Section Circumdati Research Articles

Assessment of toxigenic fungi on Argentinean medicinal herbs

This work was performed to determine the incidence of toxigenic fungi and their mycotoxins on 152 dried medicinal and aromatic herbs, belonging to 56 species, which are used as raw material for drugs. International methodologies for fungal enumeration and identification were applied as well as TLC and HPLC techniques for toxins detection. The 52% out of 152 samples were contaminated with species from Aspergillus genus, 27% belonging to the Flavi section and 25% to the Circumdati section. The 16% of the total samples was contaminated with species from Fusarium genus. Aspergillus flavus and A. parasiticus ( Flavi section), were the predominant species isolated, 50% out of 40 isolates were toxigenic. Aflatoxin concentrations ranged from 10 to 2000 ng/g. Only 26% of isolates from the Circumdati section ( A. alliaceus, A. ochraceus and A. sclerotiorum) produced ochratoxin A in low concentrations between 0.12 and 9 ng/g. From a total of 29 strains of Fusarium spp., 27.5% were Fusarium verticillioides and F. proliferatum, which produced fumonisin B1and fumonisin B2 ranged from 20 to 22000 μg/g and from 5 to 3000 μg/g respectively. The remaining species, F. equiseti, F. oxysporum, F. semitectum, F. compactum, F. sambucinum and F. solani were able to produce neither group A and B trichothecenes nor zearalenone. The incidence of A. ochraceus and Fusarium spp. and their toxigenic capacities on medicinal herbs were studied for the first time in Argentina. It would be important to look for natural contamination to define acceptability limits which allow the control of sanitary quality of medicinal herbs used as phytotherapic medicines in several countries.

Occurrence of ochratoxin A and toxigenic potential of fungal isolates from Spanish grapes

AbstractThis paper reports the results from an extensive survey of filamentous fungi isolated from wine‐producing grapes and their capacity to produce ochratoxin A (OTA) on Czapek Yeast Autolysate agar (CYA), in order to assess their potential for producing this toxin in grapes. Grapes were sampled from four Spanish wine‐producing regions during 2001. The fungal infection in berries increased with time, reaching 100% at harvest. A total of 386 isolates of Aspergillus section Nigri and 10 of Aspergillus section Circumdati were isolated and tested for their ability to produce OTA in CYA. 21 strains produced OTA (18 Aspergillus section Nigri and 3 Aspergillus section Circumdati). Aspergillus section Circumdati isolates produced higher amounts of OTA than Aspergillus section Nigri ones, with means of 10.76 µg g−1 CYA and 1.42 µg g−1 CYA, respectively. Despite this, black aspergilli are believed to be highly responsible for the OTA levels found in musts and wines, as it is more widespread in grapes. Musts (n = 40) produced from the grapes collected were analysed. 15% were found to contain OTA, concentrations ranging from 0.091 to 0.813 ng ml−1 (detection limit: 0.07 ng ml−1), but no correlation was found with the ochratoxigenic moulds isolated from grapes. Copyright © 2004 Society of Chemical Industry

Phylogenetic and morphological analysis of Aspergillus ochraceoroseus

Aspergillus ochraceoroseus produces the yellow-gold conidia and other characteristics of Aspergillus subgenus Circumdati section Circumdati. However, this species produces aflatoxin, a secondary metabolite characteristic of some members of subgenus Circumdati section Flavi and sterigmatocystin, a related secondary metabolite usually associated with subgenus Nidulantes sections Nidulantes and Versicolores, as well as members of several other genera. Our morphological data support the placement of A. ochraceoroseus in subgenus Circumdati. Sequence data from A. ochraceoroseus aflatoxin and sterigmatocystin genes aflR and nor-1/stcE, as well as 5.8S ITS and beta tubulin genes, were compared to those of aspergilli in sections Circumdati, Flavi, Nidulantes and Versicolores. In the sequence comparisons, A. ochraceoroseus was related more closely to the species in subgenus Nidulantes than to species from subgenus Circumdati.

Phylogenetic and Morphological Analysis of Aspergillus ochraceoroseus

Aspergillus ochraceoroseus produces the yellow-gold conidia and other characteristics of Aspergillus subgenus Circumdati section Circumdati. However, this species produces aflatoxin, a secondary metabolite characteristic of some members of subgenus Circumdati section Flavi and sterigmatocystin, a related secondary metabolite usually associated with subgenus Nidulantes sections Nidulantes and Versicolores, as well as members of several other genera. Our morphological data support the placement of A. ochraceoroseus in subgenus Circumdati. Sequence data from A. ochraceoroseus aflatoxin and sterigmatocystin genes aflR and nor-1/stcE, as well as 5.8S ITS and beta tubulin genes, were compared to those of aspergilli in sections Circumdati, Flavi, Nidulantes and Versicolores. In the sequence comparisons, A. ochraceoroseus was related more closely to the species in subgenus Nidulantes than to species from subgenus Circumdati.

Toxigenic fungi associated with processed (green) coffee beans ( Coffea arabica L.)

Processed (green) coffee beans from Coffea arabica in Brazil were assessed for the presence of Aspergillus and Penicillium species both before and after surface sterilisation, the aflatoxigenic and ochratoxigenic potential of the isolates and ochratoxin A levels. Contamination by Aspergillus and Penicillium species was found on 96% and 42%, respectively, of 45 samples from 11 localities. After disinfection with 1% sodium hypochlorite, the levels fell to 47% and 24%, respectively. One hundred and eighty isolates were identified to species level and comprised Aspergillus sections Circumdati (10 species), Flavi (3), Nigri (3), Versicolores (4), while two were teleomorphic species. Eight species of Penicillium were isolated. Within section Circumdati, 75% of the isolates produced ochratoxin A and all except Aspergillus elegans and Aspergillus insulicola have previously been reported to produce ochratoxin A. One-third of the 18 isolates of Aspergillus flavus produced aflatoxin B 1 and B 2. None of the isolates belonging to Aspergillus section Nigri or Penicillium produced ochratoxin A. Of the 40 bean samples analysed, 58% were infected with potentially ochratoxigenic fungi but only 22% of these were contaminated with ochratoxin A at levels that varied from 0.47 to 4.82 ng/g, with an average contamination level of 2.45 ng/g.

Ochratoxin production by the Aspergillus ochraceus group and Aspergillus alliaceus.

Ochratoxin A is a toxic and carcinogenic fungal secondary metabolite; its presence in foods is increasingly regulated. Various fungi are known to produce ochratoxins, but it is not known which species produce ochratoxins consistently and which species cause ochratoxin contamination of various crops. We isolated fungi in the Aspergillus ochraceus group (section Circumdati) and Aspergillus alliaceus from tree nut orchards, nuts, and figs in California. A total of 72 isolates were grown in potato dextrose broth and yeast extract-sucrose broth for 10 days at 30 degrees C and tested for production of ochratoxin A in vitro by high-pressure liquid chromatography. Among isolates from California figs, tree nuts, and orchards, A. ochraceus and Aspergillus melleus were the most common species. No field isolates of A. ochraceus or A. melleus produced ochratoxin A above the level of detection (0.01 microg/ml). All A. alliaceus isolates produced ochratoxin A, up to 30 microg/ml. We examined 50,000 figs for fungal infections and measured ochratoxin content in figs with visible fungal colonies. Pooled figs infected with A. alliaceus contained ochratoxin A, figs infected with the A. ochraceus group had little or none, and figs infected with Penicillium had none. These results suggest that the little-known species A. alliaceus is an important ochratoxin-producing fungus in California and that it may be responsible for the ochratoxin contamination occasionally observed in figs.

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.

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.

Aflatoxin and cyclopiazonic acid production by a sclerotium-producing Aspergillus tamarii strain.

The production of aflatoxins B1 and B2 by Aspergillus tamarii (subgenus Circumdati section Flavi) is reported for the first time. The fungus was isolated from soil collected from a tea (Camellia sinensis) field in Miyazaki Prefecture, Japan. Three single-spore cultures, NRRL 25517, NRRL 25518, and NRRL 25519, were derived from subcultures of the original isolate 19 (MZ2). Each of these single-spore cultures of A. tamarii produced aflatoxins B1 and B2 and cyclopiazonic acid, as well as black, pear-shaped sclerotia. The demonstration of aflatoxin production by A. tamarii is examined in connection with A. tamarii phylogenetic relationships, chemical ecology, and potential use in food fermentations.

AspergillusSpecies and Mycotoxins in Figs from California Orchards

Although 23 different Aspergillus spp. decayed figs in California orchards, only A. niger occurred in more than 0.2% of the figs. The black-spored Aspergillus isolates that caused the disease fig smut were classified as A. niger var. niger, A. niger var. awamori, A. japonicus, and A. carbonarius. Different fungi differed in their association with Aspergillus Section Nigri (causal agents of fig smut) infections in figs. For example, most figs infected with Aspergillus Section Flavi (potential aflatoxin producers) also had infections by Section Nigri. For other fungi, there was either no significant relationship between fig infections by these fungi and Section Nigri or simultaneous infections by Section Nigri were fewer than expected. Insect damage to the fig fruit, predominantly by navel orangeworm (Amyelois transitella), did not significantly increase the colonization of figs by Aspergillus spp. The incidences of infection by Aspergillus (Sections Nigri, Aspergillus, Flavi, and Circumdati) in figs differed little for different harvests. Figs naturally infected with A. alliaceus, A. melleus, A. ochraceus, and A. sclerotiorum of Aspergillus Section Circumdati contained ochratoxin up to 9,600 ng/g, although only 40% of the figs with these fungi had more than a trace amount of ochratoxin. Aflatoxin contamination in figs naturally infected with Aspergillus Section Flavi varied according to the species involved. No aflatoxins were detected in all figs infected with A. tamarii and in most figs infected with A. flavus. High levels of aflatoxin (>100 ng/g) were detected in 83% of the figs infected by A. parasiticus, but in only 32% of the figs infected by A. flavus. Section Flavi isolates from fig orchard soils were tested for their ability to produce the mycotoxins aflatoxin and cyclopiazonic acid. Aspergillus parasiticus isolates always produced aflatoxin but never cyclopiazonic acid; A. flavus strain S (producers of small sclerotia) isolates always produced both aflatoxin and cyclopiazonic acid, but strain L (producers of large sclerotia) isolates frequently did not produce aflatoxin or cyclopiazonic acid; and A. tamarii isolates never produced aflatoxin but always produced cyclopiazonic acid. Aspergillus flavus was recovered from the soil, at fewer than 6 CFU/g of dry soil of every fig orchard assayed in 1992 and 1993. Although A. parasiticus was rarer in fig fruit than was A. flavus for each year, in orchard soil A. parasiticus was more frequent than A. flavus. Isolates of A. flavus strain L were much more common in the orchard soil and fig fruit than those of strain S. Figs in commercial orchards seem to be a favorable substrate for infection by and growth of Aspergillus spp.

Electrophoretic comparison of enzymes as a chemotaxonomic aid among Aspergillus taxa: (3) The identity of the xerophilic species A. penicillioides in subgenus Aspergillus section Restricti.

The identity of the xerophilic species Aspergillus penicillioides and related taxa has been studied chemotaxonomically using polyacrylamide slab gel electrophoresis. In a dendrogram based on the calculated similarity values of five enzymes, 14 isolates of A. penicillioides in subgenus Aspergillus section Restricti and 28 isolates of related taxa in subgenus Aspergillus formed 22 clusters at a 60% similarity level. A. penicillioides isolates were scattered over the 11 clusters. In the ubiquinone systems, all isolates of A. penicillioides, including IFO 8155 (originally described as A. vitricolae [sic]), had the Q-9 system, which was the same ubiquinone system as Eurotium spp. A case study based on comparisons between the similarity values in enzyme patterns and the homology values in DNA relatedness in subgenus Circumdati section Flavi clearly indicates that Aspergillus isolates at a 60% or greater similarity level in enzyme patterns are considered to be conspecific. According to this guideline, A. penicillioides isolates are extremely diverse and presumably assigned to several species and we revive the name A. vitricola for IFO 8155 (ex holotype). Further molecular systematic studies are needed to clarify the identity and speciation of A. penicillioides.

Phylogenetic analysis of Aspergillus sections Cremei and Wentii, based on ribosomal DNA sequences

Large subunit ribosomal DNA from Aspergillus species in sections Cremei, Wentii, and some morphologically related species in subgenus Circumdati, was amplified using PCR techniques. The nucleotide sequences were determined from the amplified DNA and phylogenetic species relationships were inferred from parsimony analysis of the data. On the basis of the analysis, section Wentii is abandoned. All species formerly assigned to section Wentii are phylogenetically related to species in other sections of subgenus Circumdati (e.g. sections Cremei, Flavi). Aspergillus gorakhpurensis (section Sparsi), A. dimorphicus (section Circumdati) and A. pulvinus (section Versicolores) are transferred to section Cremei. Genetic and morphological analysis support these changes in Aspergillus subgeneric classification. A key to the species in the modified section Cremei is provided.