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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.
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.
Mycobiota and aflatoxigenic profile of Portuguese almonds and chestnuts from production to commercialisation
Publication . Rodrigues, Paula
Aflatoxin (AF) contamination of nuts is an increasing concern to the consumer’s health. Portugal is a big producer of almonds and chestnuts, but there is no scientific knowledge on the safety of those nuts. AFs B1, B2, G1 and G2 are produced mainly by some species of Aspergillus belonging to section Flavi, which is composed of a large number of very closely related species. While these species are difficult to differentiate morphologically and even genetically, they differ in a characteristic that is of paramount importance for food safety, as only some are responsible for the production of the highly toxigenic AFs. Taxonomy and species identification are therefore subject of great interest, and the establishment of schemes for species and for aflatoxigenic strains identification that are simultaneously accurate, sensitive, robust and expedite is mandatory.
This work had three major goals: the first was to provide knowledge on the general mycobiota, aflatoxigenic fungi and AF contamination of Portuguese almonds and chestnuts, and its evolution throughout the various stages of production (field, storage and processing). For this matter, 45 chestnut samples were collected from orchards from Trás-os-Montes. Forty-seven almond samples were collected in Trás-os-Montes at different stages of production: field, storage and processing. All fungi belonging to genus Aspergillus were isolated and identified to the section level, and all isolates belonging to section Flavi were further tested for their aflatoxigenic ability. Fungi representative of other genera were identified to the genus level. Almond samples were tested for AF contamination.
The mycobiota of almonds and chestnut was found to vary in terms of both matrix and stage of production. Chestnuts were mainly contaminated with the genera Fusarium, Cladosporium, Alternaria and Penicillium, and the genus Aspergillus was only rarely found, whereas almonds were more contaminated with Aspergillus. No Aspergillus section Flavi were isolated from chestnuts. In almonds, Fusarium, Cladosporium, Alternaria and Penicillium decreased from field to the end of processing, whereas Aspergillus increased significantly, including those from section Flavi. In total, 352 fungi belonging to section Flavi were isolated from Portuguese almonds, of which 231 isolates (66%) were aflatoxigenic. Even so, only one sample from storage was found to be contaminated with AFs (4.97 µg/kg) at a level below the maximum levels recently imposed by the Commission Regulation (EU) No 165/2010.
The second goal of this work was to characterise and identify the isolates of Aspergillus section Flavi by applying a polyphasic approach including classic phenotypic and molecular methods as well as the innovative technology protein spectral analysis Matrix-Assisted Laser Desorption/Ionisation-Time of Flight Intact-Cell Mass Spectrometry (MALDI-TOF ICMS), and to devise accurate and sensitive schemes for species identification. For the morphological analysis, fungi were cultured on different media and were characterised for several macro and micro morphological features. Morphological analysis was complemented with biochemical analyses, which consisted of determining the extrolite profiles relative to AFs and cyclopiazonic acid. A group of selected isolates was identified molecularly based on the sequencing of the ITS region and partial calmodulin gene. Spectral analysis was made by MALDI-TOF ICMS to obtain spectra of protein masses. Dendrograms of relatedness were obtained for each set of data and used to compare sensitivity and accurateness of the different approaches.
From the preliminary morphological analysis, three morphotypes were identified: as “A. flavus morphotype” (36.4% of the isolates), “A. parasiticus morphotype” (55.4%), and “A. tamarii morphotype” (8.2%). The 3 morphotypes were then divided into 9 phenotypes based on their extrolite profile. Genotypic and spectral analyses clustered the selected isolates into the same 3 groups created by morphological analysis. Furthermore, all sets of data, including the morphological complemented with extrolite profile, were able to further resolve the isolates into more restrictive clusters. They all positioned two of the 9 phenotypes in two unidentified terminal clades closely related to A. parasiticus.
The third goal was to test a molecular method based on multiplex PCR and RT-PCR for the ability to differentiate aflatoxigenic and non-aflatoxigenic isolates. Two genes of the AF biosynthetic pathway, aflD (= nor1) and aflQ (= ord1= ordA), were tested for presence and expression (by PCR and RT-PCR, respectively). The presence of both genes did not correlate with aflatoxigenicity. In terms of gene expression, aflD was not considered a good marker for differentiating aflatoxigenic from non-aflatoxigenic isolates, but aflQ showed a good correlation between expression and AF-production ability.
In conclusion, Portuguese almonds and chestnuts seem to be generally safe in terms of AF contamination. Nevertheless, the majority of the isolates of Aspergillus section Flavi obtained from Portuguese almonds was found to be aflatoxigenic, which may constitute a problem in terms of food safety if storage and processing conditions are not effectively controlled. At present, these conditions seem to be guaranteed, since only one almond sample was found to be contaminated. At the species identification level, good agreement was obtained between the 3 methods of analysis since they all generated similar dendrograms with concordant strain clustering. Morphological analysis has shown sensitive and reliable as a preliminary method for species identification only when complemented with the extrolite profile. The calmodulin gene showed to be more robust and reliable as genomic marker for this group of fungi than the ITS region, providing good DNA barcoding potential. MALDI-TOF ICMS results confirmed that this technique is highly reliable for fungal identification, and is faster and less expensive in terms of labour and consumables when compared with other biological techniques, which is essential whenever there is a paucity of characters for defining many fungal species and when high numbers of isolates are involved. Expression analysis of the aflQ gene seems to be a good method for the differentiation of aflatoxigenic and non-aflatoxigenic isolates.
Species identification of Aspergillus section Flavi isolates from Portuguese almonds using phenotypic, including MALDI-TOF ICMS, and molecular approaches
Publication . Rodrigues, Paula; Venâncio, Armando; Lima, Nelson
Section Flavi is one of the most significant Sections in the genus Aspergillus. Taxonomy of this section currently depends on multivariate approaches, entailing phenotypic and molecular traits. This work aimed to identify isolates from section Flavi by combining various classic phenotypic and genotypic methods as well as the novel approach based on spectral analysis by MALDI-TOF ICMS, and to evaluate the discriminatory power of the various approaches in species identification.
Methods and Results Aspergillus section Flavi isolates obtained from Portuguese almonds were characterised in terms of macro- and micromorphology, mycotoxin pattern, calmodulin gene sequence and MALDI-TOF protein fingerprint spectra. For each approach, dendrograms were created and results were compared. All data sets divided the isolates into 3 groups, corresponding to taxa closely related to A. flavus, A. parasiticus and A. tamarii. In the A. flavus clade, molecular and spectral analyses were not able to resolve between aflatoxigenic and non-aflatoxigenic isolates. In the A. parasiticus cluster, two well-resolved clades corresponded to unidentified taxa, corresponding to those isolates with mycotoxin profile different from that expected for A. parasiticus.
Conclusions Good agreement was obtained between methods on species level identification. The incongruences detected in classic phenotypic analysis were generally well resolved with the molecular and/or spectral analyses. MALDI–TOF ICMS demonstrated to be sensitive and accurate in species discrimination.
Significance and Impact of Study MALDI–TOF ICMS when compared with other identification methods currently used can be regarded as an objective and fast analytical methodology suitable for applications which have particular needs in high-throughput and highly accurate identification.
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European Commission
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FP7
Número da atribuição
228310