Browsing by Author "Santos, Cledir"
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- Calmodulin gene as good voucher as MALDI-TOF ICMS to identify Portuguese isolates of Aspergillus section FlaviPublication . Rodrigues, Paula; Santos, Cledir; Venâncio, Armando; Lima, NelsonAspergillus 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 (internal transcribed spacer (ITS) and partial LSU-rRNA regions), calmodulin and ~-tubulin genes, have been widely reported as good markers for Aspergillus species identification, because they are rapid and cost-effective.
- Characterization and identification of aspergillus section flavi isolates from portuguese almonds using a polyphasic approach including MALDI-TOF ICMSPublication . Rodrigues, Paula; Santos, Cledir; Venâncio, Armando; Lima, NelsonAspergillus is a large genus, with a complex and ever evolving taxonomy. Section Flavi is one of the most significant sections in the genus, and is one of the best studied among fungi, for the numerous industrial applications as well as for food safety issues. Section Flavi 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 some are responsible for the production of the highly toxigenic aflatoxins. Taxonomy and species identification are therefore subject of great interest for scientists aiming to clarify the species concept and limits within the section. In this sense, the establishment of schemes for species and for aflatoxigenic strains identification that are simultaneously accurate, sensitive, robust and expedite is mandatory. At present, reliable identification schemes typically imply the analysis of a wide 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, which result in a taxa specific fingerprint. This technique has already shown high potentialities to discriminate very closely related taxa, but has rarely been used in fungal species identification, either on its one or as part of a polyphasic scheme of identification. This work aimed to: i) characterize the population of Aspergillus section Flavi collected from Portuguese almonds in relation to their aflatoxigenic potential; ii) identify the isolates by applying a set of morphological, biochemical, molecular and spectral analyses (polyphasic approach); iii) compare the data obtained from the various approaches in terms of sensitivity, reliability and user-friendliness; and iii) determine the validity of MALDI-TOF technique for the identification of closely related field isolates of section Flavi.
- Conservation status of the threatened Iberian Peninsula narrow endemic Antirrhinum lopesianum Rothm. (Scrophulariaceae)Publication . Bernardos, Sónia; Amado, Anabela; Aguiar, Carlos; Santos, Cledir; Fernández Diez, Javier; Gonzalez-Talavan, A.; Amich, FranciscoAntirrhinum lopesianum Rothm. is a narrow endemic of the Lusitan Duriensean biogeographical sector (central western Spain and north-eastern Portugal). The species is listed as threatened in several Spanish documents, although it does not figure as such in any Portuguese document. This paper provides a detailed study of its distribution, estimates of the sizes of its populations, the threats it faces, and its current conservation status. The total number of individuals thought to exist is only 768, distributed along the valley of the River Duero on the Spanish – Portuguese border (562, 71.2%), and in the Portuguese Sabor River valley (206, 26.8%). The main threat to the species is loss of habitat: about one third of the Iberian populations can be considered threatened; one population containing 37.6% of all these plants (289) is severely threatened. To determine the Area of Occupancy and the Extent of Occurrence, an exhaustive bibliographical survey was carried out, and herbarium specimens deposited in several institutions were revised. It is, therefore, classifiable as Critically Endangered in Portugal and Endangered in Spain
- Contributions of Micoteca da Universidade do Minho to food mycologyPublication . Rodrigues, Paula; Soares, Célia; Freitas-Silva, Otniel; Santos, Cledir; Paterson, Russell; Kozakiewicz, Zofia; Venâncio, Armando; Lima, NelsonMicoteca da Universidade do Minho (MUM) is a collection of fungi established in 1996. Research involving secondary metabolites in the form of mycotoxins is paramount. An objective is to provide authentic strains for research in biotechnology, life sciences and challenge testing. Knowledge, information and training in mycology are promulgated by the biological resource centre. MUM is involved in projects which address the health and quality risks from fungal contamination of grape products (e.g. wine), apples, cheese, chilies, nuts and corn: Fungi in drinking water is of particular concern. Aspergillus ochraceous, Aspergillus flavus, Penicillium expansum and Fusarium graminearum especially are relevant to our work: Associated mycotoxins are ochratoxin A, aflatoxins, patulin, citrinin and zearalenone. We study the earthy off-flavours in wine from Botrytis cinearum and P. expansum. The analytical techniques employed are PCR, HPLC, GC-MS and MALDITOF MS for strain characterization. MUM participates in several European and national projects. Post-graduate studies on mycotoxigenic fungi and food security particularly are important. A key objective is international collaboration. MUM strains are well characterized for toxigenicity, making the collection relevant to its mission and industry. This presentation will describe the progress made in this innovative Portuguese laboratory over 10 years.
- Integrated morphologic, metabolic, molecular and spectral methods for identification of Aspergillus section Flavi isolated from Portuguese almonds.Publication . Rodrigues, Paula; Santos, Cledir; Venâncio, Armando; Lima, NelsonAspergillus 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 food industry. Taxonomy of Section Flavi currently depends on multivariate approaches entailing both phenotypic (morphology, extrolite profiles) and molecular traits. No method works flawlessly in recognising species, so polyphasic approaches should be given emphasis in taxonomic decision-making. This work aims to identify strains from Section Flavi by combining various methods, including morphological, biochemical and molecular ones, as well as the novel approach based on spectral analysis by MALDI-TOF-ICMS. About 350 fungal isolates from Portuguese almonds, all belonging to Section Flavi, were characterised morphologically and biochemically. From those, 26 were selected for further analysis. Mycotoxigenic patterns, partial calmodulin gene sequences and MALDI-TOF protein spectra were analysed, dendrograms were created and results were compared. We obtained good agreement between methods on species level identification. Additionally, the few incongruencies detected between morphological and biochemical data were well resolved with the molecular and spectral analyses.
- MALDI-TOF ICMS as a modern approach to identify potential aflatoxigenic fungiPublication . Rodrigues, Paula; Santos, Cledir; Venâncio, Armando; Lima, NelsonThe 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 potentialities and limits to characterise aflatoxigenic species of Aspergillus Section FlaviPublication . Rodrigues, Paula; Santos, Cledir; Kallow, Wibke; Erhard, Marcel; Welker, Martin; Kozakiewicz, Zofia; Lima, Nelson; Venâncio, Armando
- MALDI-TOF MS: improved methods for the identification/characterisation and authentication of fungal strainsPublication . Santos, Cledir; Rodrigues, Paula; Venâncio, Armando; Lima, NelsonThe 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.
- Micotoxinas: contributos da micoteca da Universidade do Minho (mum) para a segurança alimentarPublication . Rodrigues, Paula; Soares, Célia; Freitas-Silva, Otniel; Santos, Cledir; Paterson, Russell; Kozakiewicz, Zofia; Venâncio, Armando; Lima, NelsonA Micoteca da Universidade do Minho (MUM), fundada em 1996, tem como missão ser uma colecção de fungos filamentosos com o objectivo principal de manter e fornecer estirpes com qualidade e autenticidade para a investigação em biotecnologia e ciências da vida, e laboratórios de ensaio, actuando também como um centro de conhecimento, informação e formação na área da micologia. Dentro desta missão, a MUM tem estado envolvida em projectos que procuram dar resposta aos riscos alimentares derivados da contaminação fúngica.
- Micotoxinas: contributos da Micoteca da Universidade do Minho (MUM) para a segurança alimentar.Publication . Rodrigues, Paula; Soares, Célia; Freitas-Silva, Otniel; Santos, Cledir; Paterson, Russell; Venâncio, Armando; Lima, NelsonA Micoteca da Universidade do Minho (MUM), fundada em 1996, tem como missão ser uma colecção de fungos filamentosos com o objectivo principal de manter e fornecer estirpes com qualidade e autenticidade para a investigação em biotecnologia e ciências da vida, e laboratórios de ensaio, actuando também como um centro de conhecimento, informação e formação na área da micologia. Dentro desta missão, a MUM tem estado envolvida em projectos que procuram dar resposta aos riscos alimentares derivados da contaminação fúngica.