Zofia Kozakiewicz

A polyphasic approach to the identification of aflatoxigenic and non-aflatoxigenic strains of Aspergillus Section Flavi isolated from Portuguese almonds

A polyphasic approach consisting of morphological, chemical and molecular characterization was applied to 31 isolates of Aspergillus Section Flavi originating from Portuguese almonds, with the aim of characterizing and identifying aflatoxigenic and non-aflatoxigenic strains. On the basis of morphological characters (mainly colony color on Czapek-Dox agar and conidia morphology), we found two distinct groups among the population under study: 18 isolates (58%) had dark-green colonies and rough conidia, and were classified as Aspergillus parasiticus; the remaining 13 isolates (42%) had yellow-green colonies and smooth to finely rough globose conidia, and were classified as Aspergillus flavus. Chemical characterization involved the screening of the isolates for aflatoxins B (AFB) and G (AFG), and also for cyclopiazonic acid (CPA), by HPLC with fluorescence and UV detection, respectively. All A. parasiticus isolates were strong AFB and AFG producers, but no CPA production was detected, showing a consistent mycotoxigenic pattern. The A. flavus isolates showed to be more diversified, with 77% being atoxigenic, whereas 15% produced CPA and low levels of AFB and 8% produced the 3 groups of mycotoxins. Aflatoxin production was also screened on Coconut Agar Medium (CAM), and the results were consistent with the HPLC analysis. Sclerotia production showed no correlation to aflatoxigenicity. Molecularly, two genes of the aflatoxin biosynthetic pathway, aflD (=nor1) and aflQ (=ord1=ordA) were tested for presence and expression (by PCR and RT-PCR, respectively). The presence of both genes did not correlate with aflatoxigenicity. aflD expression was not considered a good marker for differentiating aflatoxigenic from non-aflatoxigenic isolates, but aflQ showed a good correlation between expression and aflatoxin-production ability.

Black Aspergillus species as ochratoxin A producers in Portuguese wine grapes

To evaluate the incidence of fungi producing ochratoxin A (OA) in Portuguese wine grapes, a survey was conducted in 11 vineyards, from four winemaking regions each with distinct climatic conditions. From setting to the harvesting period, a total of 1,650 berries were sampled by plating methods. Out of 370 aspergilli and 301 Penicillium strains isolated, 14% of the aspergilli were OA-producing strains. None of the penicillia were OA-producing strains. The black aspergilli were predominant (90%). All Aspergillus strains were tested in vitro for OA production and all were preserved in the Micoteca da Universidade do Minho (MUM) culture collection. Most of the Aspergillus carbonarius (97%) and 4% of the Aspergillus niger aggregate strains were OA producers. Almost all ochratoxigenic strains were isolated at harvest time, mainly in the regions with a Mediterranean climate. In the vineyards sampled, the percentage of colonized berries with ochratoxigenic strains was up to 38%. The vineyards from the region with Atlantic influences, with high rainfall, exhibited the lowest occurrence of Aspergillus and ochratoxigenic strains, 0% to 10% and 0% to 2% colonized berries, respectively. Data obtained here supports the hypothesis that A. carbonarius and occasionally A. niger, are the main producers of OA in grapes. In this study, the highest incidence of these fungi occurred in vineyards with a Mediterranean climate.

Mycotoxin production from fungi isolated from grapes

Aims: In order to assess the potential for producing mycotoxins, fungi were isolated from wine producing grapes.

Mycoflora and mycotoxins in Brazilian black pepper, white pepper and Brazil nuts.

A wide range of field and storage fungi were isolated from black pepper, white pepper and Brazil nut kernels from Amazonia. A total of 42 species were isolated from both peppers. Aspergillus flavus and A. niger were isolated more frequently from black than from white pepper. Other potential mycotoxigenic species isolated included: A. ochraceus, A. tamarii, A. versicolor, Emericella nidulans and Chaetomium globosum, Penicillium brevicompactum, P. citrinum, P. islandicum and P. glabrum. Species isolated from pepper for the first time were Acrogenospora sphaerocephala, Cylindrocarpon lichenicola, Lacellinopsis sacchari, Microascus cinereus, Petriella setifera and Sporormiella minima. Seventeen species were isolated from Brazil nut kernels. A. flavus was the dominant species followed by A. niger.P. citrinum and P. glabrum were the only penicillia isolated. Species isolated for the first time included Acremonium curvulum, Cunninghamella elegans, Exophiala sp., Fusarium oxysporum, Pseudoallescheria boydii, Rhizopusoryzae, Scopulariopsis sp., Thielavia terricola and Trichoderma citrinoviride. Considerably more metabolites were detected from black than white pepper in qualitative analyses. Chaetocin, penitrem A, and xanthocillin were identified only from black pepper, and tenuazonic acid was identified from both black and white pepper. Aflatoxin G2, chaetoglobosin C, and spinulosin were identified from poor quality brazil nuts. Aflatoxin B1 and B2 were also only detected in poor quality brazil nuts at concentrations of 27.1 μg kg−1 and 2.1 μg kg−1 respectively (total 29.2 μg kg−1).

Restriction fragment length polymorphisms in the mitochondrial DNAs of the Aspergillus niger aggregate

Restriction fragment length polymorphisms (RFLPs) were detected in the mitochondrial and nuclear DNAs of isolates belonging to the Aspergillus niger aggregate. These strains can be classified into two well-defined groups according to the electrophoretic patterns of their mtDNAs obtained with some restriction enzymes, such as EcoR I, Pvu II, Hae III and Bgl II. Strains belonging to both groups show some further minor variation. Estimates of the molecular weight of these mitochondrial DNAs were between 30 and 32·5 kb. The patterns produced by the nuclear ribosomal DNA repeats in digests of total DNA made it possible to classify the black Aspergillus strains into two groups, each further subdivided into two. The two main groups established from the mitochondrial and ribosomal DNA patterns coincide with each other. A rapid and easy method was also worked out to classify large numbers of field isolates of black aspergilli into one of the groups described using mtDNA patterns obtained by the double-digestion of total DNA samples with Hae III and Bgl II.

Penicillium populations in dry-cured ham manufacturing plants

Seven ham manufacturing plants were sampled for 1 year to assess the mycoflora present in the air and on hams, with special attention given to potential mycotoxin producers. Temperature and relative humidity were recorded in the ripening rooms. Maturing rooms held hams from 2 to 3 through 6 to 7 ripening months, and aging rooms held hams for the following 6 to 7 months, until the 14-month ripening point, when they were ready for the market. Mean temperatures and relative humidities registered during the study were 14.9 degrees C and 62.4%, respectively, in maturing rooms and 16.3 degrees C and 57.6% in aging rooms. Aspergilli and penicillia, potential mycotoxin producers, were isolated in all the plants from the air and the ham. Aspergilli represented 5% of the isolates, while penicillia were largely dominant, with Penicillium nalgiovense being the most represented species (around 60% of the penicillia), followed by Penicillium nordicum, with 10 and 26% of the penicillia isolated, respectively, from the air or the ham. Ochratoxin A production ability, checked in vitro at 250C, was observed in 50% of the P. nordicum isolates obtained both from the air and the ham. Air and ham surface contamination by penicillia was greater in the ripening rooms, where higher temperatures were registered. A certain correlation was also observed between air and ham surface contamination. On the basis of this study, P. nordicum, the ochratoxin A producer that is notable on proteinaceous substrates, is normally present in ham manufacturing plants in Italy, even though not a dominant species. Further studies are necessary to clarify and ensure if dry-curing conditions minimize the potential risk of ochratoxin A formation in the product.

Aspergillus uvarum sp nov., an uniseriate black Aspergillus species isolated from grapes in Europe

A novel species, Aspergillus uvarum sp. nov., is described within Aspergillus section Nigri. This species can be distinguished from other black aspergilli based on internal transcribed spacers (ITS), beta-tubulin and calmodulin gene sequences, by AFLP analysis and by extrolite profiles. Aspergillus uvarum sp. nov. isolates produced secalonic acid, common to other Aspergillus japonicus-related taxa, and geodin, erdin and dihydrogeodin, which are not produced by any other black aspergilli. None of the isolates were found to produce ochratoxin A. The novel species is most closely related to two atypical strains of Aspergillus aculeatus, CBS 114.80 and CBS 620.78, and was isolated from grape berries in Portugal, Italy, France, Israel, Greece and Spain. The type strain of Aspergillus uvarum sp. nov. is IMI 388523T=CBS 127591T=ITEM 4834T=IBT26606T.

Molecular Diversity of Agriculturally Important Aspergillus Species

Although Aspergillus species are not usually considered as serious plant pathogens, Aspergilli are frequently encountered in plant products. The most important consequence of their presence is mycotoxin contamination. The main mycotoxins produced by Aspergilli are the aflatoxins, ochratoxin A and patulin, which are produced by a variety of Aspergillus species in different plant commodities. Phylogenetic analysis of sequences of the ribosomal RNA gene cluster is useful for clarifying taxonomic relationships among toxigenic Aspergilli causing pre- and postharvest contamination of agricultural products. Molecular data has enabled us to clarify the taxonomy of black Aspergilli, A. flavus and its relatives, and sections Circumdati and Clavati, which include ochratoxin and patulin-producing species. Phylogenetically unrelated species were found to produce the same mycotoxins, indicating that mycotoxin-producing abilities of the isolates have been lost (or gained) several times during the evolution of the genus. The data also indicate that biosynthetic gene-based probes are necessary for molecular detection of these mycotoxin-producing organisms. The organisation of the biosynthetic genes of patulin and ochratoxins is unknown, although experiments are in progress in several laboratories to clarify the genetic background of biosynthesis of these mycotoxins. Identification of biosynthetic genes responsible for mycotoxin production is essential for clarifying the evolution of mycotoxin biosynthesis in Aspergilli, and to develop specific gene probes for the detection of mycotoxin-producing Aspergilli in agricultural products.

Fourier transform infrared as a powerful technique for the identification and characterization of filamentous fungi and yeasts

Fourier transform infrared is considered a powerful technique for characterizing chemical compositions of complex probes such as microorganisms. It has successfully been applied to fungal identification. In this paper, the current state of identification and characterization of filamentous fungi and yeasts by Fourier transform infrared is reviewed.

Mycotoxin production and evolutionary relationships among species of Aspergillus section Clavati.

Aspergillusclavatus is a commonly encountered fungus in the environment, producing a number of mycotoxins including patulin, kojic acid, cytochalasins and tremorgenic mycotoxins. A. clavatus belongs to Aspergillus section Clavati together with six other species, all of which possess clavate-shaped vesicles. Patulin production was analysed by thin layer chromatography and high performance liquid chromatography, while a primer pair developed for the detection of an iso-epoxydon dehydrogenase gene involved in the biosynthesis of patulin in penicillia was used to detect the ability of patulin production in the isolates examined. A good correlation was observed between patulin producing properties, and the presence of an iso-epoxydon dehydrogenase gene fragment among the isolates tested. A. longivesica was found for the first time to produce patulin. Ribotoxin production was also examined using a PCR-based approach. Ribotoxins were detected for the first time in an A. pallidus and a Hemicarpenteles acanthosporus isolate. A phylogenetic analysis of intergenic transcribed spacer sequence data indicated that most isolates belong to two main clades that have also been identified earlier based on 26 S rDNA sequence data. A. pallidus isolates clustered together with A. clavatus strains. Although A. clavatus isolates produced highly homogeneous random amplified polymorphic DNA profiles, phylogenetic analysis of these data let us cluster A. clavatus isolates into distinct clades. Correlations were not observed between either patulin or ribotoxin production, and the taxonomic position of the isolates tested, indicating that patulin and ribotoxin producing abilities were lost several times during evolution of Aspergillus section Clavati. Although patulin was earlier found to inhibit mycovirus replication, one of the mycovirus carrying isolates also produced patulin, and both carried the iso-epoxydon dehydrogenase gene.