Aspergillus bertholletius sp. nov. from Brazil Nuts

During a study on the mycobiota of brazil nuts (Bertholletia excelsa) in Brazil, a new Aspergillus species, A. bertholletius, was found, and is described here. A polyphasic approach was applied using morphological characters, extrolite data as well as partial β-tubulin, calmodulin and ITS sequences to characterize this taxon. A. bertholletius is represented by nineteen isolates from samples of brazil nuts at various stages of production and soil close to Bertholletia excelsa trees. The following extrolites were produced by this species: aflavinin, cyclopiazonic acid, kojic acid, tenuazonic acid and ustilaginoidin C. Phylogenetic analysis using partial β-tubulin and camodulin gene sequences showed that A. bertholletius represents a new phylogenetic clade in Aspergillus section Flavi. The type strain of A. bertholletius is CCT 7615 ( = ITAL 270/06 = IBT 29228).


Introduction
Brazil nuts are one of the most important products extracted from the Amazon rainforest region. Trees of Bertholletia excelsa grow wild, reaching up to 60 meters, take 12 years to bear fruit and may live up to 500 years. The trees are found in groves of 50-100 individuals and the groves are separated by up to 1 km. Pollination is by wild, large bodied bees, especially Euglossinae species [1]. The Amazon rainforest has multiple ecosystems with a huge biodiversity. It has an important role in the global weather balance and is the location of many native peoples. The equatorial climate is hot and humid, with an average temperature of 26uC and relative humidity of 80-95%.
Among these species, a major concern relates to those within Aspergillus section Flavi, because some of them have the potential for aflatoxin production. A major challenge in brazil nut production is controlling the high rate of contamination by species within Aspergillus section Flavi and hence the potential for high aflatoxin production.
The taxonomy of this section is still highly complex and continually evolving. Phylogenetic analysis based on sequence data from b-tubulin and calmodulin genes revealed that Aspergillus section Flavi includes seven main clades (A. flavus clade, A. tamarii clade, A. nomius clade, A. alliaceus clade, A. togoensis clade, A. leporis clade, and A. avenaceus clade), with 20 or more taxa [6]. According to these authors, the main clades are well defined. However, many subclades are represented by a single isolate and further collections and studies are needed to clarify speciation in this section.
During a course of studies on the mycobiota of brazil nuts, a new Aspergillus taxon in Aspergillus section Flavi was found in soil and several brazil nut samples collected at various stages of the production chain. This species is described here as Aspergillus bertholletius sp. nov. It does not produce aflatoxin.

Fungal isolation from brazil nuts samples and soil
A total of 290 brazil nut samples (174 nuts and 116 shells) each of approximately 2 kg were collected in the Amazon region and São Paulo State, Brazil. Besides that, 28 samples of soil (each approximately 200 g) were collected from Amazon rainforest close to Bertholletia excelsa trees. The sampling was carried out together with the Brazilian Ministry of Agriculture and all necessary permits were obtained for the described field studies. The taxonomic study is supported by Brazilian Resolution of Genetic Heritage Management Council (MMA/CGEN 21/06).
Approximately 100 g of shelled nuts and 100 g of shells were disinfected separately by immersion in 0.4% sodium hypochlorite solution for 1 min. Fifty pieces of nuts or shells were plated onto Dichloran 18% Glycerol agar (DG18), according to the methodology of Pitt and Hocking [7]. Plates were incubated for 5 days at 25uC.
For soil samples, under aseptic conditions, samples (25 g) were weighed and sterile peptone water (0.1%; 225 ml) was added. Aliquots were serially diluted and spread plated onto Dichloran 18% Glycerol agar. The plates were incubated at 25uC for 7 days, according to Pitt and Hocking [7]. All isolateswith the appearance of belonging to Aspergillus section Flavi were isolated onto Czapek yeast extract agar [7]and incubated at 25uC for 7 days.

Morphological examination
The fungi were examined on standard identification media for Aspergillus species, namely Czapek yeast extract agar (CYA), malt extract agar (MEA), Aspergillus flavus and parasiticus agar (AFPA) and 25% glycerol nitrate agar (G25N) [7] at 25uC and also at 37uC and 42uC on CYA. The incubation time for all media and conditions was 7 days.
The standard conditions used for the description of Aspergillus bertholletius are taken from Pitt and Hocking [7]. Capitalised colours are from the Methuen Handbook of Colour [8].

Molecular analysis
Isolates were cultivated in yeast extract and lactose (YEL) solid medium for seven days. From each culture, a suspension of approximately 10 7 conidia suspended in Tween 80 (2.5 ml) was inoculated into bottles containing YEL liquid (50 ml), and incubated in a shaker (180 rpm) at 28uC for 16 to 24 h. After incubation, mycelia were collected by vacuum filtration and washed in sterile water. Nucleic acids were extracted according to Azevedo et al. [9], and treated with ribonucleaseA (20 mg/ml). Partial amplification of the ß-tubulin gene was performed using standard amplification reactions and the following primer pair: Bt2a (59 GGT AAC CAA ATC GGT GCT TTC 39) and Bt2b (59 ACC CTC AGT GTA GTG ACC CTT GGC 39), as described by Glass and Donaldson [10]. Part of the calmodulin gene region was amplified using the cmd5 (59 CCG AGT ACA AGG AGG  CCT TC 39) and cmd6 (59 CCG ATA GAG GTC ATA ACG TGG 39) primers previously reported by Hong et al. [11]. Similarly, the ITS1-5.8S-ITS2 region of rDNA was amplified with the primer ITS1 (59 TCCGTAGGTGAACCTGCGG39) and ITS4 (TCCTCCGCTTATTGATATGC39) [12]. Fragments generated by PCRwere purified with WizardH SV Gel and PCR Clean-Up System (Promega). The amplicons were submitted to direct sequencing in both directions (forward and reverse) with a The software package MEGA5 [14] was used to construct a neighbour joining tree [15].

Extrolite analysis
The cultures were analysed by HPLC with diode arraydetection according to the method of Frisvad and Thrane [16] as modified by Houbraken et al. [17]. The isolates were analysed on CYA and YES agar using three agar plugs. Five plugs of each agar medium were taken and pooled into same vial for extraction with 0.75 ml of a mixture of ethyl acetate/dichloromethane/methanol (3:2:1)

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Results and Discussion
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 Table 1, and the incidence of A. bertholletius in the samples throughout the brazil nut chain in Table 2. 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 8.0610 3 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 37uC, where colonies of A. bertholletius are 5 to 15 mm in diameter, while those of A. tamariii are 50 mm or more in diameter [7]. 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 [7].
Apart from very slow growth at 37uC, the morphology of strains of A. bertholletius are consistent with placement within Aspergillus section Flavi. However, that striking difference in growth rate at 37uC 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 b-tubulin and calmodulin genes matched in showing that the A. bertholletius isolates represent a new phylogenetic clade in Aspergillus section Flavi (Figure 1 and 2). 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 (Figure 3).
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.
Aspergillus bertolletius 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 Omethylsterigmatocystin with all aflatoxin producers. It shares kojic acid with all species in Aspergillus section Flavi, except A. avenaceus [6]. Figure 4 shows the morphology of A. bertholletius colonies on Czapek yeast extract agar and malt extract agar after 7 days incubation at 25uC and the conidial heads. Diagnosis. This species differs from of species in Aspergillus section Flavi by slow growth on CYA at 37uC, 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.

Aspergillus bertholletius
Description. 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 37uC 5-15 mm in diameter, sometimes with brown sporulation.
Other isolates examined.

Conclusion
A. bertholletius represents a new important phylogenetic clade in Aspergillus section Flavi applying a polyphasic approach using morphological characters, extrolite data, b-tubulin and calmodulin partial gene sequences.