Molecular analysis of variability within the toxigenic Aspergillus ochraceus species

Genetic variability of Aspergillus ochraceus was examined at the DNA level. Based on the HaeIII-Bg/II generated mitochondrial DNA restriction profiles, most isolates could be classified into two distinct groups. These two groups could also be distinguished by the random amplified polymorphic DNA technique, and with telomeric PCR amplifications. Phylogenetic analysis of sequences of the intergenic transcribed spacer region of some of the strains resulted in a dendrogram with the same topology as that based on mitochondrial DNA and amplified DNA data. None of the isolates with type 2 mtDNA profiles produce ochratoxins. Some strains (e.g., A. ochraceus ICMP 939) displayed strain-specific mitochondrial DNA patterns, and their amplified DNA profiles were also different from all other A. ochraceus strains examined.     

Phylogenetic analysis of Aspergillus section Circumdati based on sequences of the internal transcribed spacer regions and the 5.8 S rRNA gene

Phenotypic features and sequences of the internal transcribed spacer (ITS) regions and the 5.8 S rRNA gene of type or neotype strains and other isolates of the 17 species currently assigned to Aspergillus section Circumdati and some potentially related species were analyzed. Parsimony analysis of sequence data indicated that Aspergillus section Circumdati is paraphyletic. Aspergillus campestris, A. lanosus, and A. dimorphicus with A. sepultus were found to be more closely related to Aspergillus sections Candidi, Flavi, and Cremei, respectively. These results were also supported by phenotypic data. A. robustus and A. ochraceoroseus were found not to be related to any of the species examined. Species of the proposed revised Aspergillus section Circumdati formed two main clades, which could also be distinguished based on phenotypic methods. Phylogenetic analysis of sequence data of other isolates assigned to species of the revised section indicates that either some of these isolates were misidentified or species concepts of A. ochraceus, A. melleus, and A. petrakii should be reconsidered.     

Production of Ochratoxins A and B on Country Cured Ham

Two strains of Aspergillus ochraceus and six of Penicillium viridicatum isolated from country cured hams were screened for production of ochratoxins A and B. None of the isolated P. viridicatum strains yielded detectable amounts of ochratoxin A or B, whereas both strains of A. ochraceus produced ochratoxins A and B on rice, defatted peanut meal, and country cured ham. After 21 days of incubation on ham, one-third of the toxin was found in the mycelial mat on the ham surface, whereas two-thirds had penetrated into the meat to a distance of 0.5 cm.     

Utilization of AFLP markers for PCR-based identification of Aspergillus carbonarius and indication of its presence in green coffee samples

AIMS: The objective of this work was to test whether ochratoxin A (OTA) production of Aspergillus niger and A. carbonarius is linked to a certain genotype and to identify marker sequences with diagnostic value aiding identification of A. carbonarius, a fungus of major concern regarding OTA production in food and food raw materials. METHODS AND RESULTS: Aspergillus niger and A. carbonarius were isolated mainly from Brazilian coffee sources. The ability of isolates to produce OTA was tested by thin layer chromatography (TLC). Strains were genetically characterized by AFLP fingerprinting and compared with each other and with reference strains. Cluster analysis of fingerprints showed clear separation of A. niger from A. carbonarius strains. To obtain marker sequences, AFLP fragments were isolated from silver stained polyacrylamide gels, cloned and sequenced. Sequences obtained were used to develop species- specific PCR primers for the identification of A. carbonarius in pure culture and in artificially and naturally infected samples of green coffee. CONCLUSIONS: No clear correlation between genetic similarity of the strains studied and their potential to produce OTA was found. The PCR assays designed are a useful and specific tool for identification and highly sensitive detection of A. carbonarius. SIGNIFICANCE AND IMPACT OF THE STUDY: The developed PCR assays allow specific and sensitive detection and identification of A. carbonarius, a fungus considered to be one of the major causative agents for OTA in coffee and grape-derived products. Assays may provide powerful tools to improve quality control and consumer safety in the food processing industry.     

Genetic relationships among Brazilian strains of Aspergillus ochraceus based on RAPD and ITS sequences

Ochratoxin A (OA) is a mycotoxin that has been found in coffee beans and coffee beverages. Its toxicological profile includes carcinogenicity, nephrotoxicity, and immunotoxicity. Aspergillus ochraceus is the major species responsible for OA production in Brazilian coffee beans. The genetic relationships among 25 A. ochraceus strains collected from Brazilian coffee-bean samples were determined based on RAPD and internal transcribed spacer (ITS) sequence data. The isolates were resolved into 2 distinct groups, one with 4 strains (group A) and the other with 21 strains (group B). Specific nucleotide variations characterizing group A and B were found for both ITS1 and ITS2 regions. Group B is a new group proposed here to accommodate the majority of the Brazilian isolates. Each group was found to contain both toxigenic and nontoxigenic strains, indicating that there is no association between molecular genotypes and the ability to produce OA.     

Cytotoxicity of Aspergillus fungi isolated from hospital environment

The majority of mycotoxins produced by Aspergillus fungi are immunosuppressive agents, and their cytotoxicity may impair defense mechanisms in humans. The objective of the study was evaluation of the cytotoxicity of fungi isolated from an environment where inpatients with impaired immunity were present. The materials comprised 57 fungal strains: Aspergillus fumigatus, Aspergillus niger: Aspergillus ochraceus, Aspergillus flavus, Aspergillus versicolor and Aspergillus ustus isolated from hospital rooms in Cracow. The cytotoxicity of all the strains was evaluated using the MTT test (3-(4,5-dimethylthiazol-2-yl) 2,5 diphenyltetrazolium bromide). To emphasize the differences in cytotoxicity among the particular strains, variance analysis (ANOVA) and Tukey's difference test were used. Out of 57 Aspergillus strains tested, 48 (84%) turned out to be cytotoxic. The cytyotoxicity was high (+++) in 21 strains, mainly in A. fumigatus. The least cytotoxic were A. niger fungi, this being statistically significant (p<0,05). To protect a patient from the adverse effects of mycotoxins, not only his or her immunity status should be evaluated but also the presence of fungi in hospital environment and their cytotoxicity should be monitored (possible exposure).     

Production of Ochratoxin A by Aspergillus ochraceus Isolated in Japan from Moldy Rice

A simple thin-layer chromatography-fluorodensitometric method for quantitative analysis of ochratoxin A was developed. This method proved to be of use in investigating the production of the toxin and the nutritional factors affecting the toxin production by two strains of Aspergillus ochraceus isolated from moldy rice in Japan. These fungi produced large amounts of ochratoxin A in a nutrient solution containing 1% l-phenylalanine and 2% yeast extract.     

Ochratoxin production by Aspergillus species.

Ochratoxin production was tested in 172 strains representing species in sections Fumigati, Circumdati, Candidi, and Wentii of the genus Aspergillus by an immunochemical method using a monoclonal antibody preparation against ochratoxin A. Ochratoxin A was detected in Aspergillus ochraceus, A. alliaceus, A. sclerotiorum, A. sulphureus, A. albertensis, A. auricomus, and A. wentii strains. This is the first report of production of ochratoxins in the latter three species. Ochratoxin production by these species was confirmed by high-performance thin-layer chromatography and by high-performance liquid chromatography. The chemical methods also indicated the production of ochratoxin B by all of the Aspergillus strains mentioned above.     

Evidence of RIP (repeat-induced point mutation) in transposase sequences of Aspergillus oryzae

A DNA methyl-binding column was used to isolate genomic fragments enriched for DNA-methylation from Aspergillus parasiticus. One of the isolated sequences presented 67% identity at the protein level with the transposase from the transposable element Tan1 of Aspergillus niger var. awamori, and was found to be present in at least 20 copies in the Aspergillus oryzae database. Analysis of four copies showed evidence of C:G to T:A transitions in at least 98.2% of the mutations found over a 1,032-1,180 bp region spanning a large part of the transposase sequence. Using copy specific primers three sequences were amplified from a different strain of A. oryzae and a similar pattern of C:G to T:A transitions was found. These transitions are similar to those observed in RIP, in Neurospora crassa, where cytosine-methylation is believed to be involved. Using methylation-sensitive Southern blotting, no evidence of methylation was found in the transposase sequences in these two A. oryzae strains as well as one A. parasiticus and one Aspergillus flavus strain.     

Curvature of mouse satellite DNA and condensation of heterochromatin

Cloned, sequenced mouse satellite DNA exhibits properties characteristic of molecules that possess a stable curvature. Circularly permuted fragments containing the region predicted to bend were used to map the curvature relative to DNA sequence. The altered mobility of these fragments in polyacrylamide gels is reversed when gels are run in the presence of distamycin A, a drug that binds preferentially to AT-rich DNA. Treatment of living mouse cells with this drug dramatically reduces the condensation of centromeric heterochromatin, the exclusive location of satellite sequences. In situ hybridization of satellite probes to extended chromosomes at the electron microscope level shows that satellite does not comprise a single block but is distributed throughout the centromere region. Based on these experiments, we hypothesize that the structure of mouse satellite DNA is an important feature of centromeric heterochromatin condensation.     

Localization of pyruvate carboxylase in organic acid-producing Aspergillus strains.

The localization of pyruvate carboxylase (cytosolic or mitochondrial) was studied in nine different Aspergillus species (14 strains). In some species (A. aculeatus, A. flavus, A. foetidus, A. nidulans, A. ochraceus, and A. sojae), the pyruvate carboxylase activity could be detected only in the cytosolic fraction of the cells. Pyruvate carboxylase has been found only in the mitochondrial fraction of two strains of Aspergillus wentii. In Aspergillus oryzae and in five strains of Aspergillus niger, pyruvate carboxylase activity was detected both in the mitochondrial fraction and in the cytosol. There was no quantitative or qualitative correlation between the activities of pyruvate carboxylase in the mitochondrial and cytosolic fractions of the cells and the ability of the various Aspergillus strains to accumulate different organic acids.     

Detection, sequence patterns and function of unusual DNA structures.

Unusual DNA structures were detected by an electrophoretic procedure in which DNA fragments were separated according to size on agarose gels and then by shape on polyacrylamide gels. Fragments from yeast centromeres migrated faster in polyacrylamide than predicted from their base composition and size and this property was attributed to a nonrandom distribution of oligomeric A tracts that exhibited minima at 10-11 base intervals. Fragments from seven loci in 107 kb of DNA migrated anomalously slow and these fragments contained blocks of A2-6 in a 10-11 base periodicity which is indicative of bent DNA. The most pronounced bent sequences were found within yeast ARS1 and centered at 245 and 240 bp from the left and right ends of the adenovirus genome. Each sequence is approximately 150 bp away from a replication origin and the adenovirus sequences are within 50 bp of enhancers. Nuclear matrix attachment sites, which are also adjacent to enhancers, contain sequences characteristic of bent DNA. These results suggest that bent structures reside at the base of DNA loops in chromosomes.     

Immunochemical identification of fungi using polyclonal antibodies raised in rabbits to exoantigens from Aspergillus ochraceus

Aspergillus ochraceus Wilhelm is a producer of highly toxic mycotoxin, ochratoxin A, and is known to contribute to the damage of grain on storage. Rabbit antibodies were produced against the exoantigens of A. ochraceus , and ELISA and immunoblotting procedures were used to characterize their reactivity towards fungal exoantigens of four Aspergillus , seven Penicillium , three Fusarium , one Mucor and one Alternaria species. The antibodies were specific for A. ochraceus and essentially cross-reacted neither with other fungi nor with the water-soluble grain components. These antibodies, therefore, have the potential of being utilized for the discriminative monitoring of A. ochraceus in foodstuffs and feeds.     

Localization of pyruvate carboxylase in organic acid-producing Aspergillus strains

The localization of pyruvate carboxylase (cytosolic or mitochondrial) was studied in nine different Aspergillus species (14 strains). In some species (A. aculeatus, A. flavus, A. foetidus, A. nidulans, A. ochraceus, and A. sojae), the pyruvate carboxylase activity could be detected only in the cytosolic fraction of the cells. Pyruvate carboxylase has been found only in the mitochondrial fraction of two strains of Aspergillus wentii. In Aspergillus oryzae and in five strains of Aspergillus niger, pyruvate carboxylase activity was detected both in the mitochondrial fraction and in the cytosol. There was no quantitative or qualitative correlation between the activities of pyruvate carboxylase in the mitochondrial and cytosolic fractions of the cells and the ability of the various Aspergillus strains to accumulate different organic acids.     

Restriction endonuclease analysis of the genomes of virulent and avirulent Marek's disease viruses

The DNAs from virulent strain BC-1 and avirulent strains C2(A) of Marek's disease virus (MDV) were compared by electrophoresis on 0.5% agarose gels of the products obtained with the restriction endonucleases Bam H1, Sal 1, and Sma 1. The patterns of the fragments of the DNAs from these two strains were very similar, but showed some significant differences in the number and mobility of the DNA bands. The DNA fragments obtained with the restriction endonucleases, and especially Sma 1, were mostly present in equal molar ratios, but a few of those obtained with Bam H1 and Sal 1 were present in submolar amounts. In addition, several fragments obtained with Bam H1 and Sal 1 were present in greater than molar quantities, suggesting the presence of reiterated sequences in MDV DNA. The terminal fragments of MDV DNA were identified by their sensitivity to lambda 5'-exonuclease. The terminal fragments obtained with Bam H1 A and Sal 1B showed heterogeneous electrophoretic mobility and contained sequences with high content of guanosine and cytosine, suggesting the presence of reiterated sequences at the end of the MDV DNA molecule.     

Preparations of extracellular proteinases from Aspergillus ochraceus 513 and Aspergillus alliaceus 7dN1

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.     

Detection and quantification of Aspergillus ochraceus in green coffee by PCR

AIMS: The aim of this study was to detect and quantify DNA of the ochratoxinogenic fungus Aspergillus ochraceus in green coffee and to compare the results with the ochratoxin A content of naturally contaminated samples. METHODS AND RESULTS: A DNA extraction protocol based on a combination of ultrasonification and a commercial kit was tested for the recovery of fungal DNA. PCR and real-time PCR protocols were established for the detection of A. ochraceus. Sensitivity of the PCR was checked by the addition of inoculated green coffee and pure fungal DNA to uncontaminated green coffee samples. The A. ochraceus DNA content of 30 naturally contaminated green coffee samples was determined and compared with the ochratoxin A concentrations. CONCLUSIONS: Aspergillus ochraceus can be rapidly and specifically detected in green coffee by PCR. A positive correlation between the ochratoxin A content and the DNA quantity was established. Significance and Impact of the Study: This work offers a quick alternative to the conventional mycological detection and quantification of A. ochraceus in green coffee.     

Mycoflora and mycotoxin-producing fungi of air-dust particles from Egypt

Using the dilution-plate method, 27 genera and 64 species were collected from 20 air-dust samples on glucose — (24 genera and 57 species) and cellulose — (21 genera and 45 species) Czapek's agar at 28 °C. There are basic similarities between the mycoflora of air-dust on the two media and the most prevalent species were Aspergillus niger, A. flavus, A. ochraceus, A. terreus, A. versicolor, Penicillium chrysogenum, P. funiculosum, Alternaria alternata, Cladosporium herbarum, Fusarium oxysporum, Rhizopus stolonifer and Trichoderma viride. Chaetomium globosum, Stachybotrys chartarum, Humicola grisea and Arthrobotrys oligospora were common only on cellulose agar plates.Extracts of mycelium from 25 isolates were tested with brine schrimp (Artemia salina); of these 23 displayed varying degrees of toxicity. Thin layer Chromatographic analysis of 12 isolates of Aspergillus flavus revealed that 4 strains were producing detectable aflatoxin. Zearalenone production was noted for 3 out of 5 strains of Fusarium oxysporum and 2 out of 5 strains of F. solani.     

我国部分地区自然发酵豆酱中的曲霉菌及其有性型

Twenty species of Aspergillus and their teleomorphs were isolated from naturally fermented soybean pastes collected in 42 regions of China. Of which A. tubingensis, A. flavus, A. niger, A. ochraceus, A. candidus and A. fumigatus were predominant. The high frequency of A. flavus from the samples indicates that the traditional making method of soybean paste might possibly be harmful to food safety. Some Aspergillus species or strains isolated from the traditional food samples may be hopeful of using in pure leaven making and industrial production of soybean paste with special flavor.     

我国部分地区自然发酵豆酱中的曲霉菌及其有性型

从我国42个地区的自然发酵酱醅和酱醪样品中分离得到曲霉Aspergillus及其有性型真菌20种,其中优势曲霉菌种类为A.tubingensis、A.flavus、A.niger、A.ochraceus、A.candidus和A.fumigatus.调查研究发现,从酱醅和酱醪样品中分离出黄曲霉的频率较高,表明自然发酵豆酱存在一定的安全隐患.研究中分离获得的曲霉菌株有望用于纯种制曲或特殊风味豆酱的工业化生产.