Portuguese isolates of Aspergillus section Flavi unraveled by the calmodulin gene
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abstract
Aspergillus is a large genus, with a complex and ever evolving taxonomy. Section Flavi is one of the most significant sections in the genus. Taxonomy and species identification is subject of great interest for scientists aiming to clarify the species concept and limits within the section. Furthermore, this section comprises both toxigenic and non-toxigenic species/strains, with great interest to biotechnology and food industry.
Various genes, namely the rRNA (ITS region), calmodulin and β-tubulin genes, have been widely reported as good markers for Aspergillus species identification, because they are rapid and cost-effective.
In the present study, we evaluated the discriminatory power of the ITS region and the calmodulin gene to distinguish closely related taxa within Aspergillus section Flavi. For this purpose, 26 isolates of Aspergillus section Flavi obtained from Portuguese almonds were characterized at various levels: i) phenotypic, regarding various aspects of morphology and physiology; ii) spectral, using MALDI-TOF ICMS to obtain protein fingerprinting; and iii) genotypic, by sequence analysis of a 730 bp segment of the calmodulin gene and a 908 bp segment of the ITS region. For the various methods, dendrograms were created and results were compared.
Both genotypic and spectral analyses divided the isolates in 3 groups corresponding to closely related taxa of A. flavus, A. parasiticus and A. tamarii. Except for the ITS region, all sets of analysis positioned 5 of the 26 isolates in two unidentified clades close to A. parasiticus, and divided the A. flavus group in two distinct clades. The phylogenetic analysis of the calmodulin sequences resulted in very similar dendrograms when using various methods of analysis (Neighbor-Joining, Maximum Likelihood, Bayesian Inference), and altering the analytical parameters did not result in significant changes. Furthermore, the genetic dendrograms were strongly supported by the phenotypic and spectral analyses.
These results confirm the calmodulin gene as a robust and reliable genomic marker for this group of fungi. The unsolved isolate identifications are currently under further analysis.
