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.     

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.     

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.     

Ochratoxigenic species from Spanish wine grapes

The ochratoxigenic mycobiota of grapes belonging to representative wine regions located along the Mediterranean coast of Spain at different developmental stages was identified. During the development of the berries, the occurrence of Aspergillus spp. increased while the percentage of berries contaminated by non-ochratoxin A (OTA) producing species such as Alternaria spp. and Cladosporium spp. decreased. Penicillium verrucosum, the only confirmed Penicillium spp. that is able to produce OTA, was not isolated. The contamination by OTA-producing species comes from the surface of the berries and not from the inner fruit. Black aspergilli were predominant among the different Aspergillus spp. isolated. All the Aspergillus carbonarius isolates were able to produce OTA at different concentrations. None of the isolates belonging to Aspergillus niger aggregate and to Aspergillus japonicus var. aculeatus were able to produce OTA. These results are a strong evidence of the contribution of A. carbonarius in the OTA contamination in wine grapes, mainly at the last developmental stages of the berries.     

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.     

Occurrence of ochratoxigenic fungi and ochratoxin A in grapes from a Tunisian vineyard

The occurrence of ochratoxin A (OTA) and the identification of the ochratoxigenic microbiota in Tunisian grapes were studied for the first time. Black aspergilli were the dominant genus among the filamentous fungi isolated from grapes and were the only potential OTA-producing fungi found. The most abundant species were member of Aspergillus niger aggregate (63%) and Aspergillus carbonarius (36%). Uniseriate aspergilli were rarely present (1%). Of the A. carbonarius isolates, 97% were OTA positive but only 3% of the A. niger aggregate isolates were OTA positive. During grape maturation, the frequency of black aspergilli increased due to increase of the numbers of A. carbonarius. Musts (n=24) obtained from grapes collected at the different sampling times were analyzed for their OTA content. Up to 37% of the musts contained OTA at levels varying between 0.59 and 2.57 microg/l. The amounts of OTA in musts increased as grapes matured. These results indicate that A. carbonarius is the main cause of OTA contamination of Tunisian grapes.     

Kinetics of Ochratoxin A Production and Accumulation by Aspergillus carbonarius on Synthetic Grape Medium at Different Temperature Levels

ABSTRACT:  The effects of incubation time (up to 10 d) and temperature (7, 15, 20, 25, 30, 35, and 42 °C) on mean ochratoxin A (OTA)-producing capacity and OTA accumulation by 4 Aspergillus carbonarius strains isolated from Spanish and Tunisian grapes were studied on grape-like medium. The limits of growth and OTA production have been identified. OTA production was significantly higher at 20 °C, followed by 25, 15, 30, 35 °C. No growth was observed at 7 °C and 42 °C after 10 d of incubation and, consequently, no OTA was detected at these temperature levels. In general, maximum mean OTA-producing capacity was found earlier with increasing incubation temperatures. However, at 35 °C, OTA was rarely detected although growth was maximum at this temperature. OTA accumulation was maximum after 10 d of incubation for all the temperatures except at 30 °C, were the maximum was detected at earlier incubation time (6 to 8 d) and then remained stable. Colony diameters and OTA accumulation along time were modeled by the Gompertz sigmoidal model and an estimation of maximum OTA accumulation rate and the delay until OTA production could be established. At high temperatures OTA accumulation starts earlier but increases slowly and becomes constant in a few days.     

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.     

Toxigenic fungi isolated from dried vine fruits in Argentina

To evaluate the potential for mycotoxin production by fungi in dried vine fruits, the mycobiota was determined both before and after surface disinfection. Predominant genera were Aspergillus (50.2%), Eurotium (21.4%) and Penicillium (13.5%). Aspergillus section Nigri ("black aspergilli") were isolated with relatively high frequency. Aspergillus niger was the most common species but only 3 of 293 isolates screened were ochratoxin A (OTA) producers. Aspergillus carbonarius was less common but 96% of 48 strains screened were ochratoxigenic. OTA was not produced by A. japonicus. Other toxigenic fungi detected were A. ochraceus (3 strains produced OTA), Aspergillus flavus (5 strains produced cyclopiazonic acid but not aflatoxins), P. citrinum (19 strains were strong citrinin producers) and Alternaria alternata (15 strains were producers of tenuazonic acid, alternariol and alternariol methyl ether). In spite of the high incidence of A. carbonarius capable of producing OTA, low levels of this toxin were detected in the samples analysed.     

Australian research on ochratoxigenic fungi and ochratoxin A

The presence of the mycotoxin, ochratoxin A (OTA), has been reported in Australian grape products. Comprehensive surveys of Australian wines have determined that the frequency and level of OTA contamination are low. Aspergillus carbonarius is the primary OTA-producing species associated with grapes in Australia, and all isolates tested to date produce OTA. Aspergillus niger is isolated more frequently from vineyards, however, few strains produce OTA. A. carbonarius and A. niger exist as saprophytes in the top layer of soil beneath vines, from where they are thought to be blown onto bunches. The level of A. carbonarius in soil may be reduced by temperatures above or below the optimum temperature for survival (25 degrees C), by high soil moisture content, and by modifications to tillage and mulching practices. A. carbonarius is an opportunistic pathogen of damaged berries. In the absence of damage, spores may exist on berry surfaces without causing visible rots. Aspergillus rots are associated with black Aspergillus species, primarily A. niger, A. carbonarius and A. aculeatus. The potential for such rots is increased with berry damage, inoculum coverage and berry maturity. Susceptibility to berry splitting is related, in part, to bunch structure, and may be variety-dependent or influenced by rainfall, irrigation and canopy management. Black Aspergillus spp. are closely associated with berries near the main stem of the bunch. During winemaking, around 80% of the OTA initially present in grapes is removed, primarily with the skins and pulp during pressing. Additional reductions occur with the removal of precipitated grape and yeast solids. Bentonite in white wine and yeast hulls in red wine were the most effective non-carbonaceous fining agents for the removal of OTA.     

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.     

Influence of nitrogen and carbon sources on the production of ochratoxin A by ochratoxigenic strains of Aspergillus spp. isolated from grapes

This work studies the influence of nitrogen and carbon source on ochratoxin A production by three Aspergillus isolates A. ochraceus (Aso2), A. carbonarius (Ac25) and A. tubingensis (Bo66), all isolated from grapes. A basal medium (0.01 g/l FeSO4.7H2O, 0.5 g/l MgSO4.7H2O, 0.5 g/l Na2HPO4.2H2O, 1.0 g/l KCl) was prepared. This medium was supplemented with different nitrogen sources, both inorganic [(NH4)3PO(4), 0.3 g/l plus NH4NO3, 0.2 g/l] and organic (histidine, proline, arginine, phenylalanine, tryptophan or tyrosine) at two concentrations (0.05 g/l or 0.3 g/l), and different carbon sources (sucrose, glucose, maltose, arabinose or fructose) at three concentrations (10 g/l, 50 g/l or 150 g/l). A medium with sucrose (18 g/l) and glucose (1 g/l) was also tested. After a 10-day incubation period at 25 degrees C the highest levels of OTA (44.0 ng/ml, 13.5 ng/ml and 0.49 ng/ml for A. ochraceus, A. carbonarius and A. tubingensis, respectively) were obtained in the cultures containing 150 g/l of arabinose and 0.05 g/l of phenylalanine. Analysis of variance of the data showed that there were significant differences (p-value 0.05) among the OTA levels in the cultures with regard to carbon source and isolate. No significant differences were detected in OTA production regarding nitrogen source, although 0.05 g/l of phenylalanine generally favoured OTA production in the cultures of the three isolates. The dynamics of toxin production in the cultures of each isolate using the optimized basal medium supplemented with 0.05 g/l of phenylalanine and 150 g/l of arabinose for a period of 42 days at 25 degrees C was also studied. The maximum level of OTA was detected on the 3rd day of incubation in A. tubingensis cultures and on the 35th and 43(rd) days of incubation in A. ochraceus and A. carbonarius, respectively. This is the first study in which defined media have been used to assess the influence of carbon and nitrogen sources on OTA production by isolates of OTA-producing species isolated from grapes and to analyse the dynamics of toxin production in these species in a defined culture medium. This optimized medium for OTA production is being used in current studies aimed at elucidating its biosynthetic pathway.     

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.     

Study of the effect of water activity and temperature on ochratoxin A production by Aspergillus carbonarius

The effect of water activity (aw) (0.78-0.99) and temperature (15 and 30 degrees C) on growth and production of ochratoxin A (OTA) of six Aspergillus carbonarius strains was studied in two culture media: Czapek yeast autolysate (CYA) agar and yeast extract sucrose (YES) agar, during a period of 30 days. The strains were selected to include different sources and different reported abilities to produce OTA and were characterized by RAPD and ITS-5.8S rDNA sequencing. CYA showed to be better culture medium than YES for OTA production in the isolates tested. OTA concentration was higher at 15 degrees C than at 30 degrees C. At 30 degrees C, ranges for OTA production were more restrictive than those for growth. OTA was produced from 0.86, 0.90 or 0.94 aw depending on the strain. At 15 degrees C, growth and OTA production were detected only in the 0.94-0.99 aw range. The molecular study performed showed that five of the strains were conspecific and no correlation was found between molecular data and the OTA production level or origin. The remaining strain had never been able to produce OTA and will probably represent a new species in the Aspergillus section Nigri. Our results show that A. carbonarius is able to grow and produce OTA in a wide range of water activities at both high and low temperatures.     

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.     

Influence of pH and incubation time on ochratoxin A production by Aspergillus carbonarius in culture media

The effect of pH (2 to 10) and temperature (15 and 30 degrees C) on growth and production of ochratoxin A (OTA) of six strains of Aspergillus carbonarius was studied in two culture media: Czapek yeast autolysate agar and yeast extract sucrose agar. Isolates were selected by their different source and different reported ability to produce OTA. Regardless of the initial pH or the temperature tested, Czapek yeast autolysate agar has been shown to be the best culture medium for OTA production by A. carbonarius. In this medium, OTA was produced from pH 2 to 10 at the two incubation temperatures tested. The results obtained show the ability of A. carbonarius to not only grow but also produce OTA over a wide pH range at high or low temperatures. This may help explain why this species is considered the main OTA source in some substrata.     

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.     

控制葡萄及其制品中赭曲霉毒素A的研究进展

赭曲霉毒素A(OTA)是一种有毒的真菌次级代谢产物,在葡萄及其制品中经常被检出。本文综述了控制葡萄及其制品中OTA的主要措施,包括抑制OTA产生菌的生长及其产毒和去除已产生的OTA两方面。葡萄中的致病菌主要是青霉和曲霉,而炭黑曲霉是其中最主要OTA产生菌,通过模拟葡萄生长及葡萄制品的生产过程,发现温度、光照、湿度等因素是影响OTA产生菌的生长及其产毒的重要因素。去除OTA的方法概括的分为物理方法、化学方法、生物方法,其中生物降解OTA是目前研究的热点。      

Occurrence of toxigenic fungi in ochratoxin A contaminated liquorice root

Fungi associated with ochratoxin A (OTA)-contaminated liquorice root and their capabilities for OTA production were investigated. Medicinal materials of mouldy liquorice root were collected from herbal markets located in Jiangxi, Zhejiang, Henan provinces and Beijing, China, respectively. Sixteen fungal species belonging to Penicillium, Aspergillus, Eurotium, Fusarium, Mucor and Scopulariopsis were isolated; the fungal composition was different in each liquorice root sample. Penicillium polonicum was predominant, comprising 54% of the total isolates in the liquorice root sample from Jiangxi province, which was contaminated with OTA at the highest level. In other samples with lower OTA contents, species of Aspergillus and Eurotium were predominant. OTA production of representative strains on rice media was detected by LC-MS/MS; all Penicillium polonicum isolates and a P. chrysogenum were ochratoxigenic; OTA concentrations ranged from 6.94 to 217.37?ng?g(-1). This is the first study to report P. polonicum as an OTA-producing fungus. OTA contamination of mouldy liquorice root constitutes a major health hazard in consumption. This situation demands urgent and undivided attention.     

Managing ochratoxin A risk in the grape-wine food chain

The main source of ochratoxin A (OTA) in the wine food chain is the infection of grapes by "black aspergilli" in the field. OTA-producing black aspergilli include principally Aspergillus carbonarius, followed by A. niger and possibly A. tubingensis. They are opportunistic fungi that develop particularly on damaged berries at ripening, although they may occur and form OTA on grapes from veraison to harvest. Climatic conditions (high humidity and temperature) and geographical location are important factors favouring OTA accumulation in grape berries. The severity of aspergillus rot is influenced by excessive irrigation and rainfall prior to harvest, which causes berry splitting. In addition, berry wounds caused by insect attack provide preferential entries for black aspergilli. High OTA levels occur in grapes severely damaged by the grape moth, Lobesia botrana, particularly in Mediterranean areas. Some grape varieties display greater susceptibility to aspergillus rot due to intrinsic genetic characteristics and bunch conformation (i.e. compact>sparse). Control measures for toxigenic mycoflora in the vineyards must consider these critical control points. Proper fungicidal and insecticidal treatments can reduce OTA contamination. Nevertheless, knowledge about the fate of OTA and its distribution in wine and winery by-products is important to manage OTA risk in contaminated stock. In our wine-making experiments, only 4% of the OTA present in grapes remained in the wine--the majority is retained in pressed grape pomaces. OTA concentration remained unchanged in wine after a 1-year aging as well as in all liquid fractions collected during vinification (i.e. must, free run wine, and wine after first and second decantation). Activated carbon can reduce OTA levels in wine but negatively affects wine quality.