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

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

Study of Spanish grape mycobiota and ochratoxin A production by Isolates of Aspergillus tubingensis and other members of Aspergillus section Nigri

The native mycobiota of five grape varieties grown in Spain has been studied. Four (Bobal, Tempranillo, Garnacha, and Monastrell) were red varieties and one (Moscatel) was white. The main fungal genera isolated were Alternaria, Cladosporium, and Aspergillus. The isolation frequency of Aspergillus spp. section Nigri in contaminated samples was 82%. Ochratoxin A (OTA) production was assessed using yeast extract-sucrose broth supplemented with 5% bee pollen. Cultures of 205 isolates from this section showed that 74.2% of Aspergillus carbonarius and 14.3% of Aspergillus tubingensis isolates produced OTA at levels ranging from 1.2 to 3,530 ng/ml and from 46.4 to 111.5 ng/ml, respectively. No Aspergillus niger isolate had the ability to produce this toxin under the conditions assayed. Identification of the A. niger aggregate isolates was based on PCR amplification of 5.8S rRNA genes and its two intergenic spacers, internal transcribed spacer 1 (ITS1) and ITS2, followed by digestion with restriction endonuclease RsaI of the PCR products. The restriction patterns were compared with those from strains of A. niger CECT 2807 and A. tubingensis CECT 20393, held at the Spanish Collection of Type Cultures. DNA sequencing of the ITS1-5.8S rRNA gene-ITS2 region of the OTA-producing isolates of A. tubingensis matched 99 to 100% with the nucleotide sequence of strain A. tubingensis CBS 643.92. OTA determination was accomplished by liquid chromatography with fluorescence detection. OTA confirmation was carried out by liquid chromatography coupled to ion trap mass spectrometry. The results showed that there are significant differences with regard to the isolation frequency of ochratoxinogenic fungi in the different grape varieties. These differences were uncorrelated to berry color. The ability of A. tubingensis to produce OTA and the influence of grape variety on the occurrence of OTA-producing fungi in grapes are described in this report for the first time.     

Aspergillus carbonarius growth and ochratoxin A production on a synthetic grape medium in relation to environmental factors

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.     

Efficacy of natamycin for control of growth and ochratoxin A production by Aspergillus carbonarius strains under different environmental conditions

AIMS: To examine the efficacy of natamycin produced by Streptomyces natalensis against strains of Aspergillus carbonarius growth and ochratoxin A (OTA) production under different environmental factors on a grape juice-based medium. METHODS AND RESULTS: Detailed studies in the range 0-20 ng ml(-1) for control of growth and ochratoxin production by strains of A. carbonarius at 0.98, 0.96 and 0.94 water availabilities (a(w)) and 15-25 degrees C on a fresh red grape extract medium were examined. Inhibition of growth was depending on temperature and a(w) level. At 15 degrees C, 5-10 ng ml(-1) natamycin was effective in reducing growth almost completely. However, at 20-25 degrees C and all the three a(w) levels, growth was only slightly inhibited by 5-10 ng ml(-1) natamycin. There were strain differences with regard to inhibition of OTA production. At 15 degrees C and 0.98 a(w), 10 ng ml(-1) was required to inhibit production by >90%. However, at 0.96 and 0.94 a(w), almost complete inhibition occurred. At 20 degrees C, OTA production was only significantly inhibited by 10 ng ml(-1) natamycin at 0.94 a(w). At 0.96 and 0.98 a(w), some inhibition occurred with 5-10 ng ml(-1), but greater concentrations would be required for effective inhibition. At 25 degrees C, 5 ng ml(-1) was effective at all a(w) levels. However, at 15 degrees C and 25 degrees C and a wide range of a(w) levels, natamycin effectively controlled OTA production. CONCLUSIONS: Natamycin appears to be a very effective for controlling growth and OTA production by strains of A. carbonarius over a range of a(w) and temperature conditions on grape-based media. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first detailed study to demonstrate the impact of natamycin against A. carbonarius. This study suggests that use of natamycin at 50-100 ng ml(-1) can give complete inhibition of growth of A. carbonarius and OTA production over a range of environmental conditions. Natamycin could be an important component of a system to prevent OTA contamination of wine as well during the drying and production of vine fruits.     

Effect of preharvest fungicides and interacting fungi on Aspergillus carbonarius growth and ochratoxin A synthesis in dehydrating grapes

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.     

Incubation time and water activity effects on ochratoxin A production by Aspergillus section Nigri strains isolated from grapes

AIMS: 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. METHODS AND RESULTS: Two Aspergillus carbonarius and two Aspergillus niger aggregate strains isolated from grapes were tested in vitro for OTA accumulation at 25 degrees 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). CONCLUSIONS: Provided that A. section Nigri, and mainly A. carbonarius, play the main role in OTA presence in grapes, it would be critical to adjust the harvest and processing time to significantly reduce the chances for OTA accumulation. SIGNIFICANCE AND IMPACT OF THE STUDY: Ochratoxin A production by A. section Nigri has been shown for the first time to occur optimally after as little as 5 days on a grape-like medium.     

Effect of temperature and water activity on growth and ochratoxin A production boundaries of two Aspergillus carbonarius isolates on a simulated grape juice medium

Aims: To develop and validate a logistic regression model to predict the growth and ochratoxin A (OTA) production boundaries of two Aspergillus carbonarius isolates on a synthetic grape juice medium as a function of temperature and water activity (a_w). Methods and Results: A full factorial design was followed between the factors considered. The a_w levels assayed were 0·850, 0·880, 0·900, 0·920, 0·940, 0·960, 0·980 and the incubation temperatures were 10, 15, 20, 25, 30, 35 and 40°C. Growth and OTA production responses were evaluated for a period of 25 days. Regarding growth boundaries, the degree of agreement between predictions and observations was >99% concordant for both isolates. The erroneously predicted growth cases were 3·4–4·1% false‐positives and 0·7–1·4% false‐negatives. No growth was observed at 10°C and 40°C for all a_w levels assayed, with the exception of 0·980 a_w/40°C, where weak growth was observed. Similarly, OTA production was correctly predicted with a concordance rate >98% for the two isolates with 0·7–1·4% accounting for false‐positives and 2·0–2·7% false‐negatives. No OTA production was detected at 10°C or 40°C regardless of a_w, and at 0·850 a_w at all incubation temperatures. With respect to time, the OTA production boundary shifted to lower temperatures (15–20°C) as opposed to the growth boundary that shifted to higher temperature levels (25–30°C). Using two literature datasets for growth and OTA production of A. carbonarius on the same growth medium, the logistic model gave one false‐positive and three false‐negative predictions out of 68 growth cases and 13 false‐positive predictions out of 45 OTA production cases. Conclusions: The results of this study suggest that the logistic regression model can be successfully used to predict growth and OTA production interfaces for A. carbonarius in relation to temperature and a_w. Significance and Impact of the Study: The proposed modelling approach helps the understanding of fungal‐food ecosystem relations and it could be employed in risk analysis implementation plans to predict the risk of contamination of grapes and grape products by A. carbonarius.     

Predictive assessment of ochratoxin A accumulation in grape juice based-medium by Aspergillus carbonarius using neural networks

Aims:  To study the ability of multi-layer perceptron artificial neural networks (MLP-ANN) and radial-basis function networks (RBFNs) to predict ochratoxin A (OTA) concentration over time in grape-based cultures of Aspergillus carbonarius under different conditions of temperature, water activity ( a _w) and sub-inhibitory doses of the fungicide carbendazim.
Methods and Results:  A strain of A. carbonarius was cultured in a red grape juice-based medium. The input variables to the network were temperature (20–28°C), a _w (0·94–0·98), carbendazim level (0–450 ng ml⁻¹) and time (3–15 days after the lag phase). The output of the ANNs was OTA level determined by liquid chromatography. Three algorithms were comparatively tested for MLP. The lowest error was obtained by MLP without validation. Performance decreased when hold-out validation was accomplished but the risk of over-fitting is also lower. The best MLP architecture was determined. RBFNs provided similar performances but a substantially higher number of hidden nodes were needed.
Conclusions:  ANNs are useful to predict OTA level in grape juice cultures of A. carbonarius over a range of a _w, temperature and carbendazim doses.
Significance and Impact of the Study:  This is a pioneering study on the application of ANNs to forecast OTA accumulation in food based substrates. These models can be similarly applied to other mycotoxins and fungal species.     

Brief in vitro study on Botrytis cinerea and Aspergillus carbonarius regarding growth and ochratoxin A

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.     

Ochratoxin A production and amplified fragment length polymorphism analysis of Aspergillus carbonarius, Aspergillus tubingensis, and Aspergillus niger strains isolated from grapes in Italy

Ochratoxin A is a potent nephrotoxin and a possible human carcinogen that can contaminate various agricultural products, including grapes and wine. The capabilities of species other than Aspergillus carbonarius within Aspergillus section Nigri to produce ochratoxin A from grapes are uncertain, since strain identification is based primarily on morphological traits. We used amplified fragment length polymorphisms (AFLPs) and genomic DNA sequences (rRNA, calmodulin, and beta-tubulin genes) to identify 77 black aspergilli isolated from grape berries collected in a 2-year survey in 16 vineyards throughout Italy. Four main clusters were distinguished, and they shared an AFLP similarity of <25%. Twenty-two of 23 strains of A. carbonarius produced ochratoxin A (6 to 7,500 microg/liter), 5 of 20 strains of A. tubingensis produced ochratoxin A (4 to 130 microg/liter), 3 of 15 strains of A. niger produced ochratoxin A (250 to 360 microg/liter), and none of the 19 strains of Aspergillus "uniseriate" produced ochratoxin A above the level of detection (4 microg/liter). These findings indicate that A. tubingensis is able to produce ochratoxin and that, together with A. carbonarius and A. niger, it may be responsible for the ochratoxin contamination of wine in Italy.     

Influence of the interaction of temperature and water activity on the production of ochratoxin A and the growth of Aspergillus niger, Aspergillus carbonarius and Aspergillus ochraceus on coffee-based culture medium

In the present study, the effect of temperature and water activity on fungal growth and ochratoxin production on coffee-based medium was assessed. Optimal growth of three Aspergillus strains was observed in the same ecological conditions, namely 30 degrees C and 0.99 water activity. Maximal daily growth is 11.2, 6.92, and 7.22 mm/day for Aspergillus niger, Aspergillus carbonarius, and Aspergillus ochraceus, respectively. However, ecological conditions for optimal ochratoxin production vary according to the toxinogenic strain, with water activity as a limiting factor. Such an ochratoxin A production is inhibited at 42 degrees C and 0.75 water activity. Correspondence between laboratory tested water activity and that measured on a sun-dried ripe cherry batch shows that the first 5 days of drying are critical for fungal growth and ochratoxin A production. Accordingly, artificial drying of cherries at temperatures above 42 degrees C will impede not only fungal growth but also contamination with ochratoxin A.     

Kinetics of ochratoxin A production in different Aspergillus species

Kinetics of ochratoxin A production was examined in a number of ochratoxin producing isolates representing different sections of the Aspergillus genus. Both weak and high ochratoxin producers were tested using immunochemical or high-performance liquid chromatograhic methods. All isolates were found to produce the highest amounts of ochratoxin A after 7-10 days of incubation. Ochratoxin production varied between 30 - 5 x l0(5) ng ml(-1) among the Aspergillus isolates tested. The A. albertensis and A. melleus isolates examined were found to produce ochratoxin A constitutively. A. albertensis produced the highest amounts of ochratoxin A at 30 degrees C after 7 days' incubation in YES liquid medium. Ergosterol content and ochratoxin production of A. albertensis cultures were in good correlation.     

Identification of ochratoxin A producing Aspergillus carbonarius and A. niger clade isolated from grapes using the loop‐mediated isothermal amplification (LAMP) reaction

Aims

To develop two assays based on the loop‐mediated isothermal amplification (LAMP) of DNA for the quick and specific identification of Aspergillus carbonarius and ochratoxigenic strains of the Aspergillus niger clade isolated from grapes.

Methods and Results

Two sets of primers were designed based on the polyketide synthase genes involved or putatively involved in ochratoxin A (OTA) biosynthesis in A. carbonarius and A. niger clade. Hydroxynaphthol blue was used as indirect method to indicate DNA amplification. The limit of detection of both assays was comparable to that of a PCR reaction. Specificities of the reactions were tested using DNA from different black aspergilli isolated from grapes. The two LAMP assays were then used to identify A. carbonarius and ochratoxigenic A. niger and A. awamori grown in pure cultures without a prior DNA extraction.

Conclusions

The two LAMP assays permitted to quickly and specifically identify DNA from OTA‐producing black aspergilli, as well as isolates grown in pure culture.

Significance and Impact of the Study

Monitoring vineyards for the presence of OTA‐producing strains is part of the measures to minimize the occurrence of OTA in grape products. The two LAMP assays developed here could be potentially used to speed the screening process of vineyards for the presence of OTA‐producing black aspergilli.     

Inhibition by antimicrobial food additives of ochratoxin A production by Aspergillus sulphureus and Penicillium viridicatum

The effects of antimicrobial food additives on growth and ochratoxin A production by Aspergillus sulphureus NRRL 4077 and Penicillium viridicatum NRRL 3711 were investigated. At pH 4.5, growth and toxin production by both A. sulphureus and P. viridicatum were completely inhibited by 0.02% potassium sorbate, 0.067% methyl paraben, 0.0667% methyl paraben, and 0.2% sodium propionate. At pH 5.5, 0.134% potassium sorbate and 0.067% methyl paraben completely inhibited growth and ochratoxin A production by both fungi. Sodium bisulfite at 0.1%, the maximum level tested, was found to inhibit growth of A. sulphureus and P. viridicatum by 45 and 89%, respectively. Toxin production was inhibited by 97 and 99%, respectively. Sodium propionate (0.64%) at pH 5.5 inhibited growth of A. sulphureus and P. viridicatum by 76 and 90%, respectively. Toxin production was inhibited by greater than 99% for each fungus. Antimicrobial agents were ranked as to effectiveness by comparing the level required for complete inhibition of ochratoxin A production to the highest antimicrobial agent level normally used in food. At pH 4.5, the most effective inhibitor of growth and toxin production was potassium sorbate, followed by sodium propionate, methyl paraben, and sodium bisulfite, respectively, for both fungi. However, at pH 5.5, the most effective antimicrobial agents for inhibiting ochratoxin production were methyl paraben and potassium sorbate, followed by sodium propionate. Sodium bisulfite was not highly inhibitory to these toxigenic fungi at the higher pH value tested.     

Temperature and incubation time effects on growth and ochratoxin A production by Aspergillus sclerotioniger and Aspergillus lacticoffeatus on culture media

Aims: As there is no knowledge of the influence of abiotic factors on the two new ochratoxin A (OTA)‐producing species Aspergillus sclerotioniger and Aspergillus lacticoffeatus, the aim of this study was to evaluate the effect of temperature and incubation time on growth and OTA production by these species on culture media. Methods and Results: The study was carried out on yeast extract sucrose agar (YES) and Czapek yeast extract agar (CYA) incubated at ten different temperatures from 5 to 50°C (at 5°C intervals). Growth assessment and OTA production were determined after 5, 10, 15, 20 and 30 days of incubation at each temperature. Aspergillus sclerotioniger grew from 10 to 35°C; OTA was detected from 10 to 35°C and the highest concentration was achieved at 15°C in CYA. Aspergillus lacticoffeatus grew from 10 to 45°C; OTA was detected from 15 to 45°C, and the maximum concentration was produced after 5 days at 25°C in YES. Conclusions: The studied species can produce OTA over a wide range of temperatures and significant amounts can be produced in only 5 days. Significance and Impact of the Study: This is the first report on the influence of ecophysiological factors on these two ochratoxigenic species. The pattern of effects of temperature on growth and OTA production by A. sclerotioniger and A. lacticoffeatus was similar to those reported for the closely related species Aspergillus carbonarius and Aspergillus niger, respectively. The two new OTA‐producing species have both been isolated from coffee beans, and the closely related ochratoxigenic species of section Nigri, A. carbonarius and A. niger are important sources of OTA in this substrate.     

Uptake of radiolabelled ochratoxin A from soil by coffee plants

[3H, 14C] Ochratoxin A, prepared biosynthetically, was applied in dilute NaHCO3 solution to the soil in which coffee plants had grown to four pairs of leaves. Three weeks later the compound, isolated from dilute NaHCO3 extract of leaves by immunoaffinity chromatography, was detected by scintillation counting as a 1-2 ppm component of leaf dry weight, greatly exceeding the trace (ppb) occurrence of ochratoxin A in some green coffees, which therefore might arise in the field directly from fungal activity in soil rather than from fungal infection of cherries or processed green coffee.     

Effects of cysteine and structurally related compounds on ochratoxin production by Aspergillus ochraceus

Addition of 10(-2) M L-cysteine, L-cystine, or S-ethyl-L-cysteine to a synthetic medium containing xylose as the sole carbon source did not decrease ochratoxin production by Aspergillus ochraceus. At that concentration, DL-homocysteine thiolactone HC1, DL-cysteine HC1, L-ethionine, S-methyl-L-cysteine, and glutathione (reduced) strongly inhibited ochratoxin production. DL-Homocysteine thiolactone HC1, DL-cysteine HC1, and L-ethionine also strongly inhibited fungal growth. At lower concentrations (10(-3) and 10(-4) M) only L-ethionine decreased the toxin production. Ochratoxin inhibition caused by DL-homocysteine thiolactone HC1, DL-cysteine HC1, and glutathione was observed only in cases where the pH of the media was below 5.0. The inhibition caused by 10(-3) M ethionine was partially prevented by the addition of 10(-3) M methionine but this was not the case after the addition of S-methyl-L-cysteine to the medium.