Discrimination of Aspergillus niger and other Aspergillus species belonging to section Nigri by PCR assays

Aspergillus species included in section Nigri are common in plant products and processed food, such as grapes, cereals, coffee and derivatives, particularly in warm and tropical climates. Two of these species, A. carbonarius and A. niger, are known to produce ochratoxin A (OTA), a potent nephrotoxin and carcinogenic to human (group 2B). Recognition of the several species of this section is difficult and requires considerable expertise using conventional methods based on morphological features. In this work we describe rapid, sensitive and robust assays based on the PCR technique to discriminate the main species included in section Nigri: A. japonicus, A. heteromorphus, A. ellipticus and the two morphologically indistinguishable species of the A. niger aggregate: A. niger and A. tubingensis. The species-specific primers have been designed on the basis of ITS (internal transcribed spacers of rDNA units) sequence comparisons obtained from several Aspergillus strains and have been tested in a number of strains from different origins and hosts. These PCR assays, based on multi-copy sequences, are highly sensitive and specific and represent a good tool for an early detection of OTA-producing Aspergillus species in order to prevent OTA from entering the food chain.     

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.     

A Molecular Method for Detection of <Emphasis Type="Italic">Aspergillus carbonarius</Emphasis> in Coffee Beans

Ochratoxin A (OTA) is a carcinogenic and nephrotoxic mycotoxin that has been detected in a variety of food products, including green coffee beans. About 80% of Aspergillus carbonarius strains collected from coffee beans are able to produce OTA on this substrate. The rapid identification of this fungal species would be desirable. RAPD assays were applied to identify amplification products specific for A. carbonarius. One selected fragment, denoted OPX7₈₀₉, was cloned and sequenced. Based on the nucleotide sequence obtained, specific oligonucleotides (OPX7F₈₀₉ and OPX7R₈₀₉) were designed and used as primers for DNA amplification. One amplified band of 809 bp was obtained from A. carbonarius genomic DNA, whereas no amplified fragment from DNA of other Aspergillus species was detected. This PCR analysis was also successfully employed to detect A. carbonarius in coffee beans. This PCR assay could contribute to the early and rapid detection of the potential presence of OTA in coffee samples.     

Aspergillus ibericus: a new species of section Nigri isolated from grapes

As part of a study on the ochratoxin producing mycoflora of grapes, several Aspergillus strains were isolated and tested for their ochratoxin A (OTA) producing abilities. Aspergillus strains of the section Nigri, which did not produce detectable amounts of OTA but which had a similar morphology to A. carbonarius, were isolated from wine grapes and/or dried vine fruit in Portugal and Spain. These strains, however, have characters that allow morphological distinction from the other species in the section, particularly the conidia size (5-7 microm), which allows separation of the species from the two most common biseriate species in section Nigri: A. carbonarius (7-9 microm) and A. niger and its aggregate species (3-5 microm). The strains are described here as belonging to a new species, named A. ibericus. The validation of this new taxon is supported further by analysis of the ITS-5.8S rDNA and calmodulin gene sequences and by analysis of the amplified fragment length polymorphism (AFLP) patterns, which were consistent in separating these strains from other species in the section. A. ibericus strains do not produce OTA therefore they are interesting for biotechnological exploration because many metabolites with commercial value are produced by other species in the section.     

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.     

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.     

Utility of Microsatellite Markers and Amplified Fragment Length Polymorphism in the Study of Potentially Ochratoxigenic Black Aspergilli

Microsatellite markers and the results of amplified fragment length polymorphism (AFLP) were compared in the characterization of 68 Aspergillus carbonarius and A. niger aggregate strains of differing ochratoxin-producing ability and from different geographic areas, isolated mainly from grapes and soil. AFLP was applied to both A. carbonarius and A. niger aggregate strains, and it clearly differentiated these species. Microsatellite markers were only applied to A. niger aggregate strains because of the species-specific nature of these markers. Both AFLP and microsatellite marker analyses were able to divide A. niger aggregate strains into the two recognized internal transcribed spacer (ITS)-5.8S rDNA RFLP types, N and T. Clustering of A. niger aggregate strains was similar in both AFLP and microsatellite analyses, yielding an additional separation of N type strains into two groups. Both microsatellite marker and AFLP analyses showed high levels of polymorphism in the A. niger aggregate (index of discriminatory power 0.991 and 1.0, respectively). Of the two techniques, microsatellite marker analysis was quicker and more straightforward to perform. In addition, microsatellite marker analysis is more reproducible, and the results can be expressed as quantitative data, making microsatellite markers a good candidate for use in large-scale studies of genetic diversity in A. niger aggregate species.     

Ochratoxin A-producing Aspergilli in Vietnamese green coffee beans

AIMS: To determine the incidence and severity of infection by ochratoxin A (OA)-producing fungi in Vietnamese green coffee beans. METHODS AND RESULTS: Aspergillus carbonarius, A. niger and yellow Aspergilli (A. ochraceus and related species in section Circumdati) were isolated by direct plating of surface-disinfected Robusta (65 samples) and Arabica (11 samples) coffee beans from southern and central Vietnam. Significantly, more Robusta than Arabica beans were infected by fungi. Aspergillus niger infected 89% of Robusta beans, whereas A. carbonarius and yellow Aspergilli each infected 12-14% of beans. OA was not produced by A. niger (98 isolates) or A. ochraceus (77 isolates), but was detected in 110 of 113 isolates of A. carbonarius, 10 isolates of A. westerdijkiae and one isolate of A. steynii. The maximum OA observed in samples severely infected with toxigenic species was 1.8 microg kg(-1); however, no relationship between extent of infection and OA contamination was observed. CONCLUSIONS: Aspergillus niger is the dominant species infecting Vietnamese coffee beans, yet A. carbonarius is the likely source of OA contamination. SIGNIFICANCE AND IMPACT OF STUDY: Vietnamese green coffee beans were more severely infected with fungi than the levels reported for beans from other parts of the world, yet OA contamination appears to be infrequent.     

Partitioning of ochratoxin A in mycelium and conidia of Aspergillus carbonarius and the impact on toxin contamination of grapes and wine

AIMS: Aspergillus carbonarius is an important ochratoxin A (OTA)-producing fungus which is responsible for toxin contamination of grapes and wine. The objectives of this study were to examine the partitioning of OTA in mycelium and conidia of a range of A. carbonarius strains on artificial grape juice and defined media, to determine the excretion patterns of OTA from these spores, and the effect of organic acids used in wine production on OTA excretion from conidia. METHODS AND RESULTS: The results showed that 60-70% of the OTA was accumulated in the conidia of a number of different isolates of A. carbonarius. Calculations showed that on different defined media, an amount of 0.011- to 0.1-pg OTA was present per conidium. The OTA in spores was found to be rapidly excreted into the medium during the initial few hours after conidial germination leading to an increase of OTA in must during maceration for wine production. The presence of tartaric acid inhibited OTA production, but malic acid enhanced this production during mycelial growth. These acids were also shown to affect the time course of germination and the rate of OTA excretion from conidia during germination. CONCLUSIONS: This study is the first to examine and show the partitioning of OTA into spores of strains of A. carbonarius and that rapid excretion of OTA from spores could be a reason for OTA accumulation in musts during wine production. SIGNIFICANCE AND IMPACT OF THE STUDY: Conidia of A. carbonarius could be a major source of OTA contamination of grapes used in wine production. This information could help in the development of effective prevention strategies to minimize wine contamination with this important mycotoxin.     

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.     

OTA-producing fungi isolated from stored cocoa beans

Aims:  The aim of this study was to identify fungal populations in unroasted cocoa beans stored in Spain in order to evaluate the ochratoxin A (OTA)-production ability of certain Aspergillus isolates.
Methods and Results:  Twenty batches of cocoa beans from different origins and with different OTA content were selected for this study. Three Aspergillus carbonarius and 13 Aspergillus niger aggregate strains isolated from these cocoa bean samples were selected to evaluate their OTA synthesis ability, being the only A. carbonarius isolates which are OTA producers [−1 culture medium; LOD = 6  μ g kg⁻¹ culture medium].
Conclusions:  No correspondence was found between the OTA levels in cocoa beans and the presence of OTA-producing fungi. Nonetheless, some samples contained A. carbonarius with a high OTA-producing ability and, consequently, specific fungal controls should be set up during storage to avoid this toxin.
Significance and Impact of the Study:  Toxigenic fungi in cocoa beans are not well understood. This study attempted to identify these fungi and evaluate their OTA-producing ability.     

Specific detection of Aspergillus carbonarius by SYBR® Green and TaqMan® quantitative PCR assays based on the multicopy ITS2 region of the rRNA gene

Agroproducts contaminated by ochratoxin A (OTA) represent a risk for human and animal health and, therefore, maximum limits have been established by Food Safety Authorities. Reduction of OTA contamination may be accomplished by early detection of OTA-producing fungal species using rapid, specific and sensitive detection and quantification by PCR-based methods. Aspergillus carbonarius is one of the most important OTA-producing species, in particular in grapes and derivatives from Mediterranean regions. In this work, highly efficient quantitative PCR assays using SYBR Green I and TaqMan methods were developed for specific detection of A. carbonarius to be used in grapes. The primers and the TaqMan probe were based on the internal transcribed region 2 multicopy region (internal 2 sequence of the rRNA gene). The specificity and sensitivity of both assays were tested on genomic DNA mixtures of several A. carbonarius strains and other fungal species frequently present in grapes. Both methods were also compared using grapes inoculated with different spore concentrations of A. carbonarius , detecting up to 0.4 pg DNA g⁻¹ grape berries. The efficiency and sensitivity of both methods were comparable and only the lower cost of SYBR Green might favour its use in routine screenings.     

Aspergillus species producing ochratoxin A: isolation from vineyard soils and infection of Semillon bunches in Australia

AIMS: The incidence of toxigenicity among Australian isolates of Aspergillus niger and Aspergillus carbonarius was assessed. Aspergillus rot and concomitant production of ochratoxin A (OA) in bunches inoculated with A. carbonarius were also investigated. METHODS AND RESULTS: Aspergillus niger and A. carbonarius were isolated from vineyard soils. Aspergillus niger was more widespread than A. carbonarius, and two restriction fragment length polymorphism types of A. niger, N and T, were present. Three of 113 A. niger isolates and all 33 A. carbonarius isolates produced OA. Aspergillus carbonarius was inoculated onto Semillon bunches with and without damage in the month before harvest. Damaged berries at greater than 12.3 (o) Bx were particularly susceptible to Aspergillus rot and production of OA, which was concentrated in severely mouldy berries. CONCLUSIONS: OA in Australian grapes results mainly from infection of berries by A. carbonarius. It is concentrated in discoloured, shrivelled berries. The potential for Aspergillus rot and OA production appears to commence after veraison and increase with berry damage and ripeness. SIGNIFICANCE AND IMPACT OF THE STUDY: Minimizing damage to grapes between veraison and harvest significantly reduces Aspergillus rot and OA formation. Monitoring the extent of Aspergillus rot in bunches infected with toxigenic Aspergillus spp. may give some indication of OA contamination.     

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.     

Molecular typing of Aspergillus ochraceus and construction of species specific SCAR-primers based on AFLP

70 strains of Aspergillus ochraceus mainly isolated from Brazilian coffee related sources were investigated for genetic relatedness using automated laser fluorescence analysis of AFLP fragments. Cluster analysis of fingerprints revealed a very close relationship among most of the strains. Based on these results, a sub-set of characteristic A. ochraceus strains was chosen for the detection of marker sequences. These sequences were obtained from silver stained AFLPs separated on polyacrylamide gels. A number of bands characteristic for A. ochraceus were detected and cut out from the gels. DNA was reamplified, cloned and fragments were sequenced. Based on these sequences a set of SCAR PCR-primers was constructed. PCRs were optimised for specificity and subsequently tested against a panel of Aspergillus species. Using this approach a PCR specific for Aspergillus ochraceus was developed.     

Fungi and ochratoxin A detected in healthy grapes for wine production

AIMS: The mycoflora of healthy grapes (i.e. without visible symptoms of rot) for wine production in Portuguese wine-making regions was assessed and its potential for ochratoxin A (OTA) production evaluated. The OTA content of grapes was also determined. METHODS AND RESULTS: A total of 386 fungal strains were isolated by plating methods. The most frequent genera found in grapes were non-ochratoxigenic species: Cladosporium (28%), Penicillium (24%), Botrytis (13%) and Aspergillus (9%). Two OTA-producing strains were isolated, belonging to the species Aspergillus carbonarius and Aspergillus ochraceus. OTA was detected in three of four grape samples, up to 116 ng l(-1). CONCLUSIONS: OTA is being produced in healthy berries by Aspergillus species, namely A. carbonarius, at levels below the maximum recommended limit of 2,000 ng l(-1) in wine. SIGNIFICANCE AND IMPACT OF THE STUDY: The OTA concentration detected in healthy Portuguese grapes does not represent a risk to wine regarding the legal limit established.     

Molecular characterization of Aspergillus section Flavi isolates collected from peanut fields in Argentina using AFLPs

AIMS: The objectives of this study were: (i) to evaluate genetic relatedness among Aspergillus section Flavi strains isolated from soil and peanut seeds in Argentina; (ii) to determine if AFLP molecular markers could be useful to identify isolates up to species level, and to correlate these markers with the isolates' toxigenic potentials and/or vegetative compatibility group (VCG) affiliations. METHODS AND RESULTS: Amplified fragment length polymorphism (AFLPs) analysis was applied to compare 82 isolates of Aspergillus section Flavi. Cluster analysis showed a clear separation of A. flavus and A. parasiticus, and comparison of fingerprints revealed several specific markers for each group of isolates. AFLP analysis indicates that no genotypical differences can be established between aflatoxigenic and nonaflatoxigenic producers in both species analysed. In addition, candidate AFLP markers associated with a particular VCG were not found. CONCLUSIONS: There was a concordance between morphological identification and separation up to species level using molecular markers. The findings of specific bands for A. flavus and A. parasiticus may be useful for the design of specific PCR primers in order to differentiate these species and detect them in food. SIGNIFICANCE AND IMPACT OF THE STUDY: The present study provides new data on molecular characterization of Aspergillus section Flavi in Argentina.     

Genetic improvement of Aspergillus carbonarius for pectinase overproduction during solid state growth

Low pectinase production by Aspergillus carbonarius growing on wheat bran solid substrate was found to be due to reduced colonizational ability of the fungus. Since A. niger showed higher growth rates on wheat bran, strain improvement to obtain higher pectinase production in solid state was carried out by inter-specific fusion of protoplasts of A. carbonarius and A. niger. One of the mutants selected for higher activities of alpha-glucosidase showed improved growth rates on wheat bran solid substrate together with increased pectinase production. Size similarities of amplified polymorphic DNA of the mutant with the two parents and identification of a 66 kDa polygalacturonase specific to A. niger suggested genetic recombination in the mutant.     

Taxonomy and significance of black aspergilli

Members of Aspergillus section Nigri (formerly A. niger group) are distributed worldwide and are regarded as common food spoilage fungi. Some of them are widely used and studied for industrial purposes. They are common sources of extracellular enzymes and organic acids to be used in food processing and are also used in the production of traditional foods, especially in the Orient. Products produced by strains of Aspergillus niger hold the GRAS (Generally Recognised As Safe) status from the FDA. However some species in Aspergillus section Nigri can produce ochratoxin A, a nephrotoxic mycotoxin. In spite of their industrial importance, the taxonomy of black aspergilli ( Aspergillus section Nigri ) is not clear and many attempts have been made in order to find suitable taxonomic criteria. The aim of this paper is to provide an overview of the significance of black aspergilli focusing on all the approaches made in the taxonomy of this group of fungi. Some species, such as A. carbonarius and uniseriate species can be easily recognised. In the A. niger aggregate, although speciation at molecular level has been proposed, no morphological differences can be observed and species identification will therefore remain problematic. Phylogenetic analyses of ITS and 5.8S rDNA gene region of representative black Aspergillus species and a simple key to the most common species that can be easily distinguished by morphological criteria are also included.     

Mycelial growth and ochratoxin A production by Aspergillus section Nigri on simulated grape medium in modified atmospheres

Aims:  To evaluate the impact of modified atmosphere packaging on in vitro growth of Aspergillus carbonarius and Aspergillus niger , and possible effects on ochratoxin A (OTA) biosynthesis.
Methods and Results:  Ochratoxigenic isolates belonging to the species A. carbonarius and A. niger were grown on a synthetic grapejuice medium (SNM) and packaged in combinations of controlled O₂ (1% and 5%) and CO₂ levels (0% and 15%), and in air as a control. Colony diameters were recorded every 3 days up to 21 days, and OTA was analysed after 7, 14 and 21 days. The greatest reductions in mycelial growth rate were observed at 1% O₂ followed by 1% O₂/15% CO₂, whereas 5% O₂ stimulated the growth of all isolates. OTA production by A. carbonarius and A. niger isolates was minimized at 1% O₂/15% CO₂ and 1% O₂, respectively, after 7 days of incubation. Maximal OTA accumulation after 7 days was observed for all isolates in the control pack and at 5% O₂.
Conclusions:  Of the atmospheres tested, only 1% O₂ combined with 15% CO₂ consistently reduced fungal growth and OTA synthesis by A. carbonarius and A. niger .
Significance and Impact of the Study:  Storage under modified atmospheres is unlikely to be suitable as the sole method for OTA minimization and grape preservation; other inhibitory factors are necessary.