Lipoperoxidation affects ochratoxin A biosynthesis in Aspergillus ochraceus and its interaction with wheat seeds

In Aspergillus nidulans, Aspergillus flavus, and Aspergillus parasiticus, lipoperoxidative signalling is crucial for the regulation of mycotoxin biosynthesis, conidiogenesis, and sclerotia formation. Resveratrol, which is a lipoxygenase (LOX) and cyclooxygenase inhibitor, downmodulates the biosynthesis of ochratoxin A (OTA) in Aspergillus ochraceus. In the genome of A. ochraceus, a lox-like sequence (AoloxA; National Center for Biotechnology Information (NCBI) accession number: DQ087531) for a lipoxygenase-like enzyme has been found, which presents high homology (100 identities, 100 positives %, score 555) with a lox gene of Aspergillus fumigatus (NCBI accession number: XM741370). To study how inhibition of oxylipins formation may affect the A. ochraceus metabolism, we have used a ΔAoloxA strain. This mutant displays a different colony morphology, a delayed conidia formation, and a high sclerotia production. When compared to the wild type, the ΔAoloxA strain showed a lower basal activity of LOX and diminished levels of 13-hydroperoxylinoleic acid (HPODE) and other oxylipins derived from linoleic acid. The limited oxylipins formation corresponded to a remarkable inhibition of OTA biosynthesis in the ΔAoloxA strain. Also, wheat seeds (Triticum durum cv Ciccio) inoculated with the ΔAoloxA mutant did not accumulate 9-HPODE, which is a crucial element in the host defence system. Similarly, the expression of the pathogenesis-related protein 1 (PR1) gene in wheat seeds was not enhanced. The results obtained contribute to the current knowledge on the role of lipid peroxidation governed by the AoloxA gene in the morphogenesis, OTA biosynthesis, and in host–pathogen interaction between wheat seeds and A. ochraceus.     

Mycotoxin production by different ochratoxigenic <Emphasis Type="Italic">Aspergillus</Emphasis> and <Emphasis Type="Italic">Penicillium</Emphasis> species on coffee- and wheat-based media

Ochratoxin A (OTA) is one of the most widespread mycotoxins, and is produced by several Aspergillus or Penicillium species. Human exposure to OTA is mainly by intake of contaminated food, with cereal products, followed by coffee and red wine as the main sources of OTA. In this study, the OTA production of four ochratoxigenic fungi (two Aspergillus and two Penicillium species) was investigated in four different media, i.e. wheat and coffee model media as food-based media and two standard laboratory media (malt extract glucose agar, MEA and yeast extract sucrose agar, YES). Colony growth was documented and OTA concentrations in cultures were determined at day 2, 4 and 8 of incubation at 25°C by high-performance thin-layer chromatography (HPTLC) and high-performance liquid chromatography (HPLC). OTA production clearly depended upon time of incubation, fungal species, and medium composition. On coffee based medium, moderate OTA levels were produced by A. ochraceus BFE635 (9.8 μg/g) and by A. niger BFE632 (10.6 μg/g) on day 8 of incubation. In wheat-based medium, these strains produced much more OTA than in coffee. The highest OTA concentration (83.8 μg/g on day 8) was formed by A. ochraceus BFE635 followed by the other Aspergillus niger BFE632 (49 μg/g). Lower OTA levels were produced by P. verrucosum BFE550 and P. nordicum BFE487, in both wheat and in YES medium, whilst OTA was hardly detectable in coffee and in MEA in case of P. nordicum. Colony growth of the tested strains on different media was not indicative of OTA production. Guttation droplets developed on wheat-based medium with the Aspergillus strains within a week, and this phenomenon coincided with the high OTA amounts formed by these species. Results from this study add to our knowledge on the behaviour of ochratoxigenic fungal species when cultured on food based media.     

Molecular diagnosis of ochratoxinogenicAspergillus species

Toxigenic and non-toxigenic black aspergilli belonging to theAspergillus niger aggregate and toA. carbonarius were compared to each other and to strains of other species by DNA fingerprinting. AFLPs showed a clear separation ofA. niger andA. carbonarius. However, no clear correlation between the genetic similarity of the strains and the ability to produce ochratoxin A (OTA) was detected. Based on AFLP, marker sequences were chosen for the construction of SCAR-PCR primers for the detection ofA. carbonarius. A similar approach was used forA. ochraceus, another fungus of concern regarding ochratoxin A contamination of coffee. Cluster analysis ofA. ochraceus isolates mainly from Brazilian coffee showed a very close genetic similarity. Three species specific primer pairs were developed and one of these was used for the PCR and realtime PCR (RT-PCR) based detection of the mould in green coffee.A. ochraceus was specifically and rapidly detected and quantified in green coffee. A positive correlation between the amount of DNA and OTA content was established.     

Antifungal properties of selected lactic acid bacteria and application in the biological control of ochratoxin A producing fungi during cocoa fermentation

Thirteen Lactic acid bacteria strains isolated from fermenting cocoa and seven reference strains were used in order to assess their antifungal properties towards three ochratoxin A (OTA) producing fungi (Aspergillus carbonarius, Aspergillus niger and Aspergillus ochraceus). Furthermore, two of the isolates strains (A19 and A21) identified as belonging to the genus of Pediococcus as well as Lactobacillus plantarum B4496, Lactobacillus brevis 207 and Lactobacillus sanfranciscensis BB12 showed interesting in vitro broad antifungal activities towards the three ochratoxin-producing fungi with inhibition percentages ranging from 15% to 66.7%. Treatment of cell-free supernatant at 100°C affected antifungal activity suggesting that the main compounds responsible for this activity were of proteic nature, and hence could be bacteriocins. Application of isolate A19 in cocoa fermentation as starter inhibited the growth of each of the OTA-producing species. At the end of fermentation in boxes inoculated with A19, A. niger was not detectable while A. carbonarius concentration was found to be 2 Log CFU/g of wet beans. The assessment of the ochratoxin produced during fermentation of cocoa inoculated with A. carbonarius indicated that the use of isolate A19 as starter could reduce their level of growth so as to have only a toxin production of 0.0012 ± 0.0005 μg/kg after 40 days of storage, while this was 2.45 ± 0.35 μg/kg of fermented and dried cocoa beans in the absence of A19. This work is a contribution for the application of biological control of OTA-producing fungi during cocoa production.     

Inhibitory Effect of Essential Oils on Aspergillus ochraceus Growth and Ochratoxin A Production

Ochratoxin A (OTA) is a mycotoxin which is a common contaminant in grains during storage. Aspergillus ochraceus is the most common producer of OTA. Essential oils play a crucial role as a biocontrol in the reduction of fungal contamination. Essential oils namely natural cinnamaldehyde, cinnamon oil, synthetic cinnamaldehyde, Litsea citrate oil, citral, eugenol, peppermint, eucalyptus, anise and camphor oils, were tested for their efficacy against A. ochraceus growth and OTA production by fumigation and contact assays. Natural cinnamaldehyde proved to be the most effective against A. ochraceus when compared to other oils. Complete fungal growth inhibition was obtained at 150–250 µL/L with fumigation and 250–500 µL/L with contact assays for cinnamon oil, natural and synthetic cinnamaldehyde, L. citrate oil and citral. Essential oils had an impact on the ergosterol biosynthesis and OTA production. Complete inhibition of ergosterol biosynthesis was observed at ≥100 µg/mL of natural cinnamaldehyde and at 200 µg/mL of citral, but total inhibition was not observed at 200 µg/mL of eugenol. But, citral and eugenol could inhibit the OTA production at ≥75 µg/mL and ≥150 µg/mL respectively, while natural cinnamaldehyde couldn’t fully inhibit OTA production at ≤200 µg/mL. The inhibition of OTA by natural cinnamaldehyde is mainly due to the reduction in fungal biomass. However, citral and eugenol could significant inhibit the OTA biosynthetic pathway. Also, we observed that cinnamaldehyde was converted to cinnamic alcohol by A. ochraceus, suggesting that the antimicrobial activity of cinnamaldehyde was mainly attributed to its carbonyl aldehyde group. The study concludes that natural cinnamaldehyde, citral and eugenol could be potential biocontrol agents against OTA contamination in storage grains.     

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.     

Isolation and identification of ochratoxin A-producing Aspergillus section Nigri strains from California raisins

Aims: To determine incidence and levels of ochratoxin A (OTA) in California raisins and to isolate and characterize OTA‐producing fungi from California raisin vineyard populations. Methods and Results: Forty raisin clusters sampled from four California vineyards in the San Joaquin Valley were analysed for OTA content using immunoaffinity and HPLC methods. OTA was detected in 93% of the samples, at levels from 0·06 to 11·4 ng g^?1. From these raisin samples, a total of 400 strains of Aspergillus were isolated and analysed for OTA production. Twelve isolates (3%), from five raisin samples, produced OTA. These isolates were identified as Aspergillus carbonarius, based on morphological characteristics and multilocus sequence analysis. Levels of OTA produced by these isolates on raisin agar ranged from 0·9 to 15 μg g^?1. Conclusions: OTA is a common contaminant of raisin vineyards, but average levels are much lower than EU regulatory limits for dried fruit. The primary species responsible for OTA contamination in California raisins is A. carbonarius. Significance and Impact of the Study: This study illustrates that low‐level OTA contamination of raisins occurs in California and that ecological studies of A. carbonarius within the Aspergillus section Nigri population on raisins are warranted to monitor ochratoxigenic potential of the crop.     

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.     

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.     

Occurrence of ochratoxin A and ochratoxigenic mycoflora in corn and corn based foods and feeds in some South American countries

Cereals and cereal- derived products constitute the base of human and animal feeding in South American countries. This review attempts to give an overview of the ochratoxin A (OTA) occurrence and potential sources of OTA contamination in those products. The environmental conditions as humidity and temperature in the colonization of the substrates by Aspergillus section Nigri isolated from corn kernels were also discussed. The available information on the ochratoxigenic mycoflora and OTA presence in corn, corn based food and feed is limited. Only few surveys have been carried out in Argentina, Ecuador and Brazil; which showed that Aspergillus niger aggregate and A. ochraceus species would be the main source of OTA. It’s possible to emphasize that, the species A. carbonarius has not been isolated from these substrates and Penicillium verrucosum was isolated only from pig feeds of Argentinean samples in low percentage. Studies about the ecophysiology of ochratoxigenic fungi and OTA occurrence are in progress in Latin America to reduce the impact of this toxin in the food chain. Carina E. Magnoli, Stella M. Chiacchiera, Ana M. Dalcero—Members of the Research Career Andrea L. Astoreca—Fellowship of CONICET     

Inhibition of ochratoxin A production and growth of <Emphasis Type="Italic">Aspergillus</Emphasis> species by phenolic antioxidant compounds

The phenolic antioxidants, gallic acid, vanillic acid, protocatechuic acid, 4-hydroxybenzoic acid, catechin, caffeic acid, and chlorogenic acid were studied for their effects on ochratoxin A (OTA) production and fungal growth of ochratoxigenic Aspergilli. Of the 12 strains tested, which included A. alliaceus, A. lanosus, A. ochraceus, A. albertensis, A. melleus, A. sulphureus, A. carbonarius, A. elegans, and A. sclerotiorum, the greatest inhibition of OTA production was seen in A. sulphureus, A. elegans, and A. lanosus. Vanillic acid and 4-hydroxybenzoic acid were the most inhibitory to both OTA production and growth of most of the strains tested. However, A.␣ochraceus was not inhibited by either compound, and A. carbonarius was not inhibited by vanillic acid. The effect of each compound on OTA production and growth differed among strains and generally was variable, suggesting that species-specific OTA production and response to phenolic compounds may be influenced by different ecological and developmental factors. In addition, inhibition of OTA production by antioxidant compounds may be useful in determining biosynthetic and regulatory genes involved in both OTA production and stress response in ochratoxigenic Aspergilli.     

Occurrence of Ochratoxin A-Producing Fungi in Commercial Corn Kernels in Argentina

Potentially ochratoxigenic Aspergillus and Penicillium species were identified and the natural occurrence of ochratoxin A (OTA) in corn kernels was evaluated. Likewise, the capacity to produce OTA by Aspergillus section Nigri and Circumdati was investigated. A total of 50 corn samples for human consumption was collected in the south of Córdoba Province. The surface-disinfected method for mycobiota determination was used. The OTA detection was performed by HPLC. OTA production was tested in strains belonging to section Nigri and Circumdati. Statistical analysis demonstrated that the specie A. flavus was isolated in higher frequency (p<0.01) from corn kernels in DRBC and DG18 media. The percentage of corn kernels contaminated by A. niger var. niger was similar in DRBC and DG18 media. The frequency of grains contaminated by A. flavus and A. niger var. awamori was higher than A. niger var. niger and A. japonicus var. japonicus (p<0.01) in DG18 media. The other potentially ochratoxigenic species, A. ochraceus, was isolated between 5% and 10% of the corn kernels in DG18 and DRBC media, respectively. The OTA producing species P. verrucosum was not isolated. All samples of corn were OTA negative (<1 ng g⁻¹). Thirty strains (25%) of the black Aspergillus were OTA producers. From four strains of A. ochraceus isolated, only one produced OTA. Due to the storage variable conditions could not be adequate in this substrate, the presence of ochratoxigenic strains of section Nigri and OTA needs to be evaluated for a longer time to establish the toxicological risk for human beings. The contamination of stored corn kernels with A. flavus and Aspergillus section Nigri was significant.     

Molecular detection of ochratoxin A producers: an updated review

Ochratoxin A (OTA) can be detected worldwide from various food and feed sources. It is produced byPenicillium nordicum andP. verrucosum as well as by variousAspergillus species, withA. ochraceus andA. carbonarius as the predominant producers. Various pairs of PCR primers based on AFLP, RAPD as well as primers specific to ribosomal RNA and genes coding for calmodulin and OTA biosynthetic pathway components were recently developed to detect and identify OTA producers in conventional and real-time PCR assays. Application of such assays in contaminated samples was demonstrated only in few cases. The current review gives an updated overview over the methods at hand.     

Impact of some environmental factors on growth and production of ochratoxin A of/by <Emphasis Type="Italic">Aspergillus tubingensis,A. niger</Emphasis>, and <Emphasis Type="Italic">A. carbonarius</Emphasis> isolated from moroccan grapes

The effects of temperature, water activity (a_w), incubation time, and their combinations on radial growth and ochratoxin A (OTA) production of/by eight Aspergillus niger aggregate strains (six A. tubingensis and two A. niger) and four A. carbonarius isolated from Moroccan grapes were studied. Optimal conditions for the growth of most studied strains were shown to be at 25°C and 0.95 a_w. No growth was observed at 10°C regardless of the water activity and isolates. The optimal temperature for OTA production was in the range of 25°C∼30°C for A. carbonarius and 30°C∼37°C for A. niger aggregate. The optimal a_w for toxin production was 0.95∼0.99 for A. carbonarius and 0.90∼0.95 for A. niger aggregate. Mean OTA concentration produced by all the isolates of A. niger aggregate tested at all sampling times shows that maximum amount of OTA (0.24 μg/g) was produced at 37°C and 0.90 a_w. However, for A. carbonarius, mean maximum amounts of OTA (0.22 μg/g) were observed at 25°C and 0.99 a_w. Analysis of variance showed that the effects of all single factors (a_w, isolate, temperature and incubation time) and their interactions on growth and OTA production were highly significant.     

Comparative proteomics and physiological characterization of Arabidopsis thaliana seedlings in responses to Ochratoxin A

Ochratoxin A (OTA) is a mycotoxin that is primarily produced by Aspergillus ochraceus and Penicillium verrucosum. This mycotoxin is a contaminant of food and feedstock worldwide and may induce cell death in plants. To investigate the dynamic growth process of Arabidopsis seedlings in response to OTA stress and to obtain a better understanding of the mechanism of OTA toxicity towards Arabidopsis, a comparative proteomics study using 2-DE and MALDI-TOF/TOF MS/MS was performed. Mass spectrometry analysis identified 59 and 51 differentially expressed proteins in seedlings exposed to 25 and 45 μM OTA for 7 days, respectively. OTA treatment decreased root elongation and leaf area, increased anthocyanin accumulation, damaged the photosynthetic apparatus and inhibited photosynthesis. Treatment of the seedlings with 25 μM OTA enhanced energy metabolism, whereas higher concentration of OTA (45 μM) inhibited energy metabolism in the seedlings. OTA treatment caused an increase of ROS, an enhancement of antioxidant enzyme defense responses, disturbance of redox homeostasis and activation of lipid oxidation. Glutamine and S-adenosylmethionine metabolism may also play important roles in the response to OTA. In conclusion, our study provided novel insights regarding the response of Arabidopsis to OTA at the level of the proteome. These results are expected to be highly useful for understanding the physiological responses and dissecting the OTA response pathways in higher plants.     

Preparations of Extracellular Proteinases from Aspergillus ochraceus 513 and Aspergillus alliaceus 7 dN1

Preparations of extracellular proteolytic enzymes with high anticoagulant activity resembling protein C activators were isolated from the culture liquids of Aspergillus ochraceus 513 and Aspergillus alliaceus 7 dN1 by precipitation with ammonium sulfate and subsequent purification from ammonium ions by gel filtration on a column with Sephadex G-25. The pH and temperature activity optima and stability of the proteolytic enzymes from A. ochraceus 513 and A. alliaceus 7 dN1 were determined.     

Production and properties of xylanases from Aspergillus terricola Marchal and Aspergillus ochraceus and their use in cellulose pulp bleaching

Aspergillus terricola and Aspergillus ochraceus, isolated from Brazilian soil, were cultivated in Vogel and Adams media supplemented with 20 different carbon sources, at 30 °C, under static conditions, for 120 and 144 h, respectively. High levels of cellulase-free xylanase were produced in birchwood or oat spelt xylan-media. Wheat bran was the most favorable agricultural residue for xylanase production. Maximum activity was obtained at 60 °C and pH 6.5 for A. terricola, and 65 °C and pH 5.0 for A. ochraceus. A. terricola xylanase was stable for 1 h at 60 °C and retained 50% activity after 80 min, while A. ochraceus xylanase presented a t ₅₀ of 10 min. The xylanases were stable in an alkali pH range. Biobleaching of 10 U/g dry cellulose pulp resulted in 14.3% delignification (A. terricola) and 36.4% (A. ochraceus). The brightness was 2.4–3.4% ISO higher than the control. Analysis in SEM showed defibrillation of the microfibrils. Arabinase traces and β-xylosidase were detected which might act synergistically with xylanase.     

Survey of Philippine coffee beans for the presence of ochratoxigenic fungi

In 2012 to 2014, Philippine green coffee beans from Coffea arabica in Benguet and Ifugao; Coffea canephora var. Robusta in Abra, Cavite, and Ifugao; and Coffea liberica and Coffea excelsea from Cavite were collected and assessed for the distribution of fungi with the potential to produce ochratoxin A (OTA). The presence of fungal species was evaluated both before and after surface sterilization. There were remarkable ecological and varietal differences in the population of OTA-producing species from the five provinces. Aspergillus ochraceus, A. westerdijkiae, and Penicillium verruculosum were detected from Arabica in Benguet and Ifugao while Aspergillus carbonarius, Aspergillus niger, and Aspergillus japonicus were isolated in Excelsa, Liberica, and Robusta varieties from Abra, Cavite, and Davao. Contamination by Aspergillus and Penicillium species was found on 59 and 19 %, respectively, of the 57 samples from five provinces. After disinfection with 1 % sodium hypochlorite, the levels of infection by Aspergillus and Penicillium fell to 40 and 17 %, respectively. A total of 1184 fungal isolates were identified to species level comprising Aspergillus sections Circumdati (four species), Clavati (one), Flavi (one), Fumigati (one), Nigri (three), and Terrie (one). Within section Circumdati, 70 % of A. ochraceus produced OTA as high as 16238 ng g^?1 while 40 % of A. westerdijkiae produced maximum OTA of 36561 ng g^?1 in solid agar. Within section Nigri, 16.76 % of A. niger produced OTA at the highest 18439 ng g^?1, 10 % of A. japonicus at maximum level of 174 ng g^?1, and 21.21 % of A. carbonarius yielded maximum OTA of 1900 ng g^?1. Of the 12 species of Penicillium isolated, P. verruculosum was ochratoxigenic, with a maximum OTA production of 12 ng g^?1.     

Ochratoxin A in airborne dust and fungal conidia

Farm workers are often exposed to high concentrations of airborne organic dust and fungal conidia, especially when working with plant materials. The purpose of this investigation was to study the possibility of exposure to the mycotoxin ochratoxin A (OTA) through inhalation of organic dust and conidia. Dust and aerosol samples were collected from three local cowsheds. Aerosol samples for determination of total conidia and dust concentrations were collected by stationary sampling on polycarbonate filters. Total dust was analysed by gravimetry, and conidia were counted using scanning electron microscopy. A method was developed for extraction and determination of OTA in small samples of settled dust. OTA was extracted with a mixture of methanol, chloroform, HCI, and water, purified on immunoaffinity column, and analysed by ion-pair HPLC with fluorescence detection. Recovery of OTA from spiked dust samples (0.9–1.0 μg/kg) was 74% (quantitation limit 0.150 μg/kg). OTA was found in 6 out of 14 settled dust samples (0.2–70 μg/kg). The total concentration of airborne conidia ranged from < 1.1 × 10⁴ to 3.9 × 15⁵ per m³, and the airborne dust concentration ranged from 0.08 to 0.21 mg/m³. Conidia collected from cultures of Penicillium verrucosum and Aspergillus ochraceus contained 0.4–0.7 and 0.02–0.06 pg OTA per conidium, respectively. Testing of conidial extracts from these fungi in a Bacillus subtilis bioassay indicated the presence of toxic compounds in addition to OTA. The results show that airborne dust and fungal conidia can be sources of OTA. Peak exposures to airborne OTA may be significant, e.g., in agricultural environments. This revised version was published online in August 2006 with corrections to the Cover Date.     

Potential of ochratoxin A production by Aspergillus carbonarius strains isolated from grapes at different ecological factors

Aspergillus carbonarius is known to colonize and produce ochratoxin A (OTA) on grapes and its derived products which is harmful to humans. We screened and tested A. carbonarius strains which isolated from grapes for production of OTA and selected three high OTA producing strains (ACSP1, ACSP2, ACSP3) for this study. These strains were further tested for their ability to produce OTA at different ecological factors [temperature 15, 25, 30, 35°C; water activity (a_w) 0.98, 0.95, 0.90, 0.88; and pH 4.0, 7.0, 9.0, 10.0]. Out of the three strains tested, A. carbonarius ACSP3 produced high levels of OTA than ACSP2 and ACSP1 in all the ecological factors. At 30°C A. carbonarius strains produced the highest OTA compared with other temperature regimes. With reference to water activity, a_w 0.98 favoured mycelial growth and accumulation of more OTA with all the three A. carbonarius strains. Further, pH 4.0 was encouraged the greatest production of OTA in all the strains. No growth was observed at a_w 0.88 and pH 10.0 in all the three strains except the strain ACSP3 at high pH. Our work demonstrated that temperature 30°C, a_w 0.98 and pH 4.0 is optimum for growth and production of OTA by A. carbonarius strains. Maximum amounts of OTA were found at earlier growth stages (7–9 days of incubation) in all the strains of A. carbonarius. The present study revealed that different ecological factors had great impact on OTA production by A. carbonarius which is useful for understanding OTA contamination and to develop proper management practices in future research programmes.