Diversity within Aspergillus niger Clade and Description of a New Species: Aspergillus vinaceus sp. nov.

Diversity of species within Aspergillus niger clade, currently represented by A. niger sensu stricto and A. welwitshiae, was investigated combining three-locus gene sequences, Random Amplified Polymorphic DNA, secondary metabolites profile and morphology. Firstly, approximately 700 accessions belonging to this clade were investigated using calmodulin gene sequences. Based on these sequences, eight haplotypes were clearly identified as A. niger (n = 247) and 17 as A. welwitschiae (n = 403). However, calmodulin sequences did not provide definitive species identities for six haplotypes. To elucidate the taxonomic position of these haplotypes, two other loci, part of the beta-tubulin gene and part of the RNA polymerase II gene, were sequenced and used to perform an analysis of Genealogical Concordance Phylogenetic Species Recognition. This analysis enabled the recognition of two new phylogenetic species. One of the new phylogenetic species showed morphological and chemical distinguishable features in comparison to the known species A. welwitschiae and A. niger. This species is illustrated and described as Aspergillus vinaceus sp. nov. In contrast to A. niger and A. welwitschiae, A. vinaceus strains produced asperazine, but none of them were found to produce ochratoxin A and/or fumonisins. Sclerotium production on laboratory media, which does not occur in strains of A. niger and A. welwitschiae, and strictly sclerotium-associated secondary metabolites (14-Epi-hydroxy-10,23-dihydro-24,25-dehydroaflavinine; 10,23-Dihydro-24,25-dehydroaflavinine; 10,23-Dihydro-24,25-dehydro-21-oxo-aflavinine) were found in A. vinaceus. The strain type of A. vinaceus sp. nov. is ITAL 47,456 (T) (=IBT 35556).

Occurrence and diversity of Aspergillus in commercial yerba mate elaborated for the Brazilian beverage 'chimarrão'.

Dried leaves and stems of Ilex paraguariensis St. Hil. (yerba mate) are used to make a popular beverage in some countries of South America, commonly known as "chimarrão". The present study was designed to evaluate the occurrence of toxigenic Aspergillus in yerba mate in order to define the mycotoxin risk associated with this foodstuff. All samples tested were positive for fungal contamination, and the fungal load per sample ranged from 2.0 × 102 to 1.6 × 104 CFU/g. Aspergillus section Nigri was found in all samples and represented 76.5% of the total fungi isolated. Aspergillus section Circumdati, Aspergillus section Flavi and Aspergillus section Cremei were found at low frequencies. Thirteen different Aspergillus species were identified. The most common species found was A. luchuensis, which does not produce any harmful toxin for humans. A. niger, A. welwitschiae, A. flavus and A. novoparasiticus, all potentially toxigenic species, were found only in small quantities. The A. niger and A. welwitschiae strains were cultured to test for ochratoxin A and fumonisin B2 production. Only one strain producing ochratoxin A was found, but approximately 29% of the strains were positive for fumonisin B2. The A. flavus and A. novoparasiticus strains were tested for aflatoxins production, and 63% were positive. A. pallidofulvus, recently assigned to A. section Circumdati, was reported for the first time in herbs. All A. pallidofulvus strains analyzed in this study were negative for ochratoxin A production. In conclusion, A. section Nigri occurs with high frequency in yerba mate, and A. luchuensis is the predominant species. Although toxigenic species were found in this herb, the incidence was low.

Aspergillus labruscus sp. nov., a new species of Aspergillus section Nigri discovered in Brazil.

A novel fungal species, Aspergillus labruscus sp. nov., has been found in Brazil during an investigation of the fungal species present on the surface of grape berries (Vitis labrusca L.) for use in the production of concentrated grape juice. It seems to be associated to V. labrusca, and has never been recovered from Vitis vinifera. This new species belonging to Aspergillus subgenus Circumdati section Nigri is described here using morphological characters, extrolite profiling, partial sequence data from the BenA and CaM genes, and internal transcribed spacer sequences of ribosomal DNA. Phenotypic and molecular data enabled this novel species to be clearly distinguished from other black aspergilli. A. labruscus sp. nov. is uniseriate, has yellow mycelium, poor sporulation on CYA at 25 °C, abundant salmon to pink sclerotia and rough conidia. Neoxaline and secalonic acid D were consistently produced by isolates in this taxon. The type strain of A. labruscus sp. nov. is CCT 7800 (T) = ITAL 22.223 (T) = IBT 33586 (T).

Occurrence and fumonisin B2 producing potential of Aspergillus section Nigri in Brazil nuts.

Bertholletia excelsa is the tree that produces Brazil nuts which have vast economic importance in the Amazon region and as an export commodity. The aim of this study was to assess the presence of Aspergillus section Nigri in Brazil nut samples at different stages of its production chain and to verify the toxigenic potential for fumonisin B2 (FB2) production of these isolates along with the presence of this mycotoxin in Brazil nut samples. The fungal infection ranged from 0 to 80% at the different stages of the harvest and processing chain and the water activity of the nuts from 0.273 to 0.994. A total of 1052 A. section Nigri strains were isolated from Brazil nuts and 200 strains were tested for their ability to produce FB2: 41 strains (20.5%) were FB2 producers with concentrations ranging from 0.09 to 37.25 mg/kg; 2 strains (1%) showed traces of FB2, less than the detection limit (0.08 mg/kg); and 157 (78.5%) were not FB2 producers. Although several samples showed high contamination by A. section Nigri, no sample was contaminated by FB2.

Biodiversity of mycobiota throughout the Brazil nut supply chain: From rainforest to consumer.

A total of 172 Brazil nut samples (114 in shell and 58 shelled) from the Amazon rainforest region and São Paulo state, Brazil was collected at different stages of the Brazil nut production chain: rainforest, street markets, processing plants and supermarkets. The mycobiota of the Brazil nut samples were evaluated and also compared in relation to water activity. A huge diversity of Aspergillus and Penicillium species were found, besides Eurotium spp., Zygomycetes and dematiaceous fungi. A polyphasic approach using morphological and physiological characteristics, as well as molecular and extrolite profiles, were studied to distinguish species among the more important toxigenic ones in Aspergillus section Flavi and A. section Nigri. Several metabolites and toxins were found in these two sections. Ochratoxin A (OTA) was found in 3% of A. niger and 100% of A. carbonarius. Production of aflatoxins B and G were found in all isolates of A. arachidicola, A. bombycis, A. nomius, A. pseudocaelatus and A. pseudonomius, while aflatoxin B was found in 38% of A. flavus and all isolates of A. pseudotamarii. Cyclopiazonic acid (CPA) was found in A. bertholletius (94%), A. tamarii (100%), A. caelatus (54%) and A. flavus (41%). Tenuazonic acid, a toxin commonly found in Alternaria species was produced by A. bertholletius (47%), A. caelatus (77%), A. nomius (55%), A. pseudonomius (75%), A. arachidicola (50%) and A. bombycis (100%). This work shows the changes of Brazil nut mycobiota and the potential of mycotoxin production from rainforest to consumer, considering the different environments which exist until the nuts are consumed.