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

Abstract

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.