A. niger, A. awamori and A. carbonarius isolates. These data are relevant since these species are isolated in high frequency on numerous substrates for human and animal consumption in Argentina.     

Occurrence of ochratoxin A-producing fungi in grapes grown in Italy

A study was carried out to investigate fungi present on grapes grown in Italy. Aspergillus and Penicillium spp. isolates were identified and studied in vitro, and their ability to produce ochratoxin A (OA) was investigated. The survey involved nine vineyards, three located in northern Italy and six located in southern Italy. In 1999 and 2000, bunches of grapes at different growth stages were collected from all nine vineyards, and berry samples were placed in moist chambers and incubated. The resultant fungal colonies were then transferred to petri dishes containing Czapek yeast agar and incubated at 25 degrees C for 7 days; the fungal isolates were identified and then cultivated in liquid Czapek yeast medium and evaluated for their ability to produce OA. During the survey, 508 isolates were collected, with 477 belonging to Aspergillus spp. and 31 belonging to Penicillium spp. Among the aspergilli, species of the Fumigati, Circumdati, and Nigri sections were identified, with species of the Nigri section (464 isolates) largely predominating; for species of the Nigri section, 108 isolates were uniseriate, 270 were biseriate, and 86 were identified as Aspergillus carbonarius. Black aspergilli isolated over the 2 years of the study showed a very similar pattern. On average, the biseriates represented about 60% of the isolates collected in both years and were followed by uniseriates (21%) and A. carbonarius (19%). The most toxigenic strains proved to be those of A. carbonarius; about 60% of these isolates were OA producers and produced the highest levels of OA. A. carbonarius was more frequent in the south, but in both areas the percentages of OA-producing isolates remained the same.     

Studies on the interaction between grape-associated filamentous fungi on a synthetic medium

Eleven fungi isolated from grapes and sun-dried grapes (Alternaria alternata, Cladosporium herbarum, Eurotium amstelodami, Penicillium janthinellum, P. decumbens, Trichoderma harzianum, Candida sp., Aspergillus carbonarius OTA-negative, A. carbonarius OTA-positive, A. niger var. niger. and A. japonicus var. aculeatus), were grown in SNM medium at different water activities (0.82-0.97) and temperature (20-40 degrees C) levels for 18 days. Pairs of one Aspergillus faced with one non-Aspergillus were grown at 0.87-0.97 a(w) and at 20 and 30 degrees C. In single cultures, daily radii were recorded. In paired cultures radii were recorded and each A. section Nigri isolate was given a dominance score. At high temperatures and low water activities, Penicillium isolates, E. amstelodami and A. niger var. niger showed higher growth rates, while T. harzianum only grew well at the highest water activity. In addition, A. section Nigri was dominant in most paired assays, being only surpassed by T. harzianum at 0.97 a(w) and 20 degrees C. Thus, prevalence of A. section Nigri in sun-dried grapes can be explained by its adaptation to environmental conditions of sun-drying, and by its capability to dominate other fungal species when coming into contact with them.     

Effects of fungal interaction on ochratoxin A production by A. carbonarius at different temperatures and a(w)

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.     

Effect of carbendazim and physicochemical factors on the growth and ochratoxin A production of Aspergillus carbonarius isolated from grapes

Carbendazim is a systemic fungicide that is commonly used on several crops (tobacco, fruit, vegetables, cereals, etc.). This fungicide is used to control fungal infections in vineyards. It is indicated against Botrytis cinerea, Uncinula necator, Plasmopara viticola and other fungi and can be used either alone or coupled with other fungicides. However, there is a lack of in-depth studies to evaluate its effectiveness against growth of Aspergillus carbonarius isolated from grapes and OTA production. A medium based on red grape juice was used in this study. Preliminary studies were performed at 0.98 a(w) and 25 degrees C using carbendazim concentrations over a wide range (1-2000 ng/ml medium) to control both growth of a strain of A. carbonarius isolated from grape and its ability to produce OTA. As the lag phase increased considerably at levels > 1000 ng/ml of medium, detailed studies were carried out in the range 50-450 ng/ml of medium at 0.98-0.94 a(w) and 20-28 degrees C. Statistical analysis (multifactor ANOVA) of the data revealed that the three factors assayed and the interactions a(w)-carbendazim concentration and a(w)-temperature had significant effects on lag phase duration. The highest lag-times were observed at 0.94 a(w,) 20 degrees C, and with 450 ng carbendazim/ml. The three factors also had significant effects of the growth rate and there was an interaction between a(w) and temperature. The growth rate of A. carbonarius in these cultures is favoured by high water availability and relatively high temperatures. However, addition of carbendazim at the assayed levels did not significantly influenced fungal growth rate. Accumulation of OTA was studied as a function of four factors (the three previously considered, and time). All factors had significant effects on the accumulation of OTA. There were also two significant interactions (a(w)-temperature and temperature-time). On the basis of the results obtained, carbendazim does not increase the lag phase of A. carbonarius except at relatively low a(w) and temperatures. It does not substantially decrease fungal growth rate once growth is apparent but it appears to cause an increase in OTA accumulation in the medium at the doses assayed. Carbendazim, which is widely used against fungal infections in grape, can positively influence OTA production by A. carbonarius in field, which can increase OTA content in grape juices and wines.     

Kinetics and spatial distribution of OTA in Aspergillus carbonarius cultures

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.     

Production of ochratoxin A by Aspergillus carbonarius on coffee cherries

Robusta coffee cherries collected before and during sun drying from two coffee farms in Thailand were examined for moulds producing ochratoxin A (OA). Aspergillus ochraceus was only detected in one sample, whereas Aspergillus carbonarius was isolated from 7 out of 14 samples. On gamma-irradiated coffee cherries, each of the six tested A. carbonarius strains produced OA. More than 4800 microg kg(-1) of toxin were detected under optimal conditions (25 degrees C, a(w) 0.99). OA production was strongly reduced (230 microg kg(-1)) at an a(w) of 0.94.     

Fungi and mycotoxins in vineyards and grape products

Many fungi may occur on grapes during growth in the vineyard, but the main concern from the viewpoint of mycotoxin contamination is the black Aspergilli, Aspergillus carbonarius and A. niger. These fungi are capable of producing ochratoxin A (OA) which may contaminate grapes and grape products such as wine, grape juice and dried vine fruit. Understanding the ecology and physiology of the black Aspergilli can provide tools for management of OA at all stages of grape production and processing. In the vineyard, careful management of cultivation, irrigation and pruning can assist in minimising the levels of black Aspergilli in the soil, which in turn, can minimise contamination of grapes by these fungi. Minimising damage to grapes on the vine by the use of open vine canopies, grape varieties with resistance to rain damage and by the management of insect pests and fungal diseases (e.g., mildew, Botrytis bunch rot) can reduce the incidence of Aspergillus rot in mature berries. The risk of OA in table grapes can be minimised by careful visual inspection to avoid damaged and discoloured berries. In wine, harvesting grapes with minimal damage, rapid processing and good sanitation practices in the winery assist in minimising OA. During vinification, pressing of grapes, and clarification steps which remove grape solids, grape proteins and spent yeast can also remove a significant proportion of OA. For dried vine fruit production, avoiding berry damage, rapid drying, and final cleaning and sorting to remove dark berries can reduce overall OA levels in finished products.     

Effect of intra and interspecific interaction on OTA production by A. section Nigri in grapes during dehydration

The aim of this study was to assess the colonizing capability and OTA production of different populations of Aspergillus section Nigri spp. in grapes, as affected by the interactions with other fungi, during a simulated in vitro sun-drying. Mature white grapes were divided into two lots of healthy and artificially injured grapes and inoculated with A. section Nigri spp. (A. carbonarius OTA producer, A. niger aggregate OTA producer, A. niger aggregate OTA non-producer), Eurotium amstelodami and Penicillium janthinellum, in different combinations. The drying process was simulated adjusting water activity firstly at 0.98 a(w) and gradually decreasing it to 0.76 a(w) for a total of 20 days. Colonizing grape percentages were recorded after 5, 10, 15 and 20 days of dehydration and OTA content was measured after 5, 7, 10, 12, 15, 17 and 20 days. Colonization of grapes increased with time in all treatments. A. niger aggregate OTA-positive showed the highest colonization percentage, followed by A. carbonarius, and finally their mixed inoculum. When the two OTA-producing strains were combined, addition of any other microorganism increased the percentage of infection by A. section Nigri. A. carbonarius was the highest OTA producer in pure culture, followed by A. niger aggregate OTA-positive. In general, when competing fungi were added to A. carbonarius inoculum, the OTA content was reduced. E. amstelodami was the only competing fungus which increased OTA accumulation. The sun-drying process may be conducive to OTA accumulation in dried grapes. The complex fungal interactions which may take place during this process, may act as an additional control factor, given that the higher presence of A. niger aggregate OTA-negative inhibits OTA accumulation by OTA producing species.     

Real-time PCR based procedures for detection and quantification of Aspergillus carbonarius in wine grapes

Aspergillus carbonarius is the main species responsible for ochratoxin A accumulation in wine grapes and consequently, its rapid and sensitive detection is increasingly investigated. A new real-time PCR (RTi-PCR) based procedure was developed for the rapid and specific detection and quantification of A. carbonarius in wine grapes. The procedure includes the use of the pulsifier equipment to remove conidia from grapes which prevents releasing of PCR inhibitors, and DNA extraction with the EZNA Fungal DNA kit. It reduced the time for A. carbonarius DNA extraction from grapes to 30 min. Two specific primers (AcKS10L/AcKS10R) delimiting a 161 bp fragment, and a probe were designed and directed to the beta-ketosynthase domain of a polyketide synthase from A. carbonarius. Specificity was confirmed by testing primers towards purified DNA from 52 fungal strains, including reference and food isolates. Quantification was linear over at least 5 log units using both serial dilutions of purified DNA and calibrated conidial suspensions from A. carbonarius. The SYBR-Green I and TaqMan RTi-PCR approaches established were able to detect at least 2.4 and 24 genomic equivalents, respectively, using purified DNA. Results obtained from conidial suspensions, after DNA extraction, showed that at least 5 conidia per reaction should be present for a positive result with SYBR-Green I and 50 in the case of TaqMan. The quantification of fungal genomic DNA in artificially inoculated wine grapes performed successfully, with a minimum threshold of 10(3) conidia mL(-1) for accurate quantification. The developed RTi-PCR assay is a promising tool in the prediction of potential ochratoxigenic risk, even in the case of low-level infections, and suitable for a rapid, automated and high throughput analysis.     

What is the source of ochratoxin A in wine?

During a microvinification trial using natural mouldy grapes from a research experimental vineyard, ochratoxin A (OTA) contaminated white wine was obtained. Potential OTA-producing mycobiota of grape samples used in this microvinification process was assessed. Only Aspergillus carbonarius isolates were detected as producers of OTA. Our report is a strong evidence of the contribution of A. carbonarius in the OTA contamination in wine.     

The effect of juice clarification, static or rotary fermentation and fining on ochratoxin A in wine

Semillon and Shiraz grapes containing ochratoxin A (OA) were produced by inoculation of bunches on the vine with Aspergillus carbonarius . Small scale vinification of these grapes was performed and the OA content at various stages was analysed. Semillon must was treated with pectolytic enzymes and the juice after pressing was fined with bentonite. Neither pectolytic enzymes nor bentonite influenced the OA content of the clarified juice. Shiraz must was vinified by either static or rotary fermentation. No differences in the OA content of wine after pressing were noted. OA was concentrated in juice lees and gross lees. However, additional juice or wine recovered from lees after centrifugation did not display increased OA contamination compared to racked juice or wine. Addition of bentonite at 2.5 g/L to a Semillon wine containing 56 mg grape-derived protein/L and ca. 8 μg OA/kg resulted in a 67% reduction in OA concentration. Potassium caseinate showed some effect, whereas gelatin, isinglass and PVPP did not significantly diminish OA in white wine compared to wine settled without fining agents. Addition of yeast hulls at 5.0 g/L to a Shiraz wine containing ca. 5 μg OA/kg yielded a 43% reduction. Gelatin was also slightly effective. Storage of wine for 10–14 months resulted in a decrease in OA concentration of 22% and 29% for white and red wines, respectively.     

Occurrence of ochratoxin A in wine and ochratoxigenic mycoflora in grapes and dried vine fruits in South America

Grape and wine production in South America represents about 6.6% and 10% respectively of the world grape and wine production. The available information on the ochratoxigenic mycoflora and ochratoxin A (OTA) presence in wine grapes, wines, grape juices and dried vine fruits is limited. Surveys have been carried out in Argentina and Brazil which showed that Aspergillus niger aggregate are predominant in the Argentinean varieties while from the Brazilian varieties the species A. niger, Aspergillus ochraceus and Aspergillus carbonarius were isolated. A mycobiota survey from wine grapes in Argentina showed that while Alternaria alternata was predominant, Aspergillus section Nigri species were isolated from 60% of samples. About 41% of black Aspergilli isolates produced OTA with levels ranging from 2 to 24.5 ng mL(-1). In another study, about 83% of A. carbonarius isolates from dried vine fruits produced OTA, with levels ranging from 2 to 5200 ng mL(-1). A survey of grape juices and wines of Brazilian, Argentinean and Chilean origin were found to contain very low levels of OTA. Studies are in progress in Latin America on the ecophysiology of ochratoxigenic fungi and OTA occurrence to reduce the impact of this toxin in the food chain.     

European research on ochratoxin A in grapes and wine

European wine production represents about 70% of world production and thus is an important export commodity. Ochratoxin A (OTA) was first detected as a wine contaminant in 1996 and the role of Aspergillus section Nigri and A. carbonarius in OTA production discovered in Europe in 1999. Subsequently Europe-wide surveys have shown that A. carbonarius is predominantly responsible for OTA contamination of grapes, wine and vine fruits. Analyses of wine samples throughout Europe have shown that there is a gradient in OTA concentration with a decrease from red, to rose and to white wines. The latitude of production is an important factor in determining risk from OTA contamination. Some geographic regions in Southern Europe are more prone to contamination with the toxigenic species and OTA. Ochratoxin A has also been found in much higher concentrations (max. 53 mug/kg) in dried vine fruit than in wine suggesting that A. carbonarius can dominate the drying vine fruit ecosystem. There is a significant lack of knowledge in Europe on conducive climatic conditions preharvest and their relationship with levels of risk from OTA contamination in grapes and their fate in wine production. This needs to be integrated with cultivation system to maximise the prevention of OTA entering this food chain.     

Black Aspergillus species as ochratoxin A producers in Portuguese wine grapes

To evaluate the incidence of fungi producing ochratoxin A (OA) in Portuguese wine grapes, a survey was conducted in 11 vineyards, from four winemaking regions each with distinct climatic conditions. From setting to the harvesting period, a total of 1,650 berries were sampled by plating methods. Out of 370 aspergilli and 301 Penicillium strains isolated, 14% of the aspergilli were OA-producing strains. None of the penicillia were OA-producing strains. The black aspergilli were predominant (90%). All Aspergillus strains were tested in vitro for OA production and all were preserved in the Micoteca da Universidade do Minho (MUM) culture collection. Most of the Aspergillus carbonarius (97%) and 4% of the Aspergillus niger aggregate strains were OA producers. Almost all ochratoxigenic strains were isolated at harvest time, mainly in the regions with a Mediterranean climate. In the vineyards sampled, the percentage of colonized berries with ochratoxigenic strains was up to 38%. The vineyards from the region with Atlantic influences, with high rainfall, exhibited the lowest occurrence of Aspergillus and ochratoxigenic strains, 0% to 10% and 0% to 2% colonized berries, respectively. Data obtained here supports the hypothesis that A. carbonarius and occasionally A. niger, are the main producers of OA in grapes. In this study, the highest incidence of these fungi occurred in vineyards with a Mediterranean climate.     

An ITS-RFLP method to identify black Aspergillus isolates responsible for OTA contamination in grapes and wine

Ochratoxigenic mycobiota in grapes from representative wine regions in Valencia was identified. Black aspergilli were predominant among the different Aspergillus spp. isolated. Restriction digestion analysis of the ITS products was tested as a rapid method to identify isolates of black Aspergillus species from grapes. Restriction endonuclease digestion of the ITS products using the endonucleases HhaI, NlaIII and RsaI, distinguished five types of restriction fragment length polymorphism (RFLP) corresponding to Aspergillus niger, Aspergillus tubingensis, Aspergillus carbonarius and Aspergillus aculeatus species. In addition, a new RFLP type in the A. niger aggregate was identified. The fragments obtained by digestion with the endonuclease NlaIII could be used to identify these new isolates. Black Aspergillus isolates were tested for their ability to produce OTA. Most of the isolates that produced ochratoxin A in YES medium belonged to A. carbonarius species. These results support evidence that A. carbonarious greatly contributes to OTA contamination in grapes and consequently in wine. The ITS-RFLP assay is proposed as a rapid and easy method to identify black Aspergillus species isolated from grapes, especially in studies that involve a large number of isolates.     

Effect of temperature and water activity on growth and ochratoxin A production by Australian Aspergillus carbonarius and A. niger isolates on a simulated grape juice medium

The effect of water activity (0.92, 0.95, 0.965 and 0.98) and temperature (15 °C, 25 °C, 30 °C and 35 °C) on growth rate and ochratoxin A (OA) production by five strains of Aspergillus carbonarius and two strains of A. niger isolated from Australian vineyards was characterised on a synthetic grape juice medium. Maximum growth for A. carbonarius occurred at ca 0.965 a_w and 30 °C, and for A. niger, at ca 0.98 a_w and 35 °C. The optimum temperature for OA production was 15 °C and little was produced above 25 °C. The optimum a_w for toxin production was 0.95–0.98 for A. carbonarius and 0.95 for A. niger. Toxin was produced in young colonies after and, typically, did not continue to accumulate the entire surface area of the plate was colonised. Rather, the amount decreased as colonies aged. Trends for growth and OA production were similar among Australian isolates and those from European grapes, as reported in the literature.     

Prediction of fungal growth and ochratoxin A production by Aspergillus ochraceus on irradiated barley grain as influenced by temperature and water activity

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 (a(w), 0.80-0.99), temperature (10, 20, 30 degrees 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 a(w) and 30 degrees C, with a marked decrease of growth rates and OTA production at the lowest levels of a(w) and temperature assayed. The minimum a(w) level for growth, observed in this study, was 0.85 and 0.90 a(w) 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.     

Effect of photoperiod and day-night temperatures simulating field conditions on growth and ochratoxin A production of Aspergillus carbonarius strains isolated from grapes

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.