White-rot fungi capable of decolourising textile dyes under alkaline conditions.

Twelve white-rot fungal strains belonging to seven different species were screened on plates under alkaline condition to study the decolourisation of the textile dyes Reactive Black 5 and Poly R-478. Three strains of Trametes versicolor (Micoteca da Universidade do Minho (MUM) 94.04, 04.100 and 04.101) and one strain of Phanerochaete chrysosporium (MUM 94.15) showed better decolourisation results. These four strains were used for decolourisation studies in liquid culture medium. All four selected strains presented more efficient decolourisation rates on Reactive Black 5 than on Poly R-478. For both dyes on solid and liquid culture media, the decolourisation capability exhibited by these strains depended on dye concentration and pH values of the media. Finally, the decolourisation of Reactive Black 5 by T. versicolor strains MUM 94.04 and 04.100 reached 100 %. In addition, the highest white-rot fungi ligninolytic enzyme activities were found for these two strains.

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.

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.

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.

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 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.

Fungi in bottled water: a case study of a production plant.

A one-year fungal survey of a water bottling plant was conducted in order to evaluate the incidence and fluctuations of the mycobiota. The dominant fungal genera in order of highest numbers isolated were Penicillium, Cladosporium and Trichoderma followed by Aspergillus, Paecilomyces, and others. As expected, the highest number of isolates were collected during the warmer months, particularly May and June. Indeed during these two months there were more fungi present in the water, indicating that during those times of the year when fungal contamination is high, 0.4 mm filters should be changed on a more regular basis. In order to assess whether contamination was single or multi-loci, molecular methods based on the PCR were used for Penicillium brevicompactum. Overall, fungal contamination arose from multiple sources. Some P. brevicompactum strains were very "alike" and were detected during different sampling times, indicating that they were endemic to the plant. There was no evidence to suggest that fungi detected in the source water passed through to other parts of the plant. However, there was evidence that fungal strains isolated from the water filter were detected elsewhere in the factory, confirming the need to change filters more regularly during periods of high fungal contamination. In order to improve quality control a HACCP programme was implemented and Best Practice Guidelines introduced.

Evolutionary relationships among Aspergillus terreus isolates and their relatives.

Aspergillus terreus is a ubiquitous fungus in our environment. It is an opportunistic human pathogen and economically important as the main producer of lovastatin, a cholesterol lowering drug. Our aim was to examine the genetic variability of A. terreus and closely related species using molecular and analytical techniques. Lovastatin production was examined by HPLC. Lovastatin was produced by seven isolates belonging to the species A. terreus. RAPD analyses were carried out using 25 different random primers. Neighbor-joining analysis of RAPD data (120 characters) resulted in clustering of the A. terreus isolates into distinct groups. Some correlation was observed between lovastatin producing abilities of the isolates and their position on the dendrogram based on RAPD profiles. The internal transcribed spacer region and the 5.8S rRNA gene of A. terreus and related isolates was also sequenced. Phylogenetic analysis of sequence data let us classify the isolates into different clades which mostly correspond to the species Aspergillus terreus, Aspergillus flavipes, Aspergillus niveus, Aspergillus carneus and Aspergillus janus/A. janus var. brevis. Aspergillus allahabadii, A. terreus var. aureus and A. niveus var. indicus belonged to the A. niveus clade, while an Aspergillus isolate previously classified as A. niveus was most closely related to A. flavipes isolates. Aspergillus anthodesmis formed a distinct branch on the tree. Although it was previously suggested based on 28S rDNA sequence data that Aspergillus section Terrei should include A. carneus and A. niveus isolates, phylogenetic analysis of ITS sequences indicate that A. flavipes isolates are more closely related to A. terreus than A. carneus isolates. Our data suggest that sections Terrei and Flavipedes should be merged. However, further loci should be analysed to draw more definite conclusions.

Use of ozone to reduce molds in a cheese ripening room.

Cheese ripening rooms have an unusual environment, an environment that encourages mold growth. Ozone has been applied in various ways in the food industry. One useful advantage of ozone is that it inactivates molds. In this study, a cheese ripening room was ozonated, and the effectiveness of this treatment was evaluated both in air and on surfaces through sampling on a weekly basis over a 3-month period. The results obtained indicate that ozone treatment reduced the viable airborne mold load but did not affect viable mold on surfaces. Only by wiping the surfaces with a commercial sanitizer was it possible to decrease the viable mold load on surfaces. To improve overall hygiene in the ripening room, a combination of cleaning regimes is recommended. The mold genera occurring most frequently in the air of the cheese ripening room were Penicillium, Cladosporium, and Aspergillus, which accounted for 89.9% of the mold isolates. Penicillium and Aspergillus were identified to the species level, and data showed that P. brevicompactum and P. aurantiogriseum, as well as A. versicolor, were the species most frequently isolated.

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 and evolutionary relationships among species of Aspergillus section Clavati.

Aspergillus clavatus 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.

Occurrence of ochratoxin A-producing fungi in grapes grown in Italy.

A study was carried out to investigate fungi present on grapes grown in Italy. Aspergillus and Penicillium spp. isolates were identified and studied in vitro, and their ability to produce ochratoxin A (OA) was investigated. The survey involved nine vineyards, three located in northern Italy and six located in southern Italy. In 1999 and 2000, bunches of grapes at different growth stages were collected from all nine vineyards, and berry samples were placed in moist chambers and incubated. The resultant fungal colonies were then transferred to petri dishes containing Czapek yeast agar and incubated at 25 degrees C for 7 days; the fungal isolates were identified and then cultivated in liquid Czapek yeast medium and evaluated for their ability to produce OA. During the survey, 508 isolates were collected, with 477 belonging to Aspergillus spp. and 31 belonging to Penicillium spp. Among the aspergilli, species of the Fumigati, Circumdati, and Nigri sections were identified, with species of the Nigri section (464 isolates) largely predominating; for species of the Nigri section, 108 isolates were uniseriate, 270 were biseriate, and 86 were identified as Aspergillus carbonarius. Black aspergilli isolated over the 2 years of the study showed a very similar pattern. On average, the biseriates represented about 60% of the isolates collected in both years and were followed by uniseriates (21%) and A. carbonarius (19%). The most toxigenic strains proved to be those of A. carbonarius; about 60% of these isolates were OA producers and produced the highest levels of OA. A. carbonarius was more frequent in the south, but in both areas the percentages of OA-producing isolates remained the same.

Evolutionary relationships within Aspergillus section Flavi based on sequences of the intergenic transcribed spacer regions and the 5.8S rRNA gene.

Sequences of the intergenic transcribed spacer regions and the 5.8S rRNA gene (455 nucleotides) of type strains or representative isolates of 23 species and subspecies either currently assigned to Aspergillus subgenus Circumdati section Flavi or other closely related sections, were analyzed. Parsimony analysis of sequence data indicated that species of Aspergillus section Flavi form distinct clades. The three main clades identified based on sequence data could also be distinguished based on colony color, and their ubiquinone systems. The 'A. flavus' clade includes species characterized with Q-10(H(2)) as their main ubiquinone, conidial colors in shades of green, and dark sclerotia. The 'A. tamarii' clade involves species with ubiquinone system Q-10(H(2)), and conidia in shades of olive to brown, while the 'A. alliaceus' clade consists of species with Q-10 ubiquinone system, and conidia in shades of ocher. The synnematous species A. coremiiformis was found to be closely related to species in the 'A. tamarii' clade. A. thomii and A. terricola var. americana were found to be related to the 'A. flavus' clade in spite of producing brownish colonies. Three species, A. nomius, A. avenaceus, and A. leporis were found to form separate lineages not closely related to any of the main clades identified. It is suggested that A. clavatoflavus and A. zonatus be excluded from Aspergillus section Flavi. Phylogenetic analysis of partial 26S rRNA gene sequences (564 nucleotides) supported our findings.