Intraspecific variability of growth and ochratoxin A production by Aspergillus carbonarius from different foods and geographical areas.

Ochratoxin A (OTA) is a nephrotoxic mycotoxin naturally found in a wide range of food commodities throughout the world. Aspergillus carbonarius is the most important source of OTA in food commodities such as wine, grapes and dried vine fruits and is also responsible for the formation of OTA in coffee. The aim of this study was to determine the simultaneous effect of three culture media (Czapek Yeast Extract Broth (CYB); Synthetic Grape Juice Medium (SGM) and White grape juice (WGJ)) at three water activity (aw) levels (0.90; 0.95 and 0.98-0.99), and three incubation temperatures (15 °C, 25 °C and 35 °C) on the growth and OTA production by 16 strains of A. carbonarius. The strains were mainly isolated from grapes from areas with a Mediterranean climate. All the strains were confirmed for identity by sequencing of the calmodulin gene. The assay was performed in microtiter plates, determining the absorbance at 530 nm and the concentration of OTA after 1, 2, 4 and 10 days of incubation. No significant differences were observed in absorbance values between the strains. The highest absorbance values were recorded in CYB at 0.99 aw and at 0.95 aw after 10 days of incubation at 25 °C and 35 °C. None of the strains were able to grow at 0.90 aw and 15 °C in any culture media after 10 days of incubation. OTA concentration was statistically higher at 15 °C than at 25 °C or 35 °C. The highest significant OTA values were obtained at 0.98-0.99 aw and the best culture media for OTA production was CYB, followed by WGJ and SGM. While strains isolated from Mediterranean climate foods had a similar behavior despite being isolated from different geographical areas, OTA concentration produced by one Robusta coffee strain from Thailand was statistically higher at 25 °C than at 15 °C. This would suggest that the type of food matrices and consequently the adaptation of A. carbonarius strains to different climatic conditions would have a greater influence on the ecophysiology of the strains than only their geographical origin.

Impact of some environmental factors on growth and ochratoxin A production by Aspergillus niger and Aspergillus welwitschiae.

Ochratoxin A (OTA) is a nephrotoxic mycotoxin which may contaminate various foods and feed products worldwide. Aspergillus niger is one of the species responsible for OTA contamination in grapes and derived products. This species has recently been split into A. niger and Aspergillus welwitschiae. Both species can not be distinguished by phenotypic or extrolite profiles and to date there is no ecophysiological information of A. welwitschiae. The aim of this study was to determine the effects of water activity (aw) (0.90; 0.95 and 0.98-0.99), culture media (Yeast Extract Sucrose Broth (YESB); Synthetic Grape Juice Medium (SGM); White grape juice (WGJ)) and temperature (15 °C, 25 °C and 35 °C) on the growth and OTA production of four strains of A. niger and six strains of A. welwitschiae. The assay was performed in microtiter plates, determining the absorbance at 530 nm and the concentration of OTA at 1, 2, 4 and 10 days. No significant differences were observed in absorbance and OTA values between the two species under study. The highest absorbance values were recorded in YESB, followed by SGM and WGJ. Absorbance values increased with increasing aw and temperature. The highest OTA values were obtained at 0.98-0.99 aw and the best culture media for OTA production was YESB, followed by WGJ and SGM. The studied strains of A. niger produced the highest mean OTA level at 25 °C whereas A. welwitschiae strains produced the highest mean OTA concentration at 15 °C, although not differing significantly from concentration produced at 25 °C. To our knowledge, this is the first report on the impact of some environmental factors on growth and OTA production by A. welwitschiae.

A new in vitro method to detect growth and ochratoxin A-producing ability of multiple fungal species commonly found in food commodities.

The aim of the study was to develop a new screening method to detect growth and ochratoxin A (OTA) production by multiple fungi growing in a small quantity of culture media, using microtiter plates. Eight ochratoxigenic species were included in the study. The strains were inoculated in sterile 96-well flat-bottom microtiter plates containing Yeast Extract Sucrose broth and Czapek Yeast Extract broth and incubated at 25 °C. Growth was daily monitored by absorbance measurements for 4 days and extended to 7 and 10 days for Penicillium spp. The entire experiment was repeated twice on different days. On each sampling time, five of the seven replicate wells inoculated for each strain and culture media were randomly selected and the content of each well was removed, extracted and injected into the HPLC. No statistically significant differences were observed for absorbance and OTA values, neither between replicates nor between experiments. Quantifiable OTA levels were detected after 48 h of incubation in Aspergillus alliaceus, Aspergillus carbonarius and Aspergillus niger, after 72 h in Aspergillus flocculosus, Aspergillus steynii and Aspergillus westerdijkiae and after 7 days in Penicillium nordicum and Penicillium verrucosum. The method offers the necessary tools for a rapid detection of growth and OTA production avoiding the use of plate cultures and can be very useful when many fungal isolates need to be screened.

Characterization of nonochratoxigenic strains of Aspergillus carbonarius from grapes.

Aspergillus carbonarius is the main responsible source of ochratoxin A (OTA) in food commodities such as wine, grapes or dried vine fruits from main viticultural regions worldwide. Besides, OTA production is a very consistent property of this species and for this reason atoxigenic isolates of A. carbonarius are very rarely found in natural environments. In the present study, for the first time, three nonochratoxigenic wild strains of A. carbonarius have been discovered, unambiguously identified, characterized in deep and compared to ochratoxigenic strains of the same species. In addition, polyketide synthase (pks) genes suggested to be involved in OTA biosynthesis were also screened in these strains. The identification of the strains was confirmed by ITS-5.8S rRNA, ß-tubulin and calmodulin gene sequencing. The three atoxigenic strains did not produce OTA in a conducive culture medium at any of the temperatures and times of incubation tested. Five ketosynthase domains from pks genes previously described in A. carbonarius were detected both in ochratoxigenic and in nonochratoxigenic strains. Atoxigenic strains of A. carbonarius could be useful as biotechnological agents to be used in food industry and as biological agents for control of OTA production in vineyards and other crops.

Mycobiota and mycotoxin contamination of maize flours and popcorn kernels for human consumption commercialized in Spain.

Mycobiota and co-occurrence of aflatoxins, citrinin, ochratoxin A and zearalenone in 30 samples of maize flours and 30 of popcorn kernels purchased in Spain for human consumption were determined. The mycotoxin-producing ability of Aspergillus, Fusarium and Penicillium spp. was also studied. Total fungal counts of maize flours ranged from <10 to 8.4 × 10(4) CFU/g and predominant mycobiota belonged to Aspergillus spp. and Penicillium spp. In popcorn kernels samples the most frequent species were Aspergillus spp., Mucorales, Fusarium spp. and Penicillium spp. Aflatoxins were produced by Aspergillus flavus and Aspergillus parasiticus, citrinin by Penicillium citrinum and Penicillium verrucosum, ochratoxin A by Aspergillus niger and patulin by Aspergillus clavatus and Penicillium griseofulvum. Identification of all the mycotoxin-producing strains as well as some Aspergillus spp. difficult to identify using phenotypic characters only was also performed by molecular methods. Aflatoxins were detected in 14 maize flours and 2 popcorn kernels samples, while ochratoxin A was detected in 4 maize flours and 10 popcorn samples. Co-occurrence of aflatoxins and ochratoxin A was found in the 4 ochratoxin-positive maize flour samples. Citrinin and zearalenone were not detected. This is the first report of aflatoxins and ochratoxin A contamination in maize flours and popcorn kernels commercialized in Spain.

Effect of water activity, temperature and incubation time on growth and ochratoxin A production by Aspergillus niger and Aspergillus carbonarius on maize kernels.

The aim of this study was to determine the effects of water activity (a(w)) (0.92-0.98), temperature (5-45 °C) and incubation time (5-60 days) on growth and ochratoxin A (OTA) production by Aspergillus niger and Aspergillus carbonarius on maize kernels using a simple method. Colony diameters of both strains at 0.92 a(w) were significantly lower than those at 0.96 and 0.98 a(w) levels. The optimum growth temperature range for A. niger was 25-40 °C and for A. carbonarius 20-35 °C. A. niger produced OTA from 15 to 40 °C, and the highest OTA level was recorded at 15 °C. The concentration of OTA produced at 0.92 a(w) was significantly lower than those at 0.96 and 0.98 a(w). A. carbonarius produced OTA from 15 to 35 °C and the maximum concentration was achieved at 15 °C, although not differing statistically from the concentration detected at 20 °C. At 0.98 a(w) the OTA concentration was significantly higher than at 0.96 and 0.92 a(w). Our results show that maize supports both growth and OTA production by A. niger and A. carbonarius. The studied strains were able to produce OTA in maize kernels from the fifth day of incubation over a wide range of temperatures and water availabilities. Although the limit of quantification of our method was higher than that required for the analysis of OTA in food commodities, it has proved to be a useful and rapid way to detect OTA production by fungi inoculated onto natural substrates, in a similar way as for pure culture. Both species could be a source of OTA in this cereal in temperate and tropical zones of the world.

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.

Comparison of two selective culture media for the detection of Fusarium infection in conventional and transgenic maize kernels.

AIMS: To assess differences between two recommended selective culture media, Nash and Snyder medium (NS) and malachite green agar 2.5 (MGA 2.5), for the detection of Fusarium infection in conventional and transgenic maize kernels. METHODS AND RESULTS: In total, 10 800 kernels from commercial varieties grown in Spain were analysed using these Fusarium selective culture media. Fusarium verticillioides was predominant in both selective culture media. Mean percentages of Fusarium infected kernels were significantly lower in transgenic maize kernels than in conventional maize kernels. There were no significant differences in percentage of Fusarium infection between the two selective culture media used, although the total mean value on MGA 2.5 (18.8%) was slightly lower than on NS (19.1%). CONCLUSIONS: MGA 2.5 performed as a potent selective medium for the detection of Fusarium infection in maize kernels using the direct plating technique. SIGNIFICANCE AND IMPACT OF THE STUDY: NS with pentachloronitrobenzene (PCNB) as fungal inhibitor is one of the most widely employed selective culture medium for Fusarium spp. However, PCNB has been reported to be carcinogenic. MGA 2.5 can be used as an alternative to NS in the detection of Fusarium infection in grain samples using the direct plating technique.

Comparison of methods to detect resistance of Penicillium expansum to thiabendazole.

AIMS: Because of the lack of a standard method, the aim of this work is to evaluate the suitability of the broth microdilution method CLSI M38-A in determining the resistance level of some Penicillium expansum isolates to thiabendazole (TBZ). The ability of the isolates to produce patulin (PAT) and citrinin (CIT) has been also assessed. METHODS AND RESULTS: Penicillium expansum isolates (128) were assayed (apples, pears, grapes and five reference strains). It was observed that 69.4% of the strains isolated from apples and pears were resistant to TBZ. Sensitive isolates were inhibited at 0.25-0.5 microg ml(-1) whilst resistant isolates still grew at 512 microg ml(-1). PAT was produced by all P. expansum isolates. CIT was detected in 98.8% of TBZ-resistant isolates and in 89.1% of the TBZ-sensitive isolates. CONCLUSIONS: The preliminary screening method combined with the adaptation of the method CLSI M38-A, can be a good strategy to be used in assessing the in vitro activity of TBZ against a large number of isolates. SIGNIFICANCE AND IMPACT OF THE STUDY: The proposed methodology can be a contribution to the standardization of susceptibility tests to fungicides against P. expansum.

In vitro activity of imazalil against Penicillium expansum: comparison of the CLSI M38-A broth microdilution method with traditional techniques.

Penicillium expansum is one of the most important pathogens that cause blue mold in stored apples and is regarded as the major producer of the mycotoxin patulin. Imazalil is one of the fungicides used in Spain to control postharvest blue mold, but development of fungal resistance has been reported in P. digitatum and P. italicum. The most common used methods to detect antifungal susceptibility of fungal crop pathogens in vitro, are direct-plating isolates in media amended with various concentrations of fungicide and determining inhibition of growth and/or spore germination. These techniques are time- and labor-intensive and are not suitable if a large number of isolates has to be evaluated. On the other hand, the broth microdilution method M38-A is the reference method developed by the Clinical and Laboratory Standards Institute (CLSI) for antifungal susceptibility testing in some clinical fungi, but Penicillium spp. are not included. Due to the lack of a standard method, the aim of this work is to evaluate the suitability of an adaptation of the CLSI M38-A method to monitor P. expansum susceptibility to imazalil in comparison with other techniques. A total of 128 P. expansum strains have been studied (118 isolates from apples and pears, 5 from grapes and 5 reference strains). Imazalil has shown to be highly active in vitro against all the P. expansum isolates tested, as all the evaluated parameters were in the range reported for imazalil sensitive Penicillium spp. The mean minimum inhibitory concentration determined by broth microdilution method and by agar dilution method (48-72 h readings) was 0.0625 microg/ml and 0.11-0.12 microg/ml respectively. The mean concentration that inhibited the size of colonies (48-72 h) and spore germination by 50% was 0.05-0.06 and 0.04 microg/ml respectively. Our results highlight that the broth microdilution method CLSI M38-A is a good alternative to be used in screening the in vitro activity of imazalil against a large number of isolates.

Ochratoxin A and citrinin producing species of the genus Penicillium from feedstuffs.

In Spain, low ochratoxin A (OTA) levels have been detected in several pork products but there is no information published about the fungi involved in this OTA contamination. It is well known that P. verrucosum is much more frequently found on cereals in countries where they occasionally have OTA problems as in North European countries compared with South Europe where levels of OTA generally seem to be lower or not detected. Much less information is available about citrinin (CIT) and CIT producing species in cereals and their by products. The aim of this study was to determine, identify and characterize the occurrence of potential OTA and CIT producing Penicillium spp. from mixed feeds and raw materials purchased in the Spanish market and used as feedstuffs. A total of 155 Penicillium spp. isolates belonging to 34 species were analyzed in order to know if they are able to produce OTA and/or CIT. From these isolates, 11 P. verrucosum which were characterized by RAPD analyses, produced OTA. Fourteen isolates were CIT producers, 10 isolates of P. verrucosum and 4 of P. citrinum. Although the occurrence and abundance of OTA and CIT Penicillium producing species have been low in our study, our results confirm the potential risk of OTA and CIT production in feeds if stored improperly. Our results also confirm the occurrence of P. verrucosum in South European countries and that it is the only OTA producing Penicillium species in these substrates.

Occurrence of Penicillium verrucosum in retail wheat flours from the Spanish market.

In Spain, low ochratoxin A (OTA) levels have been detected in wheat and different wheat products but no information has been published about the fungi involved in this OTA contamination. Some species of the genera Penicillium and Aspergillus are known to form OTA but few of them are known to contaminate foods with this mycotoxin. Penicillium verrucosum, an important OTA producer typical of temperate and cold climates, is much more frequently found on cereals in countries where they occasionally have OTA problems as in North European countries compared with South Europe, where levels of OTA generally seem to be lower or is not detected. The aim of this study was to determine, identify and characterize the occurrence of potential OTA-producing Aspergillus spp. and Penicillium spp. from retail wheat flours purchased in the Spanish market and used for human consumption. A total of 105 Aspergillus isolates were analyzed in order to know whether they are able to produce OTA and/or citrinin (CIT). None of these isolates were able to produce these mycotoxins. However, 17 suspected P. verrucosum isolates were recovered and confirmed by RAPD analyses. Eleven isolates were OTA producers and 14 isolates produced CIT. Our results confirm the potential risk of OTA and CIT production in wheat flours if stored improperly and the occurrence of P. verrucosum in South European countries. This was the only species able to produce these mycotoxins.

Low occurrence of patulin- and citrinin-producing species isolated from grapes.

AIMS: To assess the ability of fungi isolated from grapes to produce patulin and citrinin. METHODS AND RESULTS: A total of 446 Aspergillus isolates belonging to 20 species and 101 Penicillium isolates were inoculated in Czapek yeast extract agar and yeast extract sucrose agar and incubated for 7 days at 25 degrees C. Extracts were analysed for patulin and citrinin by thin-layer chromatography. None of the isolates of Aspergillus spp. produced either patulin or citrinin. Patulin was produced by three isolates of Penicillium expansum and two of Penicillium griseofulvum. Citrinin was produced by five isolates of P. expansum, two of Penicillium citrinum and one of Penicillium verrucosum. CONCLUSIONS: Our results show that the Aspergillus and Penicillium species commonly isolated from grapes are not a source of the mycotoxins, patulin and citrinin. SIGNIFICANCE AND IMPACT OF THE STUDY: The possibility of co-occurrence of patulin and citrinin with ochratoxin A in grapes and grape products remain low, owing to the low frequency of isolation of potentially producing species.

Ochratoxin A-producing fungi from grapes intended for liqueur wine production.

The ochratoxigenic mycobiota of grapes intended for liqueur wines from four Spanish vineyards were studied. The specific wine-making technology of these wines requires overripening of the grapes on the vine or extended post-harvest exposure of the grapes in the sun. In every vineyard, samples were taken at three different developmental stages: veraison, harvesting time and after over-ripening. With the maturation of the berries there was a clear increase of Aspergillus spp. In the last sampling time studied, they were isolated from the 90.3% of the plated berries. Black aspergilli (mainly A. niger aggregate and A. carbonarius) were predominant among the different Aspergillus spp. isolated and constituted 98.5% of the total Aspergillus strains isolated. At harvesting time and after over-ripening, the percentage of colonized berries with A. carbonarius exceeded that of Aspergillus niger aggregate. Due to their low frequency of isolation, Penicillium spp. and Aspergillus spp. outside black aspergilli are not an important source of ochratoxin A in grapes for liqueur wine production. On the contrary, 98.5% of the A. carbonarius isolates screened were able to produce ochratoxin A. Although the possible participation of different ochratoxin A-producing species may occur, our results confirm that A. carbonarius is the most important source of ochratoxin A in liqueur wines, increasing its occurrence along the ripening of grapes.

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 pH on ochratoxin A production by Aspergillus niger aggregate species.

The effect of pH (2-10) on growth and ochratoxin A (OTA) production by 12 Aspergillus niger aggregate strains was studied in two culture media: Czapek yeast autolysate agar (CYA) and yeast extract sucrose agar (YES), over 30 days. The strains were selected to include different sources, different reported abilities to produce OTA and different ITS-5.8S rDNA RFLP patterns. YES was a better culture medium than CYA for OTA production. In this medium, OTA was produced from pH 2 or 3 to 10 depending on the strain. The results show the ability of A. niger aggregate strains not only to grow, but also to produce OTA over a wide pH range. The results will lead to a better understanding of the role of A. niger aggregate strains in the OTA contamination of several food commodities.

Mycobiota and ochratoxin A producing fungi from Spanish wine grapes.

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

RFLP characterization of Aspergillus niger aggregate species from grapes from Europe and Israel.

In order to characterize by molecular methods the Aspergillus niger aggregate species involved in the ochratoxin A (OTA) contamination of European wine grapes and table grapes from Israel, a total of 173 strains were studied. The ITS-5.8S rDNA fragments of 173 A. niger agreggate strains from grapes included in this study were amplified and their PCR amplicons were RsaI digested in order to classify the strains in the RFLP types, N and T. All of the strains belonging to the A. niger aggregate were classified into the two RFLP types previously defined: type N (43%) and type T (57%). Twenty out of the 173 strains of A. niger aggregate produced OTA (0.1 to 10.5 mug g(-1)). All the OTA producing species belonged to the N-RFLP type.

Effect of water activity on ochratoxin A production by Aspergillus niger aggregate species.

The effect of water activity (a(w)) (0.82-0.99) on growth and ochratoxin A (OTA) production by twelve Aspergillus niger aggregate strains, cultured in Czapek Yeast Autolysate agar (CYA) and Yeast Extract Sucrose agar (YES), was studied for an incubation period of 30 days. The strains were selected to include diverse sources, different reported abilities to produce OTA and different ITS-5.8 S rDNA Restriction Fragment Length Polymorphism (RFLP) pattern. They were characterized by Random Amplification of Polymorphic DNA (RAPD) and ITS-5.8 S rDNA and 28 S rDNA (D1/D2) sequencing. Regardless of the a(w) value tested, YES was a better culture medium than CYA for OTA production. The a(w) range for OTA production was narrower than that for growth. OTA was produced from 0.90, 0.92, 0.94 or 0.96 to 0.99 a(w) depending on the strain and the culture medium. The molecular study differentiated strains into two groups which corresponded to the RFLP types N and T although it did not distinguish them by their source of isolation or OTA producing abilities. Our results show that A. niger aggregate strains are able to grow and produce OTA over a wide a(w) range. These results will lead to a better understanding of the contribution of A. niger aggregate in OTA contamination of food and feed.

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.