kambarensis, A. subolivaceus and A. thomii[7]), and for the A. tamarii synonym A. terricola[7]). These sequences showed the same two conserved DraI restriction sites, in contrast to distinct RFLP profiles observed in sequences for Aspergillus species not belonging to section Flavi (Additional file 1), as well as
in the WZB117 supplier Aspergillus teleomorphs and non-target genera Mycena, Monascus and Leiothecium. In order to validate the restriction mapping data, PCR RFLP analysis was conducted on PCR-amplified specific mtDNA SSU rRNA amplicons across the different Aspergillus species isolated. PCR-RFLPs with DraI confirmed differentiation of these three section Flavi members from the other Aspergillus species, with digest patterns in agreement with in silico data (Figure 3). Figure 3 Dra I restriction digest profiles of the specific mtDNA
SSU rRNA amplicon for differentiation of Aspergillus section Flavi species members from other aspergilli. M: Low DNA Mass Ladder; 1–3: Aspergillus flavus; 4–5: Aspergillus nomius; 6: Aspergillus tamarii; 7–8: Aspergillus fumigatus; 9–10: Aspergillus niger. Discussion Morphology-based methods for identification of species of the genus Aspergillus can be unreliable as a result of both intraspecific similarities and differences [16]. In this present study, identification of Aspergillus species on Brazil nut from different states in the Brazilian Amazon region was conducted according to Samson and Varga [6] and Baquião et al. [14], through morphological and molecular characterization, buy SHP099 together with extrolite profile (aflatoxins and CPA). As observed in previous studies for section Flavi[24, 31], species identifications based upon analyses of rDNA ITS, β-tubulin and calmodulin gene sequence identities against sequences for ex-type strains available through the NCBI nucleotide nr database provided results in agreement with morphology-based identification and extrolite production. The frequency we observed of aflatoxigenic Aspergillus section Flavi species
from Brazil nut shell material confirmed recent reports that A. nomius and A. flavus are abundant species on Brazil nut across production areas in the Brazilian Amazonian region [14, 32]. In our study, these two species represented over 85% of all Aspergillus species many isolated. Qualitative analysis of mycotoxin production in strains of the Selleckchem IWP-2 mycotoxigenic species representative of the different states of origin supported the identifications, with A. flavus strains producing AFB and CPA, and A. nomius producing AFB and AFG, without CPA production. The extrolite profiles are in agreement with expected chemical characterization data for these member species in the section [16, 33]. Given the documented widespread occurrence of both A. flavus and A. nomius on Brazil nut, together with the known capacity to produce mycotoxins AFB and CPA, and AFB and AFG, respectively, the presence of these species on husk materials represents a threat to safe production of Brazil nut.