European Consortium for Microbial Resource Centres

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MALDI-TOF ICMS as a modern approach to identify potential aflatoxigenic fungi

Publication . Rodrigues, Paula; Santos, Cledir; Venâncio, Armando; Lima, Nelson

The Aspergillus section Flavi is among the best studied fungi, having different commercial applications, but also causing biodeterioration of commodities and food spoilage. Fungi from this Section are also responsible for the production of highly toxic secondary metabolites – the aflatoxins. They are morphologically and genetically very similar, and can be difficult to differentiate by both cultural and molecular biology methods. Besides that, new species are continuously being described in this Section. A reliable identification typically implies the analyses of a variety of morphological, biochemical and molecular traits. Recently, Matrix-Assisted Laser Desorption/Ionisation Time-Of-Flight Intact Cell Mass Spectrometry (MALDI-TOF ICMS) has been used to generate spectra of protein masses in a range of 2,000 to 20,000 Da that are a taxa specific fingerprinting. This technique has already shown high potentialities to discriminate very closely related taxa and, it has been used as a new tool in the polyphasic approach to identify potential aflatoxigenic fungi. Aim: This work aims to validate the MALDI-TOF ICMS technique on Aspergillus Section Flavi identification. As a matter of consequence, obtained results by spectral analysis were compared to those obtained by morphological, biochemical and molecular biology methods. Materials and Methods: 1. Morphological analysis: fungi were cultured on three different media (Malt Extract Agar [MEA], Czapek Yeast Autolysate [CYA] and CYA supplemented with 20% sucrose [CY20S]); 2. Biochemical analysis: Aflatoxins and Cyclopiazonic Acid analyses were performed by HPLC; 3. Molecular biology analysis: Partial calmodulin gene was sequenced; 4. MALDI-TOF ICMS analysis: spectra of protein masses, on 2,5-dihydroxybenzoic acid (DHB) in a range of 2,000 to 20,000 Da, were obtained using Shimadzu Axima-LNR equipment and treated for fungal identification using SARAMISTM Package. Results and Discussion: 1. A good agreement between methods on species level identification was obtained; 2. Molecular biology and spectral data analyses generated similar dendrograms with concomitant strains clustering; 3. Under the experimental conditions used spectral analyses were able to identify potential aflatoxigenic species. Conclusion: MALDI-TOF ICMS has shown a very good resolution on the identification of Aspergillus Section Flavi species. Results obtained with MALDI‐TOF ICMS were similar to those obtained by DNA sequence analysis, with the advantage of being (a) rapid, (b) inexpensive in terms of labour and consumables, and (c) reliable when compared with other biological techniques. Using MALDI‐TOF ICMS the results showed a great potential to the fungal identification and it is another additional step for our polyphasic fungal identification approach. However, even with the polyphasic approach fungal identifications remain in some situations time‐consuming and decisions regarding what represents a species tend to be subjective.

2010Conference object

Open access

MALDI-TOF MS: improved methods for the identification/characterisation and authentication of fungal strains

Publication . Santos, Cledir; Rodrigues, Paula; Venâncio, Armando; Lima, Nelson

The identification of species is an important goal in taxonomic mycology. Information about each fungus (e.g. morphological description, physiological and biochemical properties, ecological roles, and societal risks or benefits) is key element in this process. Identifications can be a long and seemingly never-ending process with frequent revisions of the taxonomic schemes. These changes make identifications even more complicated for the non-specialised researchers as each taxonomic group has specialised literature, terminology and characters. This occurs to the extent that identifications can only be undertaken by a narrow group of scientists especially skilled in the “art”, which can make the procedures appear to be subjective. Aspergillus is a large fungal genus, with a complex and ever evolving taxonomy. Section Flavi is one of the most significant sections in this 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. In the present study, from 352 isolates of Aspergillus section Flavi obtained from Portuguese almonds and identified based on morphological, biochemical and MALDI-TOF MS profiles, 24 isolates were further characterised through molecular analyses by use of ITS region and calmodulin gene. Molecular results confirm that ITS gene was not able to resolve differences at the species-level on this particular taxonomic group. In contrast, calmodulin gene was a robust and reliable genomic marker for this taxon. In conclusion, the results obtained from MALDI-TOF MS confirm that this technique is as good as calmodulin gene analysis for fungal identification. Another important output of this work was the clear evidence that two putative new species were present among these isolates. Finally, MALDI-TOF MS technique is rapid, reliable and inexpensive in terms of labour and consumables when compared with molecular techniques. At present, it adds an additional step for polyphasic identification which is essential when there is a paucity of characters for defining many fungal species.

2012Conference object

Open access