Of 290 brazil nut samples (nuts and shells), 15 samples showed the presence of Aspergillus bertholletius
, with an incidence ranging from 2 to 46% infection after direct plating on DG18. In total, 65 isolates were found from shells and nuts and from soil close to Bertholletia excelsa
trees. The origin of the Aspergillus bertholletius
isolates is shown in , and the incidence of A. bertholletius
in the samples throughout the brazil nut chain in . Most samples were not infected by A. bertholletius
. However, one sample from a street market in the Amazon region was highly infected, with 46% and 36% of nuts and shells infected. Of 28 samples of soil from areas adjacent to B. excelsa
trees, only one was contaminated with A. bertholletius,
showing a count of 8.0×103
CFU/g. Isolation of A. bertholletius
from brazil nuts and soil may have been underestimated because colonies of A. bertholletius
on DG18 are similar to those of A. tamarii
. Distinctions were found after incubating isolates on CYA at 37°C, where colonies of A. bertholletius
are 5 to 15 mm in diameter, while those of A. tamariii
are 50 mm or more in diameter 
. When cultured on AFPA, A. bertholletius
is readily recognised from A. tamarii
on AFPA by a cream colony reverse, unlike the dark brown characteristic of A. tamarii.
On the other hand, A. bertholletius
differs from A. flavus
and A. parasiticus
which give an orange reverse colour on AFPA due to the production of aspergilic acid or noraspergillic acid which react with ferric ammonium citrate present in the medium 
Aspergillus bertholletius isolates from brazil nuts (nuts and shell) and soil from Amazonian rainforest.
Incidence of A. bertholletius in brazil nut samples.
Apart from very slow growth at 37°C, the morphology of strains of A. bertholletius are consistent with placement within Aspergillus section Flavi. However, that striking difference in growth rate at 37°C correlates well with the distinct separation of A. bertholletius from other species in section Flaviin a neighbour joining tree derived from b-tubulin and calmodulin sequences. The nucleotide sequence data of β-tubulin and calmodulin genes matched in showing that the A. bertholletius isolates represent a new phylogenetic clade in Aspergillus section Flavi ( and ). In addition, A. bertholletius was also differentiated from all other known Aspergillus when analyzing the ITS1–5.8S–ITS2 region. A comparison of a 459-bp fragment from this region of A. bertholletius relative to A. pseudotamarii, the taxon with the most similar sequence indicated by BLASTn tool, revealed 6 nucleotide substitutions and 3 insertion/deletions ().
Neighbour joining tree reconstructed from the β-tubulin gene sequences aligned with corresponding sequences of Aspergillus section Flavi type species deposited in public databases.
Neighbour joining tree reconstructed from the calmodulin partial gene sequences aligned with corresponding sequences of Aspergillus section Flavi type species deposited in public databases.
Nucleotide sequence alignment of a 459-bp fragment of the ITS-5.8S-ITS region of Aspergillus bertholletius (accession no. JX 198673, present study) and A. pseudotamarii (AF004931).
Metabolite analysis indicated that A. bertholletius does not produce aflatoxins. However, one strain, the ex type strain, CCT 7615, produced O-methylsterigmatocystin, indicating that A. bertholletius may have silent genes for aflatoxin production. All strains produced the mycotoxin cyclopiazonic acid or its precursors and five of 18 strains examined produced the mycotoxin tenuazonic acid. Other metabolites produced were kojic acid (17/18 strains), ustilaginoidin C (9/18 strains) and indole alkaloids (16/18 strains). The isolates exhibited a unique profile of metabolites, consistent with production by an undescribed species.
shares the production of cyclopiazonic acid with A. flavus, A. minisclerotigenes, A. oryzae, A. parvisclerotigenus, A. pseudocaelatus, A. pseudotamarii
, and A. tamarii
. It shares the ability to produce tenuazonic acid with A. caelatus
and A. nomius
and O-methylsterigmatocystin with all aflatoxin producers. It shares kojic acid with all species in Aspergillus
, except A. avenaceus
shows the morphology of A. bertholletius colonies on Czapek yeast extract agar and malt extract agar after 7 days incubation at 25°C and the conidial heads.
Aspergillus bertholletius Taniwaki, Pitt & Frisvad sp. nov. [urn:lsid:mycobank.org: 800125]
Named from the generic epithet of the brazil nut tree Bertholletia excelsa, the known habitat for this species.
CCT 7615 in Coleção de Cultura Tropical (Campinas, Brazil) is designated as the holotype of Aspergillus bertholletius. It was isolated from soil close to Bertholletia excelsa trees, Instituto de Tecnologia de Alimentos, Campinas, Brazil, 2006. Cultures derived from type include ITAL 270/06 (where ITAL is the culture collection of Instituto de Tecnologia de Alimentos, Campinas, Brazil), and IBT 29228 (where IBT is the culture collection of the Technical University of Denmark, Lyngby, Denmark).
This species differs from of species in Aspergillus section Flavi by slow growth on CYA at 37°C, linoleum brown conidia en masse, a unique profile of secondary metabolites and a distinct DNA sequence in the region of the ß-tubulin and calmodulin genes.
Colonies on CYA 60–70 mm in diameter, often almost covering the Petri dish, deep but velutinous; margins entire, narrow; mycelium inconspicuous; conidiogenesis heavy, brown near Linoleum Brown (M. 5E7); exudate and soluble pigment absent; reverse uncoloured to pale brown. Colonies on MEA 60–70 mm in diameter, similar to on CYA, but conidia slightly more green than on CYA, olive brown near Khaki (M. 4D5-E7); reverse pale.
Colonies on G25N 25 mm in diameter, low, often heavily sporing in colours near those on MEA; reverse pale.
Colonies on CYA at 37°C 5–15 mm in diameter, sometimes with brown sporulation.
Conidiophores borne from surface or aerial hyphae, 70–150×6–8 µm, with very thin, smooth walls; vesicles spherical, 10–17(–20) µm in diameter, bearing uncrowded phialides; phialides ampulliform, large and broad, 10–14×5–6(–7) µm; conidia uniform in size and shape, spherical, 5.5–6.5 µm in diameter, with finally spinose walls, borne in long, tangled chains. Sclerotia are not produced on any media.
Other isolates examined
ITAL 116 (
CCT 7612), ITAL 259 (
CCT 7613),ITAL 262(
IBT 31739),ITAL 271/06 (
IBT 30618), ITAL 272/06 (
IBT 30617), ITAL 273/06 (
IBT 30619), ITAL 275/06 (
IBT 29227), ITAL 7157 (
IBT 31548), ITAL 7179 (
IBT 31554), ITAL 7180 (
IBT 31555), ITAL 7189 (
IBT 31151), ITAL 7191 (
IBT 31553), ITAL 7192 (
CCT 7619), ITAL 7193(
IBT 31549), ITAL 7194 (
IBT 31556), ITAL 7195 (
IBT 31557), ITAL 7196 (
IBT 31546) and ITAL 7197(
IBT 31500), all from nuts of Bertholletia excelsa,
the brazil nut tree and soil close to the tree.